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Increased growth hormone response to clonidine in nonobese normoinsulinemic patients with polycystic ovary syndrome
POLYCYSTIC OVARY DISEASE
FERTILITY AND STERILITY威 VOL. 81, NO. 1, JANUARY 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Increased growth hormone response to clonidine in nonobese normoinsulinemic patients with polycystic ovary syndrome Fa´bio Vasconcellos Comim, M.D.,a and Poli Mara Spritzer, M.D., Ph.D.a,b Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clı´nicas de Porto Alegre; and Department of Physiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
Received December 18, 2002; revised and accepted May 28, 2003. Supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), FAPERGS (Fundac¸a˜o de Amparo a` Pesquisa do Rio Grande do Sul), Porto Alegre, Brazil, and PRONEX 26/98 (Programa de Apoio aos Nu´cleos de Exceleˆncia em Pesquisa, Brasilia, Brazil). Presented in part at the 5th International Congress of Neuroendocrinology, Bristol, United Kingdom, July 31 to August 4, 2002. Reprint requests: Poli Mara Spritzer, M.D., Ph.D., Department of Physiology, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, 90050-170-Porto Alegre RS, Brazil (FAX: ⫹55-513316-3453; E-mail: [email protected]). a Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clinicas de Porto Alegre. b Department of Physiology, Universidade Federal do Rio Grande do Sul. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 05.027
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Objective: To assess growth hormone (GH) levels in response to acute clonidine stimulation in nonobese patients with polycystic ovary syndrome (PCOS) in comparison to patients with idiopathic hirsutism (IH) and normal women without hirsutism. Design: Cross-sectional study. Setting: Outpatient clinic, Porto Alegre, Brazil. Patient(s): Fourteen patients with PCOS, 11 women with IH, and 10 age- and weight-matched normal women without hirsutism were studied. All subjects presented normal body mass index (⬍25 kg/m2) and insulin levels (⬍25 IU/mL). Intervention(s): Growth hormone levels were assessed in all patients before and 30, 60, 90, and 120 minutes after oral administration of 0.3 mg of clonidine. Main Outcome Measure(s): Growth hormone levels before and after clonidine administration. Result(s): Delta GH and GH levels at 30, 60, and 120 minutes were significantly higher in the PCOS group than in the IH and control groups. Conclusion(s): The greater GH response to clonidine in nonobese normoinsulinemic PCOS patients observed in this study suggests a dysregulation in GH secretion in these patients. Further studies are required to elucidate the role of GH in the pathogenesis of PCOS and to investigate the existence of an association between androgens, IGF-I, and GH modulation in PCOS. (Fertil Steril威 2004;81:108 –13. ©2004 by American Society for Reproductive Medicine.) Key Words: Hirsutism, PCOS, clonidine, growth hormone
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, with prevalence ranging from 5% to 10% (1, 2). Polycystic ovary syndrome is a heterogeneous clinical condition, characterized by hirsutism, irregular menstrual cycles, infertility, and endocrine abnormalities such as hyperandrogenism. Moreover, a considerable percentage of women with PCOS present insulin resistance and compensatory hyperinsulinemia. Normal-weight women with PCOS present a lower incidence of hyperinsulinemia/insulin resistance than do obese women (3, 4). Obesity is associated with higher hypothalamic secretion of somatostatin and, in consequence, lower basal GH levels and lower GH response to stimulation tests (5, 6). In addition, some in-
vestigators have proposed the existence of different pathophysiological models in PCOS patients with or without insulin resistance: hyperinsulinemic and normoinsulinemic PCOS patients would present a cogonadotropic action associated predominantly with either insulin (hyperinsulinemic patients) or GH (normoinsulinemic patients) (7–10). There is some evidence that the somatotropic axis plays a role in the pathophysiology of PCOS, especially when only normal-weight normoinsulinemic subjects are considered (7–9, 11). Morales et al. (8) have observed an increase in GH pulsatility in normal-weight PCOS patients compared with control individuals of similar weight. Zournatti et al. (11) described an increase in GH in lean PCOS patients after the insulin hypoglycemia test
when compared with healthy volunteers. Moreover, there is controversy concerning the GH response to administration of growth hormone–releasing hormone (GHRH) in normalweight PCOS patients and controls: some investigators have not detected a difference (8, 12), whereas others have observed an increase in GH in the PCOS group (13). Growth hormone response to clonidine has been used to test GH deficiency, particularly in children. This test is also occasionally used in adults to evaluate the somatotropic axis in the absence of frank GH deficiency, as in anovulatory women with normal or poor ovarian response to gonadotropin stimulation (14) or in women who are resistant to clomiphene citrate stimulation (15). Clonidine seems to stimulate GH secretion by acting on GHRH via postsynaptic ␣-2 adrenergic receptors. Some evidence, however, suggests an additional inhibition of somatostatinergic pathways (16). Therefore, the aim of this study was to assess GH levels in response to acute clonidine stimulation in nonobese patients with PCOS in comparison to patients with idiopathic hirsutism (IH) and normal women with regular ovulatory cycles paired for age, body mass index (BMI), and insulin levels.
MATERIALS AND METHODS Patients Two study groups were set up with women treated for hirsutism. These women were seen consecutively between October 2000 and September 2001 at the Gynecological Endocrinology Unit at Hospital de Clı´nicas de Porto Alegre, Brazil. Late-onset (nonclassic) congenital adrenal hyperplasia patients were excluded on the basis of a high plasma level of 17-hydroxyprogesterone (⬎5 ng/dL) and a marked increase in this level after ACTH stimulation (⬎12 ng/dL) (17, 18). Patients with hyperprolactinemia (serum prolactin levels ⬎20 g/L on 2 different occasions) were also excluded. Fourteen patients with PCOS and 11 patients with IH were selected for the study. All presented normal BMI (⬍25 kg/m2) and normal insulin levels (⬍25 IU/mL). None had received any drugs known to interfere with hormonal levels for ⱖ3 months before the study. The mean score for hirsutism, assigned by the original method of Ferriman and Gallwey (19), was 13 ⫾ 7. Ten normal women with regular cycles and without hirsutism were also selected as a control group. The diagnosis of PCOS was based on the physical features of hyperandrogenism, disturbed menstrual cycles, elevated serum LH levels or LH-FSH ratio, increased levels of either or both serum T or free androgen index (FAI), and absence of ovarian or adrenal neoplasm or Cushing’s syndrome. Idiopathic hirsutism was diagnosed, as described elsewhere (20), in hirsute patients with regular, ovulatory cycles FERTILITY & STERILITY威
(luteal-phase P levels of ⬎3.0 ng/mL), normal androgen levels, and absence of known underlying disease. The study protocol was approved by the local ethics committee (equivalent to an institutional review board), and written informed consent was obtained from all subjects.
Study Protocol Anthropometric measurements included body weight, height, waist-to-hip ratio (waist circumference was recorded at the narrowest point or at the umbilicus, and hip circumference was recorded at the level of the greater trochanter), and BMI (current measured weight in kilograms divided by height in square meters). Hormonal and metabolic parameters were assessed between days 2 and 10 of the menstrual cycle or on any day when the patients were amenorrheic. After an overnight fast, blood samples were drawn from an antecubital vein for determination of plasma glucose and insulin. Blood samples were also drawn for LH, FSH, sex hormone– binding globulin, and total T determinations. All samples were obtained between 8 and 10 AM. The FAI was estimated by dividing total serum T (nmol/L) by sex hormone– binding globulin (nmol/L) ⫻ 100. The clonidine test consisted of determining GH levels before and 30, 60, 90, and 120 minutes after the oral administration of clonidine, 0.3 mg (Atensina; Boehringer Ingelheim, Sa˜ o Paulo, Brazil). Any increase in GH levels was considered to be a positive response to clonidine. Delta GH was estimated as the highest GH value at any time minus the basal GH value before clonidine administration. Potential confounding factors for the GH response to clonidine, such as age, administration of other drugs, and phase of menstrual cycle (10, 21–23) were controlled by pairing the three groups for age, by selecting women who had had no other medical treatment for ⱖ3 months, and by performing the clonidine test during the follicular phase in IH and control women.
Assays Glucose was measured by the glucose oxidase technique using Mega Merck Kits (Merck, Darmstadt, Germany). Serum LH and FSH were measured by specific immunofluorimetric assays (Wallac, Turku, Finland) with intra-assay and interassay coefficients of variation (CV) of 6.7% and 11%, respectively, for LH, and 6.6% and 10.2% for FSH. The sensitivity of the assays was 0.12 IU/L for LH and 0.05 IU/L for FSH. Total serum T levels were measured with the RIA method (DPC, Los Angeles, CA), with an assay sensitivity of 0.04 ng/mL and intra-assay and interassay CV of 8.5% and 10.3%, respectively. Sex hormone– binding globulin was measured by chemiluminescent enzyme immunoassay (DPC), with an assay sensitivity of 0.2 nmol/L, and intra-assay and interassay CV of 6.1% and 8.0%, respectively. The assay sensitivity of double-antibody RIA (CIS 109
TABLE 1 Characteristics of patients with PCOS and IH and control women. Parameter
PCOS (n ⫽ 14)
IH (n ⫽ 11)
Controls (n ⫽ 10)
P
Age (y) BMI (kg/m2) Waist-hip ratio Insulin (IU/mL) Glucose-insulin ratio (mIU/mg) Free androgen indexb T (ng/mL) SHBG (nmol/L)a PRL (ng/mL) LH-FSHa LH (mIU/mL)a
20.71 ⫾ 4.81 21.53 ⫾ 2.43 0.77 ⫾ 0.06 16.09 ⫾ 5.52 21 (11–24)a 15.2 ⫾ 8.8c 0.98 ⫾ 0.45c 28.0 (16.3–45.9) 11.75 ⫾ 4.46 1.40 (0.90–2.25) 6.7 (3.1–9.6)
21.36 ⫾ 5.81 22.19 ⫾ 2.80 0.73 ⫾ 0.11 13.19 ⫾ 6.46 19 (10–25) 6.7 ⫾ 4.9 0.63 ⫾ 0.18 41.8 (22.8–46.4) 10.69 ⫾ 3.70 0.86 (0.7–1.24) 3.3 (1.3–5.1)
23.7 ⫾ 5.25 20.14 ⫾ 2.21 0.71 ⫾ 0.02 14.10 ⫾ 2.46 18 (15–20) 4.6 ⫾ 2.0 0.66 ⫾ 0.18 48.6 (41.9–52.0) 8.22 ⫾ 1.78 0.82 (0.7–1.24) 4.3 (3.2–8.2)
.38 .34 .29 .37 .81 .014 .03 .24 .19 .19 .10
Note: Data without superscripts are presented as mean ⫾ SD. Data are presented as median (25th–75th interquartile range). b Calculated as TT ⫻ 3.48/SHBG. c Compared with IH and control groups. a
Comim. GH in nonobese PCOS patients. Fertil Steril 2004.
Bio International, Gif-Sur-Ivette, France) to measure serum insulin levels was 2.0 IU/mL. The intra-assay and interassay CV were 7.5% and 9%, respectively. Growth hormone levels were measured with chemiluminescent enzyme immunoassay (Immulite; DPC), with an assay sensitivity of 0.01 ng/mL and intra-assay and interassay CV of 3.8% and 5.5%, respectively.
and 120 minutes and the initial value were analyzed as an increase in relation to the basal value of zero. All analyses were performed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL). Data were considered to be significant at P⬍.05.
Statistical Analysis
Results are presented as means ⫾ SD, unless otherwise noted. Comparisons between group means were analyzed by two-way analysis of variance, followed by the StudentNewman-Keuls test; comparisons between median values were analyzed using the Kruskal-Wallis and Dunn tests. Log 10 transformation was used to normalize the distribution of non-Gaussian variables related to GH levels. Mean values were back-transformed for presentation. For standardization of the GH response curve to clonidine stimulation, the basal values for each group were considered to be zero; the difference between log-transformed GH values at 30, 60, 90,
RESULTS Table 1 shows the clinical and hormonal characteristics of the patients with PCOS and IH and of the control group. No differences were observed in terms of age, BMI, or waist-hip ratio among the groups. Testosterone and FAI were significantly higher in PCOS patients than in IH or control women. Table 2 summarizes data related to the response of GH to the clonidine test for the studied groups. No significant differences were observed among the three groups in terms of the number of positive responses to clonidine, GH levels before clonidine administration, or area under the curve for
TABLE 2 Levels of GH after stimulation with clonidine. Parameter Responsive to clonidine Basal GH levels GH area under the curve Delta GH (ng/mL)
PCOS (n ⫽ 14)
Controls (n ⫽ 10)
IH (n ⫽ 11)
P
13/14 1.4 (0.45–2.42) 534.1 (226.9–888.1) 6.27 (4.80–10.40)a
6/10 4.45 (1.65–7.1) 465.7 (323.8–617.6) 3.10 (1.37–5.45)
6/11 4.2 (0.5–7.7) 278.7 (168.0–462.0) 1.40 (0.30–3.37)
.11 .07 .20 .014
Note: Data are presented as median (25th–75th interquartile range). a Compared with IH and control groups. Comim. GH in nonobese PCOS patients. Fertil Steril 2004.
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Vol. 81, No. 1, January 2004
FIGURE 1 Delta log GH values. The line across each box represents the median. The upper and lower lines show the interquartile range. *P⫽.014.
Delta GH was significantly higher in the PCOS group than in the IH and control groups (Table 2; Fig. 1). Figure 2 shows the pattern of GH response to clonidine in all groups. Polycystic ovary syndrome patients presented higher GH levels at 30, 60, and 120 minutes in comparison to IH and control women (P30⫽.04; P60⫽.03; P120⫽.01). These statistically significant differences in the GH response to clonidine were maintained even when the analysis included only women with positive responses (data not shown).
DISCUSSION Normal-weight women with PCOS are characterized by hyperandrogenism and increased LH levels; however, they present a lower incidence of hyperinsulinemia/insulin resistance than do obese women with PCOS (3, 20). Thus, normal-weight normoinsulinemic PCOS patients may serve as a reliable model for assessing a possible role of GH on the pathophysiology of this syndrome.
Comim. GH in nonobese PCOS patients. Fertil Steril 2004.
GH. However, these variables presented a relatively high dispersion, as shown in Table 2.
FIGURE 2 Growth hormone after clonidine stimulation. Filled squares, PCOS; filled circles, IH; filled triangles, controls. Values are expressed as means of log GH ⫾ SEM; *P⫽.05 for 30, 60, and 120 minutes.
Comim. GH in nonobese PCOS patients. Fertil Steril 2004.
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In the present study, women with PCOS were assessed and compared with a group of IH women. We believe that these ovulatory IH women can be considered an especially apt control group, because their hirsutism is similar to that of PCOS patients, but without the hormonal alterations that distinguish PCOS (4, 20, 24). We also studied a group of normal women because the response to the clonidine test has not been well characterized in the literature for adult female subjects, and only few studies have been published about this subject (10, 23). We observed a higher frequency of positive responses to clonidine in our lean patients with PCOS, although this was without statistical significance. The lack of significance may have been due to the size of the sample, as evidenced by the retrospective statistical power analysis, which was ⬍50% for this variable; therefore, the trend observed would probably be confirmed, and not invalidated, had we been able to include a larger number of women. In contrast, a higher response of GH to clonidine was observed in PCOS patients without insulin resistance, as compared with IH patients and normal controls who were paired for age, BMI, and insulin levels with the PCOS group. It is interesting to observe that the pattern of GH response in both the IH and control groups was very similar, which supports the notion that IH patients may be used as controls in comparative studies with PCOS patients. Clonidine was chosen for the stimulation test essentially because of the large amount of experience with this drug, low incidence of severe side effects, simplicity of use, and low cost. Although the clonidine test is usually not used to assess GH deficiency in adults (because there are other tests with higher sensitivity for low GH levels), it is considered to be a useful tool to detect differences in GH secretion in adult subjects without GH deficiency (15, 25). However, even with a conventional oral dose of 300 mg, the GH response may be minimal or absent, as previously observed (14). 111
Although no difference in GHRH-stimulated GH secretion between lean PCOS patients and controls has been observed in other studies (8, 12, 13), in the present study, a higher GH response to clonidine in PCOS patients vs. IH patients or control women was recorded. The main mechanism behind the positive action of clonidine on GH secretion seems to occur via GHRH neuronal stimulation (16). However, an inhibition on the somatostatinergic pathway may also take place under some conditions (26 –28). This could explain, at least partially, the differences between our results with clonidine and other results concerning GHRH. The present data represent new and valuable evidence concerning the influence of GH on lean patients with PCOS. On the basis of this type of evidence, further studies are necessary before the clinical relevance of the clonidine test can be established and before this test can be recognized as a diagnostic tool; such studies should investigate, among other aspects, other stimuli to GH secretion. Some studies have examined the relationship between the control of GH secretion and the secretion of other hormones in PCOS. Morales et al. (8) described an association between GH and LH pulse amplitudes in normal-weight PCOS patients, whereas Garcia-Rudaz et al. (9) have shown a positive association of GH secretion with LH and androgen levels in nonobese, normoinsulinemic women with PCOS. In nonobese PCOS patients, GH could act as a cogonadotropic factor stimulating androgen production by the ovary (7). Moreover, although obese PCOS patients frequently present high free insulin-like growth factor-I (IGF-I) levels that are associated with low hepatic production of IGF– binding protein-1, caused by hyperinsulinemia (29 –31), no consistent change has been described for IGF-I or IGF– binding proteins in nonobese, normoinsulinemic PCOS patients (7, 9, 32). Several studies indicate that GH may influence reproductive activity by increasing gonadotropin secretion at the hypothalamic and pituitary level and by enhancing gonadotropin responsiveness at the gonadal level. Moreover, evidence from human and in vitro studies suggests an important role for GH, along with IGF-I and IGF-II, in the regulation of folliculogenesis (33) and steroidogenesis (34). Thus, it could be hypothesized that inadequate GH response to clonidine in PCOS is associated with a defect in the hypothalamic regulation of gonadotropin release. An effect of GH on theca cells is also possible, inducing the local production of IGF-I and acting in synergism with LH to promote increased androgen production. In the present study, the higher T and FAI concentrations observed in PCOS patients may have contributed toward the increased GH response to clonidine. Recent studies have shed some light on the possible effects of androgen levels on GH secretion. Testosterone (but not dihydrotestosterone) may increase serum GH concentrations in boys, possibly by 112
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conversion to E2 (35). Rizvi et al. (36) have shown an increase in the area under the curve for GH in response to N-methyl-aspartate administration in orchiectomized rhesus monkeys supplemented with T in comparison to intact or orchiectomized groups. In contrast, no differences were observed using GHRH. Wu et al. (37) have shown that bilateral ovarian wedge resection was able to reduce the levels of androgen as well as the levels of IGF-I in lean PCOS patients. In conclusion, our data showing a greater GH response to clonidine in nonobese normoinsulinemic PCOS patients are suggestive of a dysregulation in GH secretion. Further studies are required to clarify the role of GH in the pathogenesis of PCOS and to specifically investigate the proposed association between androgens, IGF-I, and GH modulation in PCOS.
Acknowledgments: The authors thank Angela D’Avila, M.D., and Mariana Ughini, M.D., for their contribution to data collection and thank the Graduate Research Group (GPPG) at Hospital de Clı´nicas de Porto Alegre for providing editorial support.
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Increased growth hormone response to clonidine in nonobese normoinsulinemic patients with polycystic ovary syndrome Fa´bio Vasconcellos Comim, M.D.,a and Poli Mara Spritzer, M.D., Ph.D.a,b Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clı´nicas de Porto Alegre; and Department of Physiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
Received December 18, 2002; revised and accepted May 28, 2003. Supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), FAPERGS (Fundac¸a˜o de Amparo a` Pesquisa do Rio Grande do Sul), Porto Alegre, Brazil, and PRONEX 26/98 (Programa de Apoio aos Nu´cleos de Exceleˆncia em Pesquisa, Brasilia, Brazil). Presented in part at the 5th International Congress of Neuroendocrinology, Bristol, United Kingdom, July 31 to August 4, 2002. Reprint requests: Poli Mara Spritzer, M.D., Ph.D., Department of Physiology, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, 90050-170-Porto Alegre RS, Brazil (FAX: ⫹55-513316-3453; E-mail: [email protected]). a Gynecological Endocrinology Unit, Division of Endocrinology, Hospital de Clinicas de Porto Alegre. b Department of Physiology, Universidade Federal do Rio Grande do Sul. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 05.027
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Objective: To assess growth hormone (GH) levels in response to acute clonidine stimulation in nonobese patients with polycystic ovary syndrome (PCOS) in comparison to patients with idiopathic hirsutism (IH) and normal women without hirsutism. Design: Cross-sectional study. Setting: Outpatient clinic, Porto Alegre, Brazil. Patient(s): Fourteen patients with PCOS, 11 women with IH, and 10 age- and weight-matched normal women without hirsutism were studied. All subjects presented normal body mass index (⬍25 kg/m2) and insulin levels (⬍25 IU/mL). Intervention(s): Growth hormone levels were assessed in all patients before and 30, 60, 90, and 120 minutes after oral administration of 0.3 mg of clonidine. Main Outcome Measure(s): Growth hormone levels before and after clonidine administration. Result(s): Delta GH and GH levels at 30, 60, and 120 minutes were significantly higher in the PCOS group than in the IH and control groups. Conclusion(s): The greater GH response to clonidine in nonobese normoinsulinemic PCOS patients observed in this study suggests a dysregulation in GH secretion in these patients. Further studies are required to elucidate the role of GH in the pathogenesis of PCOS and to investigate the existence of an association between androgens, IGF-I, and GH modulation in PCOS. (Fertil Steril威 2004;81:108 –13. ©2004 by American Society for Reproductive Medicine.) Key Words: Hirsutism, PCOS, clonidine, growth hormone
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, with prevalence ranging from 5% to 10% (1, 2). Polycystic ovary syndrome is a heterogeneous clinical condition, characterized by hirsutism, irregular menstrual cycles, infertility, and endocrine abnormalities such as hyperandrogenism. Moreover, a considerable percentage of women with PCOS present insulin resistance and compensatory hyperinsulinemia. Normal-weight women with PCOS present a lower incidence of hyperinsulinemia/insulin resistance than do obese women (3, 4). Obesity is associated with higher hypothalamic secretion of somatostatin and, in consequence, lower basal GH levels and lower GH response to stimulation tests (5, 6). In addition, some in-
vestigators have proposed the existence of different pathophysiological models in PCOS patients with or without insulin resistance: hyperinsulinemic and normoinsulinemic PCOS patients would present a cogonadotropic action associated predominantly with either insulin (hyperinsulinemic patients) or GH (normoinsulinemic patients) (7–10). There is some evidence that the somatotropic axis plays a role in the pathophysiology of PCOS, especially when only normal-weight normoinsulinemic subjects are considered (7–9, 11). Morales et al. (8) have observed an increase in GH pulsatility in normal-weight PCOS patients compared with control individuals of similar weight. Zournatti et al. (11) described an increase in GH in lean PCOS patients after the insulin hypoglycemia test
when compared with healthy volunteers. Moreover, there is controversy concerning the GH response to administration of growth hormone–releasing hormone (GHRH) in normalweight PCOS patients and controls: some investigators have not detected a difference (8, 12), whereas others have observed an increase in GH in the PCOS group (13). Growth hormone response to clonidine has been used to test GH deficiency, particularly in children. This test is also occasionally used in adults to evaluate the somatotropic axis in the absence of frank GH deficiency, as in anovulatory women with normal or poor ovarian response to gonadotropin stimulation (14) or in women who are resistant to clomiphene citrate stimulation (15). Clonidine seems to stimulate GH secretion by acting on GHRH via postsynaptic ␣-2 adrenergic receptors. Some evidence, however, suggests an additional inhibition of somatostatinergic pathways (16). Therefore, the aim of this study was to assess GH levels in response to acute clonidine stimulation in nonobese patients with PCOS in comparison to patients with idiopathic hirsutism (IH) and normal women with regular ovulatory cycles paired for age, body mass index (BMI), and insulin levels.
MATERIALS AND METHODS Patients Two study groups were set up with women treated for hirsutism. These women were seen consecutively between October 2000 and September 2001 at the Gynecological Endocrinology Unit at Hospital de Clı´nicas de Porto Alegre, Brazil. Late-onset (nonclassic) congenital adrenal hyperplasia patients were excluded on the basis of a high plasma level of 17-hydroxyprogesterone (⬎5 ng/dL) and a marked increase in this level after ACTH stimulation (⬎12 ng/dL) (17, 18). Patients with hyperprolactinemia (serum prolactin levels ⬎20 g/L on 2 different occasions) were also excluded. Fourteen patients with PCOS and 11 patients with IH were selected for the study. All presented normal BMI (⬍25 kg/m2) and normal insulin levels (⬍25 IU/mL). None had received any drugs known to interfere with hormonal levels for ⱖ3 months before the study. The mean score for hirsutism, assigned by the original method of Ferriman and Gallwey (19), was 13 ⫾ 7. Ten normal women with regular cycles and without hirsutism were also selected as a control group. The diagnosis of PCOS was based on the physical features of hyperandrogenism, disturbed menstrual cycles, elevated serum LH levels or LH-FSH ratio, increased levels of either or both serum T or free androgen index (FAI), and absence of ovarian or adrenal neoplasm or Cushing’s syndrome. Idiopathic hirsutism was diagnosed, as described elsewhere (20), in hirsute patients with regular, ovulatory cycles FERTILITY & STERILITY威
(luteal-phase P levels of ⬎3.0 ng/mL), normal androgen levels, and absence of known underlying disease. The study protocol was approved by the local ethics committee (equivalent to an institutional review board), and written informed consent was obtained from all subjects.
Study Protocol Anthropometric measurements included body weight, height, waist-to-hip ratio (waist circumference was recorded at the narrowest point or at the umbilicus, and hip circumference was recorded at the level of the greater trochanter), and BMI (current measured weight in kilograms divided by height in square meters). Hormonal and metabolic parameters were assessed between days 2 and 10 of the menstrual cycle or on any day when the patients were amenorrheic. After an overnight fast, blood samples were drawn from an antecubital vein for determination of plasma glucose and insulin. Blood samples were also drawn for LH, FSH, sex hormone– binding globulin, and total T determinations. All samples were obtained between 8 and 10 AM. The FAI was estimated by dividing total serum T (nmol/L) by sex hormone– binding globulin (nmol/L) ⫻ 100. The clonidine test consisted of determining GH levels before and 30, 60, 90, and 120 minutes after the oral administration of clonidine, 0.3 mg (Atensina; Boehringer Ingelheim, Sa˜ o Paulo, Brazil). Any increase in GH levels was considered to be a positive response to clonidine. Delta GH was estimated as the highest GH value at any time minus the basal GH value before clonidine administration. Potential confounding factors for the GH response to clonidine, such as age, administration of other drugs, and phase of menstrual cycle (10, 21–23) were controlled by pairing the three groups for age, by selecting women who had had no other medical treatment for ⱖ3 months, and by performing the clonidine test during the follicular phase in IH and control women.
Assays Glucose was measured by the glucose oxidase technique using Mega Merck Kits (Merck, Darmstadt, Germany). Serum LH and FSH were measured by specific immunofluorimetric assays (Wallac, Turku, Finland) with intra-assay and interassay coefficients of variation (CV) of 6.7% and 11%, respectively, for LH, and 6.6% and 10.2% for FSH. The sensitivity of the assays was 0.12 IU/L for LH and 0.05 IU/L for FSH. Total serum T levels were measured with the RIA method (DPC, Los Angeles, CA), with an assay sensitivity of 0.04 ng/mL and intra-assay and interassay CV of 8.5% and 10.3%, respectively. Sex hormone– binding globulin was measured by chemiluminescent enzyme immunoassay (DPC), with an assay sensitivity of 0.2 nmol/L, and intra-assay and interassay CV of 6.1% and 8.0%, respectively. The assay sensitivity of double-antibody RIA (CIS 109
TABLE 1 Characteristics of patients with PCOS and IH and control women. Parameter
PCOS (n ⫽ 14)
IH (n ⫽ 11)
Controls (n ⫽ 10)
P
Age (y) BMI (kg/m2) Waist-hip ratio Insulin (IU/mL) Glucose-insulin ratio (mIU/mg) Free androgen indexb T (ng/mL) SHBG (nmol/L)a PRL (ng/mL) LH-FSHa LH (mIU/mL)a
20.71 ⫾ 4.81 21.53 ⫾ 2.43 0.77 ⫾ 0.06 16.09 ⫾ 5.52 21 (11–24)a 15.2 ⫾ 8.8c 0.98 ⫾ 0.45c 28.0 (16.3–45.9) 11.75 ⫾ 4.46 1.40 (0.90–2.25) 6.7 (3.1–9.6)
21.36 ⫾ 5.81 22.19 ⫾ 2.80 0.73 ⫾ 0.11 13.19 ⫾ 6.46 19 (10–25) 6.7 ⫾ 4.9 0.63 ⫾ 0.18 41.8 (22.8–46.4) 10.69 ⫾ 3.70 0.86 (0.7–1.24) 3.3 (1.3–5.1)
23.7 ⫾ 5.25 20.14 ⫾ 2.21 0.71 ⫾ 0.02 14.10 ⫾ 2.46 18 (15–20) 4.6 ⫾ 2.0 0.66 ⫾ 0.18 48.6 (41.9–52.0) 8.22 ⫾ 1.78 0.82 (0.7–1.24) 4.3 (3.2–8.2)
.38 .34 .29 .37 .81 .014 .03 .24 .19 .19 .10
Note: Data without superscripts are presented as mean ⫾ SD. Data are presented as median (25th–75th interquartile range). b Calculated as TT ⫻ 3.48/SHBG. c Compared with IH and control groups. a
Comim. GH in nonobese PCOS patients. Fertil Steril 2004.
Bio International, Gif-Sur-Ivette, France) to measure serum insulin levels was 2.0 IU/mL. The intra-assay and interassay CV were 7.5% and 9%, respectively. Growth hormone levels were measured with chemiluminescent enzyme immunoassay (Immulite; DPC), with an assay sensitivity of 0.01 ng/mL and intra-assay and interassay CV of 3.8% and 5.5%, respectively.
and 120 minutes and the initial value were analyzed as an increase in relation to the basal value of zero. All analyses were performed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL). Data were considered to be significant at P⬍.05.
Statistical Analysis
Results are presented as means ⫾ SD, unless otherwise noted. Comparisons between group means were analyzed by two-way analysis of variance, followed by the StudentNewman-Keuls test; comparisons between median values were analyzed using the Kruskal-Wallis and Dunn tests. Log 10 transformation was used to normalize the distribution of non-Gaussian variables related to GH levels. Mean values were back-transformed for presentation. For standardization of the GH response curve to clonidine stimulation, the basal values for each group were considered to be zero; the difference between log-transformed GH values at 30, 60, 90,
RESULTS Table 1 shows the clinical and hormonal characteristics of the patients with PCOS and IH and of the control group. No differences were observed in terms of age, BMI, or waist-hip ratio among the groups. Testosterone and FAI were significantly higher in PCOS patients than in IH or control women. Table 2 summarizes data related to the response of GH to the clonidine test for the studied groups. No significant differences were observed among the three groups in terms of the number of positive responses to clonidine, GH levels before clonidine administration, or area under the curve for
TABLE 2 Levels of GH after stimulation with clonidine. Parameter Responsive to clonidine Basal GH levels GH area under the curve Delta GH (ng/mL)
PCOS (n ⫽ 14)
Controls (n ⫽ 10)
IH (n ⫽ 11)
P
13/14 1.4 (0.45–2.42) 534.1 (226.9–888.1) 6.27 (4.80–10.40)a
6/10 4.45 (1.65–7.1) 465.7 (323.8–617.6) 3.10 (1.37–5.45)
6/11 4.2 (0.5–7.7) 278.7 (168.0–462.0) 1.40 (0.30–3.37)
.11 .07 .20 .014
Note: Data are presented as median (25th–75th interquartile range). a Compared with IH and control groups. Comim. GH in nonobese PCOS patients. Fertil Steril 2004.
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Vol. 81, No. 1, January 2004
FIGURE 1 Delta log GH values. The line across each box represents the median. The upper and lower lines show the interquartile range. *P⫽.014.
Delta GH was significantly higher in the PCOS group than in the IH and control groups (Table 2; Fig. 1). Figure 2 shows the pattern of GH response to clonidine in all groups. Polycystic ovary syndrome patients presented higher GH levels at 30, 60, and 120 minutes in comparison to IH and control women (P30⫽.04; P60⫽.03; P120⫽.01). These statistically significant differences in the GH response to clonidine were maintained even when the analysis included only women with positive responses (data not shown).
DISCUSSION Normal-weight women with PCOS are characterized by hyperandrogenism and increased LH levels; however, they present a lower incidence of hyperinsulinemia/insulin resistance than do obese women with PCOS (3, 20). Thus, normal-weight normoinsulinemic PCOS patients may serve as a reliable model for assessing a possible role of GH on the pathophysiology of this syndrome.
Comim. GH in nonobese PCOS patients. Fertil Steril 2004.
GH. However, these variables presented a relatively high dispersion, as shown in Table 2.
FIGURE 2 Growth hormone after clonidine stimulation. Filled squares, PCOS; filled circles, IH; filled triangles, controls. Values are expressed as means of log GH ⫾ SEM; *P⫽.05 for 30, 60, and 120 minutes.
Comim. GH in nonobese PCOS patients. Fertil Steril 2004.
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In the present study, women with PCOS were assessed and compared with a group of IH women. We believe that these ovulatory IH women can be considered an especially apt control group, because their hirsutism is similar to that of PCOS patients, but without the hormonal alterations that distinguish PCOS (4, 20, 24). We also studied a group of normal women because the response to the clonidine test has not been well characterized in the literature for adult female subjects, and only few studies have been published about this subject (10, 23). We observed a higher frequency of positive responses to clonidine in our lean patients with PCOS, although this was without statistical significance. The lack of significance may have been due to the size of the sample, as evidenced by the retrospective statistical power analysis, which was ⬍50% for this variable; therefore, the trend observed would probably be confirmed, and not invalidated, had we been able to include a larger number of women. In contrast, a higher response of GH to clonidine was observed in PCOS patients without insulin resistance, as compared with IH patients and normal controls who were paired for age, BMI, and insulin levels with the PCOS group. It is interesting to observe that the pattern of GH response in both the IH and control groups was very similar, which supports the notion that IH patients may be used as controls in comparative studies with PCOS patients. Clonidine was chosen for the stimulation test essentially because of the large amount of experience with this drug, low incidence of severe side effects, simplicity of use, and low cost. Although the clonidine test is usually not used to assess GH deficiency in adults (because there are other tests with higher sensitivity for low GH levels), it is considered to be a useful tool to detect differences in GH secretion in adult subjects without GH deficiency (15, 25). However, even with a conventional oral dose of 300 mg, the GH response may be minimal or absent, as previously observed (14). 111
Although no difference in GHRH-stimulated GH secretion between lean PCOS patients and controls has been observed in other studies (8, 12, 13), in the present study, a higher GH response to clonidine in PCOS patients vs. IH patients or control women was recorded. The main mechanism behind the positive action of clonidine on GH secretion seems to occur via GHRH neuronal stimulation (16). However, an inhibition on the somatostatinergic pathway may also take place under some conditions (26 –28). This could explain, at least partially, the differences between our results with clonidine and other results concerning GHRH. The present data represent new and valuable evidence concerning the influence of GH on lean patients with PCOS. On the basis of this type of evidence, further studies are necessary before the clinical relevance of the clonidine test can be established and before this test can be recognized as a diagnostic tool; such studies should investigate, among other aspects, other stimuli to GH secretion. Some studies have examined the relationship between the control of GH secretion and the secretion of other hormones in PCOS. Morales et al. (8) described an association between GH and LH pulse amplitudes in normal-weight PCOS patients, whereas Garcia-Rudaz et al. (9) have shown a positive association of GH secretion with LH and androgen levels in nonobese, normoinsulinemic women with PCOS. In nonobese PCOS patients, GH could act as a cogonadotropic factor stimulating androgen production by the ovary (7). Moreover, although obese PCOS patients frequently present high free insulin-like growth factor-I (IGF-I) levels that are associated with low hepatic production of IGF– binding protein-1, caused by hyperinsulinemia (29 –31), no consistent change has been described for IGF-I or IGF– binding proteins in nonobese, normoinsulinemic PCOS patients (7, 9, 32). Several studies indicate that GH may influence reproductive activity by increasing gonadotropin secretion at the hypothalamic and pituitary level and by enhancing gonadotropin responsiveness at the gonadal level. Moreover, evidence from human and in vitro studies suggests an important role for GH, along with IGF-I and IGF-II, in the regulation of folliculogenesis (33) and steroidogenesis (34). Thus, it could be hypothesized that inadequate GH response to clonidine in PCOS is associated with a defect in the hypothalamic regulation of gonadotropin release. An effect of GH on theca cells is also possible, inducing the local production of IGF-I and acting in synergism with LH to promote increased androgen production. In the present study, the higher T and FAI concentrations observed in PCOS patients may have contributed toward the increased GH response to clonidine. Recent studies have shed some light on the possible effects of androgen levels on GH secretion. Testosterone (but not dihydrotestosterone) may increase serum GH concentrations in boys, possibly by 112
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conversion to E2 (35). Rizvi et al. (36) have shown an increase in the area under the curve for GH in response to N-methyl-aspartate administration in orchiectomized rhesus monkeys supplemented with T in comparison to intact or orchiectomized groups. In contrast, no differences were observed using GHRH. Wu et al. (37) have shown that bilateral ovarian wedge resection was able to reduce the levels of androgen as well as the levels of IGF-I in lean PCOS patients. In conclusion, our data showing a greater GH response to clonidine in nonobese normoinsulinemic PCOS patients are suggestive of a dysregulation in GH secretion. Further studies are required to clarify the role of GH in the pathogenesis of PCOS and to specifically investigate the proposed association between androgens, IGF-I, and GH modulation in PCOS.
Acknowledgments: The authors thank Angela D’Avila, M.D., and Mariana Ughini, M.D., for their contribution to data collection and thank the Graduate Research Group (GPPG) at Hospital de Clı´nicas de Porto Alegre for providing editorial support.
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