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Plasma adrenomedullin levels in patients with polycystic ovary syndrome
Plasma adrenomedullin levels in patients with polycystic ovary syndrome Banu Ucar, M.D.,a Volkan Noyan, M.D.,a Osman Caglayan, M.D.,b Aykan Yucel, M.D.,a and Nevin Sagsoz, M.D.a a
Department of Obstetrics and Gynecology and b Department of Biochemistry and Clinical Biochemistry, Kirikkale University School of Medicine, Kirikkale, Turkey
Objective: To evaluate adrenomedullin levels in patients with polycystic ovary syndrome (PCOS). Design: Prospective study. Setting: Department of obstetrics and gynecology in a university hospital. Patient(s): Thirty-eight women with PCOS and 29 healthy control subjects were enrolled in the study. Intervention(s): Plasma adrenomedullin, serum androstenedione, free T, T, DHEAS, SHBG, thyrotropin, PRL, FSH, LH, and E2 were measured in each subject. Insulin resistance was estimated by fasting insulin level, fasting glucose:insulin ratio and 75-g glucose tolerance test for 2 hours. Main Outcome Measure(s): Plasma adrenomedullin levels and correlations among adrenomedullin and gonadotropins, female sex steroids, androgens, and insulin resistance. Result(s): There was no significant difference concerning plasma adrenomedullin concentrations between the groups. In patients with PCOS, fasting glucose, fasting insulin, body mass index, and free T were inversely correlated with the plasma adrenomedullin. Plasma adrenomedullin was significantly correlated with glucose: insulin ratio. After controlling for body mass index, there were no significant correlations between the abovementioned parameters. Conclusion(s): Adrenomedullin may play a role in regulating the insulin metabolism in patients with PCOS. (Fertil Steril威 2006;86:942– 8. ©2006 by American Society for Reproductive Medicine.) Key Words: Polycystic ovary syndrome, adrenomedullin, insulin resistance, obesity
Polycystic ovary syndrome (PCOS) is characterized by menstrual dysfunction, hyperandrogenism, and frequent insulin resistance. Polycystic ovary syndrome is the most common endocrinopathy among women of reproductive age. The estimate of incidence of PCOS differs between 3% and 10% (1–3), and its etiopathogenesis is still controversial (4, 5). Hypothalamus-pituitary-ovary axis changes, obesity, insulin resistance, and ovarian and adrenal androgen excess due to steroidogenic dysfunction play roles in the pathophysiology (6). The pulsatile emission of GnRH is increased in PCOS. In patients with PCOS, the amplitude and frequency of LH pulses increase (6). The changes of gonadotropin dynamics can develop secondarily to central or peripheral hormonal deviations (5). In patients with PCOS, the prevalence of cardiovascular disorders, insulin resistance, and diabetes mellitus (DM) has been found to be increased (1, 7). Adrenomedullin (AM) was first found in 1993 in human pheochromocytoma tissue (8). Adrenomedullin is a vasodilator peptide with 52 amino acids (8). Not only is the AM mRNA expressed in pheochromocytoma tissue, but AM gene products have been detected also in tissues including
Received September 27, 2005; revised and accepted February 25, 2006. Supported by the Department of Scientific Research Projects, Kirikkale University, Kirikkale, Turkey. Reprint requests: Banu Ucar, M.D., Karargah tepe mah. Atıs cad., Komut. Sok. 14/4, Meteoroloji, Kecioren, Ankara, Turkey (FAX: ⫹90-3182252819; E-mail: [email protected]).
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adrenal medulla, lung, kidney, and heart (8). In addition, AM has been found in the hypothalamus-pituitary-adrenal axis (9). Adrenomedullin has local paracrine or autocrine effects on the tissue. It can trigger many physiologic events by remaining in the plasma like the circulating hormone (8 –10). In the last couple of years, AM levels have been studied in many clinical conditions, such as endocrine disorders (11). The level of plasma AM increases during the follicular phase and decreases during the luteal phase of the menstrual cycle (12). These changes show a pattern similar to that of gonadotropins. During the menstrual cycle, the changes in plasma AM levels have been demonstrated to be in correlation with LH and E2 levels (12). These changes in the AM levels could be hypothalamic or pituitary in origin (12). Estrogens may exert a gonadotropin-mediated regulatory role on AM (9). Plasma adrenomedullin concentration is increased in pancreas function disorders such as type I DM, type II DM, and insulinoma (9), and AM could play a role in regulation of insulin secretion (13). Adrenomedullin-deficient mice (AM⫹/⫺) developed insulin resistance (14). In disorders such as DM and obesity, AM may have the role in regulating the endocrine physiology of the pancreas (13). The relation of AM with pituitary, ovarian hormones, and insulin resistance led us to consider that AM may play a role in the etiopathogenesis of PCOS.
Fertility and Sterility姞 Vol. 86, No. 4, October 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2006.02.119
The hypothesis of the present research is that adrenomedullin may play a role in the etiology and pathogenesis of PCOS and that AM may correlate with various hormonal and biochemical parameters. The aim of the present study was to compare plasma AM levels in patients with PCOS and in healthy control subjects and to evaluate the correlations between AM and gonadotropins, female sex steroids, androgens, and insulin resistance. MATERIALS AND METHODS Among women aged 18 –35 years who applied to our clinic, 38 women with PCOS and 29 healthy control subjects were included in this study. The study was approved by the local ethics committee. Written informed consent was obtained from all women. Diagnosis of PCOS was made by the 1990 criteria of the National Institute of Child Health and Human Development (15). All women with PCOS were recruited according to the following criteria: oligoamenorrhea (cycle length ⬎35 days) and clinical and/or biochemical hyperandrogenism. Clinical hyperandrogenism was assessed in all women by one examiner using the Ferriman-Gallwey score, in which a score ⬎8 indicates hirsutism. The total T level ⬎0.75 ng/mL, the free T level ⬎3.18 pg/mL or the androstenedione level ⬎3.08 ng/mL were defined as biochemical hyperandrogenism. The increased LH/FSH ratio, sonographically viewed polycystic ovaries, acanthosis nigricans, or existing acne was not considered in the diagnosis of PCOS. The control group consisted of 29 healthy women aged 18 –35 years with regular menstrual cycles (cycle length 21–35 days). All subjects were nonsmokers. Exclusion criteria from the study were as follows: 1. Endocrinopathies including diabetes mellitus, thyroid dysfunction, hyperprolactinemia, and Cushing’s disease. 2. Congenital adrenal hyperplasia. 3. Systemic diseases such as hypertension, heart disease, acute asthma, renal disorders, liver disease, and collagen tissue disorder. 4. History of neoplasm. 5. Using any medication such as oral contraceptive, corticosteroids, and GnRH agonists and antagonists in the preceding 6 months.
fasting glucose and insulin, 75 g glucose was ingested and a second blood sample was obtained for glucose level after 2 hours. The glucose-insulin ratio (GIR) was calculated through the simultaneous testing of fasting glucose and fasting insulin. Adrenomedullin blood samples were collected into Lavender Vacutainer tubes which contained EDTA and aprotinin (0.6 TIU/mL of blood). The blood samples were centrifuged at 1600g for 15 minutes at 4 °C, and plasma was collected. Plasma was stored at ⫺70°C until required for analysis. After extraction of the plasma, AM concentration was measured by enzyme immunoassay (EIA) kit (Phoenix Pharmaceuticals, Belmont, CA). All samples were run in the same assay. The minimum detectable concentration of AM by the EIA kit was 0.15 ng/mL. The interassay error was ⬍14%, and the intraassay error was ⬍5%. For AM (human) the peptide specifity was 100%. No cross-reactivity was reported with rat AM (1–50), human AM (13–50), endothelin-1, amilin, calcitonin gene–related peptide (CGRP) (human), or CGRP-2. Total T, FSH, LH, E2, and DHEAS were measured with chemiluminescence methods using Elecsys kits (Roche Diagnostics, Mannheim, Germany). Free T and androstenedione were measured by EIA (Diagnostics Systems Laboratories, Webster, TX). The SHBG was measured by EIA (Biosource, Camarillo, CA). Serum glucose was measured with commercial kits (Roche Hitachi, Roche Diagnostics, Mannheim, Germany). The statistical analysis was performed using the Statistical Package for Social Sciences for Windows 11.0 program (SPSS, Chicago, IL). The data are given as mean ⫾ standard deviation from descriptive statistics. The independent t test was used to compare the demographic characteristics, biochemical parameters, and plasma AM concentration between the groups. Correlation analysis of plasma AM concentration and the other parameters was performed using Spearman correlation coefficients (r). Partial correlation coefficients were calculated between plasma AM concentration and other parameters using BMI as covariate. Statistical significance was set at P⬍.05.
On the third day of the menstrual cycle, all blood samples were obtained in the morning between 8:00 and 9.00 after an overnight fast. Serum androstenedione, free T, testosterone, DHEAS, SHBG, TSH, PRL, FSH, LH, E2, and plasma AM levels were measured.
RESULTS The demographic and biochemical characteristics of the 38 patients with PCOS and 29 control subjects are shown in Table 1. Age, WHR, and BMI were not significantly different between the groups. Serum androstenedione, free T, total testosterone, fasting insulin levels, and Ferriman-Gallwey scores were significantly higher in cases with PCOS compared with control subjects. The GIR and SHBG were significantly lower in the PCOS group compared with the control group.
A 75-g 2-hour glucose tolerance test was performed for each woman. After a venous blood sample was drawn for
The mean plasma AM concentration was found to be 84.85 ⫾ 28.02 pg/mL in patients with PCOS, whereas it was
Age, height, weight, body mass index (BMI; kg/m2), waist-hip ratio (WHR), and the menstrual cycle patterns were recorded for each subject. The WHR was calculated as waist circumference divided by hip circumference.
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TABLE 1 Demographic and biochemical characteristic of enrolled subjects (mean ⴞ SD). Characteristic
PCOS (n ⴝ 38)
Control (n ⴝ 29)
P value
Age (y) BMI (kg/m2) Waist/hip ratio FSH (mlU/mL) LH (mlU/mL) FSH/LH ratio E2 (pg/mL) TSH (mlU/l) PRL (ng/ml) SHBG (nmol/L) Androstenedione (ng/mL) Free T (pg/mL) T (ng/mL) DHEAS (g/dL) Insulin (IU/mL) Glucose (mg/dL) Glucose-insulin ratio 2nd-hour glucose (mg/dL) Ferriman-Gallwey score Adrenomedullin (pg/mL)
23.53 ⫾ 5.51 25.20 ⫾ 5.37 0.78 ⫾ 0.047 6.13 ⫾ 1.76 7.67 ⫾ 4.47 1.07 ⫾ 0.50 53.54 ⫾ 38.23 2.37 ⫾ 1.11 18.49 ⫾ 10.28 47.26 ⫾ 42.32 3.13 ⫾ 1.54 2.60 ⫾ 2.47 0.66 ⫾ 0.24 251.34 ⫾ 123.80 10.81 ⫾ 6.04 87.87 ⫾ 8.69 10.70 ⫾ 5.60 98.47 ⫾ 21.73 7.47 ⫾ 3.33 84.85 ⫾ 28.02
25.69 ⫾ 5.15 23.78 ⫾ 3.91 0.75 ⫾ 0.055 7.55 ⫾ 2.57 5.70 ⫾ 3.80 1.60 ⫾ 0.59 42.33 ⫾ 28.42 2.10 ⫾ 1.02 18.74 ⫾ 11.40 69.19 ⫾ 38.68 2.27 ⫾ 0.90 0.94 ⫾ 1.14 0.47 ⫾ 0.29 235.20 ⫾ 116.89 6.96 ⫾ 3.62 88.69 ⫾ 8.42 17.94 ⫾ 12.64 98.38 ⫾ 20.55 0.90 ⫾ 1.39 89.62 ⫾ 32.09
NS NS NS .014 .057 .0001 NS NS NS .031 .006 .001 .006 NS .002 NS .007 NS .0001 .527
Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
89.62 ⫾ 32.09 pg/mL in the control group. There was no significant difference concerning plasma AM concentrations between the groups (P⫽.527). In patients with PCOS, Spearman correlation coefficients between plasma AM concentration and age, BMI, WHR, fasting insulin and glucose, GIR, and 2nd-hour glucose level
are shown in Table 2. Plasma AM concentration was found to be negatively correlated with BMI and fasting insulin and glucose (Figs. 1, 2, and 3, respectively). Plasma AM concentration was found to be positively correlated with GIR (Fig. 4).
FIGURE 1 TABLE 2 Spearman correlation coefficients (r) between plasma adrenomedullin concentration and age, BMI, WHR, fasting insulin and glucose, glucose/insulins ratio, and 2nd-hour glucose in patients with PCOS. Characteristic
Adrenomedullin P value
Age BMI (kg/m2) Waist/hip ratio Insulin (IU/mL) Glucose (mg/dL) Glucose-insulin ratio 2nd-hour glucose (mg/dL) Note:
a
⫺0.257 ⫺0.327 ⫺0.109 ⫺0.414 ⫺0.342 0.364 0.092
.119 .045a .517 .010a .036a .025a .582
P⬍.05.
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Correlation between plasma adrenomedullin concentration and BMI in patients with PCOS.
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Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
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FIGURE 2
FIGURE 4
Correlation between plasma adrenomedullin concentration and fasting insulin in patients with PCOS.
Correlation between plasma adrenomedullin concentration and glucose-insulin ratio in patients with PCOS.
Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
In the control group, plasma AM concentration was not found to be significantly correlated with BMI (r ⫽ ⫺0.052; P⫽.790), fasting insulin (r ⫽ ⫺0.009; P⫽.962), fasting glucose (r ⫽ ⫺0.240; P⫽.209), or GIR (r ⫽ 0.001; P⫽.997). In patients with PCOS, there was no significant correlation between plasma AM concentration and fasting insulin
FIGURE 3 Correlation between plasma adrenomedullin concentration and fasting glucose in patients with PCOS.
(r ⫽ ⫺0.2445; P⫽.145) and glucose (r ⫽ ⫺0.1473; P⫽.413) levels or GIR (r ⫽ 0.1473; P⫽.384) after controlling for BMI. In patients with PCOS, Spearman correlation coefficients between plasma AM concentration and gonadotropins, E2, TSH, PRL, SHBG, and androgens are shown in Table 3. Plasma AM concentration was found to be negatively correlated with free T (r ⫽ ⫺0.458; P⫽.004).
TABLE 3 Spearman correlation coefficients (r) between plasma adrenomedullin concentration and gonadotropins, E2, TSH, PRL, SHBG, and androgens in patients with PCOS.
FSH (mlU/mL) LH (mIU/mL) FSH/LH ratio E2 (pg/mL) TSH (mlU/l) PRL (ng/mL) SHBG (nmol/L) Androstenedione (ng/mL) Free T (pg/mL) T (ng/mL) DHEAS (g/dl) Note: Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
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a
Adrenomedullin
P value
⫺0.093 0.001 0.077 0.036 ⫺0.144 0.110 0.141 ⫺0.315
.577 .997 .646 .830 .388 .511 .400 .054
⫺0.458 ⫺0.097 ⫺0.002
.004a .561 .992
P⬍.05.
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In the control group, plasma AM concentration was found to be positively correlated with free T (r ⫽ 0.407; P⫽.029). In patients with PCOS, there was no correlation between plasma AM concentration and free T after controlling for BMI (r ⫽ ⫺0.2964; P⫽.075). DISCUSSION We found that plasma AM concentration was not significantly different between patients with PCOS and control subjects. Fasting insulin and androgen levels (androstenedione, free T, and total T) were significantly higher in patients with PCOS than in control subjects. The GIR and SHBG were significantly lower in the PCOS group compared with the control group. Our results demonstrate that plasma AM concentration was negatively correlated with fasting glucose and insulin and positively correlated with GIR in the PCOS group. Nakamura et al. (16) reported that plasma AM levels were elevated in patients with type 2 diabetes and were not affected by plasma glucose levels (17, 18). The production of AM may not be directly affected by circulating glucose levels, and this situation could be dependent to the development of microangiopathy (17, 18). In contrast, other studies have shown that in patients with uncontrolled type 2 DM without renal failure, plasma AM concentration was increased. In cultured vascular smooth muscle cells, AM mRNA expression was increased by glucose (9). Martinez et al. (19) have shown that a subset of patients had higher levels of AM than other patients with type 2 DM. In this subset, physiologic elevations of AM could trigger the disease in predisposed individuals (17). In the literature, the relationship between type 2 DM and AM is controversial (20). In another study, Kinoshita et al. (21) could not find statistically significant differences in the plasma AM levels between diabetic patients without complications and healthy subjects (21). Letizia et al. (10) have observed a correlation between insulin levels and plasma AM concentrations in obese subjects during euglycemic hyperinsulinemic clamp. Katsuki et al. (22) have demonstrated that in patients with type 2 DM without evidence of microangiopathy or macroangiopathy, a significant increase in plasma AM levels was observed concomitantly with the increase in the blood concentrations of insulin during euglycemic hyperinsulinemic clamp. Changes of plasma AM were not found to be correlated with fasting glucose, fasting insulin, and glucose infusion rate in patients with type 2 DM (22). With respect to these findings, increased AM in obese or type 2 DM patients may be an adaptive mechanism for decreasing insulin levels (10, 22). Letizia et al. (23) have demonstrated that plasma AM concentrations increased in patients with insulinoma. The increased blood levels of AM may be induced in response to hyperinsulinemia in an attempt to reduce insulin concen946
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trations or may be determined by excessive secretion by neoplastic pancreatic islet cells. But their study does not prove that the increased plasma AM concentration is related directly to hyperinsulinemia (23). In normal subjects, acute hyperinsulinemia does not affect plasma AM concentration (10, 22). The anti-AM antibodies stimulate basal insulin secretion, yielding the result that endogenous AM inhibits insulin secretion (13). It has been demonstrated that AM⫹/⫺ mice were characterized by insulin resistance, which was corrected by low-dose AM infusion. Recent studies support that AM deficiency can cause dysfunction in the carbohydrate metabolism (20). Shimosawa et al. (14) have demonstrated that aged AM⫹/⫺ mice had increased body weight, developed insulin resistance, and tended to have higher blood pressure. These findings were similar with phenotypic characteristics of syndrome X (14). In the present study, although plasma AM concentration was found to be negatively correlated with fasting insulin and positively correlated with GIR in patients with PCOS who have higher insulin levels compared with control subjects, there was no correlation between plasma AM concentration and fasting insulin and GIR in the control group. In patients with PCOS, the endogenous AM may not be sufficient to suppress insulin levels. AM deficiency may be one of the causes of hyperinsulinemia in PCOS. In the present study, a significant negative correlation was detected between plasma AM concentration and BMI in patients with PCOS. There was no correlation between BMI and plasma AM in the control group. No relation was detected between plasma AM concentration and fasting glucose and insulin levels or GIR after adjusting for BMI in patients with PCOS. Letizia et al. (10) found that plasma AM concentration was significantly higher in obese subjects than in nonobese subjects (P⬍.001). In obese patients with essential hypertension, plasma AM concentration reduces with the hypocaloric diet (17). But in aged AM⫹/⫺ mice, the body weight increases (14). Katsuki et al. (22) determined that plasma AM levels were not significantly correlated with visceral fat area or subcutaneous fat area in patients with type 2 DM. The relationship between AM and BMI can reflect the dysfunction of glucose and lipid metabolism (24). Our findings indicate that the relationship between AM concentration and insulin resistance may be dependent on obesity in patients with PCOS. Similarly to many previous reports, the FSH/LH ratio was also significantly lower in PCOS patients compared with control subjects. In the PCOS group, no statistically significant correlations were detected between serum LH, FSH levels, or FSH/LH ratio and plasma AM. Marinoni et al. (12) reported that AM could have direct effects on gonadotropin release or ovarian activity. In women with a normal menstrual cycle, AM may play a role in the Vol. 86, No. 4, October 2006
regulation of pituitary-gonadal functions. It was demonstrated that during the menstrual cycle, the changes of plasma AM concentration were correlated with the changes of LH concentration. Samson et al. (25) did not observe any changes in levels of LH from isolated rat pituitary cells in response to AM. Meeran et al. (26) reported that LH and FSH did not change after infusion of AM in men. In the present study also, no relationship was established between endogenous plasma AM concentration and gonadotropins in patients with PCOS. In women with a normal menstrual cycle, correlations between plasma AM concentration and fluctuations of gonadotropins could be related to ovulation. The ovulatory follicle contains AM, and AM increases in the ovulatory phase in the circulation (27). In the human brain, the highest AM concentration is found in the hypophysis (17). It should be considered that these effects mediated by this peptide could be local paracrine. In the present study, no significant correlation was detected between E2 levels and plasma AM concentration in patients with PCOS. It has been reported that E2 had no effect on the AM mRNA expression in the vascular endothelial cells or in the vascular smooth muscle cells (28). Manau et al. (29) did not establish a significant correlation between plasma AM levels and serum E2 in women with normal menstrual cycles. The follicular fluid concentrations of AM and E2 were significantly higher than their plasma concentrations, and no significant correlation was detected between AM and E2 concentration in the follicular fluid. Marinoni et al. (12) found the changes in the plasma AM concentration to be in correlation with the changes in E2 concentration during the menstrual cycle. According to another hypothesis based on the correlation between the E2 and AM in the follicular phase, estrogens represent a physiologic stimulation for AM secretion (9). Although estrogens do not induce AM mRNA expression in cultured human endometrial cells, one must not exclude that these hormones may exert a gonadotropin-mediated regulatory role on AM (9). We found a significant negative correlation between plasma AM concentration and free T in patients with PCOS. No statistically significant correlation was observed between the other androgens and plasma AM concentration. The relationship between AM and free T is not clear. In the rat ventral prostate, AM expression is dependent on androgen, with a 25-fold reduction in mRNA after castration, which is fully reversible by androgen administration (11). Adrenomedullin is expressed at a very low level in zona reticularis (20). In cultured cells, it was demonstrated that adrenocortical sex steroids led to a very small increase in AM levels (8, 30). It was reported that T had no effect on AM mRNA expression in the vascular endothelial cells or in the vascular smooth muscle cells (28). In PCOS, hyperinsulinemia leads to the production of ovarian and adrenal androgen, stimulates proliferation of the pilosebaceous unit, and supresses SHBG production (31). Fertility and Sterility姞
Administration of insulin to women with polycystic ovaries increases circulating androgen levels (32). It has been reported in many studies that metformin increased peripheral tissue sensitivity to insulin and inhibited hepatic glucose production (32). In women with PCOS who received metformin, serum free T was reduced (32). In the present study, the cause of the negative correlation between plasma AM and serum free T could be that the endogenous AM concentration was not sufficient to suppress hyperinsulinemia in patients with PCOS. In conclusion, in the present study there was no significant difference concerning plasma AM concentrations between patients with PCOS and control subjects. In the PCOS group, a significant negative correlation was observed between plasma AM concentration and BMI, fasting insulin, fasting glucose, and free T, and a significant positive correlation was observed between plasma AM concentration and GIR. In patients with PCOS, AM may play a role in regulating the insulin metabolism. Further studies are needed to evaluate the role of AM in PCOS. REFERENCES 1. Carmina E, Lobo RA. Polycytic ovary syndrome (PCOS): arguably the most common endocrinopathy is associated with significant morbidity in woman. J Clin Endocrinol Metab 1999;84:1897–9. 2. Taylor AE, Mccourt B, Martin KA, Anderson EJ, Adams JM, Schoenfeld D, et al. Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab 1997;82:2248 –56. 3. Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R. Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 1998;83:3078 – 82. 4. Arroyo A, Laughlin GA, Morales AJ, Yen SSC. Inappropriate gonadotropin secretion in polycystic ovary sendrome: influence of adiposity. J Clin Endocrinol Metab 1997;82:3728 –33. 5. Doi SAR, Towers PA, Scott CJ, Al-Shoumer KAS. PCOS: an ovarian disorder that leads to dysregulation in the hypothalamic-pituitaryadrenal axis? Eur J Obstet Gynecol Reprod Biol 2005;118:4 –16. 6. Anovulation and polycystic ovary. In: Speroff L, Glass RH, Kase NG, Clinical gynecologic endocrinology and infertility. 6th ed. Baltimore, MD: Lippincott Williams & Wilkins; 1999. p. 487–522. 7. Guzick DS. Editorial: cardiovascular risk in PCOS. J Clin Endocrinol Metab 2004;89:3694 –5. 8. Samson WK. Adrenomedullin and the control of fluid and electrolyte homeostasis. Annu Rev Physiol 1999;61:363– 89. 9. Letizia C, Rossi G, Cerci S. Adrenomedullin and endocrine disorders. Panminerva Medica 2003;45:241–51. 10. Letizia C, Iacobellis G, Caliumi C, Leonetti F, Cotesta D, Ribaudo MC, et al. Acute hyperinsulinemia is associated with increased plasma adrenomedullin concentrations in uncomplicated obesity. Exp Clin Endocrinol Diabetes 2005;113:171–5. 11. Hinson PJ, Kapas S, Smith DM. Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev 2000;21:138 – 67. 12. Marinoni E, Iorio RD, Letizia C, Lucchini C, Alo P, Cosmi EV. Changes in plasma adrenomedullin levels during the menstrual cycle. Regul Pept 2000;87:15– 8. 13. Martinez A, Weaver C, Lopez J, Bhathena SJ, Elsasser TH, Miller MJ, et al. Regulation of insulin secretion and blood glucose metabolism by adrenomedullin. Endocrinology 1996;137;2626 –32.
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Department of Obstetrics and Gynecology and b Department of Biochemistry and Clinical Biochemistry, Kirikkale University School of Medicine, Kirikkale, Turkey
Objective: To evaluate adrenomedullin levels in patients with polycystic ovary syndrome (PCOS). Design: Prospective study. Setting: Department of obstetrics and gynecology in a university hospital. Patient(s): Thirty-eight women with PCOS and 29 healthy control subjects were enrolled in the study. Intervention(s): Plasma adrenomedullin, serum androstenedione, free T, T, DHEAS, SHBG, thyrotropin, PRL, FSH, LH, and E2 were measured in each subject. Insulin resistance was estimated by fasting insulin level, fasting glucose:insulin ratio and 75-g glucose tolerance test for 2 hours. Main Outcome Measure(s): Plasma adrenomedullin levels and correlations among adrenomedullin and gonadotropins, female sex steroids, androgens, and insulin resistance. Result(s): There was no significant difference concerning plasma adrenomedullin concentrations between the groups. In patients with PCOS, fasting glucose, fasting insulin, body mass index, and free T were inversely correlated with the plasma adrenomedullin. Plasma adrenomedullin was significantly correlated with glucose: insulin ratio. After controlling for body mass index, there were no significant correlations between the abovementioned parameters. Conclusion(s): Adrenomedullin may play a role in regulating the insulin metabolism in patients with PCOS. (Fertil Steril威 2006;86:942– 8. ©2006 by American Society for Reproductive Medicine.) Key Words: Polycystic ovary syndrome, adrenomedullin, insulin resistance, obesity
Polycystic ovary syndrome (PCOS) is characterized by menstrual dysfunction, hyperandrogenism, and frequent insulin resistance. Polycystic ovary syndrome is the most common endocrinopathy among women of reproductive age. The estimate of incidence of PCOS differs between 3% and 10% (1–3), and its etiopathogenesis is still controversial (4, 5). Hypothalamus-pituitary-ovary axis changes, obesity, insulin resistance, and ovarian and adrenal androgen excess due to steroidogenic dysfunction play roles in the pathophysiology (6). The pulsatile emission of GnRH is increased in PCOS. In patients with PCOS, the amplitude and frequency of LH pulses increase (6). The changes of gonadotropin dynamics can develop secondarily to central or peripheral hormonal deviations (5). In patients with PCOS, the prevalence of cardiovascular disorders, insulin resistance, and diabetes mellitus (DM) has been found to be increased (1, 7). Adrenomedullin (AM) was first found in 1993 in human pheochromocytoma tissue (8). Adrenomedullin is a vasodilator peptide with 52 amino acids (8). Not only is the AM mRNA expressed in pheochromocytoma tissue, but AM gene products have been detected also in tissues including
Received September 27, 2005; revised and accepted February 25, 2006. Supported by the Department of Scientific Research Projects, Kirikkale University, Kirikkale, Turkey. Reprint requests: Banu Ucar, M.D., Karargah tepe mah. Atıs cad., Komut. Sok. 14/4, Meteoroloji, Kecioren, Ankara, Turkey (FAX: ⫹90-3182252819; E-mail: [email protected]).
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adrenal medulla, lung, kidney, and heart (8). In addition, AM has been found in the hypothalamus-pituitary-adrenal axis (9). Adrenomedullin has local paracrine or autocrine effects on the tissue. It can trigger many physiologic events by remaining in the plasma like the circulating hormone (8 –10). In the last couple of years, AM levels have been studied in many clinical conditions, such as endocrine disorders (11). The level of plasma AM increases during the follicular phase and decreases during the luteal phase of the menstrual cycle (12). These changes show a pattern similar to that of gonadotropins. During the menstrual cycle, the changes in plasma AM levels have been demonstrated to be in correlation with LH and E2 levels (12). These changes in the AM levels could be hypothalamic or pituitary in origin (12). Estrogens may exert a gonadotropin-mediated regulatory role on AM (9). Plasma adrenomedullin concentration is increased in pancreas function disorders such as type I DM, type II DM, and insulinoma (9), and AM could play a role in regulation of insulin secretion (13). Adrenomedullin-deficient mice (AM⫹/⫺) developed insulin resistance (14). In disorders such as DM and obesity, AM may have the role in regulating the endocrine physiology of the pancreas (13). The relation of AM with pituitary, ovarian hormones, and insulin resistance led us to consider that AM may play a role in the etiopathogenesis of PCOS.
Fertility and Sterility姞 Vol. 86, No. 4, October 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2006.02.119
The hypothesis of the present research is that adrenomedullin may play a role in the etiology and pathogenesis of PCOS and that AM may correlate with various hormonal and biochemical parameters. The aim of the present study was to compare plasma AM levels in patients with PCOS and in healthy control subjects and to evaluate the correlations between AM and gonadotropins, female sex steroids, androgens, and insulin resistance. MATERIALS AND METHODS Among women aged 18 –35 years who applied to our clinic, 38 women with PCOS and 29 healthy control subjects were included in this study. The study was approved by the local ethics committee. Written informed consent was obtained from all women. Diagnosis of PCOS was made by the 1990 criteria of the National Institute of Child Health and Human Development (15). All women with PCOS were recruited according to the following criteria: oligoamenorrhea (cycle length ⬎35 days) and clinical and/or biochemical hyperandrogenism. Clinical hyperandrogenism was assessed in all women by one examiner using the Ferriman-Gallwey score, in which a score ⬎8 indicates hirsutism. The total T level ⬎0.75 ng/mL, the free T level ⬎3.18 pg/mL or the androstenedione level ⬎3.08 ng/mL were defined as biochemical hyperandrogenism. The increased LH/FSH ratio, sonographically viewed polycystic ovaries, acanthosis nigricans, or existing acne was not considered in the diagnosis of PCOS. The control group consisted of 29 healthy women aged 18 –35 years with regular menstrual cycles (cycle length 21–35 days). All subjects were nonsmokers. Exclusion criteria from the study were as follows: 1. Endocrinopathies including diabetes mellitus, thyroid dysfunction, hyperprolactinemia, and Cushing’s disease. 2. Congenital adrenal hyperplasia. 3. Systemic diseases such as hypertension, heart disease, acute asthma, renal disorders, liver disease, and collagen tissue disorder. 4. History of neoplasm. 5. Using any medication such as oral contraceptive, corticosteroids, and GnRH agonists and antagonists in the preceding 6 months.
fasting glucose and insulin, 75 g glucose was ingested and a second blood sample was obtained for glucose level after 2 hours. The glucose-insulin ratio (GIR) was calculated through the simultaneous testing of fasting glucose and fasting insulin. Adrenomedullin blood samples were collected into Lavender Vacutainer tubes which contained EDTA and aprotinin (0.6 TIU/mL of blood). The blood samples were centrifuged at 1600g for 15 minutes at 4 °C, and plasma was collected. Plasma was stored at ⫺70°C until required for analysis. After extraction of the plasma, AM concentration was measured by enzyme immunoassay (EIA) kit (Phoenix Pharmaceuticals, Belmont, CA). All samples were run in the same assay. The minimum detectable concentration of AM by the EIA kit was 0.15 ng/mL. The interassay error was ⬍14%, and the intraassay error was ⬍5%. For AM (human) the peptide specifity was 100%. No cross-reactivity was reported with rat AM (1–50), human AM (13–50), endothelin-1, amilin, calcitonin gene–related peptide (CGRP) (human), or CGRP-2. Total T, FSH, LH, E2, and DHEAS were measured with chemiluminescence methods using Elecsys kits (Roche Diagnostics, Mannheim, Germany). Free T and androstenedione were measured by EIA (Diagnostics Systems Laboratories, Webster, TX). The SHBG was measured by EIA (Biosource, Camarillo, CA). Serum glucose was measured with commercial kits (Roche Hitachi, Roche Diagnostics, Mannheim, Germany). The statistical analysis was performed using the Statistical Package for Social Sciences for Windows 11.0 program (SPSS, Chicago, IL). The data are given as mean ⫾ standard deviation from descriptive statistics. The independent t test was used to compare the demographic characteristics, biochemical parameters, and plasma AM concentration between the groups. Correlation analysis of plasma AM concentration and the other parameters was performed using Spearman correlation coefficients (r). Partial correlation coefficients were calculated between plasma AM concentration and other parameters using BMI as covariate. Statistical significance was set at P⬍.05.
On the third day of the menstrual cycle, all blood samples were obtained in the morning between 8:00 and 9.00 after an overnight fast. Serum androstenedione, free T, testosterone, DHEAS, SHBG, TSH, PRL, FSH, LH, E2, and plasma AM levels were measured.
RESULTS The demographic and biochemical characteristics of the 38 patients with PCOS and 29 control subjects are shown in Table 1. Age, WHR, and BMI were not significantly different between the groups. Serum androstenedione, free T, total testosterone, fasting insulin levels, and Ferriman-Gallwey scores were significantly higher in cases with PCOS compared with control subjects. The GIR and SHBG were significantly lower in the PCOS group compared with the control group.
A 75-g 2-hour glucose tolerance test was performed for each woman. After a venous blood sample was drawn for
The mean plasma AM concentration was found to be 84.85 ⫾ 28.02 pg/mL in patients with PCOS, whereas it was
Age, height, weight, body mass index (BMI; kg/m2), waist-hip ratio (WHR), and the menstrual cycle patterns were recorded for each subject. The WHR was calculated as waist circumference divided by hip circumference.
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TABLE 1 Demographic and biochemical characteristic of enrolled subjects (mean ⴞ SD). Characteristic
PCOS (n ⴝ 38)
Control (n ⴝ 29)
P value
Age (y) BMI (kg/m2) Waist/hip ratio FSH (mlU/mL) LH (mlU/mL) FSH/LH ratio E2 (pg/mL) TSH (mlU/l) PRL (ng/ml) SHBG (nmol/L) Androstenedione (ng/mL) Free T (pg/mL) T (ng/mL) DHEAS (g/dL) Insulin (IU/mL) Glucose (mg/dL) Glucose-insulin ratio 2nd-hour glucose (mg/dL) Ferriman-Gallwey score Adrenomedullin (pg/mL)
23.53 ⫾ 5.51 25.20 ⫾ 5.37 0.78 ⫾ 0.047 6.13 ⫾ 1.76 7.67 ⫾ 4.47 1.07 ⫾ 0.50 53.54 ⫾ 38.23 2.37 ⫾ 1.11 18.49 ⫾ 10.28 47.26 ⫾ 42.32 3.13 ⫾ 1.54 2.60 ⫾ 2.47 0.66 ⫾ 0.24 251.34 ⫾ 123.80 10.81 ⫾ 6.04 87.87 ⫾ 8.69 10.70 ⫾ 5.60 98.47 ⫾ 21.73 7.47 ⫾ 3.33 84.85 ⫾ 28.02
25.69 ⫾ 5.15 23.78 ⫾ 3.91 0.75 ⫾ 0.055 7.55 ⫾ 2.57 5.70 ⫾ 3.80 1.60 ⫾ 0.59 42.33 ⫾ 28.42 2.10 ⫾ 1.02 18.74 ⫾ 11.40 69.19 ⫾ 38.68 2.27 ⫾ 0.90 0.94 ⫾ 1.14 0.47 ⫾ 0.29 235.20 ⫾ 116.89 6.96 ⫾ 3.62 88.69 ⫾ 8.42 17.94 ⫾ 12.64 98.38 ⫾ 20.55 0.90 ⫾ 1.39 89.62 ⫾ 32.09
NS NS NS .014 .057 .0001 NS NS NS .031 .006 .001 .006 NS .002 NS .007 NS .0001 .527
Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
89.62 ⫾ 32.09 pg/mL in the control group. There was no significant difference concerning plasma AM concentrations between the groups (P⫽.527). In patients with PCOS, Spearman correlation coefficients between plasma AM concentration and age, BMI, WHR, fasting insulin and glucose, GIR, and 2nd-hour glucose level
are shown in Table 2. Plasma AM concentration was found to be negatively correlated with BMI and fasting insulin and glucose (Figs. 1, 2, and 3, respectively). Plasma AM concentration was found to be positively correlated with GIR (Fig. 4).
FIGURE 1 TABLE 2 Spearman correlation coefficients (r) between plasma adrenomedullin concentration and age, BMI, WHR, fasting insulin and glucose, glucose/insulins ratio, and 2nd-hour glucose in patients with PCOS. Characteristic
Adrenomedullin P value
Age BMI (kg/m2) Waist/hip ratio Insulin (IU/mL) Glucose (mg/dL) Glucose-insulin ratio 2nd-hour glucose (mg/dL) Note:
a
⫺0.257 ⫺0.327 ⫺0.109 ⫺0.414 ⫺0.342 0.364 0.092
.119 .045a .517 .010a .036a .025a .582
P⬍.05.
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Correlation between plasma adrenomedullin concentration and BMI in patients with PCOS.
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Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
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FIGURE 2
FIGURE 4
Correlation between plasma adrenomedullin concentration and fasting insulin in patients with PCOS.
Correlation between plasma adrenomedullin concentration and glucose-insulin ratio in patients with PCOS.
Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
In the control group, plasma AM concentration was not found to be significantly correlated with BMI (r ⫽ ⫺0.052; P⫽.790), fasting insulin (r ⫽ ⫺0.009; P⫽.962), fasting glucose (r ⫽ ⫺0.240; P⫽.209), or GIR (r ⫽ 0.001; P⫽.997). In patients with PCOS, there was no significant correlation between plasma AM concentration and fasting insulin
FIGURE 3 Correlation between plasma adrenomedullin concentration and fasting glucose in patients with PCOS.
(r ⫽ ⫺0.2445; P⫽.145) and glucose (r ⫽ ⫺0.1473; P⫽.413) levels or GIR (r ⫽ 0.1473; P⫽.384) after controlling for BMI. In patients with PCOS, Spearman correlation coefficients between plasma AM concentration and gonadotropins, E2, TSH, PRL, SHBG, and androgens are shown in Table 3. Plasma AM concentration was found to be negatively correlated with free T (r ⫽ ⫺0.458; P⫽.004).
TABLE 3 Spearman correlation coefficients (r) between plasma adrenomedullin concentration and gonadotropins, E2, TSH, PRL, SHBG, and androgens in patients with PCOS.
FSH (mlU/mL) LH (mIU/mL) FSH/LH ratio E2 (pg/mL) TSH (mlU/l) PRL (ng/mL) SHBG (nmol/L) Androstenedione (ng/mL) Free T (pg/mL) T (ng/mL) DHEAS (g/dl) Note: Ucar. Adrenomedullin in PCOS. Fertil Steril 2006.
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a
Adrenomedullin
P value
⫺0.093 0.001 0.077 0.036 ⫺0.144 0.110 0.141 ⫺0.315
.577 .997 .646 .830 .388 .511 .400 .054
⫺0.458 ⫺0.097 ⫺0.002
.004a .561 .992
P⬍.05.
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In the control group, plasma AM concentration was found to be positively correlated with free T (r ⫽ 0.407; P⫽.029). In patients with PCOS, there was no correlation between plasma AM concentration and free T after controlling for BMI (r ⫽ ⫺0.2964; P⫽.075). DISCUSSION We found that plasma AM concentration was not significantly different between patients with PCOS and control subjects. Fasting insulin and androgen levels (androstenedione, free T, and total T) were significantly higher in patients with PCOS than in control subjects. The GIR and SHBG were significantly lower in the PCOS group compared with the control group. Our results demonstrate that plasma AM concentration was negatively correlated with fasting glucose and insulin and positively correlated with GIR in the PCOS group. Nakamura et al. (16) reported that plasma AM levels were elevated in patients with type 2 diabetes and were not affected by plasma glucose levels (17, 18). The production of AM may not be directly affected by circulating glucose levels, and this situation could be dependent to the development of microangiopathy (17, 18). In contrast, other studies have shown that in patients with uncontrolled type 2 DM without renal failure, plasma AM concentration was increased. In cultured vascular smooth muscle cells, AM mRNA expression was increased by glucose (9). Martinez et al. (19) have shown that a subset of patients had higher levels of AM than other patients with type 2 DM. In this subset, physiologic elevations of AM could trigger the disease in predisposed individuals (17). In the literature, the relationship between type 2 DM and AM is controversial (20). In another study, Kinoshita et al. (21) could not find statistically significant differences in the plasma AM levels between diabetic patients without complications and healthy subjects (21). Letizia et al. (10) have observed a correlation between insulin levels and plasma AM concentrations in obese subjects during euglycemic hyperinsulinemic clamp. Katsuki et al. (22) have demonstrated that in patients with type 2 DM without evidence of microangiopathy or macroangiopathy, a significant increase in plasma AM levels was observed concomitantly with the increase in the blood concentrations of insulin during euglycemic hyperinsulinemic clamp. Changes of plasma AM were not found to be correlated with fasting glucose, fasting insulin, and glucose infusion rate in patients with type 2 DM (22). With respect to these findings, increased AM in obese or type 2 DM patients may be an adaptive mechanism for decreasing insulin levels (10, 22). Letizia et al. (23) have demonstrated that plasma AM concentrations increased in patients with insulinoma. The increased blood levels of AM may be induced in response to hyperinsulinemia in an attempt to reduce insulin concen946
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trations or may be determined by excessive secretion by neoplastic pancreatic islet cells. But their study does not prove that the increased plasma AM concentration is related directly to hyperinsulinemia (23). In normal subjects, acute hyperinsulinemia does not affect plasma AM concentration (10, 22). The anti-AM antibodies stimulate basal insulin secretion, yielding the result that endogenous AM inhibits insulin secretion (13). It has been demonstrated that AM⫹/⫺ mice were characterized by insulin resistance, which was corrected by low-dose AM infusion. Recent studies support that AM deficiency can cause dysfunction in the carbohydrate metabolism (20). Shimosawa et al. (14) have demonstrated that aged AM⫹/⫺ mice had increased body weight, developed insulin resistance, and tended to have higher blood pressure. These findings were similar with phenotypic characteristics of syndrome X (14). In the present study, although plasma AM concentration was found to be negatively correlated with fasting insulin and positively correlated with GIR in patients with PCOS who have higher insulin levels compared with control subjects, there was no correlation between plasma AM concentration and fasting insulin and GIR in the control group. In patients with PCOS, the endogenous AM may not be sufficient to suppress insulin levels. AM deficiency may be one of the causes of hyperinsulinemia in PCOS. In the present study, a significant negative correlation was detected between plasma AM concentration and BMI in patients with PCOS. There was no correlation between BMI and plasma AM in the control group. No relation was detected between plasma AM concentration and fasting glucose and insulin levels or GIR after adjusting for BMI in patients with PCOS. Letizia et al. (10) found that plasma AM concentration was significantly higher in obese subjects than in nonobese subjects (P⬍.001). In obese patients with essential hypertension, plasma AM concentration reduces with the hypocaloric diet (17). But in aged AM⫹/⫺ mice, the body weight increases (14). Katsuki et al. (22) determined that plasma AM levels were not significantly correlated with visceral fat area or subcutaneous fat area in patients with type 2 DM. The relationship between AM and BMI can reflect the dysfunction of glucose and lipid metabolism (24). Our findings indicate that the relationship between AM concentration and insulin resistance may be dependent on obesity in patients with PCOS. Similarly to many previous reports, the FSH/LH ratio was also significantly lower in PCOS patients compared with control subjects. In the PCOS group, no statistically significant correlations were detected between serum LH, FSH levels, or FSH/LH ratio and plasma AM. Marinoni et al. (12) reported that AM could have direct effects on gonadotropin release or ovarian activity. In women with a normal menstrual cycle, AM may play a role in the Vol. 86, No. 4, October 2006
regulation of pituitary-gonadal functions. It was demonstrated that during the menstrual cycle, the changes of plasma AM concentration were correlated with the changes of LH concentration. Samson et al. (25) did not observe any changes in levels of LH from isolated rat pituitary cells in response to AM. Meeran et al. (26) reported that LH and FSH did not change after infusion of AM in men. In the present study also, no relationship was established between endogenous plasma AM concentration and gonadotropins in patients with PCOS. In women with a normal menstrual cycle, correlations between plasma AM concentration and fluctuations of gonadotropins could be related to ovulation. The ovulatory follicle contains AM, and AM increases in the ovulatory phase in the circulation (27). In the human brain, the highest AM concentration is found in the hypophysis (17). It should be considered that these effects mediated by this peptide could be local paracrine. In the present study, no significant correlation was detected between E2 levels and plasma AM concentration in patients with PCOS. It has been reported that E2 had no effect on the AM mRNA expression in the vascular endothelial cells or in the vascular smooth muscle cells (28). Manau et al. (29) did not establish a significant correlation between plasma AM levels and serum E2 in women with normal menstrual cycles. The follicular fluid concentrations of AM and E2 were significantly higher than their plasma concentrations, and no significant correlation was detected between AM and E2 concentration in the follicular fluid. Marinoni et al. (12) found the changes in the plasma AM concentration to be in correlation with the changes in E2 concentration during the menstrual cycle. According to another hypothesis based on the correlation between the E2 and AM in the follicular phase, estrogens represent a physiologic stimulation for AM secretion (9). Although estrogens do not induce AM mRNA expression in cultured human endometrial cells, one must not exclude that these hormones may exert a gonadotropin-mediated regulatory role on AM (9). We found a significant negative correlation between plasma AM concentration and free T in patients with PCOS. No statistically significant correlation was observed between the other androgens and plasma AM concentration. The relationship between AM and free T is not clear. In the rat ventral prostate, AM expression is dependent on androgen, with a 25-fold reduction in mRNA after castration, which is fully reversible by androgen administration (11). Adrenomedullin is expressed at a very low level in zona reticularis (20). In cultured cells, it was demonstrated that adrenocortical sex steroids led to a very small increase in AM levels (8, 30). It was reported that T had no effect on AM mRNA expression in the vascular endothelial cells or in the vascular smooth muscle cells (28). In PCOS, hyperinsulinemia leads to the production of ovarian and adrenal androgen, stimulates proliferation of the pilosebaceous unit, and supresses SHBG production (31). Fertility and Sterility姞
Administration of insulin to women with polycystic ovaries increases circulating androgen levels (32). It has been reported in many studies that metformin increased peripheral tissue sensitivity to insulin and inhibited hepatic glucose production (32). In women with PCOS who received metformin, serum free T was reduced (32). In the present study, the cause of the negative correlation between plasma AM and serum free T could be that the endogenous AM concentration was not sufficient to suppress hyperinsulinemia in patients with PCOS. In conclusion, in the present study there was no significant difference concerning plasma AM concentrations between patients with PCOS and control subjects. In the PCOS group, a significant negative correlation was observed between plasma AM concentration and BMI, fasting insulin, fasting glucose, and free T, and a significant positive correlation was observed between plasma AM concentration and GIR. In patients with PCOS, AM may play a role in regulating the insulin metabolism. Further studies are needed to evaluate the role of AM in PCOS. REFERENCES 1. Carmina E, Lobo RA. Polycytic ovary syndrome (PCOS): arguably the most common endocrinopathy is associated with significant morbidity in woman. J Clin Endocrinol Metab 1999;84:1897–9. 2. Taylor AE, Mccourt B, Martin KA, Anderson EJ, Adams JM, Schoenfeld D, et al. Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab 1997;82:2248 –56. 3. Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R. Prevalence of the polycystic ovary syndrome in unselected black and white women of the southeastern United States: a prospective study. J Clin Endocrinol Metab 1998;83:3078 – 82. 4. Arroyo A, Laughlin GA, Morales AJ, Yen SSC. Inappropriate gonadotropin secretion in polycystic ovary sendrome: influence of adiposity. J Clin Endocrinol Metab 1997;82:3728 –33. 5. Doi SAR, Towers PA, Scott CJ, Al-Shoumer KAS. PCOS: an ovarian disorder that leads to dysregulation in the hypothalamic-pituitaryadrenal axis? Eur J Obstet Gynecol Reprod Biol 2005;118:4 –16. 6. Anovulation and polycystic ovary. In: Speroff L, Glass RH, Kase NG, Clinical gynecologic endocrinology and infertility. 6th ed. Baltimore, MD: Lippincott Williams & Wilkins; 1999. p. 487–522. 7. Guzick DS. Editorial: cardiovascular risk in PCOS. J Clin Endocrinol Metab 2004;89:3694 –5. 8. Samson WK. Adrenomedullin and the control of fluid and electrolyte homeostasis. Annu Rev Physiol 1999;61:363– 89. 9. Letizia C, Rossi G, Cerci S. Adrenomedullin and endocrine disorders. Panminerva Medica 2003;45:241–51. 10. Letizia C, Iacobellis G, Caliumi C, Leonetti F, Cotesta D, Ribaudo MC, et al. Acute hyperinsulinemia is associated with increased plasma adrenomedullin concentrations in uncomplicated obesity. Exp Clin Endocrinol Diabetes 2005;113:171–5. 11. Hinson PJ, Kapas S, Smith DM. Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev 2000;21:138 – 67. 12. Marinoni E, Iorio RD, Letizia C, Lucchini C, Alo P, Cosmi EV. Changes in plasma adrenomedullin levels during the menstrual cycle. Regul Pept 2000;87:15– 8. 13. Martinez A, Weaver C, Lopez J, Bhathena SJ, Elsasser TH, Miller MJ, et al. Regulation of insulin secretion and blood glucose metabolism by adrenomedullin. Endocrinology 1996;137;2626 –32.
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