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Plasma metastin levels are negatively correlated with insulin resistance and free androgens in women with polycystic ovary syndrome
POLYCYSTIC OVARY SYNDROME Plasma metastin levels are negatively correlated with insulin resistance and free androgens in women with polycystic ovary syndrome Dimitrios Panidis, Ph.D.,a David Rousso, M.D.,a George Koliakos, M.D.,b Anargyros Kourtis, M.D.,a Ilias Katsikis, M.D.,a Dimitrios Farmakiotis, M.D.,a Elissavet Votsi, M.D.,a and Evanthia Diamanti-Kandarakis, M.D.c a
Division of Endocrinology and Human Reproduction, Second Department of Obstetrics and Gynecology and b Department of Biochemistry, Medical School, Aristotle University of Thessaloniki, Thessaloniki; and c Endocrine Section of the First Department of Internal Medicine, Athens University School of Medicine, Athens, Greece
Objective: This study was designed to: [1] measure, for the first time, metastin (kisspeptin) levels in women with polycystic ovary syndrome (PCOS), a condition associated with hypersecretion of LH and hyperandrogenemia; and [2] investigate the possible correlations between metastin and PCOS-related reproductive and metabolic disturbances. Design: Clinical study. Setting: University hospital. Patient(s): Twenty-eight obese and overweight (body mass index [BMI] ⬎25 kg/m2) women with PCOS, 28 normal weight (BMI ⬍25 kg/m2) women with the syndrome, and 13 obese and overweight controls (ovulatory women without clinical or biochemical hyperandrogenemia) were selected. Intervention(s): Blood samples were collected between day 3 and day 6 of a spontaneous bleeding episode in the PCOS groups and a menstrual cycle of the controls, at 9:00 AM, after an overnight fast. Main Outcome Measure(s): Circulating levels of LH, FSH, PRL, T, ⌬4-androstenedione (A), DHEAS, 17␣OH-P, sex hormone-binding globulin (SHBG), insulin, glucose, and metastin were measured. Result(s): Both normal weight women with PCOS and obese controls were less insulin resistant and had significantly higher metastin levels, compared to obese and overweight women with the syndrome. Plasma kisspeptin levels were negatively correlated with BMI, free androgen index, and indices of insulin resistance. Conclusion(s): These results indicate that metastin is negatively associated with free androgen levels. The PCOS-associated insulin resistance and consequent hyperinsulinemia probably contribute to this effect by [1] stimulating androgen synthesis by the polycystic ovary (PCO) and [2] suppressing SHBG production in the liver. (Fertil Steril威 2006;85:1778 – 83. ©2006 by American Society for Reproductive Medicine.) Key Words: Metastin, kisspeptin, PCOS, obesity, insulin resistance
Metastin is a 54-amino-acid peptide, which was first isolated from the human placenta in 2001 (1). It is encoded by the Kiss-1 gene, therefore, it is also known as Kiss-1 peptide (kisspeptin) (2– 4). Metastin action is exerted by a trans-membrane G-protein-coupled receptor, named GPR54, AXOR12, or HoT7T175 (1, 5, 6). Kiss-1 expression has been associated with antimetastatic activity in malignant melanoma (4), breast (7), papillary thyroid (3), esophageal (8) and bladder (9) carcinoma. Furthermore, kisspeptin has also been found to suppress the invasion of human trophoblast cells (10), a process sharing common pathways with tumor metastasis (11–13). A possible physiological role of this peptide in the control of trophoblast growth into adjacent Received March 24, 2005; revised and accepted November 6, 2005. Reprint requests: Anargyros Kourtis, M.D., 2nd Department of Obstetrics and Gynecology, 45 Aristotelous Str, 55236, Panorama, Greece (FAX: 30 2310 346942; E-mail: [email protected]).
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tissues could also account for the dramatic increase of plasma metastin levels in pregnancy (14). Intriguingly, loss of Kiss-1 function is associated with hypogonadotropic hypogonadism in humans (15) and in animal models (16, 17). Administration of the Kiss-1 peptide (metastin/kisspeptin) has been shown to induce maturation of the female reproductive system and precocious puberty in rodents (18). Moreover, there is strong evidence that metastin exerts these effects by stimulating GnRH release from the hypothalamus, and, as a consequence, pituitary LH secretion (19 –22). It is also interesting that a negative feedback loop might also exist, as kisspeptin has been reported to be down-regulated by sex steroids (23). The polycystic ovary syndrome (PCOS) is a common heterogeneous disorder of women of reproductive age, and the most frequent cause of hyperandrogenism combined with
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anovulatory infertility (24, 25). Its complex pathogenesis involves [1] hypothalamic–pituitary disturbances in gonadotropin secretion, specifically increased LH levels (26, 27); [2] increased resistance to insulin (28) and compensatory hyperinsulinemia, which has been associated with enhanced androgen production (29) and reduced synthesis of sex hormone-binding globulin (SHBG) (30); and [3] disturbed gonadal steroidogenesis (31). Given the complex relationship between the novel peptide metastin and the hypothalamic–pituitary– gonadal axis, the present study was designed to: [1] measure metastin levels in women with PCOS, a condition associated with aberrant gonadotropin secretion and hyperandrogenemia; and [2] investigate the possible correlations between kisspeptin and PCOS-related reproductive and metabolic disturbances. MATERIALS AND METHODS Subjects Fifty-six women with PCOS, aged 15–37 years, were recruited from the outpatient endocrine infirmary of our clinic, with at least one of the following signs: oligomenorrhea, fertility problem, hirsutism, acne, or male pattern alopecia. Diagnosis of PCOS was based on the presence of chronic anovulation (less than six cycles in 12 months) and hyperandrogenemia. Other common causes of hyperandrogenism or menstrual disorders (prolactinoma, congenital adrenal hyperplasia, Cushing syndrome, and virilizing ovarian or adrenal tumors) were excluded, in accord with the criteria proposed in 1990 by the National Institutes of Health–National Institute of Child Health and Human Development (NIHNICHHD) (32), and revised in 2003 by the Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group (33). Women with PCOS were further divided into two groups, based on body mass index (BMI) values: obese and overweight (BMI ⬎25 kg/m2) (n ⫽ 28) and normal weight (BMI ⬍25 kg/m2) women with the syndrome (n ⫽ 28). Thirteen obese and overweight healthy women, aged 18 –31 years, volunteered as controls. All controls had normal ovulating cycles (28 ⫾ 2 days, blood P levels ⬎10 ng/mL in two consequent cycles), and no signs of hyperandrogenism. None of the women studied had galactorrhea, or systemic disease that could affect their reproductive physiology. Furthermore, no woman reported use of any medication that could interfere with the normal function of the hypothalamic–pituitary– gonadal axis, during the past 6 months. Informed consent was obtained from all 69 women and the study was approved by the local ethical committee. Hormonal and Biochemical Measurements and Calculations Blood samples were collected between day 3 and day 6 of a spontaneous bleeding episode in the PCOS groups and a menstrual cycle of the controls, at 9:00 AM, after an overFertility and Sterility姞
night fast. In all women, basal serum levels of FSH, LH, T, ⌬4-androstenedione (A), and DHEAS were measured. Fasting concentrations of PRL, 17␣-OH-P, sex hormone-binding globulin (SHBG), glucose, insulin, and metastin were also measured. Free androgen index (FAI) was calculated according to the equation: T (nmol/L) ⫻ 100/SHBG (nmol/L) (34). Homeostatic model assessment–insulin resistance (HoMA-IR) was derived from the equation: Glucose (mmol/L) ⫻ Insulin (IU/mL)/22.5 (35). The glucose-to-insulin ratio was also calculated as a reliable index of insulin resistance (36). Hyperandrogenemia was defined as T levels ⬎60 ng/mL. This value was derived from the mean value ⫾ 2SD of 100 control women. On the same day that blood samples were collected, transvaginal ultrasonographic evaluation was performed. Assay Methods Plasma glucose concentrations were measured with the glucose oxidase technique using an auto-analyzer (Roche/Hitachi 902; Roche Diagnostics GmBH, Manheim, Germany). LH, FSH, PRL, androgen, and 17␣-OH-P levels were measured with the RIA method, whereas SHBG levels were measured with the IRMA method, using commercial kits (FSH: Radioisotopic Kit, Nichols Institute Diagnostics, San Juan Capistrano, CA; LH: Radioisotopic Kit, Nichols Institute Diagnostics; PRL: Radioisotopic Kit, Nichols Institute Diagnostics; T: Radioisotopic Kit, Diagnostic Systems Laboratories, Webster, TX; ⌬4A: Radioisotopic Kit, Diagnostic Systems Laboratories; DHEAS: Radioisotopic Kit, Diagnostic Systems Laboratories; 17␣-OHP: Radioisotopic Kit, Diagnostic Systems Laboratories; SHBG: Immunoradiometric Assay (IRMA) Kit, Diagnostic Systems Laboratories). Serum insulin levels were measured by enzyme immunoassay (ELISA Kit, Mercodia AB, Uppsala, Sweden). Metastin levels were also measured with an enzyme-linked immunoassay kit (ELISA kit, Phoenix Pharmaceuticals Inc., Belmond, CA), after extraction with Phoenix Peptide sep-columns (RK-Sepcol-2). An intra-assay variation of 4.5% was observed. Statistical Analyses Values are presented as mean ⫾ standard error for mean (SEM). The Kolmogorov-Smirnov test was used to test the normality of distribution. Comparison of means between women with PCOS and controls was performed with the Mann-Whitney U test for non-normal values. Comparisons between the three groups were performed with multivariate general linear model-based one-way analysis of variance (ANOVA), after log-transformation of non-normal values. Post-hoc analysis was performed with Dunnette’s T3 test. Calculation of the Spearman coefficient was used to assess the correlation of plasma kisspeptin to each parameter. All analyses were performed by SPSS software (v.13.0 SPSS, Inc., Chicago, IL). The level of statistical significance was set at 5%. 1779
TABLE 1 Anthropometric, hormonal, and metabolic features of the women studied (mean ⴞ SEM, statistical significance).
No of women (n) Age (y) BMI (kg/m2) W/H FSH (mIU/mL) LH (mIU/mL) PRL (ng/mL) T (ng/dL) FAI ⌬4A (ng/mL) DHEA-S (g/mL) 17␣-OH-progesterone (ng/mL) SHBG (nmol/L) Glucose (mg/dL) Insulin (IU/mL) HoMA-IR Glucose-to-insulin ratio Metastin (fmol/mL)
PCOS BMI >25
PCOS BMI <25
Controls BMI >25
P value (ANOVA)
28 24.30 ⫾ 1.18 33.02 ⫾ 0.56 0.84 ⫾ 0.01 6.02 ⫾ 0.33 11.41 ⫾ 1.29 12.71 ⫾ 1.52 79.37 ⫾ 3.92 14.15 ⫾ 1.76 2.74 ⫾ 0.17 2.54 ⫾ 0.22 1.15 ⫾ 0.10 24.16 ⫾ 1.83 98.64 ⫾ 2.59 16.81 ⫾ 1.18 4.16 ⫾ 0.34 6.83 ⫾ 0.60 0.24 ⫾ 0.02
28 23.64 ⫾ 0.58 20.63 ⫾ 0.26a 0.76 ⫾ 0.01a 6.04 ⫾ 0.30 14.14 ⫾ 2.14 17.10 ⫾ 1.74 88.34 ⫾ 5.14 8.29 ⫾ 0.93a 3.29 ⫾ 0.19 2.69 ⫾ 0.17 1.31 ⫾ 0.11 43.51 ⫾ 3.49a 93.18 ⫾ 2.31 7.70 ⫾ 0.71a 1.74 ⫾ 0.15a 14.83 ⫾ 1.34a 0.33 ⫾ 0.02a
13 26.85 ⫾ 1.06 32.13 ⫾ 1.85b 0.80 ⫾ 0.02 5.64 ⫾ 0.63 4.37 ⫾ 0.60ab 11.23 ⫾ 1.72 41.59 ⫾ 2.74ab 3.35 ⫾ 0.28ab 1.36 ⫾ 0.11ab 1.88 ⫾ 0.19 0.59 ⫾ 0.05ab 47.55 ⫾ 5.89a 99.54 ⫾ 3.54 11.90 ⫾ 1.98a 3.04 ⫾ 0.55a 10.70 ⫾ 1.24a 0.36 ⫾ 0.05
— ns ⬍.001 ⬍.001 ns ⬍.001 ns ⬍.001 ⬍.001 ⬍.001 ⬍.05 ⬍.001 ⬍.001 ns ⬍.001 ⬍.001 ⬍.001 ⬍.05
See text for abbreviations. a Post-hoc P⬍.05 vs obese and overweight women with PCOS. b Post-hoc P⬍.05 vs normal weight women with PCOS. Panidis. Metastin in PCOS. Fertil Steril 2006.
RESULTS Basic anthropometric, hormonal, and metabolic features of the women studied are summarized in Table 1. No significant difference in age was observed. Obese and overweight women with PCOS had significantly higher BMI and waistto-hip ratio than normal weight women with the syndrome (P⬍.001). Obese and overweight controls had significantly higher BMI, compared to lean women with PCOS, but the difference in the waist-to-hip ratio was nonsignificant (P⫽.095). No difference in BMI or waist-to-hip ratio was observed between the two groups with BMI ⬎25 kg/m2. The LH levels were significantly higher in both groups of women with PCOS, compared to controls (P⬍.001). Normal weight women with PCOS had higher LH levels, compared to obese and overweight women with the syndrome, but not significantly. No significant difference was found in FSH or PRL levels between the three groups. Women with PCOS had higher T, A, 17␣-OH-P levels and FAI values, compared to controls (P⬍.001). No difference in androgen levels was observed between the two groups of women with PCOS. Normal weight women with the syndrome had significantly lower FAI values, and higher SHBG levels, compared to women with PCOS and BMI ⬎25 kg/m2 (P⬍.001). Obese and overweight women with 1780
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PCOS also had significantly lower SHBG levels, compared to controls (P⬍.001); no difference in SHBG levels was observed between the two groups of lean women. No difference in fasting glucose levels was observed between the three groups. Insulin levels and HoMA-IR values were significantly higher, and the glucose-to-insulin ratio was significantly lower in obese and overweight women with PCOS, compared to lean women with the syndrome (P⬍.001) and obese/overweight controls (P⬍.05). No significant difference was observed between the latter two groups. Metastin levels were lower in women with PCOS (0.2846 ⫾ 0.014 fmol/ml), compared to controls (0.3564 ⫾ 0.0538 fmol/ml), but not significantly (Fig. 1).However, when women with PCOS were further divided, based on BMI values, a significant difference emerged (P⬍.05). Specifically, metastin levels were significantly higher in normal weight women with PCOS (Post-hoc P⬍.05), compared to obese and overweight women with the syndrome. No significant difference was found between the two groups of obese and overweight women, neither between the control group and obese/overweight women with PCOS, although kisspeptin levels were higher in controls (Fig. 2). Vol. 85, No. 6, June 2006
FIGURE 1
FIGURE 3
Plasma metastin levels of all women with polycystic ovary syndrome (PCOS) and controls (mean ⫾ SEM).
Scatter-plot diagram of plasma metastin levels vs. free androgen index (FAI); r ⫽ ⫺0.255, P⬍.05. PCOS ⫽ polycystic ovary syndrome.
Panidis. Metastin in PCOS. Fertil Steril 2006. Panidis. Metastin in PCOS. Fertil Steril 2006.
FIGURE 2 Plasma metastin levels of obese or overweight women with polycystic ovary syndrome (PCOS), normal weight women with PCOS, and controls (mean ⫾ SEM); *P⬍.05 vs. obese and overweight women with PCOS.
Metastin levels were found to be negatively correlated with BMI (r ⫽ ⫺0.238, P⬍.05), fasting insulin levels (r ⫽ ⫺0.308, P⬍.05), FAI (r ⫽ ⫺0.255, P⬍.05) (Fig. 3), and HoMA-IR values (r ⫽ ⫺0.238, P⬍.05) and positively correlated with SHBG levels (r ⫽ 0.249, P⬍.05) and the glucose-to-insulin ratio (r ⫽ 0.338, P⬍.005) (Fig. 4). DISCUSSION In the present study, plasma concentrations of metastin (kisspeptin), a novel peptide with putative antimetastatic and endocrine properties (1–9), were measured, for the first time, in women with PCOS. Apparently this peptide is also involved in the complex mechanisms that regulate hypothalamic–pituitary– gonadal function and reproductive physiology (14 –18). Specifically, metastin has been recently associated with increased GnRHinduced secretion of LH (19 –22). Because women with PCOS demonstrate higher LH levels, compared to ovulatory women without the syndrome (24 –26), one would expect kisspeptin levels to be significantly increased in the former. Nevertheless, in our study, no such difference was observed (Table 1, Fig. 1), although LH levels were significantly higher in both groups of women with PCOS, compared to controls (Table 1). Moreover, no significant correlation between plasma metastin and LH levels was observed.
Panidis. Metastin in PCOS. Fertil Steril 2006.
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On that basis metastin does not seem to be directly involved in the increased LH secretion observed in women 1781
FIGURE 4 Scatter-plot diagram of plasma metastin levels vs. glucose-to-insulin ratio; r ⫽ 0.338, P⬍.005. PCOS ⫽ polycystic ovary syndrome.
values), compared to normal weight women with the syndrome and controls (Table 1). Moreover, plasma kisspeptin levels were significantly correlated with FAI (Fig. 3). Because it has been reported that metastin and gonadal steroids are inversely correlated (23), we conclude that the increase in free androgen levels, as a result of insulin resistance, is associated with a relative decrease in circulating kispeptin levels. In summary, the present findings indicate that metastin, although not directly involved in PCOS-associated LH hypersecretion, is negatively correlated with insulin resistance and increased free androgen levels, which are prominent features of the syndrome. It seems improbable that metastin (kisspeptin) can be a single cause of PCOS-associated reproductive or metabolic abnormalities. However, it could be involved in the interaction between gonadotropin secretion and the negative feedback influence of sex steroids. REFERENCES
Panidis. Metastin in PCOS. Fertil Steril 2006.
with PCOS. It should be noted that most data on the interactions between gonadotropin production and this novel hormone are, so far, obtained from rodents (16 –22), whereas studies of plasma kisspeptin in humans are rather scarce (14, 15). It is, therefore, possible that different pathways determine the relationship between metastin and human physiology of reproduction. Interestingly, when women with PCOS were further divided, based on BMI values, obese and overweight women with the syndrome had significantly lower kisspeptin levels compared to normal weight women with the syndrome (Fig. 2). In the present study, the number of control women was relatively small, and no lean healthy volunteers were involved. Therefore, it is too soon to draw definite conclusions. It should be noted that metastin levels were significantly and inversely correlated with all indices of insulin resistance (Figs. 3 and 4) and women with PCOS and BMI ⬎25 kg/m2 were more insulin-resistant, compared to women with PCOS and BMI ⬍25 kg/m2, and controls with BMI ⬎25 kg/m2. From these results, it can be inferred that insulin resistance is linked to a significant decrease in plasma metastin levels. The PCOS-associated insulin resistance with compensatory hyperinsulinemia have been shown to increase free androgen levels by stimulating androgen synthesis in the PCO (29) and also by suppressing SHBG production in the liver (30). In our study, women with PCOS and BMI ⬎25 kg/m2 had significantly higher free androgen levels (FAI 1782
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1. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, et al. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001;411:613–7. 2. Hori A, Honda S, Asada M, Ohtaki T, Oda K, Watanabe T, et al. Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 2001;286:958 – 63. 3. Ringel MD, Hardy E, Bernet VJ, Burch HB, Schuppert F, Burman KD, et al. Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 2002;87:2399 – 404. 4. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, et al. KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. J Natl Cancer Inst 1996;88:1731–7. 5. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 2001;276:34631– 6. 6. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, et al. AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2001;276:28969 –75. 7. Lee JH, Welch DR. Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. Cancer Res 1997;57:2384 –7. 8. Ikeguchi M, Yamaguchi K, Kaibara N. Clinical significance of the loss of KiSS-1 and orphan G-protein-coupled receptor (hOT7T175) gene expression in esophageal squamous cell carcinoma. Clin Cancer Res 2004;10:1379 – 83. 9. Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C. Tumor suppressor role of KiSS-1 in bladder cancer: loss of KiSS-1 expression is associated with bladder cancer progression and clinical outcome. Am J Pathol 2003;162:609 –17. 10. Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, et al. Kisspeptin-10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 2004;117:1319 –28. 11. McMaster MT, Bass KE, Fisher SJ. Human trophoblast invasion. Autocrine control and paracrine modulation. Ann NY Acad Sci 1994; 734:122–31. 12. Murray MJ, Lessey BA. Embryo implantation and tumor metastasis: common pathways of invasion and angiogenesis. Semin Reprod Endocrinol 1999;17:275–90. 13. Bischof P, Meisser A, Campana A. Paracrine and autocrine regulators of trophoblast invasion—a review. Placenta 2000;21:S55–S60.
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14. Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, et al. Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans. J Clin Endocrinol Metab 2003;88:914 –9. 15. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100: 10972– 6. 16. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Shagoury JK, et al. The GPR54 gene as a regulator of puberty. N Engl J Med 2003;349:1614 –27. 17. Funes S, Hendrick JA, Vassileva G, Markowitz L, Abbondanzo S, Golovko A, et al. The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system. Biochem Biophys Res Commun 2003;312:1357– 63. 18. Navarro VM, Fernandez-Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, et al. Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. J Physiol 2004;561:379 – 86. 19. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, et al. A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 2004;145:4073–7. 20. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T. Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 2004;320:383– 8. 21. Navarro VM, Castellano JM, Fernandez-Fernandez R, Barreiro ML, Roa J, Sanchez-Criado JE, et al. Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor GPR54 in rat hypothalamus and potent LH releasing activity of KiSS-1 peptide. Endocrinology 2004;145:4565–74. 22. Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, et al. Characterization of the potent LH releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 2005;146: 156 – 63. 23. Colledge WH. GPR54 and puberty. Trends Endocrinol Metab 2004;15: 448 –53. 24. Diamanti-Kandarakis E, Dunaif A. New perspectives in polycystic ovary syndrome. Trends Endocrinol Metab 1996;7:267–71. 25. Diamanti-Kandarakis E, Kouli C, Bergiele A, Filandra F, Tsianateli T, Spina G, et al. A survey of the polycystic ovary syndrome in the Greek
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Division of Endocrinology and Human Reproduction, Second Department of Obstetrics and Gynecology and b Department of Biochemistry, Medical School, Aristotle University of Thessaloniki, Thessaloniki; and c Endocrine Section of the First Department of Internal Medicine, Athens University School of Medicine, Athens, Greece
Objective: This study was designed to: [1] measure, for the first time, metastin (kisspeptin) levels in women with polycystic ovary syndrome (PCOS), a condition associated with hypersecretion of LH and hyperandrogenemia; and [2] investigate the possible correlations between metastin and PCOS-related reproductive and metabolic disturbances. Design: Clinical study. Setting: University hospital. Patient(s): Twenty-eight obese and overweight (body mass index [BMI] ⬎25 kg/m2) women with PCOS, 28 normal weight (BMI ⬍25 kg/m2) women with the syndrome, and 13 obese and overweight controls (ovulatory women without clinical or biochemical hyperandrogenemia) were selected. Intervention(s): Blood samples were collected between day 3 and day 6 of a spontaneous bleeding episode in the PCOS groups and a menstrual cycle of the controls, at 9:00 AM, after an overnight fast. Main Outcome Measure(s): Circulating levels of LH, FSH, PRL, T, ⌬4-androstenedione (A), DHEAS, 17␣OH-P, sex hormone-binding globulin (SHBG), insulin, glucose, and metastin were measured. Result(s): Both normal weight women with PCOS and obese controls were less insulin resistant and had significantly higher metastin levels, compared to obese and overweight women with the syndrome. Plasma kisspeptin levels were negatively correlated with BMI, free androgen index, and indices of insulin resistance. Conclusion(s): These results indicate that metastin is negatively associated with free androgen levels. The PCOS-associated insulin resistance and consequent hyperinsulinemia probably contribute to this effect by [1] stimulating androgen synthesis by the polycystic ovary (PCO) and [2] suppressing SHBG production in the liver. (Fertil Steril威 2006;85:1778 – 83. ©2006 by American Society for Reproductive Medicine.) Key Words: Metastin, kisspeptin, PCOS, obesity, insulin resistance
Metastin is a 54-amino-acid peptide, which was first isolated from the human placenta in 2001 (1). It is encoded by the Kiss-1 gene, therefore, it is also known as Kiss-1 peptide (kisspeptin) (2– 4). Metastin action is exerted by a trans-membrane G-protein-coupled receptor, named GPR54, AXOR12, or HoT7T175 (1, 5, 6). Kiss-1 expression has been associated with antimetastatic activity in malignant melanoma (4), breast (7), papillary thyroid (3), esophageal (8) and bladder (9) carcinoma. Furthermore, kisspeptin has also been found to suppress the invasion of human trophoblast cells (10), a process sharing common pathways with tumor metastasis (11–13). A possible physiological role of this peptide in the control of trophoblast growth into adjacent Received March 24, 2005; revised and accepted November 6, 2005. Reprint requests: Anargyros Kourtis, M.D., 2nd Department of Obstetrics and Gynecology, 45 Aristotelous Str, 55236, Panorama, Greece (FAX: 30 2310 346942; E-mail: [email protected]).
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tissues could also account for the dramatic increase of plasma metastin levels in pregnancy (14). Intriguingly, loss of Kiss-1 function is associated with hypogonadotropic hypogonadism in humans (15) and in animal models (16, 17). Administration of the Kiss-1 peptide (metastin/kisspeptin) has been shown to induce maturation of the female reproductive system and precocious puberty in rodents (18). Moreover, there is strong evidence that metastin exerts these effects by stimulating GnRH release from the hypothalamus, and, as a consequence, pituitary LH secretion (19 –22). It is also interesting that a negative feedback loop might also exist, as kisspeptin has been reported to be down-regulated by sex steroids (23). The polycystic ovary syndrome (PCOS) is a common heterogeneous disorder of women of reproductive age, and the most frequent cause of hyperandrogenism combined with
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anovulatory infertility (24, 25). Its complex pathogenesis involves [1] hypothalamic–pituitary disturbances in gonadotropin secretion, specifically increased LH levels (26, 27); [2] increased resistance to insulin (28) and compensatory hyperinsulinemia, which has been associated with enhanced androgen production (29) and reduced synthesis of sex hormone-binding globulin (SHBG) (30); and [3] disturbed gonadal steroidogenesis (31). Given the complex relationship between the novel peptide metastin and the hypothalamic–pituitary– gonadal axis, the present study was designed to: [1] measure metastin levels in women with PCOS, a condition associated with aberrant gonadotropin secretion and hyperandrogenemia; and [2] investigate the possible correlations between kisspeptin and PCOS-related reproductive and metabolic disturbances. MATERIALS AND METHODS Subjects Fifty-six women with PCOS, aged 15–37 years, were recruited from the outpatient endocrine infirmary of our clinic, with at least one of the following signs: oligomenorrhea, fertility problem, hirsutism, acne, or male pattern alopecia. Diagnosis of PCOS was based on the presence of chronic anovulation (less than six cycles in 12 months) and hyperandrogenemia. Other common causes of hyperandrogenism or menstrual disorders (prolactinoma, congenital adrenal hyperplasia, Cushing syndrome, and virilizing ovarian or adrenal tumors) were excluded, in accord with the criteria proposed in 1990 by the National Institutes of Health–National Institute of Child Health and Human Development (NIHNICHHD) (32), and revised in 2003 by the Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group (33). Women with PCOS were further divided into two groups, based on body mass index (BMI) values: obese and overweight (BMI ⬎25 kg/m2) (n ⫽ 28) and normal weight (BMI ⬍25 kg/m2) women with the syndrome (n ⫽ 28). Thirteen obese and overweight healthy women, aged 18 –31 years, volunteered as controls. All controls had normal ovulating cycles (28 ⫾ 2 days, blood P levels ⬎10 ng/mL in two consequent cycles), and no signs of hyperandrogenism. None of the women studied had galactorrhea, or systemic disease that could affect their reproductive physiology. Furthermore, no woman reported use of any medication that could interfere with the normal function of the hypothalamic–pituitary– gonadal axis, during the past 6 months. Informed consent was obtained from all 69 women and the study was approved by the local ethical committee. Hormonal and Biochemical Measurements and Calculations Blood samples were collected between day 3 and day 6 of a spontaneous bleeding episode in the PCOS groups and a menstrual cycle of the controls, at 9:00 AM, after an overFertility and Sterility姞
night fast. In all women, basal serum levels of FSH, LH, T, ⌬4-androstenedione (A), and DHEAS were measured. Fasting concentrations of PRL, 17␣-OH-P, sex hormone-binding globulin (SHBG), glucose, insulin, and metastin were also measured. Free androgen index (FAI) was calculated according to the equation: T (nmol/L) ⫻ 100/SHBG (nmol/L) (34). Homeostatic model assessment–insulin resistance (HoMA-IR) was derived from the equation: Glucose (mmol/L) ⫻ Insulin (IU/mL)/22.5 (35). The glucose-to-insulin ratio was also calculated as a reliable index of insulin resistance (36). Hyperandrogenemia was defined as T levels ⬎60 ng/mL. This value was derived from the mean value ⫾ 2SD of 100 control women. On the same day that blood samples were collected, transvaginal ultrasonographic evaluation was performed. Assay Methods Plasma glucose concentrations were measured with the glucose oxidase technique using an auto-analyzer (Roche/Hitachi 902; Roche Diagnostics GmBH, Manheim, Germany). LH, FSH, PRL, androgen, and 17␣-OH-P levels were measured with the RIA method, whereas SHBG levels were measured with the IRMA method, using commercial kits (FSH: Radioisotopic Kit, Nichols Institute Diagnostics, San Juan Capistrano, CA; LH: Radioisotopic Kit, Nichols Institute Diagnostics; PRL: Radioisotopic Kit, Nichols Institute Diagnostics; T: Radioisotopic Kit, Diagnostic Systems Laboratories, Webster, TX; ⌬4A: Radioisotopic Kit, Diagnostic Systems Laboratories; DHEAS: Radioisotopic Kit, Diagnostic Systems Laboratories; 17␣-OHP: Radioisotopic Kit, Diagnostic Systems Laboratories; SHBG: Immunoradiometric Assay (IRMA) Kit, Diagnostic Systems Laboratories). Serum insulin levels were measured by enzyme immunoassay (ELISA Kit, Mercodia AB, Uppsala, Sweden). Metastin levels were also measured with an enzyme-linked immunoassay kit (ELISA kit, Phoenix Pharmaceuticals Inc., Belmond, CA), after extraction with Phoenix Peptide sep-columns (RK-Sepcol-2). An intra-assay variation of 4.5% was observed. Statistical Analyses Values are presented as mean ⫾ standard error for mean (SEM). The Kolmogorov-Smirnov test was used to test the normality of distribution. Comparison of means between women with PCOS and controls was performed with the Mann-Whitney U test for non-normal values. Comparisons between the three groups were performed with multivariate general linear model-based one-way analysis of variance (ANOVA), after log-transformation of non-normal values. Post-hoc analysis was performed with Dunnette’s T3 test. Calculation of the Spearman coefficient was used to assess the correlation of plasma kisspeptin to each parameter. All analyses were performed by SPSS software (v.13.0 SPSS, Inc., Chicago, IL). The level of statistical significance was set at 5%. 1779
TABLE 1 Anthropometric, hormonal, and metabolic features of the women studied (mean ⴞ SEM, statistical significance).
No of women (n) Age (y) BMI (kg/m2) W/H FSH (mIU/mL) LH (mIU/mL) PRL (ng/mL) T (ng/dL) FAI ⌬4A (ng/mL) DHEA-S (g/mL) 17␣-OH-progesterone (ng/mL) SHBG (nmol/L) Glucose (mg/dL) Insulin (IU/mL) HoMA-IR Glucose-to-insulin ratio Metastin (fmol/mL)
PCOS BMI >25
PCOS BMI <25
Controls BMI >25
P value (ANOVA)
28 24.30 ⫾ 1.18 33.02 ⫾ 0.56 0.84 ⫾ 0.01 6.02 ⫾ 0.33 11.41 ⫾ 1.29 12.71 ⫾ 1.52 79.37 ⫾ 3.92 14.15 ⫾ 1.76 2.74 ⫾ 0.17 2.54 ⫾ 0.22 1.15 ⫾ 0.10 24.16 ⫾ 1.83 98.64 ⫾ 2.59 16.81 ⫾ 1.18 4.16 ⫾ 0.34 6.83 ⫾ 0.60 0.24 ⫾ 0.02
28 23.64 ⫾ 0.58 20.63 ⫾ 0.26a 0.76 ⫾ 0.01a 6.04 ⫾ 0.30 14.14 ⫾ 2.14 17.10 ⫾ 1.74 88.34 ⫾ 5.14 8.29 ⫾ 0.93a 3.29 ⫾ 0.19 2.69 ⫾ 0.17 1.31 ⫾ 0.11 43.51 ⫾ 3.49a 93.18 ⫾ 2.31 7.70 ⫾ 0.71a 1.74 ⫾ 0.15a 14.83 ⫾ 1.34a 0.33 ⫾ 0.02a
13 26.85 ⫾ 1.06 32.13 ⫾ 1.85b 0.80 ⫾ 0.02 5.64 ⫾ 0.63 4.37 ⫾ 0.60ab 11.23 ⫾ 1.72 41.59 ⫾ 2.74ab 3.35 ⫾ 0.28ab 1.36 ⫾ 0.11ab 1.88 ⫾ 0.19 0.59 ⫾ 0.05ab 47.55 ⫾ 5.89a 99.54 ⫾ 3.54 11.90 ⫾ 1.98a 3.04 ⫾ 0.55a 10.70 ⫾ 1.24a 0.36 ⫾ 0.05
— ns ⬍.001 ⬍.001 ns ⬍.001 ns ⬍.001 ⬍.001 ⬍.001 ⬍.05 ⬍.001 ⬍.001 ns ⬍.001 ⬍.001 ⬍.001 ⬍.05
See text for abbreviations. a Post-hoc P⬍.05 vs obese and overweight women with PCOS. b Post-hoc P⬍.05 vs normal weight women with PCOS. Panidis. Metastin in PCOS. Fertil Steril 2006.
RESULTS Basic anthropometric, hormonal, and metabolic features of the women studied are summarized in Table 1. No significant difference in age was observed. Obese and overweight women with PCOS had significantly higher BMI and waistto-hip ratio than normal weight women with the syndrome (P⬍.001). Obese and overweight controls had significantly higher BMI, compared to lean women with PCOS, but the difference in the waist-to-hip ratio was nonsignificant (P⫽.095). No difference in BMI or waist-to-hip ratio was observed between the two groups with BMI ⬎25 kg/m2. The LH levels were significantly higher in both groups of women with PCOS, compared to controls (P⬍.001). Normal weight women with PCOS had higher LH levels, compared to obese and overweight women with the syndrome, but not significantly. No significant difference was found in FSH or PRL levels between the three groups. Women with PCOS had higher T, A, 17␣-OH-P levels and FAI values, compared to controls (P⬍.001). No difference in androgen levels was observed between the two groups of women with PCOS. Normal weight women with the syndrome had significantly lower FAI values, and higher SHBG levels, compared to women with PCOS and BMI ⬎25 kg/m2 (P⬍.001). Obese and overweight women with 1780
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PCOS also had significantly lower SHBG levels, compared to controls (P⬍.001); no difference in SHBG levels was observed between the two groups of lean women. No difference in fasting glucose levels was observed between the three groups. Insulin levels and HoMA-IR values were significantly higher, and the glucose-to-insulin ratio was significantly lower in obese and overweight women with PCOS, compared to lean women with the syndrome (P⬍.001) and obese/overweight controls (P⬍.05). No significant difference was observed between the latter two groups. Metastin levels were lower in women with PCOS (0.2846 ⫾ 0.014 fmol/ml), compared to controls (0.3564 ⫾ 0.0538 fmol/ml), but not significantly (Fig. 1).However, when women with PCOS were further divided, based on BMI values, a significant difference emerged (P⬍.05). Specifically, metastin levels were significantly higher in normal weight women with PCOS (Post-hoc P⬍.05), compared to obese and overweight women with the syndrome. No significant difference was found between the two groups of obese and overweight women, neither between the control group and obese/overweight women with PCOS, although kisspeptin levels were higher in controls (Fig. 2). Vol. 85, No. 6, June 2006
FIGURE 1
FIGURE 3
Plasma metastin levels of all women with polycystic ovary syndrome (PCOS) and controls (mean ⫾ SEM).
Scatter-plot diagram of plasma metastin levels vs. free androgen index (FAI); r ⫽ ⫺0.255, P⬍.05. PCOS ⫽ polycystic ovary syndrome.
Panidis. Metastin in PCOS. Fertil Steril 2006. Panidis. Metastin in PCOS. Fertil Steril 2006.
FIGURE 2 Plasma metastin levels of obese or overweight women with polycystic ovary syndrome (PCOS), normal weight women with PCOS, and controls (mean ⫾ SEM); *P⬍.05 vs. obese and overweight women with PCOS.
Metastin levels were found to be negatively correlated with BMI (r ⫽ ⫺0.238, P⬍.05), fasting insulin levels (r ⫽ ⫺0.308, P⬍.05), FAI (r ⫽ ⫺0.255, P⬍.05) (Fig. 3), and HoMA-IR values (r ⫽ ⫺0.238, P⬍.05) and positively correlated with SHBG levels (r ⫽ 0.249, P⬍.05) and the glucose-to-insulin ratio (r ⫽ 0.338, P⬍.005) (Fig. 4). DISCUSSION In the present study, plasma concentrations of metastin (kisspeptin), a novel peptide with putative antimetastatic and endocrine properties (1–9), were measured, for the first time, in women with PCOS. Apparently this peptide is also involved in the complex mechanisms that regulate hypothalamic–pituitary– gonadal function and reproductive physiology (14 –18). Specifically, metastin has been recently associated with increased GnRHinduced secretion of LH (19 –22). Because women with PCOS demonstrate higher LH levels, compared to ovulatory women without the syndrome (24 –26), one would expect kisspeptin levels to be significantly increased in the former. Nevertheless, in our study, no such difference was observed (Table 1, Fig. 1), although LH levels were significantly higher in both groups of women with PCOS, compared to controls (Table 1). Moreover, no significant correlation between plasma metastin and LH levels was observed.
Panidis. Metastin in PCOS. Fertil Steril 2006.
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On that basis metastin does not seem to be directly involved in the increased LH secretion observed in women 1781
FIGURE 4 Scatter-plot diagram of plasma metastin levels vs. glucose-to-insulin ratio; r ⫽ 0.338, P⬍.005. PCOS ⫽ polycystic ovary syndrome.
values), compared to normal weight women with the syndrome and controls (Table 1). Moreover, plasma kisspeptin levels were significantly correlated with FAI (Fig. 3). Because it has been reported that metastin and gonadal steroids are inversely correlated (23), we conclude that the increase in free androgen levels, as a result of insulin resistance, is associated with a relative decrease in circulating kispeptin levels. In summary, the present findings indicate that metastin, although not directly involved in PCOS-associated LH hypersecretion, is negatively correlated with insulin resistance and increased free androgen levels, which are prominent features of the syndrome. It seems improbable that metastin (kisspeptin) can be a single cause of PCOS-associated reproductive or metabolic abnormalities. However, it could be involved in the interaction between gonadotropin secretion and the negative feedback influence of sex steroids. REFERENCES
Panidis. Metastin in PCOS. Fertil Steril 2006.
with PCOS. It should be noted that most data on the interactions between gonadotropin production and this novel hormone are, so far, obtained from rodents (16 –22), whereas studies of plasma kisspeptin in humans are rather scarce (14, 15). It is, therefore, possible that different pathways determine the relationship between metastin and human physiology of reproduction. Interestingly, when women with PCOS were further divided, based on BMI values, obese and overweight women with the syndrome had significantly lower kisspeptin levels compared to normal weight women with the syndrome (Fig. 2). In the present study, the number of control women was relatively small, and no lean healthy volunteers were involved. Therefore, it is too soon to draw definite conclusions. It should be noted that metastin levels were significantly and inversely correlated with all indices of insulin resistance (Figs. 3 and 4) and women with PCOS and BMI ⬎25 kg/m2 were more insulin-resistant, compared to women with PCOS and BMI ⬍25 kg/m2, and controls with BMI ⬎25 kg/m2. From these results, it can be inferred that insulin resistance is linked to a significant decrease in plasma metastin levels. The PCOS-associated insulin resistance with compensatory hyperinsulinemia have been shown to increase free androgen levels by stimulating androgen synthesis in the PCO (29) and also by suppressing SHBG production in the liver (30). In our study, women with PCOS and BMI ⬎25 kg/m2 had significantly higher free androgen levels (FAI 1782
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