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Metastin levels in relation with hormonal and metabolic profile in patients with polycystic ovary syndrome
European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 56–60
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Metastin levels in relation with hormonal and metabolic profile in patients with polycystic ovary syndrome S.A. Yilmaz a, *, O.S. Kerimoglu a , A.T. Pekin a , F. Incesu a , N.U. Dogan b , C. Celik a , A. Unlu c a
Department of Obstetrics and Gynecology, Selcuk University, Faculty of Medicine, Konya, Turkey Department of Obstetrics and Gynecology, Akdeniz University, Faculty of Medicine, Antalya, Turkey c Department of Biochemistry, Selcuk University, Faculty of Medicine, Konya, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 February 2014 Received in revised form 29 April 2014 Accepted 4 June 2014
Objective: The aim of the present study was to evaluate serum concentrations of metastin in relation with hormonal and metabolic profile in patients with and without polycystic ovary syndrome (PCOS). Study design: The study was a clinical study. Eighty-three women with PCOS and 66 body mass index (BMI) matched controls were divided into two groups, based on BMI: overweight and obese (BMI 25 kg/ m2) and normal weight. (BMI < 25 kg/m2) Hirsutism scores, hormonal and metabolic profile as well as metastin levels were evaluated in each subject. Blood samples were collected in the early follicular phase (between day 2 and day 5 of the menstrual cycle) at 9:00 AM, after an overnight fast. Circulating levels of LH, FSH, PRL, TSH, T, fT, DHEAS, 17-OH-P, sex hormone-binding globulin (SHBG), insulin, glucose, lipid profile and metastin were measured. Results: Metastin levels were significantly higher in the PCOS group compared to controls (2.02 ng/ml versus 1.16 ng/ml, p < 0.001). Metastin levels correlated significantly positively with luteinizing hormone (LH), total testosterone (T), dehydroepiandrosteronesulphate (DHEA-SO4) levels, modified Ferriman– Gallwey (mFG) scores and free androgen index (FAI); however, correlated negatively with sex hormone binding globulin (SHBG) levels (p < 0.05). When overweight or obese (BMI 25 kg/m2) and normal weight (BMI < 25 kg/m2) women with PCOS were compared to body mass index (BMI) matched controls, higher metastin levels were also found in PCOS groups (1.94 ng/ml versus 1.18 ng/ml, and 2.06 ng/ml versus 1.08 ng/ml, p < 0.05, respectively). Conclusions: These findings suggest that metastin levels were higher in women with PCOS as compared to controls regardless of BMI. Furthermore, metastin levels can be used as a specific marker for androgenic profile and this marker might play a role in the pathogenesis of PCOS. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Metastin Kisspeptin PCOS Insulin resistance
Introduction Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting women, with a prevalence of 6–12% in women of reproductive age [1]. It is characterized by androgen excess, chronic oligoanovulation and polycystic ovaries (PCO) on ultrasound. Clinically, the androgen excess presents as hirsutism and acne, whereas anovulation presents as subfertility and menstrual irregularity. In addition, severe physiological abnormalities such as insulin resistance, inflammation, visceral fat, cardiovascular disease, infertility are common among women with PCOS [2].
* Corresponding author at: Department of Obstetrics and Gynecology, Selcuk University, Faculty of Medicine, Konya, 42100, Turkey. Tel.: +90 332 2245164; fax: +90 332 2416065. E-mail address: [email protected] (S.A. Yilmaz). http://dx.doi.org/10.1016/j.ejogrb.2014.06.004 0301-2115/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Metastin is a 54 amino-acid peptide, which was first isolated from the human placenta in 2001 [3]. It is encoded by the Kiss-1 gene, therefore it is also known as Kiss-1 peptide (kisspeptin) [4]. Metastin action is exerted by a trans-membrane G-protein coupled receptor, named GPR54, AXOR12, or HoT7T175 [3–6]. Loss of Kiss-1 gene function is reported to be associated with hypogonadotropic hypogonadism in humans and animal models [7,8]. Metastin has recently been shown to regulate the secretion of luteinizing hormone (LH) during the promotion of ovulation [9] by stimulating gonadotropin releasing hormone (GnRH) release from the hypothalamus [10,11]. Women with PCOS commonly display deregulated gonadotropin secretion with higher LH pulsatility and perturbed LH/FSH (follicle stimulating hormone) ratios, which likely contributes to the ovarian phenotype and might be indicative of disrupted GnRH secretory activity. Given the complex relationship between metastin and hypothalamic-pituitary gonadal axis, the present study aimed to investigate whether there was a difference in serum
S.A. Yilmaz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 56–60 Table 1 Demographic, clinical, biochemical and hormonal characteristics of women with PCOS and controls.
Age (years) BMI (kg/m2) FPG (mg/dl) Fasting insulin (mIU/ml) HOMA-IR Total cholesterol (mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl) fT(pg/ml) DHEA-SO4 (mg/dl) T (nmol/L) SHBG TSH (mIU/ml) FSH (mIU/ml) LH (mIU/ml) E2 (pg/ml) PRL (mIU/ml) 17OH-Progesterone mFG score FAI Metastin (ng/ml)
PCOS (n = 83)
Controls (n = 66)
p
21 (17–37) 24.5 (18.1–34.8) 89 (72–117) 9.5 (3.1–38.0) 2.0 (0.7–9.2) 163 (104–298) 47 (31–77) 99.5 24.1 90 (38–276) 3.2 (0.6–18.7) 303.0 101.6 1.9 (0.8–3.8) 31 (15–76.1) 2.0 (0.4–4.3) 5.1 (2.1–8.4) 9.3 (3.5–29.0) 41 (17–67) 12 (4.5–36) 1.2 (0.1–2.6) 9 (6–21) 6.0 (1.4–14.1) 2.02 (0.56–4.43)
24.5 (17–38) 23.1 (18–34.8) 91 (77–109) 7.7 (1.4–34.0) 1.8 (0.3–9.1) 165 (98–270) 47.5 (30–78) 101.227.8 93.5 (24.1–287) 1.4 (0.1–9.7) 230.5107.7 1.0 (0.3–2.8) 45.1 (21.9–138.0) 1.6 (0.4–9.0) 5.2 (3.1–8.5) 4.5 (2.0–9.2) 43 (16–76) 12 (3.2–25.4) 1.1 (0.3–2.6) 0 (0–1) 2.0 (0.6–6.5) 1.16 (0.45–2.00)
<0.001 0.125 0.034 0.039 0.101 0.645 0.950 0.687 0.677 <0.001 <0.001 <0.001 <0.001 0.057 0.097 <0.001 0.570 0.421 0.907 <0.001 <0.001 <0.001
BMI: body mass index, FPG: fasting plasma glucose, HOMA-IR: homeostasis model mssessment-mnsulin resistence, HDL: high density lipoprotein, LDL: low density lipoprotein, TG: triglycerides, fT: free testosterone, DHEA-SO4: dehydroepiandrosteronesulphate, fT3-fT4: free thyroxine levels, TSH: thyroid stimulating hormone, FSH: follicle stimulating hormone, LH: luteinizing hormone, E2:estradiol, PRL: prolactin, T:total testosterone, SHBG: sex hormone binding globulin, mFG: modified Ferriman–Gallwey score, FAI: free androgen index. Results for continous variables are expressed as mean SD or median (range). p < 0.05 was considered significant.
metastin levels between women with and without PCOS, a condition associated with aberrant gonadotropin secretion and hyperandrogenemia. This study also set out to correlate serum metastin levels in relation with hormonal and metabolic profile to evaluate the possible correlations between metastin and PCOSrelated reproductive and metabolic disturbances.
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menses and no clinical or biochemical hyperandrogenism or PCO were eligible. Detailed clinical history was taken and physical examination was performed for all participants. Hirsutism was evaluated according to the modified Ferriman–Gallwey score (greater than seven indicates hirsutism) [13]. BMI was calculated as the ratio of weight divided by height squared (kg/m2). Women’s with PCOS and controls were further divided into two groups, based on BMI values: overweight or obese (BMI 25 kg/m2) and normal weight (BMI < 25 kg/m2). Routine chemistry laboratory investigations included fasting blood glucose, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG) were measured by commercially available Abbott Diagnostics kits in Architect c16000 chemistry auto analyser (Abbott Diagnostics, USA). Routine hormonal analysis of LH, FSH, estradiol (E2), thyroid stimulating hormone (TSH), fasting insulin, dehydroepiandrosterone sulphate (DHEA-SO4), total testosterone (T) and sex hormone binding globuline (SHBG), were done by chemiluminescence methods using ADVIA Centaur1 XP Immunoassay system (Siemens Diagnostics, Germany). 17-OH-progesterone, and free testosterone (fT) levels were measured by Diametra ELISA kits (Viaapozaula, Italy). Insulin resistance (IR) was determined by the homeostasis model assessment (HOMA) index (fasting glucose (mg/dl) fasting insulin (mU/ml)/405) [14]. Free androgen index (FAI) was calculated according to the equation: T (nmol/l) 100/SHBG (nmol/l) [15]. All sampling procedures were performed in the early follicular phase (day 2–5 of the menstrual cycle) in the morning after an overnight fast. Serum metastin levels were assessed by an Enzyme Immunoassay kit (Phoenix Pharmaceuticals, Karlsruhe Germany). The intraand inter-assay coefficients of variations for metastin were <10% and <15%, respectively. The detection range for metastin was 0–1 ng/ml.
5
Materials and methods 4
3
2
Metastin (ng/mL)
The design of the present study was approved by the ethical committee and institutional review board of Selcuk University, Faculty of Medicine, where the study was conducted. Written informed consents were obtained from all participants. A total sample size of 88 (22 cases for each group) was required to detect at least 0.58 (ng/ml) difference in metastin levels between groups with a power of 80% at the 5% significance level. The difference of 0.58 (ng/ml) was taken from both pilot study and our clinical experiments. Sample size estimation was performed by using NCSS and PASS 2000i software. A total of 83 women with PCOS and 66 BMI matched controls participated in this study. The diagnosis of PCOS was made according to the Rotterdam criteria [12] in the presence of at least two of the following: (1) oligomenorrhea and/or anovulation, (2) biochemical and/or clinical hyperandrogenism, (3) ultrasound appearance of polycystic ovaries (PCO) (multiple cysts >12 in number of 2–9 mm size) with the exclusion of other etiologies such as congenital adrenal hyperplasia, virilising tumor, Cushing syndrome and prolactinoma, diabetes, hypertension and other cardiovascular diseases. Over the three months preceding the study no subject had been on hormonal contraceptives, other medications or diet which could affect lipid and carbohydrate metabolism. No subject smoked or consumed alcohol. As controls 66 body mass index (BMI) matched women who had regular
BMI
1
<25 kg/m^2 0
>=25 kg/m^2 Control
PCO
Fig. 1. Plasma metastin levels of women with regard to body mass index. The horizontal lines in the middle of each box indicates the median (50th percentile) metastin values, while the top and bottom borders of the box mark the 25th and 75th percentiles, respectively. The whiskers above and below the box mark indicates the maximum and minimum metastin levels.
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Statistical analysis
Results
Data analysis was performed by using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, IL, United States). Whether the distributions of metric discrete and continuous variables were normally or not was determined by Kolmogorov–Smirnov test. Levene test was applied for determining the homogeneity of variances. Metric discrete and continuous variables were shown as mean standard deviation (SD) or median (min–max), where applicable. While, the mean differences between groups were compared by Student’s t test, otherwise, Mann–Whitney U test was applied for comparisons of the median values. Nominal data were analyzed by Pearson’s Chi-square test. Degrees of association between continuous variables were calculated by Spearman’s correlation coefficient. Determining the best predictors which affect on PCOS was evaluated by Multiple Logistic Regression Analysis Backward LR procedure. Any variable whose univariable test had a p value < 0.25 was accepted as a candidate for the multivariable model along with all variables of known clinical importance. Odds ratio, 95% confidence interval and Wald statistic for each independent variable were also calculated. Whether the statistically significant predictivity of metastin measurements on PCO was going on or not was evaluated by Multiple Logistic Regression analysis after adjustment for BMI, LH and FAI. Odds ratios and 95% confidence intervals for each independent variable were also calculated. A p value less than 0.05 was considered statistically significant. But, for all multiple comparisons the Bonferroni adjustment was applied for controlling Type I error. The optimal cut off points of metastin to discriminate control and PCO groups was evaluated by ROC analyses calculating area under the curve as giving the maximum sum of sensitivity and specificity for the significant test. Sensitivity, specificity, positive and negative predictive values for metastin at the best cut off points were also calculated.
Demographic, clinical, biochemical and hormonal characteristics of women with PCOS and controls are summarized in Table 1. Age, fasting insulin, T, fT, DHEA-SO4, LH levels and mFG and FAI scores were significantly higher, whereas SHBG and fasting plasma glucose levels were lower in PCOS group compared to controls (p < 0.05). Metastin levels were also found to be higher in women with PCOS than controls (2.02 [056–443] ng/ml versus 1.16 [045– 200] ng/ml, p < 0.001) (Fig. 1). Table 2 shows the demographic, clinical, biochemical and hormonal characteristics of women with PCOS and controls with regard to BMI. When women were further divided based on BMI in both groups metastin levels continued to be higher in women with PCOS groups regardless of BMI (Table 2, Fig. 1). When obese and overweight (BMI 25 kg/m2, n = 66, 41 women with PCOS and 25 controls) and lean (BMI < 25 kg/m2, n = 83, 42 women with PCOS and 41 controls) women were evaluated on their own PCOS group showed higher metastin levels than controls (1.94 [056–349] ng/ml versus 1.18 [080–197] ng/ml, p < 0.001 and 2.06 [067–443] ng/ml versus 1.08 [045–200] ng/ml, p = 0.021, respectively). Metastin levels showed a positive significant correlation with LH, T, and DHEA-SO4 levels, modified Ferriman–Gallwey (mFG) scores and FAI; however were negatively correlated with SHBG levels (r: 0.33, p < 0.05). Correlations between metastin with demographic and clinical characteristics as well as hormonal and metabolic profile of all patients including controls are shown in Table 3. Determining the best predictors which affect on PCOS was evaluated by Multiple Logistic Regression Analysis Backward LR procedure. Using Multiple Logistic Regression Analysis Backward LR procedure after adjustment for all possible confounding factors we found that metastin levels (OR: 6.826; 95% CI: 2.162–21.552; p < 0.001), and FAI (OR: 1.910; 95% CI: 1.319–2.767; p < 0.001) and
Table 2 Demographic, clinical, biochemical and hormonal characteristics of women with PCOS and controls regarding BMI. BMI < 25
Age (years) FPG (mg/dl) Fasting insulin (mIU/ml) HOMA-IR TC (mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl) fT(pg/ml) DHEA-SO4 (mg/dl) T (nmol/L) SHBG TSH (mIU/ml) FSH (mIU/ml) LH (mIU/ml) E2 (pg/ml) PRL (mIU/ml) 17OH-Progesterone mFG score FAI Metastin(ng/ml)
BMI 25 a
PCOS (n = 42)
Controls (n = 41)
p
PCOS (n = 41)
Controls (n = 25)
pb
21 (17–32) 87(72–17) 8.2 (3.2–20.8) 1.7 (0.7–4.5) 159 (104–98) 47 (31–77) 95.2 25.9 77 (38–189) 2.9 (0,6–17.8) 312.1106.7 1.81 (0.79–3.78) 33.7 (20,7–76.1) 1.9 (0,9–4.2) 5.0 (2.3–7.4) 9.3 (3.5–29.0) 45 (24–67) 12.5 (5.7–34.0) 1.1 (0.1–2.6) 9.5 (7–21) 5.08(1.38–13.92) 2.06 (0.67–4.43)
22.5 (17–38) 90 (77–109) 7.2 (1.4–34.0) 1.5 (0.3–9.1) 160.5 (98–225) 50 (32–78) 93.426.9 78.5 (40–188) 1.4 (0.4–5.1) 242.6117.6 0.97 (0.27–2.84) 53.5 (24.9–138,0) 1.6 (0.5–3.6) 5.3 (3.1–8.5) 5.6 (2.0–8.7) 43.5 (16–76) 12.0 (5.8–25.4) 1.2 (0.5–2.6) 0 (0–1) 1.98 (0.59–6.54) 1.08 (0.45–2.00)
0.004 0.088 0.064 0.147 0.919 0.475 0.762 0.707 0.002 0.006 <0.001 <0.001 0.129 0.047 <0.001 0.728 0.262 0.267 <0.001 <0.001 <0.001
22 (17–37) 89 (73–106) 11.8c (3.1–38,0) 2.6c (0.7–9,2) 170 (128–248) 45 (34–67) 103.9 21.6 109c (39–276) 3.5 (1.0–18.7) 293.796.5 1.94 (0.86–3.78) 29.9c(15.0–45.0) 2.0 (0.4–4.3) 5.3 (2.1–8.4) 9.2 (5.6–18.0) 38c (17–60) 11.2 (4.5–36.0) 1.3 (0.1–2.4) 9c (6–21) 6.95c (3.25–14,10) 1.94 (0.56–3.49)
29d (20–38) 93.5 (79–107) 10.4d (4.3–31.0) 2.5d (0.8–6.8) 179d (125–270) 40d (30–56) 114.8d 24.1 116.5d (24.1–287) 1.7 (0.1–9.7) 210.587.3 1.11 (0.27–1.97) 43.0 (21.9–132.0) 1.5 (0.4–9.0) 5.2 (3.3–8.1) 3.9d (2.2–9.2) 42.5 (23–58) 12 (3.2–21.6) 1.0 (0.3–1.9) 0 (0–0) 2.11 (0.72–6.49) 1.18 (0.80–1.97)
<0.001 0.120 0.719 0.957 0.248 0.110 0.068 0.550 <0.001 <0.001 <0.001 <0.001 0.310 0.724 <0.001 0.237 0.838 0.105 <0.001 <0.001 0.021
BMI: body mass index, FPG: fasting plasma glucose, HOMA-IR: homeostasis model assessment-insulinresistence, HDL: high density lipoprotein, LDL: low density lipoprotein, TG: triglycerides, TC: totalcholesterole, fT: free testosterone, DHEA-SO4: dehydroepiandrosterone sulphate, fT3-fT4: free thyroxinelevels, TSH: thyroid stimulating hormone, FSH: follicle stimulating hormone, LH: luteinizing hormone, E2:estradiol, PRL: prolactin, T: total testosterone, SHBG: sex hormone binding globulin, mFG: modified Ferriman– Gallwey score, FAI: free androgen indexResults for continous variables are expressed as mean SD or median (range). p < 0.025 is accepted statistically significant after Bonferroni adjustment. a Difference between PCOS and controls BMI < 25. b Difference between PCOS and controls BMI 25. c Difference between obese-overweight and lean in PCOS group. d Difference between obese-overweight and lean in control group.
S.A. Yilmaz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 56–60
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Table 3 Correlations between metastin with demographic and clinical characteristics and hormonal and metabolic profile of all patients including controls. r Age (years) BMI (kg/m2) FPG (mg/dl) Fasting insulin (mIU/ml) HOMA-IR TC (mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl) fT(pg/ml) DHEA-SO4 (mg/dl) T (nmol/L) SHBG TSH (mIU/ml) FSH (mIU/ml) LH (mIU/ml) E2 (pg/ml) PRL (mIU/ml) 17OH-Progesterone mFG score FAI
p 0.148 0.076 0.089 0.097 0.078 0.088 0.019 0.098 0.028 0.161 0.166 0.172 0.330 0.081 0.063 0.237 0.060 0.011 0.018 0.474 0.309
0.074 0.363 0.284 0.244 0.350 0.288 0.817 0.240 0.736 0.052 0.045 0.038 <0.001 0.331 0.448 0.004 0.473 0.892 0.825 <0.001 <0.001
BMI: body mass index, FPG: fasting plasma glucose, HOMA-IR: homeostasis model assessment-insulin resistence, HDL: high density lipoprotein, LDL: low density lipoprotein, TG: triglycerides, TC: total cholesterole, fT: free testosterone, DHEA-SO4: dehydroepiandrosteronesulphate, fT3-fT4: free thyroxine levels, TSH: thyroid stimulating hormone, FSH: follicle stimulating hormone, LH: luteinizing hormone, E2:estradiol, PRL: prolactin, T: total testosterone, SHBG:sex hormone binding globulin, mFG: modified Ferriman– Gallwey score, FAI: free androgen index.
LH (OR: 1.997; 95% CI: 1.440–2.770; p < 0.001) were all positively associated with PCOS (Table 4). Thus, our results suggest that metastin might be an independent marker for PCOS. ROC analyses of metastine levels between the study and control groups are shown in Fig. 2. In this study population, the best cut-off point for serum metastine levels was 1.87 ng/ml. Serum metastine levels greater than 1.87 ng/ml demonstrated a sensitivity and specificity of 59% and 93.8%, respectively, for PCOS diagnosis (ROC AUC = 0.762; 95% CI: 0.685–0.840; p = 0.004). The positive predictive value was 92.5%, and the negative predictive value was 63.8 % (Fig. 2).
There are limited data in the literature with regard to changes in metastin levels and its relation with metabolic and hormonal disturbances in PCOS. So far only three studies assessed metastin in patients with PCOS, and their results are in consistent. Two previous studies [17,18] reported higher metastin levels in women with PCOS, similar to the data of the present study; however, one study [19] reported lower levels in women with PCOS as compared to controls. There are also controversial data regarding the association between metastin levels with BMI, insulin resistance, LH and androgenic profile. Chen et al. [17] measured metastin levels in 42 women with PCOS (23 adult, 19 adolescents) and 20 adolescent controls to investigate the possible correlations
Discussion The Kiss-1 system has emerged in the recent years as a fundamental player in the control of the reproductive axis, with essential roles in differentiation and pubertal activation of the reproductive system as well as key functions in the regulation of ovulation and the metabolic control of fertility. These features make it tempting to predict that alterations of this system might result in substantial perturbations of the gonadotropic axis, some of which may resemble those seen in PCOS [16]. As metastin (kisspeptin) has been defined as important for the initiation of GnRH secretion at puberty and shown to regulate the secretion of LH during the promotion of ovulation, and PCOS is a condition associated with disordered hypothalamic-pituitary-gonadal axis, the present study was designed to investigate the possible role of metastin in the pathophysiology of PCOS.
Variables
BMI > 25 kg/m2 LH FAI Metastin
OR
1.270 1.997 1.910 6.826
95% CI Minimum
Maximum
0.287 1.440 1.319 2.162
5.611 2.770 2.767 21.552
Wald
p
0.099 17.155 11.740 10.723
0.753 <0.001 <0.001 <0.001
BMI: body mass index, LH: luteinizing hormone, FAI: free androgen index, OR: Odss Ratio CI: confidence interval.
,8
,6
,4
Sensitivity
Table 4 Multiple regression analysis of possible factors affecting PCOS after adjustment for BMI, LH and FAI.
1,0
,2
0,0 0,0
,2
,4
,6
,8
1,0
1 - Specificity Fig. 2. ROC analyses of metastin levels between the study and control groups.
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between metastin and PCOS related reproductive and metabolic disturbances. Their results suggested that metastin concentrations were elevated in lean adolescent and adult women with PCOS compared with lean adolescent control subjects. Moreover metastin showed a positive correlation with T concentrations and LH similar to the findings of the present study. Jeon et al. [18] also reported higher metastin levels in women with PCOS however they did not found any correlations between metastin and other clinical, hormonal and metabolic parameters except for retinol binding protein 4 (RBP4) and a positive correlation between metastin levels and FAI in obese PCOS patients which may be reflective of the hyperandrogenism associated with this subgroup. In their series metastin levels were found to be higher in both the obese (BMI 23 kg/m2) and non-obese (BMI < 23 kg/m2) PCOS group compared with lean controls. In another study conducted by Panidis et al. [19] normal weight women with PCOS and obese control women showed less insulin resistance and higher metastin levels compared with obese women with PCOS. They also concluded that plasma metastin levels were negatively correlated with BMI and FAI. Since metastin was identified as an important factor for initiating GnRH secretion and for regulating GnRH induced LH secretion; it is reasonable to find higher metastin levels in patients with PCOS who demonstrate higher LH levels and androgenic profile compared to healthy women. Higher metastin levels in women with PCOS may result in increased LH pulsatility which in turn causes a persistant increase in androgen levels. Already two studies [17,18] reported higher metastin levels in women with PCOS similar to the data of the present study. The BMI values differed between these studies, which may explain the contradictory results regarding metastin concentrations as well as methodological differences. Additionally, small sample size and the heterogeneity of the study groups are the limitations of these previous studies. The data of the present study claimed that women with PCOS exhibited higher metastin levels than controls, even after controlling for BMI. Furthermore, metastin concentrations were found to be in a positive correlation with FAI and mFG scores as well as T, DHEA-SO4 and LH levels; however, negatively correlated with SHBG levels. There was no significant correlation between metastin and indices of insulin resistance. Eventually, the present findings indicated that increasing metastin may be a marker to recognize PCOS features especially androgenic profile. Discrepant findings among the results of the published studies may be attributed to the design and sample size and the demographic and genetic characteristics of the different populations. Likewise, differences between the studies regarding the efficiency and specificity of the metastin assays used might also contribute to the discrepancies. Prospective design of the present study may provide advantages as well as the larger sample size compared to aforementioned studies. However, one of the limitations of our study is that we used single measurement of baseline levels of metastin from one collected serum.
The mechanism of metastin for regulating gonadotropin secretion still remains unknown. As a peptide recently recognized as important for reproduction affecting hypothalamo-pituitarygonadal axis, further evaluation of kisspeptins is warranted. Since a conclusive demonstration of a pathophysiologic association between kisspeptin signaling and PCOS is still lacking, studies investigating the Kiss-1 system and associated biochemical pathways are essential to define therapeutic interventions targeting reproductive consequences associated with PCOS. References [1] Wojciechowski P, Lipowska A, Rys P, et al. Impact of FTO genotypes on BMI and weight in polycystic ovary syndrome: a systematic review and meta-analysis. Diabetologia 2012;55:2636–45. [2] Farrell K, Antoni MH. Insulin resistance, obesity, inflammation, and depression in polycystic ovary syndrome: bio behavioral mechanisms and interventions. Fertil Steril 2010;94:1565–74. [3] Ohtaki T, Shintani Y, Honda S, et al. Metastasis suppressor gene KiSS-1 encodes peptidelig and of a G-protein-coupled receptor. Nature 2001;411:613–7. [4] Hori A, Honda S, Asada M, et al. Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 2001;286:958–63. [5] Kotani M, Detheux M, Vandenbogaerde A, et al. The metastasis suppressor gene KiSS-1 encodes kiss peptins, the natural ligands of the orphan G proteincoupled receptor GPR54. J Biol Chem 2001;276:34631–6. [6] Muir AI, Chamberlain L, Elshourbagy NA, et al. AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2013;276:28969–75. [7] deRoux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100:10972–6. [8] Funes S, Hedrick JA, Vassileva G, 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. [9] Castellano JM, Gaytan M, Roa J, et al. Expression of KiSS-1 in ratovary: putativelocal regulator of ovulation? Endocrinology 2006;147:4852–62. [10] 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. [11] Navarro VM, Castellano JM, Fernández-Fernández R, et al. Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 2005;146:156–63. [12] Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19(1):41–7. [13] Hatch R, Rosenfield RL, Kim MH, Tredway D. Hirsutism: implications, etiology, and management. Am J Obstet Gynecol 1981;140:815–30. [14] Legro RS, Castracane VD, Kauffman RP. Detecting insulin resistance in polycystic ovary syndrome: purposes and pitfalls. Obstet Gynecol Surv 2004;59(2):141–54. [15] Morley JE, Patrick P, Perry 3rd. HM. Evaluation of assays available to measure free testosterone. Metabolism 2002;51(5):554–9. [16] Witched SF, Tena-Sempere M. The Kiss1 system and polycystic ovary syndrome: lessons from physiology and putative pathophysiologic implications. Fertil Steril 2013;100:12–22. [17] Chen X, Mo Y, Li L, Chen Y, Li Y, Yang D. Increased plasma metastin levels in adolescent women with polycystic ovary syndrome. Eur J Obstet Gynecol Reprod Biol 2010;149(1):72–6. [18] Jeon YE, Lee KE, Jung JA, et al. Kisspeptin, leptin, and retinol-binding protein 4 in women with polycystic ovary syndrome. Gynecol Obstet Invest 2013;75:268–74. [19] Panidis D, Rousso D, Koliakos G, et al. Plasma metastin levels are negatively correlated with insulin resistance and free androgens in women with polycystic ovary syndrome. Fertil Steril 2006;85:1778–83.
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Metastin levels in relation with hormonal and metabolic profile in patients with polycystic ovary syndrome S.A. Yilmaz a, *, O.S. Kerimoglu a , A.T. Pekin a , F. Incesu a , N.U. Dogan b , C. Celik a , A. Unlu c a
Department of Obstetrics and Gynecology, Selcuk University, Faculty of Medicine, Konya, Turkey Department of Obstetrics and Gynecology, Akdeniz University, Faculty of Medicine, Antalya, Turkey c Department of Biochemistry, Selcuk University, Faculty of Medicine, Konya, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 February 2014 Received in revised form 29 April 2014 Accepted 4 June 2014
Objective: The aim of the present study was to evaluate serum concentrations of metastin in relation with hormonal and metabolic profile in patients with and without polycystic ovary syndrome (PCOS). Study design: The study was a clinical study. Eighty-three women with PCOS and 66 body mass index (BMI) matched controls were divided into two groups, based on BMI: overweight and obese (BMI 25 kg/ m2) and normal weight. (BMI < 25 kg/m2) Hirsutism scores, hormonal and metabolic profile as well as metastin levels were evaluated in each subject. Blood samples were collected in the early follicular phase (between day 2 and day 5 of the menstrual cycle) at 9:00 AM, after an overnight fast. Circulating levels of LH, FSH, PRL, TSH, T, fT, DHEAS, 17-OH-P, sex hormone-binding globulin (SHBG), insulin, glucose, lipid profile and metastin were measured. Results: Metastin levels were significantly higher in the PCOS group compared to controls (2.02 ng/ml versus 1.16 ng/ml, p < 0.001). Metastin levels correlated significantly positively with luteinizing hormone (LH), total testosterone (T), dehydroepiandrosteronesulphate (DHEA-SO4) levels, modified Ferriman– Gallwey (mFG) scores and free androgen index (FAI); however, correlated negatively with sex hormone binding globulin (SHBG) levels (p < 0.05). When overweight or obese (BMI 25 kg/m2) and normal weight (BMI < 25 kg/m2) women with PCOS were compared to body mass index (BMI) matched controls, higher metastin levels were also found in PCOS groups (1.94 ng/ml versus 1.18 ng/ml, and 2.06 ng/ml versus 1.08 ng/ml, p < 0.05, respectively). Conclusions: These findings suggest that metastin levels were higher in women with PCOS as compared to controls regardless of BMI. Furthermore, metastin levels can be used as a specific marker for androgenic profile and this marker might play a role in the pathogenesis of PCOS. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Metastin Kisspeptin PCOS Insulin resistance
Introduction Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting women, with a prevalence of 6–12% in women of reproductive age [1]. It is characterized by androgen excess, chronic oligoanovulation and polycystic ovaries (PCO) on ultrasound. Clinically, the androgen excess presents as hirsutism and acne, whereas anovulation presents as subfertility and menstrual irregularity. In addition, severe physiological abnormalities such as insulin resistance, inflammation, visceral fat, cardiovascular disease, infertility are common among women with PCOS [2].
* Corresponding author at: Department of Obstetrics and Gynecology, Selcuk University, Faculty of Medicine, Konya, 42100, Turkey. Tel.: +90 332 2245164; fax: +90 332 2416065. E-mail address: [email protected] (S.A. Yilmaz). http://dx.doi.org/10.1016/j.ejogrb.2014.06.004 0301-2115/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Metastin is a 54 amino-acid peptide, which was first isolated from the human placenta in 2001 [3]. It is encoded by the Kiss-1 gene, therefore it is also known as Kiss-1 peptide (kisspeptin) [4]. Metastin action is exerted by a trans-membrane G-protein coupled receptor, named GPR54, AXOR12, or HoT7T175 [3–6]. Loss of Kiss-1 gene function is reported to be associated with hypogonadotropic hypogonadism in humans and animal models [7,8]. Metastin has recently been shown to regulate the secretion of luteinizing hormone (LH) during the promotion of ovulation [9] by stimulating gonadotropin releasing hormone (GnRH) release from the hypothalamus [10,11]. Women with PCOS commonly display deregulated gonadotropin secretion with higher LH pulsatility and perturbed LH/FSH (follicle stimulating hormone) ratios, which likely contributes to the ovarian phenotype and might be indicative of disrupted GnRH secretory activity. Given the complex relationship between metastin and hypothalamic-pituitary gonadal axis, the present study aimed to investigate whether there was a difference in serum
S.A. Yilmaz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 56–60 Table 1 Demographic, clinical, biochemical and hormonal characteristics of women with PCOS and controls.
Age (years) BMI (kg/m2) FPG (mg/dl) Fasting insulin (mIU/ml) HOMA-IR Total cholesterol (mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl) fT(pg/ml) DHEA-SO4 (mg/dl) T (nmol/L) SHBG TSH (mIU/ml) FSH (mIU/ml) LH (mIU/ml) E2 (pg/ml) PRL (mIU/ml) 17OH-Progesterone mFG score FAI Metastin (ng/ml)
PCOS (n = 83)
Controls (n = 66)
p
21 (17–37) 24.5 (18.1–34.8) 89 (72–117) 9.5 (3.1–38.0) 2.0 (0.7–9.2) 163 (104–298) 47 (31–77) 99.5 24.1 90 (38–276) 3.2 (0.6–18.7) 303.0 101.6 1.9 (0.8–3.8) 31 (15–76.1) 2.0 (0.4–4.3) 5.1 (2.1–8.4) 9.3 (3.5–29.0) 41 (17–67) 12 (4.5–36) 1.2 (0.1–2.6) 9 (6–21) 6.0 (1.4–14.1) 2.02 (0.56–4.43)
24.5 (17–38) 23.1 (18–34.8) 91 (77–109) 7.7 (1.4–34.0) 1.8 (0.3–9.1) 165 (98–270) 47.5 (30–78) 101.227.8 93.5 (24.1–287) 1.4 (0.1–9.7) 230.5107.7 1.0 (0.3–2.8) 45.1 (21.9–138.0) 1.6 (0.4–9.0) 5.2 (3.1–8.5) 4.5 (2.0–9.2) 43 (16–76) 12 (3.2–25.4) 1.1 (0.3–2.6) 0 (0–1) 2.0 (0.6–6.5) 1.16 (0.45–2.00)
<0.001 0.125 0.034 0.039 0.101 0.645 0.950 0.687 0.677 <0.001 <0.001 <0.001 <0.001 0.057 0.097 <0.001 0.570 0.421 0.907 <0.001 <0.001 <0.001
BMI: body mass index, FPG: fasting plasma glucose, HOMA-IR: homeostasis model mssessment-mnsulin resistence, HDL: high density lipoprotein, LDL: low density lipoprotein, TG: triglycerides, fT: free testosterone, DHEA-SO4: dehydroepiandrosteronesulphate, fT3-fT4: free thyroxine levels, TSH: thyroid stimulating hormone, FSH: follicle stimulating hormone, LH: luteinizing hormone, E2:estradiol, PRL: prolactin, T:total testosterone, SHBG: sex hormone binding globulin, mFG: modified Ferriman–Gallwey score, FAI: free androgen index. Results for continous variables are expressed as mean SD or median (range). p < 0.05 was considered significant.
metastin levels between women with and without PCOS, a condition associated with aberrant gonadotropin secretion and hyperandrogenemia. This study also set out to correlate serum metastin levels in relation with hormonal and metabolic profile to evaluate the possible correlations between metastin and PCOSrelated reproductive and metabolic disturbances.
57
menses and no clinical or biochemical hyperandrogenism or PCO were eligible. Detailed clinical history was taken and physical examination was performed for all participants. Hirsutism was evaluated according to the modified Ferriman–Gallwey score (greater than seven indicates hirsutism) [13]. BMI was calculated as the ratio of weight divided by height squared (kg/m2). Women’s with PCOS and controls were further divided into two groups, based on BMI values: overweight or obese (BMI 25 kg/m2) and normal weight (BMI < 25 kg/m2). Routine chemistry laboratory investigations included fasting blood glucose, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG) were measured by commercially available Abbott Diagnostics kits in Architect c16000 chemistry auto analyser (Abbott Diagnostics, USA). Routine hormonal analysis of LH, FSH, estradiol (E2), thyroid stimulating hormone (TSH), fasting insulin, dehydroepiandrosterone sulphate (DHEA-SO4), total testosterone (T) and sex hormone binding globuline (SHBG), were done by chemiluminescence methods using ADVIA Centaur1 XP Immunoassay system (Siemens Diagnostics, Germany). 17-OH-progesterone, and free testosterone (fT) levels were measured by Diametra ELISA kits (Viaapozaula, Italy). Insulin resistance (IR) was determined by the homeostasis model assessment (HOMA) index (fasting glucose (mg/dl) fasting insulin (mU/ml)/405) [14]. Free androgen index (FAI) was calculated according to the equation: T (nmol/l) 100/SHBG (nmol/l) [15]. All sampling procedures were performed in the early follicular phase (day 2–5 of the menstrual cycle) in the morning after an overnight fast. Serum metastin levels were assessed by an Enzyme Immunoassay kit (Phoenix Pharmaceuticals, Karlsruhe Germany). The intraand inter-assay coefficients of variations for metastin were <10% and <15%, respectively. The detection range for metastin was 0–1 ng/ml.
5
Materials and methods 4
3
2
Metastin (ng/mL)
The design of the present study was approved by the ethical committee and institutional review board of Selcuk University, Faculty of Medicine, where the study was conducted. Written informed consents were obtained from all participants. A total sample size of 88 (22 cases for each group) was required to detect at least 0.58 (ng/ml) difference in metastin levels between groups with a power of 80% at the 5% significance level. The difference of 0.58 (ng/ml) was taken from both pilot study and our clinical experiments. Sample size estimation was performed by using NCSS and PASS 2000i software. A total of 83 women with PCOS and 66 BMI matched controls participated in this study. The diagnosis of PCOS was made according to the Rotterdam criteria [12] in the presence of at least two of the following: (1) oligomenorrhea and/or anovulation, (2) biochemical and/or clinical hyperandrogenism, (3) ultrasound appearance of polycystic ovaries (PCO) (multiple cysts >12 in number of 2–9 mm size) with the exclusion of other etiologies such as congenital adrenal hyperplasia, virilising tumor, Cushing syndrome and prolactinoma, diabetes, hypertension and other cardiovascular diseases. Over the three months preceding the study no subject had been on hormonal contraceptives, other medications or diet which could affect lipid and carbohydrate metabolism. No subject smoked or consumed alcohol. As controls 66 body mass index (BMI) matched women who had regular
BMI
1
<25 kg/m^2 0
>=25 kg/m^2 Control
PCO
Fig. 1. Plasma metastin levels of women with regard to body mass index. The horizontal lines in the middle of each box indicates the median (50th percentile) metastin values, while the top and bottom borders of the box mark the 25th and 75th percentiles, respectively. The whiskers above and below the box mark indicates the maximum and minimum metastin levels.
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S.A. Yilmaz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 56–60
Statistical analysis
Results
Data analysis was performed by using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, IL, United States). Whether the distributions of metric discrete and continuous variables were normally or not was determined by Kolmogorov–Smirnov test. Levene test was applied for determining the homogeneity of variances. Metric discrete and continuous variables were shown as mean standard deviation (SD) or median (min–max), where applicable. While, the mean differences between groups were compared by Student’s t test, otherwise, Mann–Whitney U test was applied for comparisons of the median values. Nominal data were analyzed by Pearson’s Chi-square test. Degrees of association between continuous variables were calculated by Spearman’s correlation coefficient. Determining the best predictors which affect on PCOS was evaluated by Multiple Logistic Regression Analysis Backward LR procedure. Any variable whose univariable test had a p value < 0.25 was accepted as a candidate for the multivariable model along with all variables of known clinical importance. Odds ratio, 95% confidence interval and Wald statistic for each independent variable were also calculated. Whether the statistically significant predictivity of metastin measurements on PCO was going on or not was evaluated by Multiple Logistic Regression analysis after adjustment for BMI, LH and FAI. Odds ratios and 95% confidence intervals for each independent variable were also calculated. A p value less than 0.05 was considered statistically significant. But, for all multiple comparisons the Bonferroni adjustment was applied for controlling Type I error. The optimal cut off points of metastin to discriminate control and PCO groups was evaluated by ROC analyses calculating area under the curve as giving the maximum sum of sensitivity and specificity for the significant test. Sensitivity, specificity, positive and negative predictive values for metastin at the best cut off points were also calculated.
Demographic, clinical, biochemical and hormonal characteristics of women with PCOS and controls are summarized in Table 1. Age, fasting insulin, T, fT, DHEA-SO4, LH levels and mFG and FAI scores were significantly higher, whereas SHBG and fasting plasma glucose levels were lower in PCOS group compared to controls (p < 0.05). Metastin levels were also found to be higher in women with PCOS than controls (2.02 [056–443] ng/ml versus 1.16 [045– 200] ng/ml, p < 0.001) (Fig. 1). Table 2 shows the demographic, clinical, biochemical and hormonal characteristics of women with PCOS and controls with regard to BMI. When women were further divided based on BMI in both groups metastin levels continued to be higher in women with PCOS groups regardless of BMI (Table 2, Fig. 1). When obese and overweight (BMI 25 kg/m2, n = 66, 41 women with PCOS and 25 controls) and lean (BMI < 25 kg/m2, n = 83, 42 women with PCOS and 41 controls) women were evaluated on their own PCOS group showed higher metastin levels than controls (1.94 [056–349] ng/ml versus 1.18 [080–197] ng/ml, p < 0.001 and 2.06 [067–443] ng/ml versus 1.08 [045–200] ng/ml, p = 0.021, respectively). Metastin levels showed a positive significant correlation with LH, T, and DHEA-SO4 levels, modified Ferriman–Gallwey (mFG) scores and FAI; however were negatively correlated with SHBG levels (r: 0.33, p < 0.05). Correlations between metastin with demographic and clinical characteristics as well as hormonal and metabolic profile of all patients including controls are shown in Table 3. Determining the best predictors which affect on PCOS was evaluated by Multiple Logistic Regression Analysis Backward LR procedure. Using Multiple Logistic Regression Analysis Backward LR procedure after adjustment for all possible confounding factors we found that metastin levels (OR: 6.826; 95% CI: 2.162–21.552; p < 0.001), and FAI (OR: 1.910; 95% CI: 1.319–2.767; p < 0.001) and
Table 2 Demographic, clinical, biochemical and hormonal characteristics of women with PCOS and controls regarding BMI. BMI < 25
Age (years) FPG (mg/dl) Fasting insulin (mIU/ml) HOMA-IR TC (mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl) fT(pg/ml) DHEA-SO4 (mg/dl) T (nmol/L) SHBG TSH (mIU/ml) FSH (mIU/ml) LH (mIU/ml) E2 (pg/ml) PRL (mIU/ml) 17OH-Progesterone mFG score FAI Metastin(ng/ml)
BMI 25 a
PCOS (n = 42)
Controls (n = 41)
p
PCOS (n = 41)
Controls (n = 25)
pb
21 (17–32) 87(72–17) 8.2 (3.2–20.8) 1.7 (0.7–4.5) 159 (104–98) 47 (31–77) 95.2 25.9 77 (38–189) 2.9 (0,6–17.8) 312.1106.7 1.81 (0.79–3.78) 33.7 (20,7–76.1) 1.9 (0,9–4.2) 5.0 (2.3–7.4) 9.3 (3.5–29.0) 45 (24–67) 12.5 (5.7–34.0) 1.1 (0.1–2.6) 9.5 (7–21) 5.08(1.38–13.92) 2.06 (0.67–4.43)
22.5 (17–38) 90 (77–109) 7.2 (1.4–34.0) 1.5 (0.3–9.1) 160.5 (98–225) 50 (32–78) 93.426.9 78.5 (40–188) 1.4 (0.4–5.1) 242.6117.6 0.97 (0.27–2.84) 53.5 (24.9–138,0) 1.6 (0.5–3.6) 5.3 (3.1–8.5) 5.6 (2.0–8.7) 43.5 (16–76) 12.0 (5.8–25.4) 1.2 (0.5–2.6) 0 (0–1) 1.98 (0.59–6.54) 1.08 (0.45–2.00)
0.004 0.088 0.064 0.147 0.919 0.475 0.762 0.707 0.002 0.006 <0.001 <0.001 0.129 0.047 <0.001 0.728 0.262 0.267 <0.001 <0.001 <0.001
22 (17–37) 89 (73–106) 11.8c (3.1–38,0) 2.6c (0.7–9,2) 170 (128–248) 45 (34–67) 103.9 21.6 109c (39–276) 3.5 (1.0–18.7) 293.796.5 1.94 (0.86–3.78) 29.9c(15.0–45.0) 2.0 (0.4–4.3) 5.3 (2.1–8.4) 9.2 (5.6–18.0) 38c (17–60) 11.2 (4.5–36.0) 1.3 (0.1–2.4) 9c (6–21) 6.95c (3.25–14,10) 1.94 (0.56–3.49)
29d (20–38) 93.5 (79–107) 10.4d (4.3–31.0) 2.5d (0.8–6.8) 179d (125–270) 40d (30–56) 114.8d 24.1 116.5d (24.1–287) 1.7 (0.1–9.7) 210.587.3 1.11 (0.27–1.97) 43.0 (21.9–132.0) 1.5 (0.4–9.0) 5.2 (3.3–8.1) 3.9d (2.2–9.2) 42.5 (23–58) 12 (3.2–21.6) 1.0 (0.3–1.9) 0 (0–0) 2.11 (0.72–6.49) 1.18 (0.80–1.97)
<0.001 0.120 0.719 0.957 0.248 0.110 0.068 0.550 <0.001 <0.001 <0.001 <0.001 0.310 0.724 <0.001 0.237 0.838 0.105 <0.001 <0.001 0.021
BMI: body mass index, FPG: fasting plasma glucose, HOMA-IR: homeostasis model assessment-insulinresistence, HDL: high density lipoprotein, LDL: low density lipoprotein, TG: triglycerides, TC: totalcholesterole, fT: free testosterone, DHEA-SO4: dehydroepiandrosterone sulphate, fT3-fT4: free thyroxinelevels, TSH: thyroid stimulating hormone, FSH: follicle stimulating hormone, LH: luteinizing hormone, E2:estradiol, PRL: prolactin, T: total testosterone, SHBG: sex hormone binding globulin, mFG: modified Ferriman– Gallwey score, FAI: free androgen indexResults for continous variables are expressed as mean SD or median (range). p < 0.025 is accepted statistically significant after Bonferroni adjustment. a Difference between PCOS and controls BMI < 25. b Difference between PCOS and controls BMI 25. c Difference between obese-overweight and lean in PCOS group. d Difference between obese-overweight and lean in control group.
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Table 3 Correlations between metastin with demographic and clinical characteristics and hormonal and metabolic profile of all patients including controls. r Age (years) BMI (kg/m2) FPG (mg/dl) Fasting insulin (mIU/ml) HOMA-IR TC (mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl) fT(pg/ml) DHEA-SO4 (mg/dl) T (nmol/L) SHBG TSH (mIU/ml) FSH (mIU/ml) LH (mIU/ml) E2 (pg/ml) PRL (mIU/ml) 17OH-Progesterone mFG score FAI
p 0.148 0.076 0.089 0.097 0.078 0.088 0.019 0.098 0.028 0.161 0.166 0.172 0.330 0.081 0.063 0.237 0.060 0.011 0.018 0.474 0.309
0.074 0.363 0.284 0.244 0.350 0.288 0.817 0.240 0.736 0.052 0.045 0.038 <0.001 0.331 0.448 0.004 0.473 0.892 0.825 <0.001 <0.001
BMI: body mass index, FPG: fasting plasma glucose, HOMA-IR: homeostasis model assessment-insulin resistence, HDL: high density lipoprotein, LDL: low density lipoprotein, TG: triglycerides, TC: total cholesterole, fT: free testosterone, DHEA-SO4: dehydroepiandrosteronesulphate, fT3-fT4: free thyroxine levels, TSH: thyroid stimulating hormone, FSH: follicle stimulating hormone, LH: luteinizing hormone, E2:estradiol, PRL: prolactin, T: total testosterone, SHBG:sex hormone binding globulin, mFG: modified Ferriman– Gallwey score, FAI: free androgen index.
LH (OR: 1.997; 95% CI: 1.440–2.770; p < 0.001) were all positively associated with PCOS (Table 4). Thus, our results suggest that metastin might be an independent marker for PCOS. ROC analyses of metastine levels between the study and control groups are shown in Fig. 2. In this study population, the best cut-off point for serum metastine levels was 1.87 ng/ml. Serum metastine levels greater than 1.87 ng/ml demonstrated a sensitivity and specificity of 59% and 93.8%, respectively, for PCOS diagnosis (ROC AUC = 0.762; 95% CI: 0.685–0.840; p = 0.004). The positive predictive value was 92.5%, and the negative predictive value was 63.8 % (Fig. 2).
There are limited data in the literature with regard to changes in metastin levels and its relation with metabolic and hormonal disturbances in PCOS. So far only three studies assessed metastin in patients with PCOS, and their results are in consistent. Two previous studies [17,18] reported higher metastin levels in women with PCOS, similar to the data of the present study; however, one study [19] reported lower levels in women with PCOS as compared to controls. There are also controversial data regarding the association between metastin levels with BMI, insulin resistance, LH and androgenic profile. Chen et al. [17] measured metastin levels in 42 women with PCOS (23 adult, 19 adolescents) and 20 adolescent controls to investigate the possible correlations
Discussion The Kiss-1 system has emerged in the recent years as a fundamental player in the control of the reproductive axis, with essential roles in differentiation and pubertal activation of the reproductive system as well as key functions in the regulation of ovulation and the metabolic control of fertility. These features make it tempting to predict that alterations of this system might result in substantial perturbations of the gonadotropic axis, some of which may resemble those seen in PCOS [16]. As metastin (kisspeptin) has been defined as important for the initiation of GnRH secretion at puberty and shown to regulate the secretion of LH during the promotion of ovulation, and PCOS is a condition associated with disordered hypothalamic-pituitary-gonadal axis, the present study was designed to investigate the possible role of metastin in the pathophysiology of PCOS.
Variables
BMI > 25 kg/m2 LH FAI Metastin
OR
1.270 1.997 1.910 6.826
95% CI Minimum
Maximum
0.287 1.440 1.319 2.162
5.611 2.770 2.767 21.552
Wald
p
0.099 17.155 11.740 10.723
0.753 <0.001 <0.001 <0.001
BMI: body mass index, LH: luteinizing hormone, FAI: free androgen index, OR: Odss Ratio CI: confidence interval.
,8
,6
,4
Sensitivity
Table 4 Multiple regression analysis of possible factors affecting PCOS after adjustment for BMI, LH and FAI.
1,0
,2
0,0 0,0
,2
,4
,6
,8
1,0
1 - Specificity Fig. 2. ROC analyses of metastin levels between the study and control groups.
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between metastin and PCOS related reproductive and metabolic disturbances. Their results suggested that metastin concentrations were elevated in lean adolescent and adult women with PCOS compared with lean adolescent control subjects. Moreover metastin showed a positive correlation with T concentrations and LH similar to the findings of the present study. Jeon et al. [18] also reported higher metastin levels in women with PCOS however they did not found any correlations between metastin and other clinical, hormonal and metabolic parameters except for retinol binding protein 4 (RBP4) and a positive correlation between metastin levels and FAI in obese PCOS patients which may be reflective of the hyperandrogenism associated with this subgroup. In their series metastin levels were found to be higher in both the obese (BMI 23 kg/m2) and non-obese (BMI < 23 kg/m2) PCOS group compared with lean controls. In another study conducted by Panidis et al. [19] normal weight women with PCOS and obese control women showed less insulin resistance and higher metastin levels compared with obese women with PCOS. They also concluded that plasma metastin levels were negatively correlated with BMI and FAI. Since metastin was identified as an important factor for initiating GnRH secretion and for regulating GnRH induced LH secretion; it is reasonable to find higher metastin levels in patients with PCOS who demonstrate higher LH levels and androgenic profile compared to healthy women. Higher metastin levels in women with PCOS may result in increased LH pulsatility which in turn causes a persistant increase in androgen levels. Already two studies [17,18] reported higher metastin levels in women with PCOS similar to the data of the present study. The BMI values differed between these studies, which may explain the contradictory results regarding metastin concentrations as well as methodological differences. Additionally, small sample size and the heterogeneity of the study groups are the limitations of these previous studies. The data of the present study claimed that women with PCOS exhibited higher metastin levels than controls, even after controlling for BMI. Furthermore, metastin concentrations were found to be in a positive correlation with FAI and mFG scores as well as T, DHEA-SO4 and LH levels; however, negatively correlated with SHBG levels. There was no significant correlation between metastin and indices of insulin resistance. Eventually, the present findings indicated that increasing metastin may be a marker to recognize PCOS features especially androgenic profile. Discrepant findings among the results of the published studies may be attributed to the design and sample size and the demographic and genetic characteristics of the different populations. Likewise, differences between the studies regarding the efficiency and specificity of the metastin assays used might also contribute to the discrepancies. Prospective design of the present study may provide advantages as well as the larger sample size compared to aforementioned studies. However, one of the limitations of our study is that we used single measurement of baseline levels of metastin from one collected serum.
The mechanism of metastin for regulating gonadotropin secretion still remains unknown. As a peptide recently recognized as important for reproduction affecting hypothalamo-pituitarygonadal axis, further evaluation of kisspeptins is warranted. Since a conclusive demonstration of a pathophysiologic association between kisspeptin signaling and PCOS is still lacking, studies investigating the Kiss-1 system and associated biochemical pathways are essential to define therapeutic interventions targeting reproductive consequences associated with PCOS. References [1] Wojciechowski P, Lipowska A, Rys P, et al. Impact of FTO genotypes on BMI and weight in polycystic ovary syndrome: a systematic review and meta-analysis. Diabetologia 2012;55:2636–45. [2] Farrell K, Antoni MH. Insulin resistance, obesity, inflammation, and depression in polycystic ovary syndrome: bio behavioral mechanisms and interventions. Fertil Steril 2010;94:1565–74. [3] Ohtaki T, Shintani Y, Honda S, et al. Metastasis suppressor gene KiSS-1 encodes peptidelig and of a G-protein-coupled receptor. Nature 2001;411:613–7. [4] Hori A, Honda S, Asada M, et al. Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 2001;286:958–63. [5] Kotani M, Detheux M, Vandenbogaerde A, et al. The metastasis suppressor gene KiSS-1 encodes kiss peptins, the natural ligands of the orphan G proteincoupled receptor GPR54. J Biol Chem 2001;276:34631–6. [6] Muir AI, Chamberlain L, Elshourbagy NA, et al. AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2013;276:28969–75. [7] deRoux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100:10972–6. [8] Funes S, Hedrick JA, Vassileva G, 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. [9] Castellano JM, Gaytan M, Roa J, et al. Expression of KiSS-1 in ratovary: putativelocal regulator of ovulation? Endocrinology 2006;147:4852–62. [10] 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. [11] Navarro VM, Castellano JM, Fernández-Fernández R, et al. Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 2005;146:156–63. [12] Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19(1):41–7. [13] Hatch R, Rosenfield RL, Kim MH, Tredway D. Hirsutism: implications, etiology, and management. Am J Obstet Gynecol 1981;140:815–30. [14] Legro RS, Castracane VD, Kauffman RP. Detecting insulin resistance in polycystic ovary syndrome: purposes and pitfalls. Obstet Gynecol Surv 2004;59(2):141–54. [15] Morley JE, Patrick P, Perry 3rd. HM. Evaluation of assays available to measure free testosterone. Metabolism 2002;51(5):554–9. [16] Witched SF, Tena-Sempere M. The Kiss1 system and polycystic ovary syndrome: lessons from physiology and putative pathophysiologic implications. Fertil Steril 2013;100:12–22. [17] Chen X, Mo Y, Li L, Chen Y, Li Y, Yang D. Increased plasma metastin levels in adolescent women with polycystic ovary syndrome. Eur J Obstet Gynecol Reprod Biol 2010;149(1):72–6. [18] Jeon YE, Lee KE, Jung JA, et al. Kisspeptin, leptin, and retinol-binding protein 4 in women with polycystic ovary syndrome. Gynecol Obstet Invest 2013;75:268–74. [19] Panidis D, Rousso D, Koliakos G, et al. Plasma metastin levels are negatively correlated with insulin resistance and free androgens in women with polycystic ovary syndrome. Fertil Steril 2006;85:1778–83.