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Circulating ghrelin levels and the polycystic ovary syndrome: correlation with the clinical, hormonal and metabolic features
European Journal of Obstetrics & Gynecology and Reproductive Biology 155 (2011) 65–68
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Circulating ghrelin levels and the polycystic ovary syndrome: correlation with the clinical, hormonal and metabolic features Maha H. Daghestani a,*, Mazin H. Daghestani b, Akmal El-Mazny b a b
King Saud University, Department of Zoology, College of Science, P.O. Box 22452, Riyadh11495, Saudi Arabia Department of Obstetrics and Gynecology, Umm-Al-Qura University, P.O. Box 424, Makkah 21955, Saudi Arabia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2010 Received in revised form 23 October 2010 Accepted 29 November 2010
Objectives: Dysregulation of ghrelin levels may lead to physiological problems including obesity and polycystic ovary syndrome (PCOS). The aim of the study was to compare ghrelin levels in women with and without PCOS. Study design: Serum ghrelin levels (pre- and post-prandial) were compared between 30 Saudi women suffering from PCOS and 30 healthy controls. The relationship between circulating ghrelin levels and other hormones was investigated. Anthropometric measurements were made for all subjects. Biochemical and hormonal investigations included plasma glucose, insulin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17b-estradiol (E2), progesterone, testosterone and sex hormone binding globulin (SHGB), and serum ghrelin levels. The data were statistically analyzed using independent T-test and ANOVA. Correlation studies were performed between ghrelin levels and other variables. Results: No differences were observed in the levels of ghrelin during fasting and the postprandial period in the PCOS (p = 0.487) and control groups (p = 0.378). A significant inverse correlation was observed in ghrelin levels (fasting and postprandial) levels and BMI (PCOS: r = 0.529; p = 0.009, controls: r = 0.670; p = 0.005); PCOS: r = 0.421; p = 0.007, controls: r = 0.491; p = 0.004 respectively). No correlations between ghrelin levels and other parameters were observed. Conclusion: The findings of the study suggest that circulating plasma ghrelin levels were found to be normal and were inversely related to BMI in women with PCOS. No relationship between circulating ghrelin levels and the abnormal hormonal pattern of the PCOS were observed. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Fasting and postprandial ghrelin levels Polycystic ovary syndrome Body mass index
1. Introduction Polycystic ovary syndrome (PCOS) is a complex multifactorial genetic disorder with dysregulated steroidogenesis [1]. It is one of the most common causes of infertility, affecting up to 10% of women of reproductive age. The disease is characterized by chronic anovulation, functional hyperandrogenism and polycystic ovaries on ultrasound examination [2]. PCOS is considered a plurimetabolic syndrome associated with insulin resistance and central obesity [3]. Although obesity and PCOS are strongly correlated, the cause of this association remains unclear. Obesity is present in at least 30% of cases but the proportion may be as high as 75% in some series [4]. In women with PCOS, the presence of hyperinsulinemia, dyslipidemia and/or hypertension is associated with obesity. The obesity and PCOS place this group of women at
* Corresponding author. Tel.: +966 050 4157812; fax: +966 01 4767296. E-mail addresses: [email protected], [email protected] (M.H. Daghestani), [email protected] (M.H. Daghestani), [email protected] (A. El-Mazny). 0301-2115/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2010.11.019
high risk of developing adverse metabolic profiles including insulin resistance [5]. Ghrelin was discovered in the recent past as a 28 amino acid peptide hormone, produced predominantly by endocrine cells in the stomach. It plays an important role in release of growth hormone, feeding behavior, glucose metabolism and memory, and acts as an antidepressant. It is a potent orexigenic peptide that stimulates food intake and body weight gain, thus regulating energy homeostasis [6]. Several studies revealed high levels of ghrelin during fasting, which returned to basal levels after refeeding [7–9]. Plasma ghrelin levels are lower in obese subjects [10], while in lean ones, plasma ghrelin levels rise progressively during fasting and fall after eating, a pattern reciprocal to that of insulin. This observation supports the possibility that ghrelin may be acting as a ‘‘trigger’’ for meal initiation [11] and that it might play a role in the pathogenesis of human obesity [12]. In PCOS, low levels of ghrelin were observed during fasting compared to those in control groups [13–16], though not in all studies [17–22]. Furthermore, plasma ghrelin is lower in Pima Indians, a population predisposed to obesity, compared with ageand weight-matched caucasians [23]. Postprandial ghrelin was not
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examined in most of these studies. It is unclear from these studies whether ghrelin homeostasis is impaired in PCOS. The objectives of this study were to investigate fasting and postprandial ghrelin levels in Saudi women with or without PCOS and the relationship of these levels to the clinical, hormonal and metabolic features of PCOS. 2. Materials and methods 2.1. Samples Data were collected from 60 female volunteers, recruited from the out-patient clinic, Department of Obstetrics and Gynecology, Al-Noor Hospital, Makkah, Kingdom of Saudi Arabia. The purpose of the protocol was explained to the patients and the control women, and signed informed consent was obtained from all the subjects, as approved by the local ethical committee. 2.2. Subjects Thirty women were enrolled in this study fulfilling two of the following three criteria for having PCOS [2]: (1) Irregular or absent ovulation confirmed by luteal progesterone and normal serum FSH levels (normal range: 1.0–10.0 IU/l). (2) Clinical and/or biochemical signs of raised androgens; elevated serum androgen levels (total testosterone >2 nmol/l), and/or androstenedione >0.15 nmol/l, and/or dehydroepiandrosterone sulphate (DHEAS) >10 mmol/l); LH to FSH ratio >2. (3) At least one ovary examined by ultrasound contained 12 follicles measuring 2–9 mm in diameter and/or increased ovarian volume of at least 10 ml. Thirty healthy women were enrolled as the control group. They had regular ovulatory cycles, no clinical or biochemical signs of raised androgens, and normal ovarian morphology on ultrasound. The health status of the women in the control group was determined by medical history, physical and pelvic examination, and complete blood chemistry. Hormonal investigations were also done if needed. Their normal ovulatory state was confirmed by trans-vaginal ultrasound and plasma progesterone assay during the luteal phase of the cycle. The PCOS and control groups were matched for age (two years) [PCOS: 27.22 0.60 years, controls: 26.05 1.11 years; p = 0.511] and BMI (10%) [PCOS: 29.35 7.25 kg/m2, controls: 2 28.31 6.97 kg/m ; p = 0.835]. In each group, the percentage of overweight and obese women was about 70% (21/30 women). Exclusion criteria for all the subjects included pregnancy, hypothyroidism, hyperprolactinemia, Cushing’s syndrome, congenital adrenal hyperplasia, current or previous (within the last six months) use of oral contraceptives, glucocorticoids, antiandrogens, ovulation induction agents, antidiabetic and anti-obesity drugs or other hormonal drugs. None of the patients was affected by neoplastic, metabolic and cardiovascular disorder or other concurrent medical illness such as diabetes, renal disease, and hepatic disorders. All the subjects were non-smokers and had normal physical activity. 3. Anthropometric measurements For each woman weight and height were measured to calculate the BMI (weight in kg divided by height in m2). Waist circumference (the narrowest circumference between the lower costal margins and the iliac crest) and hip circumference (the maximum circumference at the level of the femoral trochanters) were also measured in the standing position to calculate the waist– hip ratio (WHR).
3.1. Biochemical measurements Blood was sampled in the morning between 08.00 h and 09.00 h after an overnight fast. During the early follicular phase (2nd or 3rd day), 5 ml of blood was drawn in plain red-top tubes for the determination of LH, FSH, E2 and insulin levels in the serum. Two millilitres of blood were collected into chilled tubes containing 1.2 mg EDTA and aprotinin (500 KIU/ml; Trasylol; Bayer Corp., Leverkusen, Germany) for total ghrelin levels and 2 ml were drawn in fluoride tubes (gray top) for glucose estimation. Following fasting blood sample collection, subjects consumed a standard mixed breakfast of about 527 kcal during 15 min. The meal consisted of 50 g white bread, 33 g black bread, 18 g margarine, 30 g cheese, 9 g jam and 200 ml of 0.5% fat milk (24.1% fat, 54.4% carbohydrate, 21.5% protein). Two millilitres of blood were collected 60 min after the meal ingestion for determination of postprandial ghrelin levels. On day 20 or 21 of the menstrual cycle, 5 ml blood samples were obtained for determination of 17OHprogesterone (17OH-P), testosterone (T), androstenedione (A) and sex-hormone binding globulin (SHBG) levels. All blood samples for each woman were immediately centrifuged, and the serum was stored at 80 8C until analysis. The basal serum levels of LH, FSH, E2, P, T, SHBG and insulin were estimated using the electrochemiluminescence immunoassay ‘‘ECLlA’’ on a Roche Elecsys 1010/2010 and MODULAR ANALYTICS E170 (Elecsys module) immunoassay analyzers (Roche Diagnostic, Mannheim, Germany). Androstenedione levels were estimated by using the Gamma Coat Androstenedione Radioimmunoassay Kit (DiaSorin Inc, Stillwater, USA). Total ghrelin levels were measured in duplicate using a commercial ghrelin (human) enzyme immunoassay kit (EIA) from (Phoenix Pharmaceuticals, Inc., Belmont, CA, USA), with a lower limit of detection of 0.06 ng/ ml. Plasma glucose levels were determined by the glucose oxidase method on a Beckman Glucose Analyzer (Fullerton, CA). 3.2. Statistical analysis Data are presented as mean SEM. The comparisons between PCOS patients and their matched control were done using independent T- and ANOVA tests. The Pearson correlation coefficient (r) was used to find out the correlation between ghrelin and other variables. A probability value p 0.05 was considered statistically significant. All analyses were run using the StatView program for Windows (version 8.0; SAS). In this study, due to the relatively small sample size, the statistical power in detecting the minimum difference = 6.1% and that of detecting the maximum difference = 50% adjusting a error at 0.05. 4. Results Clinical, hormonal and metabolic features of women with PCOS and controls are summarized in Table 1. In the PCOS group, WHR, LH, LH/FSH ratio, E2, 17OH-P, T, A, SHBG and serum insulin levels were significantly different in comparison with the control group (p < 0.05). No difference in fasting ghrelin levels was detected between the PCOS and control groups (p = 0.487). One-hour postprandial, ghrelin levels were decreasing in both groups, but the difference between the two groups was not statistically significant (p = 0.378) as shown in Fig. 1. Correlation of fasting ghrelin levels to the clinical, hormonal and metabolic features of women with PCOS and controls is presented in Table 2. In both PCOS and control groups, there was a significant inverse correlation between fasting ghrelin levels and BMI (PCOS: r = 0.529; p = 0.009, controls: r = 0.670; p = 0.005). In both groups, fasting ghrelin levels did not correlate with either age, WHR, LH, FSH, LH/FSH ratio, E2, 17OH-P, T, A, SHBG, 1-hour postprandial ghrelin, fasting glucose and insulin levels (p = NS).
M.H. Daghestani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 155 (2011) 65–68 Table 1 Clinical, hormonal and metabolic features of women with PCOS and controls.
Age (yr) BMI (kg/m2) WHR LH (IU/l) FSH (IU/l) LH/FSH ratio E2 (pmol/l) P (nmol/l) T (nmol/l) A (nmol/l) SHBG (nmol/l) Fasting ghrelin (ng/ml) 1 h ghrelin (ng/ml) Glucose (mmol/l) Insulin (pmol/l)
PCOS (n = 30)
Controls (n = 30)
p
Sig.
27.22 0.60 29.35 7.25 0.84 0.02 13.64 1.21 5.22 0.46 2.81 0.19 201.07 14.13 3.23 0.46 2.69 0.16 0.23 0.10 27.41 2.64 0.42 0.02 0.34 0.01 5.10 0.09 97.46 25.86
26.05 1.11 28.31 6.97 0.77 0.01 4.65 0.22 5.09 0.29 0.98 0.06 134.97 7.72 26.12 3.02 1.20 0.11 0.04 0.00 45.76 3.04 0.43 0.02 0.32 0.01 4.73 0.09 73.73 5.94
0.511 0.835 <0.001 <0.001 0.576 <0.001 0.001 <0.001 <0.001 0.021 0.015 0.487 0.378 0.140 0.040
NS NS S S NS S S S S S S NS NS NS S
NS = non-significant = p > 0.05. S = significant = p 0.05.
[()TD$FIG]
Fig. 1. Fasting and 1-hour postprandial ghrelin levels in women with PCOS and controls.
Table 2 Correlation of fasting ghrelin levels to the clinical, hormonal and metabolic features of women with PCOS and controls. PCOS (n = 30)
Age (yr) BMI (kg/m2) WHR LH (IU/l) FSH (IU/l) LH/FSH ratio E2 (pmol/l) P (nmol/l) T (nmol/l) A (nmol/l) SHBG (nmol/l) 1 h ghrelin (ng/ml) Glucose (mmol/l) Insulin (pmol/l)
0.057 0.529 0.044 0.034 0.124 0.086 0.122 0.088 0.189 0.205 0.024 0.751 0.038 0.144
NS = non-significant = p > 0.05 S = significant = p 0.05
Table 3 Correlation of 1-hour postprandial ghrelin levels to the clinical hormonal and metabolic features of women with PCOS and controls. PCOS (n = 30) r Age (yr) BMI (kg/m2) WHR LH (IU/l) FSH (IU/l) LH/FSH ratio E2 (pmol/l) P (nmol/l) T (nmol/l) A (nmol/l) SHBG (nmol/l) Fasting ghrelin (ng/ml) Glucose (mmol/l) Insulin (pmol/l)
0.045 0.421 0.035 0.027 0.099 0.068 0.097 0.070 0.150 0.163 0.019 0.751 0.030 0.115
Controls (n = 30)
p
Sig.
0.401 0.007 0.115 0.694 0.479 0.497 0.426 0.296 0.288 0.211 0.537 0.072 0.556 0.427
NS S NS NS NS NS NS NS NS NS NS NS NS NS
r 0.086 0.491 0.170 0.123 0.116 0.095 0.279 0.053 0.113 0.095 0.230 0.536 0.185 0.243
p
Sig.
0.450 0.004 0.4539 0.352 0.110 0.348 0.070 0.438 0.303 0.261 0.146 0.308 0.204 0.053
NS S NS NS NS NS NS NS NS NS NS NS NS NS
NS = non-significant = p > 0.05. S = significant = p 0.05.
Correlation of 1-hour postprandial ghrelin levels to the clinical, hormonal and metabolic features of women with PCOS and controls is presented in Table 3. In both PCOS and control groups, there was a significant inverse correlation between 1-hour postprandial ghrelin levels and BMI (PCOS: r = 0.421; p = 0.007, controls: r = 0.491; p = 0.004). In both groups, 1-hour postprandial ghrelin levels did not correlate with age, WHR, LH, FSH, LH/FSH ratio, E2, 17OH-P, T, A, SHBG, fasting ghrelin, fasting glucose and insulin levels (p = NS).
r
67
Controls (n = 30)
p
Sig.
0.504 0.009 0.145 0.873 0.602 0.625 0.536 0.372 0.362 0.265 0.675 0.072 0.699 0.537
NS S NS NS NS NS NS NS NS NS NS NS NS NS
r 0.117 0.670 0.232 0.168 0.158 0.129 0.381 0.072 0.154 0.130 0.313 0.536 0.252 0.332
p
Sig.
0.613 0.005 0.619 0.480 0.150 0.475 0.096 0.597 0.413 0.356 0.199 0.308 0.278 0.059
NS S NS NS NS NS NS NS NS NS NS NS NS NS
5. Comments This study investigated fasting and 1-hour postprandial ghrelin levels in patients with PCOS in comparison to those in control women matched for age and BMI. In this regard this study was different from most other reports, which have not attempted to study the postprandial ghrelin level. We estimated the total ghrelin level, which is composed of active (acyl-) and inactive (des-acyl) ghrelin. In a recent report the active/total ghrelin ratio was reported to be significantly lower in PCOS patients compared to a control group but the levels of active and total ghrelin, though lower, did not reach significance [24]. Both active and inactive ghrelin are found in the circulation, where the latter is the more abundant form. Some actions of inactive ghrelin oppose those of the active form, suggesting that the ratio of active to inactive ghrelin may play a critical role in the overall physiological response. The majority of the studies on ghrelin, however, has investigated total ghrelin, in line with this present study, and there is a discrepancy in the reported results. In the present investigation, the fasting and 1-hour postprandial ghrelin levels in patients with PCOS are comparable to those of the control group and no correlation was demonstrated between circulating total ghrelin levels and the hormonal pattern characteristic of PCOS, suggesting that ghrelin secretion is not associated with increased circulating levels of LH, 17b-estradiol, testosterone, androstenedione, or SHBG. In contrast to our findings, some previous studies [13–16] have reported decreased fasting ghrelin levels in PCOS women compared to controls, though others have failed to show any variation [17–22]. In contrast to our findings, Glintborg et al. [13] concluded that ghrelin levels were decreased in hirsute PCOS patients and showed a significant negative correlation with testosterone, independent of body composition. Micic et al. [14] demonstrated that women with PCOS had lower fasting ghrelin levels and decreased insulin sensitivity independently of their BMI, compared to controls. On the other hand, Was´ko et al. [19] concluded that PCOS women have higher ghrelin levels compared to controls. A PCOS group showed negative correlation between BMI and insulin levels. A correlation between ghrelin and SHBG was also observed. This discrepancy of results may be explained by the relatively small sample size in our study, or may be related to certain genetic or social factors that need further investigation. In agreement with our results, however, Schofl et al. [17] found that in insulin-sensitive PCOS women, ghrelin levels were similar to those in healthy controls; suggesting that ghrelin could be
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linked to the degree of insulin resistance in PCOS. Ghrelin levels did not correlate to any of the parameters of hyperandrogenemia or to the LH/FSH ratio in PCOS. Orio et al. [18] demonstrated that in women with PCOS, ghrelin concentrations are not different from those of weight-matched controls. There was no relationship between circulating ghrelin and the abnormal hormonal pattern of PCOS. In comparison to matched controls, PCOS women had greater serum insulin levels, confirming previous reports [25]. In our study, ghrelin did not significantly correlate with fasting insulin levels, suggesting that increased insulin production of PCOS women does not adversely affect ghrelin secretion. This result is apparently in contrast with observations demonstrating a possible negative feedback regulation between insulin and ghrelin in humans. An inverse relationship has been reported between the circulating levels of the two hormones [23]. It has also been shown that ghrelin administration to healthy humans at pharmacological doses reduces insulin secretion [26] and, conversely, that insulin administration in high doses is capable of reducing ghrelin secretion [27]. Our results demonstrate that, despite different circulating insulin levels, ghrelin levels were similar in PCOS and control groups, suggesting that modest changes in fasting insulin levels are not sufficient to modulate ghrelin secretion. In both groups, PCOS as well as control, fasting and 1-hour postprandial ghrelin levels were significantly and inversely correlated with BMI. The result indicates that in women with PCOS, the abundance of fat mass reduces ghrelin levels, in accordance with observations in obese subjects [23]. The inverse correlation between ghrelin levels and BMI, as well as the increase of ghrelin levels after weight loss [28], supports the hypothesis that levels of ghrelin modulate food intake through both a short-acting feedback related to meal consumption [11] and a long-term feedback related to body weight and composition [12]. In conclusion, we have reported fasting and postprandial ghrelin levels in PCOS women. Our data demonstrate that in Saudi women with PCOS, ghrelin levels are not different from those of age- and BMI-matched controls and are inversely correlated with BMI. There is no relationship between circulating ghrelin levels and the abnormal hormonal pattern of the PCOS. Acknowledgments This work Grant No. (08-MED 604-2) was supported thankfully by NPST Program at King Saud University to Dr. Maha Daghestani. We thank all the subjects for their cooperation and participation in the study. We would also like to thank all the participants (researchers, technicians and nurses) for their notable contribution. References [1] Setji TL, Brown AJ. Polycystic ovary syndrome: diagnosis and treatment. Am J Med 2007;120:128–32. [2] 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;19:41–7.
[3] Goodarzi MO, Korenman SG. The importance of insulin resistance in polycystic ovary syndrome. Fertil Steril 2003;80:255–8. [4] Ehrmann DA. Polycystic ovary syndrome. N Eng J Med 2005;352:1223–36. [5] Barber TM, Wass JA, McCarthy MI, Franks S. Metabolic characteristics of women with polycystic ovaries and oligoamenorrhoea but normal androgen levels: implications for the management of polycystic ovary syndrome. Clin Endrocrinol (oxf) 2007;66:513–7. [6] Sakata I, Sakai T. Ghrelin cells in the gastrointestinal tract. Int J Pept 2010;1–7. [7] Asakawa A, Inui A, Kaga T, et al. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 2001;120:337–45. [8] Toshinai K, Mondal MS, Nakazato M, et al. Upregulation of ghrelin expression in the stomach upon fasting, insulin-induced hypoglycemia, and leptin administration. Biochem Biophys Res Commun 2001;281:1220–5. [9] Tschop M, Smiley DL, Heiman ML. Ghrelin induces idiposity in rodents. Nature 2000;407:2510–6. [10] Ukkola O, Poykko S. Ghrelin, growth and obesity. Ann Med 2002;34(2):102–8. [11] Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001;50(8):1714–9. [12] Ukkola O, Ravussin E, Jacobson P, et al. Role of ghrelin polymorphisms in obesity based on three different studies. Obes Res 2002;10(8):782–91. [13] Glintborg D, Andersen M, Hagen C, et al. Evaluation of metabolic risk markers in polycystic ovary syndrome (PCOS). Adiponectin, ghrelin, leptin and body composition in hirsute PCOS patients and controls. Eur J Endocrinol 2006;155(2):337–45. [14] Micic D, Sumarac-Dumanovic M, Kendereski A, et al. Total ghrelin levels during acute insulin infusion in patients with polycystic ovary syndrome. J Endocrinol Invest 2007;30(10):820–7. [15] Moran LJ, Noakes M, Clifton PM, et al. Postprandial ghrelin, cholecystokinin, peptide YY, and appetite before and after weight loss in overweight women with and without polycystic ovary syndrome. Am J Clin Nutr 2007;86(6):1603–10. [16] Barber TM, Casanueva FF, Karpe F, et al. Ghrelin levels are suppressed and show a blunted response to oral glucose in women with polycystic ovary syndrome. Eur J Endocrinol 2008;158(4):511–6. [17] Schofl C, Horn R, Schill T, Schlosser HW, Muller MJ, Brabant G. Circulating ghrelin levels in patients with polycystic ovary syndrome. J Clin Endocrinol Metab 2002;87(10):4607–10. [18] Orio Jr F, Lucidi P, Palomba S, et al. Circulating ghrelin concentrations in the polycystic ovary syndrome. J Clin Endocrinol Metab 2003;88(2):942–5. [19] Was´ko R, Komarowska H, Warenik-Szymankiewicz A, Sowin´ski J. Elevated ghrelin plasma levels in patients with polycystic ovary syndrome. Horm Metab Res 2004;36:170–3. [20] Skommer J, Katulski K, Poreba E, et al. Gherlin expression in women with polycystic ovary syndrome – a preliminary study. Eur J Gynaecol Oncol 2005;26(5):553–6. [21] Komarowska H, Was´ko R, Iwanik K, et al. Ghrelin ovarian cell expression in patients with polycystic ovary syndrome: an immunohistochemical evaluation. Horm Metab Res 2006;38(12):783–8. [22] Bik W, Baranowska-Bik A, Wolinska-Witort E, Chmielowska M, Martynska L, Baranowska B. The relationship between metabolic status and levels of adiponectin and ghrelin in lean women with polycystic ovary syndrome. Gynecol Endocrinol 2007;23(6):325–31. [23] Tschop M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML. Circulating ghrelin levels are decreased in human obesity. Diabetes 2001;50(4):707–9. [24] Panidis D, Asteriadis C, Georgopoulos NA, et al. Decreased active, total and altered active to total ghrelin ratio in normal weight women with the more severe form of polycystic ovary syndrome. Eur J Obstet Gynecol Reprod Biol 2010;149:170–4. [25] Dunaif A. Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 1997;18(6):774–800. [26] Broglio F, Arvat E, Benso A, et al. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 2001;86(10):5083–6. [27] Saad MF, Bernaba B, Hwyu CM, et al. Insulin regulates plasma ghrelin concentration. J Clin Endocrinol Metab 2002;87(8):3997–4000. [28] Hansen TK, Dall R, Hosoda H, et al. Weight loss increases circulating levels of ghrelin in human obesity. Clin Endocrinol 2002;56(2):203–6.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Circulating ghrelin levels and the polycystic ovary syndrome: correlation with the clinical, hormonal and metabolic features Maha H. Daghestani a,*, Mazin H. Daghestani b, Akmal El-Mazny b a b
King Saud University, Department of Zoology, College of Science, P.O. Box 22452, Riyadh11495, Saudi Arabia Department of Obstetrics and Gynecology, Umm-Al-Qura University, P.O. Box 424, Makkah 21955, Saudi Arabia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2010 Received in revised form 23 October 2010 Accepted 29 November 2010
Objectives: Dysregulation of ghrelin levels may lead to physiological problems including obesity and polycystic ovary syndrome (PCOS). The aim of the study was to compare ghrelin levels in women with and without PCOS. Study design: Serum ghrelin levels (pre- and post-prandial) were compared between 30 Saudi women suffering from PCOS and 30 healthy controls. The relationship between circulating ghrelin levels and other hormones was investigated. Anthropometric measurements were made for all subjects. Biochemical and hormonal investigations included plasma glucose, insulin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17b-estradiol (E2), progesterone, testosterone and sex hormone binding globulin (SHGB), and serum ghrelin levels. The data were statistically analyzed using independent T-test and ANOVA. Correlation studies were performed between ghrelin levels and other variables. Results: No differences were observed in the levels of ghrelin during fasting and the postprandial period in the PCOS (p = 0.487) and control groups (p = 0.378). A significant inverse correlation was observed in ghrelin levels (fasting and postprandial) levels and BMI (PCOS: r = 0.529; p = 0.009, controls: r = 0.670; p = 0.005); PCOS: r = 0.421; p = 0.007, controls: r = 0.491; p = 0.004 respectively). No correlations between ghrelin levels and other parameters were observed. Conclusion: The findings of the study suggest that circulating plasma ghrelin levels were found to be normal and were inversely related to BMI in women with PCOS. No relationship between circulating ghrelin levels and the abnormal hormonal pattern of the PCOS were observed. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Fasting and postprandial ghrelin levels Polycystic ovary syndrome Body mass index
1. Introduction Polycystic ovary syndrome (PCOS) is a complex multifactorial genetic disorder with dysregulated steroidogenesis [1]. It is one of the most common causes of infertility, affecting up to 10% of women of reproductive age. The disease is characterized by chronic anovulation, functional hyperandrogenism and polycystic ovaries on ultrasound examination [2]. PCOS is considered a plurimetabolic syndrome associated with insulin resistance and central obesity [3]. Although obesity and PCOS are strongly correlated, the cause of this association remains unclear. Obesity is present in at least 30% of cases but the proportion may be as high as 75% in some series [4]. In women with PCOS, the presence of hyperinsulinemia, dyslipidemia and/or hypertension is associated with obesity. The obesity and PCOS place this group of women at
* Corresponding author. Tel.: +966 050 4157812; fax: +966 01 4767296. E-mail addresses: [email protected], [email protected] (M.H. Daghestani), [email protected] (M.H. Daghestani), [email protected] (A. El-Mazny). 0301-2115/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2010.11.019
high risk of developing adverse metabolic profiles including insulin resistance [5]. Ghrelin was discovered in the recent past as a 28 amino acid peptide hormone, produced predominantly by endocrine cells in the stomach. It plays an important role in release of growth hormone, feeding behavior, glucose metabolism and memory, and acts as an antidepressant. It is a potent orexigenic peptide that stimulates food intake and body weight gain, thus regulating energy homeostasis [6]. Several studies revealed high levels of ghrelin during fasting, which returned to basal levels after refeeding [7–9]. Plasma ghrelin levels are lower in obese subjects [10], while in lean ones, plasma ghrelin levels rise progressively during fasting and fall after eating, a pattern reciprocal to that of insulin. This observation supports the possibility that ghrelin may be acting as a ‘‘trigger’’ for meal initiation [11] and that it might play a role in the pathogenesis of human obesity [12]. In PCOS, low levels of ghrelin were observed during fasting compared to those in control groups [13–16], though not in all studies [17–22]. Furthermore, plasma ghrelin is lower in Pima Indians, a population predisposed to obesity, compared with ageand weight-matched caucasians [23]. Postprandial ghrelin was not
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examined in most of these studies. It is unclear from these studies whether ghrelin homeostasis is impaired in PCOS. The objectives of this study were to investigate fasting and postprandial ghrelin levels in Saudi women with or without PCOS and the relationship of these levels to the clinical, hormonal and metabolic features of PCOS. 2. Materials and methods 2.1. Samples Data were collected from 60 female volunteers, recruited from the out-patient clinic, Department of Obstetrics and Gynecology, Al-Noor Hospital, Makkah, Kingdom of Saudi Arabia. The purpose of the protocol was explained to the patients and the control women, and signed informed consent was obtained from all the subjects, as approved by the local ethical committee. 2.2. Subjects Thirty women were enrolled in this study fulfilling two of the following three criteria for having PCOS [2]: (1) Irregular or absent ovulation confirmed by luteal progesterone and normal serum FSH levels (normal range: 1.0–10.0 IU/l). (2) Clinical and/or biochemical signs of raised androgens; elevated serum androgen levels (total testosterone >2 nmol/l), and/or androstenedione >0.15 nmol/l, and/or dehydroepiandrosterone sulphate (DHEAS) >10 mmol/l); LH to FSH ratio >2. (3) At least one ovary examined by ultrasound contained 12 follicles measuring 2–9 mm in diameter and/or increased ovarian volume of at least 10 ml. Thirty healthy women were enrolled as the control group. They had regular ovulatory cycles, no clinical or biochemical signs of raised androgens, and normal ovarian morphology on ultrasound. The health status of the women in the control group was determined by medical history, physical and pelvic examination, and complete blood chemistry. Hormonal investigations were also done if needed. Their normal ovulatory state was confirmed by trans-vaginal ultrasound and plasma progesterone assay during the luteal phase of the cycle. The PCOS and control groups were matched for age (two years) [PCOS: 27.22 0.60 years, controls: 26.05 1.11 years; p = 0.511] and BMI (10%) [PCOS: 29.35 7.25 kg/m2, controls: 2 28.31 6.97 kg/m ; p = 0.835]. In each group, the percentage of overweight and obese women was about 70% (21/30 women). Exclusion criteria for all the subjects included pregnancy, hypothyroidism, hyperprolactinemia, Cushing’s syndrome, congenital adrenal hyperplasia, current or previous (within the last six months) use of oral contraceptives, glucocorticoids, antiandrogens, ovulation induction agents, antidiabetic and anti-obesity drugs or other hormonal drugs. None of the patients was affected by neoplastic, metabolic and cardiovascular disorder or other concurrent medical illness such as diabetes, renal disease, and hepatic disorders. All the subjects were non-smokers and had normal physical activity. 3. Anthropometric measurements For each woman weight and height were measured to calculate the BMI (weight in kg divided by height in m2). Waist circumference (the narrowest circumference between the lower costal margins and the iliac crest) and hip circumference (the maximum circumference at the level of the femoral trochanters) were also measured in the standing position to calculate the waist– hip ratio (WHR).
3.1. Biochemical measurements Blood was sampled in the morning between 08.00 h and 09.00 h after an overnight fast. During the early follicular phase (2nd or 3rd day), 5 ml of blood was drawn in plain red-top tubes for the determination of LH, FSH, E2 and insulin levels in the serum. Two millilitres of blood were collected into chilled tubes containing 1.2 mg EDTA and aprotinin (500 KIU/ml; Trasylol; Bayer Corp., Leverkusen, Germany) for total ghrelin levels and 2 ml were drawn in fluoride tubes (gray top) for glucose estimation. Following fasting blood sample collection, subjects consumed a standard mixed breakfast of about 527 kcal during 15 min. The meal consisted of 50 g white bread, 33 g black bread, 18 g margarine, 30 g cheese, 9 g jam and 200 ml of 0.5% fat milk (24.1% fat, 54.4% carbohydrate, 21.5% protein). Two millilitres of blood were collected 60 min after the meal ingestion for determination of postprandial ghrelin levels. On day 20 or 21 of the menstrual cycle, 5 ml blood samples were obtained for determination of 17OHprogesterone (17OH-P), testosterone (T), androstenedione (A) and sex-hormone binding globulin (SHBG) levels. All blood samples for each woman were immediately centrifuged, and the serum was stored at 80 8C until analysis. The basal serum levels of LH, FSH, E2, P, T, SHBG and insulin were estimated using the electrochemiluminescence immunoassay ‘‘ECLlA’’ on a Roche Elecsys 1010/2010 and MODULAR ANALYTICS E170 (Elecsys module) immunoassay analyzers (Roche Diagnostic, Mannheim, Germany). Androstenedione levels were estimated by using the Gamma Coat Androstenedione Radioimmunoassay Kit (DiaSorin Inc, Stillwater, USA). Total ghrelin levels were measured in duplicate using a commercial ghrelin (human) enzyme immunoassay kit (EIA) from (Phoenix Pharmaceuticals, Inc., Belmont, CA, USA), with a lower limit of detection of 0.06 ng/ ml. Plasma glucose levels were determined by the glucose oxidase method on a Beckman Glucose Analyzer (Fullerton, CA). 3.2. Statistical analysis Data are presented as mean SEM. The comparisons between PCOS patients and their matched control were done using independent T- and ANOVA tests. The Pearson correlation coefficient (r) was used to find out the correlation between ghrelin and other variables. A probability value p 0.05 was considered statistically significant. All analyses were run using the StatView program for Windows (version 8.0; SAS). In this study, due to the relatively small sample size, the statistical power in detecting the minimum difference = 6.1% and that of detecting the maximum difference = 50% adjusting a error at 0.05. 4. Results Clinical, hormonal and metabolic features of women with PCOS and controls are summarized in Table 1. In the PCOS group, WHR, LH, LH/FSH ratio, E2, 17OH-P, T, A, SHBG and serum insulin levels were significantly different in comparison with the control group (p < 0.05). No difference in fasting ghrelin levels was detected between the PCOS and control groups (p = 0.487). One-hour postprandial, ghrelin levels were decreasing in both groups, but the difference between the two groups was not statistically significant (p = 0.378) as shown in Fig. 1. Correlation of fasting ghrelin levels to the clinical, hormonal and metabolic features of women with PCOS and controls is presented in Table 2. In both PCOS and control groups, there was a significant inverse correlation between fasting ghrelin levels and BMI (PCOS: r = 0.529; p = 0.009, controls: r = 0.670; p = 0.005). In both groups, fasting ghrelin levels did not correlate with either age, WHR, LH, FSH, LH/FSH ratio, E2, 17OH-P, T, A, SHBG, 1-hour postprandial ghrelin, fasting glucose and insulin levels (p = NS).
M.H. Daghestani et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 155 (2011) 65–68 Table 1 Clinical, hormonal and metabolic features of women with PCOS and controls.
Age (yr) BMI (kg/m2) WHR LH (IU/l) FSH (IU/l) LH/FSH ratio E2 (pmol/l) P (nmol/l) T (nmol/l) A (nmol/l) SHBG (nmol/l) Fasting ghrelin (ng/ml) 1 h ghrelin (ng/ml) Glucose (mmol/l) Insulin (pmol/l)
PCOS (n = 30)
Controls (n = 30)
p
Sig.
27.22 0.60 29.35 7.25 0.84 0.02 13.64 1.21 5.22 0.46 2.81 0.19 201.07 14.13 3.23 0.46 2.69 0.16 0.23 0.10 27.41 2.64 0.42 0.02 0.34 0.01 5.10 0.09 97.46 25.86
26.05 1.11 28.31 6.97 0.77 0.01 4.65 0.22 5.09 0.29 0.98 0.06 134.97 7.72 26.12 3.02 1.20 0.11 0.04 0.00 45.76 3.04 0.43 0.02 0.32 0.01 4.73 0.09 73.73 5.94
0.511 0.835 <0.001 <0.001 0.576 <0.001 0.001 <0.001 <0.001 0.021 0.015 0.487 0.378 0.140 0.040
NS NS S S NS S S S S S S NS NS NS S
NS = non-significant = p > 0.05. S = significant = p 0.05.
[()TD$FIG]
Fig. 1. Fasting and 1-hour postprandial ghrelin levels in women with PCOS and controls.
Table 2 Correlation of fasting ghrelin levels to the clinical, hormonal and metabolic features of women with PCOS and controls. PCOS (n = 30)
Age (yr) BMI (kg/m2) WHR LH (IU/l) FSH (IU/l) LH/FSH ratio E2 (pmol/l) P (nmol/l) T (nmol/l) A (nmol/l) SHBG (nmol/l) 1 h ghrelin (ng/ml) Glucose (mmol/l) Insulin (pmol/l)
0.057 0.529 0.044 0.034 0.124 0.086 0.122 0.088 0.189 0.205 0.024 0.751 0.038 0.144
NS = non-significant = p > 0.05 S = significant = p 0.05
Table 3 Correlation of 1-hour postprandial ghrelin levels to the clinical hormonal and metabolic features of women with PCOS and controls. PCOS (n = 30) r Age (yr) BMI (kg/m2) WHR LH (IU/l) FSH (IU/l) LH/FSH ratio E2 (pmol/l) P (nmol/l) T (nmol/l) A (nmol/l) SHBG (nmol/l) Fasting ghrelin (ng/ml) Glucose (mmol/l) Insulin (pmol/l)
0.045 0.421 0.035 0.027 0.099 0.068 0.097 0.070 0.150 0.163 0.019 0.751 0.030 0.115
Controls (n = 30)
p
Sig.
0.401 0.007 0.115 0.694 0.479 0.497 0.426 0.296 0.288 0.211 0.537 0.072 0.556 0.427
NS S NS NS NS NS NS NS NS NS NS NS NS NS
r 0.086 0.491 0.170 0.123 0.116 0.095 0.279 0.053 0.113 0.095 0.230 0.536 0.185 0.243
p
Sig.
0.450 0.004 0.4539 0.352 0.110 0.348 0.070 0.438 0.303 0.261 0.146 0.308 0.204 0.053
NS S NS NS NS NS NS NS NS NS NS NS NS NS
NS = non-significant = p > 0.05. S = significant = p 0.05.
Correlation of 1-hour postprandial ghrelin levels to the clinical, hormonal and metabolic features of women with PCOS and controls is presented in Table 3. In both PCOS and control groups, there was a significant inverse correlation between 1-hour postprandial ghrelin levels and BMI (PCOS: r = 0.421; p = 0.007, controls: r = 0.491; p = 0.004). In both groups, 1-hour postprandial ghrelin levels did not correlate with age, WHR, LH, FSH, LH/FSH ratio, E2, 17OH-P, T, A, SHBG, fasting ghrelin, fasting glucose and insulin levels (p = NS).
r
67
Controls (n = 30)
p
Sig.
0.504 0.009 0.145 0.873 0.602 0.625 0.536 0.372 0.362 0.265 0.675 0.072 0.699 0.537
NS S NS NS NS NS NS NS NS NS NS NS NS NS
r 0.117 0.670 0.232 0.168 0.158 0.129 0.381 0.072 0.154 0.130 0.313 0.536 0.252 0.332
p
Sig.
0.613 0.005 0.619 0.480 0.150 0.475 0.096 0.597 0.413 0.356 0.199 0.308 0.278 0.059
NS S NS NS NS NS NS NS NS NS NS NS NS NS
5. Comments This study investigated fasting and 1-hour postprandial ghrelin levels in patients with PCOS in comparison to those in control women matched for age and BMI. In this regard this study was different from most other reports, which have not attempted to study the postprandial ghrelin level. We estimated the total ghrelin level, which is composed of active (acyl-) and inactive (des-acyl) ghrelin. In a recent report the active/total ghrelin ratio was reported to be significantly lower in PCOS patients compared to a control group but the levels of active and total ghrelin, though lower, did not reach significance [24]. Both active and inactive ghrelin are found in the circulation, where the latter is the more abundant form. Some actions of inactive ghrelin oppose those of the active form, suggesting that the ratio of active to inactive ghrelin may play a critical role in the overall physiological response. The majority of the studies on ghrelin, however, has investigated total ghrelin, in line with this present study, and there is a discrepancy in the reported results. In the present investigation, the fasting and 1-hour postprandial ghrelin levels in patients with PCOS are comparable to those of the control group and no correlation was demonstrated between circulating total ghrelin levels and the hormonal pattern characteristic of PCOS, suggesting that ghrelin secretion is not associated with increased circulating levels of LH, 17b-estradiol, testosterone, androstenedione, or SHBG. In contrast to our findings, some previous studies [13–16] have reported decreased fasting ghrelin levels in PCOS women compared to controls, though others have failed to show any variation [17–22]. In contrast to our findings, Glintborg et al. [13] concluded that ghrelin levels were decreased in hirsute PCOS patients and showed a significant negative correlation with testosterone, independent of body composition. Micic et al. [14] demonstrated that women with PCOS had lower fasting ghrelin levels and decreased insulin sensitivity independently of their BMI, compared to controls. On the other hand, Was´ko et al. [19] concluded that PCOS women have higher ghrelin levels compared to controls. A PCOS group showed negative correlation between BMI and insulin levels. A correlation between ghrelin and SHBG was also observed. This discrepancy of results may be explained by the relatively small sample size in our study, or may be related to certain genetic or social factors that need further investigation. In agreement with our results, however, Schofl et al. [17] found that in insulin-sensitive PCOS women, ghrelin levels were similar to those in healthy controls; suggesting that ghrelin could be
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linked to the degree of insulin resistance in PCOS. Ghrelin levels did not correlate to any of the parameters of hyperandrogenemia or to the LH/FSH ratio in PCOS. Orio et al. [18] demonstrated that in women with PCOS, ghrelin concentrations are not different from those of weight-matched controls. There was no relationship between circulating ghrelin and the abnormal hormonal pattern of PCOS. In comparison to matched controls, PCOS women had greater serum insulin levels, confirming previous reports [25]. In our study, ghrelin did not significantly correlate with fasting insulin levels, suggesting that increased insulin production of PCOS women does not adversely affect ghrelin secretion. This result is apparently in contrast with observations demonstrating a possible negative feedback regulation between insulin and ghrelin in humans. An inverse relationship has been reported between the circulating levels of the two hormones [23]. It has also been shown that ghrelin administration to healthy humans at pharmacological doses reduces insulin secretion [26] and, conversely, that insulin administration in high doses is capable of reducing ghrelin secretion [27]. Our results demonstrate that, despite different circulating insulin levels, ghrelin levels were similar in PCOS and control groups, suggesting that modest changes in fasting insulin levels are not sufficient to modulate ghrelin secretion. In both groups, PCOS as well as control, fasting and 1-hour postprandial ghrelin levels were significantly and inversely correlated with BMI. The result indicates that in women with PCOS, the abundance of fat mass reduces ghrelin levels, in accordance with observations in obese subjects [23]. The inverse correlation between ghrelin levels and BMI, as well as the increase of ghrelin levels after weight loss [28], supports the hypothesis that levels of ghrelin modulate food intake through both a short-acting feedback related to meal consumption [11] and a long-term feedback related to body weight and composition [12]. In conclusion, we have reported fasting and postprandial ghrelin levels in PCOS women. Our data demonstrate that in Saudi women with PCOS, ghrelin levels are not different from those of age- and BMI-matched controls and are inversely correlated with BMI. There is no relationship between circulating ghrelin levels and the abnormal hormonal pattern of the PCOS. Acknowledgments This work Grant No. (08-MED 604-2) was supported thankfully by NPST Program at King Saud University to Dr. Maha Daghestani. We thank all the subjects for their cooperation and participation in the study. We would also like to thank all the participants (researchers, technicians and nurses) for their notable contribution. References [1] Setji TL, Brown AJ. Polycystic ovary syndrome: diagnosis and treatment. Am J Med 2007;120:128–32. [2] 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;19:41–7.
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