144 Article
Authors
L. Xu1, Y. Shi2, J. Gu1, Y. Wang1, L. Wang3, L. You2, X. Qi1, Y. Ye1, Z. Chen2
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ gene ● ▶ ghrelin ● ▶ polycystic ovary syndrome ● ▶ single nucleotide ● polymorphisms
Abstract
received 06.10.2013 first decision 06.12.2013 accepted 17.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1367024 Exp Clin Endocrinol Diabetes 2014; 122: 144–148 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence Z. Chen Shandong Provincial Key Laboratory of Reproductive Medicine Center for Reproductive Medicine Provincial Hospital Affiliated to Shandong University 324 Jing-5-wei-7 Road Jinan People’s Republic of China Tel.: + 86/531/87068 226 Fax: + 86/531/87068 226 [email protected]
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Objective: To investigate the association between 2 single nucleotide polymorphisms (SNP501A/C and 604 G/A) in the promoter of the ghrelin gene and the hormonal and metabolic phenotypes of polycystic ovary syndrome (PCOS) in a Chinese population. Materials and methods: 285 patients with PCOS and 260 healthy controls were selected for a prospective, case-control study at Shandong Provincial Hospital, Jinan, China. All subjects underwent genotype analysis of the 2 single nucleotide polymorphisms of the ghrelin gene. Measurements were also taken of blood lipids, glucose, and hormone levels, and calculations of
Introduction
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Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age, and is characterized by oligomenorrhoea or amenorrhoea, hyperandrogenism, infertility, obesity, multiple small subcapsular cystic follicles in the ovary and insulin resistance [1–3]. The disorder has also been reported to be associated with metabolic syndromes such as type 2 diabetes mellitus (T2DM) and dyslipidemia [4]. PCOS is thought to be a multifactorial condition with contributing genetic, endocrine, and environmental factors. The increased risk of T2DM and obesity in women with PCOS suggests that those affected share etiologic determinants, thus prompting an investigation into the relationship between PCOS and T2DM-related genes [5] or obesity genes. Recently, ghrelin was found to associate not only with T2DM [6], but also with obesity [7], and even with insulin resistance [8]. Ghrelin is a 28-amino-acid peptide with an n-octanoylated serine-3 residue primarily expressed in the X/A-
Xu L et al. Association between ghrelin gene … Exp Clin Endocrinol Diabetes 2014; 122: 144–148
body mass index (BMI) and waist-to-hip ratio (WHR) were performed to detect hormonal and metabolic phenotypes. Results: No significant differences in polymorphism genotypes were found between PCOS patients and healthy controls. However, the frequency of the -501 A/C A allele was significantly higher in the PCOS group than in the control group. PCOS -501 A/C A carriers had significantly higher BMI and WHR than PCOS women with the CC genotype. -604 G/A polymorphisms were not associated with clinical or biochemical characteristics of PCOS. Conclusions: The -501 A/C polymorphism of the ghrelin gene is associated with metabolic features of PCOS in a Chinese population.
like oxyntic cells of the stomach, as well as in the hypothalamus [9] and pancreas [10]. The peptide stimulates growth hormone release [11] and orexigenic (appetite-stimulating) activity [12], which also be demonstrated to influence LH secretion [13]. Plasma ghrelin levels of PCOS women are lower than normal and are highly correlated to the degree of insulin resistance [14, 15], indicating that ghrelin may be involved in the pathogenesis of PCOS. Then, it can be hypothesized that ghrelin is a candidate gene for the pathogenesis of PCOS. The human ghrelin gene is located at the chromosomal locus 3p26-p25, and the preprohormone is encoded by 5 exons. Several single nucleotide polymorphisms (SNPs) in the coding region for preproghrelin have been previously described, although Arg51Gln(346 G > A) (in the coding region of the preproghrelin gene) and Leu72Met (408 C > A) (in exon 2) have no correlation with PCOS [16], associations have been found between polymorphisms in the promoter region of the ghrelin gene with body mass index and waist circumference [17], which are often
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Association between Ghrelin Gene Variations, Body Mass Index, and Waist-to-hip Ratio in Patients with Polycystic Ovary Syndrome
seen in PCOS women. The purpose of this study was to determine the relationship between the SNPs rs26802 and rs27647, in the promoter region of the ghrelin gene, and the pathogenesis of PCOS. In addition, we determined whether the ghrelin gene plays a role in endocrine and clinical traits of PCOS patients, which may further our understanding of the susceptibility of ghrelin gene variation in the development of PCOS.
insulin secretion were calculated using the software HOMA Calculator v2.2 at http://www.dtu.ox.ac.uk/homa/. The insulin index (ISI) were also calculated. The study protocols were reviewed and approved by the Ethics Committees for Human Research at Shandong Provincial Hospital, that complied with the recommendations of the Declaration of Helsinki (British Medical Journal, 1964, ii, 177). And informed consent was obtained from all subjects.
Materials and Methods
DNA preparation and SNP genotyping
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Subjects 285 PCOS patients aged between 22 and 42 years were randomly recruited between July 1, 2007 and July 1, 2010 from the reproductive centre at the Shandong Provincial Hospital affiliated to Shandong University. All patients fulfilled the revised PCOS diagnostic criteria, announced in the 2003 American Society for Reproductive Medicine/European Society for Reproduction and Embryology (ASRM/ESHRE) Rotterdam consensus. All symptoms of the revised diagnostic criteria were shared by 121 patients, including oligomenorrhoea or amenorrhoea, hyperandrogenism and polycystic ovaries (PCO), while 132 patients in the PCOS group had oligomenorrhoea or amenorrhoea and PCO, 17 had oligomenorrhoea or amenorrhoea and hyperandrogenism and 15 had hyperandrogenism and PCO. All the patients are excluded the diseases of hyperprolactinemia or thyroid dysfunction diseases or nonclassic congenital adrenal hyperplasia by clinical and laboratory examinations. The control population consisted of 260 age-matched women with some reasons which result in nonpregnancy such as fallopian tube and male factors, but with normal cycles, they were also recruited during this period and evaluated consecutively. All were evaluated carefully and none showed any of the PCOS symptoms mentioned above. They had normal hormonal status, including sex hormones, adrenal coryex hormones, thyroid hormones, they also showed no sign of galactorrhoea or thyroid dysfunction, and had no personal or family history of diabetes. No participants had used hormonal preparations, including oral contraceptives, for at least 3 months before enrolment in the study. Women in the control group were recruited from the same area as the patients with PCOS. All the subjects in the study were Chinese women. After providing their medical history and undergoing a physical examination by the same doctor, including measurement of blood pressure, weight and height, and abdominal and hip circumferences, blood sampling was performed on days 3–5 of the menstrual cycle in a fasting state in all subjects. Blood samples for molecular genetic studies were collected in tubes containing ethylenediaminetetraacetic acid as an anticoagulant and stored at − 20°C. Other samples were immediately chilled on ice, centrifuged, and frozen at − 80°C until assayed. Levels of hormones (FSH, LH, estradiol (E2), prolactin (PRL) and testosterone (T) hormone, blood lipids (low density lipoprotein cholesterol and triglycerides) and glucose were measured. The normal range of each sex hormone: FSH: 1.5–10 IU/L; LH:2.1– 10.8 IU/L; E2:24–114 pg/ML; PRL:5–25 ng/ML T: 2–15 IU/L; Body mass index (BMI) and the waist-to-hip ratio (WHR) were calculated using the following formulae: BMI = weight (kg)/height (m)2; WHR = abdominal circumference (cm)/hip circumference (cm). The homeostasis model assessment for insulin resistance (HOMA-IR) and homeostasis model assessment score for B-cell
DNA was extracted from human leukocyte nuclei isolated from whole blood (QIAamp DNA Blood Kit, QIAGEN). SNPs were detected by polymerase chain reaction-based restriction fragment length polymorphism analysis (PCR-RFLP) using the following primers: 1) 501 A/C forward primer: 5’-AAAACAAACGCC AGTCATCC-3’, reverse primer: 5’-GTCTTCCAGCCAGACAGTCC-3’; 2) 604 G/A forward primer: 5’-CACAGCAACAAAGCTGCACC-3’, reverse primer: 5’-AAGTCCAGCCAGAGCATGCC-3’. PCR was carried out in a 25 μl volume containing 0.2 μg blood leukocyte genomic DNA, 0.4 mmol/L primers, 0.2 mmol/L each of deoxyATP, -GTP, -CTP, and -TTP, 2 mM MgCl2, and 0.5 μl Taq DNA polymerase. PCR involved an initial denaturation step at 95 °C for 10 min, then 10 cycles of 95 °C for 30 s, with annealing steps of 30 s descending by 1 °C per cycle from 68 °C to 58 °C, followed by 30 cycles of denaturation at 95 °C for 30 s, primer annealing at 58 °C for 30 s and primer extension at 72 °C for 45 s, and a final extension step for 7 min at 72 °C in a GeneAmp PCR system 9 600 thermal-cycler (Perkin Elmer, United States). This procedure generated a 205 bp fragment for rs26802 501 A/C and a 912 bp fragment for rs27647 604 G/A, which were resolved by digesting with specific restriction enzymes (New England Biolabs (Beijing), Ltd) for 4 h. For 501 A/C, digestion with Scr FI at 37 °C resulted in fragments of 146, 43 and 16 bp for genotype AA, fragments of 146, 99, 47, 43 and 16 bp for genotype AC, and fragments of 99, 47, 43 and 16 bp for genotype CC. For 604 G/A, digestion with Dra I at 37 °C resulted in fragments of 664 and 248 bp for genotype AA, fragments of 912, 664 and 248 bp for genotype AG, and a 912 bp fragment for genotype GG. The fragments were separated on a 1 % agarose gel for 501 A > C and 2 % agarose gel for 604 G > A, then visualized under ultraviolet light after staining with ethidium bromide.
Statistical analysis All clinical data are expressed as means ± SD. The clinical and biochemical variable differences between PCOS patients and controls were evaluated by an independent samples t-test. The Hardy-Weinberg distribution of genotypes in the PCOS and control groups was assessed. Categorical data were expressed as frequencies and percentages. Statistical analysis of allele and genotype frequencies between women with PCOS and controls was compared using Pearson’s χ2 test. One-way analysis of variance followed by the least significant differences test for posthoc comparisons was used to analyse clinical data in different genotypes. Univariate general linear model analyses were used to evaluate the effects of ghrelin genotypes on BMI, introducing WHR as a covariate. All analyses were performed using the Statistical Package for Social Sciences software (version 13.0; SPSS Inc., Chicago, IL, USA). P < 0.05 was accepted as statistically significant.
Xu L et al. Association between ghrelin gene … Exp Clin Endocrinol Diabetes 2014; 122: 144–148
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Article 145
146 Article
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Clinical and biochemical characteristics of PCOS and control ▶ Table 1. There was no significant differwomen are shown in ● ence in age between PCOS patients and controls. However, BMI, WHR, LH, and T levels were significantly higher in the PCOS group than in the control group. In addition, FINS and HOMA-IR were higher while ISI was lower in PCOS patients compared with controls, these differences were statistically significant. We identified SNPS -501 A/C (rs26802) and -604 G/A (rs27647) and showed that both were in Hardy-Weinberg equilibrium in all data sets. All samples were double genotyped with 100 % concordance. No significant differences were found between PCOS and normal subjects regarding genotype distributions of ▶ Table 2). -604 G/A and -501 A/C (● SNP -604 G/A was shown to have no impact on hormonal or metabolic phenotypes of PCOS patients, as BMI, WHR, and FSH, LH, T, plasma glucose and insulin levels were similar between differ▶ Table 3). However, the SNP ent genotypes of PCOS subjects (● -501 A/C was associated with some metabolic variations in PCOS women. The effects of the different genotypes on clinical and bio▶ Table 4. chemical variables in women with PCOS are listed in ● Statistically significant differences in BMI and WHR were found in the SNP -501 A/C genotype when AA and AC carriers were compared with CC mutant homozygotes. The difference in BMI and WHR between A carriers after combining AA and AC (A carriers) and CC homozygotes also showed statistical significance (P = 0.03, 0.04, respectively). Association studies of other clinical parameters were also carried out with different genotypes of -501 A/C, but no significant differences were found. Table 1 Clinical, endocrine. and metabolic characteristics.
Age (years) BMI (kg/m2) Waist hip ratio FSH (IU/L) LH (IU/L) T (nmol/L) FBG (mmol/L) FINS (mIU/L) ISI HOMA-IR2 HOMAβ % CHO (mmol/L) TG (mmol/L) LDL (mmol/L)
PCOS (n = 285)
Controls (n = 260)
P-value
28.79 ± 3.41 25.87 ± 4.91 0.89 ± 0.05 6.94 ± 1.98 9.88 ± 7.79 2.36 ± 0.92 4.90 ± 0.87 8.45 ± 4.97 0.037 ± 0.029 1.87 ± 1.66 26.21 ± 59.54 4.99 ± 0.56 0.95 ± 0.53 2.79 ± 0.92
29.46 ± 3.35 22.02 ± 3.29 0.78 ± 0.04 6.67 ± 2.01 4.35 ± 3.56 1.15 ± 0.67 4.88 ± 0.95 6.26 ± 4.57 0.050 ± 0.026 1.39 ± 1.75 87.45 ± 32.67 4.72 ± 0.61 0.84 ± 0.62 2.46 ± 1.13
0.082 < 0.001 < 0.001 0.076 < 0.001 < 0.001 0.826 0.041 0.007 0.038 0.312 0.738 0.691 0.663
Values are means ± SD BMI: body mass index; FSH: follicle-stimulating hormone; LH: luteinizing hormone; T: testosterone; FBG: fasting blood glucose; FINS: fasting insulin; ISI: insulin index; HOMA-IR2: insulin resistance index; HOMA β %: β-cell function index; CHO: cholesterol; TG: triglycerides; LDL: low density lipoprotein cholesterol
Because the SNP -501 A/C was associated with WHR and BMI, a univariate general linear model analysis, introducing WHR as a covariate in the model, was performed. The results showed that SNP-501 A/C alleles significantly influenced BMI (P = 0.032). All same clinical parameters as PCOS women were tested in an association study of the control group with different genotypes of -501 A/C and 604 G/A, but no significant differences were found (P > 0.05).
Discussion
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The human ghrelin gene is located on chromosome 3p25–26 and consists of 4 exons and 3 introns [18]. In this study, we selected 2 SNPs within its promoter region, and investigated whether each SNP genotype was associated with PCOS. We showed that the SNP genotypes were similar between PCOS patients and controls, indicating that ghrelin does not involve in the pathogenesis of PCOS directly. However, the presence of the 501 A/C polymorphism in the ghrelin gene promoter appeared to be associated with some metabolic characteristics of PCOS. Significant differences were found in BMI and WHR between SNP -501 A/C genotype AA and AC carriers and CC mutant homozygotes. After AA and AC genotypes were combined, differences in BMI and WHR between A carriers and C/C homozygotes remained statistically significant. The same results were not obtained in the control group. Obesity is often associated with PCOS [19] and plays a pivotal role in the development and/or maintenance of the syndrome. In addition, BMI and WHR were previously shown to positively correlate with free androgen index and HOMA-IR [20]. Thus, while the common 501 A > C polymorphism does not affect ovarian and adrenal androgen levels or HOMA-IR, it may indirectly influence PCOS pathogenesis by controlling some metabolic phenotypes, BMI and WHR. However, further studies are necessary to confirm this and analysis how the 501 A > C polymorphism influence PCOS. No significant differences were found in the other clinical, metabolic and endocrine parameters evaluated in the present study to determine the association between PCOS and controls with different -501 A/C and -604 G/A genotypes. However, significant differences were observed, including higher HOMA-IR and FINS but lower ISI in PCOS patients compared with controls. Other ghrelin gene SNPs have previously been shown to correlate with insulin resistance [21, 22] in T2DM and obese subjects. Recently we discovered that there was no association between Arg51Gln and Leu72Met polymorphisms and insulin resistance in PCOS [16], and the present study found no correlation between SNPs 501 A/C and 604 G/A and insulin resistance in PCOS. This indicates that SNPs 501 A/C and 604 G/A are not involved in PCOS through influencing insulin and glucose metabolisms. We also found that PCOS subjects had higher blood testosterone and higher LH levels. Androgens are independent modulators of
Table 2 Allele and genotype frequencies of ghrelin gene polymorphisms. SNP rs26802 (501 A/C) rs27647 (604 G/A)
Group
N
PCOS Control
285 260
PCOS Control
285 260
Genotype AA 130 (45) 101 (39) AA 216 (76) 195 (75)
AC 121 (42) 97 (37) AG 55 (19) 52 (20)
P-value CC 34 (13) 62 (22) GG 14 (5) 13 (5)
0.06
0.79
Xu L et al. Association between ghrelin gene … Exp Clin Endocrinol Diabetes 2014; 122: 144–148
Allele A 381 (66.3 %) 299 (56.9 %) A 487 (85.44 %) 442 (85.00 %)
P-value C 189 (32.2 %) 221 (43.1 %) G 83 (14.56 %) 78 (15.00 %)
0.07
0.86
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Results
Article 147
P-value
Genotype
AA (n = 216)
AG n = 55)
GG (n = 14)
Age (years) BMI (kg/m2) FSH (IU/L) LH (IU/L) T (nmol/L) FBG (mmol/L) FINS (mIU/L) ISI HOMA-IR2 HOMAβ % CHO (mmol/L) LDL (mmol/L) TG (mmol/L)
28.36 ± 3.15 25.12 ± 3.75 6.38 ± 1.54 9.88 ± 6.13 2.52 ± 0.35 5.05 ± 1.33 9.12 ± 3.44 0.028 ± 0.021 2.01 ± 1.13 89.32 ± 76.17 4.55 ± 0.73 2.58 ± 1.32 0.77 ± 0.49
28.55 ± 2.98 25.46 ± 4.37 6.27 ± 1.65 9.76 ± 5,87 2.51 ± 0.28 4.96 ± 1.77 8.99 ± 2.94 0.033 ± 0.029 1.89 ± 1.36 87.33 ± 71.09 4.69 ± 0.12 2.63 ± 1.29 0.86 ± 0.75
27.96 ± 3.14 24.96 ± 3.86 6.53 ± 1.49 9.95 ± 5.46 2.64 ± 0.53 5.11 ± 0.81 9.03 ± 4.31 0.029 ± 0.025 1.96 ± 1.38 84.29 ± 68.39 4.48 ± 0.26 2.54 ± 1.47 0.91 ± 0.84
0.78 0.43 0.81 0.67 0.72 0.46 0.59 0.22 0.25 0.37 0.23 0.61 0.18
Table 3 Comparison of characteristics in women with PCOS according to -604 G/A genotype.
Values are means ± SD. The P values are a global comparison
Table 4 Comparison of characteristics in women with PCOS according to -501 A/C genotype. -501 A/C Genotype
AA (n = 130)
Age (y) BMI (kg/m2) WHR FSH (IU/L) LH (IU/L) T (nmol/L) FBG (mmol/L) FINS (mU/L) ISI HOMA-IR2 HOMAβ % CHO (mmol/L) LDL (mmol/L) TG (mmol/L)
28.46 ± 2.98 29.58 ± 3.42 a 0.93 ± 0.03 a 6.35 ± 1.64 10.04 ± 5.89 2.49 ± 0.78 5.18 ± 1.62 8.96 ± 4.46 0.029 ± 0.012 2.06 ± 1.02 101.23 ± 56.18 4.77 ± 0.64 2.85 ± 1.03 0.78 ± 0.62
AC(n = 121) 28.54 ± 3.12 29.71 ± 3.65 a 0.92 ± 0.04 a 6.67 ± 1.23 9.96 ± 6.25 2.50 ± 0.69 5.06 ± 1.21 8.78 ± 5.21 0.032 ± 0.036 1.94 ± 1.03 93.16 ± 46.32 4.69 ± 1.05 2.67 ± 1.22 0.98 ± 1.53
t-value
CC (n = 34) 28.38 ± 3.23 22.01 ± 2.45 0.71 ± 0.06 6.48 ± 2.05 9.47 ± 5.74 2.56 ± 0.63 4.98 ± 1.72 9.01 ± 3.57 0.034 ± 0.029 1.97 ± 1.21 99.57 ± 108.12 4.38 ± 0.87 2.74 ± 1.65 0.85 ± 0.72
0.78 0.02 0.03 0.56 0.43 0.64 0.59 0.76 0.63 0.51 0.45 0.36 0.69 0.21
Combined- 501 A > C AA and AC (n = 251)
CC (n = 34)
28.50 ± 3.01 29.64 ± 3.53 0.93 ± 0.04 6.47 ± 1.43 10.01 ± 5.79 2.49 ± 0.77 5.12 ± 1.43 8.85 ± 4.96 0.030 ± 0.022 2.01 ± 1.02 98.35 ± 49.78 4.72 ± 0.91 2.76 ± 1.19 0.87 ± 1.12
28.38 ± 3.23 22.01 ± 2.45 0.71 ± 0.06 6.48 ± 2.05 9.47 ± 5.74 2.56 ± 0.63 4.98 ± 1.72 9.01 ± 3.57 0.034 ± 0.029 1.97 ± 1.21 99.57 ± 108.12 4.38 ± 0.87 2.74 ± 1.65 0.85 ± 0.72
t-value 0.74 0.03 0.04 0.76 0.23 0.63 0.52 0.64 0.66 0.52 0.57 0.34 0.71 0.46
Values are means ± SD. The P values are a global comparison a
P < .05 vs. women with PCOS and with the CC genotype
circulating ghrelin levels [23, 24], so it is conceivable that there is a close relationship between the ghrelin gene and endocrine disorders in PCOS women. However, when we compared different genotypes of the two SNPs in PCOS subjects, we found no difference in testosterone levels. In further analysis, the levels of FSH, LH, E2, and PRL in PCOS patients of different genotypes were also similar. Thus the -501 A/C and 604 G/A polymorphisms appear not to be associated with endocrine disorders of PCOS. To the best of our knowledge, this study, which includes a relatively large number of Chinese women with PCOS, is the first of its kind to provide data concerning -501 A/C and 604 G/A polymorphisms in the ghrelin gene promoter and PCOS. Although we have not conducted a comprehensive analysis of all variants in and around the ghrelin gene and cannot exclude the role of other variants outside this region, the available evidence suggests that at least one variant of the ghrelin gene confers susceptibility to high BMI and WHR in women with PCOS.
Acknowledgements
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We thank the staff of the laboratory and the reproductive center in Shandong Provincial Hospital very much for work
performed to support this study. This work was supported by grants from the National Natural Science Foundation (China, Canada). This work was supported by the National Basic Research Program of China (973 Program-2006CB944004) and the National High-Technology Research and Development Program of China (863Program-2006AA02Z4A4). Natural Science Foundation of China (81100554), Shandong Provincial Natural Science Foundation, China (BS2012YY003), the Scientific and Technical Development Project of Department of health of Shandong Province (2011QZ007) and the Scientific and Technical Development Project of Qingdao (12-1-4-20-jch and No. 2012-1-3-2-(1)-nsh).
Conflict of interest: None. Affiliations Obstetrics and Gynecology Department, The Affiliated Hospital of Qingdao University Medical College, Qingdao, People’s Republic of China 2 Shandong Provincial Key Laboratory of Reproductive Medicine, Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, Jinan, People’s Republic of China 3 Central Laboratory, Provincial Hospital Affiliated to Shandong University, Jinan, People’s Republic of China 1
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604 G/A
References 1 Frank S. Polycystic ovary syndrome. N Engl J Med 1995; 333: 853–861 2 Diamanti KE, Kouli CR, Bergiele AT et al. A survey of the polycystic ovary syndrome in the Greek island of Lesbos: hormonal and metabolic profile. J Clin Endocrinol Metab 1999; 84: 4006–4011 3 Asuncion M, Calvo RM, San Millan JL et al. A prospective study of the prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain. J Clin Endocrinol Metab 2000; 85: 2434–2438 4 Ehrmann DA, Liljenguist DR., Kasza K et al. Prevalence and predictors of the metabolic syndrome in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2006; 91: 48–53 5 Tucci S, Futterweit W, Concepcion ES et al. Evidence for association of polycystic ovary syndrome in Caucasian women with a marker at the insulin receptor gene locus. J Clin Endocrinol Metab 2001; 86: 446–449 6 Miraglia del Giudice E, Santoro N, Cirillo G et al. Molecular screening of the ghrelin gene in Italian obese children: the Leu72Met variant is associated with an earlier onset of obesity. Int J Obes Relat Metab Disord 2004; 28: 447–450 7 Muccioli G, Tschop M, Papotti M et al. Neuroendocrine and peripheral activities of ghrelin: implications in metabolism and obesity. Eur J Pharmacol 2002; 440: 235–254 8 Katsuki A, Urakawa H, Gabazza EC et al. Circulating levels of active ghrelin is associated with abdominal adiposity, hyperinsulinemia and insulin resistance in patients with type 2 diabetes mellitus. Eur J Endocrinol 2004; 151: 546–2553 9 Howard AD, Feighner SD, Cully DF et al. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996; 273: 974–977 10 Date Y, Nakazato M, Hashiguchi S et al. Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion. Diabetes 2002; 51: 124–129 11 Takaya K, Ariyasu H, Kanamoto N et al. Ghrelin strongly stimulates growth hormone release in humans. J Clin Endocrinol Metab 2000; 85: 4908–4911 12 Lawrence CB, Snape AC, Baudoin FM et al. Acute central ghrelin and GH secretagogues induce feeding and activate brain appetite centers. Endocrinology 2002; 143: 155–162
13 Fernandez-Fernandez R, Tena-Sempere M, Aguilar E et al. Ghrelin effects on gonadotropin secretion in male and female rats. Neurosci Lett 2004; 362: 103–107 14 Panidis D, Farmakiotis D, Koliakos G et al. Comparative study of plasma ghrelin levels in women with polycystic ovary syndrome, in hyperandrogenic women and in normal controls. Hum Reprod 2005; 20: 2127–2132 15 Schöfl C, Horn R, Schill T et al. Circulating ghrelin levels in patients with polycystic ovary syndrome. J Clin Endocrinol Metab 2002; 87: 4607–4610 16 Wang K, Wang L, Zhao Y et al. No association of the Arg51Gln and Leu72Met polymorphisms of the ghrelin gene and polycystic ovary syndrome. Hum Reprod 2009; 24: 485–490 17 Vartiainen J, Kesaniemi YA, Ukkola O. Sequencing analysis of ghrelin gene 5’ flanking region: relations between the sequence variants, fasting plasma total ghrelin concentrations, and body mass index. Metabolism 2006; 55: 1420–1425 18 Kojima M, Kangawa K. Ghrelin: Structure and function. Physiol Rev 2005; 85: 495–522 19 Gambineri A, Pelusi C, Vicennati V et al. Obesity and the polycystic ovary syndrome. Int J Obes Relat Metab Disord 2002; 26: 883–896 20 Li X, Lin JF. Clinical features, hormonal profile, and metabolic abnormalities of obese women with obese polycystic ovary syndrome. Zhonghua Yi Xue Za Zhi 2005; 85: 3266–3271 21 Zavarella S, Petrone A, Zampetti S et al. A new variation in the promoter region, the -604 C > T, and the Leu72Met polymorphism of the ghrelin gene are associated with protection to insulin resistance. Int J Obes (Lond) 2008; 32: 663–668 22 Ukkola O, Ravussin E, Jacobson P et al. Mutations in the preproghrelin/ ghrelin gene associated with obesity in humans. J Clin Endocrinol Metab 2001; 86: 3996–3999 23 Pagotto U, Gambineri A, Vicennati V et al. Plasma ghrelin, obesity, and the polycystic ovary syndrome: correlation with insulin resistance and androgen levels. J Clin Endocrinol Metab 2003; 87: 5625–5629 24 Gambineri A, Pagotto U, Tschop M et al. Anti-androgen treatment increases circulating ghrelin levels in obese women with polycystic ovary syndrome. J Endocrinol Invest 2003; 26: 629–634
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148 Article
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Authors
L. Xu1, Y. Shi2, J. Gu1, Y. Wang1, L. Wang3, L. You2, X. Qi1, Y. Ye1, Z. Chen2
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ gene ● ▶ ghrelin ● ▶ polycystic ovary syndrome ● ▶ single nucleotide ● polymorphisms
Abstract
received 06.10.2013 first decision 06.12.2013 accepted 17.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1367024 Exp Clin Endocrinol Diabetes 2014; 122: 144–148 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence Z. Chen Shandong Provincial Key Laboratory of Reproductive Medicine Center for Reproductive Medicine Provincial Hospital Affiliated to Shandong University 324 Jing-5-wei-7 Road Jinan People’s Republic of China Tel.: + 86/531/87068 226 Fax: + 86/531/87068 226 [email protected]
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Objective: To investigate the association between 2 single nucleotide polymorphisms (SNP501A/C and 604 G/A) in the promoter of the ghrelin gene and the hormonal and metabolic phenotypes of polycystic ovary syndrome (PCOS) in a Chinese population. Materials and methods: 285 patients with PCOS and 260 healthy controls were selected for a prospective, case-control study at Shandong Provincial Hospital, Jinan, China. All subjects underwent genotype analysis of the 2 single nucleotide polymorphisms of the ghrelin gene. Measurements were also taken of blood lipids, glucose, and hormone levels, and calculations of
Introduction
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Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age, and is characterized by oligomenorrhoea or amenorrhoea, hyperandrogenism, infertility, obesity, multiple small subcapsular cystic follicles in the ovary and insulin resistance [1–3]. The disorder has also been reported to be associated with metabolic syndromes such as type 2 diabetes mellitus (T2DM) and dyslipidemia [4]. PCOS is thought to be a multifactorial condition with contributing genetic, endocrine, and environmental factors. The increased risk of T2DM and obesity in women with PCOS suggests that those affected share etiologic determinants, thus prompting an investigation into the relationship between PCOS and T2DM-related genes [5] or obesity genes. Recently, ghrelin was found to associate not only with T2DM [6], but also with obesity [7], and even with insulin resistance [8]. Ghrelin is a 28-amino-acid peptide with an n-octanoylated serine-3 residue primarily expressed in the X/A-
Xu L et al. Association between ghrelin gene … Exp Clin Endocrinol Diabetes 2014; 122: 144–148
body mass index (BMI) and waist-to-hip ratio (WHR) were performed to detect hormonal and metabolic phenotypes. Results: No significant differences in polymorphism genotypes were found between PCOS patients and healthy controls. However, the frequency of the -501 A/C A allele was significantly higher in the PCOS group than in the control group. PCOS -501 A/C A carriers had significantly higher BMI and WHR than PCOS women with the CC genotype. -604 G/A polymorphisms were not associated with clinical or biochemical characteristics of PCOS. Conclusions: The -501 A/C polymorphism of the ghrelin gene is associated with metabolic features of PCOS in a Chinese population.
like oxyntic cells of the stomach, as well as in the hypothalamus [9] and pancreas [10]. The peptide stimulates growth hormone release [11] and orexigenic (appetite-stimulating) activity [12], which also be demonstrated to influence LH secretion [13]. Plasma ghrelin levels of PCOS women are lower than normal and are highly correlated to the degree of insulin resistance [14, 15], indicating that ghrelin may be involved in the pathogenesis of PCOS. Then, it can be hypothesized that ghrelin is a candidate gene for the pathogenesis of PCOS. The human ghrelin gene is located at the chromosomal locus 3p26-p25, and the preprohormone is encoded by 5 exons. Several single nucleotide polymorphisms (SNPs) in the coding region for preproghrelin have been previously described, although Arg51Gln(346 G > A) (in the coding region of the preproghrelin gene) and Leu72Met (408 C > A) (in exon 2) have no correlation with PCOS [16], associations have been found between polymorphisms in the promoter region of the ghrelin gene with body mass index and waist circumference [17], which are often
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Association between Ghrelin Gene Variations, Body Mass Index, and Waist-to-hip Ratio in Patients with Polycystic Ovary Syndrome
seen in PCOS women. The purpose of this study was to determine the relationship between the SNPs rs26802 and rs27647, in the promoter region of the ghrelin gene, and the pathogenesis of PCOS. In addition, we determined whether the ghrelin gene plays a role in endocrine and clinical traits of PCOS patients, which may further our understanding of the susceptibility of ghrelin gene variation in the development of PCOS.
insulin secretion were calculated using the software HOMA Calculator v2.2 at http://www.dtu.ox.ac.uk/homa/. The insulin index (ISI) were also calculated. The study protocols were reviewed and approved by the Ethics Committees for Human Research at Shandong Provincial Hospital, that complied with the recommendations of the Declaration of Helsinki (British Medical Journal, 1964, ii, 177). And informed consent was obtained from all subjects.
Materials and Methods
DNA preparation and SNP genotyping
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Subjects 285 PCOS patients aged between 22 and 42 years were randomly recruited between July 1, 2007 and July 1, 2010 from the reproductive centre at the Shandong Provincial Hospital affiliated to Shandong University. All patients fulfilled the revised PCOS diagnostic criteria, announced in the 2003 American Society for Reproductive Medicine/European Society for Reproduction and Embryology (ASRM/ESHRE) Rotterdam consensus. All symptoms of the revised diagnostic criteria were shared by 121 patients, including oligomenorrhoea or amenorrhoea, hyperandrogenism and polycystic ovaries (PCO), while 132 patients in the PCOS group had oligomenorrhoea or amenorrhoea and PCO, 17 had oligomenorrhoea or amenorrhoea and hyperandrogenism and 15 had hyperandrogenism and PCO. All the patients are excluded the diseases of hyperprolactinemia or thyroid dysfunction diseases or nonclassic congenital adrenal hyperplasia by clinical and laboratory examinations. The control population consisted of 260 age-matched women with some reasons which result in nonpregnancy such as fallopian tube and male factors, but with normal cycles, they were also recruited during this period and evaluated consecutively. All were evaluated carefully and none showed any of the PCOS symptoms mentioned above. They had normal hormonal status, including sex hormones, adrenal coryex hormones, thyroid hormones, they also showed no sign of galactorrhoea or thyroid dysfunction, and had no personal or family history of diabetes. No participants had used hormonal preparations, including oral contraceptives, for at least 3 months before enrolment in the study. Women in the control group were recruited from the same area as the patients with PCOS. All the subjects in the study were Chinese women. After providing their medical history and undergoing a physical examination by the same doctor, including measurement of blood pressure, weight and height, and abdominal and hip circumferences, blood sampling was performed on days 3–5 of the menstrual cycle in a fasting state in all subjects. Blood samples for molecular genetic studies were collected in tubes containing ethylenediaminetetraacetic acid as an anticoagulant and stored at − 20°C. Other samples were immediately chilled on ice, centrifuged, and frozen at − 80°C until assayed. Levels of hormones (FSH, LH, estradiol (E2), prolactin (PRL) and testosterone (T) hormone, blood lipids (low density lipoprotein cholesterol and triglycerides) and glucose were measured. The normal range of each sex hormone: FSH: 1.5–10 IU/L; LH:2.1– 10.8 IU/L; E2:24–114 pg/ML; PRL:5–25 ng/ML T: 2–15 IU/L; Body mass index (BMI) and the waist-to-hip ratio (WHR) were calculated using the following formulae: BMI = weight (kg)/height (m)2; WHR = abdominal circumference (cm)/hip circumference (cm). The homeostasis model assessment for insulin resistance (HOMA-IR) and homeostasis model assessment score for B-cell
DNA was extracted from human leukocyte nuclei isolated from whole blood (QIAamp DNA Blood Kit, QIAGEN). SNPs were detected by polymerase chain reaction-based restriction fragment length polymorphism analysis (PCR-RFLP) using the following primers: 1) 501 A/C forward primer: 5’-AAAACAAACGCC AGTCATCC-3’, reverse primer: 5’-GTCTTCCAGCCAGACAGTCC-3’; 2) 604 G/A forward primer: 5’-CACAGCAACAAAGCTGCACC-3’, reverse primer: 5’-AAGTCCAGCCAGAGCATGCC-3’. PCR was carried out in a 25 μl volume containing 0.2 μg blood leukocyte genomic DNA, 0.4 mmol/L primers, 0.2 mmol/L each of deoxyATP, -GTP, -CTP, and -TTP, 2 mM MgCl2, and 0.5 μl Taq DNA polymerase. PCR involved an initial denaturation step at 95 °C for 10 min, then 10 cycles of 95 °C for 30 s, with annealing steps of 30 s descending by 1 °C per cycle from 68 °C to 58 °C, followed by 30 cycles of denaturation at 95 °C for 30 s, primer annealing at 58 °C for 30 s and primer extension at 72 °C for 45 s, and a final extension step for 7 min at 72 °C in a GeneAmp PCR system 9 600 thermal-cycler (Perkin Elmer, United States). This procedure generated a 205 bp fragment for rs26802 501 A/C and a 912 bp fragment for rs27647 604 G/A, which were resolved by digesting with specific restriction enzymes (New England Biolabs (Beijing), Ltd) for 4 h. For 501 A/C, digestion with Scr FI at 37 °C resulted in fragments of 146, 43 and 16 bp for genotype AA, fragments of 146, 99, 47, 43 and 16 bp for genotype AC, and fragments of 99, 47, 43 and 16 bp for genotype CC. For 604 G/A, digestion with Dra I at 37 °C resulted in fragments of 664 and 248 bp for genotype AA, fragments of 912, 664 and 248 bp for genotype AG, and a 912 bp fragment for genotype GG. The fragments were separated on a 1 % agarose gel for 501 A > C and 2 % agarose gel for 604 G > A, then visualized under ultraviolet light after staining with ethidium bromide.
Statistical analysis All clinical data are expressed as means ± SD. The clinical and biochemical variable differences between PCOS patients and controls were evaluated by an independent samples t-test. The Hardy-Weinberg distribution of genotypes in the PCOS and control groups was assessed. Categorical data were expressed as frequencies and percentages. Statistical analysis of allele and genotype frequencies between women with PCOS and controls was compared using Pearson’s χ2 test. One-way analysis of variance followed by the least significant differences test for posthoc comparisons was used to analyse clinical data in different genotypes. Univariate general linear model analyses were used to evaluate the effects of ghrelin genotypes on BMI, introducing WHR as a covariate. All analyses were performed using the Statistical Package for Social Sciences software (version 13.0; SPSS Inc., Chicago, IL, USA). P < 0.05 was accepted as statistically significant.
Xu L et al. Association between ghrelin gene … Exp Clin Endocrinol Diabetes 2014; 122: 144–148
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Article 145
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Clinical and biochemical characteristics of PCOS and control ▶ Table 1. There was no significant differwomen are shown in ● ence in age between PCOS patients and controls. However, BMI, WHR, LH, and T levels were significantly higher in the PCOS group than in the control group. In addition, FINS and HOMA-IR were higher while ISI was lower in PCOS patients compared with controls, these differences were statistically significant. We identified SNPS -501 A/C (rs26802) and -604 G/A (rs27647) and showed that both were in Hardy-Weinberg equilibrium in all data sets. All samples were double genotyped with 100 % concordance. No significant differences were found between PCOS and normal subjects regarding genotype distributions of ▶ Table 2). -604 G/A and -501 A/C (● SNP -604 G/A was shown to have no impact on hormonal or metabolic phenotypes of PCOS patients, as BMI, WHR, and FSH, LH, T, plasma glucose and insulin levels were similar between differ▶ Table 3). However, the SNP ent genotypes of PCOS subjects (● -501 A/C was associated with some metabolic variations in PCOS women. The effects of the different genotypes on clinical and bio▶ Table 4. chemical variables in women with PCOS are listed in ● Statistically significant differences in BMI and WHR were found in the SNP -501 A/C genotype when AA and AC carriers were compared with CC mutant homozygotes. The difference in BMI and WHR between A carriers after combining AA and AC (A carriers) and CC homozygotes also showed statistical significance (P = 0.03, 0.04, respectively). Association studies of other clinical parameters were also carried out with different genotypes of -501 A/C, but no significant differences were found. Table 1 Clinical, endocrine. and metabolic characteristics.
Age (years) BMI (kg/m2) Waist hip ratio FSH (IU/L) LH (IU/L) T (nmol/L) FBG (mmol/L) FINS (mIU/L) ISI HOMA-IR2 HOMAβ % CHO (mmol/L) TG (mmol/L) LDL (mmol/L)
PCOS (n = 285)
Controls (n = 260)
P-value
28.79 ± 3.41 25.87 ± 4.91 0.89 ± 0.05 6.94 ± 1.98 9.88 ± 7.79 2.36 ± 0.92 4.90 ± 0.87 8.45 ± 4.97 0.037 ± 0.029 1.87 ± 1.66 26.21 ± 59.54 4.99 ± 0.56 0.95 ± 0.53 2.79 ± 0.92
29.46 ± 3.35 22.02 ± 3.29 0.78 ± 0.04 6.67 ± 2.01 4.35 ± 3.56 1.15 ± 0.67 4.88 ± 0.95 6.26 ± 4.57 0.050 ± 0.026 1.39 ± 1.75 87.45 ± 32.67 4.72 ± 0.61 0.84 ± 0.62 2.46 ± 1.13
0.082 < 0.001 < 0.001 0.076 < 0.001 < 0.001 0.826 0.041 0.007 0.038 0.312 0.738 0.691 0.663
Values are means ± SD BMI: body mass index; FSH: follicle-stimulating hormone; LH: luteinizing hormone; T: testosterone; FBG: fasting blood glucose; FINS: fasting insulin; ISI: insulin index; HOMA-IR2: insulin resistance index; HOMA β %: β-cell function index; CHO: cholesterol; TG: triglycerides; LDL: low density lipoprotein cholesterol
Because the SNP -501 A/C was associated with WHR and BMI, a univariate general linear model analysis, introducing WHR as a covariate in the model, was performed. The results showed that SNP-501 A/C alleles significantly influenced BMI (P = 0.032). All same clinical parameters as PCOS women were tested in an association study of the control group with different genotypes of -501 A/C and 604 G/A, but no significant differences were found (P > 0.05).
Discussion
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The human ghrelin gene is located on chromosome 3p25–26 and consists of 4 exons and 3 introns [18]. In this study, we selected 2 SNPs within its promoter region, and investigated whether each SNP genotype was associated with PCOS. We showed that the SNP genotypes were similar between PCOS patients and controls, indicating that ghrelin does not involve in the pathogenesis of PCOS directly. However, the presence of the 501 A/C polymorphism in the ghrelin gene promoter appeared to be associated with some metabolic characteristics of PCOS. Significant differences were found in BMI and WHR between SNP -501 A/C genotype AA and AC carriers and CC mutant homozygotes. After AA and AC genotypes were combined, differences in BMI and WHR between A carriers and C/C homozygotes remained statistically significant. The same results were not obtained in the control group. Obesity is often associated with PCOS [19] and plays a pivotal role in the development and/or maintenance of the syndrome. In addition, BMI and WHR were previously shown to positively correlate with free androgen index and HOMA-IR [20]. Thus, while the common 501 A > C polymorphism does not affect ovarian and adrenal androgen levels or HOMA-IR, it may indirectly influence PCOS pathogenesis by controlling some metabolic phenotypes, BMI and WHR. However, further studies are necessary to confirm this and analysis how the 501 A > C polymorphism influence PCOS. No significant differences were found in the other clinical, metabolic and endocrine parameters evaluated in the present study to determine the association between PCOS and controls with different -501 A/C and -604 G/A genotypes. However, significant differences were observed, including higher HOMA-IR and FINS but lower ISI in PCOS patients compared with controls. Other ghrelin gene SNPs have previously been shown to correlate with insulin resistance [21, 22] in T2DM and obese subjects. Recently we discovered that there was no association between Arg51Gln and Leu72Met polymorphisms and insulin resistance in PCOS [16], and the present study found no correlation between SNPs 501 A/C and 604 G/A and insulin resistance in PCOS. This indicates that SNPs 501 A/C and 604 G/A are not involved in PCOS through influencing insulin and glucose metabolisms. We also found that PCOS subjects had higher blood testosterone and higher LH levels. Androgens are independent modulators of
Table 2 Allele and genotype frequencies of ghrelin gene polymorphisms. SNP rs26802 (501 A/C) rs27647 (604 G/A)
Group
N
PCOS Control
285 260
PCOS Control
285 260
Genotype AA 130 (45) 101 (39) AA 216 (76) 195 (75)
AC 121 (42) 97 (37) AG 55 (19) 52 (20)
P-value CC 34 (13) 62 (22) GG 14 (5) 13 (5)
0.06
0.79
Xu L et al. Association between ghrelin gene … Exp Clin Endocrinol Diabetes 2014; 122: 144–148
Allele A 381 (66.3 %) 299 (56.9 %) A 487 (85.44 %) 442 (85.00 %)
P-value C 189 (32.2 %) 221 (43.1 %) G 83 (14.56 %) 78 (15.00 %)
0.07
0.86
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Results
Article 147
P-value
Genotype
AA (n = 216)
AG n = 55)
GG (n = 14)
Age (years) BMI (kg/m2) FSH (IU/L) LH (IU/L) T (nmol/L) FBG (mmol/L) FINS (mIU/L) ISI HOMA-IR2 HOMAβ % CHO (mmol/L) LDL (mmol/L) TG (mmol/L)
28.36 ± 3.15 25.12 ± 3.75 6.38 ± 1.54 9.88 ± 6.13 2.52 ± 0.35 5.05 ± 1.33 9.12 ± 3.44 0.028 ± 0.021 2.01 ± 1.13 89.32 ± 76.17 4.55 ± 0.73 2.58 ± 1.32 0.77 ± 0.49
28.55 ± 2.98 25.46 ± 4.37 6.27 ± 1.65 9.76 ± 5,87 2.51 ± 0.28 4.96 ± 1.77 8.99 ± 2.94 0.033 ± 0.029 1.89 ± 1.36 87.33 ± 71.09 4.69 ± 0.12 2.63 ± 1.29 0.86 ± 0.75
27.96 ± 3.14 24.96 ± 3.86 6.53 ± 1.49 9.95 ± 5.46 2.64 ± 0.53 5.11 ± 0.81 9.03 ± 4.31 0.029 ± 0.025 1.96 ± 1.38 84.29 ± 68.39 4.48 ± 0.26 2.54 ± 1.47 0.91 ± 0.84
0.78 0.43 0.81 0.67 0.72 0.46 0.59 0.22 0.25 0.37 0.23 0.61 0.18
Table 3 Comparison of characteristics in women with PCOS according to -604 G/A genotype.
Values are means ± SD. The P values are a global comparison
Table 4 Comparison of characteristics in women with PCOS according to -501 A/C genotype. -501 A/C Genotype
AA (n = 130)
Age (y) BMI (kg/m2) WHR FSH (IU/L) LH (IU/L) T (nmol/L) FBG (mmol/L) FINS (mU/L) ISI HOMA-IR2 HOMAβ % CHO (mmol/L) LDL (mmol/L) TG (mmol/L)
28.46 ± 2.98 29.58 ± 3.42 a 0.93 ± 0.03 a 6.35 ± 1.64 10.04 ± 5.89 2.49 ± 0.78 5.18 ± 1.62 8.96 ± 4.46 0.029 ± 0.012 2.06 ± 1.02 101.23 ± 56.18 4.77 ± 0.64 2.85 ± 1.03 0.78 ± 0.62
AC(n = 121) 28.54 ± 3.12 29.71 ± 3.65 a 0.92 ± 0.04 a 6.67 ± 1.23 9.96 ± 6.25 2.50 ± 0.69 5.06 ± 1.21 8.78 ± 5.21 0.032 ± 0.036 1.94 ± 1.03 93.16 ± 46.32 4.69 ± 1.05 2.67 ± 1.22 0.98 ± 1.53
t-value
CC (n = 34) 28.38 ± 3.23 22.01 ± 2.45 0.71 ± 0.06 6.48 ± 2.05 9.47 ± 5.74 2.56 ± 0.63 4.98 ± 1.72 9.01 ± 3.57 0.034 ± 0.029 1.97 ± 1.21 99.57 ± 108.12 4.38 ± 0.87 2.74 ± 1.65 0.85 ± 0.72
0.78 0.02 0.03 0.56 0.43 0.64 0.59 0.76 0.63 0.51 0.45 0.36 0.69 0.21
Combined- 501 A > C AA and AC (n = 251)
CC (n = 34)
28.50 ± 3.01 29.64 ± 3.53 0.93 ± 0.04 6.47 ± 1.43 10.01 ± 5.79 2.49 ± 0.77 5.12 ± 1.43 8.85 ± 4.96 0.030 ± 0.022 2.01 ± 1.02 98.35 ± 49.78 4.72 ± 0.91 2.76 ± 1.19 0.87 ± 1.12
28.38 ± 3.23 22.01 ± 2.45 0.71 ± 0.06 6.48 ± 2.05 9.47 ± 5.74 2.56 ± 0.63 4.98 ± 1.72 9.01 ± 3.57 0.034 ± 0.029 1.97 ± 1.21 99.57 ± 108.12 4.38 ± 0.87 2.74 ± 1.65 0.85 ± 0.72
t-value 0.74 0.03 0.04 0.76 0.23 0.63 0.52 0.64 0.66 0.52 0.57 0.34 0.71 0.46
Values are means ± SD. The P values are a global comparison a
P < .05 vs. women with PCOS and with the CC genotype
circulating ghrelin levels [23, 24], so it is conceivable that there is a close relationship between the ghrelin gene and endocrine disorders in PCOS women. However, when we compared different genotypes of the two SNPs in PCOS subjects, we found no difference in testosterone levels. In further analysis, the levels of FSH, LH, E2, and PRL in PCOS patients of different genotypes were also similar. Thus the -501 A/C and 604 G/A polymorphisms appear not to be associated with endocrine disorders of PCOS. To the best of our knowledge, this study, which includes a relatively large number of Chinese women with PCOS, is the first of its kind to provide data concerning -501 A/C and 604 G/A polymorphisms in the ghrelin gene promoter and PCOS. Although we have not conducted a comprehensive analysis of all variants in and around the ghrelin gene and cannot exclude the role of other variants outside this region, the available evidence suggests that at least one variant of the ghrelin gene confers susceptibility to high BMI and WHR in women with PCOS.
Acknowledgements
▼
We thank the staff of the laboratory and the reproductive center in Shandong Provincial Hospital very much for work
performed to support this study. This work was supported by grants from the National Natural Science Foundation (China, Canada). This work was supported by the National Basic Research Program of China (973 Program-2006CB944004) and the National High-Technology Research and Development Program of China (863Program-2006AA02Z4A4). Natural Science Foundation of China (81100554), Shandong Provincial Natural Science Foundation, China (BS2012YY003), the Scientific and Technical Development Project of Department of health of Shandong Province (2011QZ007) and the Scientific and Technical Development Project of Qingdao (12-1-4-20-jch and No. 2012-1-3-2-(1)-nsh).
Conflict of interest: None. Affiliations Obstetrics and Gynecology Department, The Affiliated Hospital of Qingdao University Medical College, Qingdao, People’s Republic of China 2 Shandong Provincial Key Laboratory of Reproductive Medicine, Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, Jinan, People’s Republic of China 3 Central Laboratory, Provincial Hospital Affiliated to Shandong University, Jinan, People’s Republic of China 1
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604 G/A
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