Document Type : Original Article
Authors
1 Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran;Department of Clinical Biochemistry, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Clinical Biochemistry, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran
3 Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
4 Department of Dermatology, Kermanshah University of Medical Sciences, Kermanshah, Iran
5 Department of Clinical Biochemistry, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran;4Fertlility and Infertility Research Centre, Kermanshah University of Medical Sciences, Kermanshah, Iran
Abstract
Keywords
Polycystic ovary syndrome (PCOS) is one of the most
frequent endocrine-related gynecological disorders
among women of reproductive age (
The peroxisome proliferator-activated receptors
(PPARs) belong to the nuclear hormone receptors that
regulate the transcription of a variety of genes such as
those involved in the metabolism of lipids in adipose tissue,
liver and skin (
The common
The
The aim of this study was to assess the association of
The present cross-sectional study consisted of 50 women
with confirmed PCOS according to the Rotterdam criteria
(
Two out of three criteria of clinical and/or biochemical signs of PCOS, namely hyperandrogenism (the presence of hirsutism), acne or alopecia and ovarian dysfunction (oligo- and/or anovulation and/or polycystic ovaries detected by ultrasound scans) were sufficient to diagnose PCOS. Exclusion criteria were congenital adrenal hyperplasia, androgen-secreting tumors, and intake of any medication that may affect the endocrinal parameters along with the glucose and lipid profiles for at least 3 months prior to enrolment.
Height and weight were obtained from each individual and the body mass index (BMI) was calculated. All women in this study were from the Kermanshah province in West Iran, belonging to the Kurdish ethnicity.
All individuals agreed to participate in the study and signed a written informed consent before participation. The Ethics Committee of Kermanshah University of Medical Sciences approved the study. The study was in accordance with the principles of the Declaration of Helsinki II.
From each individual, a sample of 10 milliliters of venous blood was collected at 9 am under standard conditions. The sample was divided to two portions of six milliliters; portion one was centrifuged for 10 minutes at 1600 g in the absence of any anticoagulant and the obtained serum was used for biochemical analysis according to the standard protocol. The second portion (4 ml) was treated with EDTA and used for DNA extraction and further genetic analysis.
The levels of fasting blood sugar (FBS), triglycerides (TG), cholesterol, low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL- C) were measured using the Bionic Diagnostic Kits (Iran) on Mindray BS-480 Chemistry Analyzer (China). Serum estradiol level in the mid-follicular phase of the menstrual cycle and SHBG were measured using the chemiluminescent method by using the Abbott Architect i1000 (Abbott Laboratory, USA).
DNA was extracted from venous blood using the standard
phenol-chloroform method (
F: 5'-GCCAATTCAAGCCCAGTC-3'
R: 5'-GATATGTTTGCAGACAGTGTATCAGTGAAGGAATCGCTTTCCG- 3' primers.
The PCR reaction in a final volume of 25 µl contained
20 pmol of each primer, 100-200 ng DNA, 200 µM dNTPs,
1.5 mM MgCl2, 1 U Taq polymerase and 2.5 µl of
10X PCR buffer (SinaClon, Iran). The PCR conditions
were an initial denaturation at 94°C for 5 minutes followed
by 30 cycles of 94°C for 60 seconds, 55°C for 60
seconds and 72°C for 60 seconds, with a final extension
for 5 minutes at 72°C. Five microliters of the resulting
270 bp PCR product was examined using electrophoresis
on a 1% agarose gel containing the Gel Red (Kawsar
Biotech Company, Iran) stain and was visualized under a
UV Gel Documentation System (Quantum ST4). Fifteen
microliters of the PCR product was treated with 5 U of
the restriction enzyme BstU I at 37°C overnight and the
RFLP products were electrophoresed on a 2% agarose gel
(
Agarose gel electrophoresis (2%) pattern of digested polymerase chain reaction (PCR) products by the BstU I restriction enzyme. From left to right, lanes 1, 2, and 3 represent the PPARγ CG genotype, lane 4 indicates the CC genotype and lane 5 shows the 50 bp DNA molecular weight marker.
The PPARγ C161T SNP was detected by PCR-RFLP using specific
F: 5'-CAA GAC AAC CTG CTA CAA GC-3'
R: 5' -TCC TTG TAG ATC TCC TGC AG -3' primers.
The PCR reaction consisted of 20 pmol of each primer,
100-200 ng DNA, 200 µM dNTPs, 1.5 mM MgCl2, 1 U
Taq polymerase and 2.5 µl of 10X PCR buffer in a final
volume of 25 µl. The PCR thermal cycling conditions were
an initial denaturation at 94°C for 5 minutes, followed by
35 cycles by 94°C for 60 seconds, 55°C for 60 seconds and
72°C for 60 seconds, with a final extension for 5 minutes at
72°C. Five microliters of the resulting 200 bp PCR product
was examined using electrophoresis on a 1% agarose gel
containing Gel Red stain and visualized under a UV Gel
Documentation System (Quantum ST4). Fifteen microliters
of the PCR product were treated with 5 U of the restriction
enzyme Pml1 at 37°C overnight and the RFLP products
were electrophoresed on a 2% agarose gel (
The agarose gel electrophoresis of restriction fragment length polymorphism (RFLP)
products obtained by digestion of polymerase chain reaction (PCR) products by the
The frequency of alleles was calculated by the chromosome counting method and deviation from the Hardy-Weinberg equilibrium (HWE) was calculated using the Chi-square test. Comparison of genotype and allele frequencies of the two SNPs between PCOS patients and controls was undertaken using the Chi-square test. The SPSS logistic regression was used to calculate odds ratio (OR) as an estimate of relative risk for the disease and its 95% confidence interval (CI). The association between biochemical data and SNPs was calculated using the independent-sample t test and ANOVA. The P<0.05 was considered as statistically significant. The statistical package for social sciences (SPSS, SPSS Inc., Chicago, IL) version 16.0 was used for the statistical analysis.
Demographic and biochemical characteristics of the
participants are presented in Table 1. Patients were age-
matched with controls (P=0.09). Also, the two groups
were BMI-matched (P=0.25,
Characteristics of PCOS patients and controls
| Variable | Patient n=50 Mean ± SD | Control n=233 Mean ± SD | P value |
|---|---|---|---|
| Age (Y) | 23.6 ± 5.3 | 22.2 ± 4.2 | 0.09 |
| BMI (Kg/m2) | 23.7 ± 4.9 | 22.8 ± 5.8 | 0.25 |
| FBS (mg/dl) | 78.6 ± 13.2 | 78.5 ± 14.8 | 0.97 |
| Cholesterol (mg/dl) | 131.1 ± 32.8 | 129.7 ± 30.6 | 0.78 |
| TG (mg/dl) | 78.8 ± 43.2 | 88 ± 51.5 | 0.25 |
| HDL-C (mg/dl) | 45.6 ± 11.7 | 46.5 ± 12.8 | 0.61 |
| LDL-C (mg/dl) | 74 ± 26.6 | 74.8 ± 24.5 | 0.82 |
| Estradiol (pg/ml) | 70 ± 45.5 | 109.7 ± 91.2 | <0.001 |
| SHBG (nmol/l) | 52.2 ± 24.5 | 58.6 ± 33.9 | 0.13 |
PCOS; Polycystic ovary syndrome, BMI; Body mass index, FBS; Fasting blood sugar, TG; Triglycerides, HDL-C; High density lipoprotein-cholesterol, LDL-C; Low density lipoprotein-cholesterol, and SHBG; Sex hormone binding globulin.
The genotypic distribution of
The genotype and allele frequencies of both SNPs are given
in Tables
The frequency of PPARγ Pro12Ala (C/G) genotypes and alleles in patients and controls
| Parameter | Patient n=50 (%) | Control n=233 (%) | |
|---|---|---|---|
| Genotypes | |||
| CC | CC | 201 (86.3) | |
| CG | CG | 32 (13.7) | |
| χ2=9.75, P=0.002, OR=2.96 , (95% CI: 1.46-5.96, P=0.002) | |||
| Alleles | Alleles | ||
| C | C | 434 (93.1) | |
| G | G | 32 (6.9) | |
| χ2=9.75, P=0.002, OR=2.96 (95% CI: 1.46-5.96, P=0.002) | |||
OR; Odds ratio and CI; Confidence interval.
The effect of both polymorphisms on lipid and lipoprotein
profiles along with estradiol and SHBG levels in all
studied individuals is shown in Table 4. A significantly
higher level of TG was detected in the presence of the
The genotype and allele frequencies of PPARγ C161T in the patient and control groups
| Parameter | Patient n=50(%) | Control n=233(%) | |
|---|---|---|---|
| Genotypes | |||
| CC | 31 (62) | 155 (66.5) | |
| CT | 17 (34) | 76 (32.6) | |
| TT | 2 (4) | 2 (0.9) | |
| χ2=3.05, P=0.21 | |||
| Alleles | |||
| C | 79 (79) | 386 (82.8) | |
| T | 21 (21) | 80 (17.2) | |
| χ2=0.4, P=0.52 | |||
When each group was studied separately, the association of
the
We identified an association between the PPARγ Pro12Ala
CG genotype and the risk of PCOS in our population. We
did not detect the GG genotype among our studied individuals
because the homozygote Ala genotype is rare in the
overall population (
There are inconsistent reports on the association of
In a study from Germany, the frequency of the
Mean number of primordial, primary, growing, atretic graafian follicles, graafian follicles and corpora lutea in the ovaries of rats in the experimental and control groups
| Variable | ||||||
|---|---|---|---|---|---|---|
| CC (n=235) | CG (n=48) | CC (n=186) | CT (n=93) | TT (n=4) | ||
| FBS (mg/dl) | 78.7 ± 15 | 77.3 ± 11.6 | 79.4 ± 15.8 | 77.3 ± 11.3 | 63.3 ± 9.1 | |
| P=0.61 | P=0.47 | P=0.06 | ||||
| Cholesterol (mg/dl) | 129.0 ± 30.5 | 134.1 ± 32.9 | 130.6 ± 30.6 | 130.6 ± 30.7 | 85.5 ± 24.4 | |
| P=0.33 | P=1 | P=0.011* | ||||
| P=0.012** | ||||||
| TG (mg/dl) | 76.0 ± 40 | 101.1 ± 59.4 | 81.7 ± 46.4 | 79.8 ± 42.1 | 40.3 ± 18.4 | |
| P=0.007 | P=0.94 | P=0.16 | ||||
| HDL-C (mg/dl) | 46.8 ± 13 | 44.3 ± 10.6 | 46.9 ± 13.1 | 45.7 ± 11.7 | 36.0 ± 10.4 | |
| P=0.16 | P=0.71 | P=0.2 | ||||
| LDL-C (mg/dl) | 74.3 ± 24.4 | 76.1 ± 26.9 | 75.4 ± 24.5 | 74.5 ± 25.1 | 42.5 ± 12.8 | |
| P=0.67 | P=0.95 | P=0.023* | ||||
| P=0.031** | ||||||
| Estradiol (pg/ml) | 103.8 ± 86.6 | 96.8 ± 84.1 | 102.3 ± 86.5 | 103.6 ± 87 | 91.6 ± 57.2 | |
| P=0.61 | P=0.99 | P=0.96 | ||||
| SHBG (nmol/l) | 58.3 ± 33.8 | 53.2 ± 24.6 | 58.3 ± 34.2 | 55.2 ± 28.9 | 72.9 ± 32.1 | |
| P=0.24 | P=0.44 | P=0.4 | ||||
Data are presented as mean ± SD.*; Compared with the CC genotype, **; Compared with the CT genotype, FBS; Fasting blood sugar, TG; Triglycerides, HDL-C; High density lipoprotein-cholesterol, LDL-C; Low density lipoprotein-cholesterol, and SHBG; Sex hormone binding globulin
The PPARγ is a critical transcription factor involved in
regulating glucose and lipid metabolism (
Our study showed an association between
)
