Hum. Reprod. Advance Access originally published online on July 31, 2006
Human Reproduction 2006 21(12):3157-3161; doi:10.1093/humrep/del300
The PON1–108C/T polymorphism, and not the polycystic ovary syndrome, is an important determinant of reduced serum paraoxonase activity in premenopausal women
1 Department of Molecular Genetics 2 Department of Endocrinology, Hospital Universitario Ramón y Cajal, Madrid 28034, Spain
3 To whom correspondence should be addressed at: Department of Endocrinology & Universidad de Alcalá, Hospital Universitario Ramón y Cajal, Carretera de Colmenar KM 9.1, Madrid E-28034, Spain. E-mail: hescobarm.hrc{at}salud.madrid.org
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Abstract |
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BACKGROUND: Because serum paraoxonase activity is influenced by the –108C/T polymorphism in the PON1 gene, we studied its involvement in the decreased paraoxonase activity recently described in the polycystic ovary syndrome (PCOS). METHODS: Paraoxonase activity, PON1–108C/T genotypes and clinical, hormonal and biochemical variables were evaluated in a case–control study involving 139 consecutive PCOS patients and 85 healthy controls matched for BMI and prevalence of smoking. RESULTS: Women homozygous for –108T presented with reduced serum paraoxonase activity compared with carriers of C alleles (P < 0.001), both in PCOS patients and in controls. Although homozygosity for T alleles was more prevalent in PCOS patients than in controls (P = 0.003), serum paraoxonase activity was not significantly different in the PCOS and control groups. In a stepwise multivariate linear regression model, homozygosity for PON1–108T alleles was the only significant predictor of the logarithm of serum paraoxonase activity (
= –0.328, t = –4.176, P < 0.001). CONCLUSIONS: In premenopausal women from the Spanish population, the PON1–108C/T polymorphism, and not PCOS, is an important determinant of serum paraoxonase activity.
Key words: insulin resistance/molecular genetics/oxidative stress/polymorphism/premenopausal women
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Introduction |
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The polycystic ovary syndrome (PCOS) may be considered a human model of insulin resistance, given that both lean and obese women presenting with PCOS are insulin resistant when compared with their non-hyperandrogenic counterparts, although insulin resistance is not a universal finding in PCOS patients (Ehrmann, 2005
). Partly because of the frequent association with insulin resistance, cardiovascular risk factors cluster in women with PCOS (Solomon, 1999), although the demonstration of an increased rate of cardiovascular events in these women is still pending (Legro, 2003).
Serum paraoxonase is an antioxidant high-density lipoprotein (HDL)-associated enzyme encoded by the PON1 gene, which is mainly expressed in the liver (bin Ali et al., 2003). Considering that oxidative stress may impair insulin action (Rudich et al., 1997) and is a substantial contributor to atherosclerosis (Navab et al., 2004), the finding in PCOS patients of reduced serum paraoxonase activity (Dursun et al., 2006) might contribute to explain the insulin resistance, and increased cardiovascular risk, associated with PCOS. In conceptual agreement, reduced serum paraoxonase activity has been found in other insulin-resistant disorders such as type 2 diabetes mellitus (Sakai et al., 1998) and cardiovascular atherosclerotic disease (Mackness et al., 2004).
The –108C/T polymorphism in PON1 is responsible for 
23% of PON1 expression levels in some cell systems, in which –108TT constructs showed reduced PON1 expression compared with –108CC constructs (Brophy et al., 2001). We recently found that homozygosity for –108T alleles is more prevalent in PCOS patients compared with healthy controls (San Millan et al., 2004) and hypothesized that the resulting decrease in serum paraoxonase activity might result in a higher oxidative stress in women suffering from PCOS.
We conducted this study to evaluate the impact of the PON1–108C/T polymorphism on the serum paraoxonase activity of a series of premenopausal women presenting with or without PCOS.
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Methods |
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Subjects
One hundred and thirty-nine consecutive PCOS patients and 85 healthy control women were included in this study. The PCOS and control groups were matched for BMI, the grade of obese [lean, BMI < 25 kg/m2; overweight, BMI 25–29.9 kg/m2; obese, BMI
30 kg/m2 (National Institutes of Health, 1998)] and the prevalence of smokers. The control group was composed of lean female volunteers and consecutive patients reporting to the endocrinology outpatient clinic for dietary treatment of obesity. None of the controls had signs or symptoms of hyperandrogenism, menstrual dysfunction or history of infertility. PCOS and hyperandrogenic disorders, including idiopathic hirsutism, were actively ruled out in all the controls. All the women were Caucasian, and none had a history of disorders of glucose tolerance or hypertension or had taken hormonal medications for the last 6 months. The ethics committee of the Hospital Universitario Ramón y Cajal approved this study, and informed consent was obtained from each patient and control.
The diagnosis of PCOS was based on endocrine criteria according to the 1990 National Institute of Child Health and Human Development conference (Zawadzki and Dunaif, 1992): clinical and/or biochemical hyperandrogenism, ovulatory dysfunction, and exclusion of hyperprolactinaemia, non-classic congenital adrenal hyperplasia and androgen-secreting tumours. The ultrasound examination of the ovaries was not performed, and no patients with the new criteria added by the ESHRE/ASRM consensus meeting (The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004) were studied here. The precise description of the methods used to define hyperandrogenism and ovulatory dysfunction and to exclude secondary aetiologies has been detailed elsewhere (Villuendas et al., 2005).
Protocol
Clinical and anthropometric variables, including hirsutism score, office blood pressure (determined as the mean of two manual mercury sphygmomanometer readings in the sitting position), BMI and waist-to-hip ratio, were determined in all the subjects. Hirsutism was defined by the presence of excessive body hair with an androgen-dependent pattern and a modified Ferriman–Gallwey score above 7 (Hatch et al., 1981). The waist-to-hip ratio was calculated by dividing the minimal waist circumference by the hip circumference at the level of greater trochanters, using a non-stretchable measuring tape. Serum and plasma sampling and an oral glucose tolerance test (OGTT) were performed as previously reported (Villuendas et al., 2005). Samples were used for the measurement of total testosterone, androstenedione, dehydroepiandrosterone-sulphate, sex hormone-binding globulin, a complete lipid profile, and basal and post-OGTT insulin and glucose levels.
Serum paraoxonase activity was estimated only in the subset of women from whom we had serum aliquots stored at –30 C that have not been thawed previously. Paraoxonase activity was estimated by the rate of hydrolysis of paraoxon (1 mM, o,o-diethyl-o-p-nitrophenylphosphate, Sigma Chemical) to p-nitrophenol in 1 mM CaCl2 in 0.1 M Tris–HCl (pH 8.0) at a final concentration of 1.2 mM. The amount of p-nitrophenol generated was calculated from the increase in absorbance at 412 nm and 25 C using a spectrophotometer, considering a molar absorptivity at pH 8.0 of 17 000/mol/cm. Non-enzymatic hydrolysis was corrected by assaying a blank sample without serum under the same analytical conditions. One unit of serum paraoxonase activity was defined as 1 nmol of p-nitrophenol formed per minute (Gan et al., 1991). The intra- and inter-assay coefficients of variations in our laboratory were 4.4 and 5.4%, respectively.
The technical characteristics of the assays employed for plasma glucose, lipid profiles and serum hormone measurements have been reported elsewhere (Escobar-Morreale et al., 1997; Escobar-Morreale et al., 2000; San Millán et al., 2001). The free testosterone concentration was calculated from total testosterone and sex hormone-binding globulin concentrations (Vermeulen et al., 1999). The composite insulin sensitivity index was calculated from the circulating glucose and insulin concentrations during the OGTT (Matsuda and DeFronzo, 1999), and -cell function (HOMA-) was estimated from fasting insulin and glucose levels by the homeostasis model assessment (Matthews et al., 1985).
Genotype analysis
Genomic DNA from peripheral blood mononuclear cells was extracted using commercial DNA purification kits (Nucleon BAC C3, Amersham Pharmacia, Buckinghamshire, UK). The –108C/T variant in the PON1 gene was analysed using PCR-restriction fragment length polymorphism as previously described (Brophy et al., 2001).
Statistical analysis
Data are represented as mean ± SD unless otherwise stated. Continuous dependent variables were analysed by univariate general linear model (GLM) analyses introducing age as a covariate because controls were older than PCOS patients. Independent variables were PON1 genotypes and PCOS or control status. Before GLM analysis, the dependent variables were tested for normality using the Kolmogorov–Smirnov statistic, logarithmic or square-root transformations applied as needed to ensure a normal distribution. A multivariate linear regression model using stepwise introduction of independent variables (probability of F to enter 
0.05; probability of F to remove 0.10) was also used to delineate the influence of different variables on serum paraoxonase activity.
Pearson’s chi-square and Fisher’s exact tests were used to test the association between discontinuous variables. P < 0.05 was considered statistically significant.
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Results |
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Characterization of PCOS patients and healthy control women
Patients and controls were equally distributed according to the prevalence of smokers (34.5 and 34.8% respectively,
2 = 0.001, P = 0.999), BMI and the grade of obesity (Table I). Controls were older than PCOS patients (31 ± 8 versus 25 ± 6 years, P < 0.001), and therefore, age was introduced as a covariate in all the comparisons between PCOS patients and controls described below.
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When compared with controls, PCOS patients presented with increased hirsutism scores, waist-to-hip ratio, total and free testosterone levels, androstenedione and dehydroepiandrosterone sulphate concentrations, fasting and OGTT 2-h insulin levels, OGTT 2-h glucose levels and HOMA-
values (Table II). The insulin sensitivity index and sex hormone-binding globulin levels were, on the contrary, reduced in PCOS patients compared with controls, whereas no differences were observed in fasting glucose levels and in the lipid profile (Table II).
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PON1 genotype and serum paraoxonase activity
Homozygosity for PON1–108T alleles was more prevalent in PCOS patients than in controls (30.2 versus 12.9%,
2 = 8.714, P = 0.003), confirming our previous report in this extended series of PCOS patients and controls.
Furthermore, in the subgroup of 107 patients and 58 controls in whom serum paraoxonase activity was determined [homozygosity for –108T alleles was more prevalent in PCOS patients than in controls (29.0 versus 12.1%, 2 = 6.062, P = 0.014) also in this subgroup of subjects, which was representative of the whole cohort of subjects in all the variables and comparisons studied], women homozygous for –108T alleles presented with reduced paraoxonase activity compared with those carrying one or two –108C alleles (Table III). However, the association between homozygosity for PON1–108T alleles and PCOS was not strong enough to result in an overall reduction in serum paraoxonase activity in the PCOS group compared with the controls (Table III). Figure 1 shows the distribution of PCOS cases and controls according to the deciles of serum paraoxonase activity in the premenopausal women studied here. Visual inspection of the figure apparently shows an overrepresentation of PCOS patients in the lower deciles of paraoxonase activity (although this tendency was far from reaching statistical significance) that might be related to a higher prevalence of PON1–108T alleles, and hence of reduced serum paraoxonase activity, in a subgroup of PCOS patients. However, serum paraoxonase activity showed a large interindividual variability in our series (Table III) that might have contributed to the lack of statistically significant differences between PCOS patients and controls observed for this variable.
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Moreover, in a stepwise multivariate linear regression model (R2 = 0.107, F = 17.438, P < 0.001), including the logarithm of serum paraoxonase activity as dependent variable, and homozygosity for PON1–108T alleles, having PCOS, smoking, BMI, waist-to-hip ratio, hirsutism score, total testosterone and the insulin sensitivity index as independent variables, only homozygosity for PON1–108T alleles was maintained as a significant predictor of serum paraoxonase activity (
= –0.328, t = –4.176, P < 0.001). Finally, the PON1–108C/T variant did not influence any of the clinical, metabolic and hormonal variables studied here, either considering all the possible genotypes or considering homozygosity for PON1–108T alleles compared with carriers of –108C alleles, taking both PCOS patients and controls as a whole, or each group separately (data not shown).
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Discussion |
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Oxidative stress plays a central role in the pathogenesis of insulin resistance and cardiovascular disease. Apart from impairing insulin action (Rudich et al., 1997
), oxidative stress induces chronic inflammation (Ceriello and Motz, 2004), which further contributes to insulin resistance, type 2 diabetes, atherosclerosis and cardiovascular disease (Fernandez-Real and Ricart, 2003). Very importantly, chronic inflammation underlies the pathogenesis of PCOS (Escobar-Morreale et al., 2005).
The antioxidant HDL-associated serum enzyme paraoxonase has been shown to play an important role in lipid metabolism. Experimental studies suggest that paraoxonase lowers the risk of coronary heart disease by preventing the oxidation of low-density lipoprotein (LDL) and HDL because this oxidation is involved in the initiation and progression of atherosclerotic lesions (Li et al., 2003). Therefore, reduced paraoxonase activity is considered a risk factor for atherosclerosis and cardiovascular disease (Li et al., 2003).
Serum paraoxonase is encoded by the PON1 gene, which is expressed mainly in the liver. Liver PON1 mRNA expression is influenced by genetic and environmental factors, and both androgens and proinflammatory mediators decrease liver PON1 expression (bin Ali et al., 2003).
The –108C/T polymorphism in PON1 is responsible for 23% of PON1 expression levels in some cell systems, in which –108TT constructs showed reduced PON1 expression compared with –108CC constructs (Brophy et al., 2001). Therefore, we speculated that the association of homozygosity for –108T alleles with PCOS recently found by us in a previous report (San Millan et al., 2004), together with hyperandrogenism and proinflammatory genotypes, might contribute to reduced PON1 expression in PCOS patients, possibly resulting in a higher oxidative stress in these women. This hypothesis is supported by the recent finding by Dursun et al. (2006) of reduced serum paraoxonase activity in Turkish PCOS patients. Unfortunately, the PON1–108C/T polymorphism was not investigated in these Turkish women.
Our present results demonstrate that serum paraoxonase activity is not reduced specifically in PCOS patients and that the –108C/T polymorphism in PON1 is an important determinant of the serum activity in premenopausal women. On the one hand, our data clearly show that women homozygous for –108T alleles of PON1 present with reduced serum paraoxonase activities irrespective of the presence or absence of PCOS. On the other hand, the serum paraoxonase activities of the PCOS and control groups were not actually different in our series, despite a 2.5-fold increase in the frequency of homozygosity for –108T alleles of PON1 in PCOS patients compared with that in healthy controls. Finally, according to the results of the multivariate linear regression analysis, serum paraoxonase activity was not influenced by hyperandrogenism and insulin resistance when also considering in the model the influence of the –108C/T polymorphism in PON1.
Considering that that homozygosity for –108T alleles of PON1 is associated with a decrease in serum paraoxonase levels and is more prevalent in PCOS patients than in controls, the oxidative stress resulting from decreased paraoxonase activity possibly contributes to inflammation, insulin resistance and cardiovascular risk more frequently in PCOS patients than in controls, although, as a group, PCOS patients did not have reduced serum paraoxonase activity.
Conversely, considering that any disorder that associates insulin resistance and inflammation may favour androgen excess in predisposed women (Escobar-Morreale et al., 2005), the induction of these pathogenic mechanisms by the increased oxidative stress resulting from decreased serum paraoxonase activity might explain the association of PCOS with homozygosity for PON1–108T alleles.
Possibility exists that homozygosity for –108T alleles of PON1 was more frequent in the Turkish PCOS population, explaining the reduced serum paraoxonase activity found in these women (Dursun et al., 2006). Unfortunately, because these Turkish patients (Dursun et al., 2006) were not tested for the PON1 variant studied here, this explanation remains considerably speculative.
In conclusion, the PON1–108C/T polymorphism, and not PCOS, is an important determinant of serum paraoxonase activity, at least in premenopausal women from the Spanish population.
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Acknowledgements |
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The authors thank Ms Genoveva González, Laboratorio de Endocrinología, Hospital Universitario Ramón y Cajal, for excellent technical help. This work was supported by Grants PI020741, PI050341, PI050551 and RGDMG03/212 from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, and by Grant GR/SAL/0137/2004 from the Consejería de Educación, Comunidad de Madrid.
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Submitted on April 16, 2006; resubmitted on May 27, 2006; accepted on June 28, 2006.
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