Effect of the R92H and A379V genotypes of platelet-activating factor acetylhydrolase on its enzyme activity, oxidative stress and metabolic profile in Chinese women with polycystic ovary syndrome

The G994T polymorphism in platelet-activating factor acetylhydrolase (PAF-AH) gene is associated with the risk of polycystic ovary syndrome (PCOS). The aim of this study was to investigate the relationship between R92H and A379V variants of the PAF-AH gene and the risk of PCOS and to evaluate the effects of the genotypes on PAF-AH activities and clinical, metabolic and oxidative stress indexes in Chinese women. A total of 862 patients with PCOS based on the Rotterdam consensus criteria and 750 control women from a population of Chinese Han nationality in the Chengdu area were studied from 2006–2015. PAF-AH genotypes were determined by PCR and restriction fragment length polymorphism analysis. Plasma PAF-AH, high-density lipoprotein (HDL)-associated PAF-AH (H-PAF-AH) and apolipoprotein (apo) B-containing lipoprotein-associated PAF-AH (apoB-PAF-AH) activities were measured using the trichloroacetic acid precipitation procedure with PAF C-16 as a substrate. Circulating markers of oxidative stress, including serum total oxidant status, total antioxidant capacity, oxidative stress index and malondialdehyde levels, and clinical and metabolic parameters were also analyzed. No significant differences were observed in the frequencies of R92H and A379V genotypes and alleles of the PAF-AH gene between PCOS and control groups (P > 0.05). Compared with patients with the 92RR genotype, patients with H allele of R92H (RH + HH genotype) had significantly higher plasma PAF-AH and apoB-PAF-AH activities (P < 0.05) and tended to exhibit increased H-PAF-AH activity (P = 0.063) after adjusted for age and BMI. However, when serum LDL-C, HDL-C, TG and HOMA index were added as covariates, the comparisons no longer remained statistical significance (P > 0.05). There were no significant differences in clinical, hormonal, metabolic and circulating oxidative stress parameters and the frequencies of PAF-AH G449T genotype according to PAF-AH R92H or A379V genotyping in patients with PCOS and control women. There were no significant associations between R92H and A379V variants of PAF-AH gene and risk of PCOS in Chinese women. The increased plasma PAF-AH and apoB-PAF-AH activities in patients with H allele of R92H are related to the R92 → H variation, changes in plasma lipoprotein levels, insulin resistance, aging, and gaining weight and thus may be involved in the pathogenesis of PCOS and the increased risks of future cardiovascular diseases.

Several single-nucleotide polymorphisms (SNPs) in the exon region of the PAF-AH gene influence its activity or concentration. The V279F (G994T) SNP in exon 9 completely abolishes enzymatic activity in 279 FF homozygotes and results in a 50% decrease of catalytic activity in heterozygotes [19,21]. The V allele of the A379V SNP in exon 11 results in a two-fold decrease in the affinity of PAF-AH for PAF [22]. The H allele of the R92H SNP in exon 4 is associated with a higher PAF-AH mass [16]. Several studies reported that the PAF-AH R92H and A379V variants were associated with increased risk of coronary heart disease (CHD), but the results were inconsistent [14,15,23,24]. However, the evidence available to date strongly suggests that PAF-AH G994 → T mutation is associated with increased risk of cardiovascular diseases in Japanese and Chinese cohorts [14,24].
Previously Fan et al. demonstrated that the T allele of the G994T SNP in PAF-AH gene was one of the genetic determinants for PCOS in Chinese Han women [19]. Plasma PAF-AH hydrolyzes and inactivates PAF and PAF-like oxidized phospholipids, and is associated with circulating oxidative stress and inflammation status [14,24]. However, to date, little information is available regarding the possible connection between the PAF-AH gene A379V and R92H SNPs and PCOS or oxidative stress. In the present study, we investigated the relationship between R92H and A379V variants of PAF-AH gene and the risk of PCOS and evaluated the effects of the genotypes on PAF-AH activities and clinical, metabolic and oxidative stress indexes in Chinese women.

Study subjects
This is a case-control study, which consists of 862 cases and 755 controls. The frequencies of PAF-AH genotype and allele are main variables. The sample sizes in the present study are reasonable and practicable according to a report by B-Rao [25].
Women with or without PCOS aged 17-40 years were recruited from 2006-2015 from the Outpatient Clinic of Reproductive Endocrinology, West China Second University Hospital.
Each woman with PCOS met diagnostic criteria for PCOS based on the revised 2003 Rotterdam ESHRE/ ASRM consensus criteria [26]. Oligo-or anovulation (OA) was assessed as oligomenorrhea (i.e., fewer than eight cycles per year). Biochemical or clinical hyperandrogenism (HA) was assessed by total testosterone (TT) levels above the 95th percentile of the levels (2.60 nmol/l) detected in a group of normal menstruating women with normal cycles, hirsutism with a modified Ferriman-Gallwey (F-G) score of more than 6 and/or clinical presence of obvious acne [4,27,28]. Polycystic ovaries (PCOs) were confirmed if there were 12 or more follicles in each ovary measuring 2-9 mm in diameter and/ or increased ovarian volume (>10 mL) by ultrasonic examination. The diagnosis of PCOS was based on a patient having two of these three findings for women aged 20-40 years or having all three findings for women aged < 20 years [2], with exclusion of other etiologies, such as androgen-secreting tumors, congenital adrenal hyperplasias, and Cushing syndrome. All of the control women had regular menstrual cycles (between 21 and 35 days), exhibited normal circulating androgen levels, the absence of hirsutism or obvious acne on physical examination, and normal ovarian morphology as determined by ultrasound.
None of the subjects had clinically evident chronic or acute diseases, such as infection, tumors, cardiovascular disease, thyroid dysfunction, endometriosis, hyperprolactinemia, hypogonadotropic hypogonadism or premature ovarian failure.
For association studies between PAF-AH genotypes and hormonal, metabolic and oxidative stress parameters, the subjects were excluded if they met one of the following criteria: [i] taking medication known to affect the metabolism of carbohydrates, lipids, or hormones within 3 months before the study; [ii] pregnant or in the luteal phase; and [iii] smokers.
Clinical and anthropometrical variables, including waist circumference, hip circumference, waist-to-hip ratio, body mass index (BMI, kg/m 2 ), systolic and diastolic blood pressure (SBP and DBP), and the degree of hirsutism and acne were measured or assessed in all subjects. Ultrasound ovarian volume was also assessed using the formula [29]: 0.523 × length × width × thickness.
Blood samples were obtained in the morning after overnight fasting, placed on ice immediately and centrifuged at 1500 g for 15 min at 4°C within 2 h. Plasma or serum aliquots were stored at −80°C and blood cells were stored at 4°C.

Measurements of plasma PAF-AH, H-PAF-AH and apoB-PAF-AH activities
HDL fractions were obtained by precipitating apoBcontaining lipoproteins using 13% polyethylene glycol (PEG) 6000. The plasma PAF-AH and H-PAF-AH activities were measured via the trichloroacetic acid precipitation procedure as previously described [19,32]. The intra-and inter-assay coefficients of variation for all measurements were less than 3% and 5%, respectively. The apoB-PAF-AH activity was obtained by subtracting the H-PAF-AH activity from plasma PAF-AH activity.

Statistical analysis
Data were presented as the mean ± standard deviation (SD). Differences in variables were evaluated by the independent sample t-test between PCOS and control subjects. Variables with asymmetric distribution were evaluated by nonparametric tests (Mann-Whitney U test). X 2 analysis was used to test deviations of genotype distribution from Hardy-Weinberg equilibrium and to determine allele or genotype frequencies between patients and controls. Analysis of covariance was used to estimate the differences in metabolic and oxidative stress parameters and PAF-AH activities between two groups after correction for differences in covariates such as age, BMI and serum lipid levels. Pearson correlation was performed to define the correlations between PAF-AH activities and the other parameters in patients with PCOS. A P-value < 0.05 was considered to be statistically significant. All statistical analyses were performed using Statistical Program for Social Sciences (SPSS) 13.0 for Windows (Chicago, IL, USA).
As shown in Table 1, BMI, waist circumference, waist-to-hip ratio, F-G score, DBP, and average ovarian volume were significantly increased, and age was significantly lower in the PCOS group compared with the control group.
TT and LH levels, the ratio of LH to FSH, fasting insulin concentration, HOMA index, TG, TC, LDL-C, apoB and MDA levels, TOS, T-AOC, OSI, the ratio of apoB-PAF-AH to H-PAF-AH and the frequency of the T allele carriers (GT + TT) of PAF-AH G994T were significantly increased and FSH and HDL-C levels and H-PAF-AH activities were significantly reduced in the PCOS group compared with the control group after adjusted for age and BMI (Table 2). However, when serum LDL-C, HDL-C, TG and HOMA index were added as covariates, the comparisons of plasma PAF-AH, apoB-PAF-AH and H-PAF-AH activities as well as the ratio of apoB-PAF-AH to H-PAF-AH were statistically significant (P < 0.05).

Distribution of PAF-AH R92H and A379V genotypes and alleles
Genotypic distributions of PAF-AH R92H and A379V were in Hardy-Weinberg equilibrium in the PCOS and control groups. No significant differences were observed in the frequencies of PAF-AH R92H and A379V genotypes and alleles between PCOS and control groups (P > 0.05, Table 3).
Effects of PAF-AH R92H and A379V genetic variants on clinical, hormonal, metabolic and oxidative stress parameters and PAF-AH activities We further analyzed effects of PAF-AH R92H and A379V genetic variants on oxidative stress parameters and PAF-AH activities as well as clinical, hormonal, metabolic parameters in PCOS patients and control women. Because the sample sizes of the 92HH or 379VV homozygotes were too small, we combined them into heterozygous subgroups.
As shown in Table 4, compared with patients with 92 RR genotype, patients with H allele of R92H (RH + HH genotype) had significantly higher plasma PAF-AH (P = 0.034) and apoB-PAF-AH activities (P = 0.045) and tended to exhibit increased H-PAF-AH activity (P = 0.063) after adjusted for age and BMI. Similar alterations were also observed in the control group, but the values did not reach statistical significance (P > 0.05). In addition, when serum LDL-C, HDL-C, TG and HOMA index were added as covariates, plasma PAF-AH, apoB-PAF-AH and H-PAF-AH activities no longer remained statistical significance between patients with 92 RR genotype and patients with H allele (P = 0.054, 0.243 and 0.183, respectively). No significant differences in oxidative stress indexes and the frequencies of PAF-AH G449T genotype (Table 4) and clinical, hormonal, metabolic parameters (data not shown) according to PAF-AH R92H genotyping were detected in PCOS patients and control women (P > 0.05).
No significant differences in oxidative stress indexes, the frequencies of PAF-AH G449T genotype and PAF-AH activities (Table 5) as well as other clinical, hormonal, and metabolic parameters (data not shown) according to PAF-AH A379V genotypes were detected in PCOS patients and control women (P > 0.05).

Discussion
In the present study, we show that PAF-AH R92H and A379V genetic polymorphisms are not associated with the risk of PCOS in Chinese women. However, we found that plasma PAF-AH and apoB-PAF-AH activities in patients carrying the minor allele of R92H polymorphism of PAF-AH gene are increased compared with those homozygous for the wild-type allele, suggesting that the R92H variant may be associated with increased plasma and lipoprotein-associated PAF-AH activities in these patients. In addition, our results revealing no significant differences in TOS, OSI and MDA levels according to R92H or A379V genotypes in PCOS and control groups suggests that these genetic variants that modestly or minimally impact enzymatic activity are unlikely to significantly affect circulating oxidative stress levels.
Plasma PAF-AH specifically hydrolyzes and inactivates PAF and PAF-like oxidized phospholipids that are potent pro-inflammatory mediators and thus plays an antiinflammatory role [14,24]. Genetic studies in humans harboring an inactivating mutation (PAF-AH G994 → T mutation) indicate that loss of PAF-AH activity is a risk factor for inflammatory and cardiovascular diseases in Japanese cohorts [14,24]. Consistently, overexpression of PAF-AH has anti-inflammatory and anti-atherogenic properties in animal models [14,24,35]. However, several clinical studies have demonstrated that plasma PAF-AH activity and mass are strongly associated with atherogenic lipids and risk of cardiovascular disease [14,16,24,36]. The hydrolysis products of this enzyme, lysophosphatidylcholine (lyso-PC) and oxidatively modified nonesterified fatty acids, promote the pathogenesis of atherosclerosis [14,24,35]. Thus, controversy remains regarding whether plasma PAF-AH exerts a pro-or antiinflammatory action. Given that H-PAF-AH, one of antioxidant enzymes of HDL, plays anti-inflammatory role [14,19] and apoB-PAF-AH is associated with inflammation [13,14,24], it has recently been suggested that the relative distribution of the enzyme between apoBcontaining lipoproteins and HDL determines its pro-or anti-inflammatory actions. Previous studies indicated that H-PAF-AH activity was decreased and the ratio of LDL-associated PAF-AH to H-PAF-AH activities and MDA levels were increased in women with PCOS, and the PAF-AH G994 → T gene mutation is a risk factor for PCOS [19,20,32]. Consistent with previous studies, this study further determined that patients with PCOS had significantly higher TOS, OSI, and the ratio of apoB-PAF-AH to H-PAF-AH activities compared with the Values are presented as the mean ± SD control women, suggesting that increased circulating absolute (TOS) and relative (OSI) oxidative stress levels and chronic inflammation existed in these patients. Several studies have demonstrated that the PAF-AH 92R → H genetic variant was associated with an increased PAF-AH mass [15,16,23] and was a risk factor of CHD [23,37]. However, in meta-analyses, the PAF-AH R92H genetic polymorphism was not significantly associated with the risk of CHD [15]. In the present study, we showed that this genetic polymorphism was not associated with the risk of PCOS in Chinese women. However, the PAF-AH 92R → H variant was significantly associated with increased plasma and lipoproteinassociated PAF-AH activities in PCOS patients after adjusted for age and BMI, but when LDL-C, HDL-C, TG, and HOMA-IR were added as covariates, the statistical differences were no longer maintained. Grallert et al [15] indicated that PAF-AH activity was strongly associated with genetic variants involved in lipid metabolism. A previous study also demonstrated that the ratio of L-PAF-AH to H-PAF-AH activities was positively correlated with LDL-C, TG, HOMA-IR, fasting plasma insulin and glucose concentrations, and negatively correlated with HDL-C levels in patients with PCOS [20]. Similarly, GT + TT 20 + 0 (7.3%) 34 + 4 (13.5%) 0.020 Values are presented as the mean ± SD ApoA1 apolipoprotein A1, apoB apolipoprotein B, TOS total oxidant status, T-AOC total antioxidant capacity, OSI oxidative stress index, PAF-AH platelet activating factor acetylhydrolase, H-PAF-AH HDL-associated PAF-AH, ApoB-PAF-AH apolipoprotein B-containing lipoprotein-associated PAF-AH P a All comparisons of parameters were corrected for differences in age and BMI between the two groups a Controls (n = 275), PCOS (n = 281) the present study indicated that plasma PAF-AH and apoB-PAF-AH activities were positively correlated with LDL-C, apoB, TC, TG, age, SBP, waist circumference, waist-to-hip ratio, and HOMA-IR (P < 0.05) and negatively correlated with HDL-C levels (P < 0.05) in the patients according to the correlation analysis (data not shown). In addition, evidence suggests that PAF, oxidized LDL, and oxidized phospholipids upregulate expression of PAF-AH in vivo [24]. These results suggest that multiple factors including the 92R → H variant, changes in plasma lipoprotein levels, increased oxidative stress or/and chronic inflammation, insulin resistance, aging, and gaining weight may contribute to increased plasma and lipoprotein-associated PAF-AH activities according to PAF-AH R92H genotypes in PCOS patients.
The PAF-AH 379A → V variant results in a two-fold decrease in the affinity of PAF-AH for its substrates [22], thus prolonging the pro-inflammatory actions of PAF and PAF-like oxidized phospholipids in plasma. However, the effect of this variant on PAF-AH activity or disease is conflicting. The 379A → V variant is associated with increased [15,31,36] or decreased [38] activity and increased [38,39], decreased [40,41] or no affect [15,36] on the risk of cardiovascular diseases. Our study determined that the A379V SNP was not significantly associated with the risk of PCOS and PAF-AH activities in Chinese women.
In particular, the allele frequencies for PAF-AH genetic variants differ among ethnic groups [14,24]. For example, the G994T SNP is common in Japanese and Taiwan populations and has been reported in Korean, Chinese, Turkey populations; however, this SNP is rarely observed in Europeans [24]. In the present study, the frequency of the T allele carriers (GT + TT) of PAF-AH G994T was significantly increased in the PCOS group compared with the control group (P = 0.02, Table 2). This may be an important reason why patients with  PCOS have higher TC, TG and LDL-C levels, but have relatively lower plasma PAF-AH and apoB-PAF-AH activities. Because The G994 → T variation completely abolishes enzymatic activity in TT homozygotes and results in a 50% decrease of catalytic activity in heterozygotes [19,21]. In addition, environmental factors, such as increased oxidative stress, inflammation status, a high-fat diet, and lipoprotein levels may also affect PAF-AH expression or the distribution of this enzyme in lipoproteins [16,19,20,24]. Therefore, it is possible that the relationships between PAF-AH genetic polymorphisms and PAF-AH activities or diseases may differ among different populations.
Regarding limitations of the present study, given the low frequency of homozygosity of minor alleles, 92HH and 379VV, we could not analyze them in the form of subgroups, and a larger number of subjects are needed to properly evaluate dose dependent genotype characteristics. Second, due to insufficient sample, we did not assess plasma and lipoproteinassociated PAF-AH activities in some subjects, which might influence the power of these parameters or result in the absence of statistical significances. Further study to increase the sample size for PAF-AH activity determination may help improve statistical power of these parameters between the two different genotype subgroups.

Conclusion
The present study demonstrates that PAF-AH R92H and A379V genetic polymorphisms are not associated with the risk of PCOS and the circulating absolute and relative oxidative stress levels in Chinese women. The increased plasma PAF-AH and apoB-PAF-AH activities in patients with H allele of R92H are related to multiple factors including the R92 → H variation, changes in plasma lipoprotein levels, insulin resistance, aging, and gaining weight and thus might be involved in the pathogenesis of PCOS and the increased risks of future cardiovascular diseases in the patients.  Values are presented as the mean ± SD TOS total oxidant status, T-AOC total antioxidant capacity, OSI oxidative stress index, PAF-AH platelet activating factor acetylhydrolase, H-PAF-AH HDL-associated PAF-AH, ApoB-PAF-AH apolipoprotein B-containing lipoprotein-associated PAF-AH Comparisons of oxidative stress parameters and PAF-AH activities were corrected for differences in age and BMI between the two groups