{"paper_id":"e9bcc3bc-fba8-4b2a-b795-62cad9b79610","body_text":"Polycystic ovary syndrome (PCOS) is a common heterogeneous female endocrine metabolic disorder affecting 4–18% of reproductive-aged women [ 1 ,  2 ]. In addition to reproductive disorders, PCOS is often associated with long-term cardiovascular health risks, including obesity, visceral obesity, insulin resistance, hyperinsulinemia, dyslipidemia, increased oxidative stress, chronic low-grade inflammation, endothelial dysfunction, vascular preclinical abnormalities, elevated risks of metabolic syndrome, impaired glucose tolerance, type 2 diabetes, and future cardiovascular diseases [ 2 – 7 ]. The etiology of PCOS remains obscure, but studies have suggested that PCOS appears to have a complex, multifactorial etiology resulting from the interactions between genetic, environmental and intrauterine factors [ 8 ,  9 ].\nApolipoprotein (apo) C1 is a constituent of high-density lipoprotein (HDL) and triglyceride-rich lipoproteins and plays an important role in the regulation of lipoprotein metabolism and serum lipid levels. ApoC1 affects catabolism and clearance of apoB-containing lipoproteins by inhibiting lipoprotein lipase (LPL), liver-specific low density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) [ 10 – 12 ]. This elevates serum levels of cholesterol and triglycerides (TG) in human apoC1–transgenic mice [ 10 – 12 ].\nThe human  apoE–apoC1–apoC4–apoC2  gene cluster is located on chromosome 19. Several genome-wide association studies have demonstrated that the  rs4420638 A → G  variant of the  apoC1  gene, located in the non-coding region, is associated with increased serum total cholesterol (TC) and LDL-C levels [ 13 – 15 ], plasma glucose concentration [ 16 ] and platelet-activating factor acetylhydrolase (PAF-AH) activity [ 17 ]. Furthermore, the  rs4420638 A → G  variant is associated with elevated risks of metabolic syndrome, type 2 diabetes, and coronary heart disease [ 14 ,  16 ,  17 ]. However, the mechanism by which the  apoC 1  rs4420638A/G  variant influences plasma lipid levels and increases the risks of diseases is unclear. A 4-bp  CGTT  deletion ( H1 )/insertion ( H2 ) polymorphism (rs11568822) at the − 317 site in the  apoC 1 promoter region has been reported to affect apoC1 gene expression [ 18 ,  19 ] and is related to the risks of late-onset Alzheimer’s disease [ 20 ] and type III hyperlipoproteinemia [ 21 ]. In addition, the  rs4420638A/G  and  -317H1/H2  polymorphisms of the  apoC1  gene have been reported to exist in linkage disequilibrium with  apoE ε2/ε3/ε4  polymorphisms but exhibit a different degree of linkage disequilibrium in populations of different races [ 19 ,  22 ].\nThe elevated serum apoC1 levels in patients with PCOS, even PCOS patients with normal weight or normolipemic indexes, suggest that apoC1 may be an early protein marker of metabolic abnormality in these patients [ 23 ]. However, to date, little information is available regarding the possible connection between the  apoC1 rs4420638A/G  and  -317H1/H2  polymorphisms and PCOS and the degree of linkage disequilibrium between these variants and  apoE ε2/ε3/ε4  polymorphisms in Chinese women. In the present study, we investigated the relationship between the  rs4420638A/G  and  -317H1/H2  polymorphisms of the  apoC1  gene and the risk of PCOS, evaluated the effects of the genotypes on clinical and metabolic indexes and PAF-AH activity and defined the association of  apoC1  gene variations with  apoE  allele status in southwest Chinese women with or without PCOS using relatively large sample sizes.\n\nWomen with or without PCOS aged 17 to 40 years were recruited from 2006 to 2015 from the Outpatient Clinic of Reproductive Endocrinology at West China Second University Hospital. For the genetic association study of  apoC1  and  apoE  polymorphisms, we included 877 patients and 761 controls. For association studies between  apoC1  genotypes and hormonal levels, metabolic parameters, and PAF-AH activity, 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] being pregnant or in the luteal phase; and [iii] smoking. Finally, 575 patients and 506 controls were included in the metabolism-related study. In addition, PAF-AH activity was measured in 281 patients and 277 controls of the metabolic analysis groups.\nEach patient with PCOS met the revised 2003 Rotterdam ESHRE/ASRM consensus criteria [ 24 ]. Oligo-ovulation or anovulation (OA) was assessed as oligomenorrhea (i.e., fewer than eight cycles per year). Clinical or biochemical hyperandrogenism (HA) was assessed by three measures: hirsutism with a modified Ferriman–Gallwey (F-G) score of more than 6, clinical presence of obvious acne and/or total testosterone (TT) level ≥ 2.60 nmol/L [ 3 ,  4 ,  25 ]. Polycystic ovaries (PCOs) were confirmed if there were 12 or more follicles in each ovary measuring 2 to 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 with HA as an essential condition for women aged < 20 years [ 2 ] and exclusion of other etiologies such as androgen-secreting tumors, congenital adrenal hyperplasias, and Cushing syndrome. All the controls were clinically healthy women who had regular menstrual cycles (between 21 and 35 days), exhibited normal circulating androgen levels, did not show hirsutism or obvious acne on physical examination, and had normal ovarian morphology as determined by ultrasound.\nNone of the subjects had clinically evident acute or chronic diseases, such as infection, tumors, cardiovascular disease, thyroid dysfunction, endometriosis, hyperprolactinemia, hypogonadotropic hypogonadism or premature ovarian insufficiency.\nClinical and anthropometrical parameters, including waist circumference, waist-to-hip ratio, body mass index (BMI, kg/m 2 ), systolic and diastolic blood pressure (SBP and DBP), the degree of hirsutism and acne, and ultrasound ovarian volume [ 26 ] were measured or assessed in all subjects.\nBlood samples were obtained in the morning after overnight fasting on day 3–10 of the menstrual cycle from regularly menstruating women or at random from women with OA. Samples were placed on ice immediately and centrifuged at 1500×g for 15 min at 4 °C within 2 h. Plasma and serum samples were stored at − 80 °C. Blood cells were stored at 4 °C.\nGenomic DNA was isolated from peripheral blood leukocytes of the subjects [ 27 ,  28 ]. For the  apoC1 rs4420638  genotype, a 101-bp fragment was amplified and detected by a Taqman real-time PCR allelic discrimination assay using the forward primer, 5’-TCAGCCTAGCAATGTCACTATGC-3′, the reverse primer, 5’-GTCTGCCTCAAAACAGAAACAAAA-3′, the wild-type probe, HEX-CTTTTCCTaGTGTGGTCTA-TAMRA and the mutant probe, FAM-CACTTTTCCTgGTGTGGT-TAMRA. The 151-bp wild-type (A) or mutant (G) DNA fragments of the  apoC1  gene were used as positive controls. The primers, probes and positive controls were designed and synthesized by the Genecore Biotech Co. Ltd., Shanghai, China. For the  -317H1/H2  genotype, the 221-bp ( H1, CGTT  deletion) and 225-bp ( H2, CGTT  insertion) fragments were amplified using the forward primer: 5’-TTTGAGCTCGGCTCTTGAGACAGGAA-3′ and the reverse primer 5’-GGTCCCGGGCACTTCCCTTAGCCCCA-3′ [ 29 ]. The  -317H1/H2  PCR products were digested with HpaI (Thermo) and analyzed by electrophoresis on a 3.0% agarose gel and visualized by staining with Genecolour fluorescent dye. The enzyme digestion resulted in 159- and 66-bp fragments for the  -317H2  allele and a non-digested 221-bp fragment for the  -317H1  allele.  ApoE ε2/ε3/ε4  polymorphisms (rs429358 and rs7412) were measured as described previously [ 30 ]. For quality control, more than 30% of DNA samples were genotyped again by a different operator.\nSerum luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E 2 ), TT, triglyceride (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), apoA1, apoB, plasma glucose and insulin concentrations, plasma PAF-AH, apoB-containing lipoprotein-associated PAF-AH (apoB-PAF-AH) and HDL-associated PAF-AH (H-PAF-AH) activity were measured as previously described [ 4 ,  31 ,  32 ]. Additionally, the atherogenic index (AI) and the homeostatic model assessment of insulin resistance (HOMA index) were assessed as described previously [ 4 ,  31 ]. The intra- and inter-assay coefficients of variation for all measurements were less than 5 and 10%, respectively.\nData were presented as the mean ± standard deviation (SD). Differences in variables were evaluated by an independent sample t-test between PCOS and control subjects. Variables with asymmetric distribution were evaluated by a Mann-Whitney U test. A chi-square analysis was used to determine allele or genotype frequencies and to test deviations in the genotype distribution from Hardy-Weinberg equilibrium between patients and controls. An analysis of covariance (ANCOVA) or a two way analysis of variance (ANOVA) was used to estimate the differences in clinical parameters, hormonal levels, metabolic profile and PAF-AH activity between the two groups or genotype subgroups after correction for differences in age and BMI. A  P -value of < 0.05 was considered to be statistically significant. All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS) 13.0 for Windows (Chicago, IL, USA).\nThe analysis of linkage disequilibrium between different genetic polymorphic loci was performed by the SHEsis online software at  http://analysis.bio-x.cn/myAnalysis.php .\n\nBecause mean age and BMI were different between the PCOS and the control groups (Table  1 ), differences that could bias comparisons of other clinical parameters, the hormonal and metabolic indexes and PAF-AH activity between the two groups were adjusted for the difference in age and BMI in the following analysis. Table 1 Clinical characteristics of PCOS patients and controls Controls ( n  = 761) PCOS ( n  = 877) \n P \n \n P \n a \n Age (years) 28.19 ± 4.14 24.66 ± 3.95 < 0.001 BMI (kg/m 2 ) 21.18 ± 2.95 22.79 ± 4.04 < 0.001 Waist circumference (cm) 73.63 ± 8.23 78.74 ± 11.17 < 0.001 < 0.001 Waist-to-hip ratio 0.82 ± 0.06 0.85 ± 0.07 < 0.001 < 0.001 F-G score 0.22 ± 0.71 1.68 ± 2.03 < 0.001 < 0.001 Acne grade score 0.09 ± 0.29 0.60 ± 0.90 < 0.001 < 0.001 SBP (mmHg) 113.20 ± 11.72 114.09 ± 10.62 0.110 0.743 DBP (mmHg) 73.87 ± 9.25 75.51 ± 8.92 < 0.001 0.089 Ovarian volume (ml) 7.53 ± 2.84 9.90 ± 4.09 < 0.001 < 0.001 Values are presented as the mean ± SD P a  All comparisons were corrected for differences in age and BMI between the two groups except the parameters of age and BMI\nClinical characteristics of PCOS patients and controls\nValues are presented as the mean ± SD\nP a  All comparisons were corrected for differences in age and BMI between the two groups except the parameters of age and BMI\nAs shown in Table  1 , BMI, waist circumference, waist-to-hip ratio, F-G score, acne grade score, and average ovarian volume were significantly increased, and age was significantly decreased in the PCOS group compared with the control group.\nTT and LH levels, the ratio of LH to FSH, fasting insulin concentration, HOMA index, TG, TC, LDL-C, non-HDL-C and apoB levels, AI, the ratio of apoB to apoA1, and the ratio of apoB-PAF-AH to H-PAF-AH were significantly increased, whereas FSH and HDL-C levels and H-PAF-AH activity were significantly reduced in the PCOS group compared with the control group (Table  2 ). Table 2 Hormonal levels, metabolic profile and PAF-AH activity in PCOS patients and controls Controls ( n  = 506) PCOS ( n  = 575) \n P \n \n P \n a \n Age (years) 27.96 ± 4.19 24.66 ± 3.96 < 0.001 BMI (kg/m 2 ) 21.10 ± 2.91 23.10 ± 4.26 < 0.001 Hormonal levels E 2  (pmol/L) 333.95 ± 351.36 284.06 ± 271.26 0.014 0.373 TT (nmol/L) 1.54 ± 0.54 2.42 ± 0.77 < 0.001 < 0.001 LH (IU/L) 8.72 ± 11.33 13.89 ± 10.91 < 0.001 < 0.001 FSH (IU/L) 6.67 ± 2.98 5.99 ± 2.23 < 0.001 0.015 LH/FSH 1.30 ± 1.32 2.35 ± 1.26 < 0.001 < 0.001 Metabolic profile Fasting Ins (pmol/L) 66.98 ± 36.74 106.08 ± 74.24 < 0.001 < 0.001 Fasting Glu (mmol/L) 5.30 ± 0.73 5.37 ± 0.77 0.110 0.515 HOMA-IR 2.34 ± 1.90 3.79 ± 3.19 < 0.001 0.006 TG (mmol/L) 1.04 ± 0.90 1.42 ± 1.36 < 0.001 < 0.001 TC (mmol/L) 4.25 ± 0.70 4.41 ± 0.81 < 0.001 < 0.001 HDL-C (mmol/L) 1.51 ± 0.32 1.38 ± 0.35 < 0.001 0.006 LDL-C (mmol/L) 2.35 ± 0.61 2.55 ± 0.76 < 0.001 < 0.001 non-HDL-C (mmol/L) 2.73 ± 0.64 3.02 ± 0.81 < 0.001 < 0.001 AI 1.90 ± 0.68 2.37 ± 1.02 < 0.001 < 0.001 ApoA1 (g/L) 1.45 ± 0.21 1.42 ± 0.21 0.005 0.725 ApoB (g/L) 0.75 ± 0.17 0.82 ± 0.20 < 0.001 < 0.001 ApoB/apoA1 0.53 ± 0.14 0.60 ± 0.18 < 0.001 < 0.001 PAF-AH activity a Plasma PAF-AH (nmol/min/ml) 48.39 ± 10.81 47.44 ± 12.94 0.344 0.722 H-PAF-AH (nmol/min/ml) 5.14 ± 1.51 4.67 ± 1.91 0.001 0.031 ApoB-PAF-AH (nmol/min/ml) 43.25 ± 9.96 42.77 ± 11.89 0.604 0.967 ApoB-PAF-AH/H-PAF-AH 8.86 ± 2.48 10.04 ± 3.89 < 0.001 < 0.001 Values are presented as the mean ± SD P a  All comparisons of parameters were corrected for differences in age and BMI between the two groups except the parameters of age and BMI a  Control ( n  = 277), PCOS ( n  = 281)\nHormonal levels, metabolic profile and PAF-AH activity in PCOS patients and controls\nValues are presented as the mean ± SD\nP a  All comparisons of parameters were corrected for differences in age and BMI between the two groups except the parameters of age and BMI\na  Control ( n  = 277), PCOS ( n  = 281)\nGenotypic distributions of  apoC1 rs4420638A/G  and  -317H1/H2  were in Hardy-Weinberg equilibrium in the PCOS and control groups. No significant differences were observed in the frequencies of the  apoC1 rs4420638A/G  and  -317H1/H2  genotypes and alleles between PCOS and control groups ( P  > 0.05, Table  3 ). Table 3 Frequencies of the  apoC1  genotype and allele in PCOS patients compared with controls Controls (n = 761) PCOS (n = 877) \n X \n 2 \n \n P \n Genotype  -317 \n H1H1 \n 483 (63.5%) 570 (65.0%) \n H1H2 \n 245 (32.2%) 269 (30.7%) \n H2H2 \n 33 (4.3%) 38 (4.3%) 0.448 0.799  rs4420638 \n AA \n 578 (76.0%) 683 (77.9%) \n AG \n 176 (23.1%) 182 (20.8%) \n GG \n 7 (0.9%) 12 (1.4%) 1.954 0.375 Allele frequency  -317 \n H1 \n 0.796 0.803 \n H2 \n 0.204 0.197 0.297 0.586  rs4420638 \n A \n 0.875 0.883 \n G \n 0.125 0.117 0.419 0.518 Genotype data are presented as the number (%) of patients or controls\nFrequencies of the  apoC1  genotype and allele in PCOS patients compared with controls\nGenotype data are presented as the number (%) of patients or controls\nThe degree of linkage disequilibrium (LD) among the  apoE ε2/ε3/ε4 , and the  apoC1 rs4420638A/G and -317H1/H2  genetic polymorphisms was analyzed in patients with PCOS and the controls, with all found to be in moderate to reasonably high LD with each other:  apoC1–317H1/H2  loci and  apoE ε2/ε3/ε4  loci (D’ = 0.970, r 2  = 0.702),  apoC1–317H1/H2  loci and  rs4420638A/G  loci (D’ = 0.910, r 2  = 0.455), and  apoC1 rs4420638A/G  loci and  apoE ε2/ε3/ε4  loci (D’ = 0.758, r 2  = 0.397).\nBecause the sample sizes of the  rs4420638GG  or  -317H2H2  homozygotes were too small, we combined them into the heterozygous subgroups.\nAs shown in Table  4 , compared with  AA  homozygotes,  G  allele carriers ( AG  +  GG ) of the  rs4420638  polymorphism had significantly higher apoB levels and a higher ratio of apoB to apoA1 in patients with PCOS or the controls ( P  < 0.05). Patients with the  G  allele had higher serum LH, TC, LDL-C and non-HDL-C levels ( P  < 0.05), and tended to have a reduced waist-to-hip ratio, DBP and fasting insulin levels ( P  < 0.100) and an increased ratio of LH to FSH ( P  = 0.074) compared with patients with the  AA  genotype. The controls with the  G  allele had higher fasting insulin and glucose concentrations, HOMA indexes, AI, plasma PAF-AH and apoB-PAF-AH activity ( P  < 0.05), and tended to have increased LDL-C and non-HDL-C levels ( P  < 0.080) and decreased HDL-C concentrations ( P  < 0.070) than the controls with the  AA  genotype. Table 4 Clinical characteristics, hormonal levels, metabolic profile and PAF-AH activity of the  apoC1 rs4420638  genotypes in PCOS patients and controls Controls PCOS AA  ( n  = 379) AG + GG  ( n  = 121 + 6) AA  ( n  = 435) AG + GG  ( n  = 134 + 6) Age (yr) 28.18 ± 4.10 27.28 ± 4.41 a 24.68 ± 4.01 a, b 24.60 ± 3.81 a, b BMI (kg/m 2 ) 21.17 ± 2.96 20.88 ± 2.74 23.24 ± 4.32 a, b 22.68 ± 4.03 a, b Waist circumference (cm) 73.41 ± 8.21 73.36 ± 8.36 80.13 ± 11.67 a, b 77.96 ± 11.22 a, b Waist-to-hip ratio 0.81 ± 0.06 0.82 ± 0.06 0.86 ± 0.07 a, b 0.84 ± 0.07 a, b F-G score 0.22 ± 0.69 0.26 ± 0.82 1.73 ± 2.07 a, b 1.71 ± 2.08 a, b Acne grade score 0.10 ± 0.30 0.08 ± 0.27 0.66 ± 0.94 a, b 0.53 ± 0.86 a, b SBP (mmHg) 113.42 ± 11.55 112.98 ± 11.84 114.89 ± 10.42 a 113.78 ± 11.65 DBP (mmHg) 73.97 ± 8.88 73.40 ± 8.75 76.35 ± 9.08 a, b 74.70 ± 8.99 Ovarian volume (ml) 7.88 ± 2.76 7.47 ± 3.24 10.22 ± 3.92 a, b 9.95 ± 4.63 a, b Hormonal levels E 2  (pmol/L) 322.08 ± 341.61 367.37 ± 376.94 281.07 ± 279.42 b 293.07 ± 245.85 TT (nmol/L) 1.54 ± 0.54 1.53 ± 0.56 2.41 ± 0.77 a, b 2.45 ± 0.77 a, b LH (IU/L) 8.53 ± 10.38 9.28 ± 13.76 13.24 ± 8.57 a, b 15.85 ± 15.91 a, b, c FSH (IU/L) 6.59 ± 2.84 6.89 ± 3.34 5.95 ± 2.27 a, b 6.12 ± 2.11 b LH/FSH 1.31 ± 1.24 1.29 ± 1.54 2.28 ± 1.18 a, b 2.58 ± 1.45 a, b Metabolic profile Fasting Ins (pmol/L) 64.87 ± 32.78 73.28 ± 46.17 a 109.10 ± 77.82 a, b 96.60 ± 60.98 a, b Fasting Glu (mmol/L) 5.26 ± 0.46 5.42 ± 1.22 a 5.38 ± 0.71 a 5.36 ± 0.93 a HOMA-IR 2.22 ± 1.22 2.73 ± 3.13 a 3.89 ± 3.31 a, b 3.46 ± 2.80 a, b TG (mmol/L) 1.02 ± 0.93 1.08 ± 0.76 1.46 ± 1.50 a, b 1.29 ± 0.73 a TC (mmol/L) 4.22 ± 0.71 4.31 ± 0.66 4.35 ± 0.80 a 4.57 ± 0.82 a, b, c HDL-C (mmol/L) 1.52 ± 0.32 1.48 ± 0.29 1.37 ± 0.34 a, b 1.42 ± 0.36 a, b LDL-C (mmol/L) 2.32 ± 0.61 2.44 ± 0.60 2.51 ± 0.75 a 2.66 ± 0.77 a, b, c non-HDL-C (mmol/L) 2.70 ± 0.63 2.83 ± 0.65 2.98 ± 0.81 a, b 3.14 ± 0.78 a, b, c AI 1.87 ± 0.65 2.02 ± 0.74 a 2.37 ± 1.05 a, b 2.37 ± 0.91 a, b ApoA1 (g/L) 1.46 ± 0.22 1.44 ± 0.18 1.41 ± 0.21 a 1.43 ± 0.22 a ApoB (g/L) 0.74 ± 0.17 0.78 ± 0.17 a 0.81 ± 0.20 a, b 0.85 ± 0.20 a, b, c ApoB/apoA1 0.52 ± 0.14 0.55 ± 0.16 a 0.59 ± 0.18 a, b 0.61 ± 0.19 a, b, c PAF-AH activity d Plasma PAF-AH (nmol/min/ml) 47.80 ± 10.91 50.08 ± 10.39 a 47.54 ± 12.27 b 47.05 ± 15.17 H-PAF-AH (nmol/min/ml) 5.09 ± 1.51 5.30 ± 1.52 4.64 ± 1.89 a, b 4.77 ± 2.01 ApoB-PAF-AH (nmol/min/ml) 42.74 ± 10.09 44.78 ± 9.50 a 42.91 ± 11.34 42.28 ± 13.74 ApoB-PAF-AH/H-PAF-AH 8.85 ± 2.61 8.88 ± 2.09 10.91 ± 4.17 a, b 9.48 ± 2.60 Values are presented as the mean ± SD Comparisons of all parameters were corrected for differences in age and BMI between the two subgroups except the parameters of age and BMI a \n P  < 0.05, compared with  AA  genotype subgroup in controls b P  < 0.05, compared with  AG + GG  genotype subgroup in controls c P  < 0.05, compared with  AA  genotype subgroup in PCOS patients d  Controls:  AA  ( n  = 207),  AG + GG  ( n  = 67 + 3); PCOS:  AA  ( n  = 219),  AA + AG  ( n  = 60 + 2)\nClinical characteristics, hormonal levels, metabolic profile and PAF-AH activity of the  apoC1 rs4420638  genotypes in PCOS patients and controls\nValues are presented as the mean ± SD\nComparisons of all parameters were corrected for differences in age and BMI between the two subgroups except the parameters of age and BMI\na \n P  < 0.05, compared with  AA  genotype subgroup in controls\nb P  < 0.05, compared with  AG + GG  genotype subgroup in controls\nc P  < 0.05, compared with  AA  genotype subgroup in PCOS patients\nd  Controls:  AA  ( n  = 207),  AG + GG  ( n  = 67 + 3); PCOS:  AA  ( n  = 219),  AA + AG  ( n  = 60 + 2)\nCompared with patients with the  H1H1  genotype, patients with the  H2  allele ( H1H2  +  H2H2 ) in  -317H1/H2  polymorphism had lower acne grade score and the ratio of apoB-PAF-AH to H-PAF-AH ( P  < 0.05). The controls with the  H2  allele had lower TC and LDL-C levels ( P  < 0.05), and tended to have increased waist-to-hip ratio and HOMA index ( P  < 0.09) and reduced non-HDL-C and apoB levels ( P  < 0.09) than the controls with the  H1H1  genotype (Table  5 ). Table 5 Clinical characteristics, hormonal levels, metabolic profile and PAF-AH activity of the  apoC1–317H1/H2  genotypes in PCOS patients and controls Controls PCOS H1H1  ( n  = 318) H1H2 + H2H2  ( n  = 163 + 25) H1H1  ( n  = 370) H1H2 + H2H2  ( n  = 177 + 28) Age (yr) 28.17 ± 4.17 27.56 ± 4.21 24.68 ± 4.03 a, b 24.63 ± 3.84 a, b BMI (kg/m 2 ) 21.18 ± 2.95 20.91 ± 2.81 22.94 ± 4.23 a, b 23.41 ± 4.29 a, b Waist circumference (cm) 73.36 ± 7.96 73.35 ± 8.61 79.35 ± 11.36 a, b 80.06 ± 12.02 a, b Waist-to-hip ratio 0.81 ± 0.06 0.82 ± 0.06 0.85 ± 0.07 a, b 0.85 ± 0.08 a, b F-G score 0.25 ± 0.76 0.19 ± 0.68 1.77 ± 2.06 a, b 1.63 ± 2.10 a, b Acne grade score 0.11 ± 0.31 0.07 ± 0.26 0.68 ± 0.96  a, b 0.52 ± 0.86 a, b, c SBP (mmHg) 113.32 ± 11.26 113.32 ± 12.22 114.36 ± 10.43 a 115.08 ± 11.27 a DBP (mmHg) 73.81 ± 8.89 73.78 ± 8.76 76.27 ± 9.08 a, b 75.38 ± 9.06 a Ovarian volume (ml) 7.80 ± 2.86 7.71 ± 2.99 10.15 ± 3.99 a, b 10.15 ± 4.32 a, b Hormonal levels E 2  (pmol/L) 324.35 ± 341.91 349.79 ± 366.62 283.87 ± 285.58 284.41 ± 244.71 TT (nmol/L) 1.55 ± 0.53 1.52 ± 0.56 2.41 ± 0.77 a, b 2.43 ± 0.77 a, b LH (IU/L) 8.80 ± 11.33 8.61 ± 11.40 13.65 ± 9.01 a, b 14.33 ± 13.68 a, b FSH (IU/L) 6.64 ± 3.02 6.71 ± 2.92 5.94 ± 1.89 a, b 6.08 ± 2.74 a, b LH/FSH 1.32 ± 1.26 1.28 ± 1.41 2.32 ± 1.20 a, b 2.42 ± 1.36 a, b Metabolic profile Fasting Ins (pmol/L) 65.53 ± 34.31 69.27 ± 40.51 105.28 ± 75.66 a, b 107.54 ± 71.75 a, b Fasting Glu (mmol/L) 5.27 ± 0.44 5.35 ± 1.06 5.39 ± 0.84 a 5.35 ± 0.62 HOMA-IR 2.24 ± 1.27 2.51 ± 2.64 3.80 ± 3.40 a, b 3.77 ± 2.79 a, b TG (mmol/L) 1.03 ± 1.00 1.05 ± 0.68 1.41 ± 1.47 a, b 1.45 ± 1.13 a, b TC (mmol/L) 4.30 ± 0.68 4.15 ± 0.72 a 4.41 ± 0.81 a, b 4.39 ± 0.82 b HDL-C (mmol/L) 1.52 ± 0.32 1.49 ± 0.31 1.39 ± 0.34 a, b 1.37 ± 0.35 a, b LDL-C (mmol/L) 2.41 ± 0.57 2.26 ± 0.66 a 2.57 ± 0.76 a, b 2.51 ± 0.77 a, b non-HDL-C (mmol/L) 2.78 ± 0.60 2.66 ± 0.70 3.03 ± 0.82 a, b 3.02 ± 0.80 a, b AI 1.92 ± 0.65 1.88 ± 0.73 2.37 ± 1.03 a, b 2.37 ± 1.00 a, b ApoA1 (g/L) 1.46 ± 0.22 1.44 ± 0.19 1.42 ± 0.21 a 1.41 ± 0.22 a, b ApoB (g/L) 0.76 ± 0.15 0.73 ± 0.18 0.83 ± 0.21 a, b 0.81 ± 0.20 a, b ApoB/apoA1 0.53 ± 0.13 0.52 ± 0.17 0.60 ± 0.18 a, b 0.59 ± 0.18 a, b PAF-AH activity d Plasma PAF-AH (nmol/min/ml) 49.20 ± 10.27 47.20 ± 11.50 48.01 ± 12.50 46.39 ± 13.71 H-PAF-AH (nmol/min/ml) 5.12 ± 1.46 5.17 ± 1.59 4.61 ± 1.98 a, b 4.76 ± 1.80 ApoB-PAF-AH (nmol/min/ml) 44.08 ± 9.40 42.03 ± 10.67 43.39 ± 11.49 41.63 ± 12.56 ApoB-PAF-AH/H-PAF-AH 9.07 ± 2.59 8.55 ± 2.29 10.42 ± 4.37 a, b 9.33 ± 2.69 c Values are presented as the mean ± SD Comparisons of all parameters were corrected for differences in age and BMI between the two subgroups except the parameters of age and BMI a \n P  < 0.05, compared with  AA  genotype subgroup in controls b P  < 0.05, compared with  H1H2 + H2H2  genotype subgroup in controls c P  < 0.05, compared with  AA  genotype subgroup in PCOS patients d Controls:  H1H1  ( n  = 165),  H1H2 + H2H2  ( n  = 97 + 15); PCOS:  H1H1  ( n  = 182),  H1H2 + H2H2  ( n  = 86 + 13)\nClinical characteristics, hormonal levels, metabolic profile and PAF-AH activity of the  apoC1–317H1/H2  genotypes in PCOS patients and controls\nValues are presented as the mean ± SD\nComparisons of all parameters were corrected for differences in age and BMI between the two subgroups except the parameters of age and BMI\na \n P  < 0.05, compared with  AA  genotype subgroup in controls\nb P  < 0.05, compared with  H1H2 + H2H2  genotype subgroup in controls\nc P  < 0.05, compared with  AA  genotype subgroup in PCOS patients\nd Controls:  H1H1  ( n  = 165),  H1H2 + H2H2  ( n  = 97 + 15); PCOS:  H1H1  ( n  = 182),  H1H2 + H2H2  ( n  = 86 + 13)\nAdditionally, patients with the respective AA and AG + GG genotypes of the rs4420638 polymorphism or the respective  H1H1  and  H1H2+ H2H2  genotypes of the  -317H1/H2  polymorphism were more obese and had a higher F-G score, acne grade score and average ovarian volume, and more adverse hormonal, glucose and lipid metabolic profiles compared with the corresponding control subgroups. Patients with the  AA  or  H1H1  genotype had a decreased H-PAF-AH activity and an increased ratio of apoB-PAF-AH to H-PAF-AH compared with the corresponding  AA  or  H1H1  control subgroups ( P  < 0.05, Tables  4  and  5 ).\nThe associations of  apoC1  allele status with  apoE ε2/ε3/ε4  allele status were observed in patients with PCOS and controls, as shown in Tables  6 . A strong association of the  apoC1 H2  allele with the  apoE ε2  or  apoE ε4  allele and of the  apoC1 rs4420638 G  allele with the apo ε4  allele was revealed in patients with PCOS and controls. Of the  apoE ε2  alleles evaluated, 273 (100%) were associated with  apoC1 H2  alleles. Of the 319  apoE ε4  alleles, 303 (95%) were  H2  alleles or  rs4420638 G  alleles and only 16 (5%) were  H1  alleles or  rs4420638 A  alleles. In contrast, the  apoE ε3  allele was strongly associated with the  apoC1 H1 or rs4420638 A  alleles .  Of the 2684 apo ε3  alleles, 2604 (97%) were  H1  alleles and 2594 (96.6%) were  rs4420638 A  alleles; however, only 80 (3%) were  H2  alleles and 90 (3.4%) were  rs4420638 G  alleles. Nevertheless, these differences were not statistically significant for patients with PCOS versus the controls ( P  > 0.05, Table  6 ). Table 6 ApoC1/apoE  haplotypes in patients with PCOS and controls ApoE  controls/cases \n ApoC1–317 \n \n ApoC1 rs4420638 \n \n H1 \n \n H2 \n \n P \n \n A \n \n G \n \n P \n \n ε2 \n  Controls 0 131 (100%) 131 (100%) 0  PCOS 0 142 (100%) – 139 (97.9%) 3 (2.1%) 0.094  Total 0 273 (100%) 270 (98.9%) 3 (1.1%) \n ε3 \n  Controls 1203 (96.7%) 41 (3.3%) 1194 (96.0%) 50 (4.0%)  PCOS 1401 (97.3%) 39 (2.7%) 0.372 1400 (97.2%) 40 (2.8%) 0.075  Total 2604 (97.0%) 80 (3.0%) 2594 (96.6%) 90 (3.4%) \n ε4 \n  Controls 8 (5.4%) 139 (94.6%) 7 (4.8%) 140 (95.2%)  PCOS 8 (4.7%) 164 (95.3%) 0.747 9 (5.2%) 163 (94.8%) 0.848  Total 16 (5.0%) 303 (95.0%) 16 (5.0%) 303 (95.0%) Haplotype data are presented as the number (%) of patients or controls\nApoC1/apoE  haplotypes in patients with PCOS and controls\nHaplotype data are presented as the number (%) of patients or controls\n\nIn this study, we show that  apoC1 rs4420638A/G  and  -317H1/H2  genetic polymorphisms are not associated with a risk of PCOS in Chinese women. However, compared with patients carrying the  AA  genotype, patients carrying the  G  allele ( AG  +  GG ) of the  rs4420638  polymorphism had significantly higher LH, TC, LDL-C, non-HDL-C and apoB levels, and a higher ratio of apoB to apoA1, and tended to have an increased ratio of LH to FSH. This suggests that this polymorphism may potentially be linked to endocrine abnormalities of the reproductive axis and dyslipidemia in the patients. In addition, we found a higher acne grade score and an increased ratio of apoB-PAF-AH to H-PAF-AH activity in patients carrying the  H1H1  genotype of the  -317H1/H2  polymorphism compared with patients carrying the  H2  allele. This suggests that the  -317H1/H2  genetic polymorphism is associated with systemic chronic inflammation in these patients. Furthermore, we demonstrated that the  rs4420638A/G  or  -317H1/H2  polymorphisms of the  apoC1  gene exist in moderate to reasonably high linkage disequilibrium with  apoE ε2/ε3/ε4  polymorphisms.\nSeveral studies have found that the  rs4420638A/G  polymorphism in the  apoC1  gene is associated with serum lipid levels, chronic inflammation and disease risk. The  G  allele of rs4420638 is associated with a higher level of TC, LDL-C, TG and PAF-AH activity, abdominal obesity, and increased risk of type 2 diabetes and coronary heart disease [ 13 – 17 ]. On the other hand, the  G  allele is associated with lower serum C-reactive protein (CRP) levels [ 13 ]. In this study, we found that the  G  allele carriers were associated with not only increased serum LDL-C levels but also elevated serum non-HDL-C and apoB levels, and the ratio of apoB to apoA1 than the  AA  homozygotes in patients with PCOS or the controls. Because apoB and the apoB/apoA-I ratio have been reported to be better predictors of cardiovascular diseases than traditional lipid measurements [ 4 ,  33 ], our results provide further evidence that the  A → G  variant of rs4420638 in the  apoC1  gene is associated with cardiovascular disease risk. We also demonstrated that the controls with the  G  allele had higher fasting insulin and glucose concentrations, HOMA indexes, plasma PAF-AH and apoB-PAF-AH activity than the  AA  homozygotic controls. Additionally, patients carrying the  G  allele tended to have a reduced waist-to-hip ratio than the  AA  homozygotic patients. Consistent with our findings, Avery et al. demonstrated that the  rs4420638A/G  polymorphism of the apoC1 gene was associated with elevated plasma glucose, atherogenic dyslipidemia, vascular inflammation, and central obesity in individuals of European descent [ 34 ]. Grallert et al. reported that the  A → G  variant of  rs4420638  was associated with increased plasma PAF-AH activity [ 17 ]. Furthermore, in this study, we demonstrated that the  apoC1 rs4420638 G  allele exist in strong linkage disequilibrium with the  apoE ε4  allele, which may partially explain the effect of the  rs4420638 G  allele on lipoprotein metabolism and disease risk. The  apoE ε4  allele was associated not only with higher serum TC and LDL-C levels but also with higher oxidative stress, a more pro-inflammatory state and increased risk of cardiovascular disease and late-onset Alzheimer’s disease [ 30 ,  35 ]. For the first time, our study determined that the PCOS patients with the  G  allele had higher LH levels and tended to have increased ratio of LH to FSH than in patients with the  AA  genotype, suggesting that the  A → G  variant of rs4420638 may potentially link to endocrine abnormalities of the reproductive axis in the patients. However, the effect of this gene variant on the reproductive axis must be investigated further.\nThe  -317H1/H2  polymorphism of the  apoC1  gene, which is localized to the transcription initiation site in the promoter region, has been identified to exist in linkage disequilibrium with the  ε2/ε3/ε4  polymorphisms of the  apoE  gene and influences lipoprotein metabolism [ 19 ]. The  H2  allele of the  apoC1  gene has been reported to cause a significant 1.5-fold increase in apoC1 gene transcription in a reporter-gene assay [ 19 ]. The  H2  allele disrupts the binding of a transcription suppressor to produce a positive effect on transcription [ 19 ]. This allele also correlated with development of Alzheimer’s disease [ 20 ] and familial dysbetalipoproteinemia [ 21 ]. In this study, we found that compared with  H1H1  homozygotes, the  H2  allele was associated with lower ratio of apoB-PAF-AH to H-PAF-AH and acne grade score in patients with PCOS. Furthermore, the  H2  allele decreased or tended to decrease TC, LDL-C, non-HDL-C and apoB levels, but tended to increase waist-to-hip ratio and HOMA index in the controls. We also determined that the  apoC1 H2  allele has complete linkage disequilibrium with the  apoE ε2  allele, and to a lesser extent, the  apoE ε4  allele in Chinese women. Consistent with our findings, Xu et al. reported that the  H2  allele was associated with a decrease or tending decrease in apoB and LDL-C levels compared with  H1H1  homozygotes in African-Americans with the  apoE ε3/ε3  genotype but not the  apoE ε3/ε4  genotype [ 19 ]. Similar to apoE2, modest levels of apoC1 expression may decrease hepatic secretion of VLDL and apoB and produce lower LDL levels due to decreased partitioning of dietary lipids to the liver, relating to delayed remnant clearance, especially in the  apoE ε2  and  apoE ε3  contexts; however, higher apoC1 levels may increase plasma lipid levels via decreased remnant clearance [ 19 ]. Expression of apoC1 is upregulated and serum apoC1 levels are higher in patients with PCOS compared with the controls [ 23 ]. This may be one of the reasons why serum lipid profiles in the  apoC1–317H1/H2  polymorphism are different between patients and controls. Plasma PAF-AH specifically hydrolyzes and inactivates PAF and PAF-like oxidized phospholipids that are potent pro-inflammatory mediators [ 36 ]. Given that H-PAF-AH, an antioxidant enzyme of HDL, plays anti-inflammatory role and apoB-PAF-AH is associated with inflammation, it has recently been suggested that an increased ratio of apoB-PAF-AH to H-PAF-AH might be a marker for chronic inflammation [ 31 ,  32 ,  36 ,  37 ]. These results suggest that the  H1  allele might relate to increased systemic chronic inflammation in patients with PCOS.\nIn addition, our study also showed that the  A → G  variant and the  CGTT  insertion ( H2 ) allele of  apoC1  appeared to have a greater impact on metabolic profile in the controls relative to in patients with PCOS (Tables  4  and  5 ). It have been demonstrated that in addition to genetic variations, other factors such as obesity, insulin resistance, etc., also have a significant impact on metabolic profile [ 4 ,  5 ,  38 ]. Since patients with PCOS are more obese and have a more severe insulin resistance compared with the controls in this study (Tables  1 ,  2 ,  4  and  5 ), it is possible that the effects of  apoC1  genetic variations on metabolic profile may be disturbed or weakened by these factors in patients. Further research is needed to clarify this issue.\nIn particular, the allele frequencies for  apoC1  and  apoE  genetic variants differ among ethnic groups, and these variants exhibit an ethnically distinct linkage disequilibrium pattern [ 19 ,  20 ,  30 ]. For example, the frequency of the  apoC1 H2  allele combined with  apoE ε2, ε3 and ε4  is 0.95, 0.02 and 0.85, respectively, in European-Americans; 1.00, 0.08 and 0.55, respectively, in African-Americans [ 19 ]; and 1.00, 0.03 and 0.95, respectively, in Chinese women. Therefore, it is possible that the relationships between the  apoC1  and  apoE  genetic polymorphisms and lipoprotein levels or diseases may differ among ethnic groups.\nWe should point out that this study has some limitations. Firstly, given the low frequency of homozygosity of minor alleles  rs4420638GG  and  -317H2H2 , we could not analyze them in the form of subgroups. A larger sample size of patients and controls are needed to properly evaluate dose-dependent genotype characteristics. Second, due to insufficient sampling, we did not measure plasma and lipoprotein-associated PAF-AH activity in some subjects, which might influence the statistical power of these parameters. Third, we could not determine plasma apoC1 concentrations due to plasma or serum sample inadequacy. Further study to detect apoC1 levels may help provide clues to the mechanisms responsible for the genetic association with PCOS.\n\nThe present study demonstrates that  apoC1 rs4420638A/G  and  -317H1/H2  genetic polymorphisms are not associated with the risk of PCOS in Chinese women. However, the  apoC1 rs4420638A → G  variation may potentially be linked to elevated serum LH levels, a relatively high ratio of LH to FSH, and an adverse lipid metabolic profile; and the  -317 H1H1  genotype is associated with increased the ratio of apoB-PAF-AH to H-PAF-AH activity and the acne grade score in the patients with PCOS. Additionally, this study also indicates that  apoC1 rs4420638A/G  and  -317H1/H2  genetic variations are associated with an adverse glucose and lipid metabolic profile and the  rs4420638G  allele increases plasma PAF-AH and apoB-PAF-AH activity in the control women. These results suggest that  apoC1 rs4420638A/G  and  -317H1/H2  genetic variants are related to metabolic abnormalities and chronic inflammation in patients and controls, while the  rs4420638A/G  polymorphism might be involved in endocrine abnormalities of reproductive axis in patients.","source_license":"CC-BY-4.0","license_restricted":false}