The Rotterdam consensus outlines the criteria for diagnosing PCOS as the presence of oligo- and/or anovulation, signs of hyperandrogenism (either clinical or biochemical), and the identification of polycystic ovaries [1]. The etiopathogenesis of PCOS is multifactorial and when it continues for a long time, it has many negative effects on health. Insulin resistance, hyperinsulinemia, deterioration in pancreatic beta cell functions and an increased risk of type 2 diabetes, which are common in PCOS, cause metabolic disorders such as obesity and hyperlipidemia. These disorders pave the way for cardiovascular diseases and increase the risk of endometrial cancer [2]. Many women with PCOS have elevated insulin levels. In the normal state, after feeding, insulin is secreted into the blood by the pancreas and in this way, glucose is taken into the cell, converted into energy or stored. The insensitivity of cells to insulin is called insulin resistance (IR). As the cells become insensitive to insulin, more insulin is secreted by pancreas. The increased level of insulin causes the ovaries to overproduce androgens, known as male hormones. This condition, called hyperandrogenism, prevents the ovaries from producing eggs and leads to menstrual irregularity, one of the most characteristic symptoms of PCOS. High androgen levels are also the cause of acne, male pattern baldness and hair growth [3]. This situation, which is formed by a high insulin level together with a high androgen level, increases the risk of diabetes and cardiological diseases in women with PCOS [4,5]. The fact that insulin levels are high in patients with PCOS brings to mind the investigation of molecules associated with insulin resistance and the genes that synthesize these molecules. One of the molecules associated with insulin resistance is fetuin-A (Alpha2-Heremans-Schmid Glycoprotein, AHSG), a 55–59 kDa glycoprotein that is synthesized from hepatocytes in the liver and released into the circulation in adult individuals [6]. Studies have shown that fetuin-A causes insulin resistance by binding to insulin receptor tyrosine kinase and weakening the signalling pathway [7]. As a matter of fact, it has been reported that the level of fetuin A is increased in patients with PCOS due to its relationship with insulin resistance [8–10].

The AHSG gene contains 7 exons and 6 introns and is located at the 3q27.3 position of chromosome 3. It has been reported that some polymorphisms on the AHSG gene are associated with Fetuin-A levels in serum [11,12]. It has been reported that the 4613 T>G polymorphism (rs2077119) in the promoter region of the AHSG gene is associated with the expression level of the gene and affects the serum fetuin A level. It was determined that the 767 G>C polymorphism (rs4918) in the 7th exon of the gene caused the Ser256Thr missense mutation in the protein product and affected the serum fetuin A level [13].

In this study, we aimed to investigate whether 4613 T>G and 767 G>C polymorphisms in the AHSG gene are associated with PCOS. According to the available literature, this is the first study to investigate AHSG gene polymorphisms in patients with PCOS.

This study adhered to the Declaration of Helsinki, and its protocols were approved by the Ethical Review Committee of Harran University under the number 76244175-050.04.04. All participants provided written informed consent before taking part in the study.

The women included in the study were women who were followed up in the Harran University Faculty of Medicine, Obstetrics, and Gynaecology Outpatient Clinic and diagnosed with PCOS according to the criteria of Rotterdam. The women included in the study and who gave consent to the study were divided into two groups: 122 patients diagnosed with PCOS and 124 healthy women. The exclusion criteria for all participants in the study were patients with congenital adrenal hyperplasia, hyperprolactinemia, an androgen-secreting tumour, or thyroid dysfunction, as well as those who had other known major diseases. All participants were subjected to clinical assessment, a detailed survey and laboratory investigations.

DNA Isolation, Genotyping and Analysis of Polymorphisms in DNA Samples by Real-Time PCR

Two millilitres of peripheral venous blood samples were withdrawn from all participants and transferred into EDTA (Ethylene Diamine Tetra Acetic Acid) tubes for DNA isolation. Total genomic DNA was isolated from whole blood leukocytes using the PureLink Genomic DNA Isolation Kit K1820-02 (Invitrogen, Inc., Carlsbad, CA).

In this study, 767 C>G (rs4918) and 4613 T>G (rs2077119) polymorphisms in the AHSG gene were genotyped by real-time PCR (Rotor-Gene Q Series, Qiagen) using "TaqMan SNP Genotyping Assays" primers and probes. Real-time PCR amplification was carried out in a final volume of 20 lL of reaction mixture, including 20 ng of genomic DNA, 5 mL of TaqManVR Universal PCR Master Mix, and 0.5 mL of the 40X TaqManVR assay. Thermal cycling conditions were initial denaturing at 95 C for 3 min, 40 cycles of 95 C for 15 s, and 60 C for 1 min. The Rotor-Gene Q Series Software Version Q 2.3.1 (Rotor-Gene Q Series, Qiagen) was used for allelic discrimination.

Characteristics of SNPs Studied in the AHSG Gene

Table 1: 767 C>G polymorphism of the AHSG gene.

Table 2: 4613 T>G polymorphisms of the AHSG gene.

For AHSG (rs4918, rs2077119) gene polymorphisms, genotype analysis was performed by real-time PCR according to the "Pre-designed TaqMan Single Nucleotide Polymorphism Genotyping Assays" system for each individual from 122 PCOS patients and 124 healthy controls. After the alleles of gene polymorphisms were marked with appropriate fluorescent dyes, the genotype determination of each sample was made according to the amplification curves. In the primer-probe set used, the polymorphic allele was marked with FAM (green) and the wild allele was marked with VIC (yellow). Negative and positive examples of wild and

polymorphic alleles are shown in Figures 1 and 2.

Figure 1:  Examples of positive and negative peaks (sigmoidal  curve is positive; horizontal curve is negative) marked with the Wild allele determinant VIC (Yellow) probe.

Figure 2:  Examples of positive and negative peaks (sigmoidal curve is positive; horizontal curve is negative) marked with the polymorphic allele-determining FAM (Green) probe.

The statistical analysis of the data gained from the study was carried out by the SPSS V20.0 package programme (IBM Corp., released 2011). IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.), and P < 0.05 in the 95% confidence interval was considered statistically significant. The conformity of the variables related to demographic and routine laboratory tests to the normal distribution was examined by Kolmogrov-Smirnov and Shapiro-Wilk tests. Numerical variables were presented as mean ± standard deviation. An independent-samples t-test was used to compare the variables with a normal distribution. In cases where the numerical distribution was abnormal, the Mann-Whitney U test was used. The Pearson chi-square test was used to examine the allele frequencies, the distribution of alleles among the genotypes, and the compatibility of the distribution with the expected values (Hardy-Weinberg equilibrium) in the AHSG gene. The possible risks of genotypes and alleles were determined by calculating the odds ratio.

This study was conducted on 122 women diagnosed with Polycystic Ovary Syndrome (PCOS) and 124 healthy controls were enrolled in the study. Demographic and laboratory results of PCOS and control groups are shown in Table 1.

Table 3: Demographic and laboratory results of PCOS and control groups.

As seen in Table 1, the mean age of the patient group was 24.81 (±3.20) and the mean age of the control group was 24.96 (±3.26). There was no statistically significant difference in age distribution between the patient and control groups (p > 0.05). When the patient and control groups were compared in terms of routine laboratory results, glucose, LH, LH/FSH ratio and total testosterone were found to be significantly higher in the patient group (p<0.001). HDL levels were found to be significantly lower in the PCOS group than in the control group (p = 0.001). When the patient and control groups were compared, no statistically significant difference was found in BMI, insulin, FSH, estradiol, progesterone, DHEAS, triglyceride, or LDL levels (p > 0.01).

In this study, two polymorphisms of the AHSG gene were studied in terms of genotype distributions and allele frequencies in the patient and control groups. The first of these polymorphisms is the 767C>G polymorphism of the FNDC5 gene (rs4918). The second one is the 4613T>G polymorphism (rs2077119). In terms of the AHSG gene 767C>G (rs4918) polymorphism, in the PCOS patient group, homozygous CC genotypes were detected with a rate of 62% in 76 patients, heterozygous CG genotypes with a rate of 33% in 40 patients, and homozygous GG genotypes with a rate of 5% in 6 patients. As for the control group, homozygous CC genotypes were found in 68 individuals with a rate of 55%, heterozygous CG genotypes in 46 individuals with a rate of 37%, and homozygous GG genotypes in 10 individuals with a rate of 8%. No statistically significant difference was found between the patient and control groups in terms of the genotype distribution of the AHSG gene 767C>G (rs4918) polymorphism. (p=0.18). The ratio and distribution of the AHSG gene 767 C>G polymorphism in the PCOS patient and control groups are shown in Table 4.

Table 4: Genotype distributions of the AHSG gene 767C>G polymorphism in PCOS patients and control groups.

The allele frequency in the PCOS patient group was 79% for the C allele and 21% for the G allele, while the C allele rate was 73% and the G allele rate was 27% in the control group. No statistically significant difference was found between the patient and control groups in terms of allele frequencies (p= 0.17). Allele frequencies of the AHSG gene 767 C>G polymorphism in the patient and control groups are shown in Table 5.

Table 5: Allele frequencies of the AHSG gene 767 C>G polymorphism in the PCOS patient and control groups.

In terms of the AHSG gene 767C>G (rs4918) polymorphism, in the PCOS patient group, homozygous TT genotype was observed with a rate of 29% in 35 patients, heterozygous TG genotype was observed with a rate of 49% in 60 patients, and homozygous GG genotype was observed with a rate of 22% in 27 patients. In the control group, homozygous TT genotype was observed with a rate of 21% in 26 individuals, heterozygous TG genotype was observed with a rate of 49% in 61 individuals, and homozygous GG genotype was observed with a rate of 30% in 37 individuals. The distribution of the AHSG gene 4613 T>G polymorphism over the PCOS patient and control groups is shown in Table 6.

Table 6: Genotype distribution of the AHSG gene 4613 T>G polymorphism in the PCOS patient and control groups.

(Pearson Chi-Square test was applied)

Allele frequency in the PCOS patient group was 53% and G allele frequency was 47%. In the control group T allele frequency was 46% and G allele frequency was 64%. As a result, there was no statistically significant difference in the distribution of alleles in the patient and control groups (p=0.09). The allele distribution frequency of the AHSG gene 4613 T>G polymorphism in the PCOS patient and control groups is shown in Table 7.

Table 7: Allele frequencies of the AHSG gene 4613 T>G polymorphism in the PCOS patient and control groups.

Although PCOS is an endocrinological disease seen in women of reproductive age, it is also a metabolic disorder that may bring along long-term risks such as hypertension, diabetes and coronary artery disease. High insulin levels, insulin resistance and obesity are common in patients with PCOS and are seen as the most important factors in the development of long-term risks, especially diabetes. The mechanisms by which the negative metabolic consequences of insulin resistance and obesity occur are still unclear. It is thought that insulin sensitivity decreases by 35-40% in patients with PCOS, and this situation plays an important role in the pathogenesis of the disease [14]. The presence of insulin resistance in patients with PCOS suggests that molecules that are effective in the formation of insulin resistance may also be effective on PCOS. As a matter of fact, there are studies reporting that the increase in the level of fetuin A, one of the molecules associated with insulin resistance, is associated with the development of PCOS [8-10]. Enli et al. suggested that increased fetuin A levels in women with PCOS are associated with insulin resistance and ovarian hyperandrogenism, and that fetuin A may trigger insulin resistance and androgen increase [9]. The study by Liu et al. indicated that PCOS patients had elevated serum levels of fetuin-A. According to their results, there was a correlation between circulating concentrations of fetuin-A and dyslipidemia, insulin resistance (IR), and ovarian hyperandrogenism in women with PCOS [15]. It is known that some polymorphisms in the AHSG gene, which is responsible for the synthesis of fetuin A, have an effect on the functional activity and serum level of the relevant molecule. There are studies reporting that these polymorphisms are associated with type 2 diabetes, gestational diabetes and the metabolic syndrome through insulin resistance [16-18].

In this study, we investigated whether two important polymorphic regions in the AHSG gene are effective in treating PCOS. One of these polymorphisms is 767 C>G, which causes amino acid changes in the synthesised protein, and the other is the 4613 T>G polymorphism located in the promoter region of the gene. The AHSG 767 C>G polymorphism is located in the 7th exon of the gene and causes tyrosine amino acid replacement for serine at the 256th position of the synthesised protein. In previous studies, this polymorphism was found to be associated with many diseases. In one of these studies, Stenvinkel et al. found that the 767 G allele was more common in underweight Swedish men [11]. Siddig et al. found that the rs1071592 polymorphism in the AHSG gene was associated with type 2 diabetes, and they also found that the rs4918 (767 C>G) polymorphism that we discussed in our study was also related to type 2 diabetes [19]. Akbas et al. On the other hand, they suggested that the minor homozygous GG genotype was seen more frequently in patients with gestational diabetes compared to the control group and may have a protective effect against the development of gestational diabetes [16]. Temesszentandrási et al. found that high leptin levels and low TNF alpha and adiponectin levels were associated with 767 "G" variants in the healthy study group, and this variant was associated with positive obesity parameters in the group with a history of myocardial infarction [20].

The second polymorphism we examined in our study is the 4613 T>G polymorphism located in the promoter region of the AHSG gene. Andersen et al. reported that this polymorphism is associated with type 2 diabetes [21]. Teo et al. reported the AHSG gene 4613 T>G polymorphism as a SNP associated with obesity and metabolic syndrome [22]. Dahlman et al. found that the 4613 T>G polymorphism of the AHSG gene was associated with inhibition of insulin-mediated lipolysis [23].

In the study group with PCOS, we found that the genotype distribution and allele frequencies of the relevant polymorphisms were not associated with PCOS. In addition, although polymorphisms may have effects on gene expression and ultimately on fetuin A plasma levels, as mentioned in the above studies, they may not have an effect on the development of PCOS. The limited sample size in the study population and the fact that the polymorphic variants in question were not compared with the expression of the PCOS gene and fetuin Plasma levels are the main limitations of the study.

According to the data obtained as a result of our study, we found that the genotype distributions of the AHSG gene 767 C>G (rs4918) polymorphism and 4613 T>G (rs2077119) polymorphism was not associated with PCOS.  These results need to be confirmed by studies with higher sample volumes of different populations.

Acknowledgments

This study was supported by HUBAK (Scientific Researches Coordinating Council of Harran University) with project number of 19104

Conflicts of interest

The authors declare that they have no conflicts of interest.

1. Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod. 2004; 19: 41-47.
2. Kousta E, Efstathiadou Z, Lawrence NJ, Jeffs JAR, Godsland IF, Barrett SC, et al. The impact of ethnicity on glucose regulation and the metabolic syndrome following gestational diabetes. Diabetologia. 2006; 49: 36-40.
3. Yen BS. Jaffe’s Reproductive Endocrinology: Physiology, Pathophysiology, and Clinical Management. 5th.ed. Elsevier Saunders Publishing, p.597–632. Berlin, 2001.
4. Harris C, Cheung T. PCOS Diet Book. Thorsons Publishing, London. 2002; p: 368.
5. Hickey JT, Hickey L, Yancy WS, Hepburn J, Westman EC. Clinical use of acarbohydrate-restricted diet to treat the dyslipidemia of the metabolic syndrome. MetabSyndr Relat Disord. 2003; 1: 227-232.
6. Goustin AS, Abou-Samra AB. The "thrifty" gene encoding Ahsg/Fetuin-A meets the insulin receptor: Insights into the mechanism of insulin resistance. Cellular Signalling. 2011; 23: 980-990.
7. Mathews ST, Rakhade S, Zhou XH, Parker GC, Coscina DC, Grunberger G. Fetuin-null mice are protected against obesity and insulin resistance associated with aging. Biochem Biophys Res Commun. 2006; 350: 437–443.
8. Sak S, Uyanikoglu H, Incebiyik A, Hilali NG, Sabuncu T, Sak E, et al. Associations of serum fetuin-A and oxidative stress parameters with polycystic ovary syndrome. Clin Exp Reprod Med. 2018; 45: 116-121.
9. Enli Y, Fenkci SM, Fenkci V, Oztekin O. Serum Fetuin-A levels, insulin resistance and oxidative stress in women with polycystic ovary syndrome. Gynecol Endocrinol. 2013; 29: 1036-1039.
10. Abali R, Celik C, Tasdemir N, Guzel S, Alpsoy S, Yuksel A, et al. The serum protein α2-Heremans-Schmid glycoprotein/fetuin-a concentration and carotid intima-media thickness in women with polycystic ovary syndrome. Eur J Obstet Gynecol Reprod Biol. 2013; 169: 45-49.
11. Stenvinkel P, Pecoits-Filho R, Lindholm B; DialGene Consortium. Gene polymorphism association studies in dialysis: the nutrition-inflammation axis. Semin Dial. 2005; 18: 322-330.
12. Osawa M, Yuasa I, Kitano T, Henke J, Kaneko M, Udono T, Saitou N, Umetsu K. Haplotype analysis of the human alpha2-HS glycoprotein(fetuin) gene. Ann Hum Genet. 2001; 65: 27-34.
13. Inoue M, Takata H, Ikeda Y, Suehiro T, Inada S, Osaki F, et al. A promoter polymorphism of the alpha2-HS glycoprotein gene is associated with its transcriptional activity. Diabetes Res Clin Pract. 2008; 79: 164-170.
14. Azziz R, Carmina E, Dewailly D, Diamanti-Kandarakis E, Escobar-Morreale HF, Futterweit W, et al. The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril. 2009; 91: 456-88.
15. Liu S, Hu W, He Y, Li L, Liu G, Gao L, et al. Serum Fetuin-A levels are increased and associated with insulin resistance in women with polycystic ovary syndrome. BMC Endocrine Disorders. 2020; 20: 67.
16. Voros K, Graf L, Jr, Prohaszka Z, Graf L, Szenthe P, Kaszas E, et al. Serum fetuin-A in metabolic and inflammatory pathways in patients with myocardial infarction. European Journal of Clinical Investigation. 2011; 41: 703–709.
17. Akbas H, Kahraman S, Sak S, Akkafa F. Minor variant of AHSG gene 767C>G polymorphism may decrease the risk of gestational diabetes mellitus. J Obstet Gynaecol. 2020; 40: 303-307.
18. Xu Y, Xu M, Bi Y, Song A, Huang Y, Liu Y, et al. Serum fetuin-A is correlated with metabolic syndrome in middle-aged and elderly Chinese. Atherosclerosis. 2011; 216: 180–186.
19. Siddiq A, Lepretre F, Hercberg S, Froguel P, Gibson F. A synonymous coding polymorphism in the alpha2-Heremans-schmid glycoprotein gene is associated with type 2 diabetes in French Caucasians. Diabetes. 2005; 8: 2477–2481.
20. Temesszentandrasi GVK, Markus B, Borocz Z, Kaszas E, Prohaszka Z, Falus A, et al. Human fetuin-A Rs4918 polymorphism and its association with obesity in healthy persons and in patients with myocardial infarction in two Hungarian Cohorts. Medical Science Monitor. 2016; 22: 2742–2750.
21. Andersen G, Burgdorf KS, Sparso T, Borch-Johnsen K, Jorgensen T, Hansen T, et al. AHSG tag single nucleotide polymorphisms associate with type 2 diabetes and dyslipidemia: studies of metabolic traits in 7,683 white Danish subjects. Diabetes. 2008; 57: 1427-1432.
22. Teo AK, Gupta MK, Doria A, Kulkarni RN. Dissecting diabetes/metabolic disease mechanisms using pluripotent stem cells and genome editing tools. Mol Metab. 2015; 4: 593-604. Published 2015 Jun 20.
23. Dahlman I, Eriksson P, Kaaman M, Jiao H, Lindgren CM, Kere L, et al. Alpha2-Heremans-Schmid glycoprotein gene polymorphisms are associated with adipocyte insulin action. Diabetologia. 2004; 47: 1974-1979.