Archives of Clinical Microbiology

Keywords

Receptor; Insulin genetics; Adiponectin genetics; Polycystic ovary syndrome; Polymorphism

Background

It is known that PCOS is associated with increased risks of infertility, impaired glucose tolerance, type 2 diabetes mellitus, and metabolic syndrome [1]. While 6%-17% of reproductive-age women worldwide suffer PCOS [2,3] some ethnicities, such as South Asian, have higher incidence rates [4]. Etiological studies demonstrated significance of genetic susceptibility for PCOS pathogenesis [5]. Central adiposity play an important role in the insulin resistance of the metabolic syndrome via deregulated production of various adipocytederived cytokines and proteins (adipocytokines), including tumor necrosis factor plasminogen activator inhibitor-1, leptin, resistin, and adiponectin [6]. Adiponectin, which is a known protein secreted exclusively by differentiated adipocytes and circulates in large amounts in humans [7]. Adiponectin (ADIPOQ) is the most common gene product in adipose tissue [8,9]. Adiponectin is encoded by Adipocyte, C1q, and Collagen Domain Containing (ACDC), which is located on chromosome 3 at q27. Genome-wide scans have revealed a susceptibility locus at 3q27 for Type 2 Diabetes (T2D), Coronary Artery Disease (CAD), obesity, and metabolic syndrome [10,11]. Several Adiponectin Gene (ADIPOQ) Single Nucleotide Polymorphisms (SNPs) influence adiponectin levels and associated with incidence of obesity, insulin resistance (IR), T2DM, and CVD [12]. The adiponectin gene consists of three exons and two introns spanning a 17-kb region [13]. Sequence polymorphisms identified in humans and investigated for their possible association of insulin resistance indexes and circulating adiponectin concentrations [11,14-16]. Most studies have focused on two polymorphisms, a silent T-to-G substitution in exon 2 (45T-G) and a G-to-T substitution in intron 2 (276G-T). These polymorphisms associated with obesity, insulin resistance, and the risk of type 2 diabetes [17-20]. Furthermore, these two polymorphisms were selected because of their high frequencies in all investigated population, while other reported polymorphisms were rare. The 45T-G polymorphism was related to 4-androstenedione concentrations in PCOS [21]. Therefore, we have investigated the relation between PCOS and axon polymorphism of the adiponect in gene, and the effect of this relation on metabolic disturbance.

Materials and Methods

Group I: Sixty women with PCOS were selected, had all presented at our gynecological department of Minia university hospital from April 2015 to January 2016 with oligomenorrhea or fertility problems. None of the women had galactorrhea, or any systemic disease that could possibly affect their reproductive physiology. Any medication could interfere with the normal function of the hypothalamic–pituitary–gonadal axis. Control group (Group 2) was selected from volunteered healthy forty women, who matched with age, with regular menses and without hyperandrogenemia. All women in the study were genetically unrelated. Before the study, blood samples were drawn from each patient between 08.00 and 08.30 a.m., after an 8 h fast for determination of hormone, adiponectin and glucose levels. A standard 75 g Oral Glucose Tolerance Test (OGTT) and the insulin response to oral glucose loading were performed between 08.30 and 10.30 h, after 10-12 h of fasting. Glucose tolerance was evaluated using the criteria of the American Diabetes Association. For all women, blood samples were collected; part of them were put in EDTA tubes stored at –80°C for genetic analysis, and the remaining blood centrifuged directly and the serum was withdrawn and stored at – 80°C for estimation of F.S.H, L.H, testosterone, adiponectin and Anti-Mullarian Hormone (AMH).

For all women (Groups 1 and 2), serum level of F.S.H, L.H, total testosterone and glucose were estimated in the laboratory unit in Minia university hospital. Adiponectin was measured by commercial immunoassays (Human Adiponectin RIA Kit, Linco Research, St. Charles, MO, USA) with intra- and inter-assay coefficients of variation below 10%. AMH was measured by commercial immunoassays (Human AMH ELIZA kit, US Biological life sciences, U.S.A) and insulin was measured by ELIZA (Human ELIZA kits, Thermo Fisher Scientific), then a glucose/insulin ratio was calculated.

Genotype analysis

Isolation of genomic DNA was carried out from peripheral blood leukocytes of women with PCOS (Group 1) and the controls (Group 2). The extraction was done as the followings: Samples were collected in sterile tubes; then DNA was extracted using the QIAamp Min elute kit protocol in automated mode by a Qiacube instrument. Real Time PCR was used according to literature (OD-0002-02) from Life River. The amplification of genomic DNA was done using the following primers: F5-GAATGAGACTCTGCTGAGATGG and R5- TATCATGTGAGGAG-TGCTTGGATG. PCR products were obtained using 25 μL reactions [5 μL genomic DNA, 12.5 μL Master Mix, 2.5 μL and Primer Probe Mix for wild gene (Green VIC) and 2.5 μL Primer Probe Mix for mutant gene (Red FAM). The amplification conditions were as follows: 95°C for 10 min, followed by 40 cycles of 15 seconds at 95°C, 60 seconds at 60°C and 90 seconds at 72°C, and ending with a single 10 min extension step at 72°C. The polymorphism was typed according to the curve obtained by the real time PCR (Fast 7500).

Statistical analysis

Analyses were performed using the Statistical Package for Social Sciences (SPSS version 18.0). Hormonal data were analyzed with durations of disease and with age using Factorial experiment with completely randomized design with three factors, and were analyzed between groups using Factorial experiment with completely randomized design with two factors. Data were represented as mean ± SE. Bivariate correlations were performed using the Pearson correlation coefficient (P): a P value of <0.05 was considered statistically significant.

Results

The demographic data of our case group were as follows. The mean and standard deviation of age were 24.2167+3.63641. Descriptive analysis in both groups in Table 1 shows that the adiponectin concentrations and glucose/ insulin ratios were significantly lower with PCOS (Group 1), whereas FSH, LH, prolactin, total testosterone and AMH were higher in PCOS. There were significant differences in adiponectin, AMH, total testosterone, glucose insulin ratio between Group 1 and control group (Group 2). On the other hand, there were no significant differences in case of FSH and prolactin. 72% of cases of PCOS came from rural areas, while 28% came from urban area (Table 2). Plasma adiponectin concentration was positively correlated with glucose insulin ratio with a significance of 0.003 (Table 3). The genotyping distributions of TG, GG and TT in women with Group 1 are 22 (37%), 19 (31.5%) and 19 (31.5%), respectively (Table 4). They differ from the same quantities in Group 2: 3 (7.5%), 7 (17.5%) and 30 (75%). Finally, our results show that the correlation of adiponcetin and genotyping of both groups is of significant value (P = 0.001, Table 5).

		Mean	St. Deviation	P value
Age	Case	24.2167	3.63641	0.001
	Control	30.3750	4.99583
FSH	Case	14.76	20.9	0.897
	Control	12.87	18.7
LH	Case	20.0967	29.26833	0.002
	Control	4.6425	2.02090
LH/ FSH	Case	2.61	0.73	0.001
	Control	0.45	0.16
Prolactin	Case	14.0983	5.03373	0.914
	Control	12.8950	5.07841
T. Testosterone	Case	103.9850	116.24456	0.002
	Control	51.4225	82.66094
G. Insulin ratio	Case	9.9583	2.89115	0.001
	Control	19.2600	2.89321
AMH	Case	10.2500	2.60173	0.001
	Control	2.1000	0.35301
Adiponectin	Case	944.5667	290.36782	0.000
	Control	3846.3750	2707.96148

Table 1: Descriptive analysis of patients with PCOS and controls

	Rural	Urban
Case	43 (72%)	17 (28%)
Control	25 (62.5%)	15 (37.5%)

Table 2: Residence of patients with PCO and controls

	Mean	Std. Deviation	Correlation	Significant
Adiponectin	944.5667	290.36782	0.085	0.003
G. I. ratio	9.9583	2.89115

Table 3: Correlation of adiponectin and glucose insulin ratio

		N (%)	P value
TG	Case	22 (37%)	0.000
	Control	3 (7.5%)
GG	Case	19 (31.5%)	0.003
	Control	7 (17.5)
TT	Case	19 (31.5%)	0.004
	Control	30 (75%)

Table 4 Genotyping analysis of patients with PCO and control

	Mean	Std. Deviation	Std. Error	P
TG	875.5909	147.01625	31.34397	0.001
GG	935.6471	440.00596	106.71712
TT	2656.8235	2716.96513	380.45122

Table 5: Correlation of adiponcetin and genotyping

Discussion

In our study, it was noted that serum adiponectin levels of the phenotypes of PCOS were significantly lower than control women (Group 2). That might be attributed to the difference in hyperandrogenism of PCOS women and the body fat distribution and insulin resistance as well. Accordingly, the decreasing in serum adiponectin levels of PCOS patients may be related to any of these variables. Serum adiponectin plays an important role in the pathogenesis of insulin resistance and consequently it might deduce that hypoadiponectinemia would contribute in insulin resistance of PCOS women [22].

Our results agreed with Spain study of seventy-six PCOS patients and 40 non-hyper androgenic women related to BMI and the degree of obesity. Free testosterone levels, age and abdominal adiposity, irrespective of the degree of obesity, were found as the major determinants of hypoadiponectinemia. Reported results support the hypothesis that hyperandrogenism might indirectly lead to insulin resistance in women, by inducing abdominal adiposity and possibly decreasing adiponectin in PCOS patients [23].

Therefore the possible dysfunction of adipose tissue is the main reason of insulin resistance in PCOS [23]. A study by Elbers et al. demonstrated that hyperandrogenism lower adeponectin level and increase insulin resistance in PCOS women [24].

Several studies have shown the higher incidence of adiponectin gene polymorphisms in PCOS subjects. Possible genetic mechanisms could explain the variations in adiponectin levels in patients with PCOS [25]. On contrary of our results, Mohan et al. revealed a completely similar genotype and allele frequency distribution of SNP in the exon 245 position of the adiponectin gene between the PCOS and control groups. Similarly, a study performed in Greek women, no significant difference was detected between 45T→G polymorphism frequencies in the PCOS and control groups, and this polymorphism at position 45T→G had not been associated with a risk for development of PCOS [26,27]. Escobar-Morreale et al. [28] showed that the adiponectin gene polymorphism, 45T→G, is not associated with patients with PCOS. In another Greek trial, the TT genotype and T allele at the 45 position was detected at a lower frequency, while the TG genotype and G allele were detected at a higher frequency in obese women with PCOS compared to those of normal weight (agreed with our results); however, these differences were not statistically significant [29].

Besides that, it was also reported [29] that the SNPs, 45G 15G (T/G) in the ADIPOQ gene are associated with PCOS. Haap et al. [30] found a higher prevalence of 45T→G polymorphism in the adiponectin gene in women with PCOS compared to controls. The 45T→G polymorphism was associated with an increased risk of type 2 DM in a Japanese study [31], which reported a strong association of 45T→G polymorphism with obesity and insulin resistance. Insulin resistance plays a role in type 2 DM pathogenesis. Patients with PCOS have a higher risk of type 2 DM. The commonality between type 2 DM and PCOS pathogenesis is insulin resistance. Some studies suggested that adiponectin gene polymorphisms may be related to type 2 DM [32]. Previous studies examined the association of these and other adiponectin gene variations with type 2 DM and other components of metabolic syndrome. In our study, there was significant statistical difference in genotype distribution and allele frequencies in PCOS compared to the control group. PCOS subjects were found to have a higher risk of type 2 DM and CVD than healthy women.

Conclusion

We concluded that there is higher prevalence of adiponectin gene polymorphism in cases of PCOS with significant correlation with glucose insulin ratio.

Acknowledgement

Authors thank the staff of Minia University Children’s Hospital for their help.

Ethics

The study protocol was approved by the Council of Faculty of Medicine and its Institutional Review Board. Each subject consented before participation in the study. The study protocol was approved by the Ethical Committee of Minia Faculty of Medicine. Written informed consent was obtained formally. The study protocol was approved by the Ethical Committee of Minia Faculty of Medicine. Written informed consent was obtained from all participants prior to participation in the study. The study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki.

Consent for Publication

I confirm that all authors agree on publication and give its liability to the corresponding author.

Competing Interests

The authors declare that they have no competing interests. The authors also declare not to have any financial support. Also the work-done is financially independent.

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References

1. Moran LJ, Misso ML, Wild RA, Norman RJ (2010) Impaired glucose tolerance, type 2 diabetes and metabolic syndrome in polycystic ovary syndrome: a systematic review and meta-analysis.Hum Reprod Update 16: 347-363.
2. March WA, Moore VM, Willson KJ, Phillips DI, Norman RJ, et al. (2010) The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria.Hum Reprod 25: 544-551.
3. Baptiste CG, Battista MC, Trottier A, Baillargeon JP (2010) Insulin and hyperandrogenism in women with polycystic ovary syndrome.J Steroid BiochemMolBiol 122: 42-52.
4. Gustin S, Lee M, Westphal L (2013) Differences in fertility and assisted reproduction in South Asian women. Springer 105-113.
5. Vink JM, Sadrzadeh S, Lambalk CB, Boomsma DI (2006) Heritability of polycystic ovary syndrome in a Dutch twin-family study.J ClinEndocrinolMetab 91: 2100-2104.
6. Ahima RS, Flier JS (2000) Adipose tissue as an endocrine organ.Trends EndocrinolMetab 11: 327-332.
7. Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF (1995) A novel serum protein similar to C1q, produced exclusively in adipocytes.J BiolChem 270: 26746-26749.
8. Maeda K, Okubo K, Shimomurz I, Funahashi T, MatsuzawaY, et al. (1996) cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (Adipose Mostabundant Gene transcript 1. BiochemBiophys Res Commun 221:286-289.
9. Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, et al. (1999) Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity.BiochemBiophys Res Commun 257: 79-83.
10. Saito K, Tobe T, Minoshima S, Asakawa S, Sumiya J, et al. (1999) Organization of the gene for gelatine binding protein (GBP28). Gene 229: 67-73.
11. Takahashi M, Arita Y, Yamagata K, Matsukawa Y, Okutomi K, et al. (2000) Genomic structure and mutations in adipose-specific gene, adiponectin.Int J ObesRelatMetabDisord 24: 861-868.
12. Lujan ME, ChizenDR, PiersonRA (1992) Diagnostic Criteria for Polycystic Ovary Syndrome: Pitfalls and Controversies. J ObstetGynaecol Can 30: 671-679.
13. Kubota N, Terauchi Y, Yamauchi T, Kubota T, Moroi M, et al. (2002) Disruption of adiponectin causes insulin resistance and neointimal formation.J BiolChem 277: 25863-25866.
14. Kissebah AH, Sonnenberg GE, Myklebust J, Goldstein M, Broman K, et al. (2000) Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome.ProcNatlAcadSci USA 97: 14478-14483.
15. Vasseur F, Helbecque N, Dina C Lobbens S, Delannoy V, Gaget S (2002) Single-nucleotide polymorphism haplotypes in the both proximal promoter and exon 3 of the APM1 gene modulate adipocyte-secreted adiponectin hormone levels and contribute to the genetic risk for type 2 diabetes in French Caucasians. Hum Mol Genet 11: 2607-2614.
16. Kondo H, Shimomura I, Matsukawa Y, Kumada M, Takahashi M, et al. (2002) Association of adiponectin mutation with type 2 diabetes: a candidate gene for the insulin resistance syndrome.Diabetes 51: 2325-2328.
17. Gu HF, Abulaiti A, Ostenson CG, Humphreys K, Wahlestedt C, et al. (2004) Single nucleotide polymorphisms in the proximal promoter region of the adiponectin (APM1) gene are associated with type 2 diabetes in Swedish Caucasians. Diabetes 53: 31-35.
18. Dunaif A (1997) Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis.Endocr Rev 18: 774-800.
19. Hara K, Boutin P, Mori Y, Tobe K, Dina C, et al. (2002) Genetic variation in the gene encoding adiponectin is associated with an increased risk of type 2 diabetes in the Japanese population.Diabetes 51: 536-540.
20. Filippi E, Sentinelli F, Trischitta V, Romeo S, Arca M, et al. (2004) Association of the human adiponectin gene and insulin resistance.Eur J Hum Genet 12: 199-205.
21. Menzaghi C, Ercolino T, Di Paola R, Berg AH, Warram JH, et al. (2002) A haplotype at the adiponectin locus is associated with obesity and other features of the insulin resistance syndrome. Diabetes51: 2306-2312.
22. Karkanaki A, Piouka A, Katsilis I, Farmakiotis D, Mecut D,et al. (2009) Adiponectin Levels Reflect the Different Phenotypes of Polycystic Ovary Syndrome: Study in Normal Weight, Normoinsulinemic Patients. Fertility and Sterility 92: 2078-2081.
23. Sieminska L, Marek B, KosKudla B, Niedziolka D, Kajdaniuk D, et al. (2004) Serum Adiponectin in Women with Polycystic Ovarian Syndrome and its Relation to Clinical, Metabolic and Endo-crine Parameters. J Endocrinological Invest 27: 528-534.
24. Elbers JM, Giltay EJ, Teerlink T, Scheffer PG, Asscheman H, et al. (2003) Effects of sex steroids on components of the insulin resistance syndrome in transsexual subjects.ClinEndocrinol (Oxf) 58: 562-571.
25. Otta CF, Wior M, Iraci GS, Kaplan R, Torres D, et al. (2010) Clinical, metabolic, and endocrine parameters in response to metformin and lifestyle intervention in women with polycystic ovary syndrome: a randomized, double-blind, and placebo control trial. Gynecological Endocrinology 26: 173-178.
26. Mohan SK, Vishnu PV (2009) Lipid peroxidation, glutathione, ascorbic acid, vitamin E, antioxidant enzyme and serum homocysteine status in patients with polycystic ovary syndrome. Biol Med 1: 44-49.
27. Marioli DJ, Koika V, Adonakis GL, Saltamavros AD, Karela A, et al. (2010) No association of the G972S polymorphism of the insulin receptor substrate-1 gene with polycystic ovary syndrome in lean PCOS women with biochemical hyperandrogenemia. Arch GynecolObstet 281: 1045-1049.
28. Escobar-Morreale HF, Villuendas G, Botella-Carretero JI, Alvarez-Blasco F, Sanchón R, et al. (2006) Adiponectin and Resistin in PCOS: A Clinical, Biochemical and Molecular Genetic Study. Human Reprod 21: 2257-2265.
29. Zhang N, Shi YH, Hao CF, Gu HF, Li Y, et al. (2008) Association of +45G15G(T/G) and +276(G/T) polymorphisms in the ADIPOQ gene with polycystic ovary syndrome among Han Chinese women.Eur J Endocrinol 158: 255-260.
30. Haap M, Machicao F, Stefan N, Thamer C, Tschritter O, et al. (2005) Genetic determinants of insulin action in polycystic ovary syndrome.ExpClinEndocrinol Diabetes 113: 275-281.
31. Hara M, Alcoser SY, Qaadir A, Beiswenger KK, Cox N, et al. (2002) Insulin resistance is attenuated in women with polycystic ovary syndrome with the Pro(12)Ala polymorphism in the PPAR-gamma gene. J ClinEndocrinolMetab 87: 772-775.
32. Prapas N, Karkanaki A, Prapas I, Kalogiannidis I, Katsikis I, et al. (2009) Genetics of polycystic ovary syndrome.Hippokratia 13: 216-223.