Leptin gene polymorphism Rs7799039; G2548A, metabolic and oxidative stress markers in polycystic ovarian syndrome

1 Introduction

Polycystic ovarian syndrome (PCOS) is an endocrine-metabolic disorder with a multitude of hormonal imbalances, with a preponderance of hyperandrogenism symptoms that have both immediate and long-term implications on female health (Rojas et al., 2014). An essential component of PCOS is stress encompassing variant equivalent definitions like emotional, inflammatory, oxidative, and metabolic stress (Papalou and Diamanti-Kandarakis, 2017). PCOS symptoms are not limited to the gynaecological realm; obesity, dyslipidemia, hypertension, metabolic syndrome (MS), and type 2 diabetes mellitus (DM2) are all more common in women with PCOS than in those without the condition (Rojas et al., 2014). These characteristics, as well as additional changes such as endothelial dysfunction and a low-grade inflammatory state, all contribute to the patients' higher risk of cardiovascular disease and all-cause death (Rojas et al., 2014). Because obesity is a prevalent characteristic of PCOS patients, researchers hypothesized that leptin might play a role in the disease's development (Rojas et al., 2014; Moran et al., 2012; Calcaterra et al., 2021). Obesity causes hyperleptinemia and oxidative stress, which may have a role in developing PCOS (Calcaterra et al., 2021).

Leptin is a protein produced by the LEP gene that regulates food intake, energy balance, and reproduction (Tu et al., 2017). Leptin, a 16-kDa protein produced by adipocytes, was initially discovered in laboratory mice due to the ob (obese) gene (Tu et al., 2017; Zhou and Rui, 2013). Leptin reduces glucose absorption, glycogen synthase, inhibits lipogenesis and inhibits lipolysis in adipocytes (Pandey et al., 2015; Zhang et al., 1994; MacDougald et al., 1995). Leptin causes insulin-like actions in hepatocytes by altering the insulin signalling pathway. As a result, leptin and insulin signalling may interact, making hepatocytes more insulin sensitive (MacDougald et al., 1995; Muller et al., 1997). Leptin levels rise with rising body mass index (BMI) and body fat since it is an adipokine. However, its relation to PCOS is fascinating because PCOS individuals have higher leptin levels than healthy people (Pandey et al., 2015; Zhang et al., 1994; MacDougald et al., 1995). Oxidative stress is a major factor in the progression of PCOS (Mohammadi, 2019). Oxidative stress is now widely acknowledged as a critical factor in the pathogenesis of various diseases, including PCOS. The oxidant and antioxidant imbalance and the production of excessive reactive oxygen species (ROS) levels is referred to as oxidative stress (Sharifi-Rad et al., 2020). Many studies have found circulating oxidative indicators as a potential inducer of PCOS pathogenesis (Manna and Jain, 2015). Several theories on the pathology have been presented to explain the complete range of symptoms and indications, but no definitive answers have been found (Fratantonio et al., 2005). Genetic and environmental factors are known to play a role in the differing pathophysiology of PCOS phenotypes. As a result, gene abnormalities in women with PCOS have extensively investigated to determine if their mutations or polymorphisms are responsible for the condition. However it is unlikely to have a single cause for PCOS. Most of the studies have focused on studying leptin gene polymorphism alone but have not linked it to its interaction with metabolic and oxidative stress markers which will shed light on whether and how leptin gene polymorphism would be associated with metabolic and oxidative stress markers and could contribute to the PCOS pathogenesis and progression. With this the present study investigated the interplay between leptin gene polymorphism metabolic, oxidative stress and inflammatory markers in polycystic ovarian syndrome.

2 Materials and method

Institutional ethical clearance was obtained (NU/CEC/2018/0195 dated 16-06-2018). Individuals visiting Justice K.S. Hegde Charitable Hospital, Deralakatte, Mangaluru for regular health check-up from July 2018- June 2021 were approached for the study. Informed consent was duly signed and subjects were recruited (Control-150; PCOS-150) in 18–40 years of age as per the exclusion and inclusion criteria. The subjects with PCOS (age group 18–40 years) will be recruited based on the Rotterdam criteria (Legro et al., 2013): Oligo and/or amenorrhea or anovulation, clinical and /or biochemical signs of hyperandrogenism, the polycystic appearance of the ovary on ultrasonography. Other causes of clinically diagnosed hyperandrogenism include hyperprolactinemia, Cushing's syndrome, androgen-secreting tumors, non-classical congenital adrenal hyperplasia, and patients on drugs that alter the sex hormone level were excluded. The Control group included healthy women aged 18–40 years with regular menstrual cycles (26–34 days). Pregnant women and subjects not willing to give consent were excluded. A detailed case history was taken during the first visit. 2 mL of peripheral blood was collected in the plain tube for biochemical estimations and2mL in EDTA vacutainer tubes for DNA isolation.

3 Results

3.2

3.2 Allele and genotype frequencies for rs7799039

Fig. 1 illustrates RFLP genotyping of rs7799039 by agarose gel electrophoresis. The agarose gels revealed that each allele had a single-band pattern: allele G, which does not contain the HhaI restriction site, was defined by a 242 bp fragment, while allele A, which contains this restriction site, was defined by two digestion bands of 181 and 61 bp.

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Fig. 1

Agarose gel electrophoresis (3%) of polymerase chain reaction (PCR) product (PC-Positive Control; NC-Negative Control and P119-P123 Samples).

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The wild type genotype is GG (single band −242 bp) for rs7799039 G > A, thehomozygous recessive variant is AA (three bands- 242 bp; 181 bp and 61 bp), and the heterozygous genotype is GA (Two bands 181 bp and 61 bp). The allele frequency of G and A was 0.65 (65%) and 0.35 (35%), respectively, in the control group. In the study group, the allele frequency of G was 0.50 (50%) and 0.50 (50%), respectively. The genotype frequency of GG is 0.29 (29%), and GA is 0.71 (71%) in the control group. It is 0.5 (50%) for GG, 0.05 (5%) for GA, and 0.45 (45 %) for AA in the study group.

4 Discussion

Insulin resistance plays an essential role in the origination of both reproductive and metabolic defects and affects 50–70% of women with PCOS. Obesity, especially abdominal obesity, can contribute to the development of IR and its complications associated with it, for example, impaired glucose tolerance and T2DM (Zhu et al., 2013).

According to earlier studies, women with PCOS are more likely to develop glucose intolerance, type 2 diabetes mellitus, metabolic syndrome, and risk of cardiovascular disease with abnormal postprandial glucose levels (Zhu et al., 2013). In the present study, fasting blood sugar, insulin, and HOMA-IR levels were higher in the study group than in the control group. Most studies relate PCOS to lower HDL-C and higher triglyceride levels and lipid profile to IR. In previous studies (Talbott et al., 1995; Orio et al., 2004; Legro et al., 2001; Erel et al., 2003), it was also found that women with polycystic ovaries had higher serum lipids (total cholesterol, LDL, triglycerides) and lower HDL than healthy people. The present study is consistent with these results. Lipolysis is checked by hyperinsulinemia, which increases esterified acids (Liu et al., 2019). Non-esterified fatty acids (NEFA) lower HDL levels and raise triglyceride levels (Fenkci et al., 2003). Oxidative stress is the generation of reactive oxygen species (ROS) like superoxide anions, hydrogen peroxide (H2O2), and hydroxyl radicals not sufficiently detoxified by cellular antioxidants and causing ROS build-up. Oxidative stress is caused by the reduction of antioxidant/anti-inflammatory activity of HDL (Pandey et al., 2015; Mohammadi, 2019; Sharifi-Rad et al., 2020; Liu et al., 2019).

Total antioxidant capacity (TAC) is the ability to scavenge free radical production (Fenkci et al., 2003) compared TAC levels in PCOS patients to age, BMI, and smoking status matched controls and found that TAC levels in PCOS individuals were considerably lower. According to (Verit and Erel, 2008) TAC levels were distinctly higher in PCOS patients than in age and BMI-matched controls. The results of our study are in line with these facts. On the basis of the results, it can be hypothesized that TAC may be elevated to counteract the increased oxidative stress.

Malonaldehyde (MDA) is formed by the lipid peroxidation of polyunsaturated fatty acids. A comparison of MDA blood levels between PCOS subjects and healthy controls was made in the study by Kuşçu and Var (2009), which showed that MDA level was remarkably higher in the PCOS group but independent of obesity. The current study showed parallel results. In the PCOS group, glutathione peroxides (GPX) and myeloperoxidase (MPX) levels were higher as well. This rise in GPX and MPX levels may be a protective mechanism against oxidative damage.

Chronic low-grade inflammation is a key indicator of PCOS. It is suggested that it plays a role in the origin and progression of the disease. Inflammation has also been related to IR in PCOS. The present study observed elevated levels of CRP, TNF-alpha, and IL-2. Consistent results were shown by Papalou et al. (2015), Orio et al. (2004) and Herlihy et al. (2011).

Leptin can be described as a double-edged sword. It positively stimulates estrogen secretion, causing puberty, and negatively inhibits gonadotrophin-stimulated ovarian response in hyperleptinemia (18–20). As reported previously, the present study showed a statistically substantial difference between the two groups concerning leptin. Leptin resistance causes the loss of peripheral glucose homeostasis by lowering the hypothalamus insulin response, giving rise to overproduction of androgenic hormones and thereby contributing to insulin resistance (Martins et al., 2017; Bains et al., 2020; Mohamed et al., 2021). Several genetic variations have been studied for their predictive value for the PCOS, as prediction accuracy is affected by factors like the participating gene number, the frequency of risk alleles, and the associated risks with genotypes (Bains et al., 2020; Mohamed et al., 2021). The present study examined leptin gene polymorphism in polycystic ovarian syndrome. The additive model showed a remarkable difference between HOMA-IR, TC, TG, and inflammatory markers (TNF-alpha, IL-2) among the three groups studied. There was a statistically striking difference between BMI, prolactin, and inflammatory markers. The recessive model showed similar results. This indicates that the leptin gene may play a part in the pathogenesis of PCOS. Based on the current study findings, the suggestive mechanism enumerating the interaction between leptin insulin resistance and developing of PCOS is that insulin resistance increases insulin levels which in turn results in leptin resistance leading to hyperleptinemia. Similarly variation in leptin gene may also result in altered levels of leptin. The ovarian physiology in PCOS is directly impacted by hyperlepitemia, thereby stopping follicle formation and PCOS pathogenesis.

Though this study addressed numerous parameters, it had a limitation of small size. To validate the significance of the leptin gene polymorphism and PCOS pathogenesis, future research on a larger sample of PCOS patients is required. The explanation of this complex interplay between the leptin gene, inflammation, and metabolic stress at the molecular level is also necessary.

5 Conclusion

The findings demonstrate a functional relationship between leptin gene polymorphism, insulin resistance, and inflammatory markers. Interpreting the interaction of these factors with each other and with the onset and clinical manifestations of PCOS is a critical step in understanding the causes of the disease. It also helps identify prospective molecules and pathways to target therapeutic and preventive intervention. The present study findings can be confirmed and expanded by conducting additional studies on a larger spectrum of PCOS patients combined with genotyping of other leptin variants. These studies can also provide a detailed understanding of the leptin gene and its influence on the emergence of PCOS and its pathogenesis.