Evaluation of Lipid Ratios and Triglyceride-Glucose (TyG) Index as Risk Markers of Insulin Resistance in Iranian Polycystic Ovary Syndrome (PCOS) Woman

Background: Insulin resistance has a key role in the pathophysiology of polycystic ovary syndrome (PCOS). Previous investigations have informed that some lipid ratios could be a simple clinical indicator of insulin resistance (IR) in some disorders and ethnicities. We aimed to examine the correlation between triglyceride to HDL-cholesterol (TG/HDL-C), total cholesterol to HDL-cholesterol (TC/HDL-C) and fasting triglyceride-glucose (TyG) indices with IR (as measured by homeostasis model assessment of IR [HOMA-IR], quantitative insulin sensitivity check index [QUICKI] and fasting glucose to insulin ratio [FGIR]), and determine a good clinical predictor for IR in Iranian PCOS woman. Methods: We evaluated 305 PCOS women. After physical evaluations, biochemical parameters were measured using commercial kits and TG/HDL-C, TC/HDL-C and TyG indices were calculated using formula. Fasting insulin level measured using ELISA technique. IR was dened as a HOMA-IR value ≥ 2.63, FG-IR<8.25 and QUICKI <0.33. Results: The insulin-resistance and insulin-sensitive groups, which established by HOMA-IR, FG-IR and QUICKI values, were different in terms of TG/HDL-C, TC/HDL-C and TyG indices. These indices were associated with IR after adjusting for age and BMI. The under ROC curves (AUC) of TyG, TG/HDL-C and TC/HDL-C for predicting HOMA-IR index were 0.639, 0.619 and 0.623 respectively which were signicant, with a p-value 0.012, 0.033 and 0.027, respectively. The AUC of TC/HDL-C (0.614) was signicant (p-value 0.04) for predicting FG-IR. Conclusion: Our ndings demonstrated that the elevated TyG, TG/HDL-C and TC/HDL-C were signicantly


Introduction
Polycystic ovary syndrome (PCOS) is a complex hormonal disorder common in reproductive-aged women that characterized by ovulation dysfunction, overproduction of androgens and polycystic ovarian morphology (PCOM) under B-ultrasound [1]. The prevalence of PCOS is approximately 5-15% which is the most common cause of infertility in women [1].
Taken together, increasing insulin and androgen levels can interrupt follicle growth which in turn can lead to the irregular menstrual cycle, an-ovulatory subfertility and accumulation of immature follicles [2,5].
Although the pathogenesis of PCOS is not completely known yet, growing body of evidence has shown that IR has a central role in the pathophysiology of PCOS and is associated with great risk of metabolic disorders including type 2 diabetes mellitus (T2DM), dyslipidemia, nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) [6]. It has been demonstrated that the modulation of IR causes a signi cant improvement in PCOS complications [7,8]. Therefore, it is of paramount importance to evaluate IR in this at risk population.
The reference standard to diagnose IR is the hyperinsulinemic euglycemic glucose clamp technique [9], but it is time-consuming, labor-intensive, costly, and technically challenging [10]. So, the surrogate markers including homeostasis model assessment for insulin resistance (HOMA-IR), the quantitative insulin sensitivity check index (QUICKI) and the fasting glucose to insulin ratio (FGIR) have emerged to estimate of IR [11]. The clinical e cacy of these surrogate markers has been limited because of the absence of standardization, cost and availability of the insulin assay technique [12]. Hence, for daily clinical practice, a simple and more available marker for predicting IR can be valuable and cost-effective for early detection of people with IR for clinicians [12].
Previous evidences have been suggested that the fasting triglyceride-glucose (TyG) index and the triglyceride to high-density lipoprotein cholesterol (TG/HDL-C) concentration ratio are closely associated with IR [13][14][15]. Some studies have shown that total cholesterol to HDL-C (TC/ HDL-C) concentration ratio also related to IR and risk of CVD [15]. It is worth noting that the association between lipid ratios and IR may differ by ethnicity and some indices may not be applicable to predict IR in particular populations [16][17][18]. Furthermore, there are a few studies dealing with the association between IR and lipid ratios among PCOS subjects in the Iranian population. This rst comprehensive study was carried out to investigate the association of TG/HDL-C, TC/HDL-C and TyG indices with IR (as estimated by HOMA-IR, QUICKI and FG-IR) and to determine the diagnostic utility of these markers in recognizing IR among PCOS women in Iran.

Study participants
A total of 305 PCOS women were recruited from Shahid Bahonar Hospital, Kerman, Iran. Participants' ages ranged from 20 to 40 years, with body mass index (BMI) of 17-35 kg/m 2 . PCOS diagnosis was according to the Rotterdam criteria [19] which includes: PCOM on ultrasound, hyperandrogenism, and oligo-or an-ovulation. Exclusion criteria were including the following: the presence of hypertension, pregnancy, and history of endocrine disorders or CVD. The present research was approved by the Ethical Committee of Kerman University of Medical Sciences (IR. KUM.REC.1399.208), and carried out according to the declaration of Helsinki. Informed consent was essential before enrolling a participant in our study.

Anthropometric and laboratory measurements
BMI was calculated using a standard formula [body weight (kg)/height (m 2 )]. Blood samples were taken after an 8-hr fasting period at the follicular phase of their menstrual cycle. Biochemical parameters including, fasting blood sugar (FBS), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were evaluated using commercial kits (Pars Azmoon, Iran). Fasting insulin level was measured using ELISA kit (Monobind Inc.). Folliclestimulating hormone (FSH), luteinizing hormone (LH), free T4, and homocysteine levels were evaluated using ELISA kits (Pishtaz Teb, Iran), according to manufacturer's instructions. Details about the intraassay coe cients of variation (CV) for biochemical parameters have been previously reported [20].

Statistical analysis
Statistical analyses were performed using IBM SPSS Statistics 16.0 (IBM SPSS, Chicago, IL). The normality of the variables was determined using the Shapiro-Wilk test. Continuous variables with normal distribution were presented as mean ± SD, while skewed variables were presented as median and interquartile range (IQR). Statistical difference between IR and IS group was assessed using Student's T test and Mann-Whitney U test. For correlation analysis, Pearson or Spearman analysis was calculated. To assess the ability of lipid ratios and TyG for predicting insulin resistance according to HOMA-IR and FG-IR in PCOS patients logistic regression and receiver operator characteristic (ROC) curve analyses were used.

Clinical and biochemical parameters of the study population
General characteristics, biochemical parameters and hormonal features of the patients in the IR and the IS groups are shown in Table 1 Table 1). The results between two groups based on QUICKI not shown due to the similarity to FG-IR. Fasting insulin concentration, TC/HDL-C and HOMA-IR were signi cantly higher (p < 0.001), and FG-IR and QUICKI were signi cantly lower (p < 0.001) in the FG-IR and QUICKI positive group compared to their counterparts (Table 1). In regard to IR and IS groups based on HOMA-IR, FBS, fasting insulin concentration, TyG, TC/HDL-C, TG/HDL-C and HOMA-IR were signi cantly higher and homocysteine, HDL-C, FG-IR and QUICKI were signi cantly lower in IR group. Table 2 shows the correlations between FG-IR, QUICKI and HOMA-IR indices with clinical and biochemical parameters with and without adjusting for age and BMI. The variables including, insulin, TG, TC, TyG, TC/HDL-C and TG/HDL-C were signi cantly negatively associated with  FG-IR and QUICKI and signi cantly positively associated with HOMA-IR before and after adjusting for age and BMI. The variable, homocysteine and HDL-C were statistically signi cantly negatively associated with HOMA-IR. Homocysteine was also signi cantly positively correlated to FG-IR and QUICKI. Although FBS concentration was not correlated to FG-IR, it was signi cantly negatively correlated to QUICKI and signi cantly positively correlated to HOMA-IR. Table 3 shows the 2 models of the association between FG-IR and HOMA-IR indices with lipid pro les, TyG, TC/HDL-C, TG/ HDL-C through multiple logistic regression analyses. In model 1, the odds ratios (OR) were calculated without adjusting. In model 2, the ORs were calculated after adjusting for age and BMI.   Table 4. Although the AUC of TyG, TG/HDL-C were not signi cant, the AUC of TC/HDL-C (0.614) was signi cant (p-value 0.04) for predicting FG-IR, as shown in Table 4. In addition, the AUC of lipid pro le (TG, TC, LDL-C and HDL-C) were not signi cant for predicting HOMA-IR and FG-IR (Fig. 1S).

Discussion
A growing body of evidence indicates that PCOS is a metabolic disease which IR has a central role in the pathogenesis and complications of its [24]. Therefore, evaluation of IR in PCOS women is very critical and helpful. Direct and indirect methods (HOMA-IR, QUICKI and FG-IR) for IR assessment are complex, expensive and not suitable for epidemiological studies [24]. Hence, there is an urgent need to develop an easy-to-measure, more reasonable and cost-effective method for IR measurements to contribute to diagnosis, treatment, and prognosis of PCOS [24].
Dyslipidemia is one of the most confusing metabolic consequences with a prevalence of up to 70% in PCOS women [25]. Although, the cause of lipidemic abnormalities in PCOS is multifactorial, IR plays most important pathophysiological role in disturbed lipid metabolism and dyslipidemia that is mediated through stimulation of lipolysis and changed expression of lipoprotein lipase and hepatic lipase [25].
Recently, some studies have proposed that lipid ratios may be useful alternative markers for IR estimation in different races [7].
The ndings of the current study demonstrated that the TG/ HDL-C, TC/HDL-C and TyG indices are strongly correlated with IR as estimated by HOMA-IR, FG-IR and QUICKI in PCOS women in Iran. A high value of these ratios was positively correlated to HOMA-IR and negatively correlated to FG-IR and QUICKI before and after adjusting for BMI and age. Although dyslipidemia was not seen in IR group (except HDL-C based on HOMA-IR), TG/HDL-C, TC/HDL-C and TyG ratios were signi cantly higher in subjects with HOMA-IR index ≥ 2.63 (IR positive) as compared to those with HOMA-IR index < 2.63 (IR negative), and also TC/HDL-C ratio was signi cantly lower in IS subjects as measured with FG-IR and QUICKI. These ndings result from a comprehensive analysis between TG/HDL-C, TC/HDL-C and TyG indices with several indices of IR suggest that these ratios might be more useful indicators of IR than lipid pro les. To the best of our knowledge, this is the rst comprehensive study to investigate these relationships in Iranian PCOS patients.
In this context, some studies have also reported the performance of these ratios for IR. The positive correlation of TG/HDL-C, TC/HDL-C with HOMA-IR has been demonstrated among PCOS women in China [24] and T2DM in Thailand [26]. Results of some studies also supported that TG/HDL-C ratio could be used as an indicator of IR among some population including the middle-aged and elderly population in Taiwan [16], euthyroid normal-weight healthy adults in Peru [27], PCOS patients in India [28] and in Chinese population without diabetes [29]. A large scale cross-sectional analysis among Chinese adults also indicated that TG/HDL-C ratio and TyG index are useful markers in estimating IR and TyG index is the best indicator for assessing the risk of IR [30]. However, it is important to highlight that, in spite of clinical effectiveness of these ratios, inconsistencies have been identi ed according to ethnicity [27]. For example, researches in African-Americans have revealed that TG/HDL-C is not a reliable indicator for IR [31][32][33].
To the best of our knowledge, only one previous study has been directed in this regard on 36 infertile women with PCOS in Iranian population and con rmed a signi cant association in TC/HDL-C, TG/HDL-C and LDLC/HDL-C ratios with IR (as estimated by HOMA-IR) in PCOS patients [7]. Another Iranian study has been conducted on 5201 non diabetic persons and noted that TG/HDL-C and TC/HDL-C serve as independent predictors of incident diabetes, during ≈ 6 years follow up [34]. Although these studies demonstrated the e ciency of lipid ratios for IR prediction, there was a lack of su cient documents concerning lipid ratios and TyG index in PCOS women in the Iranian population. Moreover, most studies in this area were based on HOMA-IR and they have not considered FG-IR and QUICKI as two well-known indicators for IR. Therefore, in the present study to assess the diagnostic ability of lipid pro le, TG/HDL-C, TC/HDL-C, and TyG in classifying insulin resistance, ROC curve analysis was applied using control cutoff values for HOMA-IR and FG-IR (i.e. 2.63 and 8.25 respectively). Despite lipid pro le did not show su cient predictability for IR, the AUC of TG/HDL-C, TC/HDL-C, and TyG based on HOMA-IR and FG-IR was greater than 0.5 and considered acceptable test performance. Thus, it seemed that these ratios are effective and bene cial diagnostic indicators for IR in PCOS. These data were consistent with prior evidence in Iran supporting the diagnostic potential of TC/HDL-C and TG/HDL-C for PCOS [7]. Even though in a previous study the AUC value of TG/HDL-C was the highest based on HOMA-IR, in the current study the AUC value of TyG was the highest based on HOMA-IR and the AUC value of TC/HDL-C was the highest based on FG-IR. This discrepancy could be partially due to the larger sample size in our study. Although the diagnostic accuracy of the TyG index has been described in some populations [35-37] the current study is the rst analyzing that assessed the diagnostic ability of TyG in Iranian PCOS woman.
As a strength, this study was the rst comprehensive study, which investigated different metabolic index for estimating of IR -as assessed using HOMA-IR, FG-IR and QUICKI-in PCOS women in Iran. It must be noted that the limitations of the current study included the lack of direct IR evaluation and the fact that patients were evaluated only once, so that within-subject biological variation of biochemical parameters measurements could not therefore be minimized.
In conclusion, TG/HDL-C, TC/HDL-C and TyG indices are valuable indicators to predict IR in Iranian PCOS women. These markers can be easily calculated because lipid pro le and glucose values can be received from routine laboratory tests and analytically and nancially available to all clinical laboratories. Therefore, we suggested the application of TG/HDL-C, TC/HDL-C and TyG indices in risk assessments for IR in Iranian PCOS women and future epidemiologic researches.

Declarations Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1975 Helsinki declaration as revised in 2008.

Con ict of interest
The authors declare that they have no con ict of interest.