ABSTRACT

Objective:

C1q/tumor necrosis factor-related protein-5 (CTRP5) is a novel peptide hormone involved in the metabolism of energy regulation. Polycystic ovary syndrome (PCOS), which is a reproductive and metabolic disorder, is associated with insulin resistance. The aim of the current study was to compare circulating levels of CTRP5 in women with and without PCOS and to investigate possible associations between CTRP5 and metabolic-hormonal parameters.

Material and Methods:

The present cross-sectional study contained 80 women with PCOS and 80 age and body mass index-matched women without PCOS. Circulating levels of CTRP5 were calculated using an enzyme-linked immunosorbent assay. We also measured hormonal and metabolic parameters.

Results:

Patients with PCOS had lower levels of circulating CTRP5 compared with women without PCOS (6.90±2.64 vs 11.73±3.66 ng/mL, p<0.001). CTRP5 was negatively correlated with insulin resistance, free-androgen index, and body mass index in both the PCOS and control groups. Moreover, patients with PCOS who had insulin resistance showed lower circulating CTRP5 levels compared with those without insulin resistance. In both the control and PCOS groups, overweight subjects had lower circulating levels of CTRP5 compared with participants of normal weight. Logistic regression analyses indicated that subjects in the lowest tertile for CTRP5 level had higher risk for PCOS compared with those in the highest tertile of CTRP5.

Conclusion:

Decreased circulating levels of CTRP5 were associated with higher risk of PCOS, as well as having metabolic disturbance among women with PCOS.

Keywords: Polycystic ovary syndrome, C1q/tumor necrosis factor-related protein-5, insulin resistance, body mass index, free-androgen index

Introduction

Laboratory and clinical characteristics of the population of the study

The comparison of the clinical and laboratory parameters of the enrolled groups are presented in Table 1.

Table 1

CTRP5 levels were notably lower in women with PCOS than in those without PCOS (6.90±2.64 vs 11.73±3.66 ng/mL, p<0.001) (Figure 1a). The levels of FBG, HOMA-IR and serum insulin were meaningfully higher in patients with PCOS as compared with controls. Moreover, patients with PCOS had markedly higher circulating levels of FAI, total testosterone, hs-CRP, and DHEA-SO₄ as compared with the controls. In the comparison of CTRP5 levels in the PCOS subgroups with insulin resistance (54 of 80 subjects with PCOS had insulin resistance) and without insulin resistance, CTRP5 level was remarkably lower in among the patients with PCOS with insulin resistance (6.43±2.67 vs 7.88±2.34 ng/mL, p=0.020*) (Figure 1b).

Figure 1

In addition, the study participants were also divided into four groups based on BMI (<25 kg/m² and ≥25 kg/m²) and PCOS status. Circulating CTRP5 levels were compared between overweight and normal weight subgroups in both PCOS and control groups using t-tests. The subdivision of the PCOS and control groups based on their BMI showed that 40 participants in the PCOS group and 41 in the control group were overweight (p=0.999). In both groups, the mean values for circulating CTRP5 were meaningfully lower in overweight subjects compared with subjects with normal BMI (PCOS group: 6.17±2.50 vs 7.63±2.61 ng/mL, p=0.013*; control group: 10.89±4.10 vs 12.60±2.95 ng/mL, p=0.035*) (Figure 1c). Moreover, we compared circulating CTRP5 levels in the PCOS and control groups according to their BMI status (Figure 1d). CTRP5 levels were found to be decreased in PCOS group compared with the control group in both overweight and normal weight subjects (p<0.001*).

Correlation of CTRP5 with other parameters

Multivariate regression analysis

Linear regression analysis was focused to assess the existence of independent relationships between CTRP5 and HOMA-IR, BMI, and FAI. PCOS status, age, hs-CRP, and lipid parameters were included in the regression model to adjust for their potentially confounding effects (Table 3). According to the results of the regression analysis, CTRP5 could show an independently negative association with HOMA-IR, FAI, and BMI.

Table 3

Multivariate binary logistic regression analysis

Discussion

Ethics

Ethical approval was issued by the ethics committee of İzmir Bozyaka Training and Research Hospital for the present study (no: 2. GOA/2016) and all participants provided written informed consent. The study was conducted and accomplished according to the Declaration of Helsinki (2008).

Study design and participants

This case-control study included two groups: 80 subjects with PCOS and a group of 80 age and BMI-matched women with normal menstruation. Participants aged 18-45 years were recruited. The study was conducted between June 2016 and January 2017 in the Endocrinology Department of the Bozyaka Training and Research Hospital in İzmir, Turkey. We consecutively recruited subjects who met all of the exclusion and inclusion criteria of the study to reach to the planned population. All participants had a BMI >18.5 kg/m² and ≤35 kg/m², and none had alcohol and tobacco addiction. The same researcher performed all examinations, and obtained detailed histories. All participants were subjected to 2-h 75-g oral glucose tolerance standard test (OGTT). All participants were drug naive.

PCOS group

Control group

The control group of comprised women who had visited the endocrinology and/or gynecology clinics for checkup and volunteer employees from the hospital. All controls had normal menstrual cycles. Disorders such as problems in concomitant health issue, acne, hyperandrogenism, or hirsutism symptoms were not detected in any patients in the control group.

Exclusion criteria

Participants who were pregnant/breastfeeding or had any other causes or signs of menstrual irregularity and/or androgen excess, such as adrenal, pituitary, or thyroid disorders (including congenital adrenal hyperplasia, hyperprolactinemia, Cushing’s syndrome, hyperprolactinemia and galactorrhea) were not included. Other exclusion criteria included reduced glucose tolerance, type 1/2 diabetes, or history of gestational diabetes; history of hypertension, hyperlipidemia, liver/renal disorders, coronary artery disease, congestive heart failure, malignancy or acute infection (in the past two weeks); and chronic inflammatory or autoimmune disorders. Furthermore, participants with hormonal contraception and/or anti-androgen use in the past six months and those taking medications for treatment of hypertension, insulin resistance, hyperglycemia, dyslipidemia and obesity were excluded.

Anthropometric evaluation

Anthropometric measurements including age, waist circumference (cm), both weight (kg) and height (cm) were analyzed when the participants were wearing casual clothes and barefoot. The distance between the lower rib margin and the iliac crest at the end of a gentle expiration was used to find the waist circumference. Blood pressure was measured with the participants in a resting position after a 15 minute resting period. BMI was calculated considering the formula of weight (kg)/ height in meters squared (m²).

Biochemical evaluation

Venous blood samples were obtained from all participants in the early follicular phase (day 3 to 5) of spontaneous or progesterone-induced menses, in the morning (between 08:00-09:00) after at least a 10 hour fast. The blood samples were held at room temperature for at least 30 minutes to allow coagulation. The samples were then centrifuged at 2000×g for 15 minutes and serum aliquots were maintained at -80 °C until analysis of CTRP5. Fasting blood glucose (FBG), glycated hemoglobin A1_C (HbA1_C), serum insulin, high-density lipoprotein cholesterol (HDL-C), total amount of triglyceride, cholesterol and testosterone, DHEA-S, luteinizing hormone (LH), sex hormone of binding-globulin (SHBG), follicle-stimulating hormone (FSH), estradiol (E₂), 2-h plasma glucose following 75-g OGTT (2-h OGTT) and high-sensitivity of CRP levels were also measured. The levels of low-density lipoprotein cholesterol (LDL-C) were calculated considering the following formula: LDL-C=total cholesterol - (HDL-C + triglycerides/5). FBG, 2-h OGTT and hs-CRP of serum, serum, total cholesterol, triglycerides, and HDL-C were measured considering an auto-analyzer (Olympus AU 2700 Beckman Coulter Inc, CA, USA) with dedicated kits (Beckman Coulter Inc, CA, USA). The levels of insulin in serum were measured by means of chemiluminescent microparticle immunoassay (CMIA) with dedicated kits (Beckman Coulter Inc, CA, USA) along with auto-analyzer (UniCel DxI 800, Beckman Coulter Inc, CA, USA). High-performance liquid chromatography (Variant II Turbo, Bio-Rad, CA, USA) was used to measure HbA1_C levels. LH, FSH, E₂, DHEA-S, the levels of total testosterone and SHBG were also measured using CMIA (UniCel DXI 800, Beckman Coulter Inc., CA, USA). We calculated FAI by the following formula as (total testosteron/SHBG)×100. We used the homeostasis model assessment of insulin resistance (HOMA-IR) for the calculation of insulin resistance: fasting insulin (µU/mL)×fasting glucose (mg/dL)/405 (20).

Measurement of circulating CTRP5 by ELISA

Commercially available human ELISA kits (E-EL-H4186, Elabscience-Biotech Co. Ltd, Wuhan, China) were used to measure serum CTRP5 levels (in duplicate) in accordance with the manufacturer’s instructions. The intra-assay coefficient of variability (CV) showed a rate <6% and inter-assay CV showed a rate <8%. The detectable range for serum CTRP5 was 0.31 to 20 ng/mL.

Power analysis

The minimum number of participants required for a study power of 0.90 and α=0.05 was determined using G Power 3.0.10 G software for Windows (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) (21), based on the results of our pilot study on circulating CTRP5 levels. We evaluated 25 women with PCOS (CTRP5 levels: 5.38±3.13 ng/mL) and 25 women as controls (CTRP5 levels: 8.20±3.98 ng/mL). According to this analysis, a minimum of 68 subjects were needed in each group. The investigation of all data analyses was completed using the Statistical Package for the Social Sciences software version 18.0 (SPSS Inc. Chicago, IL, USA). Kolmogorov-Smirnov test showed that the numeric variables conformed to normal distribution. The data are stated as mean ± standard deviation. A t-test was considered for the comparison of laboratory and demographic characteristics between the two groups. The PCOS group was separated into two different subgroups: patients with insulin resistance (HOMA-IR >2.71) and those subjects without insulin resistance (HOMA-IR ≤2.71) (22). CTRP5 levels in the PCOS subgroups were also compared using the t-test. Relationships between CTRP5 and other variables were assessed using Pearson’s correlation coefficient, and a linear regression model was formed to assess the presence of independent associations between CTRP5 and metabolic-hormonal parameters including FAI, BMI, and HOMA-IR. The variance inflation factor (VIF) of independent variables was calculated to determine multicollinearity. Variables with VIF >2.5, such as FBG and waist circumference, were not used in the model. We also adjusted the model for some parameters such as age, hs-CRP, and lipid parameters, as well as PCOS status. To estimate the possible association between CTRP5 levels (in tertiles) and PCOS risk, odds ratios (OR) were calculated using multivariate logistic regression analysis. Possible confounders such as BMI, age, HOMA-IR, FAI, and lipid parameters were included in the model for adjustment. The compatibility of the model was evaluated using the Hosmer-Lemeshow test (p>0.05). Confidence intervals (CI) were calculated at 95% and two-sided p values <0.05 were accepted as statistically significant.

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Association of decreased C1q/tumor necrosis factorrelated protein-5 levels with metabolic and hormonal disturbance in polycystic ovary syndrome