Comparison of biochemical oxidative stress markers of glucose tolerance tests performed for

Abstract

This study aimed to investigated whether any differences in the oxidative stress (OS) occurred between the two different methods used in gestational diabetes mellitus (GDM) screening. In this cross-sectional case-control study, 41 women and 47 women in the 24th-28th weeks of pregnancy underwent a 50-g glucose challenge test (GCT) (Group 1) and 75-g oral glucose tolerance test (OGTT) (Group 2), respectively. The levels of sirtuin (SIRT), total thiol, native thiol, disulfide, native SS, SS total SH, IMA, albumin, and adjusted-Ischemia Modified Albumin (IMA) were tested. The OS marker values of the hour-0 and hour-1 were compared in these two groups. The levels of native thiol in hour 1, total thiol in hour 1, disulfide in hour 0 and 1, disulfide/native thiol in hour 0 and 1, disulfide/total thiol in hour 0 and 1, and adj.-IMA in hour 1 were statistically significantly lower in group 2 compared to group 1. The IMA value in hour 1 was higher in group 2 compared to group 1. The SIRT value in hour-1 was statistically significantly higher compared to the baseline in pregnant women undergoing the 50-g GCT. In pregnant women undergoing the 75-g OGTT; The SIRT value, the disulfide/total thiol ratio, and the IMA values in hour-1 were statistically significantly higher compared to the baseline and the native thiol values, the total thiol levels, and the albumin levels in hour 1 were statistically significantly lower compared to the baseline. The 75-g OGTT, but not the 50-g GCT, induced a significant acute pro-oxidant shift, altering thiol/disulfide homeostasis and increasing IMA levels.

Introduction

Gestational diabetes mellitus (GDM) refers to glucose intolerance, which starts with conception and improves after the delivery. Pregnancy is a diabetogenic process characterized by insulin resistance, compensatory β-cell proliferation, and hyperinsulinemia. Insulin sensitivity decreases by 80% during pregnancy¹. The incidence of diabetes complicating pregnancy is estimated to be as high as 6–7% and 90% of these patients are pregnant women with GDM². Pregnant women with GDM should be identified early and followed-up closely due to the risk of potential complications. The complications associated with uncontrolled GDM include congenital anomalies, large for gestational age (LGA) babies, high rates of assisted birth and birth trauma, postpartum uterine atony, urinary tract infections, polyhydramnios, prematurity and related complications, respiratory distress syndrome (RDS), metabolic complications in newborns (hypoglycemia, hypocalcemia, hyperbilirubinemia), gestational hypertension and anxiety³. The American College of Obstetricians and Gynecologists (ACOG) recommends that all pregnant women should be screened with laboratory screening tests for detecting medical history, clinical risk factors, and blood glucose levels⁴. Screening is performed in the period from the 24th to 28th weeks, when the diabetogenic state of pregnancy usually develops.

The Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study was carried out to contribute to the development of internationally accepted criteria for the diagnosis of GDM⁵. Following this study; in 2008, the International Association of Diabetes and Pregnancy Study Groups (IADPSG) held a workshop, during which diagnostic criteria for GDM were proposed. According to the defenders of the IADPSG approach, the two-step method can miss the diagnosis in almost 25% of the GDM patients with normal 50 g-glucose challenge test (GCT) results but having abnormal oral glucose tolerance⁶. Finally, The American Diabetes Association (ADA) has recently summarized the issues associated with both strategies used in GDM screening, reporting that the information demonstrating the superiority of one strategy over the other is inadequate and that further studies are needed to determine which strategy is more effective. The two-step method starts by testing plasma glucose levels one hour after oral intake of 50-g glucose at any time of the day. When the plasma glucose level at the first hour is > 140 mg/dl, a 100-g oral glucose tolerance test (OGTT) is performed to evaluate the plasma glucose levels during the following 3 h. High plasma glucose levels in at least 2 measurements lead to the diagnosis of GDM. The one-step 75-g OGTT requires at least one value higher than a threshold level to make the diagnosis of GDM⁷.

Oxidative stress (OS) is described as the imbalance between the antioxidant defense systems and the free radicals released via pathological or normal metabolic processes⁸. OS is suggested to cause diseases due to its toxic effects on the metabolisms of carbohydrates, proteins, lipids, and DNA⁹. OS modifies the normal human serum albumin (HSA), converting it to ischemia modified albumin (IMA); which shows reduced affinity to metals such as cobalt, nickel or copper¹⁰. IMA is a metabolic variant, formed due to ischemic effects on the serum albumin. The role of high levels of free IMA is examined as a novel marker of OS in some endocrine disorders, hypoxia, inflammation, and endothelial dysfunction^11–13. Another parameter, dynamic thiol/disulfide homeostasis, has recently been described as a marker of oxidative stress and it has been demonstrated that it contributes to antioxidant defense mechanisms, detoxification, and apoptosis^14,15. Thiol is an organic compound containing an –SH moiety and it is a major component of the antioxidant system. The primary targets of the reactive oxygen species (ROS) are the –SH moieties in the sulfur-containing amino acids, primarily converting the –SH moieties to reversible disulfide bonds via oxidation. The disulfide bonds can further be reduced to thiol groups, restoring the thiol/disulfide homeostasis¹⁶.

Studies in the literature have already established that diabetic states in pregnancy aggravate OS and increase the levels of associated OS markers. This situation causes hesitations in pregnant women and obstetricians about performing OGTT. In this cross-sectional case-control study, we investigated whether any differences in the OS load occurred between the two methods used in GDM screening. This way; we aimed to obtain findings, which would contribute to the debate on the type of screening methods most suitable for the physiological constitution of pregnant women.

Materials and methods

Study design

Women with single pregnancies, admitted to the gynecology and obstetrics outpatient clinics of Liv Hospital, Ankara of in the period from August 2019-February 2020, were examined prospectively. Pregnant women were excluded when it was found that they were previously diagnosed with diabetes mellitus (DM) or endocrine disorders, or when a congenital anomaly was identified in the prenatal screening program.

41 healthy non-pregnant women and 47 women in the 24th-28th weeks of pregnancy underwent a 50-g GCT (Group 1) and 75-g OGTT (Group 2), respectively. The gestational week was determined based on either the date of the last menstrual period or the pregnancy dating in the first trimester by ultrasonographic imaging. Group 1 consisted of a different cohort of pregnant women undergoing the first step of the two-step approach (50-g GCT). Women with a positive 50-g GCT proceeded to a 100-g OGTT, but only data from their initial 50-g test used for this analysis to ensure group independence. Group 2 consisted of consecutive pregnant women undergoing the one-step 75-g OGTT. Pregnant women who would undergo 50–75 g OGTT were selected by computerized lottery. The pregnant women undergoing a 50-g GCT had an oral intake of a 50-g glucose solution and the glucose levels were tested in the first hour. When the hour 1 glucose levels were ≥140 mg/dl, a 3-hour 100-g OGTT was carried out. Peripheral venous blood samples were collected at 08.00 am after a fasting period of at least 8 h but not longer than 14 h in the pregnant women undergoing a 100-g OGTT. Then, the 100-g OGTT was performed. The pregnant women, who could not tolerate the test, underwent a repeat test a week later. The pregnant women were advised to avoid engaging in high-level physical activities during the tests. The glucose levels were tested with the glucose oxidase method. A diagnosis of GDM was made when 2 or more test results were higher than the threshold levels in the four obtained samples of blood during the 100-g OGTT. When one or more blood values of glucose in the hour 0, 1, or 2 of the 2-hour 75-g test was higher than the threshold levels, a diagnosis of GDM was made¹⁷. The Carpenter and Coustan (C&C) criteria were used for evaluating the 100-g OGTT test results. The ADA and ACOG recommendations were followed to make the diagnosis.

The demographic data of the pregnant women; age, gravidity, parity; presence of DM in their first-degree relatives in the family, history of delivering a macrosomic newborn (weighing ≥4000 g) in previous pregnancies, the increases in the bodyweight during the pregnancy, the body weight at the time of conception and childbirth, the body mass index (BMI) of the pregnant woman at the time of the first diagnosis of pregnancy, and the gestational week were noted.

The levels of thiol and disulfide were analyzed with the method developed by Erel and Neslioglu¹⁴. This method briefly involved the reduction of the reducible disulfide bonds to free functional thiol groups. Half of the difference between the total and natural thiol quantities provides the dynamic disulfide quantity. The levels of sirtuin (SIRT), total thiol, native thiol, disulfide, native SS, SS total SH, IMA, albumin, and adj-IMA were tested. The tests were carried out in a blinded fashion to the clinical data and course of the patients. The results of the tests were unavailable to the clinicians treating the patients, the study team, and the investigators during the study.

Statistical analysis

All statistical analyses were performed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). The distributional characteristics of continuous variables were assessed using the Shapiro–Wilk test, and data were presented as mean±standard deviation or median (interquartile range), as appropriate. Comparisons between the 50-g GCT and 75-g OGTT groups at baseline (Hour 0) and post-loading (Hour 1) were conducted using the independent samples t-test for normally distributed variables and the Mann–Whitney U test for non-normally distributed variables. Within-group changes from Hour 0 to Hour 1 were evaluated using paired t-tests or Wilcoxon signed-rank tests, depending on normality. Correlations between oxidative stress markers and clinical variables (age, BMI at conception, weight gain, and birthweight) were analyzed using Pearson or Spearman correlation coefficients. Effect sizes were calculated using Cohen’s d for parametric tests and the r statistic for non-parametric tests. Statistical significance was defined as a two-tailed p-value <0.05. A post-hoc sample size and power calculation, based on the between-group difference observed in disulfide levels at Hour 0 (mean difference ≈10.5 µmol/L; pooled SD ≈8 µmol/L), yielded an effect size of d ≈1.32, indicating that a minimum of 12 participants per group would have been sufficient to achieve 80% power at α =0.05. Therefore, the actual sample size of 41 and 47 participants in the two groups provided more than adequate power (>90%) to detect medium-to-large effect sizes for the primary comparisons. All statistical procedures were performed using two-tailed tests.

Ethics

This study was approved by the Institutional Review Boards (IRB) of Participants were enrolled Liv Hospital Ankara, Date: 2019/004, Nu: 001.

Results

The age, weight gain during the pregnancy, the length of the delivery period, and the APGAR scores of the infants were not different between the groups of pregnant women undergoing either a 50-g GCT or 75-g OGTT (Table 1).

Table 1.

Comparison of demographic and obstetric characteristics between groups.

Variable	50-g GCT (n:41)	75-g OGTT (n:47)	p value
Age (years)	25.06 ± 3.75	25.48 ± 4.76	0.625
BMI at conception (kg/m²)	24.6 ± 5.58	23.86 ± 4.04	0.456
Fasting glucose (mg/dL)	86.2 ± 12.44	82.42 ± 11.09	0.112
1-hour glucose (mg/dL)	121.98 ± 27.34	126.42 ± 29.27	0.435
Weight at conception (kg)	64.88 ± 14.88	61.6 ± 10.94	0.212
Weight at delivery (kg)	77.62 ± 14.6	75.61 ± 12.23	0.457
Weight gain (kg)	13.04 ± 5.85	13.77 ± 6.76	0.568
Active labor (4–10 cm, min)	185.34 ± 104.86	222.37 ± 148.29	0.177
Second stage (min)	15.14 ± 14.53	18.1 ± 17.09	0.396
Birthweight (g)	3268.8 ± 509.99	3161 ± 589.18	0.330
Delivery week (weeks)	38.54 ± 1.74	38.34 ± 1.9	0.585
APGAR 1 (n)	9 ± 0	8.9 ± 0.71	0.320
APGAR 5 (n)	9.98 ± 0.14	9.88 ± 0.72	0.336

No statistically significant differences were present between the groups in the values of SIRT in hour 0, SIRT in hour 1, total thiol in hour 0, IMA in hour 0, albumin in hour 0, and adj-IMA in hour 0. The levels of native thiol in hour 1, total thiol in hour 1, disulfide in hour 0 and 1, disulfide/native thiol in hour 0 and 1, disulfide/total thiol in hour 0 and 1, and adj-IMA in hour 1 were statistically significantly lower in group 2 compared to group 1. The IMA value in hour 1 was higher in group 2 compared to group 1 (Table 2).

Table 2.

Between-group comparison of oxidative stress markers (50-g GCT vs. 75-g OGTT).

Marker	50-g GCT Hour 0 (n:41)	75-g OGTT Hour 0 (n:47)	p₃	50-g GCT Hour 1 (n:41)	75-g OGTT Hour 1 (n:47)	p₄
SIRT1	2.05 ± 1.85	2.24 ± 2.91	0.719	4.96 ± 4.57	4.19 ± 4.95	0.719
Native thiol (µmol/L)	226.21 ± 36.54	203.51 ± 74.86	0.081	225.96 ± 66.42	183.54 ± 65.05	0.081
Total thiol (µmol/L)	281.62 ± 58.17	259.34 ± 68.71	0.107	271.47 ± 83.03	226.87 ± 71.81	0.107
Disulfide (µmol/L)	31.15 ± 8.55	20.62 ± 7.18	< 0.001	31.59 ± 10.02	19.55 ± 6.29	< 0.001
Disulfide/Native thiol	—	—	< 0.001	—	—	< 0.001
Disulfide/Total thiol	—	—	< 0.001	—	—	< 0.001
IMA (IU/mL)	0.61 ± 0.06	0.62 ± 0.06	0.177	0.61 ± 0.08	0.68 ± 0.07	< 0.001
Albumin (mg/dL)	2.88 ± 0.77	2.51 ± 1.09	0.072	2.96 ± 0.99	2.12 ± 1.03	0.072
Adj-IMA (mg/dL)	0.67 ± 0.17	0.60 ± 0.25	0.140	0.72 ± 0.23	0.57 ± 0.26	0.140

Ratio values calculated from table data. IMA: Ischemia Modified Albumin, SIRT: Sirtuin.

The OS marker values of the hour-0 and hour-1 were compared in pregnant women undergoing the 50-g GCT. The SIRT value in hour-1 was statistically significantly higher compared to the baseline. There were no differences between the hour-0 and hour-1 values in the remaining OS markers. The OS marker values of the hour-0 and hour-1 were compared in pregnant women undergoing the 75-g OGTT. The SIRT value, the disulfide/total thiol ratio, and the IMA values in hour-1 were statistically significantly higher compared to the baseline. The native thiol values, the total thiol levels, and the albumin levels in hour 1 were statistically significantly lower compared to the baseline. There were no differences between the hour-0 and hour-1 values of the remaining parameters (Table 3). The comparison of the test results of the OS markers between the GDM positive and negative pregnant women revealed no statistical differences between the two groups (Table 4).

Table 3.

Within-group changes in oxidative stress markers (Hour 0 → Hour 1).

Marker	50-g GCT Hour 0	50-g GCT Hour 1	p₁	75-g OGTT Hour 0	75-g OGTT Hour 1	p₂
SIRT1	2.05 ± 1.85	4.96 ± 4.57	0.001	2.24 ± 2.91	4.19 ± 4.95	0.022
Native thiol (µmol/L)	226.21 ± 36.54	225.96 ± 66.42	0.979	203.51 ± 74.86	183.54 ± 65.05	0.018
Total thiol (µmol/L)	281.62 ± 58.17	271.47 ± 83.03	0.293	259.34 ± 68.71	226.87 ± 71.81	0.001
Disulfide (µmol/L)	31.15 ± 8.55	31.59 ± 10.02	0.739	20.62 ± 7.18	19.55 ± 6.29	0.328
Disulfide/Native thiol	—	—	0.463*	—	—	0.423
Disulfide/Total thiol	—	—	0.067*	—	—	0.043
IMA (IU/mL)	0.61 ± 0.06	0.61 ± 0.08	0.825	0.62 ± 0.06	0.68 ± 0.07	< 0.001
Albumin (mg/dL)	2.88 ± 0.77	2.96 ± 0.99	0.587	2.51 ± 1.09	2.12 ± 1.03	0.039
Adj-IMA (mg/dL)	0.67 ± 0.17	0.72 ± 0.23	0.121	0.60 ± 0.25	0.57 ± 0.26

IMA: Ischemia Modified Albumin, SIRT: Sirtuin.

Table 4.

Comparison of oxidative stress markers between GDM (+) and GDM (–) pregnancies.

Marker	GDM– Hour 0 (n:78)	GDM– Hour 1	p₁	GDM+ Hour 0 (n:10)	GDM+ Hour 1	p₂
SIRT1	2.23 ± 2.54	4.52 ± 4.74	0.384	1.51 ± 1.74	4.76 ± 5.27	0.886
Native thiol (µmol/L)	212.44 ± 63.19	201.26 ± 70.52	0.480	226.97 ± 38.26	219.25 ± 52.31	0.439
Total thiol (µmol/L)	269.33 ± 66.77	243.71 ± 81.82	0.876	272.74 ± 47.11	278.38 ± 58.07	0.199
Disulfide (µmol/L)	25.34 ± 9.57	24.59 ± 10.36	0.594	27.04 ± 8.45	29.57 ± 7.72	0.147
IMA (IU/mL)	0.61 ± 0.06	0.65 ± 0.09	0.183	0.64 ± 0.03	0.62 ± 0.04	0.312
Albumin (mg/dL)	2.67 ± 0.97	2.46 ± 1.11	0.640	2.82 ± 0.93	2.91 ± 0.93	0.222
Adj-IMA (mg/dL)	0.63 ± 0.22	0.63 ± 0.26	0.352	0.70 ± 0.21	0.74 ± 0.21	0.230

IMA: Ischemia Modified Albumin, SIRT: Sirtuin.

Correlation analysis of the baseline levels of OS markers with the age and body weight revealed a negative correlation between the age and disulfide/native thiol ratio. No other correlations were observed between the baseline OS marker levels and the other parameters. Correlation analysis of the hour-1 levels of OS markers with the age and body weight revealed a negative correlation between the weight gain during pregnancy and disulfide levels at hour 1. No other correlations were observed between the baseline OS marker levels and the other parameters (Table 5).

Table 5.

Correlation of oxidative stress markers with clinical variables.

Marker	Age r (p)	BMI r (p)	Weight gain r (p)	Birthweight r (p)
SIRT1	0.066 (0.542)	–0.002 (0.987)	0.065 (0.547)	0.093 (0.389)
Native thiol (µmol/L)	0.179 (0.095)	0.038 (0.723)	–0.203 (0.058)	–0.143 (0.183)
Total thiol (µmol/L)	0.070 (0.519)	0.004 (0.971)	–0.164 (0.127)	–0.076 (0.481)
Disulfide (µmol/L)	–0.098 (0.365)	0.031 (0.773)	–0.239 (0.025)	–0.008 (0.944)
IMA (mg/dL)	0.033 (0.760)	0.086 (0.426)	0.056 (0.605)	0.129 (0.230)
Albumin (mg/dL)	–0.025 (0.819)	0.093 (0.391)	–0.198 (0.065)	–0.050 (0.641)
Adj-IMA (mg/dL)	–0.018 (0.868)	0.131 (0.224)	–0.192 (0.074)	–0.087 (0.419)

→ Weight gain → Disulfide (r= − 0.239, p = 0.025). IMA: Ischemia Modified Albumin, SIRT: Sirtuin.

Discussion

ROS act on several stages of atherogenesis; including endothelial activation, matrix remodeling, and LDL oxidation. ROS acts as an intracellular messenger physiologically, mainly targeting protein thiols. High levels of ROS activate MDA, leading to lipid peroxidation and cell injury.

A study on diabetic patients investigated serum levels of thiol/disulfide homeostasis and IMA levels by assigning the study patients to three groups as follows: Patients without retinopathy (group 1), patients with non-proliferative neuropathy (group 2), and patients with proliferative retinopathy (group 3). The study groups were similar in age and sex distribution. The native and total thiol levels and the native/total thiol ratio was significantly higher in group 3 compared to groups 1 and 2. The levels of disulfide, the disulfide/total thiol ratio, and the disulfide/native thiol ratio were significantly lower in group 3. Furthermore, the mean IMA levels in the study were significantly higher in the patient group with diabetic proliferative retinopathy. The study found out that the IMA levels and the parameters of thiol/disulfide homeostasis increased proportionally with the progression of diabetic retinopathy¹⁸. A review published in 2023 examined studies on insulin-like growth factor-1 receptor changes associated with DM up to 2022. The review found that IMA levels are increased in type 1, type 2, and GDM. Additionally, IMA was shown to be closely associated with the severity of DM complications, including hyperglycemia, dyslipidemia, diabetic retinopathy, diabetic nephropathy, peripheral arterial disease, and diabetic foot ulcer¹⁹.

A study from India evaluated the IMA levels in 100 patients with type 2 DM, comparing the patients with poor glycemic control with the DM patients with well-controlled blood glucose levels. The study found significantly higher levels of IMA in the patient group with poor glycemic control compared to the patients with well-controlled DM. The high serum levels of IMA were observed to be correlated with the HbA1c levels and the lipid profile. The study reported that high levels of IMA could be used as an indicator of OS in patients with DM²⁰. A similar study on children in diabetic ketoacidosis reported results supporting those of the former study²¹. Another study argued that serum albumin in diabetic patients was modified and IMA was formed in the chronic hypoxic state provoked by hyperglycemia and OS²². Many studies from Turkey have detected increased serum levels of thiol oxidation and a shift in the thiol/disulfide balance towards disulfide in both prediabetic and diabetic patients^23,24. These studies provided further support to the other studies in the literature demonstrating the OS conditions in diabetic patients.

In 2006, Biri et al. demonstrated the increased OS load in patients with GDM by measuring the oxidant and antioxidant parameters in the maternal blood, cord plasma blood, and the placenta. The authors observed that the antioxidant system was impaired, the free radical generating enzyme xanthine oxidase was activated, and oxidation reactions were facilitated in those patients with GDM²⁵. Özler et al. examined the maternal blood samples in pregnant women receiving a GDM diagnosis in the 24th -28th weeks of gestation. They found out significant relationships of insulin resistance and poor perinatal outcomes to the levels of ADAMTS5 and Total OS. Parallel to the findings reported by previous studies; they found that the aggravated OS, the levels of ADAMTS5, and elevated levels of TOS in GDM were related to the disease. Furthermore, the authors associated aggravated OS with poor perinatal outcomes²⁶. The same authors examined the aggravated OS in patients with GDM and gestational hypertension (GHT) in another study. They found even hyperglycemia or hypertension-induced by pregnancy can cause increases in DNA injury, chromosomal aberrations, and OS²⁷. That study and similar studies in the literature provides evidence for the relationship of GDM to aggravated OS.

Aktun et al. evaluated the thiol/disulfide homeostasis in the umbilical cord blood collected from pregnant women with GDM. Of the 62 GDM patients in their study, the disease was regulated by diet in 43 patients and 19 patients used insulin regularly. While the disulfide levels and the ratio of disulfide/native thiol and disulfide/total thiol were found to be elevated, the native thiol/total thiol ratio decreased. Furthermore, the study results were different between the insulin-receiving patient group and the patient group, whose diseases were regulated by diet. The levels of disulfide and HbA1c and the ratio of disulfide/native thiol and disulfide/total thiol were found to be higher in the patient group with GDM not using insulin. The study concluded that infants born to diabetic mothers were exposed to high levels of OS. The blood glucose levels were better controlled and the neonatal intensive care need was less in the patient group receiving insulin²⁸. A study in 2012 found higher levels of serum IMA levels in pregnant women with GDM compared to pregnant women with no GDM. The IMA levels were positively correlated with the levels of plasma glucose. They observed that serum IMA levels significantly decreased in the postpartum 6th week. The treatment outcomes with insulin therapy were better compared to the regulation of diabetes only with diet. A linear regression analysis demonstrated that fasting blood glucose levels determined the levels of IMA independently. GDM was found to be related to aggravated OS and pregnancy complications²⁹. Later, a study from Egypt on a total of 80 women in 2018, tested the mean platelet volumes (MPV) and IMA levels to evaluate OS in GDM. That study found statistically significantly high levels of MPV and IMA in the patient group with GDM. The study concluded that those markers tested in the cord blood were useful predictors of OS and postnatal complications in infants born to diabetic mothers³⁰. Those study results supported each other. Ozler et al. examined the thiol/disulfide balance in the cord blood samples collected from women with GDM in a prospective case-control study. They found elevated levels of disulfide and found out that the ratios of disulfide/total thiol and disulfide/native thiol were high. The study found out that the native thiol/total thiol ratio was low in the cord blood samples of infants born to obese or diabetic mothers. Those ratios were positively correlated with increased incidences of poor perinatal outcomes^31,32.

In our study, comparison of the OS marker values found in hour-0 and hour-1 in the 50-g GCT receiving pregnant women revealed that the levels of SIRT in hour-1 was statistically significantly higher compared to the baseline; however, there were no differences between the hour-0 and hour-1 values in the remaining OS markers. Comparison of the OS marker levels of the hour-0 and hour-1 in pregnant women undergoing the 75-g OGTT revealed that the levels of SIRT, the disulfide/total thiol ratio, and the IMA values in hour-1 after the OGTT were statistically significantly higher compared to the baseline levels. The native thiol values, the total thiol levels, and the albumin levels in hour 1 were statistically significantly lower compared to the baseline. We found two recent studies in the literature similar to our study. One of those studies was performed on 100 pregnant women undergoing a 50-g GCT in the 24th-28th gestational week to evaluate the effects of GCT on the thiol/disulfide balance. Comparison of the baseline levels (hour 0) with those levels in the first hour following the GCT revealed that the total thiol levels and the native/total thiol ratio showed a tendency to decline, while the disulfide levels and the ratios of disulfide/total thiol and disulfide/native thiol tended to increase in pregnant women with positive GCT results. It was concluded that OS was aggravated in women with positive GCT results because the glucose load led to alterations in the thiol/disulfide homeostasis. No such effects were observed in healthy pregnant women³³. Another study investigated the effects of the OGTT-induced hyperglycemia peak on the oxidant/antioxidant levels in the body. OS index was calculated using the TAS (total antioxidant capacity)/TOS ratio. The levels of glucose, TOS, TAS, superoxide dismutase (SOD), and lipid hydroperoxide (LOOH) were measured in minutes 0, 60, and 120 following a 75-g OGTT. The study reported that the parameters indicating OS (LOOH and TOS) increased, while the antioxidant parameters (TAS and SOD) decreased during OGTT³⁴. Our study results support the results reported by other studies in the literature investigating this subject. The age, weight gain during the pregnancy, the length of the delivery period, and the APGAR scores of the infants were similar across the groups of pregnant women undergoing either a 50-g or 75-g OGTT.

We classified the patients as GDM when they had blood glucose levels of ≥140 mg/dl and ≥180 mg/dl in the first hours after a 50-g GCT and 75-g OGTT, respectively. The patients with lower blood glucose levels at these respective time points were assigned to the non-GDM group. Of the total of 88 pregnant women, 10 were assigned to the GDM group and 78 were assigned to the non-GDM group. GDM-related complications; including perineal lacerations, neonatal intensive care unit admissions, dystocia, and polyhydramnios, were compared between these two study groups. Only one patient with perineal laceration was observed in the GDM group. No other GDM-related complications were observed in the study groups. The comparison of the baseline and hour-1 levels of the OS markers between the GDM and non-GDM pregnant women groups revealed no statistically significant differences.

Limitations

The results in Table 3 (GDM vs. non-GDM) are reported as non-significant, but with a total of only 10 GDM patients, the study is almost certainly underpowered to detect a difference. Also, because of our study is single-centered and includes relatively old data is a significant limitation.

Conclusion

Based on these results, it was concluded that 75-g OGTT aggravated the OS load in pregnant women by creating a glucose load; which eventually altered the thiol/disulfide homeostasis and IMA levels. The oxidant/antioxidant ratio was found unaltered after a 50-g GCT but it was found out that the oxidant/antioxidant levels were affected by the hyperglycemia peak induced by the 75-g OGTT. However, these increases were transient and remained within the range of normal postprandial physiological responses. Literature also shown that such acute oxidative responses following glucose challenges do not translate into short- or long-term clinical risks in healthy pregnancies. Also, further large-scale comprehensive studies investigating this subject are warranted in order to achieve certain conclusions about the oxidative stress load induced by OGTT in pregnant women and their fetuses.

Abbrevations

GDM

Gestational Diabetes Mellitus

OS

Oxidative Stress

OGTT

Oral Glucose Torelance Test

GCT

Glucose Challenge Test

IMA

Ischemia Modified Albumin

SIRT

Sirtuin

ACOG

American College of Obstetricians and Gynecologists

LGA

Large for Gestational Age

RDS

Respiratory Distress Syndrome

HAPO

Hyperglycemia and Adverse Pregnancy Outcome

HSA

Human Serum Albumin

ADA

American Diabetes Association

IADPSG

International Association of Diabetes and Pregnancy Study Groups

ROS

Reactive Oxygen Species

BMI

Body Mass Index

SPSS

Statistical Package for the Social Sciences

DM

Diabetes Mellitus

GHT

Gestational Hypertension

MPV

Mean Platelet Volumes

TAS

Total Antioxidant Capacity

SOD

superoxide dismutase

LOOH

lipid hydroperoxide

Author contributions

Bo.C.: This author made substantial contributions to the conception and design of the study; the acquisition of data; drafting the manuscript and approved the submitted version. Bu.C.: This author made substantial contributions to the conception and design of the study, drafting the manuscript and approved the submitted version. As the guarantor, this author accepts full responsibility for the fnished work and/or the conduct of the study, had access to the data, and controlled the decision to publish. She has agreed to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which he was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. B.T.: This author made substantial contributions to the interpretation of data; drafting the manuscript and approved the submitted version. T.E.: This author made substantial contributions to the conception and design of the study; the acquisition of data; drafting the manuscript and approving the submitted version. B.K.: This author made substantial contributions to the interpretation of data; drafting the manuscript and approving the submitted version. M.Ö.: This author made substantial contributions to the conception and design of the study; the acquisition of data; drafting the manuscript and approving the submitted version. C.Ş.: This author made substantial contributions to the conception and design of the study; performed the analysis and interpretation of data; drafting the manuscript and approving the submitted version. R.E.P.: This author made substantial contributions to the conception and design of the study; the acquisition of data; drafting the manuscript and approving the submitted version. B.G.: This author made substantial contributions to the conception and design of the study; the acquisition of data; drafting the manuscript and approving the submitted version.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was approved by the Institutional Review Boards (IRB) of Participants were enrolled Liv Hospital Ankara, Date: 2019/004, Nu: 001. All methods were performed in accordance with the Declaration of Helsinki. All patient provided written informed consent to participate. Competing interests The authors declare no competing interests.

Consent for publication

All authors consent for publication.