Investigation of maternal serum endocan concentrations in pregnant women with gestational diabetes mellitus; a prospective case-control study

Abstract Objective We aimed to investigate the maternal serum endocan concentrations in pregnant women diagnosed with Gestational Diabetes Mellitus (GDM) and to investigate the usability of serum endocan in GDM screening. Methods This prospective case-control study was conducted with 160 pregnant women. The GDM group consisted of 80 pregnant women who had 75 g OGTT between the 24th and 28th weeks of pregnancy and were diagnosed with GDM. The control group consisted of 80 healthy pregnant women who were matched with the GDM group in terms of age and body mass index (BMI) and had a normal 75 g OGTT result. Serum endocan concentrations were evaluated between 24 and 28 weeks of gestation in all participants and the groups were compared in terms of serum endocan concentrations. Results The median maternal serum endocan concentration was found to be significantly higher in the GDM group than in the control group (498 ng/L, and 467 ng/L, respectively, p = 0.024). In the subgroup analysis according to the BMI of the participants, the highest median maternal serum endocan concentration (513 ng/L) was found in the overweight GDM group. ROC analysis was performed to determine the value of maternal serum endocan concentration in predicting GDM. AUC analysis of maternal serum endocan for estimation of GDM was 0.603 (p = 0.024, 95% CI = 0.515 − 0.691). The optimal threshold value for maternal serum endocan concentration was determined as 376 ng/L with 88.75% sensitivity and 32.5% specificity. Conclusion Although serum endocan does not have high enough specificity to be used as an alternative to OGTT in GDM screening between 24 and 28 weeks of gestation, we think that it is somehow involved in the pathogenesis of GDM. The contribution of placental endocan expression to the serum concentration and the effect of blood glucose regulation on serum endocan concentration in GDM remain to be investigated.


Introduction
Gestational diabetes mellitus (GDM) is traditionally defined as glucose intolerance that occurs during pregnancy or is first detected during pregnancy [1].After the official definition of GDM by O'Sullivan in 1964, different protocols have been described in the screening of GDM until today [2,3].There is no clear consensus on a single screening tool for preexisting diabetes mellitus (DM) in early pregnancy.Fasting blood glucose, HbA1c, and 75 g 2-h oral glucose tolerance test (OGTT) are currently used to screen for preexisting DM in early pregnancy [1].For GDM screening in advanced weeks of gestation, the International Association of Diabetes in Pregnancy Working Groups (IADPSG) and the World Health Organization (WHO) have approved a "One-step" 2-h 75 g OGTT between 24 and 28 weeks of gestation [4,5].However diagnostic thresholds for GDM also differ slightly between committees and associations in the "One step" 2-h 75 g OGTT [4].
Endocan, previously called endothelial cell-specific molecule 1 (ESM-1), was first identified by Lasalle et al. in 1996 by cloning human umbilical vein endothelial cells [6].It was later shown that endocan is a soluble dermatan sulfate proteoglycan molecule expressed by vascular endothelial cells and can freely circulate in the bloodstream [7].The endocan gene is located at position 5q11.2 on chromosome 5 in humans, and the mature endocan molecule is a 50 kDa polypeptide consisting of 165 amino acids [7].Subsequent studies have shown that the endocan molecule is a key player in the regulation of processes such as cell adhesion, inflammatory disorders, and tumor progression [8,9].TNF-a and pro-angiogenic growth factors such as VEGF, FGF-2, and HGF/SF potently increase the expression, synthesis, or secretion of endocan by human endothelial cells [10].
Endocan expression in the placental tissue was also investigated and it was shown that endocan is expressed in fetal endothelial cells, maternal endothelial cells, cytotrophoblasts, syncytiotrophoblasts, and decidual cells in normal healthy pregnancies.When the relationship between high blood pressure in pregnancy and endocan expression in the placenta was evaluated, it was found that placental endocan expression was significantly higher in pregnant women with hypertension or preeclampsia compared to normotensive controls [11,12].
An interesting study regarding the placental expression of endocan was conducted by Murthi et al.They investigated endocan expression in the term placenta of lean and obese pregnant women with normal glucose tolerance (NGT) and GDM.Maternal obesity did not affect placental endocan expression.Endocan expression was not different between women with NGT and body mass index (BMI) matched GDM groups.Endocan expression in the placenta of obese women with GDM was significantly higher compared to women with the BMI-matched NGT group [13].
In different studies in non-pregnant individuals, it has been suggested that high serum endocan concentrations are associated with Type 1 and Type 2 DM.While higher serum endocan concentrations were detected in diabetic patients with poor glycemic control, serum endocan concentrations were also decreased in parallel with hyperglycemia, which improved with lifestyle changes and medical treatment [14,15].
Unfortunately, nowadays, a considerable number of pregnant women refuse to have an OGTT for some reason.In a study conducted in our country, the rate of those who had OGTT for GDM screening was reported as 79.4%.The most common reason for rejecting the OGTT is the belief that the OGTT is unnecessary, unimportant, or harmful to the fetus [16].In addition, considering the special patient groups in which OGTT causes serious adverse effects, such as those who become pregnant after bariatric surgery, new molecules that can be an alternative to OGTT for GDM screening have begun to be investigated [17].Considering the information in the literature, we hypothesized that serum endocan concentrations can be used as an alternative tool in pregnant women who do not want to have OGTT for GDM screening and that high serum endocan concentrations can be used to differentiate pregnant women with GDM from those with NGT.Thus, we aimed to investigate maternal serum endocan concentrations in pregnant women with GDM.

Materials and methods
This prospective case-control study was conducted with 160 pregnant women aged between 18 and 40 years who applied to the Umraniye Training and Research Hospital, Department of Obstetrics and Gynecology between September 2022 and November 2022 and had their pregnancy follow-up and delivery in our hospital.
The GDM group consisted of 80 pregnant women who had 75 g OGTT between the 24th and 28th weeks of pregnancy and were diagnosed with GDM according to the test result.The control group consisted of 80 healthy pregnant women who had a normal 75 g OGTT result.To avoid confounder factors on maternal serum endocan concentration, the control group was formed by matching the GDM group in terms of age, BMI, and the gestational week at blood sampling.
Multiple pregnancies, those who conceive by in vitro fertilization, and those with any pregestational disease were not included in the study.Smokers, those with a history of GDM in previous pregnancies, and those who developed pregnancy-related diseases such as hypertension, preeclampsia, eclampsia, or intrahepatic cholestasis were also not included in the study.
The 75 g OGTT was applied to all participants between 24 and 28 weeks of gestation.OGTT results were evaluated according to the criteria recommended by the IADPSG.Accordingly, GDM was diagnosed when a single threshold value was met or exceeded; fasting value, 92 mg/dL; 1-h value, 180 mg/dL; 2-h value, 153 mg/dL [4].
Participants' age, BMI, obstetric histories, laboratory and ultrasound findings, and perinatal outcomes were recorded.
In a study published in 2016, endocan expression in the placenta of obese women with GDM was found to be significantly higher than in women with a BMImatched NGT group [13].Considering this, we divided the control and GDM groups into two according to the participants' BMI.Those with a BMI below 25 kg/m2 were classified as normal weight, and those with a BMI of 25 kg/m 2 and above were classified as overweight.First, the GDM group and the control group, and then four subgroups were compared in terms of maternal serum endocan concentrations.
To investigate the maternal serum endocan concentration after 8 h of fasting, approximately 5 ccs of blood samples were drawn from the antecubital vein of the participants in non-anticoagulant biochemistry tubes.Blood samples were kept at room temperature for 1 h and then centrifuged at 1000 rpm for 20 min.After centrifugation, the remaining serum in the upper part of the biochemistry tube was transferred to the Eppendorf tube and stored at À 80 degrees.Endocan concentrations in blood samples were studied with the Human Endothelial Cell-Specific Molecule 1 (ESM-1) ELISA Kit (Sunredbio, Room212

Statistical analysis
Power analysis was performed using the G � Power (v3.1.9.2) program to determine the number of samples.The power of the study is expressed as 1-b (b ¼ probability of type II error) and in general, studies should have 80% power.According to Cohen's effect size coefficients, it was calculated that there should be at least 64 people in the groups at the level of a ¼ 0.05, according to the calculation made by assuming that the evaluations to be made between two independent groups would have a medium effect size (d ¼ 0.5).Considering that there may be dropouts during the study, it was decided to include 80 participants in both groups.Since there was no dropout at the end of the study, statistics were performed on 160 participants, 80 of whom were in the GDM group and 80 of them were in the control group.
Statistical analysis was performed with the NCSS (Number Cruncher Statistical System) 2020 Statistical Software (NCSS LLC, Kaysville, UT) program.While evaluating the study data, quantitative variables were shown with mean, standard deviation, median, min, and max values, and qualitative variables were shown with descriptive statistical methods such as frequency and percentage.Shapiro Wilks test and Box Plot graphics were used to evaluate the conformity of the data to the normal distribution.Student's t-test was used for quantitative two-group evaluations with normal distribution.Mann Whitney-U test for the evaluation of non-normally distributed variables according to two groups; Kruskal Wallis test was used in the comparison of more than two groups, and the Dunn test was used to determine the group that caused the difference.Wilcoxon Signed Rank test was used for ingroup evaluations.Spearman's correlation analysis was performed according to distribution in the evaluation of the relations between the variables.The chi-square test, Fisher's Exact test, and Fisher Freeman Halton test were used to compare qualitative data.Statistical significance was accepted as p < 0.05 for all values.

Results
In this study, 80 pregnant women with GDM and 80 healthy pregnant women with normal 75-g OGTT results were compared in terms of serum endocan concentrations.
Both groups were similar in terms of age, BMI, gravida, and parity (p > 0.05, for each) (Table 1).Fasting glucose level, 1-st hour glucose level, and 2-nd hour glucose level were significantly higher in the GDM group compared to the control group in 75 g OGTT (p ¼ 0.001, for each).The median fasting insulin level, median HOMA-IR and mean HbA1c level were found to be significantly higher in the GDM group compared to the control group (p ¼ 0.037, p ¼ 0.014, p ¼ 0.001, respectively) (Table 2).
Both groups were similar in terms of mode of delivery, gestational week at birth, newborn birth weight and birth height, 1st and 5th min Apgar scores, and NICU admission (p > 0.05, for each) (Table 3).
Gestational week at blood sampling for endocan was similar for the two groups (p ¼ 0.373).The median serum endocan concentration was found to be significantly higher in the GDM group than in the control group (498 ng/L, 467 ng/L, respectively, p ¼ 0.024) (Figure 1 and Table 4).
When a significant difference was detected between the two groups in terms of serum endocan concentration, the control and GDM groups were divided into two groups according to the BMI of the participants.Normal weight control, overweight control, normal weight GDM, and overweight GDM groups were also compared in terms of serum endocan concentrations.Although the gestational week at blood sampling for endocan concentration showed a statistically significant difference between the four groups, this difference was not clinically significant (p ¼ 0.030).There was no significant difference between the four groups in terms of maternal serum endocan concentrations (p ¼ 0.157).However, the highest median endocan concentration was detected in the GDM group with overweight, followed by the GDM group with normal weight, the control group with normal weight, and the control group with overweight, respectively (513 ng/L, 497 ng/L, 467.5 ng/L, 467 ng/L, respectively) (Figure 2 and Table 5).
According to the Pearson correlation analysis, no significant correlation was found between maternal age, BMI, parity, 75 g OGTT fasting, 1st and 2nd-hour   6).ROC analysis was performed to determine the value of maternal serum endocan concentration in predicting GDM.AUC analysis of maternal serum endocan for estimation of GDM was 0.603 (p ¼ 0.024, 95% CI ¼ 0.515 À 0.691).The optimal threshold value for maternal serum endocan concentration was determined as 376 ng/L with 88.75% sensitivity and 32.5% specificity (Figure 3).

Discussion
This is the first study in the literature to investigate serum endocan concentrations in pregnant women with GDM.Serum endocan concentrations were found to be significantly higher in the GDM group than in the non-GDM control group.When the groups were divided into two subgroups as normal weight and overweight according to the BMI of the participants, the highest serum endocan concentration was found to be in the overweight GDM group, although there was no significant difference.In this subgroup analysis,   we attributed the insignificance of the difference in serum endocan concentration between the groups to the small number of participants in the subgroups.
As is known, proteoglycans are complex proteins found in the extracellular matrix, usually on the surface of cells [18].Unlike these traditional proteoglycans, endocan is also a dermatan sulfate proteoglycan but circulates freely in the blood [7].Endocan is produced specifically by the vascular endothelium and predominantly by pulmonary and renal endothelial cells.[10].However, endocan is not only expressed by vascular endothelial cells, but also by epithelial cells of the kidneys and lungs and cardiac muscle cells, and also in the placenta [9,11].
To understand the molecular mechanism of endocan in endothelial cells, endocan knockout mouse models were used.Decreased vascular permeability and leukocyte migration, delayed vascular growth in sprouting vessels, reduced filopodia extensions, and phosphorylated Erk1/2 (extracellular-signal-regulated kinases 1/2) were demonstrated in these endocan knockout mice.It was also determined that endocan exerts its effect on endothelial cells mainly through vascular endothelial growth factor (VEGF).Endocan competes with VEGF-A for binding to fibronectin, which increases VEGF-A bioavailability, thereby promoting VEGF signaling to regulate vascular growth and permeability [19].
Endocan has also been implicated as a proinflammatory mediator that binds to lymphocyte functionrelated antigen-1 (LFA-1) on leukocytes and regulates the inflammatory process [8].Associated with this, increased serum endocan concentrations have been observed in many diseases, including chronic kidney disease, cardiovascular disease, respiratory disease, sepsis, rheumatoid arthritis, systemic sclerosis, Behc¸et's disease, and some types of cancer [9].
It's known that transient or prolonged or acute hyperglycemia in both animal models and humans causes deterioration in vascular endothelial function through many different mechanisms [20].This information prompted scientists to investigate the relationship between diabetes mellitus and endocan.Anık et al. investigated serum endocan concentrations in children with Type 1 DM and found that serum endocan concentrations were significantly higher in children and adolescents with Type 1 DM compared to the control group without DM.In the group with Type 1 DM, serum endocan concentrations were found to be higher in patients with poor metabolic control.In addition, a significant positive correlation was reported between HbA1c levels and endocan concentrations in this study [14].Similarly, in our study, we found the serum endocan concentration to be significantly higher in the group with GDM compared to the control group without GDM.However, we did not detect any correlation between maternal serum endocan concentration and HbA1c level.
In a different study, Arman et al. investigated the effect of glycemic regulation on serum endocan concentration in patients with Type 2 DM.It was determined that the serum endocan concentrations in patients with Type 2 DM showed a significant reduction compared to the basal period, just like HbA1c levels, after three months of lifestyle change and medical treatment.In addition, the authors stated that HbA1c level tended to be independently associated with serum endocan concentration, but the relationship was not significant [15].
In a different study published in 2020, the relationship between serum endocan concentrations and lipoproteins in patients with Type 2 DM was investigated and the serum endocan concentrations of patients with Type 2 DM were found to be significantly higher than the diabetes-free group.In addition, a positive correlation was found between serum endocan concentration and HbA1c level according to Spearman correlation analysis.Similarly, in our study, we found that the maternal serum endocan concentration was significantly higher in the GDM group, but we could not show any relationship between serum endocan concentration and HbA1c level [21].
In the study conducted by Anık et al. in children with Type 1 DM and by Klisic et al. in adults with Type 2 DM, mean HbA1c levels were high in the DM groups (8.5%, 7.0%, respectively) [14,21].In our study, the mean HbA1c level in the group with GDM was 5.19%.Unlike the results of the two studies above, we attributed the reason why we could not show a positive relationship between maternal serum endocan concentration and HbA1c level in our study, because the mean HbA1c level at the time of diagnosis in the GDM group in our study was within normal range.
The only study in the literature that has been reported to investigate endocan in GDM so far is Murthi et al.'s study.In this study, it was determined that maternal obesity did not affect the placental endocan expression and the expression of endocan in the placenta was not different between the lean pregnant women with NGT and BMI-matched pregnant women with GDM.However, endocan expression in the placenta of obese women with GDM was significantly higher compared to pregnant women with BMImatched NGT.In support of this study, we also found the highest median serum endocan concentration in the overweight GDM group.It is not clear whether the higher serum endocan concentration in the overweight GDM group in our study is due to the shedding of endocan, which is overexpressed in the placenta, into the blood circulation, or whether it is due to increased endocan expression in vascular endothelial cells secondary to hyperglycemia caused by GDM itself.
Studies investigating the relationship between endocan and obesity have also been conducted in non-pregnant individuals, and contradictory results have been reported so far.Mercantepe et al. evaluated the serum endocan concentration in obese and normal-weight individuals.The serum endocan concentration in obese individuals was found to be similar to that in normal-weight individuals [22].In a different study conducted on prepubertal obese and normalweight children, the serum endocan concentration in obese children was found to be significantly higher than in normal-weight children [23].
Unfortunately, we did not find the specificity of the cutoff value of serum endocan, which we suggest could be used to predict GDM, and the AUC in ROC analysis high enough.However, we believe that endocan is involved in the GDM pathogenesis in a way that has not yet been clarified.
This single-center study has some limitations.This study was conducted with a limited number of participants, and the serum endocan concentrations of the participants were evaluated only once at the initial time of diagnosis and the changes in serum endocan concentrations after treatment in the GDM group were not evaluated.In addition, the inability to determine the expression of endocan in the placenta and its contribution to the serum concentration in an ongoing pregnancy was another important limiting factor.
To the best of our knowledge, this is the first study in the literature to examine maternal serum endocan concentration in pregnant women with GDM.
In conclusion, maternal serum endocan concentration was found to be significantly higher in pregnant women diagnosed with GDM between 24 and 28 weeks of gestation compared to the non-GDM group.Also, the highest serum endocan concentration was found in the overweight GDM group.Although the serum endocan concentration evaluated between 24 and 28 weeks of gestation does not have such a high specificity that it can be used as an alternative to OGTT, whether endocan can be used in early GDM screening in high-risk pregnant women is a subject of research.The contribution of placental endocan expression to serum endocan concentrations and the effect of blood glucose regulation on serum endocan concentrations remain to be investigated.

Figure 1 .
Figure 1.Maternal serum endocan concentrations of the control and the GDM groups.

Figure 2 .
Figure 2. Maternal serum endocan concentrations of the control group with normal weight, the control group with overweight, the GDM group with normal weight, and the GDM group with overweight.

Figure 3 .
Figure 3. ROC analysis for sensitivity, specificity, and positive and negative predictive value of maternal serum endocan in GDM.
, MeiLan Building, No.6497 HuTai Road, Baoshan District, Shanghai, China, Catalog No: 201-12-1978) using the enzymelinked immunosorbent assay method.For the Human ESM-1 ELISA Kit used in the study, a measurement value of 8-2000 ng/L and a sensitivity of 7.506 ng/L were determined.Istanbul, Umraniye Training and Research Hospital Local Ethics Committee approved this study (Ethics Committee Approval Number: B.10.1.TKH.4.34.H. GP.0.01/248).The study protocol was maintained by the Declaration of Helsinki.Informed and written consent was obtained from all participants.

Table 1 .
Demographic characteristics of the GDM and control groups.Test; �� Man Whitney U Test; ��� Chi-Square test; BMI: body mass index.

Table 2 .
Comparison of GDM and control groups in terms of laboratory findings.Man Whitney U test; �� Student T Test; OGTT: oral glucose tolerance test; HOMA IR: homeostatic model assessment for insulin resistance; HbA1c: hemoglobin A1c.Bold values are statistically significant at p < 0.05.

Table 3 .
Comparison of GDM and control groups in terms of perinatal outcomes.Man Whitney U test; �� Student T Test; ��� Chi-Square test; NICU: neonatal intensive care unit.

Table 4 .
Comparison of GDM and control groups in terms of the gestational week at blood sampling and maternal serum endocan concentrations.
Student T test.Bold values are statistically significant at p < 0.05.

Table 5 .
Comparison of the control group with normal weight, control group with overweight, GDM group with normal weight, and GDM group with overweight in terms of BMI, gestational week at blood sampling, and maternal serum endocan concentrations.
Kruskal Wallis Test; Post hoc evaluation was performed when statistical significance was determined between the groups.'a' represents the statistical difference between the control group with normal weight and the control group with overweight.'b' represents the statistical difference between the control group with normal weight and the GDM group with normal weight.'c' represents the statistical difference between the control group with normal weight and the GDM group with overweight.'d' represents the statistical difference between the control group with overweight and the GDM group with normal weight.'e' represents the statistical difference between the control group with overweight and the GDM group with overweight.'f' represents the statistical difference between the GDM group with normal weight and the GDM group with overweight.Bold values are statistically significant at p < 0.05.

Table 6 .
Correlation between maternal serum endocan concentrations and GDM-related parameters.