# Abstract

 Our aim was to study the expression of adipokine-encoding genes (leptin, adiponectin, and angiopoietin-like protein 4 (ANGPTL4)) in human umbilical vein endothelial cells (HUVECs) and adipokine concentration in cord blood from women with gestational diabetes mellitus (GDM) depending on glycaemic targets. GDM patients were randomised to 2 groups per target glycaemic levels: GDM1 (tight glycaemic targets, fasting blood glucose \< 5.1 mmol/L and \<7.0 mmol/L postprandial, *N* = 20) and GDM2 (less tight glycaemic targets, \<5.3 mmol/L and \< 7.8 mmol/L, respectively, *N* = 21). The control group included 25 women with normal glucose tolerance. ANGPTL4 expression was decreased in the HUVECs from GDM patients versus the control group (23.11 ± 5.71, 21.47 ± 5.64, and 98.33 ± 20.92, for GDM1, GDM2, and controls; *p* < 0.001) with no difference between GDM1 and GDM2. The level of adiponectin gene expression was low and did not differ among the groups. Leptin gene expression was undetectable in HUVECs. In cord blood, leptin/adiponectin ratio (LAR) was increased in GDM2 compared to controls and GDM1 ( *p* = 0.038) and did not differ between GDM1 and controls. Tight glycaemic targets were associated with normalisation of increased LAR in the cord blood. ANGPTL4 expression was downregulated in HUVECs of newborns from GDM mothers and was not affected by the intensity of glycaemic control. # 3. Results 

## 3.1. Characteristics of the Participants Baseline characteristics of the participants are described in [Table 1](#) . The women from all three groups did not differ in terms of age and prepregnancy BMI. The GDM1 group had higher diastolic BP compared to controls ( *p* = 0.003). The GDM1 and GDM2 groups had higher levels of fasting PG ( *p* = 0.004 and *p* = 0.003, resp.) and higher levels of PG 1 h and 2 h in OGTT ( *p* < 0.001 in comparison with controls). Mean levels of fasting, 1-hour postprandial and average blood glucose measured by the participants during the study are described in [Table 2](#) . The GDM1 group achieved significantly lower average and 1-hour postprandial glucose levels compared to GDM2. Mean postprandial BG was lower in GDM1 even compared to the control group, though the difference did not reach statistical significance ( *p* = 0.088). Gestational weight gain did not differ between GDM1 and GDM2 groups and was lower in both groups compared to controls ( [Table 2](#) ). The percentage of women treated with insulin was 40% and 29% in the GDM1 and GDM2 groups, respectively, and did not significantly differ ( *p* = 0.495). 

## 3.2. Pregnancy Outcomes Pregnancy outcomes and biochemical markers in cord blood are shown in [Table 3](#) . There was no statistically significant difference among the groups in terms of pregnancy outcomes (percent of large for gestational age (LGA) and small for gestational age (SGA) newborns, delivery by caesarean section) and the level of C-peptide, adiponectin, and ANGPTL4 in cord blood serum and glucose in cord blood plasma. The level of leptin in cord blood serum was higher in the GDM2 group than in the GDM1 ( *p* = 0.036) and the control group, but the difference from the control group did not reach statistical significance ( *p* = 0.066). After adjustment by insulin therapy, age, and prepregnancy BMI, the difference in the level of leptin between GDM1 and GDM2 remained significant ( *p* = 0.01). The leptin/adiponectin ratio (LAR) in cord blood serum was higher in the GDM2 group compared to controls ( *p* = 0.011) with no difference between the GDM1 and control group ( *p* = 0.404). 

## 3.3. HUVEC Characterisation HUVECs were obtained from the umbilical vein, expanded in vitro, and characterised for expression of endothelial markers by flow cytometry and immunohistochemistry. All samples demonstrated characteristic endothelial morphology and immunophenotype CD45−/CD144+/CD31+/CD146+/CD105+ and stained positively for endothelial markers, von Willebrand factor, and CD146 (as demonstrated in our previous work \[ [20](#) \]). There was no difference in the parameters of viability and replicative aging of HUVEC cultures from different patient groups (data not presented). 

## 3.4. Gene Expression in HUVECs ANGPTL4 expression was downregulated in the HUVECs derived from GDM patients compared to control group (23.11 ± 5.71, 21.47 ± 5.64, and 98.33 ± 0.92, respectively, for GDM1, GDM2, and control groups; *p* < 0.001 for comparison among the 3 groups) while no difference between GDM1 and GDM2 groups was observed ( [Figure 1(a)](#) ). We did not detect the expression of LEP in HUVEC but found out that they expressed LEPR, and the expression of LEPR demonstrated a decline in the GDM1/GDM2 groups compared to the control group, though the differences were not statistically significant ( [Figure 1(c)](#) ). The expression of LEPR did not correlate with the level of LEP in cord plasma ( [Figure 1(d)](#) ) which indicates that there is no reciprocal regulation between LEP and its receptor in HUVECs. The expression of ADIPOQ was detected in HUVEC samples, but the level of its expression was as low as in negative control samples and lower by four orders of magnitude in HUVEC compared to adipocytes (positive control) ( [Figure 1(b)](#) ). -------------------------------------------------------------------------------------------------- GDM 1 GDM2 Control *p* ( *N* = 20) ( *N* = 21) ( *N* = 25) --------------------------------------- ----------------- ----------------- -------------- ------- Gestational age at delivery, weeks 39.2 ± 1.5 39.3 ± 1.0 39.7 ± 1.0 0.261 Caesarean section, % ( *N* ) 30% (6) 19% (4) 20% (5) 0.723 Birth weight, g 3572 ± 488 3584 ± 577 3513 ± 555 0.856 Height, cm 52.1 ± 2.5 52.4 ± 2.3 52.1 ± 2.5 0.990 LGA, % ( *N* ) 20% (4) 23% (5) 12% (3) 0.235 SGA, % ( *N* ) 5% (1) 9.5% (2) 4% (1) 0.819 Apgar score 1 min 7.5 ± 0.7 7.7 ± 1.1 7.7 ± 0.6 0.204 Apgar score 5 min 8.6 ± 0.5 8.7 ± 0.9 8.8 ± 0.4 0.208 Glucose, mmol/L 4.7 ± 1.2 5.3 ± 1.3 4.5 ± 1.2 0.203 C-peptide, ng/mL 0.8 ± 0.5 1.0 ± 0.6 0.9 ± 0.4 0.379 Leptin, ng/mL 8.8 ± 6.6 ^a^ 18.3 ± 16.1 10.6 ± 10.4 0.042 Adiponectin, ng/mL 15.9 ± 11.5 16.3 ± 14.4 18.3 ± 14.3 0.843 LAR 0.97 ± 1.31 1.70 ± 1.66 ^b^ 0.72 ± 0.46 0.038 ANGPTL4 in cord serum, ng/mL 19.9 ± 15.0 14.1 ± 4.5 13.9 ± 5.2 0.248 ANGPTL4 relative expression in HUVECs 23.1 ± 25.6 ^c^ 21.5 ± 25.8 ^c^ 98.3 ± 104.6 0.001 -------------------------------------------------------------------------------------------------- Notes: LAR: leptin/adiponectin ratio; LGA: large for gestational age; SGA: small for gestational age. LGA was defined by a birth weight exceeding the 90th percentile on standard charts. SGA was defined by a birth weight below the 10th percentile on standard charts. ^a^*p* < 0.05 versus GDM2; ^b^*p* < 0.05 versus the control group; ^c^*p* < 0.01 versus the control group.


Extracted trial result:
Outcome: leptin/adiponectin ratio (LAR) (continuous)
Intervention: GDM2 (less tight glycaemic targets, <5.3 mmol/L and < 7.8 mmol/L)
Comparator: GDM1 (tight glycaemic targets, fasting blood glucose_<_5.1_mmol/L and <7.0_mmol/L postprandial) postprandial,), control group- normal glucose tolerance
Intervention group size: 21
Intervention mean: 1.7
Intervention standard deviation: 1.66
Comparator group size: 45
Notes: combined means and stds not given