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Association of low maternal levels of salusins with gestational diabetes mellitus and with small-for-gestational-age fetuses
European Journal of Obstetrics & Gynecology and Reproductive Biology 167 (2013) 29–33
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Association of low maternal levels of salusins with gestational diabetes mellitus and with small-for-gestational-age fetuses Ebru Celik a,*, Onder Celik a, Ercan Yilmaz a, Ilgin Turkcuoglu a, Abdullah Karaer a, Ugur Turhan a, Suleyman Aydin b a b
Department of Obstetrics and Gynecology, Inonu University School of Medicine, Malatya, Turkey Firat University, Medical School, Department of Medical Biochemistry, Elazig, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 August 2012 Received in revised form 3 October 2012 Accepted 29 October 2012
Objectives: To evaluate maternal and cord serum concentrations of salusin-a and salusin-b in women with gestational diabetes mellitus (GDM) and with small-for-gestational age (SGA) fetuses. Study design: Pregnant women with GDM (n = 25), women with SGA (n = 20) and maternal age-matched normal healthy pregnant subjects (n = 25) participated in the study. Maternal serum and cord blood salusin-a and salusin-b levels at the time of birth were measured using ELISA, and their relation with metabolic parameters was also assessed. Results: Mean concentrations of maternal and fetal serum salusin-a in the GDM and SGA groups were significantly lower than those of the controls (P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively). Mean concentrations of maternal and cord blood salusin-b also decreased in both the GDM and the SGA groups in comparison to the control group (P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively). The concentrations of maternal serum salusin-a and salusin-b were strongly positively correlated with the concentrations of cord blood salusin-a and salusin-b (R = 0.92, P < 0.001 and R = 0.94, P < 0.001, respectively). Conclusions: The low levels of maternal serum salusin-a and salusin-b may have negative impact on metabolic disorders and vascular dysfunction. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gestational diabetes mellitus Small-for-gestational age Cord blood Salusin-a Salusin-b HOMA-IR
1. Introduction Many recent studies have concentrated on the vascular endothelium as a possible target organ in gestational diabetes mellitus (GDM) and disease related to placental insufficiency such as small-for-gestational-age (SGA) [1–4]. A low birth weight appears to be an indicator of fetal adaptations to a suboptimal intrauterine environment. A possible mechanism for fetal adaptive responses, which contribute to SGA, is a negative impact on endothelial function [5]. Endothelial cells have an essential effect on the regulation of vascular tone through the release of vasoactive substances [6]. In pathological pregnancies, such as GDM, SGA or preeclampsia, the synthesis of vasoactive substances is altered, leading to changes in uteroplacental circulation, which could induce slowing of fetal growth and development [7,8]. Salusin-a and salusin-b are recently recognized endogenous bioactive peptides, derived from 28- and 20-amino-acid precursors
respectively [9]. Salusins are secreted in various tissues such as blood vessels, kidneys, monocytes and macrophages, as well as being detected in human body fluids [9]. They mainly play a role in the cardiovascular system. An experimental study has indicated that infusion of either salusin-a or salusin-b results in low blood pressure and a marked decrease in heart rate and cardiac output [10]. It has also been reported that salusins may play a key role in promoting mild proliferation in vascular smooth muscle and fibroblast cells, and inhibit cardiomyocyte apoptosis [11]. Salusinb, however, has more potent effects than salusin-a. The development of vascular endothelial dysfunction may be relevant in women with gestational diabetes who suffer from increased insulin resistance and SGA [12,13]. We therefore evaluated salusin-a and salusin-b levels in such women. The objective of this study was to evaluate maternal and cord serum concentration of salusin-a and salusin-b in women with GDM, SGA and normal healthy pregnancies.
2. Materials and methods * Corresponding author at: Department of Obstetrics and Gynecology, Inonu University School of Medicine, Turgut Ozal Medical Center, 44315 Malatya, Turkey. Tel.: +90 422 3410660; fax: +90 422 3410660. E-mail address: [email protected] (E. Celik). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.10.032
Twenty-five pregnant women who were diagnosed with GDM and 20 pregnant women with fetal SGA diagnosed in the outpatient clinic of the Obstetrics and Gynecology Department
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in Inonu University, Turgut Ozal Medical Center, were recruited as the study groups. From an unselected population of pregnant women undergoing their routine pregnancy follow-up, 25 agematched pregnant women with normal glucose tolerance test were selected as the control group. The study protocol was approved by the Institutional Ethical Committee for Research on Human Subjects. Informed written consent form was obtained from all the women. 2.1. Participant selection criteria The inclusion criteria for women with a normal healthy pregnancy were (1) no pre-existing diabetes mellitus, (2) absence of clinical evidence of any major disease such as maternal cardiac disease, connective tissue disorders and renal and liver failure, (3) absence of medical treatment that may alter glucose tolerance, and (4) fetal estimated weight between 10th and 90th customized centiles confirmed at birth. The inclusion criteria for the pregnant women with GDM were: (1) newly diagnosed GDM cases, (2) no previous use of oral hypoglycemic agents, (3) no history of substance abuse or psychiatric illness, and (4) maternal age between 18 and 40 years. SGA was defined as a fetal estimated weight below 10th centile according to fetal sex, gestational age, maternal parity and reference standards [14] confirmed at birth. Pregnancies were dated according to the first-trimester crown-rump length measurements [15]. In the SGA group, women with gestational hypertensive disease (maternal systolic blood pressure 140/ 90 mm Hg; significant proteinuria > 300 mg/l/24 h) were excluded. The exclusion criteria from the study were the presence of (1) pre-existing diabetes mellitus (type-1 and -2), (2) macrovascular and/or microvascular complications, (3) multi-fetal gestation, (4) fetuses with chromosomal, genetic or structural defects and (5) chronic medical diseases such as urolithiasis, cirrhosis, congestive heart failure, hypertensive disorders or other known major diseases. 2.2. Oral glucose tolerance test and diagnosis of GDM Participants underwent a 1-h 50 g oral glucose challenge test between 24 and 28 gestational weeks as recommended by ACOG [16]. After ingestion of a drink containing 50 g of glucose, venous blood glucose level measured at 1 h. A 100 g oral glucose tolerance test (OGTT) was performed to diagnose gestational diabetes when the blood glucose after the 50 g glucose challenge test was raised (the threshold value is often set as 140 mg/dl). After ingestion of a drink containing 100 g of glucose, venous glucose levels were measured before and at 1, 2 and 3 h. The threshold values were defined as 105, 190, and 165, 145 mg/dl for fasting, 1, 2 and 3-h after 100 g OGTT, respectively. Pregnant women with two or more high serum glucose values were diagnosed as having GDM. Patients with a value of 200 mg/dl or higher after the 50 g glucose challenge test (GCT) were considered to have GDM and did not undergo the 100 g GTT. Normal glucose tolerance was diagnosed when the 50 g GCT value was at or under 140 mg/dl. Maternal age, body mass index (BMI) at delivery, blood pressure, birth weights, Apgar score and gestational ages at birth were evaluated in the study. Maternal BMI (kg/m2) was calculated as the ratio of the weight (kg) to the square of the height (m). Maternal blood pressure was measured in the right arm after the participants remained at rest for 10 min, with the subjects being in a sitting position and relaxed. Newborns who were delivered by cesarean section or spontaneous vaginal delivery were weighed, and first and fifth minute Apgar scores were also recorded subsequent to birth.
2.3. Biochemical analysis Fasting venous blood was obtained from an arm of each woman in the study groups and healthy pregnant woman, after giving birth but before delivery of the placenta. Cord blood samples were obtained from the umbilical cord immediately after delivery from all newborns in the GDM, SGA and control groups. The blood sample was delivered to the laboratory within 20 min, centrifuged (2000 g/min for 10 min at 4 8C) and the serum was stored at 80 8C until assayed. Serum salusin-a concentration was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit with a minimum detectable concentration less than 4.75 pg/ml, from Uscn Life Science Inc. (Cat No: E9189Hu, Wuhan, P.R. China). The intra- and inter-assay coefficients of variance (CV) for salusin-a ranged from 4.1% to 7.2% and 4.6% to 9.7%, respectively. Serum salusin-b concentration was measured using an ELISA kit with a minimum detectable level less than 8.2 pg/ml, from Uscn Life Science Inc. (Cat No: E92026Hu, Wuhan, P.R. China). The intra- and inter-assay CV ranged from 5.6% to 6.9% and 7.8% to 10.9%, respectively. All samples were read using Bio-Tek Instruments ELx800 Microplate Reader (Vermont, USA). The biochemist was blind to the identity of samples during processing. The results are presented as ng/ml. Serum insulin levels were measured using a competitive chemiluminescent enzyme immunoassay method with the same trademark kits (Immulite 2000 Analyzer, Diagnostic Products Corporation; DPC, Los Angeles, CA, USA). The respective inter- and intra-assay CV was 5.7% and 4.3% for insulin. Fasting glucose concentration was assessed by enzymatic colorimetric assay methods using an Abbott Architect C16000 auto analyzer (Abbott Diagnostic Lab., USA) and commercially available kits. The interand intra-assay CV were 3.4% and 3.0% for fasting glucose. For assessment of insulin resistance, the homeostasis model assessment insulin resistance index (HOMA-IR) was used [17], given as: HOMA-IR = fasting insulin (mU/ml) fasting glucose (mg/dl)/405. 2.4. Statistical analysis Comparison between the outcome groups was performed using chi square test for categorical variables. The normality of distributions was assessed using the Kolmogorov–Smirnov test. Variables (salusin-a and salusin-b in maternal and fetal serum) with a skewed distribution were log-transformed. Since there was a statistically significant difference in the BMI among the SGA, GDM and control groups, comparisons were performed using ANCOVA (BMI and gestational age were as a covariates) for the continuous variables. Correlation analysis was used to determine the significance of association (Pearson’s coefficient) between maternal serum and cord blood salusin-a and salusin-b levels with maternal and cord blood insulin, glucose levels, HOMA-IR, maternal age, BMI, gestational age and birth weight in the outcome groups. The results are presented as mean and standard deviation (SD). For all comparisons, a probability of <0.05 was considered to be significant. The data were analyzed using the Statistical Package for Social Sciences software 19.0 for Windows package software (SPSS Inc., Chicago, IL). 3. Results The maternal characteristics of each of outcome groups are compared in Table 1. In comparison with the controls, the mean BMI was significantly higher in the GDM group than the control and SGA groups (P = 0.02 and P = 0.01, respectively). The rate of cesarean section (C/S) was 60.0% (n = 15) in the GDM group, 65.0% (n = 13) in the SGA group and 56.0% (n = 14) in the control group (P = 0.73 and P = 0.49, respectively).
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Table 1 Comparison of maternal and neonatal characteristics among the GDM, SGA and control groups. Baseline variables
GDM (n = 25)
Controls (n = 25)
SGA (n = 20)
P1
P2
P3
Maternal age in years Maternal body mass index at delivery (kg/m2) Maternal fasting blood glucose (mg/dl) 1 h blood glucose (mg/dl) Maternal fasting insulin (mIU/ml) Maternal HOMA-IR Maternal glycosylated hemoglobin (HbA1c) Maternal systolic blood pressure (mmHg) Maternal diastolic blood pressure (mmHg) Gestational age at delivery (days) Birth-weight (g) Fetal length (cm) Cord blood glucose (mg/dl) Placental weight (g)
31.38 4.75 31.28 7.0 81.58 8.05 211.72 27.34 18.24 4.44 3.64 0.99* 6.08 0.71 127.31 20.39 79.20 11.15 267.19 15.89 3337.23 628.41 51.35 3.53 78.04 7.62* 666.54 183.09*
29.17 5.09 25.56 3.56 81.65 9.43 87.74 18.90 11.72 3.30 2.29 0.68 – 109.04 8.46 71.96 7.01 367.63 9.77 3076.25 332.92 51.60 4.06 75.33 10.20 670.83 179.17
27.45 6.07 26.22 4.74 78.75 8.82 92.05 12.46 – – – 121.53 16.51 77.22 13.46 243.79 29.61 1967.47 468.92* 49.68 2.77 57.95 10.05 418.61 92.39
0.38 0.002* 0.87 0.001* 0.001* 0.001* – 0.001* 0.03* 0.99 0.15 0.88 0.32 0.99
0.47 0.99 0.17 0.41 – – – 0.001* 0.33 0.001* 0.001* 0.17 0.001* 0.001*
0.05 0.008* 0.36 0.001* – – – 0.25 0.60 0.001* 0.001* 0.37 0.001* 0.001*
Values are mean SD or n (%) and otherwise stated. Comparison among the outcome groups was by Mann–Whitney U test for continuous variables. P1 represented for comparison between the GDM and control groups; P2 represented for comparison between the SGA and control groups; P3 represented for comparison between the GDM and SGA groups. * P < 0.05.
The data were divided into two groups according to the mode of delivery as follows: Group 1: spontaneous vaginal delivery (n = 28) and Group 2: cesarean delivery (n = 42). There were no significant differences between two groups regarding serum concentrations of maternal and fetal salusin-a (1.44 1.13 vs. 1.61 1.23 and 1.68 1.34 vs. 1.68 1.32, P = 0.36 and P = 0.84, respectively). There were also no significant differences in the mean serum concentrations of maternal and fetal salusin-b between Group 1 and Group 2 (2.74 2.19 vs. 2.99 2.56 and 2.98 2.55 vs. 3.11 2.61, P = 0.92 and P = 0.83, respectively). Within Group 2, there were no significant differences in serum concentrations of maternal and fetal salusin-a between women with elective indications for C/S (n = 28) and those with fetal distress as the indication (n = 14) (1.77 1.23 vs. 1.24 1.34 and 1.78 1.16, P = 0.33 and P = 0.62, respectively). The mean serum concentrations of maternal and fetal salusin-b in women who underwent C/S due to elective indications were also similar to those who underwent C/S due to fetal distress (3.23 1.62 vs. 2.64 1.91 and 3.32 1.84 vs. 2.66 2.21, P = 0.36 and P = 0.35, respectively). There were no differences between the elective C/S and spontaneous vaginal delivery groups regarding maternal and fetal salusin-a and salusin-b (P = 0.42, P = 0.87, P = 0.94 and P = 0.90, respectively). The means of HOMA-IR, fasting insulin and 1 h blood glucose after a 50 g glucose load in the GDM group were higher than those in the control group (P < 0.0001, P = 0.0001 and P = 0.0001, respectively). There was no significant difference between the GDM and the control groups with regard to gestational age (days) and neonatal birthweight (P = 0.78 and P = 0.10, respectively). Further, the mean cord serum glucose was not different in the GDM group from the control group (P = 0.88).
In the SGA group, the mean gestational age and neonatal birthweight were significantly lower than in the control group (P < 0.001 and P < 0.001, respectively). Mean cord serum glucose was lower in the SGA group when compared to the control and the GDM groups (P < 0.001 and P < 0.001, respectively). The concentrations of maternal serum and cord blood salusin-a and salusin-b in the outcome groups are presented in Table 2. The mean level of maternal serum salusin-a in the GDM group was significantly lower than in the control group (2.11 1.63 vs. 2.84 0.88 ng/ml, P < 0.001). The mean and SD level of cord blood salusin-a in the GDM group were also significantly lower than the control group (1.89 0.73 vs. 2.90 1.04 ng/ml, P = 0.001). Similarly, the mean and SD level of maternal serum salusin-b was significantly lower in the GDM than the control group (2.65 0.67 vs. 6.06 0.92 ng/ml, P < 0.001). In the GDM group, the mean and SD level of cord blood salusin-b was decreased compared to the control group (2.76 0.72 vs. 6.47 0.78 ng/ml, P < 0.001). The data were divided into four subgroups according to the maternal BMI: (1) GDM with BMI < 27 (subgroup 1), (2) GDM with BMI 27 (subgroup 2), (3) Normal healthy pregnant (control) with BMI < 27 (subgroup 3), (4) Normal healthy pregnant (control) with BMI 27 (subgroup 4). The results are shown in Table 3. The mean level of maternal serum salusin-a was considerably decreased in the SGA group compared to the control group (0.34 0.62 vs. 2.84 0.88 ng/ml, P < 0.001). In SGA group, the mean and SD level of cord blood salusin-a was significantly lower than in the control group (0.29 0.69 vs. 2.90 1.04 ng/ml, P < 0.001). Further, reduced mean levels of maternal and cord blood salusin-b were observed in the SGA group in comparison to the control group (0.35 1.34 vs. 6.06 0.92 ng/ml and 0.33 1.25 vs. 6.47 0.78 ng/ml, P < 0.001 and P < 0.001, respectively).
Table 2 Comparison of maternal serum and cord blood salusin-a and salusin-b concentrations among GDM, SGA and control groups. Variables
GDM (n = 25)
Controls (n = 25)
SGA (n = 20)
P1
P2
P3
Maternal serum salusin-a (ng/ml) Maternal serum salusin-b (ng/ml) Cord blood salusin-a (ng/ml) Spontaneous vaginal delivery Elective C/S Fetal distress Cord blood salusin-b (ng/ml) Spontaneous vaginal delivery Elective C/S Fetal distress
2.11 1.63 2.65 0.67 1.89 0.73 1.66 0.27 1.84 0.39 2.05 0.55 2.76 0.72 2.80 0.66 2.74 0.78 2.83 0.05
2.84 0.88 6.06 0.92 2.90 1.04 3.20 0.29 3.11 0.77 2.16 0.68 6.47 0.78 6.18 1.46 5.76 0.91 6.12 0.58
0.34 0.62 0.35 1.34 0.29 0.69 0.11 0.01 0.13 0.07 0.25 0.33 0.33 1.25 0.04 0.02 0.10 0.16 0.04 0.01
<0.001* <0.001* <0.001* 0.003* 0.001* 0.65 <0.001* 0.001* 0.001* 0.001*
<0.001* <0.001* <0.001* 0.004* 0.001* 0.001* <0.001* 0.004* 0.001* 0.001*
<0.001* <0.001* <0.001* 0.003* 0.003* 0.03* <0.001* 0.002* 0.003* 0.03*
Values are mean SD or n (%) and otherwise stated. Comparison among the outcome groups was by Mann–Whitney U test for continuous variables. P1 represented for comparison between the GDM and control groups; P2 represented for comparison between the SGA and control groups; P3 represented for comparison between the GDM and SGA groups. * P < 0.05.
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Table 3 Comparison of maternal serum and cord blood salusin-a and salusin-b concentrations among the subgroups, defined according to the maternal BMI. Variables
Maternal BMI < 27
Maternal BMI 27
GDM (n = 8)
Controls (n = 16)
P
GDM (n = 17)
Controls (n = 9)
P
Maternal serum salusin-a (ng/ml) Maternal serum salusin-b (ng/ml) Cord blood salusin-a (ng/ml) Cord blood salusin-b (ng/ml)
1.85 0.75 2.89 0.29 1.76 0.31 2.72 0.68
2.84 0.99 5.89 1.11 3.14 0.73 6.56 0.76
0.04* 0.001* 0.02* 0.001*
1.78 0.47 2.70 0.37 1.80 0.39 2.93 0.39
2.73 0.73 5.91 1.28 3.02 1.11 5.88 1.42
0.001* <0.001* 0.001* <0.001*
Values are mean SD. Comparison among the outcome groups was by Mann–Whitney U test for continuous variables. * P < 0.05.
Table 4 Pearson correlations of maternal serum salusin-a and salusin-b levels with the clinical features, cord blood salusin-a and salusin-b. Variables
Maternal salusin-a Correlation coefficient
BMI (kg/m2) Maternal fasting glucose (mg/dl) Maternal HOMA-IR Maternal systolic blood pressure Maternal diastolic blood pressure Gestational age, weeks Cord glucose (mg/dl) Birth weight (g) Placental weight (g) Maternal serum salusin-a (ng/ml) Maternal serum salusin-b (ng/ml) Cord blood salusin-a (ng/ml) Cord blood salusin-b (ng/ml) *
0.14 0.14 0.38 0.26 0.05 0.33 0.68 0.42 0.42 – 0.88 0.92 0.84
Maternal salusin-b P NS NS 0.01* 0.04* NS 0.008* <0.001* 0.001* 0.001* – <0.001* <0.001* <0.001*
Correlation coefficient 0.08 0.10 0.56 0.42 0.06 0.34 0.68 0.44 0.44 0.88 – 0.86 0.94
P NS NS <0.001* 0.001* NS 0.006* <0.001* <0.001* <0.001* <0.001* – <0.001* <0.001*
Statistically significant.
Within the GDM group, there was no significant difference in mean maternal serum salusin-a and salusin-b levels between those treated with insulin (n = 17) and with diet (n = 8) during pregnancy (1.90 0.58 vs. 1.60 0.42 ng/ml and 2.72 0.41 vs. 2.84 0.18 ng/ml, P = 0.36 and P = 0.38, respectively). There was no significant difference between the two treatment groups regarding the mean neonatal serum salusin-a and salusin-b (2.05 0.75 vs. 1.78 0.31 ng/ml and 2.79 0.53 vs. 3.02 0.32 ng/ml, P = 0.52 and P = 0.41, respectively). There were strong positive correlations between maternal serum and cord blood levels of salusin-a and salusin-b (Table 3). Maternal serum salusin-a and salusin-b levels correlated negatively with the maternal HOMA-IR and systolic blood pressure, but positively with neonatal birthweight and gestational weeks (Table 4). Neonatal salusin-a and salusin-b were positively correlated with mean neonatal birthweight, gestational weeks at delivery, placental weight and cord blood glucose level (R = 0.45, P < 0.001; R = 0.46, P < 0.001; R = 0.35, P = 0.005; R = 0.36, P = 0.003; R = 0.31, P = 0.01; R = 0.44, P < 0.001; R = 0.70, P < 0.001 and R = 0.68, P < 0.001, respectively). 4. Comments The key findings of current study were the reduced concentrations of maternal serum salusin-a and salusin-b in the GDM and SGA groups in comparison to women with healthy pregnancies. A further observation was the correlation of maternal serum salusina and salusin-b values with cord blood salusin-a and salusin-b values. As yet, the variation of salusin levels in pregnant women has not been clearly defined. Of note it is demonstrated that elevated insulin resistance is associated with vascular endothelial dysfunction, which contributes to vascular disease [18]. Maternal diabetes is a major factor that results in fetal growth abnormalities [19,20]. Fetal hyperinsulinemia induced by maternal hyperglycemia is widely accepted to be the cause of diabetes-induced fetal overgrowth,
but in spite of a considerable improvement in glycemic control of pregnant women with gestational diabetes, a high percentage of large-for-gestational-age neonates are still born to mothers with diabetes [21]. In our study, we found a positive correlation between maternal salusin-a and salusin-b with neonatal birthweight and cord blood glucose levels. Further, we observed an interesting negative relationship between the maternal serum concentrations of salusin-a and salusin-b with HOMA-IR. These variables may not be a regulatory marker in maternal insulin homeostasis per se, but rather a factor that reduces as a consequences of the condition of maternal vascular endothelium and feto-placental circulation. We found that mean maternal systolic blood pressure was inversely correlated with maternal serum salusin-b, but not salusin-a. A previous study has observed that the concentrations of maternal serum salusins were not different in women with preeclampsia from the normotensive pregnant women [22]. Conversely, another study on the association of salusins with blood pressure reported that salusin-b plays a crucial role in the regulation of blood pressure and is more potent than salusin-a [23]. Our data are in agreement with the latter finding. Many previous studies have demonstrated that endothelial dysfunction is one of the reasons for low birth weight during pregnancy [14,24]. It has been shown that body size is positively correlated with endothelial vasodilation in newborns and that low birth weight may result in endothelial dysfunction later in life [14,24]. Evidence from previous studies suggests that salusin-a and salusin-b induce the proliferation of vascular smooth muscle cells, fibroblasts and muscle cells in addition to their hemodynamic regulatory, anti-atherosclerotic and anti-apoptotic properties [10,25]. The low cord blood salusin-a and salusin-b in the SGA group may imply that the risk of hypertension is increased [13,24]. Since the results of this study represent only one measurement in the third trimester of pregnancy, the impact of salusin-a and salusin-b on endothelial function has not yet been clearly identified. Hence there is a necessity for longitudinal studies on
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the precise effect salusin-a and salusin-b on fetal growth and development. The other possible explanation for low cord blood salusin-a and salusin-b in the SGA and GDM groups in comparison to the control group may be a strong positive correlation between maternal and neonatal concentrations of salusin-a and salusin-b. Further, these may also indicate that the maternal salusins are a source of neonatal salusins. Reduced levels of salusins were observed in SGA group than GDM group. The explanation for these findings may be occurrence of more severe vascular endothelial dysfunction in SGA than GDM. Thus, the vasculo-endothelial dysfunction may result in low salusins. We acknowledge that our study involves a relatively small number of participants, making it difficult to speculate on whether decreased salusins are the underlying mechanism in the pathogenesis of vascular endothelial dysfunction associated with GDM and SGA or vice versa. The regulatory pathway, synthesis and secretion of salusins in pregnant women have not yet been defined. In conclusion, we demonstrated low levels of maternal and neonatal serum salusins in women with small-for-gestational age fetuses and with gestational diabetes. This study suggests that low levels of salusins may occur as a result of reaction to metabolic and placental diseases involving vascular dysfunction, but the relations of salusins with gestational diabetes mellitus and small-forgestational-age remain to be determined. Conflict of interest The authors report no conflicts of interest. References [1] Sobrevia L, Abarzu´a F, Nien JK, et al. Review: differential placental macrovascular and microvascular endothelial dysfunction in gestational diabetes. Placenta 2011;32:159–64. [2] Norman M, Martin H. Preterm birth attenuates association between low birth weight and endothelial dysfunction. Circulation 2003;108:996–1001. [3] Ness RB, Sibai BM. Shared and disparate components of the pathophysiologies of fetal growth restriction and preeclampsia. American Journal of Obstetrics and Gynecology 2006;195:40–9. [4] Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. American Journal of Obstetrics and Gynecology 1989;161:1200–4. [5] Ligi I, Grandvuillemin I, Andres V, Dignat-George F, Simeoni U. Low birth weight infants and the developmental programming of hypertension: a focus on vascular factors. Seminars in Perinatology 2010;34:188–92. [6] Leach L, Babawale MO, Anderson M, Lammiman M. Vasculogenesis, angiogenesis and the molecular organisation of endothelial junctions in the early human placenta. Journal of Vascular Research 2002;39:46–259.
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[7] Westermeier F, Puebla C, Vega JL, et al. Equilibrative nucleoside transporters in fetal endothelial dysfunction in diabetes mellitus and hyperglycaemia. Current Vascular Pharmacology 2009;7:435–49. [8] Johnson MR, Anim-Nyame N, Johnson P, Sooranna SR, Steer PJ. Does endothelial cell activation occur with intrauterine growth restriction? BJOG An International Journal of Obstetrics and Gynaecology 2002;109:836–9. [9] Shichiri M, Ishimaru S, Ota T, Nishikawa T, Isogai T, Hirata Y. Salusins: newly identified bioactive peptides with hemodynamic and mitogenic activities. Nature Medicine 2003;9:1166–72. [10] Izumiyama H, Tanaka H, Egi K, Sunamori M, Hirata Y, Shichiri M. Synthetic salusins as cardiac depressors in rat. Hypertension 2005;45:419–25. [11] Yu F, Zhao J, Yang J, et al. Salusins promote cardiomyocyte growth but does not affect cardiac function in rats. Regulatory Peptides 2004;122:191–7. [12] Zawiejska A, Wender-Oz˙egowska E, Pietryga M, Bra˛zert J. Maternal endothelial dysfunction and its association with abnormal fetal growth in diabetic pregnancy. Diabetic Medicine 2011;28:692–8. [13] Touwslager RN, Houben AJ, Gielen M, et al. Endothelial vasodilatation in newborns is related to body size and maternal hypertension. Journal of Hypertension 2012;30:124–31. [14] Figueras F, Meler E, Iraola A, et al. Customized birthweight standards for a Spanish population. European Journal of Obstetrics Gynecology and Reproductive Biology 2008;136:20–4. [15] Robinson HP, Fleming JE. A critical evaluation of sonar ‘‘crown-rump length’’ measurements. British Journal of Obstetrics and Gynaecology 1975;82:702– 10. [16] ACOG Practice Bulletin. Antepartum fetal surveillance. Number 9, October 1999. Clinical management guidelines for obstetrician-gynecologists. International Journal of Gynaecology and Obstetrics 2000;68:175–85. [17] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. [18] Guzma´n-Gutie´rrez E, Abarzu´a F, Belmar C, et al. Functional link between adenosine and insulin: a hypothesis for fetoplacental vascular endothelial dysfunction in gestational diabetes. Current Vascular Pharmacology 2011;9:750–62. [19] Langer O, Conway DL. Level of glycemia and perinatal outcome in pregestational diabetes. The Journal of Maternal-Fetal Medicine 2000;9:35–41. [20] Mitkowska-Woz´niak H, Brazert J, Wender-Ozegowska E, et al. Prediction of fetal macrosomia using sonographically measured abdominal subcutaneous tissue thickness in pregnancies complicated by diabetes mellitus. Ginekologia Polska 2003;74:1444–9. [21] Zawiejska A, Wender-Ozegowska E, Mitkowska H, Pietryga M, Brazert J. Factors influencing birthweight in diabetic pregnancy. Archives of Perinatal Medicine 2004;10:37–41. [22] Simsek Y, Celik O, Yilmaz E, et al. Serum levels of apelin, salusin-alpha, and salusin-beta in normal pregnancy and preeclampsia. Journal of Maternal-Fetal and Neonatal Medicine 2012;25:1705–8. [23] Watanabe T, Suguro T, Sato K, et al. Serum salusin-alpha levels are decreased and correlated negatively with carotid atherosclerosis in essential hypertensive patients. Hypertension Research 2008;31:463–8. [24] Martin H, Hu J, Gennser G, Norman M. Impaired endothelial function and increased carotid stiffness in 9-year-old children with low birthweight. Circulation 2000;102:2739–44. [25] Xiao-Hong Y, Li L, Yan-Xia P, et al. Salusins protect neonatal rat cardiomyocytes from serum deprivation induced cell death through upregulation of GRP78. Journal of Cardiovascular Pharmacology 2006;48:41–6.
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Association of low maternal levels of salusins with gestational diabetes mellitus and with small-for-gestational-age fetuses Ebru Celik a,*, Onder Celik a, Ercan Yilmaz a, Ilgin Turkcuoglu a, Abdullah Karaer a, Ugur Turhan a, Suleyman Aydin b a b
Department of Obstetrics and Gynecology, Inonu University School of Medicine, Malatya, Turkey Firat University, Medical School, Department of Medical Biochemistry, Elazig, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 August 2012 Received in revised form 3 October 2012 Accepted 29 October 2012
Objectives: To evaluate maternal and cord serum concentrations of salusin-a and salusin-b in women with gestational diabetes mellitus (GDM) and with small-for-gestational age (SGA) fetuses. Study design: Pregnant women with GDM (n = 25), women with SGA (n = 20) and maternal age-matched normal healthy pregnant subjects (n = 25) participated in the study. Maternal serum and cord blood salusin-a and salusin-b levels at the time of birth were measured using ELISA, and their relation with metabolic parameters was also assessed. Results: Mean concentrations of maternal and fetal serum salusin-a in the GDM and SGA groups were significantly lower than those of the controls (P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively). Mean concentrations of maternal and cord blood salusin-b also decreased in both the GDM and the SGA groups in comparison to the control group (P < 0.001, P < 0.001, P < 0.001 and P < 0.001, respectively). The concentrations of maternal serum salusin-a and salusin-b were strongly positively correlated with the concentrations of cord blood salusin-a and salusin-b (R = 0.92, P < 0.001 and R = 0.94, P < 0.001, respectively). Conclusions: The low levels of maternal serum salusin-a and salusin-b may have negative impact on metabolic disorders and vascular dysfunction. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gestational diabetes mellitus Small-for-gestational age Cord blood Salusin-a Salusin-b HOMA-IR
1. Introduction Many recent studies have concentrated on the vascular endothelium as a possible target organ in gestational diabetes mellitus (GDM) and disease related to placental insufficiency such as small-for-gestational-age (SGA) [1–4]. A low birth weight appears to be an indicator of fetal adaptations to a suboptimal intrauterine environment. A possible mechanism for fetal adaptive responses, which contribute to SGA, is a negative impact on endothelial function [5]. Endothelial cells have an essential effect on the regulation of vascular tone through the release of vasoactive substances [6]. In pathological pregnancies, such as GDM, SGA or preeclampsia, the synthesis of vasoactive substances is altered, leading to changes in uteroplacental circulation, which could induce slowing of fetal growth and development [7,8]. Salusin-a and salusin-b are recently recognized endogenous bioactive peptides, derived from 28- and 20-amino-acid precursors
respectively [9]. Salusins are secreted in various tissues such as blood vessels, kidneys, monocytes and macrophages, as well as being detected in human body fluids [9]. They mainly play a role in the cardiovascular system. An experimental study has indicated that infusion of either salusin-a or salusin-b results in low blood pressure and a marked decrease in heart rate and cardiac output [10]. It has also been reported that salusins may play a key role in promoting mild proliferation in vascular smooth muscle and fibroblast cells, and inhibit cardiomyocyte apoptosis [11]. Salusinb, however, has more potent effects than salusin-a. The development of vascular endothelial dysfunction may be relevant in women with gestational diabetes who suffer from increased insulin resistance and SGA [12,13]. We therefore evaluated salusin-a and salusin-b levels in such women. The objective of this study was to evaluate maternal and cord serum concentration of salusin-a and salusin-b in women with GDM, SGA and normal healthy pregnancies.
2. Materials and methods * Corresponding author at: Department of Obstetrics and Gynecology, Inonu University School of Medicine, Turgut Ozal Medical Center, 44315 Malatya, Turkey. Tel.: +90 422 3410660; fax: +90 422 3410660. E-mail address: [email protected] (E. Celik). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.10.032
Twenty-five pregnant women who were diagnosed with GDM and 20 pregnant women with fetal SGA diagnosed in the outpatient clinic of the Obstetrics and Gynecology Department
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E. Celik et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 167 (2013) 29–33
in Inonu University, Turgut Ozal Medical Center, were recruited as the study groups. From an unselected population of pregnant women undergoing their routine pregnancy follow-up, 25 agematched pregnant women with normal glucose tolerance test were selected as the control group. The study protocol was approved by the Institutional Ethical Committee for Research on Human Subjects. Informed written consent form was obtained from all the women. 2.1. Participant selection criteria The inclusion criteria for women with a normal healthy pregnancy were (1) no pre-existing diabetes mellitus, (2) absence of clinical evidence of any major disease such as maternal cardiac disease, connective tissue disorders and renal and liver failure, (3) absence of medical treatment that may alter glucose tolerance, and (4) fetal estimated weight between 10th and 90th customized centiles confirmed at birth. The inclusion criteria for the pregnant women with GDM were: (1) newly diagnosed GDM cases, (2) no previous use of oral hypoglycemic agents, (3) no history of substance abuse or psychiatric illness, and (4) maternal age between 18 and 40 years. SGA was defined as a fetal estimated weight below 10th centile according to fetal sex, gestational age, maternal parity and reference standards [14] confirmed at birth. Pregnancies were dated according to the first-trimester crown-rump length measurements [15]. In the SGA group, women with gestational hypertensive disease (maternal systolic blood pressure 140/ 90 mm Hg; significant proteinuria > 300 mg/l/24 h) were excluded. The exclusion criteria from the study were the presence of (1) pre-existing diabetes mellitus (type-1 and -2), (2) macrovascular and/or microvascular complications, (3) multi-fetal gestation, (4) fetuses with chromosomal, genetic or structural defects and (5) chronic medical diseases such as urolithiasis, cirrhosis, congestive heart failure, hypertensive disorders or other known major diseases. 2.2. Oral glucose tolerance test and diagnosis of GDM Participants underwent a 1-h 50 g oral glucose challenge test between 24 and 28 gestational weeks as recommended by ACOG [16]. After ingestion of a drink containing 50 g of glucose, venous blood glucose level measured at 1 h. A 100 g oral glucose tolerance test (OGTT) was performed to diagnose gestational diabetes when the blood glucose after the 50 g glucose challenge test was raised (the threshold value is often set as 140 mg/dl). After ingestion of a drink containing 100 g of glucose, venous glucose levels were measured before and at 1, 2 and 3 h. The threshold values were defined as 105, 190, and 165, 145 mg/dl for fasting, 1, 2 and 3-h after 100 g OGTT, respectively. Pregnant women with two or more high serum glucose values were diagnosed as having GDM. Patients with a value of 200 mg/dl or higher after the 50 g glucose challenge test (GCT) were considered to have GDM and did not undergo the 100 g GTT. Normal glucose tolerance was diagnosed when the 50 g GCT value was at or under 140 mg/dl. Maternal age, body mass index (BMI) at delivery, blood pressure, birth weights, Apgar score and gestational ages at birth were evaluated in the study. Maternal BMI (kg/m2) was calculated as the ratio of the weight (kg) to the square of the height (m). Maternal blood pressure was measured in the right arm after the participants remained at rest for 10 min, with the subjects being in a sitting position and relaxed. Newborns who were delivered by cesarean section or spontaneous vaginal delivery were weighed, and first and fifth minute Apgar scores were also recorded subsequent to birth.
2.3. Biochemical analysis Fasting venous blood was obtained from an arm of each woman in the study groups and healthy pregnant woman, after giving birth but before delivery of the placenta. Cord blood samples were obtained from the umbilical cord immediately after delivery from all newborns in the GDM, SGA and control groups. The blood sample was delivered to the laboratory within 20 min, centrifuged (2000 g/min for 10 min at 4 8C) and the serum was stored at 80 8C until assayed. Serum salusin-a concentration was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit with a minimum detectable concentration less than 4.75 pg/ml, from Uscn Life Science Inc. (Cat No: E9189Hu, Wuhan, P.R. China). The intra- and inter-assay coefficients of variance (CV) for salusin-a ranged from 4.1% to 7.2% and 4.6% to 9.7%, respectively. Serum salusin-b concentration was measured using an ELISA kit with a minimum detectable level less than 8.2 pg/ml, from Uscn Life Science Inc. (Cat No: E92026Hu, Wuhan, P.R. China). The intra- and inter-assay CV ranged from 5.6% to 6.9% and 7.8% to 10.9%, respectively. All samples were read using Bio-Tek Instruments ELx800 Microplate Reader (Vermont, USA). The biochemist was blind to the identity of samples during processing. The results are presented as ng/ml. Serum insulin levels were measured using a competitive chemiluminescent enzyme immunoassay method with the same trademark kits (Immulite 2000 Analyzer, Diagnostic Products Corporation; DPC, Los Angeles, CA, USA). The respective inter- and intra-assay CV was 5.7% and 4.3% for insulin. Fasting glucose concentration was assessed by enzymatic colorimetric assay methods using an Abbott Architect C16000 auto analyzer (Abbott Diagnostic Lab., USA) and commercially available kits. The interand intra-assay CV were 3.4% and 3.0% for fasting glucose. For assessment of insulin resistance, the homeostasis model assessment insulin resistance index (HOMA-IR) was used [17], given as: HOMA-IR = fasting insulin (mU/ml) fasting glucose (mg/dl)/405. 2.4. Statistical analysis Comparison between the outcome groups was performed using chi square test for categorical variables. The normality of distributions was assessed using the Kolmogorov–Smirnov test. Variables (salusin-a and salusin-b in maternal and fetal serum) with a skewed distribution were log-transformed. Since there was a statistically significant difference in the BMI among the SGA, GDM and control groups, comparisons were performed using ANCOVA (BMI and gestational age were as a covariates) for the continuous variables. Correlation analysis was used to determine the significance of association (Pearson’s coefficient) between maternal serum and cord blood salusin-a and salusin-b levels with maternal and cord blood insulin, glucose levels, HOMA-IR, maternal age, BMI, gestational age and birth weight in the outcome groups. The results are presented as mean and standard deviation (SD). For all comparisons, a probability of <0.05 was considered to be significant. The data were analyzed using the Statistical Package for Social Sciences software 19.0 for Windows package software (SPSS Inc., Chicago, IL). 3. Results The maternal characteristics of each of outcome groups are compared in Table 1. In comparison with the controls, the mean BMI was significantly higher in the GDM group than the control and SGA groups (P = 0.02 and P = 0.01, respectively). The rate of cesarean section (C/S) was 60.0% (n = 15) in the GDM group, 65.0% (n = 13) in the SGA group and 56.0% (n = 14) in the control group (P = 0.73 and P = 0.49, respectively).
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Table 1 Comparison of maternal and neonatal characteristics among the GDM, SGA and control groups. Baseline variables
GDM (n = 25)
Controls (n = 25)
SGA (n = 20)
P1
P2
P3
Maternal age in years Maternal body mass index at delivery (kg/m2) Maternal fasting blood glucose (mg/dl) 1 h blood glucose (mg/dl) Maternal fasting insulin (mIU/ml) Maternal HOMA-IR Maternal glycosylated hemoglobin (HbA1c) Maternal systolic blood pressure (mmHg) Maternal diastolic blood pressure (mmHg) Gestational age at delivery (days) Birth-weight (g) Fetal length (cm) Cord blood glucose (mg/dl) Placental weight (g)
31.38 4.75 31.28 7.0 81.58 8.05 211.72 27.34 18.24 4.44 3.64 0.99* 6.08 0.71 127.31 20.39 79.20 11.15 267.19 15.89 3337.23 628.41 51.35 3.53 78.04 7.62* 666.54 183.09*
29.17 5.09 25.56 3.56 81.65 9.43 87.74 18.90 11.72 3.30 2.29 0.68 – 109.04 8.46 71.96 7.01 367.63 9.77 3076.25 332.92 51.60 4.06 75.33 10.20 670.83 179.17
27.45 6.07 26.22 4.74 78.75 8.82 92.05 12.46 – – – 121.53 16.51 77.22 13.46 243.79 29.61 1967.47 468.92* 49.68 2.77 57.95 10.05 418.61 92.39
0.38 0.002* 0.87 0.001* 0.001* 0.001* – 0.001* 0.03* 0.99 0.15 0.88 0.32 0.99
0.47 0.99 0.17 0.41 – – – 0.001* 0.33 0.001* 0.001* 0.17 0.001* 0.001*
0.05 0.008* 0.36 0.001* – – – 0.25 0.60 0.001* 0.001* 0.37 0.001* 0.001*
Values are mean SD or n (%) and otherwise stated. Comparison among the outcome groups was by Mann–Whitney U test for continuous variables. P1 represented for comparison between the GDM and control groups; P2 represented for comparison between the SGA and control groups; P3 represented for comparison between the GDM and SGA groups. * P < 0.05.
The data were divided into two groups according to the mode of delivery as follows: Group 1: spontaneous vaginal delivery (n = 28) and Group 2: cesarean delivery (n = 42). There were no significant differences between two groups regarding serum concentrations of maternal and fetal salusin-a (1.44 1.13 vs. 1.61 1.23 and 1.68 1.34 vs. 1.68 1.32, P = 0.36 and P = 0.84, respectively). There were also no significant differences in the mean serum concentrations of maternal and fetal salusin-b between Group 1 and Group 2 (2.74 2.19 vs. 2.99 2.56 and 2.98 2.55 vs. 3.11 2.61, P = 0.92 and P = 0.83, respectively). Within Group 2, there were no significant differences in serum concentrations of maternal and fetal salusin-a between women with elective indications for C/S (n = 28) and those with fetal distress as the indication (n = 14) (1.77 1.23 vs. 1.24 1.34 and 1.78 1.16, P = 0.33 and P = 0.62, respectively). The mean serum concentrations of maternal and fetal salusin-b in women who underwent C/S due to elective indications were also similar to those who underwent C/S due to fetal distress (3.23 1.62 vs. 2.64 1.91 and 3.32 1.84 vs. 2.66 2.21, P = 0.36 and P = 0.35, respectively). There were no differences between the elective C/S and spontaneous vaginal delivery groups regarding maternal and fetal salusin-a and salusin-b (P = 0.42, P = 0.87, P = 0.94 and P = 0.90, respectively). The means of HOMA-IR, fasting insulin and 1 h blood glucose after a 50 g glucose load in the GDM group were higher than those in the control group (P < 0.0001, P = 0.0001 and P = 0.0001, respectively). There was no significant difference between the GDM and the control groups with regard to gestational age (days) and neonatal birthweight (P = 0.78 and P = 0.10, respectively). Further, the mean cord serum glucose was not different in the GDM group from the control group (P = 0.88).
In the SGA group, the mean gestational age and neonatal birthweight were significantly lower than in the control group (P < 0.001 and P < 0.001, respectively). Mean cord serum glucose was lower in the SGA group when compared to the control and the GDM groups (P < 0.001 and P < 0.001, respectively). The concentrations of maternal serum and cord blood salusin-a and salusin-b in the outcome groups are presented in Table 2. The mean level of maternal serum salusin-a in the GDM group was significantly lower than in the control group (2.11 1.63 vs. 2.84 0.88 ng/ml, P < 0.001). The mean and SD level of cord blood salusin-a in the GDM group were also significantly lower than the control group (1.89 0.73 vs. 2.90 1.04 ng/ml, P = 0.001). Similarly, the mean and SD level of maternal serum salusin-b was significantly lower in the GDM than the control group (2.65 0.67 vs. 6.06 0.92 ng/ml, P < 0.001). In the GDM group, the mean and SD level of cord blood salusin-b was decreased compared to the control group (2.76 0.72 vs. 6.47 0.78 ng/ml, P < 0.001). The data were divided into four subgroups according to the maternal BMI: (1) GDM with BMI < 27 (subgroup 1), (2) GDM with BMI 27 (subgroup 2), (3) Normal healthy pregnant (control) with BMI < 27 (subgroup 3), (4) Normal healthy pregnant (control) with BMI 27 (subgroup 4). The results are shown in Table 3. The mean level of maternal serum salusin-a was considerably decreased in the SGA group compared to the control group (0.34 0.62 vs. 2.84 0.88 ng/ml, P < 0.001). In SGA group, the mean and SD level of cord blood salusin-a was significantly lower than in the control group (0.29 0.69 vs. 2.90 1.04 ng/ml, P < 0.001). Further, reduced mean levels of maternal and cord blood salusin-b were observed in the SGA group in comparison to the control group (0.35 1.34 vs. 6.06 0.92 ng/ml and 0.33 1.25 vs. 6.47 0.78 ng/ml, P < 0.001 and P < 0.001, respectively).
Table 2 Comparison of maternal serum and cord blood salusin-a and salusin-b concentrations among GDM, SGA and control groups. Variables
GDM (n = 25)
Controls (n = 25)
SGA (n = 20)
P1
P2
P3
Maternal serum salusin-a (ng/ml) Maternal serum salusin-b (ng/ml) Cord blood salusin-a (ng/ml) Spontaneous vaginal delivery Elective C/S Fetal distress Cord blood salusin-b (ng/ml) Spontaneous vaginal delivery Elective C/S Fetal distress
2.11 1.63 2.65 0.67 1.89 0.73 1.66 0.27 1.84 0.39 2.05 0.55 2.76 0.72 2.80 0.66 2.74 0.78 2.83 0.05
2.84 0.88 6.06 0.92 2.90 1.04 3.20 0.29 3.11 0.77 2.16 0.68 6.47 0.78 6.18 1.46 5.76 0.91 6.12 0.58
0.34 0.62 0.35 1.34 0.29 0.69 0.11 0.01 0.13 0.07 0.25 0.33 0.33 1.25 0.04 0.02 0.10 0.16 0.04 0.01
<0.001* <0.001* <0.001* 0.003* 0.001* 0.65 <0.001* 0.001* 0.001* 0.001*
<0.001* <0.001* <0.001* 0.004* 0.001* 0.001* <0.001* 0.004* 0.001* 0.001*
<0.001* <0.001* <0.001* 0.003* 0.003* 0.03* <0.001* 0.002* 0.003* 0.03*
Values are mean SD or n (%) and otherwise stated. Comparison among the outcome groups was by Mann–Whitney U test for continuous variables. P1 represented for comparison between the GDM and control groups; P2 represented for comparison between the SGA and control groups; P3 represented for comparison between the GDM and SGA groups. * P < 0.05.
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Table 3 Comparison of maternal serum and cord blood salusin-a and salusin-b concentrations among the subgroups, defined according to the maternal BMI. Variables
Maternal BMI < 27
Maternal BMI 27
GDM (n = 8)
Controls (n = 16)
P
GDM (n = 17)
Controls (n = 9)
P
Maternal serum salusin-a (ng/ml) Maternal serum salusin-b (ng/ml) Cord blood salusin-a (ng/ml) Cord blood salusin-b (ng/ml)
1.85 0.75 2.89 0.29 1.76 0.31 2.72 0.68
2.84 0.99 5.89 1.11 3.14 0.73 6.56 0.76
0.04* 0.001* 0.02* 0.001*
1.78 0.47 2.70 0.37 1.80 0.39 2.93 0.39
2.73 0.73 5.91 1.28 3.02 1.11 5.88 1.42
0.001* <0.001* 0.001* <0.001*
Values are mean SD. Comparison among the outcome groups was by Mann–Whitney U test for continuous variables. * P < 0.05.
Table 4 Pearson correlations of maternal serum salusin-a and salusin-b levels with the clinical features, cord blood salusin-a and salusin-b. Variables
Maternal salusin-a Correlation coefficient
BMI (kg/m2) Maternal fasting glucose (mg/dl) Maternal HOMA-IR Maternal systolic blood pressure Maternal diastolic blood pressure Gestational age, weeks Cord glucose (mg/dl) Birth weight (g) Placental weight (g) Maternal serum salusin-a (ng/ml) Maternal serum salusin-b (ng/ml) Cord blood salusin-a (ng/ml) Cord blood salusin-b (ng/ml) *
0.14 0.14 0.38 0.26 0.05 0.33 0.68 0.42 0.42 – 0.88 0.92 0.84
Maternal salusin-b P NS NS 0.01* 0.04* NS 0.008* <0.001* 0.001* 0.001* – <0.001* <0.001* <0.001*
Correlation coefficient 0.08 0.10 0.56 0.42 0.06 0.34 0.68 0.44 0.44 0.88 – 0.86 0.94
P NS NS <0.001* 0.001* NS 0.006* <0.001* <0.001* <0.001* <0.001* – <0.001* <0.001*
Statistically significant.
Within the GDM group, there was no significant difference in mean maternal serum salusin-a and salusin-b levels between those treated with insulin (n = 17) and with diet (n = 8) during pregnancy (1.90 0.58 vs. 1.60 0.42 ng/ml and 2.72 0.41 vs. 2.84 0.18 ng/ml, P = 0.36 and P = 0.38, respectively). There was no significant difference between the two treatment groups regarding the mean neonatal serum salusin-a and salusin-b (2.05 0.75 vs. 1.78 0.31 ng/ml and 2.79 0.53 vs. 3.02 0.32 ng/ml, P = 0.52 and P = 0.41, respectively). There were strong positive correlations between maternal serum and cord blood levels of salusin-a and salusin-b (Table 3). Maternal serum salusin-a and salusin-b levels correlated negatively with the maternal HOMA-IR and systolic blood pressure, but positively with neonatal birthweight and gestational weeks (Table 4). Neonatal salusin-a and salusin-b were positively correlated with mean neonatal birthweight, gestational weeks at delivery, placental weight and cord blood glucose level (R = 0.45, P < 0.001; R = 0.46, P < 0.001; R = 0.35, P = 0.005; R = 0.36, P = 0.003; R = 0.31, P = 0.01; R = 0.44, P < 0.001; R = 0.70, P < 0.001 and R = 0.68, P < 0.001, respectively). 4. Comments The key findings of current study were the reduced concentrations of maternal serum salusin-a and salusin-b in the GDM and SGA groups in comparison to women with healthy pregnancies. A further observation was the correlation of maternal serum salusina and salusin-b values with cord blood salusin-a and salusin-b values. As yet, the variation of salusin levels in pregnant women has not been clearly defined. Of note it is demonstrated that elevated insulin resistance is associated with vascular endothelial dysfunction, which contributes to vascular disease [18]. Maternal diabetes is a major factor that results in fetal growth abnormalities [19,20]. Fetal hyperinsulinemia induced by maternal hyperglycemia is widely accepted to be the cause of diabetes-induced fetal overgrowth,
but in spite of a considerable improvement in glycemic control of pregnant women with gestational diabetes, a high percentage of large-for-gestational-age neonates are still born to mothers with diabetes [21]. In our study, we found a positive correlation between maternal salusin-a and salusin-b with neonatal birthweight and cord blood glucose levels. Further, we observed an interesting negative relationship between the maternal serum concentrations of salusin-a and salusin-b with HOMA-IR. These variables may not be a regulatory marker in maternal insulin homeostasis per se, but rather a factor that reduces as a consequences of the condition of maternal vascular endothelium and feto-placental circulation. We found that mean maternal systolic blood pressure was inversely correlated with maternal serum salusin-b, but not salusin-a. A previous study has observed that the concentrations of maternal serum salusins were not different in women with preeclampsia from the normotensive pregnant women [22]. Conversely, another study on the association of salusins with blood pressure reported that salusin-b plays a crucial role in the regulation of blood pressure and is more potent than salusin-a [23]. Our data are in agreement with the latter finding. Many previous studies have demonstrated that endothelial dysfunction is one of the reasons for low birth weight during pregnancy [14,24]. It has been shown that body size is positively correlated with endothelial vasodilation in newborns and that low birth weight may result in endothelial dysfunction later in life [14,24]. Evidence from previous studies suggests that salusin-a and salusin-b induce the proliferation of vascular smooth muscle cells, fibroblasts and muscle cells in addition to their hemodynamic regulatory, anti-atherosclerotic and anti-apoptotic properties [10,25]. The low cord blood salusin-a and salusin-b in the SGA group may imply that the risk of hypertension is increased [13,24]. Since the results of this study represent only one measurement in the third trimester of pregnancy, the impact of salusin-a and salusin-b on endothelial function has not yet been clearly identified. Hence there is a necessity for longitudinal studies on
E. Celik et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 167 (2013) 29–33
the precise effect salusin-a and salusin-b on fetal growth and development. The other possible explanation for low cord blood salusin-a and salusin-b in the SGA and GDM groups in comparison to the control group may be a strong positive correlation between maternal and neonatal concentrations of salusin-a and salusin-b. Further, these may also indicate that the maternal salusins are a source of neonatal salusins. Reduced levels of salusins were observed in SGA group than GDM group. The explanation for these findings may be occurrence of more severe vascular endothelial dysfunction in SGA than GDM. Thus, the vasculo-endothelial dysfunction may result in low salusins. We acknowledge that our study involves a relatively small number of participants, making it difficult to speculate on whether decreased salusins are the underlying mechanism in the pathogenesis of vascular endothelial dysfunction associated with GDM and SGA or vice versa. The regulatory pathway, synthesis and secretion of salusins in pregnant women have not yet been defined. In conclusion, we demonstrated low levels of maternal and neonatal serum salusins in women with small-for-gestational age fetuses and with gestational diabetes. This study suggests that low levels of salusins may occur as a result of reaction to metabolic and placental diseases involving vascular dysfunction, but the relations of salusins with gestational diabetes mellitus and small-forgestational-age remain to be determined. Conflict of interest The authors report no conflicts of interest. References [1] Sobrevia L, Abarzu´a F, Nien JK, et al. Review: differential placental macrovascular and microvascular endothelial dysfunction in gestational diabetes. Placenta 2011;32:159–64. [2] Norman M, Martin H. Preterm birth attenuates association between low birth weight and endothelial dysfunction. Circulation 2003;108:996–1001. [3] Ness RB, Sibai BM. Shared and disparate components of the pathophysiologies of fetal growth restriction and preeclampsia. American Journal of Obstetrics and Gynecology 2006;195:40–9. [4] Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. American Journal of Obstetrics and Gynecology 1989;161:1200–4. [5] Ligi I, Grandvuillemin I, Andres V, Dignat-George F, Simeoni U. Low birth weight infants and the developmental programming of hypertension: a focus on vascular factors. Seminars in Perinatology 2010;34:188–92. [6] Leach L, Babawale MO, Anderson M, Lammiman M. Vasculogenesis, angiogenesis and the molecular organisation of endothelial junctions in the early human placenta. Journal of Vascular Research 2002;39:46–259.
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