Placenta 35 (2014) 411e416
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Oxidative stress and maternal obesity: Feto-placental unit interaction N. Malti a, H. Merzouk a, *, S.A. Merzouk b, B. Loukidi a, N. Karaouzene a, A. Malti c, M. Narce d a Laboratory of Physiology, Physiopathology and Biochemistry of Nutrition, Department of Biology, Faculty of Natural and Life Sciences, Earth and Universe, University ABOU-BEKR BELKAÏD, Tlemcen 13000, Algeria b Department of Technical Sciences, Faculty of Engineering, University ABOU-BEKR BELKAÏD, Tlemcen 13000, Algeria c Gynecology and Obstetrics Department, Mother e Infant Hospital Center, University of Tlemcen, 13000, Algeria d INSERM UMR 866, ‘Lipids Nutrition Cancer’, University of Burgundy, Faculty of Life, Earth and Environment Sciences, Dijon 21000, France
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 10 March 2014
Objective: To determine oxidative stress markers in maternal obesity during pregnancy and to evaluate feto-placental unit interaction, especially predictors of fetal metabolic alterations. Patients and methods: 40 obese pregnant women (prepregnancy BMI > 30 kg/m2) were compared to 50 control pregnant women. Maternal, cord blood and placenta samples were collected at delivery. Biochemical parameters (total cholesterol and triglycerides) and oxidative stress markers (malondialdehyde, carbonyl proteins, superoxide anion expressed as reduced Nitroblue Tetrazolium, nitric oxide expressed as nitrite, reduced glutathione, catalase, superoxide dismutase) were assayed by biochemical methods. Results: Maternal, fetal and placental triglyceride levels were increased in obese group compared to control. Maternal malondialdehyde, carbonyl proteins, nitric oxide and superoxide anion levels were high while reduced glutathione concentrations and superoxide dismutase activity were low in obesity. In the placenta and in newborns of these obese mothers, variations of redox balance were also observed indicating high oxidative stress. Maternal and placental interaction constituted a strong predictor of fetal redox variations in obese pregnancies. Discussion: Maternal obesity compromised placental metabolism and antioxidant status which strongly impacted fetal redox balance. Oxidative stress may be one of the key downstream mediators that initiate programming of the offspring. Conclusion: Maternal obesity is associated with metabolic alterations and dysregulation of redox balance in the mother-placenta e fetus unit. These perturbations could lead to maternal and fetal complications and should be carefully considered. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Pregnancy Obesity Mother Newborn Placenta Oxidative stress
1. Introduction Obesity is a public health problem in most industrialized countries, and is now expanding to developing countries [1]. The rise in the prevalence of obesity might result from the increasingly sedentary lifestyle associated with a reduction in daily physical activity and/or from changes in eating behavior, both quantitatively and qualitatively that may play a role in early childhood [2]. Obesity is a risk factor for the development of several chronic diseases such as cardiovascular and respiratory diseases, diabetes type II, hypertension and some forms of cancer [3]. The importance of the intrauterine and neonatal metabolic environment as possible
* Corresponding author. Tel.: þ213 778303645. E-mail address: hafi[email protected] (H. Merzouk). http://dx.doi.org/10.1016/j.placenta.2014.03.010 0143-4004/Ó 2014 Elsevier Ltd. All rights reserved.
teratogenic determinants for the predisposition of obesity is widely supported [4]. Furthermore, obesity during pregnancy is associated to several maternal and fetal complications [5e7]. In addition, maternal obesity is important in promoting obesity in offspring, reflecting epigenetic programming [8,9]. Placental metabolic functions are not immune to this imprint [10,11]. Placental dysfunction is implicated in most of the poor pregnancy outcomes associated with maternal obesity, and is also known to be involved in developmental programming of later-life diseases [12,13]. Several studies have reported that obesity is associated with oxidative stress related to inadequate antioxidant defenses and increased rates of free radical formation [14]. The impact of oxidative stress on the fetus and the newborn is well known [15]. Pregnancy is a state of oxidative stress [16]. Placenta tissue is rich in hormones and is an important source of pro-oxidant agents as well as antioxidant enzymes [17]. Moreover, oxidative stress may affect
412
N. Malti et al. / Placenta 35 (2014) 411e416
the embryonic development [18]. Women with preeclampsia have an increased oxidative stress and lipid peroxidation and at the same time have a deficiency in several important antioxidants [19]. In this pathologic pregnancy, a heightened level of oxidative stress is encountered leading to placental oxidative damage which can affect placental function [20]. Oxidative stress may lead to a change in insulin sensitivity, oxidation of lipoproteins, lipid alterations and adult diseases, such as diabetes and atherosclerosis in the offspring of obese mothers. Maternal and fetal oxidative stress was enhanced during obesity [21]. Nevertheless, the simultaneous existence of a placental oxidative stress and maternal-feto-placental interactions during maternal obesity are still not well understood. The aim of the present work was to determine oxidative stress markers during pregnancy associated with maternal obesity, and to assess the impact on the maternal-placental-fetal unit. Therefore, it would be possible to characterize maternal and placental oxidant/ antioxidant imbalance and its impact on fetal redox status in obesity. 2. Patients and methods 2.1. Study population The study population included 50 normal weight (prepregnancy BMI between 19 and 25 kg/m2) and 40 obese (prepregnancy BMI 30 kg/m2) women who gave birth at the Obstetrics and Gynecology department of Tlemcen Hospital, Tlemcen city (West Algeria). The rigorous selection, recruitment and monitoring of pregnant women was done by clinicians of the service. These women are aged between 20 and 35 years and have full-term pregnancies (37 weeks). None of the subjects selected in this study had history of chronic diseases, hypertension, eclampsia, infections or fetal anomalies. All women were routinely seen by an obstetrician and an endocrinologist. They were tested for gestational diabetes according to the World Health Organization criteria and all had normal glucose tolerance test during the third trimester and within 48 h of delivery. All women had uncomplicated singleton pregnancies and delivered by caesarean section. The indications for elective Caesarean section at term were breech presentation, placenta praevia and previous Caesarean section. Maternal and neonatal characteristics are shown in Table 1. The study was performed according to the Declaration of Helsinki. All participants in this study were informed about the goals and the work in progress, and were asked to give their written consent beforehand. Investigations of patients as well as blood and placenta sampling conditions were subjected to a strict code of ethics. The protocol was approved by the Tlemcen Hospital Committee for Research on Human Subjects. 2.2. Blood samples Fasting maternal blood samples were obtained from the arm veins of the mothers, at the time of delivery. Cord blood samples were obtained from the umbilical vein immediately following delivery and after the cutting of the umbilical cord. Blood samples were collected in EDTA tubes, were centrifuged and plasma was separated for assessing lipids and plasma oxidative stress markers. The remaining
Table 1 Maternal and neonate characteristics. Characteristics
Control
Obese
Number Age (years) Prepregnancy BMI (kg/m2) Number of gestations Parity Gestational age (weeks) Birth weight (Kg) M/F sex ratio Total cholesterol Mothers (g/L) Newborns (g/L) Placenta (mg/g) Triglycerides Mothers (g/L) Newborns (g/L) Placenta (mg/g)
50 29 5.25 22.61 2.13 2.81 1.33 2.69 1.30 38 1 3.34 0.31 28/22
40 31.31 5.91 33.17 3.40** 3.56 2.00 3.12 1.63 38 1 4.28 0.35* 23/17
1.88 0.35 0.55 0.06 5.18 0.64
1.79 0.21 0.46 0.05 5.73 0.53
1.43 0.20 0.41 0.06 6.59 0.51
2.28 0.11** 0.68 0.07* 9.94 0.47*
Values are means SD. BMI: body mass index (weight/height2); M/F: males/females. Significant differences between obese and control groups are indicated as: *P < 0.05; **P < 0.01.
erythrocytes were washed and hemolysed by the addition of cold distilled water (1/ 4). The hemolysates were appraised for erythrocyte oxidant/antioxidant status. 2.3. Placental samples Placentas were collected immediately following delivery, within 10 min of delivery, and washed with saline water to eliminate the blood. Each placenta was weighed and sampled in three main regions (central region, mid placenta and peripheral region). Placental samples were snap frozen and stored at 80 C until further analysis. All samples were homogenized in four volumes of phosphate buffered saline (PBS) containing proteolytic enzyme inhibitors (Complete-Mini; Roche), using an Ultra-Turex homogenizer (Bioblock Scientific, Illkirch, France). Samples were then centrifuged for 30 min at 4000 rpm and the supernatant was collected for biochemical analysis. 2.4. Determination of biochemical parameters Plasma or placental homogenate cholesterol and triglycerides were determined by enzymatic methods (Kits Sigma Chemical Company, St Louis, MO, USA). 2.5. Determination of markers of the oxidant/antioxidant status Nitric oxide (NO) was determined by the method of Guevara et al. [22], after plasma or placental homogenate deproteinizing procedure (using methanol:diethylether; 3:1 mixture v/v). Nitrite and nitrate levels were measured together; nitrate being previously transformed to nitrite by cadmium reduction. Nitrite was assayed directly spectrophotometrically at 492 nm, using the colorimetric method of Griess (Griess reagent: 1 g/L sulfanilamide, 25 g/L phosphoric acid, and 0.1 g/L N-1-naphthylethylenediamine). Calibration curves were made with sodium nitrite in concentrations from 1 to 50 mmol/L. The inter- and intra-assay variations were 3.40% and 2.50%, respectively. The determination of the superoxide anion (O2) was based on Nitro Blue Tetrazolium (NBT) reduction in monofarmazan by O2 [23]. The blue formazan was dissolved using 2M potassium hydroxide and dimethylsulfoxide and its formation was monitored spectrophotometrically at 560 nm using the molar extinction coefficient (1.5 104 M1 $ cm1). The catalase activity (CAT, EC 1.11.1.6) was measured by spectrophotometric analysis of the decomposition rate of hydrogen peroxide according to the method of Aebi [24]. The reaction was initiated by addition of placental homogenate or hemolysate to the reaction mixture containing phosphate buffer (0.05 M, pH 7.2) and H2O2. Change in absorbance was recorded spectrophotometrically at 240. The results were expressed as unit of catalase activity corresponding to mmol of H2O2 decomposed per minute using the H2O2 standard curve. The inter- and intra-assay variations were 2.40% and 3%, respectively. The assessment of the enzymatic activity of superoxide dismutase (SOD, EC 1.15.1.1) was based on the ability to inhibit pyrogallol autoxidation, with one unit of SOD activity the amount that causes 50% inhibition of the oxidation of pyrogallol [25]. SOD activity was measured every 5 min over 1 h at 405 nm, for 1/20 dilution of placental homogenate and 1/5 dilution of hemolysate. The inter- and intra-assay variations were 3% and 3.80%, respectively. Hemolysate or placental homogenate reduced glutathione (GSH) levels were assayed by a colorimetric method based on the reduction of 5,5-dithiobis-(2nitrobenzoic) acid (DTNB) by GSH to generate 2-nitro-5-thiobenzoic acid which has yellow color, according a Sigma Aldrich Kit (Saint Louis, MO, USA). The absorbance at 412 nm was measured, and the GSH concentration was then determined with the GSH standard curve. The inter- and intra-assay variations were 1% and 0.50%, respectively. Plasma or placental homogenate carbonyl proteins (marker of protein oxidation) by the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) leading to the formation of stable dinitrophenyl (DNP) hydrazone adducts, which can be detected spectrophotometrically at 375 nm (Sigma Aldrich Kit (Saint Louis, MO, USA). Oxidized BSA standard was used for the standard curve. The interand intra-assay variations were 0.40% and 0.60%, respectively. Plasma or placental homogenate malondialdehyde (MDA, marker of lipid peroxidation) was estimated by the method of Draper and Hadley et al. [26] using thiobarbituric acid (TBA). Absorbance was measured at 532 nm. The results were expressed as micromoles of MDA, using the molar extinction coefficient of chromophore (1.56 105 M1 cm1). The inter- and intra-assay variations were 3.50% and 3%, respectively. 2.6. Statistical analysis The results are presented as means and standard deviations. A priori power analysis was performed to determine the sample size, using power and sample size calculator (Statistical solutions, Sigma). We calculated that a sample of 50 normal pregnant and 40 obese women would give us a 95% power of detecting a 25% difference in measurements. The results were tested for normal distribution using the ShapiroeWilk test. After variance analysis, Student’s t-test was used to compare means between normal weight and obese groups, for different parameters. The multiple regression analysis was performed with dependent variables (lipid parameters and markers of oxidant/antioxidant status in neonates) and independent
N. Malti et al. / Placenta 35 (2014) 411e416 variables (lipid parameters, and maternal-placental markers of oxidant/antioxidant status). B(ES) are the correlation coefficients (standard error) of each independent variable with the dependent variable. R2 is the coefficient of determination; it provides the percentage of variance expressed by all variables. Relationships were significant for p < 0.05. All tests were performed using STATISTICA4.1program (StatSoft, Tulsa, OK).
3. Results 3.1. Lipids and redox balance Maternal, fetal and placental cholesterol levels showed no significant difference in the obese group compared to the normal weight group. In contrast, a significant increase in maternal, fetal and placental triglyceride levels was found in the obese group (Table 1). In obese mothers, oxidant/antioxidant status alterations were marked by a significant increase in plasma concentrations of malondialdehyde, carbonyl proteins, nitrite (reflecting nitric oxide) and reduced NBT (reflecting anion superoxide production), and a significant decrease in reduced glutathione levels and in erythrocyte superoxide dismutase activity compared to control values (Figs. 1 and 2). In their newborns, the levels of malondialdehyde, nitrite, reduced NBT as well as erythrocyte activities of catalase and superoxide dismutase were significantly high compared to the control values (Figs.1 and 2). Furthermore, in the placenta of obese mothers, a significant increase was noted in the levels of malondialdehyde, carbonyl protein, reduced glutathione and also in antioxidant enzyme activities (superoxide dismutase and catalase) compared to values obtained with placentas from control mothers (Figs. 1 and 2). 3.2. Predictors of fetal metabolic alterations In order to investigate the influence of maternal and placental metabolic changes on fetal metabolism, a multiple regression
413
analysis was performed with independent variables or predictors (maternal-placental parameters) and dependent variables (neonate parameters). Maternal and placental cholesterol and triglyceride levels were not significantly correlated to those of the newborn while the interaction between these two predictors explained, respectively 38% and 39% of the variation of these parameters in control group. Regarding markers of oxidant/antioxidant status, no correlation was found between maternal, placental and fetal parameters. The mother-placenta interaction did not alter these observations (Table 2). In the obese group, maternal and placental cholesterol levels were not correlated with those of the newborn. However, the mother-placenta association influenced fetal plasma cholesterol levels and explained 35% of their variation (Table 3). There was a strong correlation between, on the one hand, maternal and fetal plasma levels of triglycerides and malondialdehyde (P ¼ 0.007 and P ¼ 0.008, respectively), and on the other hand, placental and fetal triglyceride and malondialdehyde levels (P ¼ 0.005). The mothere placenta interaction increased the power of the model (P ¼ 0.001 and P ¼ 0.004, respectively) and explained 64% and 47%, respectively, of the variation of these parameters in the newborn. Maternal Nitrite and reduced NBT levels were significantly correlated with those of newborns in obese group (P ¼ 0.01). The interaction mother-placenta could significantly predict the changes in fetal Nitrite and reduced NBT levels (42% and 43%, respectively). Regarding the antioxidant enzyme activities, though the maternal values were not associated with those of newborns, the activities of placental catalase and superoxide dismutase were positively correlated to the activities of these fetal enzymes (P ¼ 0.030 and P ¼ 0.039, respectively). The mother-placenta interaction was highly significant; it explained the respective changes (44% and 43%) of catalase and superoxide dismutase activities in newborns of obese mothers (Table 3).
Fig. 1. Antioxidant status in mothers and their newborns and placentas. Each value represents the mean standard deviation. GSH: reduced glutathione; SOD: superoxide dismutase. The significance of the differences between obese and control group was determined by the student’s t-test after analysis of variance:*P < 0.05. **P < 0.01.
414
N. Malti et al. / Placenta 35 (2014) 411e416
Fig. 2. Oxidant status in mothers and their newborns and placentas. Each value represents the mean standard deviation. CP: carbonyl proteins; MDA: malondialdehyde; NO: nitric oxide; O2: superoxide anion. The significance of the differences between obese and control group was determined by the student’s t-test after analysis of variance:*P < 0.05. **P < 0.01.
4. Discussion This study revealed changes in maternal, fetal and placental redox status as well as the maternal-placental-fetal interactions during obesity. Obesity is commonly associated with metabolic changes with high cardiovascular risk [3]. These metabolic alterations increase during pregnancy [5,6]. In our study, neither the obese women nor their neonates had any complications. These obese women presented elevated plasma triglyceride levels; whereas total plasma cholesterol levels were similar to those of normal weight women. Similar anomalies were noted in newborns and in placentas of the obese group. Our findings were in agreement with those of other authors who showed high serum triglyceride levels in obese women and their newborns compared with those of control group [20,27]. Increased estrogens levels and insulin resistance during pregnancy are responsible for this hypertriglyceridemia [28]. High triglyceride levels in newborns of obese mothers were correlated to their birth weights which were increased compared to controls [27]. We have previously found that normal weight newborns of obese mothers had triglyceride levels similar to those found in control newborns [21]. It has been shown that placental lipoprotein lipase (LPL) activity is high in obese pregnancies [29]. In the placenta, LPL hydrolyzes triglycerides from maternal circulation to release free fatty acids (FFA), which are transferred to the placenta, and then esterified to triglycerides. Other authors have noted an accumulation of triglycerides in the placenta of obese women [30].The multiple regression analysis in the control group showed that although maternal or placental lipid levels are not independently correlated to those of the fetus, the maternaleplacental
interaction was a substantial predictor of changes in fetal lipids. This was quite normal because the fetus is dependent on fat intake from the mother and transferred via the placenta. In the obese group, the correlations between maternal, placental and fetal triglycerides were more important, and the impact of the maternaleplacental interaction was stronger. This supposed the concept that in the presence of high maternal triglyceride levels, the placental transfer was more important, leading to fetal hypertriglyceridemia. Previous studies revealed also a highly significant correlation between maternal and fetal triglycerides [27]. It has been shown that high levels of triglycerides in maternal circulation may create a steep concentration gradient across the placenta, which accelerates their transport and deposition in fetal tissues [9]. Our results provided evidence that oxidant/antioxidant status was altered in the maternal fetoplacental unit during obesity. In addition, the multivariate analysis clearly revealed that fetal changes in lipid peroxidation, free radicals and antioxidant enzymes were directly related to maternal and/or placental changes in obesity. Oxidative stress is characterized by an imbalance between pro-oxidants (free radicals, peroxides) and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, antioxidant vitamins) [31,32]. Today, it is well established that oxidation phenomena are involved in the development of metabolic and neurodegenerative diseases, as well as aging. Vincent et al. . [33] showed that obesity increases oxidative stress by raising lipid peroxidation proportionally to the degree of adiposity and low antioxidant defense. Significant changes of the oxidant/antioxidant balance still exist during normal pregnancy, due to increased basal oxygen and energy consumption in different organs including the fetoplacental unit [16].
N. Malti et al. / Placenta 35 (2014) 411e416 Table 2 Multivariate analysis in the control group. Dependent Variables (newborns) Cholesterol B (ES) P R2 Triglycerides B (ES) P R2 Malondialdhyde B (ES) P R2 Carbonyl proteins B (ES) P R2 Nitrite B(ES) P R2 red. NBT B (ES) P R2 Catalase B (ES) P R2 Superoxide dismutase B (ES) P R2 Reduced glutathione B (ES) P R2
Mothers variables
Placenta variables
Interaction Mother-placenta
0.132 (0.061) 0.215 e
0.184 (0.053) 0.119 e
0.416 (0.065) 0.015 0.38
0.136 (0.040) 0.138 e
0.194 (0.020) 0.140 e
0.424 (0.069) 0.014 0.39
0.082 (0.050) 0.102 e
0.102 (0.041) 0.147 e
0.155 (0.077) 0.125 0.07
0.147 (0.032) 0.139 e
0.125 (0.051) 0.108 e
0.199 (0.078) 0.089 0.10
0.187 (0.071) 0.107 e
0.117 (0.064) 0.114 e
0.188 (0.027) 0.095 0.11
0.111 (0.037) 0.175 e
0.157 (0.027) 0.116 e
0.158 (0.063) 0.115 0.09
0.142 (0.070) 0.104 e
0.181 (0.030) 0.111 e
0.182 (0.083) 0.101 0.12
0.201 (0.073) 0.108 e
0.128 (0.076) 0.143 e
0.199 (0.051) 0.168 0.07
0.102 (0.052) 0.222 e
0.152 (0.075) 0.177 e
0.165 (0.077) 0.125 0.06
B (ES) are the correlation coefficients (standard error) of each independent variable with the dependent variable. R2 is the coefficient of determination and provides the percentage of variance explained by all variables. Relationships are significant at p < 0.05.
Antioxidants are extremely important in maintaining cell function during normal pregnancy [34]. According to Mueller et al. [17], during normal pregnancy, the placenta is a major source of prooxidants and antioxidant systems; it is capable of keeping lipid peroxidation under control. The placenta is rich in mitochondria, highly vascular and is exposed to high maternal oxygen partial pressure, therefore resulting in increased production of superoxide. The nitric oxide (NO) is also locally produced by the placenta from a substrate, L-arginine, by NO synthases (NOS) [35]. The formation of these two free radicals is tightly controlled by a highly effective antioxidant defense system during normal pregnancies. However, under pathological conditions, the redox balance is disturbed, thus inducing an oxidative stress. In our study, obese mothers showed high levels of pro-oxidant markers (superoxide anion as expressed by reduced NBT, nitric oxide as expressed by nitrite, malondialdehyde, carbonyl proteins) and a reduction in antioxidant defenses (reduced glutathione, superoxide dismutase). In our study, conventionally methods were used to estimate superoxide anion and nitric oxide. The reduction of NBT to insoluble blue formazan was used as a probe for superoxide generation, although it is not entirely specific for O2. Nitric oxide production was estimated from determining the concentrations of nitrite end products. A reduction in SOD, the primary enzyme that inactivates the superoxide radical and in GSH which is involved in glutathionedependent enzyme reactions, would lead to increased numbers of free radicals and this could thereafter be responsible for the
415
increased levels of malondialdehyde and carbonyl proteins in obese mothers. Antioxidant enzymes may also be consumed or inactivated in high oxidative conditions. Several studies reported that oxidative stress status in obese women during pregnancy was always associated with a pro-inflammatory state [36]. Our results proved the presence of oxidative stress in placentas of obese mothers. It is important to note that measurements were taken on term placentas at a single time point. The oxidative stress was characterized by high levels of malondialdehyde and carbonyl proteins despite elevated levels of reduced glutathione. Placental levels of nitric oxide (nitrite) and superoxide anion (reduced NBT) did not change significantly between the two groups. This might have resulted from the parallel increase in the activities of antioxidant enzymes, catalase and superoxide dismutase. High placental antioxidant activities might be a compensatory mechanism that limits oxidative stress in the placenta, during obesity [37]. Maintaining normal placental nitric oxide concentrations is an important aspect for a good pregnancy development, as nitric oxide plays a significant role in placental development, angiogenesis and vasodilatation [35]. However, in the placenta, excess superoxide and nitric oxide production can result in peroxynitrite formation, leading to nitrative stress. Peroxynitrite is a powerful pro-oxidant capable of initiating lipid peroxidation and nitro-tyrosine formation [19,37]. Further studies are then needed to confirm this hypothesis in the placenta of obese mothers. In the present study, infants of obese mothers were also subjected to oxidative stress. There was a significant increase in malondialdehyde, superoxide anion and nitric oxide levels in newborns of obese mothers compared to those of normal-weight mothers. There is some evidence that maternal obesity induced mitochondrial dysfunction with an increase in mitochondrial reactive oxygen species and oxidative stress in oocytes, zygotes and embryonic life [9]. Our findings are in agreement with the hypothesis that mitochondrial injury due to maternal obesity could compromise metabolism in the developing fetus and may even impact fetal mitochondrial function prior to conception. In newborns of obese mothers, the increase in pro-oxidants was associated with a parallel increase in antioxidant enzymes activities, catalase and superoxide dismutase. The over expression of antioxidant activities in these infants might be an adaptive response with an induction to counter the effect of increased oxidative stress. Our results suggested that these infants were exposed to greater oxidative stress despite higher antioxidant enzyme activities. The multivariate analysis showed that, during normal pregnancy, fetal redox status was not correlated with that of the mother or with that of the placenta, suggestive of a maximum protection of the fetus against free radicals. Pro-oxidants from the mother or from the placenta itself are usually destroyed before they reach child development [38]. In contrast, during obesity, the multivariate analysis indicated that the correlations between maternal or placental redox status and that of the fetus were significant. The maternaleplacental interaction was an important predictor of changes in fetal oxidant/antioxidant status during obese pregnancies. It was clear that oxidative stress in obese mother induced placental and fetal oxidative stress despite high placental antioxidant defense. Oxidative stress associated with obesity during pregnancy may be a contributing factor in postnatal consequences of the neonate and the induction of programmed phenotypes in the adult offspring. In fact, oxidative stress has also been identified as a contributing factor in epigenetic mechanisms [7e9,18,19,37,38]. However, only term pregnancies and placentas were considered in this study. We are then limited in our ability to identify redox alterations during the different trimesters of pregnancy. Further studies are needed to determine time course of change in maternal and placental oxidant/antioxidant status during the association of obesity and pregnancy.
416
N. Malti et al. / Placenta 35 (2014) 411e416
Table 3 Multivariate analysis in the Obese group. Dependent variables (newborn) Cholesterol B (ES) P R2 Triglycerides B (ES) P R2 Malondialdehyde B (ES) P R2 Carbonyl proteins B (ES) P R2 Nitrite B (ES) P R2 red. NBT B (ES) P R2 Catalase B (ES) P R2 Superoxide dismutase B (ES) P R2 Reduced glutathione B (ES) P R2
Mother variables
Placenta variables
Interaction mother e placenta
0.111 (0.039) 0.108 e
0.124 (0.026) 0.192 e
0.413 (0.081) 0.019 0.35
0.483 (0.053) 0.007 e
0.547 (0.095) 0.005 e
0.829 (0.028) 0.001 0.64
0.443 (0.065) 0.008 e
0.487 (0.055) 0.005 e
0.526 (0.028) 0.004 0.47
0.142 (0.045) 0.112 e
0.125 (0.054) 0.115 e
0.146 (0.038) 0.107 0.05
0.358 (0.067) 0.010 e
0.136 (0.071) 0.123 e
0.458 (0.044) 0.008 0.42
0.339 (0.046) 0.012 e
0.190 (0.045) 0.118 e
0.480 (0.031) 0.007 0.43
0.172 (0.060) 0.226 e
0.303 (0.082) 0.030 e
0.496 (0.055) 0.005 0.44
0.284 (0.052) 0.070 e
0.328 (0.040) 0.039 e
0.485 (0.078) 0.006 0.43
0.184 (0.078) 0.140 e
0.166 (0.078) 0.116 e
0.209 (0.037) 0.155 0.06
B (ES) are the correlation coefficients (standard error) of each independent variable with the dependent variable. R2 is the coefficient of determination and provides the percentage of variance explained by all variables. Relationships are significant at p < 0.05.
In conclusion, several metabolic and redox status changes appeared in the mother, the fetus and the placenta during obesity. Disturbances in the oxidant/antioxidant status that affected the entire unit mother-placenta-fetus may be responsible, during pregnancy, of a range of maternal and fetal complications and may be one of the key downstream mediators that initiate programming of the offspring. Reducing maternal oxidative stress will be important for developing therapeutic strategies for alleviating long-term programmed consequences associated with obesity. Acknowledgments The present work was realized with the financial support of the National Agency for the Development of Health Research (PNR ANDRS). Our thanks go to all volunteers. None of the authors has any financial or personal conflicts of interest. References [1] WHO. The World health report. working together for health. Geneva: World Health Organization; 2006. [2] Tounian P. Body-weight regulation in children: a key to obesity physiopathology understanding. Arch Pediatr 2004;11(3):240e4. [3] Viner RM, Segal TY, Lichtarowicz KE, Hindmarsh P. Prevalence of the insulin resistance syndrome in obesity. Arch Dis Child 2005;90:10e4. [4] Vickers MH, Sloboda DM. Prenatal nutritional influences on obesity risk in offspring. Nutrition Diet Suppl 2010;2:137e49.
[5] Dixit A, Girling JC. Obesity and pregnancy. J Obstet Gynecol 2008;28(1):14e23. [6] Deruelle P. Obesity and pregnancy. Gynecol Obstet Fertil 2011;39(2):100e5. [7] Crane JM, White J, Murphy P, Burrage L, Hutchens D. The effect of gestational weight gain by body mass index on maternal and neonatal outcomes. J Obstet Gynaecol Can 2009;31(1):28e35. [8] Catalano PM, Presley L, Minium J. Fetuses of obese mothers develop insulin resistance in utero. Diabetes Care 2009;32:1076e80. [9] Heerwagen MJR, Miller MR, Barbour LA, Friedman JE. Maternal obesity and fetal metabolic programming: a fertile epigenetic soil. Am J Physiol Regul Integr Comp Physiol 2010;299:711e22. [10] Godfrey K, Robinson S. Maternal nutrition, placental growth and fetal programming. Proc Nutr Soc 1998;57:105e11. [11] Thornburg KL, O’Tierney PF, Louey S. The placenta is a programming agent for cardiovascular disease. Placenta 2010;31:54e9. [12] Catalano PM, Thomas A, Huston-Presley L, Amini SB. Increased fetal adiposity: a very sensitive marker of abnormal in utero development. Am J Obstet Gynecol 2003;189:1698e704. [13] Reynolds LP, Borowicz PP, Caton JS, Vonnahme KA, Luther JS, Hammer JC, et al. Developmental programming: the concept, large animal models, and the key role of uteroplacental vascular development. J Anim Sci 2010;88:61e72. [14] Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 2004;114(12):1752e61. [15] Buonocore G, Perrone S. Biomarkers of oxidative stress in the fetus and newborn. Hematology 2006;10:103e7. [16] Ademuyiwa O, Odusoga OL, Adebawo OO, Ugbaja RN. Endogenous antioxidant defences in plasma and erythrocytes of pregnant women during different trimesters of pregnancy. Acta Obstet Gynecol Scand; 2007:1e6. [17] Mueller A, Koebnick C, Binder H, Hoffmann I, Schild RL, Beckmann MW, et al. Placental defense is considered sufficient to control lipid peroxidation in pregnancy. Med Hypotheses 2005;64(3):553e7. [18] Dennery PA. Effects of oxidative stress on embryonic development. Birth Defects Res C Embryo Today 2007;81(3):155e62. [19] Siddiqui IA, Jaleel A, Tamimi W, Al Kadri HMF. Role of oxidative stress in the pathogenesis of preeclampsia. Arch Gynecol Obstet 2010;282:469e74. [20] Redman CW, Sargent IL. Placental stress and preeclampsia: a revised view. Placenta 2009;30:38e42. [21] Malti N, Merzouk H, Baba Ahmed FZ, Merzouk S, Malti A, Tessier C, et al. Oxidative stress biomarkers in obese mothers and their appropriate for gestational age newborn. J Clin Diag Res 2010;4:2237e45. ska-Kie [22] Guevara I, Iwanejko J, Dembin c A, Pankiewicz J, Wanat A, Anna P, et al. Determination of nitrite/nitrate in human biological material by the simple Griess reaction. Clin Chim Acta 1998;274:177e88. [23] Auclair C, Voisin E. Nitroblue-tetrazolium reduction. In: Greenwald RA, editor. Handbook of methods for oxygen radical research. Boca Raton: CRC Press, Inc; 1985. pp. 123e32. [24] Aebi H, Catalase. In: Bergmeyer, editor. In methods of enzymatic analysis. 2nd ed.vol. 2. Weinheim: Verlagchimie Gmmbb; 1974. pp. 673e84. [25] Marklund SL. Pyrogallol autooxidation. In: Greenwald RA, editor. Handbook of methods for oxygen radical. Boca raton, Fla: CRC Press; 1985. pp. 243e7. [26] Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186:421e8. [27] Merzouk H, Meghelli-Bouchenak M, Loukidi B, Prost J, Belleville J. Impaired serum lipids and lipoproteins in fetal macrosomia related to maternal obesity. Biol Neonate 2000;77:17e24. [28] Neary RH, Kilby MD, Kumpatula P, Game FL, Bhatnagar D, Dirrington PN, et al. Fetal and maternal lipoprotein metabolism in human pregnancy. Clin Sci 1995;88:311e8. [29] Guerre-Millo M, Hauguel de Mouzon S. Placenta et obésité. Médecine Thérapeutique/médecine de la reproduction 2005;7(4):287e91. [30] Colomiere M, Permezel M, Riley C, Desoye G, Lappas M. Defective insulin signaling in placenta from pregnancies complicated by gestational diabetes mellitus. Eur J Endocrinol 2009;160:567e78. [31] Haleng J, Pincemail J, Defraigne JO, Charlier C, Chapelle JP. Oxidative stress. Rev Med Liege 2007;62(10):628e38. [32] Bonnefont-Rousselot D, Bastard JP, Jaudon MC, Delattre J. Consequences of the diabetic statut on the oxidant/antioxidant balance. Diabetes Metab 2000;26: 163e76. [33] Vincent HK, Innes KE, Vincent KR. Oxidative stress and potential interventions to reduce oxidative stress in overweight and obesity. Diabetes Obes Metab 2007;9(6):813e39. [34] Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 2010;42(10):1634e50. [35] Myatt L. Reactive oxygen and nitrogen species and functional adaptation of the placenta. Placenta 2010;31:66e9. [36] Dandona P, Aljada A, Chaudhuri A, Mohanty P, Garg R. Metabolic syndrome: a comprehensive perspective based on interactions between obesity, diabetes, and inflammation. Circulation 2005;111:1448e54. [37] Myatt L, Cui X. Oxidative stress in the placenta. Histochem Cell Biol 2004;122(4):369e82. [38] Qanungo S, Mukherjea M. Ontogenic profile of some antioxidants and lipid peroxidation in human placental and fetal tissues. Mol Cell Biochem 2000;215:11e9.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Oxidative stress and maternal obesity: Feto-placental unit interaction N. Malti a, H. Merzouk a, *, S.A. Merzouk b, B. Loukidi a, N. Karaouzene a, A. Malti c, M. Narce d a Laboratory of Physiology, Physiopathology and Biochemistry of Nutrition, Department of Biology, Faculty of Natural and Life Sciences, Earth and Universe, University ABOU-BEKR BELKAÏD, Tlemcen 13000, Algeria b Department of Technical Sciences, Faculty of Engineering, University ABOU-BEKR BELKAÏD, Tlemcen 13000, Algeria c Gynecology and Obstetrics Department, Mother e Infant Hospital Center, University of Tlemcen, 13000, Algeria d INSERM UMR 866, ‘Lipids Nutrition Cancer’, University of Burgundy, Faculty of Life, Earth and Environment Sciences, Dijon 21000, France
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 10 March 2014
Objective: To determine oxidative stress markers in maternal obesity during pregnancy and to evaluate feto-placental unit interaction, especially predictors of fetal metabolic alterations. Patients and methods: 40 obese pregnant women (prepregnancy BMI > 30 kg/m2) were compared to 50 control pregnant women. Maternal, cord blood and placenta samples were collected at delivery. Biochemical parameters (total cholesterol and triglycerides) and oxidative stress markers (malondialdehyde, carbonyl proteins, superoxide anion expressed as reduced Nitroblue Tetrazolium, nitric oxide expressed as nitrite, reduced glutathione, catalase, superoxide dismutase) were assayed by biochemical methods. Results: Maternal, fetal and placental triglyceride levels were increased in obese group compared to control. Maternal malondialdehyde, carbonyl proteins, nitric oxide and superoxide anion levels were high while reduced glutathione concentrations and superoxide dismutase activity were low in obesity. In the placenta and in newborns of these obese mothers, variations of redox balance were also observed indicating high oxidative stress. Maternal and placental interaction constituted a strong predictor of fetal redox variations in obese pregnancies. Discussion: Maternal obesity compromised placental metabolism and antioxidant status which strongly impacted fetal redox balance. Oxidative stress may be one of the key downstream mediators that initiate programming of the offspring. Conclusion: Maternal obesity is associated with metabolic alterations and dysregulation of redox balance in the mother-placenta e fetus unit. These perturbations could lead to maternal and fetal complications and should be carefully considered. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Pregnancy Obesity Mother Newborn Placenta Oxidative stress
1. Introduction Obesity is a public health problem in most industrialized countries, and is now expanding to developing countries [1]. The rise in the prevalence of obesity might result from the increasingly sedentary lifestyle associated with a reduction in daily physical activity and/or from changes in eating behavior, both quantitatively and qualitatively that may play a role in early childhood [2]. Obesity is a risk factor for the development of several chronic diseases such as cardiovascular and respiratory diseases, diabetes type II, hypertension and some forms of cancer [3]. The importance of the intrauterine and neonatal metabolic environment as possible
* Corresponding author. Tel.: þ213 778303645. E-mail address: hafi[email protected] (H. Merzouk). http://dx.doi.org/10.1016/j.placenta.2014.03.010 0143-4004/Ó 2014 Elsevier Ltd. All rights reserved.
teratogenic determinants for the predisposition of obesity is widely supported [4]. Furthermore, obesity during pregnancy is associated to several maternal and fetal complications [5e7]. In addition, maternal obesity is important in promoting obesity in offspring, reflecting epigenetic programming [8,9]. Placental metabolic functions are not immune to this imprint [10,11]. Placental dysfunction is implicated in most of the poor pregnancy outcomes associated with maternal obesity, and is also known to be involved in developmental programming of later-life diseases [12,13]. Several studies have reported that obesity is associated with oxidative stress related to inadequate antioxidant defenses and increased rates of free radical formation [14]. The impact of oxidative stress on the fetus and the newborn is well known [15]. Pregnancy is a state of oxidative stress [16]. Placenta tissue is rich in hormones and is an important source of pro-oxidant agents as well as antioxidant enzymes [17]. Moreover, oxidative stress may affect
412
N. Malti et al. / Placenta 35 (2014) 411e416
the embryonic development [18]. Women with preeclampsia have an increased oxidative stress and lipid peroxidation and at the same time have a deficiency in several important antioxidants [19]. In this pathologic pregnancy, a heightened level of oxidative stress is encountered leading to placental oxidative damage which can affect placental function [20]. Oxidative stress may lead to a change in insulin sensitivity, oxidation of lipoproteins, lipid alterations and adult diseases, such as diabetes and atherosclerosis in the offspring of obese mothers. Maternal and fetal oxidative stress was enhanced during obesity [21]. Nevertheless, the simultaneous existence of a placental oxidative stress and maternal-feto-placental interactions during maternal obesity are still not well understood. The aim of the present work was to determine oxidative stress markers during pregnancy associated with maternal obesity, and to assess the impact on the maternal-placental-fetal unit. Therefore, it would be possible to characterize maternal and placental oxidant/ antioxidant imbalance and its impact on fetal redox status in obesity. 2. Patients and methods 2.1. Study population The study population included 50 normal weight (prepregnancy BMI between 19 and 25 kg/m2) and 40 obese (prepregnancy BMI 30 kg/m2) women who gave birth at the Obstetrics and Gynecology department of Tlemcen Hospital, Tlemcen city (West Algeria). The rigorous selection, recruitment and monitoring of pregnant women was done by clinicians of the service. These women are aged between 20 and 35 years and have full-term pregnancies (37 weeks). None of the subjects selected in this study had history of chronic diseases, hypertension, eclampsia, infections or fetal anomalies. All women were routinely seen by an obstetrician and an endocrinologist. They were tested for gestational diabetes according to the World Health Organization criteria and all had normal glucose tolerance test during the third trimester and within 48 h of delivery. All women had uncomplicated singleton pregnancies and delivered by caesarean section. The indications for elective Caesarean section at term were breech presentation, placenta praevia and previous Caesarean section. Maternal and neonatal characteristics are shown in Table 1. The study was performed according to the Declaration of Helsinki. All participants in this study were informed about the goals and the work in progress, and were asked to give their written consent beforehand. Investigations of patients as well as blood and placenta sampling conditions were subjected to a strict code of ethics. The protocol was approved by the Tlemcen Hospital Committee for Research on Human Subjects. 2.2. Blood samples Fasting maternal blood samples were obtained from the arm veins of the mothers, at the time of delivery. Cord blood samples were obtained from the umbilical vein immediately following delivery and after the cutting of the umbilical cord. Blood samples were collected in EDTA tubes, were centrifuged and plasma was separated for assessing lipids and plasma oxidative stress markers. The remaining
Table 1 Maternal and neonate characteristics. Characteristics
Control
Obese
Number Age (years) Prepregnancy BMI (kg/m2) Number of gestations Parity Gestational age (weeks) Birth weight (Kg) M/F sex ratio Total cholesterol Mothers (g/L) Newborns (g/L) Placenta (mg/g) Triglycerides Mothers (g/L) Newborns (g/L) Placenta (mg/g)
50 29 5.25 22.61 2.13 2.81 1.33 2.69 1.30 38 1 3.34 0.31 28/22
40 31.31 5.91 33.17 3.40** 3.56 2.00 3.12 1.63 38 1 4.28 0.35* 23/17
1.88 0.35 0.55 0.06 5.18 0.64
1.79 0.21 0.46 0.05 5.73 0.53
1.43 0.20 0.41 0.06 6.59 0.51
2.28 0.11** 0.68 0.07* 9.94 0.47*
Values are means SD. BMI: body mass index (weight/height2); M/F: males/females. Significant differences between obese and control groups are indicated as: *P < 0.05; **P < 0.01.
erythrocytes were washed and hemolysed by the addition of cold distilled water (1/ 4). The hemolysates were appraised for erythrocyte oxidant/antioxidant status. 2.3. Placental samples Placentas were collected immediately following delivery, within 10 min of delivery, and washed with saline water to eliminate the blood. Each placenta was weighed and sampled in three main regions (central region, mid placenta and peripheral region). Placental samples were snap frozen and stored at 80 C until further analysis. All samples were homogenized in four volumes of phosphate buffered saline (PBS) containing proteolytic enzyme inhibitors (Complete-Mini; Roche), using an Ultra-Turex homogenizer (Bioblock Scientific, Illkirch, France). Samples were then centrifuged for 30 min at 4000 rpm and the supernatant was collected for biochemical analysis. 2.4. Determination of biochemical parameters Plasma or placental homogenate cholesterol and triglycerides were determined by enzymatic methods (Kits Sigma Chemical Company, St Louis, MO, USA). 2.5. Determination of markers of the oxidant/antioxidant status Nitric oxide (NO) was determined by the method of Guevara et al. [22], after plasma or placental homogenate deproteinizing procedure (using methanol:diethylether; 3:1 mixture v/v). Nitrite and nitrate levels were measured together; nitrate being previously transformed to nitrite by cadmium reduction. Nitrite was assayed directly spectrophotometrically at 492 nm, using the colorimetric method of Griess (Griess reagent: 1 g/L sulfanilamide, 25 g/L phosphoric acid, and 0.1 g/L N-1-naphthylethylenediamine). Calibration curves were made with sodium nitrite in concentrations from 1 to 50 mmol/L. The inter- and intra-assay variations were 3.40% and 2.50%, respectively. The determination of the superoxide anion (O2) was based on Nitro Blue Tetrazolium (NBT) reduction in monofarmazan by O2 [23]. The blue formazan was dissolved using 2M potassium hydroxide and dimethylsulfoxide and its formation was monitored spectrophotometrically at 560 nm using the molar extinction coefficient (1.5 104 M1 $ cm1). The catalase activity (CAT, EC 1.11.1.6) was measured by spectrophotometric analysis of the decomposition rate of hydrogen peroxide according to the method of Aebi [24]. The reaction was initiated by addition of placental homogenate or hemolysate to the reaction mixture containing phosphate buffer (0.05 M, pH 7.2) and H2O2. Change in absorbance was recorded spectrophotometrically at 240. The results were expressed as unit of catalase activity corresponding to mmol of H2O2 decomposed per minute using the H2O2 standard curve. The inter- and intra-assay variations were 2.40% and 3%, respectively. The assessment of the enzymatic activity of superoxide dismutase (SOD, EC 1.15.1.1) was based on the ability to inhibit pyrogallol autoxidation, with one unit of SOD activity the amount that causes 50% inhibition of the oxidation of pyrogallol [25]. SOD activity was measured every 5 min over 1 h at 405 nm, for 1/20 dilution of placental homogenate and 1/5 dilution of hemolysate. The inter- and intra-assay variations were 3% and 3.80%, respectively. Hemolysate or placental homogenate reduced glutathione (GSH) levels were assayed by a colorimetric method based on the reduction of 5,5-dithiobis-(2nitrobenzoic) acid (DTNB) by GSH to generate 2-nitro-5-thiobenzoic acid which has yellow color, according a Sigma Aldrich Kit (Saint Louis, MO, USA). The absorbance at 412 nm was measured, and the GSH concentration was then determined with the GSH standard curve. The inter- and intra-assay variations were 1% and 0.50%, respectively. Plasma or placental homogenate carbonyl proteins (marker of protein oxidation) by the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) leading to the formation of stable dinitrophenyl (DNP) hydrazone adducts, which can be detected spectrophotometrically at 375 nm (Sigma Aldrich Kit (Saint Louis, MO, USA). Oxidized BSA standard was used for the standard curve. The interand intra-assay variations were 0.40% and 0.60%, respectively. Plasma or placental homogenate malondialdehyde (MDA, marker of lipid peroxidation) was estimated by the method of Draper and Hadley et al. [26] using thiobarbituric acid (TBA). Absorbance was measured at 532 nm. The results were expressed as micromoles of MDA, using the molar extinction coefficient of chromophore (1.56 105 M1 cm1). The inter- and intra-assay variations were 3.50% and 3%, respectively. 2.6. Statistical analysis The results are presented as means and standard deviations. A priori power analysis was performed to determine the sample size, using power and sample size calculator (Statistical solutions, Sigma). We calculated that a sample of 50 normal pregnant and 40 obese women would give us a 95% power of detecting a 25% difference in measurements. The results were tested for normal distribution using the ShapiroeWilk test. After variance analysis, Student’s t-test was used to compare means between normal weight and obese groups, for different parameters. The multiple regression analysis was performed with dependent variables (lipid parameters and markers of oxidant/antioxidant status in neonates) and independent
N. Malti et al. / Placenta 35 (2014) 411e416 variables (lipid parameters, and maternal-placental markers of oxidant/antioxidant status). B(ES) are the correlation coefficients (standard error) of each independent variable with the dependent variable. R2 is the coefficient of determination; it provides the percentage of variance expressed by all variables. Relationships were significant for p < 0.05. All tests were performed using STATISTICA4.1program (StatSoft, Tulsa, OK).
3. Results 3.1. Lipids and redox balance Maternal, fetal and placental cholesterol levels showed no significant difference in the obese group compared to the normal weight group. In contrast, a significant increase in maternal, fetal and placental triglyceride levels was found in the obese group (Table 1). In obese mothers, oxidant/antioxidant status alterations were marked by a significant increase in plasma concentrations of malondialdehyde, carbonyl proteins, nitrite (reflecting nitric oxide) and reduced NBT (reflecting anion superoxide production), and a significant decrease in reduced glutathione levels and in erythrocyte superoxide dismutase activity compared to control values (Figs. 1 and 2). In their newborns, the levels of malondialdehyde, nitrite, reduced NBT as well as erythrocyte activities of catalase and superoxide dismutase were significantly high compared to the control values (Figs.1 and 2). Furthermore, in the placenta of obese mothers, a significant increase was noted in the levels of malondialdehyde, carbonyl protein, reduced glutathione and also in antioxidant enzyme activities (superoxide dismutase and catalase) compared to values obtained with placentas from control mothers (Figs. 1 and 2). 3.2. Predictors of fetal metabolic alterations In order to investigate the influence of maternal and placental metabolic changes on fetal metabolism, a multiple regression
413
analysis was performed with independent variables or predictors (maternal-placental parameters) and dependent variables (neonate parameters). Maternal and placental cholesterol and triglyceride levels were not significantly correlated to those of the newborn while the interaction between these two predictors explained, respectively 38% and 39% of the variation of these parameters in control group. Regarding markers of oxidant/antioxidant status, no correlation was found between maternal, placental and fetal parameters. The mother-placenta interaction did not alter these observations (Table 2). In the obese group, maternal and placental cholesterol levels were not correlated with those of the newborn. However, the mother-placenta association influenced fetal plasma cholesterol levels and explained 35% of their variation (Table 3). There was a strong correlation between, on the one hand, maternal and fetal plasma levels of triglycerides and malondialdehyde (P ¼ 0.007 and P ¼ 0.008, respectively), and on the other hand, placental and fetal triglyceride and malondialdehyde levels (P ¼ 0.005). The mothere placenta interaction increased the power of the model (P ¼ 0.001 and P ¼ 0.004, respectively) and explained 64% and 47%, respectively, of the variation of these parameters in the newborn. Maternal Nitrite and reduced NBT levels were significantly correlated with those of newborns in obese group (P ¼ 0.01). The interaction mother-placenta could significantly predict the changes in fetal Nitrite and reduced NBT levels (42% and 43%, respectively). Regarding the antioxidant enzyme activities, though the maternal values were not associated with those of newborns, the activities of placental catalase and superoxide dismutase were positively correlated to the activities of these fetal enzymes (P ¼ 0.030 and P ¼ 0.039, respectively). The mother-placenta interaction was highly significant; it explained the respective changes (44% and 43%) of catalase and superoxide dismutase activities in newborns of obese mothers (Table 3).
Fig. 1. Antioxidant status in mothers and their newborns and placentas. Each value represents the mean standard deviation. GSH: reduced glutathione; SOD: superoxide dismutase. The significance of the differences between obese and control group was determined by the student’s t-test after analysis of variance:*P < 0.05. **P < 0.01.
414
N. Malti et al. / Placenta 35 (2014) 411e416
Fig. 2. Oxidant status in mothers and their newborns and placentas. Each value represents the mean standard deviation. CP: carbonyl proteins; MDA: malondialdehyde; NO: nitric oxide; O2: superoxide anion. The significance of the differences between obese and control group was determined by the student’s t-test after analysis of variance:*P < 0.05. **P < 0.01.
4. Discussion This study revealed changes in maternal, fetal and placental redox status as well as the maternal-placental-fetal interactions during obesity. Obesity is commonly associated with metabolic changes with high cardiovascular risk [3]. These metabolic alterations increase during pregnancy [5,6]. In our study, neither the obese women nor their neonates had any complications. These obese women presented elevated plasma triglyceride levels; whereas total plasma cholesterol levels were similar to those of normal weight women. Similar anomalies were noted in newborns and in placentas of the obese group. Our findings were in agreement with those of other authors who showed high serum triglyceride levels in obese women and their newborns compared with those of control group [20,27]. Increased estrogens levels and insulin resistance during pregnancy are responsible for this hypertriglyceridemia [28]. High triglyceride levels in newborns of obese mothers were correlated to their birth weights which were increased compared to controls [27]. We have previously found that normal weight newborns of obese mothers had triglyceride levels similar to those found in control newborns [21]. It has been shown that placental lipoprotein lipase (LPL) activity is high in obese pregnancies [29]. In the placenta, LPL hydrolyzes triglycerides from maternal circulation to release free fatty acids (FFA), which are transferred to the placenta, and then esterified to triglycerides. Other authors have noted an accumulation of triglycerides in the placenta of obese women [30].The multiple regression analysis in the control group showed that although maternal or placental lipid levels are not independently correlated to those of the fetus, the maternaleplacental
interaction was a substantial predictor of changes in fetal lipids. This was quite normal because the fetus is dependent on fat intake from the mother and transferred via the placenta. In the obese group, the correlations between maternal, placental and fetal triglycerides were more important, and the impact of the maternaleplacental interaction was stronger. This supposed the concept that in the presence of high maternal triglyceride levels, the placental transfer was more important, leading to fetal hypertriglyceridemia. Previous studies revealed also a highly significant correlation between maternal and fetal triglycerides [27]. It has been shown that high levels of triglycerides in maternal circulation may create a steep concentration gradient across the placenta, which accelerates their transport and deposition in fetal tissues [9]. Our results provided evidence that oxidant/antioxidant status was altered in the maternal fetoplacental unit during obesity. In addition, the multivariate analysis clearly revealed that fetal changes in lipid peroxidation, free radicals and antioxidant enzymes were directly related to maternal and/or placental changes in obesity. Oxidative stress is characterized by an imbalance between pro-oxidants (free radicals, peroxides) and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, antioxidant vitamins) [31,32]. Today, it is well established that oxidation phenomena are involved in the development of metabolic and neurodegenerative diseases, as well as aging. Vincent et al. . [33] showed that obesity increases oxidative stress by raising lipid peroxidation proportionally to the degree of adiposity and low antioxidant defense. Significant changes of the oxidant/antioxidant balance still exist during normal pregnancy, due to increased basal oxygen and energy consumption in different organs including the fetoplacental unit [16].
N. Malti et al. / Placenta 35 (2014) 411e416 Table 2 Multivariate analysis in the control group. Dependent Variables (newborns) Cholesterol B (ES) P R2 Triglycerides B (ES) P R2 Malondialdhyde B (ES) P R2 Carbonyl proteins B (ES) P R2 Nitrite B(ES) P R2 red. NBT B (ES) P R2 Catalase B (ES) P R2 Superoxide dismutase B (ES) P R2 Reduced glutathione B (ES) P R2
Mothers variables
Placenta variables
Interaction Mother-placenta
0.132 (0.061) 0.215 e
0.184 (0.053) 0.119 e
0.416 (0.065) 0.015 0.38
0.136 (0.040) 0.138 e
0.194 (0.020) 0.140 e
0.424 (0.069) 0.014 0.39
0.082 (0.050) 0.102 e
0.102 (0.041) 0.147 e
0.155 (0.077) 0.125 0.07
0.147 (0.032) 0.139 e
0.125 (0.051) 0.108 e
0.199 (0.078) 0.089 0.10
0.187 (0.071) 0.107 e
0.117 (0.064) 0.114 e
0.188 (0.027) 0.095 0.11
0.111 (0.037) 0.175 e
0.157 (0.027) 0.116 e
0.158 (0.063) 0.115 0.09
0.142 (0.070) 0.104 e
0.181 (0.030) 0.111 e
0.182 (0.083) 0.101 0.12
0.201 (0.073) 0.108 e
0.128 (0.076) 0.143 e
0.199 (0.051) 0.168 0.07
0.102 (0.052) 0.222 e
0.152 (0.075) 0.177 e
0.165 (0.077) 0.125 0.06
B (ES) are the correlation coefficients (standard error) of each independent variable with the dependent variable. R2 is the coefficient of determination and provides the percentage of variance explained by all variables. Relationships are significant at p < 0.05.
Antioxidants are extremely important in maintaining cell function during normal pregnancy [34]. According to Mueller et al. [17], during normal pregnancy, the placenta is a major source of prooxidants and antioxidant systems; it is capable of keeping lipid peroxidation under control. The placenta is rich in mitochondria, highly vascular and is exposed to high maternal oxygen partial pressure, therefore resulting in increased production of superoxide. The nitric oxide (NO) is also locally produced by the placenta from a substrate, L-arginine, by NO synthases (NOS) [35]. The formation of these two free radicals is tightly controlled by a highly effective antioxidant defense system during normal pregnancies. However, under pathological conditions, the redox balance is disturbed, thus inducing an oxidative stress. In our study, obese mothers showed high levels of pro-oxidant markers (superoxide anion as expressed by reduced NBT, nitric oxide as expressed by nitrite, malondialdehyde, carbonyl proteins) and a reduction in antioxidant defenses (reduced glutathione, superoxide dismutase). In our study, conventionally methods were used to estimate superoxide anion and nitric oxide. The reduction of NBT to insoluble blue formazan was used as a probe for superoxide generation, although it is not entirely specific for O2. Nitric oxide production was estimated from determining the concentrations of nitrite end products. A reduction in SOD, the primary enzyme that inactivates the superoxide radical and in GSH which is involved in glutathionedependent enzyme reactions, would lead to increased numbers of free radicals and this could thereafter be responsible for the
415
increased levels of malondialdehyde and carbonyl proteins in obese mothers. Antioxidant enzymes may also be consumed or inactivated in high oxidative conditions. Several studies reported that oxidative stress status in obese women during pregnancy was always associated with a pro-inflammatory state [36]. Our results proved the presence of oxidative stress in placentas of obese mothers. It is important to note that measurements were taken on term placentas at a single time point. The oxidative stress was characterized by high levels of malondialdehyde and carbonyl proteins despite elevated levels of reduced glutathione. Placental levels of nitric oxide (nitrite) and superoxide anion (reduced NBT) did not change significantly between the two groups. This might have resulted from the parallel increase in the activities of antioxidant enzymes, catalase and superoxide dismutase. High placental antioxidant activities might be a compensatory mechanism that limits oxidative stress in the placenta, during obesity [37]. Maintaining normal placental nitric oxide concentrations is an important aspect for a good pregnancy development, as nitric oxide plays a significant role in placental development, angiogenesis and vasodilatation [35]. However, in the placenta, excess superoxide and nitric oxide production can result in peroxynitrite formation, leading to nitrative stress. Peroxynitrite is a powerful pro-oxidant capable of initiating lipid peroxidation and nitro-tyrosine formation [19,37]. Further studies are then needed to confirm this hypothesis in the placenta of obese mothers. In the present study, infants of obese mothers were also subjected to oxidative stress. There was a significant increase in malondialdehyde, superoxide anion and nitric oxide levels in newborns of obese mothers compared to those of normal-weight mothers. There is some evidence that maternal obesity induced mitochondrial dysfunction with an increase in mitochondrial reactive oxygen species and oxidative stress in oocytes, zygotes and embryonic life [9]. Our findings are in agreement with the hypothesis that mitochondrial injury due to maternal obesity could compromise metabolism in the developing fetus and may even impact fetal mitochondrial function prior to conception. In newborns of obese mothers, the increase in pro-oxidants was associated with a parallel increase in antioxidant enzymes activities, catalase and superoxide dismutase. The over expression of antioxidant activities in these infants might be an adaptive response with an induction to counter the effect of increased oxidative stress. Our results suggested that these infants were exposed to greater oxidative stress despite higher antioxidant enzyme activities. The multivariate analysis showed that, during normal pregnancy, fetal redox status was not correlated with that of the mother or with that of the placenta, suggestive of a maximum protection of the fetus against free radicals. Pro-oxidants from the mother or from the placenta itself are usually destroyed before they reach child development [38]. In contrast, during obesity, the multivariate analysis indicated that the correlations between maternal or placental redox status and that of the fetus were significant. The maternaleplacental interaction was an important predictor of changes in fetal oxidant/antioxidant status during obese pregnancies. It was clear that oxidative stress in obese mother induced placental and fetal oxidative stress despite high placental antioxidant defense. Oxidative stress associated with obesity during pregnancy may be a contributing factor in postnatal consequences of the neonate and the induction of programmed phenotypes in the adult offspring. In fact, oxidative stress has also been identified as a contributing factor in epigenetic mechanisms [7e9,18,19,37,38]. However, only term pregnancies and placentas were considered in this study. We are then limited in our ability to identify redox alterations during the different trimesters of pregnancy. Further studies are needed to determine time course of change in maternal and placental oxidant/antioxidant status during the association of obesity and pregnancy.
416
N. Malti et al. / Placenta 35 (2014) 411e416
Table 3 Multivariate analysis in the Obese group. Dependent variables (newborn) Cholesterol B (ES) P R2 Triglycerides B (ES) P R2 Malondialdehyde B (ES) P R2 Carbonyl proteins B (ES) P R2 Nitrite B (ES) P R2 red. NBT B (ES) P R2 Catalase B (ES) P R2 Superoxide dismutase B (ES) P R2 Reduced glutathione B (ES) P R2
Mother variables
Placenta variables
Interaction mother e placenta
0.111 (0.039) 0.108 e
0.124 (0.026) 0.192 e
0.413 (0.081) 0.019 0.35
0.483 (0.053) 0.007 e
0.547 (0.095) 0.005 e
0.829 (0.028) 0.001 0.64
0.443 (0.065) 0.008 e
0.487 (0.055) 0.005 e
0.526 (0.028) 0.004 0.47
0.142 (0.045) 0.112 e
0.125 (0.054) 0.115 e
0.146 (0.038) 0.107 0.05
0.358 (0.067) 0.010 e
0.136 (0.071) 0.123 e
0.458 (0.044) 0.008 0.42
0.339 (0.046) 0.012 e
0.190 (0.045) 0.118 e
0.480 (0.031) 0.007 0.43
0.172 (0.060) 0.226 e
0.303 (0.082) 0.030 e
0.496 (0.055) 0.005 0.44
0.284 (0.052) 0.070 e
0.328 (0.040) 0.039 e
0.485 (0.078) 0.006 0.43
0.184 (0.078) 0.140 e
0.166 (0.078) 0.116 e
0.209 (0.037) 0.155 0.06
B (ES) are the correlation coefficients (standard error) of each independent variable with the dependent variable. R2 is the coefficient of determination and provides the percentage of variance explained by all variables. Relationships are significant at p < 0.05.
In conclusion, several metabolic and redox status changes appeared in the mother, the fetus and the placenta during obesity. Disturbances in the oxidant/antioxidant status that affected the entire unit mother-placenta-fetus may be responsible, during pregnancy, of a range of maternal and fetal complications and may be one of the key downstream mediators that initiate programming of the offspring. Reducing maternal oxidative stress will be important for developing therapeutic strategies for alleviating long-term programmed consequences associated with obesity. Acknowledgments The present work was realized with the financial support of the National Agency for the Development of Health Research (PNR ANDRS). Our thanks go to all volunteers. None of the authors has any financial or personal conflicts of interest. References [1] WHO. The World health report. working together for health. Geneva: World Health Organization; 2006. [2] Tounian P. Body-weight regulation in children: a key to obesity physiopathology understanding. Arch Pediatr 2004;11(3):240e4. [3] Viner RM, Segal TY, Lichtarowicz KE, Hindmarsh P. Prevalence of the insulin resistance syndrome in obesity. Arch Dis Child 2005;90:10e4. [4] Vickers MH, Sloboda DM. Prenatal nutritional influences on obesity risk in offspring. Nutrition Diet Suppl 2010;2:137e49.
[5] Dixit A, Girling JC. Obesity and pregnancy. J Obstet Gynecol 2008;28(1):14e23. [6] Deruelle P. Obesity and pregnancy. Gynecol Obstet Fertil 2011;39(2):100e5. [7] Crane JM, White J, Murphy P, Burrage L, Hutchens D. The effect of gestational weight gain by body mass index on maternal and neonatal outcomes. J Obstet Gynaecol Can 2009;31(1):28e35. [8] Catalano PM, Presley L, Minium J. Fetuses of obese mothers develop insulin resistance in utero. Diabetes Care 2009;32:1076e80. [9] Heerwagen MJR, Miller MR, Barbour LA, Friedman JE. Maternal obesity and fetal metabolic programming: a fertile epigenetic soil. Am J Physiol Regul Integr Comp Physiol 2010;299:711e22. [10] Godfrey K, Robinson S. Maternal nutrition, placental growth and fetal programming. Proc Nutr Soc 1998;57:105e11. [11] Thornburg KL, O’Tierney PF, Louey S. The placenta is a programming agent for cardiovascular disease. Placenta 2010;31:54e9. [12] Catalano PM, Thomas A, Huston-Presley L, Amini SB. Increased fetal adiposity: a very sensitive marker of abnormal in utero development. Am J Obstet Gynecol 2003;189:1698e704. [13] Reynolds LP, Borowicz PP, Caton JS, Vonnahme KA, Luther JS, Hammer JC, et al. Developmental programming: the concept, large animal models, and the key role of uteroplacental vascular development. J Anim Sci 2010;88:61e72. [14] Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 2004;114(12):1752e61. [15] Buonocore G, Perrone S. Biomarkers of oxidative stress in the fetus and newborn. Hematology 2006;10:103e7. [16] Ademuyiwa O, Odusoga OL, Adebawo OO, Ugbaja RN. Endogenous antioxidant defences in plasma and erythrocytes of pregnant women during different trimesters of pregnancy. Acta Obstet Gynecol Scand; 2007:1e6. [17] Mueller A, Koebnick C, Binder H, Hoffmann I, Schild RL, Beckmann MW, et al. Placental defense is considered sufficient to control lipid peroxidation in pregnancy. Med Hypotheses 2005;64(3):553e7. [18] Dennery PA. Effects of oxidative stress on embryonic development. Birth Defects Res C Embryo Today 2007;81(3):155e62. [19] Siddiqui IA, Jaleel A, Tamimi W, Al Kadri HMF. Role of oxidative stress in the pathogenesis of preeclampsia. Arch Gynecol Obstet 2010;282:469e74. [20] Redman CW, Sargent IL. Placental stress and preeclampsia: a revised view. Placenta 2009;30:38e42. [21] Malti N, Merzouk H, Baba Ahmed FZ, Merzouk S, Malti A, Tessier C, et al. Oxidative stress biomarkers in obese mothers and their appropriate for gestational age newborn. J Clin Diag Res 2010;4:2237e45. ska-Kie [22] Guevara I, Iwanejko J, Dembin c A, Pankiewicz J, Wanat A, Anna P, et al. Determination of nitrite/nitrate in human biological material by the simple Griess reaction. Clin Chim Acta 1998;274:177e88. [23] Auclair C, Voisin E. Nitroblue-tetrazolium reduction. In: Greenwald RA, editor. Handbook of methods for oxygen radical research. Boca Raton: CRC Press, Inc; 1985. pp. 123e32. [24] Aebi H, Catalase. In: Bergmeyer, editor. In methods of enzymatic analysis. 2nd ed.vol. 2. Weinheim: Verlagchimie Gmmbb; 1974. pp. 673e84. [25] Marklund SL. Pyrogallol autooxidation. In: Greenwald RA, editor. Handbook of methods for oxygen radical. Boca raton, Fla: CRC Press; 1985. pp. 243e7. [26] Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186:421e8. [27] Merzouk H, Meghelli-Bouchenak M, Loukidi B, Prost J, Belleville J. Impaired serum lipids and lipoproteins in fetal macrosomia related to maternal obesity. Biol Neonate 2000;77:17e24. [28] Neary RH, Kilby MD, Kumpatula P, Game FL, Bhatnagar D, Dirrington PN, et al. Fetal and maternal lipoprotein metabolism in human pregnancy. Clin Sci 1995;88:311e8. [29] Guerre-Millo M, Hauguel de Mouzon S. Placenta et obésité. Médecine Thérapeutique/médecine de la reproduction 2005;7(4):287e91. [30] Colomiere M, Permezel M, Riley C, Desoye G, Lappas M. Defective insulin signaling in placenta from pregnancies complicated by gestational diabetes mellitus. Eur J Endocrinol 2009;160:567e78. [31] Haleng J, Pincemail J, Defraigne JO, Charlier C, Chapelle JP. Oxidative stress. Rev Med Liege 2007;62(10):628e38. [32] Bonnefont-Rousselot D, Bastard JP, Jaudon MC, Delattre J. Consequences of the diabetic statut on the oxidant/antioxidant balance. Diabetes Metab 2000;26: 163e76. [33] Vincent HK, Innes KE, Vincent KR. Oxidative stress and potential interventions to reduce oxidative stress in overweight and obesity. Diabetes Obes Metab 2007;9(6):813e39. [34] Al-Gubory KH, Fowler PA, Garrel C. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 2010;42(10):1634e50. [35] Myatt L. Reactive oxygen and nitrogen species and functional adaptation of the placenta. Placenta 2010;31:66e9. [36] Dandona P, Aljada A, Chaudhuri A, Mohanty P, Garg R. Metabolic syndrome: a comprehensive perspective based on interactions between obesity, diabetes, and inflammation. Circulation 2005;111:1448e54. [37] Myatt L, Cui X. Oxidative stress in the placenta. Histochem Cell Biol 2004;122(4):369e82. [38] Qanungo S, Mukherjea M. Ontogenic profile of some antioxidants and lipid peroxidation in human placental and fetal tissues. Mol Cell Biochem 2000;215:11e9.
