AMERICAN JOURNAL OF HUMAN BIOLOGY 27:822–831 (2015)

Original Research Article

Relationship Between Maternal and Newborn Endothelial Function and Oxidative Stress 2,3   GUILLERMO ORTEGA–AVILA,  CASTILLO,5 ISABELLA ECHEVERRI,1,2* JOSE MILDREY MOSQUERA,2,4 ANDRES 2,4 2,6    ELIECER JIMENEZ, MILTON FABIAN SUAREZ-ORTEGON, * JULIO CESAR MATEUS,7,8 AND CECILIA AGUILAR-DE PLATA2,4 1 Faculty of Health Sciences, Universidad ICESI, Cali, Colombia 2 Nutrition Group, Universidad del Valle, Cali, Colombia 3 Department of Basic Sciences, Research Group on Basic and Clinical Health Sciences, School of Medicine, Pontificia Universidad Javeriana, Cali, Colombia 4 Department of Physiological Sciences, Universidad del Valle, Cali, Colombia 5 Department of Biological Sciences, Faculty of Natural Sciences, Universidad ICESI, Cali, Colombia 6 Centre for Population Health Sciences, University of Edinburgh, Edinburgh, United Kingdom 7 School of Public Health, Universidad del Valle, Cali, Colombia 8 Fundacion FES, Cali, Colombia

Objective: To evaluate the relationship between maternal and newborn endothelial function and oxidative stress. Methods: Forty-three pregnant women and their offspring were evaluated. As markers of endothelial function, the flow-mediated dilation (FMD) was measured in pregnant women in the second and third trimesters, and nitric oxide (NO) was quantified in the endothelial cells of the umbilical cord vein. Malondialdehyde (MDA), as a marker of oxidative stress, was measured in the maternal plasma (second and third trimesters) and plasma from umbilical cord blood. Gestational age and birth weight were recorded. Correlations between variables were estimated, and adjustments were made for specific gestational week of measurement, gestational age at birth, and complications during pregnancy and/ or at delivery. Results: Maternal FMD at second trimester correlated positively with newborn MDA, although with marginal significance (P 5 0.090). The change in maternal FMD was positively correlated with newborn NO (P 5 0.039), although adjustment for gestational age and specific week of gestation attenuated this relationship (P 5 0.070). Maternal MDA at second trimester correlated positively with newborn MDA independently of gestational age at birth, specific week of gestation of the measurement, and having complications during pregnancy or at delivery (P 5 0.032). After adjustments, the change in maternal MDA correlated with newborn MDA but marginally (P 5 0.077). Conclusion: Study findings suggest that under physiological conditions, enhanced endothelial function and/or oxidative stress in the mother may impact on normal fetal development. Future studies are recommended, employing larger sample sizes, a more extensive set of markers of oxidative stress, and comparisons of complicated versus normal C 2015 Wiley Periodicals, Inc. V pregnancies. Am. J. Hum. Biol. 27:822–831, 2015.

During pregnancy, many changes in maternal physiology provide optimum conditions for the proper development of the embryo. Included in the major changes are those at the cardiovascular level including increased cardiac output and decreased peripheral vascular resistance due to increased endothelial vasodilator activity (Weissgerber et al., 2011). The endothelium is critical in the cardiovascular physiology of pregnancy for regulating the production of various factors responsible for tissue vasodilatation, such as the endothelium-derived hyperpolarizing factors, prostacyclin, and nitric oxide (NO) (Saarelainen et al., 2006). Alterations in maternal endothelial function during pregnancy could affect the development of the uterine and placental vascular bed, with consequent complications, such as preeclampsia and intrauterine growth restriction (IUGR) (Jansson et al, 2006). NO is involved in the process of implantation and trophoblast invasion and in the regulation of the placental vascular tone (Krause et al., 2011). The blood flow to the placenta and transport across the trophoblast membrane largely determines the intake of oxygen and nutrients by the fetus (Lewis et al., 2006). IUGR infants have an increased risk of chronic adult diseases, such as cardiovascular disease, type 2 diabetes mellitus, and metabolic syndrome (Delisle, 2002). This risk was initially demonstrated in retrospective C 2015 Wiley Periodicals, Inc. V

epidemiological studies by Barker (2002), in which the association between birth weight and adult cardiovascular disease was found. A history of low birth weight was associated with a higher risk of coronary heart disease in the sixth decade of life (Barker et al., 1989). Barker hypothesizes the existence of “fetal origins of adult diseases,” suggesting that events occurring during fetal development could have a permanent functional and/or structural lifetime effect on individual susceptibility to disease. Additionally, other studies have shown effects of

Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: Colombian Department for Science and Technology development (COLCIENCIAS); Contract grant number: 110645921540. *Correspondence to: Isabella Echeverri; Universidad ICESI, Calle 18 # 122-135, Edificio L, Quinto piso, Cali, CO; E-mail: [email protected] or Milton Fabian Su arez-Ortegon; Centre for Population Health Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, United Kingdom; E-mail: Milton.Suarez@ ed.ac.uk Received 18 September 2014; Revision received 24 March 2015; Accepted 7 April 2015 DOI: 10.1002/ajhb.22733 Published online 6 May 2015 in Wiley Online Library (wileyonlinelibrary.com).

MATERNAL AND NEWBORN ENDOTHELIAL FUNCTION AND OXIDATIVE STRESS

birth weight on postnatal characteristics in the normal range of birth weight (Holzhauer et al., 2009). With respect to the biological mechanisms implied in the developmental origins of health and disease (DOHaD), it has been reported, mainly in animal model studies, that impaired endothelial function at early stages of development might explain the observed relationship between a decrease in fetal growth and an increased risk of cardiovascular disease in adulthood (Armitage et al., 2004). This relationship could be explained by endothelial dysfunction in one or more of the factors contributing to cardiovascular disease during adulthood (e.g., regulation of blood pressure, cardiac contractility). The DOHaD observations also suggest that pregnancy-related changes in endothelial function might also play a role. Endothelial dysfunction is present in a number of pathologies such as preeclampsia during pregnancy and is typically associated with vascular oxidative stress due to decreased NO bioavailability from either decreased synthesis or its sequestration by reactive oxygen species (Davignon and Ganz, 2004). Recent studies have shown an association between: preeclampsia and decreased endothelial function and higher oxidative stress levels in young offspring (Jayet et al., 2010); maternal undernutrition and pulmonary vascular dysfunction in offspring of rats (Rexhaj et al., 2011); and prematurity and very low birth weight with high blood pressure in late life (de Jong et al., 2012; Lazdam et al., 2010). Although a variety of markers of endothelial function and oxidative stress in the adult and the relationship of endothelial function with chronic diseases have been studied ( Adams et al., 2000; Davignon and Ganz, 2004; Saarelainen et al., 2006), neither the relationship of maternal endothelial function and oxidative stress during pregnancy nor their possible association with newborn endothelial function and oxidative stress have been previously explored. Decreased endothelial function during pregnancy could be related to higher oxidative stress in mothers and newborns. The aim of this study was to evaluate this relationship between pregnant women and their newborns, and additionally the relationship of these markers with birth weight. METHODS Patients A cohort study of pregnant women and their newborns was conducted in Cali, Colombia (altitude 1,000 meters). The inclusion criteria were the following: 16- to 30-yearold primiparous women; gestational age at the time of initial study between 16 and 20 weeks (determined by means of a first trimester ultrasound or reliable last menstrual period); without detectable major malformations on ultrasound; participating in an institutional prenatal control program (supported by a health insurance plan); evaluation by a OB-GYN physician; willingness to participate in the study as demonstrated by signing the informed consent form. The Universidad del Valle Research Ethics Committee approved the study. At enrollment, the women underwent the following procedures: medical history, physical examination, and ultrasound by an OB-GYN, anthropometric measurements (weight and height), dietary assessment by a nutritionist, blood pressure, flow-mediated dilatation (FMD), and blood sampling (to measure malondialdehyde (MDA) levels). Body mass index (BMI) was calculated as weight/height2

823

(classification by BMI: underweight < 18.5; normal 18.5– 24.9; overweight 25–29.9; obesity  30). FMD measurement and blood sampling were repeated at third trimester (32–34 gestational weeks). Newborn complications of hospitalization, shortness of breath, hydrocele, myelomeningocele, IUGR, and corioamnionitis were recorded as well as information about pregnancy complications such as pregnancy-induced hypertension, preeclampsia and gestational diabetes. Additionally, FMD and MDA were measured during the third trimester in 36 and 16 of the pregnant women involved in the study. The women in this study belonged to the control group of a randomized controlled trial (Ramirez-Velez et al., 2011). In this clinical trial, 102 pregnant women were randomly selected for the control group, and 71 were followed up until delivery. Thirty-one women were unable to be followed because they could not be located (n 5 12), changed city (n 5 4), or had incomplete newborn data (n 5 15). Samples of umbilical cord vein cells (HUVECs) were obtained from the remaining 71 neonates. Of these, 43 cases were chosen, as there were technical problems in the primary culture, which resulted in the loss of 21 samples, and further, seven samples were lost in passage three of the cell culture. NO and MDA were measured in subsamples calculated for each intervention group from the clinical trial, since they were restricted with regard to the investigation possibilities. NO quantification was performed in 20 of the 43 HUVECs, MDA was successfully measured in 25 cases, and both NO and MDA in a subset of 10 cases (due to technical problems with cell conservation). At delivery, anthropometric measures (height and weight) of the 71 neonates were taken, the weight for gestational age was calculated using the curves of Fenton (Fenton, 2003), and infants whose weight was less than the 10th percentile for sex and gestational age were classified as small for gestational age. Procedures in human umbilical vein endothelial cells An experimental study of cells from the human umbilical cord endothelial vein (HUVEC) was conducted. After delivery, the obstetrician double clamped the umbilical cord (Armstrong and Stenson, 2006) and a sample of approximately 20 cm in length was obtained from the newborn side. This was kept in PBS (phosphate-buffered saline solution) 1 penicillin/2% streptomycin, at a temperature of 48C until laboratory processing which was performed within the first 12 hours after delivery. Isolation of the vein endothelial cells was performed using a laminar flow chamber under sterile conditions, according to the technique described by Baudin et al. (2007). A solution of collagenase (Sigma C9891) was perfused in the umbilical vein for 15 min. at 378C. Subsequently, the vein was perfused with a neutralizing solution of PBS 1 fetal bovine serum (FBS) at 20%, and the cells were collected in sterile tubes and isolated by centrifugation for 10 minutes (min) at 1,500 rpm. The isolated cells were cultivated at 378C in the presence of 5% CO2 in culture flasks (surface area 25 cm2), supported on 0.2% gelatin (Sigma G1393) in the presence of endothelial cell basal medium (EBM, Lonza CC 3121) to obtain a confluent monolayer. The stage 2 cells were preserved in culture medium 1 fetal bovine serum (FBS) and dimethylsulfoxide (DMSO) at 10% in liquid nitrogen until the biochemical measurements were made. The cells were heated at 378C, resuspended in an American Journal of Human Biology

824

I. ECHEVERRI ET AL

EBM 1 FBS culture medium at 20% and cultured in 24well plates (30,000 to 50,000 cells/well) for quantification of the NO production. To confirm that the culture consisted of endothelial cells, an immunofluorescence assay was performed with antiCD31 (Platelet Endothelial Cell Adhesion Molecule-1: PECAM-1, abcam 9498). The cells at a confluence of approximately 60% were fixed in 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS and adding blocking buffer for 60 minutes, the cells were incubated for 12 hours at 48C with primary antibody anti-CD31. After washing, the cells were incubated for 2 hours with the secondary antibody (abcam 96879) at room temperature in a darkroom. The cells were visualized with a Carl Zeiss confocal microscope. The cell viability was assessed by counting cells in a hemocytometer with trypan blue 0.4%. In stage 3, the concentration of NO was determined in the supernatant of the culture of HUVEC using high performance liquid chromatography (HPLC) with fluorescence detection. We used fluorochrome 4.5 diaminofluorescein (DAF-2, D224 Sigma); NO-derived nitrosation of the diamino group or oxidative nitrosylation (by NO22) of DAF-2 results in a nitrosamine which through an internal rearrangement forms the fluorescent triazole (Bryan and Grisham, 2007), and this molecule fluorescence was quantified (Leikert et al., 2001). The confluent cells were washed with PBS and incubated for 30 minutes with 1 ml of DAF-2 at a concentration of 0.1 mM. All procedures were performed using sterile materials, and all buffers were made fresh daily with ultrapure MilliPore water to avoid any nitrite contamination. For measurement by HPLC, a C18 column was used as follows: 4.6 3 150 mm, 5 mM 3 80 A, mobile phase: 1X PBS (pH 7.41)/acetonitrile 95:5 at 1 ml/min flow. The fluorescence excitation was 490 nanometres (nm), and the emission was 515 nm in a run time of 30 minutes. As the negative control, the cells were incubated with the inhibitor of endothelial NO synthase, NG-methyl-L-arginine acetate salt (L-NMMA, Sigma M7033), 500 ml to 100 lM for 30 minutes before incubation with the DAF-2 fluorochrome. The calibration curve was performed with MAHMA NONOate (Sigma M1555), a NO donor. Plasma markers The products of lipid peroxidation are widely used in the measurement of oxidative stress because polyunsaturated lipids of the cell membrane are very susceptible to attack by oxygen free radicals. One of these products is malondialdehyde (MDA), which is relatively easy to measure, and, together with F2-isoprostanes, is one of the most common and reliable markers of oxidative stress (Grune and Berger, 2007). MDA was quantified by HPLC in the maternal and newborn plasma using a procedure based on the method described by Steghens et al., (2001). The derivatization of MDA was made with diaminonaphthalene (DAN) in an acidic medium at 378C. The MDADAN compound was separated through HPLC in a C18 column, with ammonium acetate [30 mM, pH 2] as the mobile phase and 10.5% acetonitrile [v/v], flow: 0.23 ml/min., diode array detection at 311 nanometers. Flow-mediated vasodilation The estimation of FMD was made with a Siemens SG60 ultrasound, according to the technique described by American Journal of Human Biology

Celermajer et al.(1992) and the International Brachial Artery Reactivity Task Force (Corretti et al., 2002). The women were studied in the morning (7:00–9:00 am) after an overnight fast in the Department of Physiological Sciences, Faculty of Health, University of Valle. They were located in a room without noise or visual stimuli and with a controlled temperature of about 208C; the test was performed after 15 minutes of rest. The blood pressure and heart rate were measured in the right arm with an automatic sphygmomanometer. A cuff was placed at the proximal end of the left arm, and the brachial artery was located at the distal end of the arm using an ultrasound transducer from 7.5 to 13 Hz for measuring baseline brachial artery diameter in mm. Hyperemia was produced by occluding the brachial artery by inflating the cuff to 200 mmHg. In the first 15 seconds after the cuff was deflated, the peak hyperemic systolic velocity (cm/s) in the center of the vessel was measured, and after 60 seconds the peak hyperemic diameter was measured. We used an insonation angle of  608, and the baseline and hyperemic diameter was the average of three measurements of the artery. The FMD was expressed as the percent change in the diameter of the brachial artery before and after hyperemia and was calculated as follows: [(diameter of the brachial artery posthyperemia 2 diameter of the brachial artery at rest/diameter of the brachial artery at rest) 3 100] (Thijssen et al., 2011). The intra-observer and interobserver coefficients of variation were 0.13% and 0.88%, respectively. Data analysis The continuous variables were described as the mean and its interquartile range. Differences for variables between trimesters of gestation were estimated by the Wilcoxon signed-rank test. Proportions were used to describe categorical variables. The changes in maternal FMD and MDA between second and third trimester were calculated as follows: measurement 1 – measurement 2. The relationship between the maternal and newborn variables of endothelial function and oxidative stress, as well as between these markers and birth weight was evaluated using the Pearson correlation. Maternal variables were the following: FMD and MDA levels at second trimester, FMD and MDA at third trimester, and the change between these measurements. Newborn variables were NO levels in HUVEC and MDA in cord blood. Associations were adjusted for specific gestational week of measurement, and additionally for having had newborn and/or pregnancy complication, by using multiple linear regression analyses. For three cases which had missing values for specific gestational week of measurement at third trimester, the average value (32.5) between the minimum (week 31) and maximum value (week 34) for this variable was used, and most of the cases (n 5 30, 75%) with no missing value had week 32 as specific gestational week of measurement at third trimester. In the case of relationships with the changes in maternal FMD and MDA, the covariate for specific week of measurements was the difference in weeks between the two measurements. Skewed variables were transformed to approximate normal distribution as follows: logarithm of MAD in cord blood, NO in HUVEC, and specific gestational week for measurements at third trimester; square root of MDA at second and third trimesters, and gestational age. A value < 0.05 was considered statistically significant, and results were

825

MATERNAL AND NEWBORN ENDOTHELIAL FUNCTION AND OXIDATIVE STRESS

considered as trends when 0.05< P < 0.10; all the analyses were carried out using SPSS 15.0 software. RESULTS Subject characteristics

were lower at third trimester than in second trimester (P 5 0.041), whereas no difference between second and third trimester was found for MDA levels. Relationship between measures of endothelial function and oxidative stress in mothers and newborns (intragroup)

Table 1 describes the study population. Five (11.6%) of the women were underweight, and 16.2% of the women were overweight/obese. However, these figures could be slightly underestimated for underweight and overestimated for overweight, since BMI was based on body weight at the time of the study. Nine women developed complications during pregnancy and/or at delivery (Table 1). Most of the women had normal vaginal delivery. Maternal and newborn measures of endothelial function and oxidative stress The variables of endothelial function and oxidative stress are shown in Table 2. Maternal values of FMD TABLE 1. Maternal and neonatal characteristics Pregnant women (n 5 43) Age (years) 21(18–23) Weight (Kg) 54(49–61) Height (cms) 155 (154–161) BMI category (n[%]) Underweight 5[11.63] Normal 31[72.09] Overweight 6[13.95] Obese 1[2.33] Neonates (n 5 43) Gestational age (weeks) 39(38–40) Birthweight (g) 3130(2930–3500) Size at birth (cm) 50(49–51) Mode of delivery (n[%]) Vaginal delivery 31[72.1] Ceasarea section 12[27.9] Complications during pregnancy and/or at delivery (n[%]) None 34[79.1] Some complication* 9[10.3] Specific complications Premature membrane rupture 4[9.3] Hospitalization 2[4.6] Shortness of breath 1[2.3] Hydrocele 1[2.3] Myelomeningocele 1[2.3] IUGR 5[11.6] Pregnancy-induced hypertension 1[1.1] Peeclampsia 2[2.3] Gestational diabetes 1[1.1] Chorioamnionitis 1[1.1] Data are median (interquartile range). BMI, body mass index. SBP, systolic blood pressure. DBP, diastolic blood pressure. IUGR, Intrauterine Growth Restriction. *Some cases had more than one complication, and therefore, the sum of frequencies by each specific condition is higher than having some complication.

No significant correlations (whether or not they were adjusted for newborn gestational age or gestational week of measurement) were found between maternal values of FMD and MDA at second and third trimesters and between their respective changes, and between NO and MDA levels of the newborns (data not shown). Relationship between maternal FMD and newborn measures of endothelial function and oxidative stress Maternal FMD at second trimester correlated positively with newborn MDA, although with marginal significance (Fig. 1B), and the change in maternal FMD between second and third trimester was significantly and positively correlated with newborn NO (Fig. 1E). In subsequent multivariate linear regression analyses, where adjustments had been made for gestational age at birth and week of gestation when the measurements were made (Table 3 – Model 1), the significant association between change in maternal FMD and NO in newborns was attenuated [b 5 0.017(20.001 to 0.036), P 5 0.070]. Further adjustment for complications during pregnancy and/or at delivery (Model 2) did not provide additional explanation for this relationship. The marginal significance of correlation between maternal FMD at second trimester and newborn MDA remained similar after the adjustments mentioned above (P 5 0.098). The others nonsignificant correlations (Figs. 1A, C, D, 1F) remained nonsignificant in the multivariate analysis (Table 3). Relationship between maternal MDA and newborn measures of endothelial function and oxidative stress Maternal MDA at second trimester correlated positively with newborn MDA (Fig. 2B), and this relationship was independent of gestational age at birth, specific week of gestation at the time of measurement, and having complications during pregnancy or at delivery [b 5 0.013(20.001 to 0.026), P 5 0.032] (Table 3). Although the change in maternal MDA levels tended to correlate inversely with NO in newborn with a marginal significance (Fig. 2E), this relationship disappeared after adjustments [b= 20.0009(20.0003 to 0.0001), P 5 0.291] (Table 3). The nonsignificant association between the change in maternal MDA levels and newborn MDA (Fig. 2F) became marginally significant after adjustments [b 5 0.00009(0.00003

TABLE 2. Maternal and neonatal markers for oxidative stress and endothelial function Mothers Oxidative stress Endothelial function

MDA (nM) 2nd trimester (n 5 33) 1523(456.8–2274) FMD (%) 2nd trimester (n 5 43) 13.9(10.7–16.1)

MDA (nM) 3rd trimester (n 5 15) 1572(843–2427) FMD (%) 3rd trimester (n 5 36) 10.7(7.2–14.3)a

Neonates Change in MDA (nM) (n 5 15) 39(2881 to 763) Change in FMD (%) (n 5 36) 1.2(22.08 to 6.47)

MDA in cord blood (nM) (n 5 25) 1709(577.8–2582) NO in HUVEC (nmol/mg protein) (n 5 20) 0.54(0.36–0.75)

Data are median (interquartile range). a P 5 0.041 versus second trimester. NO, nitric oxide. MDA, malondialdehyde. FMD, flow-mediated dilatation. HUVEC, cells from human umbilical cord endothelial vein.

American Journal of Human Biology

826

I. ECHEVERRI ET AL

Fig. 1. Pearson correlations of neonatal MDA and NO concentrations with FMD in third (A and B) and second trimesters (C and D) and its change between these trimesters (E and F). NO, nitric oxide. FMD, flow-mediated dilatation. MDA, malondialdehyde. HUVEC, cells from human umbilical cord endothelial vein. log, logarithm. sqrt, square root.

to 0.0001), P 5 0.077], whereas the residual nonsignificant associations (Figs. 2A, C, D) remained nonsignificant in the multivariate analysis (Table 3). American Journal of Human Biology

Correlations with birth weight Birth weight was only modestly associated with the maternal MDA levels at second trimester (r 5 0.34,

827

MATERNAL AND NEWBORN ENDOTHELIAL FUNCTION AND OXIDATIVE STRESS TABLE 3. Adjusted linear regressions for the relationship of NO and MDA levels in newborns (dependent variables)

with maternal MDA levels and FMD log-NO in HUVEC (nmol/mg protein) Model 1 Beta (95% CI) Maternal endothelial function FMD at second trimester FMD at third trimester Change in FMD Maternal oxidative stress sqrt-MDA (nM) at second trimester sqrt-MDA (nM) at third trimester Change in MDA (nM)

log-MDA in cord blood (nM)

Model 2 P value

0.09 (20.01 to 0.03) 20.01 (20.04 to 0.01) 0.01 (20.001 to 0.03)

0.370

20.0008 (20.009 to 0.007) 0.002 (20.009 to 0.01) 20.0001 (20.0001 to 0.00009)

0.813

0.222 0.070

0.587 0.200

Beta (95% CI)

Model 1 P value

0.01 (20.01 to 0.03) 20.01 (20.04 to 0.01) 0.01 (20.003 to 0.03)

0.329

20.0008 (20.009 to 0.007) 0.0025 (20.012 to 0.017) 20.00009 (20.0003 to 0.0001)

0.832

0.252 0.088

0.630 0.291

Beta (95% CI)

Model 2 P value

0.03 (20.005 to 0.07) 0.005 (20.02 to 0.039) 0.01 (20.01 to 0.05)

0.089

0.01 (0.001 to 0.02) 20.002 (20.01 to 0.006) 0.00008 (0.00 to 0.0001)

0.026

0.731 0.312

0.487 0.030

Beta (95% CI)

P value

0.03 (20.006 to 0.07) 0.05 (20.02 to 0.04) 0.01 (20.02 to 0.05)

0.098

0.01 (0.001 to 0.02) 20.002 (20.01 to 0.006) 0.00006 (29 3 1026 to 0.0001)

0.032

0.729 0.326

0.492 0.077

Data are beta coefficients (95% confidence interval). Model 1: Adjusted for specific gestational week of measurements (or difference between specific weeks of measurements if change in FMD or MDA was evaluated) and gestational age. Model 2: Adjusted for specific gestational week of measurements, gestational age, and complications during pregnancy and/or at delivery. NO, nitric oxide. MDA, malondialdehyde. FMD, flow-mediated dilatation. HUVEC, cells from human umbilical cord endothelial vein. log, logarithm . sqrt, square root. Significant relationships are shown in bold.

P 5 0.051). This trend disappeared when adjustment for Model 1 (gestational age at birth, week of gestation of the measurement) or Model 2 (Model 1 plus pregnancy complications) were performed (Table 4). These adjustments did not show significant relationships between birth weight and other variables (Table 4). The R2 of each multivariate model performed to adjust the associations from Tables 3 and 4 are shown in Supporting Information Tables S1 and S2, respectively. DISCUSSION In this exploratory study of the association between maternal and newborn markers of endothelial function state and oxidative stress, we described three novel findings. First, maternal measurements of FMD and MDA at second trimester, but not at third trimester, were positively related to newborn MDA levels. Second, a trend existed for a positive second to third trimester change in maternal FMD to be associated with a higher newborn NO. Third, the maternal MDA plasma levels between gestational weeks 16 to 20 were positively and independently associated with the MDA levels in umbilical cord blood, whereas the change in maternal MDA levels tended to correlate positively with newborn MDA after considering the effect of covariates of gestational age, specific gestational week of measurement, and complications during pregnancy and/or at delivery. The observed tendency of a positive association between the change in maternal FMD during pregnancy and newborn NO could indicate that progressive changes in endothelial function during pregnancy would reciprocally affect newborn endothelial function. Against this possibility, the inclusion of gestational age at the time of study attenuated statistical significance, suggesting that gestational age rather than maternal FMD was responsible. We did not find studies regarding the association

between maternal FMD and markers of endothelial function in HUVEC. Meanwhile, a case–control study described higher plasma levels of markers of endothelial dysfunction (adhesion molecules of sE-selectin, sVCAM1 and sFlt-1) in the third trimester in pregnant women with severe preeclampsia, compared with controls, and decreased production of NO (quantified by fluorometry) in endothelial cells from the umbilical cord of the children of pregnant women with preeclampsia (Veas et al., 2011). Although several authors have found that the FMD, as a marker of endothelial function, reflects the production of NO in the endothelium (Celermajer et al., 1992; Jones et al., 2010; Mullen et al., 2001), others, including Stoner et al. (2012), and Tschakovsky and Pyke (2005), found that endothelial mediators other than NO are the mediators of vasodilatation. We theorized that a lower vascular function due to lower vascular function in the mothers would be related to higher oxidative stress in the newborns. Similarly, in animal models, oxidative stress in pregnancy has been proposed as a possible triggering factor for vascular dysfunction in offspring (Jayet et al., 2010; Rexhaj et al., 2011). However, we found an unexpected tendency of positive correlation between maternal FMD at second trimester and newborn MDA. The specific mechanism for this association remains unclear, but it would be related to normal physiological conditions in the course of the pregnancy (Hung et al., 2010). Such a physiological role would be consistent with a gradual increase in oxygen partial pressure between 7 and 16 weeks of pregnancy, in which a burst of oxidative stress at the end of the first trimester is important in the formation of fetal membranes among other functions (Jauniaux et al., 2006; Yang et al., 2012; Al-Gubory et al., 2010). Recent findings have shown that free radicals are required for cell differentiation during fetal development and that oxygen plays a role in establishing cell epigenotypes by regulating the enzymes American Journal of Human Biology

828

I. ECHEVERRI ET AL

Fig. 2. Pearson correlations of neonatal MDA and NO concentrations with maternal MDA in third (A and B) and second trimesters (C and D) and its change between these trimesters (E and F). NO, nitric oxide. FMD, flow-mediated dilatation. MDA, malondialdehyde. HUVEC, cells from human umbilical cord endothelial vein. log, logarithm. sqrt, square root.

responsible for the initiation and maintenance of gene expression (Hitchler and Domann, 2007). This shows that proper endothelial function and physiological oxidative American Journal of Human Biology

stress are required for a normal pregnancy and fetal development. On the other hand, an excessive increase in the concentration of pro-oxidant molecules could be

829

MATERNAL AND NEWBORN ENDOTHELIAL FUNCTION AND OXIDATIVE STRESS

TABLE 4. Adjusted linear regressions for the relationship of birth weight (dependent variable) with maternal and newborn variables of endo-

thelial function and oxidative stress Birth weight Model 1

Maternal endothelial function FMD at second trimester FMD at third trimester Change in FMD Maternal oxidative stress sqrt-MDA (nM) at second trimester sqrt-MDA (nM) at third trimester Change in MDA (nM)

Model 2

Beta (95% CI)

P value

Beta (95% CI)

P value

0.57 (234.2 to 35.3) 23.72 (240.2 to 32.7) 3.09 (229.3 to 35.5)

0.974 0.836 0.848

21.10 (234.9 to 32.7) 24.42 (241.1 to 32.2) 3.30 (229.2 to 35.8)

0.948 0.807 0.837

9.83 (25.25 to 24.9) 20.13 (213.8 to 13.5) 0.07 (20.09 to 0.23)

0.193 0.983 0.367

10.35 (24.32 to 25.0) 20.35 (28.69 to 7.97) 0.04 (20.09 to 0.18)

0.160 0.926 0.498

Data are beta coefficients (95% confidence interval). Model 1: Adjusted for specific gestational week of measurements (or difference between specific weeks of measurements if change in FMD or MDA was evaluated) and gestational age. Model 2: Adjusted for specific gestational week of measurements, gestational age, and complications during pregnancy and/or at delivery. NO, nitric oxide. MDA, malondialdehyde. FMD, flow-mediated dilatation. HUVEC, cells from human umbilical cord endothelial vein. sqrt, square root.

harmful to the mother and fetus, but the level at which maternal oxidative stress status could become pathological is unclear. Thus, there is a state of redox homeostasis in the body in which there is a balance between the beneficial and harmful effects of free oxygen radicals, which is a biological event of importance to consider as a biological mechanism involved in the early origin of diseases in adults (Droge, 2002). Other markers of oxidative stress should be included to determine whether such findings are characteristic of all or just some markers. In addition, further exploration is needed to explain whether, and how, maternal endothelial function in second trimester rather than at third trimester could be considered as a predictor of newborn oxidative stress at delivery. It is possible that the increase in diameter of the brachial artery, that has been shown to increase during the course of pregnancy, affects the vascular response of the FMD in the third trimester and consequently its association with newborn variables. The maternal MDA plasma levels between gestational weeks 16 to 20 were positively and independently associated with the umbilical cord blood MDA levels, whereas the change in maternal MDA levels correlated positively and significantly with newborn MDA once covariates of number of weeks between the two measurements and gestational age were taken into account. This finding is in agreement with that reported by Koklu et al. (2007), in a study of 100 healthy pregnant women and their offspring, in which the maternal MDA was measured at the time of delivery. In addition, we found a positive correlation between the maternal levels of MDA and birth weight which did not remain significant after adjustments. Osorio et al. (2011) found a significant and positive correlation between maternal antioxidant capacity markers (superoxide dismutase activity and glutathione) and birth weight; however, a study by Kim et al., (2005) of 261 pregnant women, reported a negative correlation between birth weight and MDA levels in urine at the moment of delivery. The explanation for the differences between this negative association and the positive association between maternal MDA levels and birth weight seen in the present study may be due to the timing of sample collection or the type of specimen analyzed. The values found in plasma MDA in mothers and their newborns were consistent with the levels found in healthy

pregnant women and newborns from other studies (AlSaleh et al., 2014; Calderon et al., 2008; Babu et al., 2012). Although, cutoff points for defining abnormal values of FMD and oxidative stress are not available. With respect to endothelial function in pregnancy, Kamat et al. (2011) studied a cohort of 81 pregnant women at risk of developing pregnancy-induced hypertension; they measured FMD in the second and third trimester of pregnancy. Lower values of FMD were found in those who developed pregnancy-induced hypertension and the values found in the second trimester were a good predictor of risk. In the second and third trimester, the average values of FMD among those who developed pregnancy-induced hypertension were much lower than those found in our study (mean 6 SD: 6.72 6 3.24 and 6.04 6 4.37, respectively). Miyague et al., (2013) measured FMD in 42 healthy pregnant women during the third trimester of pregnancy and found similar levels to those found in our study (mean 6 SD: 10.39% 6 5.57). Taken together with the associations between maternal FMD and newborn NO production reported here, a progressive increase in maternal endothelial function may help improve NO production in the newborn in healthy pregnancies. Regardless of events during pregnancy, however, alterations in endothelial function and oxidative stress markers could appear once the newborn begins to interact with the environment. Strengths and weaknesses Several limitations should be mentioned. This study did not evaluate other markers of newborn endothelial function, such as skin perfusion by laser Doppler and maternal biochemical markers of endothelial function, which would have allowed a more robust analysis. Information about uterine/vaginal obstruction, uterine contractions, maternal breathing patterns, and whether or not the pregnancy was induced was not recorded. The statistical power could have been affected by the fact that the measurement of MDA and NO in newborn samples was not performed in all of the subjects, and by missing values for maternal variables at third trimester, mainly MDA levels. The multiple tests performed in this exploratory analysis may have identified chance findings and our findings should be viewed with caution since further American Journal of Human Biology

830

I. ECHEVERRI ET AL

epidemiological studies are needed to confirm or contrast with them. The strengths of our study are mainly related to the novelty of measuring maternal FMD and MDA levels earlier and more frequently during pregnancy, and correlating them with directly measured NO production in newborn HUVECs at delivery. In summary, we found positive associations between measures of maternal oxidative stress and FMD during pregnancy with NO production and oxidative stress in newborns. Since pregnancy complications were infrequent, these findings suggest that under physiological conditions, enhanced endothelial function and/or oxidative stress in the mother may have an impact on normal fetal development. To confirm our findings, further research is required in larger samples with a wider set of markers of endothelial function and of oxidative stress, as well as comparisons between the changes in such variables in complicated and noncomplicated pregnancies. ACKNOWLEDGMENTS Isabella Echeverri wishes to thank COLCIENCIAS for providing scholarship. LITERATURE CITED Adams MR, Kinlay S, Blake GJ, Orford J L, Ganz P, Selwyn AP. 2000. Atherogenic lipids and endothelial dysfunction: mechanisms in the genesis of ischemic syndromes. Annu Rev Med 51:149–167. Al-Gubory KH, Fowler PA, Garrel C. 2010. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 42:1634–1650. Al-Saleh I, Al-Rouqi R, Obsum CA, Shinwari N, Mashhour A, Billedo G, Al-Sarraj Y, Rabbah A. 2014. Mercury (Hg) and oxidative stress status in healthy mothers and its effect on birth anthropometric measures. Int J Hyg Environ Health 217: 567–85. Armitage JA, Khan IY, Taylor PD, Nathanielsz PW, Poston L. 2004. Developmental programming of the metabolic syndrome by maternal nutritional imbalance: how strong is the evidence from experimental models in mammals? J Physiol 561:355–377. Armstrong L, Stenson B. 2006. Effect of delayed sampling on umbilical cord arterial and venous lactate and blood gases in clamped and unclamped vessels. Arch Dis Child Fetal Neonatal Ed 91:342–345. Babu MS, Bobby Z, Habeebullah S. 2012. Increased inflammatory response and imbalance in blood and urinary oxidant-antioxidant status in South Indian women with gestational hypertension and preeclampsia. Clin Biochem 45(10–11):835–838. Barker DJ. 2002. Fetal programming of coronary heart disease. Trends Endocrin Met 13:364–368. Barker DJP, Osmond C, Winter PD, Margetts B, Simmonds SJ. 1989. Weight in infancy and death from ischaemic heart disease. Lancet 334: 577–80. Baudin B, Bruneel A, Bosselut N, Vaubourdolle M. 2007. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc 2: 481–485. Bryan NS, Grisham MB. 2007. Methods to detect nitric oxide and its metabolites in biological samples. Free Radic Biol Med 43(5):645–657. Calderon TC, Wu W, Rawson RA, Sakala EP, Sowers LC, Boskovic DS, Angeles DM. 2008. Effect of mode of birth on purine and malondialdehyde in umbilical arterial plasma in normal term newborns. J Perinatol 28(7):475–481. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. 1992. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 340:1111–1115. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, Vogel R; International Brachial Artery Reactivity Task Force. 2002. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the international brachial artery reactivity task force. J Am Coll Cardiol 39:257–265. Davignon J, Ganz P. 2004. Role of endothelial dysfunction in atherosclerosis. Circulation 109:27–32.

American Journal of Human Biology

Delisle H. 2002. Programming of chronic disease by impaired fetal nutrition. Evidence and implications for policy and intervention strategies. Geneva: World Health Organization. De Jong F, Monuteaux MC, van Elburg RM, Gillman MW, Belfort MB. 2012. Systematic review and meta-analysis of preterm birth and later systolic blood pressure. Hypertension 59:226–234. Droge W. 2002. Free radicals in the physiological control of cell function. Physiol Rev 82:47–95. Fenton TR. 2003. A new growth chart for preterm babies: Babson and Benda’s chart updated with recent data and a new format. BMC Pediatr 3:13. Grune T, Berger MM. 2007. Markers of oxidative stress in ICU clinical settings: present and future. Curr Opin Clin Nutr Metab Care 10(6):712– 717. Hitchler MJ, Domann FE. 2007. An epigenetic perspective on the free radical theory of development. Free Radic Biol Med 43:1023–1036. Holzhauer S, Hokken Koelega AC, Ridder Md, Hofman A, Moll HA, Steegers EA, Witteman JC, Jaddoe VW. 2009. Effect of birth weight and postnatal weight gain on body composition in early infancy: the generation R study. Early Hum Dev 85(5):285–290. Hung TH, Lo LM, Chiu TH, Li MJ, Yeh YL, Chen SF, Hsieh TT. 2010. A longitudinal study of oxidative stress and antioxidant status in women with uncomplicated pregnancies throughout gestation. Reprod Sci 17: 401–409. Jansson T, Powell TL. 2006. IFPA 2005 Award in placentology lecture. Human placental transport in altered fetal growth: does the placenta function as a nutrient sensor? – a review. Placenta 27:S91–S97. Jauniaux E, Poston L, Burton GJ. 2006. Placental-related diseases of pregnancy: involvement of oxidative stress and implications in human evolution. Hum Reprod Update 12(6):747–755. Jayet PY, Rimoldi SF, Stuber T, Salm on CS, Hutter D, Rexhaj E, Thalmann S, Schwab M, Turini P, Sartori-Cucchia C, Nicod P, Villena M, Allemann Y, Scherrer U, Sartori C. 2010. Pulmonary and systemic vascular dysfunction in young offspring of mothers with preeclampsia. Circulation 122:488–494. Jones H, Green DJ, George K, Atkinson G. 2010. Intermittent exercise abolishes the diurnal variation in endothelial-dependent flow-mediated dilation in humans. Am J Physiol Regul Integr Comp Physiol 298:R427– R432. Kamat R, Jain V, Bahl A. 2011. Serial estimation of flow mediated dilatation in women at risk of hypertensive disorders of pregnancy. Int J Cardiol 19;149(1):17–22. Kim YJ, Hong YC, Lee KH, Park HJ, Park EA, Moon HS, Ha EH. 2005. Oxidative stress in pregnant women and birth weight reduction. Reprod Toxico 19: 487–492. Koklu E, Akcakus M, Narin F, Saraymen R. 2007. The relationship between birth weight, oxidative stress and bone mineral status in newborn infants. J Paediatr Child Health 43:667–672. Krause BJ, Hanson MA, Casanello P. 2011. Role of nitric oxide in placental vascular development and function. Placenta 32:797–805. Lazdam M, de la Horra A, Pitcher A, Mannie Z, Diesch J, Trevitt C, Kylintireas I, Contractor H, Singhal A, Lucas A, Neubauer S, Kharbanda R, Alp N, Kelly B, Leeson P. 2010. Elevated blood pressure in offspring born premature to hypertensive pregnancy: is endothelial dysfunction the underlying vascular mechanism? Hypertension 56:159– 165. Leikert JF, R€ athel TR, M€ uller C, Vollmar AM, Dirsch VM .2001. Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein. Febs Lett 506:131–134. Lewis RM, Poore KR, Godfrey KM. 2006. The role of the placenta in the developmental origins of health and disease—implications for practice. Rev Gynaecol Perinat Pract 6(1):70–79. Miyague AH, Martins WP, Machado JS, Palei AC, Amaral LM, Teixeira DM, Sandrim VC, Sertorio JT, Tanus-Santos JE, Duarte G, Cavalli RC. 2013. Maternal flow-mediated dilation and nitrite concentration during third trimester of pregnancy and postpartum period. Hypertens Pregnancy 32(3):225–234. Mullen MJ, Kharbanda RK, Cross J, Donald AE, Taylor M, Vallance P, Deanfield JE, MacAllister RJ. 2001. Heterogenous nature of flow mediated dilatation in human conduit arteries in vivo: relevance to endothelial dysfunction in hypercholesterolemia. Circ Res 88:145–151. Osorio JC, Cruz E, Milan es M, Ramırez Y, Sierra M, Cruz M, Sanfiel L.2011. Influence of maternal redox status on birth weight. Reprod Toxicol 31(1):35–40. Ramırez-V elez R, Romero M, Echeverri I, Ortega JG, Mosquera M, on SL, Saldarriaga W, Aguilar de Plata AC, Mateus JC. Salazar B, Gir 2011. A factorial randomized controlled trial to evaluate the effect of micronutrients supplementation and regular aerobic exercise on maternal endothelium-dependent vasodilatation and oxidative stress of the newborn. Trials 12:60.

MATERNAL AND NEWBORN ENDOTHELIAL FUNCTION AND OXIDATIVE STRESS Rexhaj E, Bloch J, Jayet PY, Rimoldi SF, Dessen P, Mathieu C, Tolsa JF, Nicod P, Scherrer U, Sartori C. 2011. Fetal programming of pulmonary vascular dysfunction in mice: role of epigenetic mechanisms. Am J Physiol Heart Circ Physiol 301:H247–H252. Saarelainen H, Laitinen T, Raitakari OT, Juonala M, Heiskanen N, LyyraLaitinen T, Viikari JS, Vanninen E, Heinonen S. 2006. Pregnancyrelated hyperlipidemia and endothelial function in healthy women. Circ J 70:768–772. Steghens JP, van Kappel AL, Denis I, Collombel C. 2001. Diaminonaphtalene, a new highly specific reagent for HPLC-UV measurement of total and free malondialdehyde in human plasma or serum. Free Radic Biol Med 31:242–249. Stoner L, Erickson ML, Young JM, Fryer S, Sabatier MJ, Faulkner J, Lambrick DM, McCully KK. 2012. There’s more to flow-mediated dilation than nitric oxide. J Atheroscler Thromb 19:589–600. Thijssen DH, Black MA, Pyke KE, Padilla J, Atkinson G, Harris RA, Parker B, Widlansky ME, Tschakovsky ME, Green DJ. 2011. Assess-

831

ment of flow-mediated dilation in humans: a methodological and physiological guideline. Am J Physiol Heart Circ Physiol 300(1):H2–H12. Tschakovsky M, Pyke K E. 2005. Point: flow-mediated dilation does reflect nitric oxide-mediated endothelial function. J Appl Physiol 99:1235– 1237. Veas CJ, Aguilera VC, Mu~ noz IJ, Gallardo VI, Miguel PL, Gonz alez MA, Lamperti LI, Escudero CA, Aguayo CR. 2011. Fetal endothelium dysfunction is associated with circulating maternal levels of sE-selectin, sVCAM1, and sFlt-1 during pre-eclampsia. J Matern Fetal Neonatal Med 24:1371–1377. Weissgerber TL, Davies GAL, Tschakovsky ME. 2011. Brachial artery flow-mediated dilation is not affected by pregnancy or regular exercise participation. Clin Sci 121:355–365. Yang X, Guo L, li H, Chen X, Tong X. 2012. Analysis of the original causes of placental oxidative stress in normal pregnancy and pre-eclampsia: a hypothesis. Review article. J Matern Fetal Neonatal Med 25(7):884– 888.

American Journal of Human Biology