Oxidized low-density lipoproteins in cord blood from neonates with intra-uterine growth restriction

European Journal of Obstetrics & Gynecology and Reproductive Biology 156 (2011) 46–49

Contents lists available at ScienceDirect

European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb

Oxidized low-density lipoproteins in cord blood from neonates with intra-uterine growth restriction Line Leduc a,*, Edgard Delvin b, Annie Ouellet c, Carole Garofalo d, Emilie Grenier d, Lucie Morin a, Johanne Dube´ a, Maurice Bouity-Voubou d, Jean-Marie Moutquin c, Jean-Claude Fouron e, Stephanie Klam f, Emile Levy d a

Department of Obstetrics and Gynaecology, Universite´ de Montre´al and Research Centre, CHU Sainte-Justine, Quebec, Canada Department of Biochemistry, Universite´ de Montre´al and Research Centre, CHU Sainte-Justine, Quebec, Canada c Department of Obstetrics and Gynaecology, Centre Hospitalier Universitaire de Sherbrooke, Quebec, Canada d Department of Nutrition, Universite´ de Montre´al and Research Centre, CHU Sainte-Justine, Quebec, Canada e Department of Cardiology service, Universite´ de Montre´al and Research Centre, CHU Sainte-Justine, Quebec, Canada f Department of Obstetrics and Gynaecology, Jewish General hospital, Quebec, Canada b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 September 2010 Received in revised form 22 December 2010 Accepted 7 January 2011

Objective: We verified whether oxidative stress indices (oxidized low-density lipoproteins and malondialdehyde) and inflammatory biomarkers (circulating C-reactive protein, interleukin-6, tumour necrosis factor-a, serum amyloid A and soluble intercellular vascular cell adhesion molecule) are increased in the umbilical vein of placental insufficiency induced intra-uterine growth restricted neonates. Study design: The prospective cohort study, involving 3 tertiary care centers, consists of 200 consecutively recruited pregnant women carrying twins. We chose the twin pregnancy model because both fetuses share the same maternal environment, thereby avoiding potential confounding factors when comparing oxidative stress and inflammation biomarkers. We analysed only twin pairs with one with intra-uterine growth restriction (N = 38) defined as fetal growth < 10th percentile with abnormal Doppler of the umbilical artery. Blood samples were taken at birth from the umbilical vein. Intra-pair comparisons on the biomarkers were performed using the Student paired t-test. Results: We observed increased cord blood levels of oxidized low-density lipoproteins, (2.394  .412 vs 1.296  .204, p = .003) but not of malondialdehyde in growth restricted neonates when compared to their normal counterparts. Although indices of inflammation tended to be increased in cord blood from growth restricted newborns, the difference did not reach statistical significance. Conclusion: In the twin model, intra-uterine growth restriction is associated with low-density lipoprotein oxidation without apparent dysregulation of inflammation biomarkers. Condensation: Increased oxidized low-density lipoproteins are observed in growth restricted twins compared to their co-twins with normal growth at birth. ß 2011 Elsevier Ireland Ltd. All rights reserved.

Keywords: Oxidative stress Fetal programming Intrauterine growth restriction Inflammation

1. Introduction Fetal growth restriction, occurring in 3–10% of all pregnancies, is recognized as a major cause of perinatal morbidity and mortality [1,2] and could possibly be related to diseases observed later in life. Barker was the first to propose the fetal origin hypothesis of adult coronary heart disease, type 2 diabetes (T2D), stroke and hypertension, now known as the ‘‘Barker hypothesis’’ [3]. An in

* Corresponding author at: Department of Obstetrics & Gynaecology, MaternalFoetal Medicine Division, CHU Sainte-Justine, 3175 Coˆte Sainte-Catherine, Montreal, QC, H3T 1C5, Canada. Tel.: +1 514 345 4706; fax: +1 514 345 4648. E-mail address: [email protected] (L. Leduc). 0301-2115/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2011.01.007

utero event operating at a critical or sensitive period may result in a long-term change in the structure or function of the organism that will predispose to disease in adult life. Endothelial vascular dysfunction, capillary rarefaction and reduced arterial diameter [4–6] have all been shown to be associated with impaired fetal growth, suggesting that placental insufficiency is an early and relevant process that triggers IUGR and endothelial cell injury, leading to subsequent manifestations of cardiovascular disease (CVD). Thus, we propose that the vascular process occurring in the growth-restricted fetus is similar to that in atherosclerotic vascular disease of later life. Since oxidative stress and inflammation are mechanistically involved in atherosclerosis development and progression, we examined whether oxidative stress indices (oxidized LDL and malondialdehyde) and inflammatory biomarkers,

L. Leduc et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 156 (2011) 46–49

(circulating C-reactive protein, interleukin-6, tumour necrosis factor-a, serum amyloid A and soluble intercellular vascular cell adhesion molecule) were increased at birth in the umbilical vein of IUGR from placental insufficiency. We elected to study twin pregnancies, reasoning that twins represent a unique model as both fetuses share the same intra-uterine environment and thereby potential confounding factors should be attenuated in intra-pair comparisons. 2. Materials and methods 2.1. Patients The Ethics Committees of the participating institutions approved this prospective cohort study and patients gave their informed consent. Women over 18 years old recruited from 3 tertiary hospital centers were invited to participate in the study at time of diagnosis of a twin pregnancy routinely established by ultrasonography at either 11–14 weeks or 16–20 weeks of gestation. Participants were followed-up until delivery. Gestational age was either calculated from the date of the last menstrual period or estimated by ultrasonography before 20 weeks. Participants’ medical history, health behaviour and socio-economic status were collected with a questionnaire. Fetal growth was assessed using the population-based Canadian twin fetal growth curve [7] and IUGR was defined as fetal growth less than the 10th percentile. For pregnancies with appropriate fetal growth, considering that the mean gestational age at delivery varies between 36 and 37 weeks [8], ultrasound was performed at 2–4 week intervals starting at 24 weeks of gestation up to 34–35 weeks [1]. Umbilical artery vascular flow resistance was assessed by Doppler at 32–34 weeks of gestation or close to the time of delivery for appropriate fetal growth, but once or twice a week in IUGR cases. Placental insufficiency was defined as a higher placental vascular resistance with a decreased (Pulsatility index > 95th percentile), absent or reversed diastolic flow of the umbilical artery. In order to document the severity of the circulatory compromise, other fetal vessels were assessed, such as the middle cerebral artery and the ductus venosus. Pregnant women with gestational diabetes or hypertension were included in the study. The exclusion criteria were: twin–twin transfusion syndrome (TTTS), fetal death of one co-twin, congenital or chromosomal anomaly, twins with reversed arterial perfusion syndrome (TRAP), and alteration in fetal cardiac ventricular function (e.g. cardiomegaly). An antenatal diagnosis was suggestive of TTTS according to ultrasound [9] and echocardiographic findings that were previously reported by our institution [10,11]. Chorionicity was confirmed with a pathologic examination of the placenta. 2.2. Blood collection, process and storage Two to 20 ml of blood were collected from the umbilical vein at the time of delivery by aspiration with a syringe, and immediately refrigerated at 4 8C or alternatively placed in melting ice into an isothermal container. Blood samples were transferred into VacutainerTM tubes (Becton-Dickinson Canada, Mississauga, Ontario) containing EDTA for plasma collection, and into dry tubes for serum collection and centrifuged within 15 min at 2000  g for 15 min at 4 8C. The serum and plasma were transferred into 0.5 ml cryovials for storage at 80 8C until used. 2.3. Biochemical tests High sensitivity C-Reactive Protein (hsCRP) was measured by immunonephelometry on the ImageTM (Beckman-Coulter, Brea,

47

CA). All other variables were measured by ELISA: interleukin-6 (IL-6), tumour necrosis factor-alpha (TNFa) and soluble intracellular adhesion molecule-1 (sICAM-1) (R&D Systems, Minneapolis, MN) and serum amyloid antigen (SAA) (Autogen Bioclear, Calne, Wilshire, UK), oxidized low-density lipoprotein (ox-LDL) were also measured by ELISA (Cayman, Ann Arbor, MI). Free malondialdehyde (MDA) was determined by HPLC, as previously described [12]. Briefly, proteins were precipitated with a 10% (w/ v) sodium tungstate (Na2WO4) (Aldrich, Milwaukee, WI) and the protein-free supernatants reacted with 0.5% (w/v) thiobarbituric acid (TBA; Sigma, St. Louis, MO) at 90 8C for 60 min. After cooling to room temperature, the pink TBA-MDA complex was extracted with 1-butanol and dried over a stream of nitrogen at 37 8C. The dry extract was dissolved in the KH2PO4/methanol (70:30, pH 7.0) and analysed by HPLC. 2.4. Statistics Statistical analysis was performed using the SPSS software version 16.0 (SPSS, Chicago, IL, USA). Normally distributed data, tested with the Shapiro–Wilk test, were expressed as mean  standard deviation (SD). The significance of the difference between the paired normal and IUGR cord blood variables was tested using Student paired t-test. A p-value < 0.01 was considered statistically significant. 3. Results Of the 200 patients originally enrolled, 38 were pregnancies with one IUGR co-twin, of which 32 were dichorionic–diamniotic and 6 monochorionic twins. The mean maternal age was 30.8  5.9 years and gestational age at delivery was 35.7  3.4 weeks. Most of them were delivered by elective cesarean section (30/ 38). There were no cases with diabetes and three women presented with severe pre-eclampsia. All IUGR neonates were less than the 10th percentile from placental insufficiency with increased umbilical artery resistance and abnormal umbilical blood flow. The mean birth weight (g) for the normal and growth restricted fetuses were respectively 2328  607 (710–3140) and 1946  598 (500–2775), p < 0.01. Table 1 summarizes the data obtained for the cord blood vascular endothelium damage, inflammation and oxidative stress biomarker concentrations. Of all the variables measured, only oxLDL concentration was statistically higher, in IUGR neonates, being almost twice that of their normal counterparts (2.394  0.412 vs 1.296  0.0204, p = 0.003), irrespective of mode of delivery.

Table 1 Cord blood vascular endothelium damage, inflammation and oxidative stress biomarkers. Oxidative stress and i nflammation biomarkers

N

Control

IUGR

p

ICAM-1, ng/ml SAA, ng/ml IL-6, pg/ml TNF-a, pg/ml hsCRP, mg/l ox-LDL, U/ml MDA, pmoles/ml

37 37 31 32 27 37 34

390.2  17.2 47.6  7.5 33.2  2.9 129.6  43.7 0.021  0.003 1.296  0.204 282.8  18.8

397.6  15.8 52.1  10.7 40.3  5.5 137.3  44.8 0.020  0.002 2.394  0.412 305.9  19.8

0.546 0.655 0.228 0.308 0.771 0.003* 0.326

sICAM-1: soluble intercellular adhesion molecule type-1; SAA: serum amyloid antigen; IL-6: interleukin-6; TNF-a: tumour necrosis factor-alpha; hsCRP: high sensitivity C-reactive protein; ox-LDL: oxidized low-density lipoprotein; MDA: malondialdehyde. The significance of the difference between the 2 groups was assessed with the Student t-test for paired variates.

48

L. Leduc et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 156 (2011) 46–49

4. Discussion Inconsistent data on the variation in growth between twin pairs and adult mortality rates from cardiovascular disease have until now been available. Twin data on biomarkers reflecting critical exposure are often problematic. The use of recalled birth weight data, questionnaire-based classification of twin type, the absence of information on chorionicity and the absence of information on utero-placental perfusion quality contribute to inconsistencies. Our twin model is unique to study fetal programming as it enables control for a host of confounding maternal factors, such as height, pre-pregnancy weight, weight gain during pregnancy, smoking, occupational and environmental exposures, chronic diseases in pregnancy, and socioeconomic status, that all have been shown to have the potential of blurring the relationship between fetal growth and health [13]. Our data obtained from carefully selected twin pairs demonstrate, for the first time to our knowledge, that IUGR neonates are exposed, in utero, to an environment conducive to lipoprotein oxidation, a prerequisite for a later development of atherosclerosis. Since ox-LDL have a wide range of atherogenic properties ranging from early lesion formation to plaque rupture [14], this observation is of potential clinical importance. We did not, however, observe increased plasma MDA levels in IUGR neonates. This apparent contradiction can be explained by the fact that mechanisms leading to lipid hydroperoxidation, the surrogate marker of which is the MDA-thiobarbituric adduct, are different from those leading to oxidized LDL in which lysine residues are the targets for oxidation. Also, circulating oxidized polyunsaturated fatty acids may have much shorter fractional catabolism than oxLDL. They are also more likely to be imbedded in cell plasma membranes. Our data differ from those of Hracsko et al. [15] who observed increased lipid peroxidation products in cord blood from 29 IUGR babies compared to 120 controls. The method for MDA measurement used in our study was based on MDA and thiobarbituric acid condensation followed by high performance chromatography, thereby diminishing the risk of measuring nonspecific compounds. They also are different from those of Gupta et al. [16] who, in a case–control study, studied singleton term small for gestational age neonates born to undernourished mothers. Despite the increase in ox-LDL, no differences were noted in ICAM-1 and SAA, markers of endothelial damage, or inflammation biomarkers. This observation leads us to propose that no endothelium damage occurred and that the inflammation biomarkers, being short-lived, might have been efficiently cleared by the placenta or the mother. One likely explanation would be that, as opposed to other studies [17,18], most of our patients were neither preeclamptic nor diabetic. These latter conditions have been associated with intense maternal or placental oxidative stress and inflammation [19–23]. Our data suggest that in our group of patients, the placental antioxidant properties might be effective in removing MDA from the circulation, thus explaining the lack of difference between the IUGR and normal twins. Indeed, we know that during normal pregnancy, lipid peroxidation is induced in the human placenta [24,25], and increased indices of oxidative stress have been reported in the placenta of IUGR [26,27], The plasma concentration of MDA at birth might be a marker of overwhelmed oxidative condition. This needs to be confirmed with future analyses on the placenta. The present study has limitations. Data were analysed and reported as a pool without distinction for zygosity, due to the small number of monozygotic twins, and thus the genetic component in birth weight was not determined. A study population including only monozygotic twins (after exclusion of TTS) would be ideal to perfectly control for the genetic influence on birth weight. Any other changes would be attributable to the quality of the

intrauterine environment. This study is currently ongoing and further analyses will consider zygosity. Various substances are linked to oxidative stress and lipid peroxidation. For instance, inducible heat shock protein 70 has been shown to be up-regulated during oxidative stress [28,29]. Increased circulating heat shock protein 70 levels were observed in pre-eclampsia and associated with systemic inflammation, oxidative stress and hepatocellular injury [30]. The role of these protective proteins in structural and functional damage following exposure to adverse environmental conditions has not yet been addressed and deserves further investigation. Our results support a role for systemic oxidative stress in the pathogenesis of fetal growth restriction. An imbalance in the oxidant/antioxidant activity seems to be central but the mechanisms have not been completely clarified. One mechanism would be a gene polymorphism of the antioxidant system. In fact, an association of extracellular superoxide dismutase Ala40Thr gene polymorphism with pre-eclampsia complicated by severe fetal growth restriction has been reported [31]. Indices of inflammation tended to be increased in cord blood from growth restricted newborns, but the difference did not reach statistical significance. This might be due to the relatively low number of patients enrolled in this study. Active recruitment is ongoing and will probably help to get the sample size needed or at least to clarify this issue. This study shows that there is evidence of oxidative stress in growth-restricted fetuses. The twin model offers a good opportunity for that kind of investigation because both fetuses are under the same maternal environment and thereby intra-pair comparisons will avoid potential confounding factors. From the present study, it is difficult to infer whether oxidative stress is a cause or effect of intrauterine growth restriction. Future studies are needed to provide new insights into mechanisms that underlie the fetal origins hypothesis. Acknowledgments This work was supported by a financial grant from the CIHR # 158179. References [1] Miller J, Turan S, Baschat AA. Fetal growth restriction. Semin Perinatol 2008;32(4):80–274. [2] Petersen SG, Wong SF, Urs P, Gray PH, Gardiner GJ, et al. Early onset, severe fetal growth restriction with absent or reversed end-diastolic flow velocity waveform in the umbilical artery: perinatal and long-term outcomes. Aust N Z J Obstet Gynaecol 2009;49(1):45–51. [3] Barker DJ. The developmental origins of chronic adult disease. Acta Paediatr Suppl 2004;93(446):26–33. [4] Halvorsen CP, Andolf E, Hu J, Pilo C, Windbladh B, Norman M, et al. Discordant twin growth in utero and differences in blood pressure and endothelial function at 8 years of age. J Intern Med 2006;259(2):63–155. [5] Pesonen E, Johnsson J, Berg A. Intimal thickness of the coronary arteries in lowbirthweight infants. Acta Paediatr 2006;95(10):8–1234. [6] Pladys P, Sinnlaub F, Brault S, et al. Microvascular rarefaction and decreased angiogenesis in rats with fetal programming of hypertension associated with exposure to a low-protein diet in utero. Am J Physiol Regul Integr Comp Physiol 2005;289(6):R1580–8. [7] Arbuckle TE, Wilkins R, Sherman GJ. Birth weight percentiles by gestational age in Canada. Obstet Gynecol 1993;81(1):39–48. [8] Leduc L, Takser L, Rinfret D. Persistance of adverse obstetric and neonatal outcomes in monochorionic twins after exclusion of disorders unique to monochorionic placentation. Am J Obstet Gynecol 2005;193(5):5–1670. [9] Quintero RA, Morales WJ, Allen MH, Bormick PW, Johnson PK, kruger M, et al. Staging of twin–twin transfusion syndrome. J Perinatol 1999;19(8 Pt 1):5–550. [10] Bensouda B, Fouron JC, Raboisson MJ, Lachance C, Leduc L. Relevance of measuring diastolic time intervals in the ductus venosus during the early stages of twin–twin transfusion syndrome. Ultrasound Obstet Gynecol 2007;30(7):7–983. [11] Raboisson MJ, Fouron JC, Lamoureux J, et al. Early intertwin differences in myocardial performance during the twin-to-twin transfusion syndrome. Circulation 2004;110(19):8–3043.

L. Leduc et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 156 (2011) 46–49 [12] Bernotti S, Seidman E, Sinnett D, et al. Inflammatory reaction without endogenous antioxidant response in Caco-2 cells exposed to iron/ascorbate-mediated lipid peroxidation. Am J Physiol Gastrointest Liver Physiol 2003;285(5):G898–906. [13] Kramer MS. The epidemiology of adverse pregnancy outcomes: an overview. J Nutr 2003;133(5 Suppl. 2):1592S–6. [14] Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherogenesis. Free Radic Biol Med 1996;20(5):27–707. [15] Hracsko Z, Orvos H, Novak Z, Pal A, Varga IS. Evaluation of oxidative stress markers in neonates with intra-uterine growth retardation. Redox Rep 2008;13(1):6–11. [16] Gupta P, Narang M, Banerjee BD, Basu S. Oxidative stress in term small for gestational age neonates born to undernourished mothers: a case control study. BMC Pediatr 2004;4:14. [17] Boutsikou T, Mastorakos G, Kyriakakou M, et al. Circulating levels of inflammatory markers in intrauterine growth restriction. Mediators Inflamm 2010;2010:790605. [18] Kinalski M, Sledziewski A, Telejko B, et al. Lipid peroxidation, antioxidant defence and acid-base status in cord blood at birth: the influence of diabetes. Horm Metab Res 2001;33(4):31–227. [19] Anastasakis E, Papantoniou N, Daskalakis G, Mesogitis S, Antsaklis A, et al. Screening for pre-eclampsia by oxidative stress markers and uteroplacental blood flow. J Obstet Gynaecol 2008;28(3):9–285. [20] LaMarca BD, Ryan MJ, Gilbert JS, Murphy SR, Granger JP. Inflammatory cytokines in the pathophysiology of hypertension during preeclampsia. Curr Hypertens Rep 2007;9(6):5–480. [21] Sitras V, Paulssen RH, Gronaas H, et al. Differential placental gene expression in severe preeclampsia. Placenta 2009;30(5):33–424. [22] Gu Y, Lewis DF, Deere K, Groone LJ, Wang Y, et al. Elevated maternal IL-16 levels, enhanced IL-16 expressions in endothelium and leukocytes, and increased IL-16

[23] [24] [25]

[26]

[27]

[28]

[29]

[30]

[31]

49

production by placental trophoblasts in women with preeclampsia. J Immunol 2008;181(6):22–4418. Redman CW, Sargent IL. Placental stress and pre-eclampsia: a revised view. Placenta 2009;30(Suppl. A):S38–42. Walsh SW, Wang Y. Secretion of lipid peroxides by the human placenta. Am J Obstet Gynecol 1993;169(6):6–1462. Walsh SW, Wang Y. Trophoblast and placental villous core production of lipid peroxides, thromboxane, and prostacyclin in preeclampsia. J Clin Endocrinol Metab 1995;80(6):93–1888. Biri A, Bozkurt N, Turp A, Kavutcu M, Himmetoglu O, Durak I. Role of oxidative stress in intrauterine growth restriction. Gynecol Obstet Invest 2007;64(4):92–187. Takagi Y, Nikaido T, Toki T, et al. Levels of oxidative stress and redox-related molecules in the placenta in preeclampsia and fetal growth restriction. Virchows Arch 2004;444(1):49–55. Hnat MD, Meadows JW, Brookman DE, Pitzer B, Lyall F, Myatt L. Heat shock protein-70 and 4-hydroxy-2-nonenal adducts in human placental villous tissue of normotensive, preeclamptic and intrauterine growth restricted pregnancies. Am J Obstet Gynecol 2005;193(3 Pt 1):40–836. Liu Y, Li N, You L, Liu X, Li H, Wang X. HSP70 is associated with endothelial activation in placental vascular diseases. Mol Med 2008;14(9–10): 6–561. Molvarec A, Rigo Jr J, Lazar L, et al. Increased serum heat-shock protein 70 levels reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. Cell Stress Chaperones 2009;14(2):9–151. Rosta K, Molvarec A, Enzsoly A, et al. Association of extracellular superoxide dismutase (SOD3) Ala40Thr gene polymorphism with pre-eclampsia complicated by severe fetal growth restriction. Eur J Obstet Gynecol Reprod Biol 2009;142(2):8–134.