Clinical and Experimental Hypertension
ISSN: 1064-1963 (Print) 1525-6006 (Online) Journal homepage: http://www.tandfonline.com/loi/iceh20
Increased oxidative stress from early pregnancy in women who develop preeclampsia Vandita D’Souza, Alka Rani, Vidya Patil, Hemlata Pisal, Karuna Randhir, Savita Mehendale, Girija Wagh, Sanjay Gupte & Sadhana Joshi To cite this article: Vandita D’Souza, Alka Rani, Vidya Patil, Hemlata Pisal, Karuna Randhir, Savita Mehendale, Girija Wagh, Sanjay Gupte & Sadhana Joshi (2016): Increased oxidative stress from early pregnancy in women who develop preeclampsia, Clinical and Experimental Hypertension, DOI: 10.3109/10641963.2015.1081226 To link to this article: http://dx.doi.org/10.3109/10641963.2015.1081226
Published online: 28 Jan 2016.
Submit your article to this journal
Article views: 9
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iceh20 Download by: [University of California, San Diego]
Date: 01 February 2016, At: 13:34
http://tandfonline.com/iceh ISSN: 1064-1963 (print), 1525-6006 (electronic) Clin Exp Hypertens, Early Online: 1–8 ! 2016 Taylor & Francis Group, LLC. DOI: 10.3109/10641963.2015.1081226
Increased oxidative stress from early pregnancy in women who develop preeclampsia Vandita D’Souza1, Alka Rani1, Vidya Patil1, Hemlata Pisal1, Karuna Randhir1, Savita Mehendale2, Girija Wagh2, Sanjay Gupte3, and Sadhana Joshi1 1
Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra, India, Department of Obstetrics and Gynaecology, Bharati Medical College and Hospital, Bharati Vidyapeeth University, Pune, Maharashtra, India, and 3 Department of Obstetrics and Gynaecology, Gupte Hospital and Research Center, Pune, Maharashtra, India
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
2
Abstract
Keywords
Preeclampsia (PE) is a pregnancy-specific disorder, defined as new onset of maternal hypertension and proteinuria after 20 weeks of gestation. Our earlier study has shown increased maternal oxidative stress at delivery to be associated with poor birth outcome in PE. However, these results were observed when the pathology had progressed and may have been secondary to the effects of the disorder. To understand the role of antioxidant defense mechanisms in PE right from early pregnancy, in this prospective study, we measured malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH) concentrations in maternal blood at 3 time-points of gestation [16–20 weeks (T1), 26–30 weeks (T2), at delivery (T3)] and in cord blood. Gene expression of SOD and GPx and protein levels of endothelial nitric oxide synthase (eNOS) enzyme were also analyzed in the placenta. MDA levels were higher at T1 (p50.01) and T2 (p50.01) in women with PE as compared with control. GPx levels were higher at T3 (p50.05) while SOD levels were lower at T2 (p50.05), T3 (p50.01) and in cord (p50.01) in PE. GSH levels at T1 (p50.05) and expression of GPx in the placenta were lower in PE as compared with control. In conclusion, this study demonstrates that women who develop PE exhibit increased oxidative stress right from 16 to 20 weeks of gestation. This may alter placental development and lead to fetal programming of adult non-communicable disease in the offspring.
Antioxidants, early pregnancy, longitudinal study, oxidative stress, placenta, preeclampsia
Introduction It is well established that a certain amount of oxidative stress during pregnancy is necessary for embryonic and fetal growth (1). Oxidative stress is induced by reactive oxygen species (ROS) including superoxide anion (O 2 ), hydroxyl anion (OH) and hydrogen peroxide (H2O2) (2). A balance between ROS and antioxidants is necessary for fertilization, embryogenesis, embryonic implantation and placental growth and differentiation (3). However, excessive production of ROS damages the cell structures including lipids, proteins and DNA (4). An internal protection system comprised of both enzymatic and non-enzymatic antioxidant defenses such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH) are known to be helpful in combating excess ROS (5). Free ROS degrades polyunsaturated lipids to form malondialdehyde (MDA) which is used as a biomarker to measure the level of oxidative stress in an organism (6).
Correspondence: Dr. Sadhana Joshi, Scientist ‘G’ and Head, Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth Deemed University, Pune 411043, Maharashtra, India. Tel: 91-20-24366929/31. Fax: 91-20-24366931. E-mail: [email protected]
History Received 28 May 2015 Revised 23 July 2015 Accepted 27 July 2015 Published online 11 January 2016
In addition, there also exists an association between ROS and the enzyme endothelial nitric oxide synthase (eNOS) which produces nitric oxide in endothelial cells. Nitric oxide (NO) is a potent vasodilator and helps in maintaining vascular health and blood pressure (BP) (7) (Figure 1). Several studies have illustrated the effects of ROS on kidney function and BP in hypertensive animal models. ROS enhances vasoconstriction by reducing endothelium-dependent relaxation factor/NO (8). Preeclampsia (PE) is a pregnancy complication characterized by hypertension and proteinuria and has extensively been shown to be associated with elevated oxidative stress (9,10). Further, children born to mothers with PE are known to be at increased risk for endocrine, nutritional, and metabolic derangements during adolescence and early-adulthood (11). We have earlier reported an imbalance between oxidative stress and antioxidants in women with PE at delivery (12,13,14). However, these results were observed when the pathology had progressed and may have been secondary to the effects of the disorder. In addition to this, our earlier study in rats suggests that the antioxidant defense system of the brain may be programmed during pregnancy leading to impaired cognition in the offspring in later life (15). With this background, we hypothesize that an abnormal antioxidant
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
2
V. D’Souza et al.
Clin Exp Hypertens, Early Online: 1–8
Figure 1. Mechanism of oxidative stress leading to preeclampsia. SOD: Superoxide dismutase; GPx: Glutathione peroxidase; CAT: Catalase; GSH: Oxidised glutathione; GSSG: Reduced glutathione; H2O2: Hydrogen peroxide; H2O: Water; O2: Oxygen; O 2 : Superoxide anion; OH : hydroxyl ion; eNOS: Endothelial nitric oxide synthase; NO: Nitric oxide; ONOO: Peroxynitrite.
defense mechanism exists from early pregnancy in women who develop PE. There are few longitudinal studies which have examined changes in the oxidative stress parameters across gestation in women with PE. However, these studies are either on a small sample size, varying ethnic population or included women who smoke, all of which may confound the levels of oxidative stress (16,17,18,19,20). Further, there are limited studies associating the levels of maternal oxidative stress with birth outcome (21). Hence, this study was designed with an appropriate power and the data was analyzed after adjusting for potential confounders. This prospective study reports MDA, SOD, GPx and GSH levels at different gestational time-points in women who developed PE and in cord as compared with normotensive control (NC). The association of these biochemical markers with birth weight and maternal systolic and diastolic BP are also reported.
Materials and methods Study design Pregnant women were enrolled for this longitudinal and comparative study from two major hospitals, Bharati Hospital and Gupte Hospital in Pune, MH, India. Bharati Vidyapeeth Medical College Institutional Ethical Committee approved the research protocols and consent forms. This study is a part of a large ongoing departmental study which recruits all consecutive pregnant women at 16–20 weeks of gestation who are willing to participate and give a written consent. The detailed study design and recruitment criteria have been described by us earlier (22). Briefly, blood samples from pregnant women were collected at three different time-points across gestation, i.e. 16–20 weeks of gestation (T1), 26–30 weeks of gestation (T2) and at delivery (T3). All women were followed until delivery and categorized as PE if there was a presence of proteinuria (1 + protein on a dipstick test) and high BP (systolic BP4140 Hg and/or diastolic BP490 Hg). We calculated the power of the study based on our earlier cross sectional study where we reported MDA levels in mothers with PE. Power was calculated using the Power and Sample Size Calculation software (version 3.0.43) (14). For 3 controls per PE subject, we required 46 PE and 138 NC subjects to be able to reject the null hypothesis. The incidence
of PE in this population is about 8–10% of the total pregnancies. In view of this, during the period of our sample collection, 148 women for NC group were selected randomly among which 123 had complete data of all three gestational time-points with cord blood samples. In the case of PE, a total of 63 women were recruited out of which 34 women had complete data of all the gestational time-points including cord blood samples. Sample collection, processing and storing Ten milliliters of maternal venous blood was drawn into tubes containing EDTA at each gestational time points and cord blood was drawn at the time of delivery. Blood was processed as described by us earlier (23). Placental samples were obtained on a sub sample from NC and PE pregnancies right after delivery. The fetal membranes were trimmed off and small pieces were randomly cut out from the placental cotyledons from decidua plate. Tissue was rinsed in 1X phosphate-buffered saline to wash off the traces of blood. All the samples were stored at 80 C until assayed. Biochemical estimations Biochemical analysis was performed by blinding to PE classification and samples remained coded until analysis was completed. MDA levels were measured from maternal and cord plasma using the Oxis Research TM BioxytechÕ MDA-586TM Spectrophotometric Assay (Foster City, CA) and are expressed as mM/mL. Levels of GPx were measured from maternal and cord erythrocytes using Oxis Research TM BioxytechÕ GPx-340TM Colorimetric Assay for Cellular Glutathione Peroxidase (Foster City, CA) and are expressed as mU/ml. SOD levels were measured from maternal and cord erythrocytes using Cayman’s Superoxide Dismutase Assay Kit (Ann Arbor, MI). SOD activity was expressed as U/ml. GSH levels were measured in maternal and cord erythrocytes using Cayman’s Glutathione Assay Kit (Ann Arbor, MI) and was expressed as mM/mL and the method has been reported by us earlier (24). The eNOS levels were measured in placental homogenates using Quantikine Human eNOS Quantikine ELISA Kit by R&D Systems (Minneapolis, MN) and expressed as pg/ml.
Maternal oxidative stress in PE
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
DOI: 10.3109/10641963.2015.1081226
Measurement of placental mRNA levels
Table 1. Maternal and neonatal characteristics.
Isolation of total RNA followed by cDNA synthesis and estimation of mRNA levels of SOD, GPx, GAPDH genes from NC and PE placental samples were performed. Total RNA from placenta samples was isolated using Trizol method and quantified by the Eppendorf Biophotometer Plus (Hamburg, Germany) as described by us earlier (25). For each placental sample, 2 mg of RNA was reverse transcribed to cDNA using the High-Capacity cDNA reverse transcription Kit from Applied Biosystems (Foster City, CA). The qRTPCR for SOD, GPx and GAPDH were performed with the TaqMan Universal PCR Master Mix (Carlsbad, CA) using 7500 Standard Real Time PCR system from Applied Biosystems (Foster City, CA). The relative expression level of the gene of interest was examined with respect to GAPDH which has been standardized in our laboratory as housekeeping gene. Relative expression levels of genes were calculated and expressed as 2DCt. The following TaqManÕ primers were used in this study: GAPDH (Hs99999905_m1); SOD (Hs00829989_gH); GPx (Hs00166575_m1).
Maternal Characteristics
Statistical analysis SPSS/PC+ package software (Chicago, IL) was used to analyze the data (Version 20.0). Continuous variables are reported as mean ± SD and discrete variables as percent (%). Log10 transformation was used for skewed distributions. Mean values were compared at conventional levels of significance (p50.05 and p50.01) using the independent t- test and oneway analysis of variance (ANOVA). The data were also analyzed after adjusting for gestational age and socioeconomic status (SES). Association between different parameters with birth outcome and BP were done after adjusting for maternal age, BMI, gestational age and SES. In the current study, the variable sample numbers for different time-points and different parameters are due to either loss in the follow up of subjects at various time-points or insufficient sample volume available.
Results Maternal and neonatal demographic characteristics The maternal body mass index (BMI) at all gestational timepoints in women with PE were higher (p50.01 for all) as compared with the NC women. Systolic BP of women with PE were higher (p50.01 for all) at all time-points as compared with the NC women. Diastolic BP were higher at T2 and T3 (p50.01 for both) in women with PE as compared with the NC women. The percentage of nulliparous women was higher in the PE group compared with the NC group (70% vs. 45%, p50.01). Neonatal characteristics show that birth weight (p50.01), baby chest circumference (p50.05) and baby head circumference (p50.05) were lower in the PE group as compared with the NC group (Table 1). Maternal and cord MDA levels Maternal plasma MDA levels at T1 (p50.01), T2 (p50.01) and T3 (p50.05) were higher in women with PE than those in the NC women. No difference in cord
Age in years BMI (kg/m2) T1 T2 T3 Gestation (weeks) T1 T2 T3 Sys BP (mmHg) T1 T2 T3 Dias BP (mmHg) T1 T2 T3 Education (%) Lower Higher Graduation Post graduation Parity (%) Nulliparous Multiparous Neonatal Characteristics Birth weight (kg) Baby length (cm) Baby head circumference (cm) Baby chest circumference (cm)
NC (n ¼ 148)
3
PE (n ¼ 63)
26 ± 4
26 ± 8
22 ± 3 24 ± 4 25 ± 4
25 ± 5** 28 ± 6** 30 ± 6**
19 ± 2 29 ± 3 39 ± 1
18 ± 2** 30 ± 2 37 ± 3**
112 ± 8 113 ± 8 120 ± 9
117 ± 12** 122 ± 13** 142 ± 18**
73 ± 7 72 ± 7 78 ± 6
74 ± 8 76 ± 9** 94 ± 12**
35 12 36 18
22 14 43 21
45 55
70 30
2.9 ± 0.3 48.2 ± 2.8 33.7 ± 1.3 32.1 ± 1.6
2.8 ± 0.6** 47.4 ± 2.3 33.2 ± 1.3* 31.5 ± 2.5*
NC: Normotensive control; PE: Preeclampsia; BMI: Body mass index; Sys BP: Systolic blood pressure; Dias BP: Diastolic blood pressure; T1: 16–20 weeks of gestation; T2: 26–30 weeks of gestation; T3: At delivery; n: Number; *p50.05, **p50.01 as compared with NC; Type of analysis: Independent Student’s t-test, values given are mean ± SD.
plasma MDA levels were observed in the PE group as compared to NC group. However, after adjusting for age and SES the levels of MDA were lower at only T1 (p50.01) and T2 (p50.01) in women with PE as compared with NC (Figure 2–A1 and A2). In the NC group, the MDA levels at T3 were significantly higher (p50.01 for both) than T1 and T2. Similarly, cord levels were also higher (p50.01 for both) than T1 and T2. However, in women with PE, the levels of MDA did not vary across gestation. Maternal and cord GPx protein levels There was higher maternal erythrocyte GPx levels at T1 (p50.05) and T3 (p50.01) in women with PE as compared with NC women (Figure 2–B1). However, after adjustment for gestational age and SES, the levels of GPx were higher at only T3 (p50.05) in women with PE as compared with NC women (Figure 2–B2). In the NC group, the GPx levels were significantly lower (p50.01) at T3 than T2. Cord GPx levels were lowest (p50.01 for all) as compared to all the three time—points, i.e. T1, T2 and T3. However, in women with PE, the levels of GPx did not show any difference across gestation but was lowest (p50.01 for all) in the cord as compared to all the three time-points.
V. D’Souza et al.
Clin Exp Hypertens, Early Online: 1–8
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
4
Figure 2. Levels of MDA, SOD, GPx and GSH in maternal blood at different time-points and in cord blood in preeclampsia and normotensive control groups. T1: 16–20 weeks of gestation; T2: 26–30 weeks of gestation; T3: At delivery; C: Cord; PE: Preeclampsia; NC: Normotensive control; MDA: Malondialdehyde; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; GSH: Glutathione; (1) A1, B1, C1 and D1 are levels of MDA, SOD, GPx and GSH respectively without adjusting for gestational age and socio-economic status (SES); (2) A2, B2, C2 and D2 are levels of MDA, SOD, GPx and GSH respectively after adjusting for gestational age and SES; *p50.05; **p50.01 as compared to NC; Type of analysis: Independent Student’s t-test, values expressed as mean ± SD.
Maternal and cord SOD protein levels The levels of maternal erythrocyte SOD were lower at T2 (p50.05), T3 (p50.05) and in cord (p50.05) in the PE group as compared with NC group (Figure 2–C1). Similar to
this, after adjusting for gestational age and SES the SOD levels were lower at T2 (p50.05), T3 (p50.01) and in cord (p50.05) in PE group as compared with the NC group (Figure 2–C2). There were no longitudinal differences observed across gestation within both NC and PE groups.
Maternal oxidative stress in PE
DOI: 10.3109/10641963.2015.1081226
5
Figure 3. Placental SOD and GPx gene expressions in preeclampsia and normotensive control groups. PE: Preeclampsia; NC: Normotensive control; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; *p50.05 as compared to NC; Type of analysis: Independent Student’s t-test, values expressed as mean ± SD.
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
Maternal and cord GSH levels The maternal erythrocyte GSH levels were lower at T1 (p50.05) and T2 (p50.05) in women with PE as compared with NC women (Figure 2–D1). However, after adjusting for gestational age and SES, the GSH levels were lower only at T1 (p50.05) in women with PE as compared with NC women (Figure 2–D2). In the NC group, GSH levels showed reducing trend towards term where the levels at T3 was lower (p ¼ 0.05) than T2, although it was not statistically significant. However, in women with PE, the levels of GSH did not show any difference across gestation. Placental SOD and GPx gene expression In the PE group, placental GPx mRNA levels were lower (p50.05) as compared with NC. However, placental SOD mRNA levels were comparatively higher in PE as compared with NC, although it was not statistically significant (Figure 3). Placental eNOS protein levels Placental levels of eNOS were comparatively lower in PE as compared with NC group although it was not statistically significant (Figure 4). Association of MDA with blood pressure and birth weight Maternal MDA was positively (r ¼ 0.176, p ¼ 0.049, n ¼ 129) associated with diastolic BP at T2 in the NC group but not in the PE group. However, there were no associations of MDA with systolic BP and birth weight at any time-point. Association of SOD with blood pressure and birth weight Maternal SOD was positively (r ¼ 0.267, p ¼ 0.043, n ¼ 62) associated with diastolic BP at T1 in the PE group. However, there were no associations of SOD with systolic BP and birth weight at any time-point. Association of GPx with blood pressure and birth weight Maternal GPx was negatively (r ¼ 0.22, p ¼ 0.02, n ¼ 115) associated with diastolic BP at T2 in NC group. Maternal GPx
Figure 4. Placental levels of eNOS in preeclampsia and normotensive control groups. PE: Preeclampsia; NC: Normotensive control; eNOS: Endothelial nitric oxide synthase; Type of analysis: Independent Student’s t-test, values expressed as mean ± SD.
at T3 was negatively (r ¼ 0.195, p ¼ 0.035, n ¼ 122) associated with birth weight in the NC group while at T2 it was positively (r ¼ 0.386, p ¼ 0.026, n ¼ 37) associated with birth weight in PE group. However, there were no associations of GPx with systolic BP at any time-point.
Discussion This study demonstrates several important findings after adjusting for SES and gestation which are as follows. In the PE group the following changes were observed as compared to NC group, (a) higher plasma MDA levels at T1 and T2, (b) higher erythrocyte GPx levels at T3, (c) lower erythrocyte SOD levels at T2, T3 and in cord, (d) lower erythrocyte GSH levels at T1 and (e) lower expression of GPx in the placenta. Further, in the PE group there was a positive association of maternal SOD with diastolic BP at T1. In addition, in the NC group, there was a positive association between MDA and diastolic BP but a negative association between GPx and diastolic BP at T2. We also observed an association between GPx of T3 with birth weight at T3 in NC. In the current study, maternal plasma MDA levels were higher in women with PE at T1, and T2 as compared to NC. MDA is a byproduct of lipid peroxidation which has been shown to induce endothelial dysfunction, damage cell membranes directly and reduce endothelium-dependent vasorelaxation (28). It has been suggested that higher MDA levels
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
6
V. D’Souza et al.
parallel the severity of PE (29).MDA levels have been reported higher during 20–23 weeks of gestation in PE women (26,20).Further, studies have also shown higher MDA levels in serum, plasma and placental tissue samples in women with PE at delivery (30,31,32). Similar result was observed in our study when data was not adjusted for gestational age and SES. In the NC group, MDA levels increased at term and in cord blood. MDA level in umbilical cord blood is suggested to serve as an indication of perinatal oxidative stress (33). However, in women with PE the levels of MDA were high right from T1 and remained similar throughout suggesting that this may be affecting the early developmental processes. Reports indicate that oxidative stress is a cause of apoptosis in feto-placental unit which predates abnormalities in the fetus (34). In the NC group, MDA levels at T2 were positively associated with diastolic BP which supports the studies indicating oxidative stress increases mid gestation possibly elevating blood pressure in normal pregnancy(35). In this study, levels of SOD are lower at T2 and T3 and also in cord in PE as compared to control. SOD is the first barrier and antioxidant defense against ROS (36). Lower levels of SOD in women who develop PE has been previously reported at 10–14 and 20–24 weeks of gestation (17). Reports from cross-sectional studies also indicate lower maternal and cord SOD levels in the case of PE (37,38). Further, we also observed a positive association between maternal SOD and diastolic BP at T1 in the PE group. SOD is responsible for the production of H2O2 which if not converted to H2O results into the production of harmful radicals (39). It is reported that accumulation of H2O2 in kidney results in sustained hypertension and arterial BP (40). Treatment with SOD mimetic has been reported to reduce BP in rat models (41). In this study maternal erythrocyte GPx levels were higher at delivery in women with PE as compared with NC. There are several studies which report higher GPx levels in mothers with PE at term (42,43,44). In contrast, lower maternal serum levels of GPx have also been reported (37). GPx is directly involved in the elimination of ROS to inhibit lipid peroxidation especially in the membrane phospholipids. The elevated levels may be an adaptive response to elevated levels of oxidative stress in later stages of PE. The current study demonstrates reduced maternal GSH levels at T1 in PE. Developing embryos are sensitive to oxidative stress and mitochondria are known to provide ATP for GSH production to participate in the regeneration of NADPH (2). Reports indicate that glutathione and glutathione-related enzymes are involved in the metabolism and detoxification of cytotoxic compounds (45). However, no change in placental or serum GSH levels in women with PE has been reported (46). A study in animals reports that reduced GSH causes significant increase in BP (47). In the NC group, the GPx levels and GSH reduced at term. This supports other studies which report lower antioxidants activity at term (48,49). GPx levels were negatively while MDA levels were positively associated with BP at T2 in NC group which indicates that the level of oxidative stress during this period of pregnancy affects endothelial functioning of the mothers.
Clin Exp Hypertens, Early Online: 1–8
In this study, there was a lower expression of GPx in the placenta in PE. This is in accordance with other studies that show highly significant reductions in expression of GPx in placenta of women with PE (50,51). The placenta is known to synthesize extracellular glutathione peroxidase (27). Reduced expression in the placenta and increased levels in the maternal blood observed in this study may be a compensatory mechanism by the placenta in PE. In our study, the levels of eNOS were comparatively lower in PE than NC group but it was not statistically significant possibly due to small sample size. Our findings support earlier studies which report lower placental tissue of nitric oxide synthase in PE (52,53). Reduced placental nitric oxide synthesis by eNOS may adversely affect hemodynamic reducing the blood flow in PE placenta (52). This study reports results before and after adjusting for SES and gestation as confounders and suggests the need to identify confounders and use appropriate statistical tools while analyzing the data. Most of the studies reported in literature have not defined confounders while comparing means of the MDA and antioxidants. One limitation of this study is that we could not collect blood samples earlier than 16–20 weeks of gestation. In conclusion, women with PE demonstrate increased oxidative stress right from 16 to 20 weeks of gestation which leads to impaired fetal development. It is likely that the antioxidant enzymes are unable to cope with the increased oxidative stress levels in women with PE right from early pregnancy. This study contributes to a better understanding of the consequences of this disorder for the mother and the fetus. We have earlier demonstrated and discussed that increased levels of homocysteine is a consequence of altered one carbon cycle leading to increase in oxidative stress (54,55). It is likely that there may be epigenetic changes at the gene specific level for genes transcribing to antioxidant enzymes which can be studied further. Studies in our laboratory are currently focusing on the above mechanisms to explain the increased oxidative stress in women with PE. These changes can help explain the link between altered placental development and fetal programming of non-communicable diseases.
Acknowledgements The authors thank all the subjects who volunteered in this study and nurses of Bharati and Gupte Hospitals who helped in collection of the samples for this study.
Declaration of interest We declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding This research work was partially funded by Department of Biotechnology, Government of India.
References 1. Dennery PA. Oxidative stress in development: nature or nurture? Free Radical Biol Med 2010;49:1147–51.
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
DOI: 10.3109/10641963.2015.1081226
2. Dumollard R, Carroll J, Duchen MR, et al. Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol 2009; 20:346–53. 3. 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;42:1634–50. 4. Golbidi S, Laher I. Antioxidant therapy in human endocrine disorders. Med Sci Monitor 2010; 16:RA9–24. 5. Finkel T. Redox-dependent signal transduction. FEBS Lett 2000; 476:52–4. 6. Del Rio D, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis 2005;15:316–28. 7. Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Investigation 1997;100:2153–7. 8. Wilcox CS. Reactive oxygen species: roles in blood pressure and kidney function. Curr Hypertens Rep 2002;4:160–6. 9. Martinez-Varea A, Pellicer B, Perales-Marin A, Pellicer A. Relationship between maternal immunological response during pregnancy and onset of preeclampsia. J Immunol Res 2014;2014: 210241. 10. Poston L, Igosheva N, Mistry HD, et al. Role of oxidative stress and antioxidant supplementation in pregnancy disorders. Am J Clin Nutr 2011;94:1980S–5S. 11. Wu CS, Nohr EA, Bech BH, et al. Health of children born to mothers who had preeclampsia: a population-based cohort study. Am J Obstet Gynecol 2009;201:e261–9. 12. Dangat KD, Mehendale SS, Yadav HR, et al. Long-chain polyunsaturated fatty acid composition of breast milk in pre-eclamptic mothers. Neonatology 2010;97:190–4. 13. Kulkarni AV, Mehendale SS, Yadav HR, et al. Circulating angiogenic factors and their association with birth outcomes in preeclampsia. Hypertens Res 2010;33:561–7. 14. Mehendale S, Kilari A, Dangat K, et al. Fatty acids, antioxidants, and oxidative stress in pre-eclampsia. Int J Gynaecol Obstet 2008; 100:234–8. 15. Roy S, Sable P, Khaire A, et al. Effect of maternal micronutrients (folic acid and vitamin B) and omega 3 fatty acids on indices of brain oxidative stress in the offspring. Brain Dev 2013;36:219–27. 16. Atiba AS, Abbiyesuku FM, Adekanle DA, et al. Malondialdehyde and antioxidant enzymes in second and third trimesters of preeclamptic Nigerian women. Nigerian Postgrad Med J 2014;21: 150–4. 17. Genc H, Uzun H, Benian A, et al. Evaluation of oxidative stress markers in first trimester for assessment of preeclampsia risk. Arch Gynecol Obstet 2011;284:1367–73. 18. Loverro G, Greco P, Capuano F, et al. Lipoperoxidation and antioxidant enzymes activity in pregnancy complicated with hypertension. Eur J Obstet Gynecolo Reprod Biol 1996;70: 123–7. 19. Medrano Rodrı´guez JC, Mendoza PY, Bernal MP, Alvarado Acosta JL. Grado de estre´s oxidativo y cambios histolo´gicos placentarios durante la preeclampsia. Ginecol Obstet Mexico 2008;76:319–26. 20. Rudra CB, Qiu C, David RM, et al. A prospective study of earlypregnancy plasma malondialdehyde concentration and risk of preeclampsia. Clin Biochem 2006;39:722–6. 21. Min J, Park B, Kim YJ, et al. Effect of oxidative stress on birth sizes: consideration of window from mid pregnancy to delivery. Placenta 2009;30:418–23. 22. Sahay AS, Patil VV, Sundrani DP, et al. A longitudinal study of circulating angiogenic and antiangiogenic factors and AT1-AA levels in preeclampsia. Hypertens Res 2014;37:753–8. 23. Sundrani D, Khot V, Pisal H, et al. Gestation dependant changes in angiogenic factors and their associations with fetal growth measures in normotensive pregnancy. PloS One 2013;8:e54153. 24. Roy S, Dhobale M, Dangat K, et al. Differential oxidative stress levels in mothers with preeclampsia delivering male and female babies. J Maternal-Fetal Neonatal Med 2015;28:1973–80. 25. Sundrani DP, Reddy US, Joshi AA, et al. Differential placental methylation and expression of VEGF, FLT-1 and KDR genes in human term and preterm preeclampsia. Clin Epigenetics 2013;5:6. 26. Anastasakis E, Papantoniou N, Daskalakis G, et al. Screening for pre-eclampsia by oxidative stress markers and uteroplacental blood flow. J Obstet Gynaecol 2008;28:285–9.
Maternal oxidative stress in PE
7
27. Avissar N, Eisenmann C, Breen JG, et al. Human placenta makes extracellular glutathione peroxidase and secretes it into maternal circulation, Am J Physiol. 1994;267:E68–76. 28. Molyneux CA, Glyn MC, Ward BJ. Oxidative stress and cardiac microvascular structure in ischemia and reperfusion: the protective effect of antioxidant vitamins. Microvasc Res 2002;64: 265–77. 29. Wiktor H and Kankofer M. [Assessment of lipid peroxidation intensification in normal and preeclamptic placentas]. Ginekologia Polska 2001;72:1217–21. 30. Can M, Guven B, Bektas S, Arikan I. Oxidative stress and apoptosis in preeclampsia. Tissue Cell 2014;46:477–81. 31. Siddiqui IA, Jaleel A, Al’Kadri HM, et al. Biomarkers of oxidative stress in women with pre-eclampsia. Biomarkers Med 2013;7: 229–34. 32. Suhail M, Suhail S, Gupta BK, Bharat V. Malondialdehyde and antioxidant enzymes in maternal and cord blood, and their correlation in normotensive and preeclamptic women. J Clin Med Res 2009;1:150–7. 33. Gulbayzar S, Arica V, Hatipoglu S, et al. Malondialdehyde level in the cord blood of newborn infants. Iranian J Pediat 2011;21: 313–19. 34. Nakatsukasa Y, Tsukahara H, Tabuchi K, et al. Thioredoxin-1 and oxidative stress status in pregnant women at early third trimester of pregnancy: relation to maternal and neonatal characteristics. J Clin Biochem Nutr 2013;52:27–31. 35. Casanueva E, Viteri FE. Iron and oxidative stress in pregnancy. J Nutr 2003;133:1700S–8S. 36. Surh Y-J. Oxidative stress, inflammation, and health. Boca Raton, FL: Taylor & Francis, 2005. 37. Negi R, Pande D, Karki K, et al. Trace elements and antioxidant enzymes associated with oxidative stress in the pre-eclamptic/ eclamptic mothers during fetal circulation. Clin Nutr 2012;31: 946–50. 38. Patil SB, Kodliwadmath MV, Kodliwadmath M. Lipid peroxidation and antioxidant activity in complicated pregnancies. Clin Exp Obstet Gynecol 2009;36:110–12. 39. Ray G, Husain SA. Oxidants, antioxidants and carcinogenesis. Indian J Exp Biol 2002;40:1213–32. 40. Makino A, Skelton MM, Zou AP, Cowley Jr AW,. Increased renal medullary H2O2 leads to hypertension. Hypertension 2003;42: 25–30. 41. Rodriguez-Iturbe B, Zhan CD, Quiroz Y, et al. Antioxidant-rich diet relieves hypertension and reduces renal immune infiltration in spontaneously hypertensive rats. Hypertension 2003;41: 341–6. 42. Krishna Mohan S, Venkataramana G. Status of lipid peroxidation, glutathione, ascorbic acid, vitamin E and antioxidant enzymes in patients with pregnancy-induced hypertension. Indian J Physiol Pharmacol 2007;51:284–8. 43. Llurba E, Grataco´s E, Martı´n-Galla´n P, et al. A comprehensive study of oxidative stress and antioxidant status in preeclampsia and normal pregnancy. Free Radical Biol Med 2004;37:557–70. 44. Sharma JB, Sharma A, Bahadur A, et al. Oxidative stress markers and antioxidant levels in normal pregnancy and pre-eclampsia. Int J Gynecol Obstet 2006;94:23–7. 45. Knapen MF, Zusterzeel PL, Peters WH, Steegers EA. Glutathione and glutathione-related enzymes in reproduction: a review. Eur J Obstet Gynecol Reprod Biol 1999;82: 171–84. 46. Das B, Saha-Roy S, Das Gupta A, et al. Assessment of placental oxidative stress in pre-eclampsia. J Obstet Gynaecol India 2012;62: 39–42. 47. Vaziri ND, Wang XQ, Oveisi F, Rad B. Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats. Hypertension 2000;36:142–6. 48. Davidge ST, Hubel CA, Brayden RD, et al. Sera antioxidant activity in uncomplicated and preeclamptic pregnancies. Obstet Gynecol 1992;79:897–901. 49. Patil SB, Kodliwadmath M, Kodliwadmath SM. Study of oxidative stress and enzymatic antioxidants in normal pregnancy. Indian J Clin Biochem 2007;22:135–7. 50. Mistry HD, Kurlak LO, Williams PJ, et al. Differential expression and distribution of placental glutathione peroxidases 1, 3
8
V. D’Souza et al.
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
and 4 in normal and preeclamptic pregnancy. Placenta 2010;31: 401–8. 51. Roland-Zejly L, Moisan V, St-Pierre I, Bilodeau JF. Altered placental glutathione peroxidase mRNA expression in preeclampsia according to the presence or absence of labor. Placenta 2011;32: 161–7. 52. Brennecke SP, Gude NM, Di Iulio JL, King RG. Reduction of placental nitric oxide synthase activity in pre-eclampsia. Clin Sci 1997; 93:51–5.
Clin Exp Hypertens, Early Online: 1–8
53. Morris NH, Sooranna SR, Learmont JG, et al. Nitric oxide synthase activities in placental tissue from normotensive, pre-eclamptic and growth retarded pregnancies. BJOG 1995;102:711–14. 54. D’Souza V, Chavan-Gautam P, Joshi S. Counteracting oxidative stress in pregnancy through modulation of maternal micronutrients and omega-3 fatty acids. Curr Med Chem 2013;20:4777–83. 55. Kulkarni A, Mehendale S, Pisal H, et al. Association of omega-3 fatty acids and homocysteine concentrations in pre-eclampsia. Clin Nutr 2011;30:60–4.
ISSN: 1064-1963 (Print) 1525-6006 (Online) Journal homepage: http://www.tandfonline.com/loi/iceh20
Increased oxidative stress from early pregnancy in women who develop preeclampsia Vandita D’Souza, Alka Rani, Vidya Patil, Hemlata Pisal, Karuna Randhir, Savita Mehendale, Girija Wagh, Sanjay Gupte & Sadhana Joshi To cite this article: Vandita D’Souza, Alka Rani, Vidya Patil, Hemlata Pisal, Karuna Randhir, Savita Mehendale, Girija Wagh, Sanjay Gupte & Sadhana Joshi (2016): Increased oxidative stress from early pregnancy in women who develop preeclampsia, Clinical and Experimental Hypertension, DOI: 10.3109/10641963.2015.1081226 To link to this article: http://dx.doi.org/10.3109/10641963.2015.1081226
Published online: 28 Jan 2016.
Submit your article to this journal
Article views: 9
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iceh20 Download by: [University of California, San Diego]
Date: 01 February 2016, At: 13:34
http://tandfonline.com/iceh ISSN: 1064-1963 (print), 1525-6006 (electronic) Clin Exp Hypertens, Early Online: 1–8 ! 2016 Taylor & Francis Group, LLC. DOI: 10.3109/10641963.2015.1081226
Increased oxidative stress from early pregnancy in women who develop preeclampsia Vandita D’Souza1, Alka Rani1, Vidya Patil1, Hemlata Pisal1, Karuna Randhir1, Savita Mehendale2, Girija Wagh2, Sanjay Gupte3, and Sadhana Joshi1 1
Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra, India, Department of Obstetrics and Gynaecology, Bharati Medical College and Hospital, Bharati Vidyapeeth University, Pune, Maharashtra, India, and 3 Department of Obstetrics and Gynaecology, Gupte Hospital and Research Center, Pune, Maharashtra, India
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
2
Abstract
Keywords
Preeclampsia (PE) is a pregnancy-specific disorder, defined as new onset of maternal hypertension and proteinuria after 20 weeks of gestation. Our earlier study has shown increased maternal oxidative stress at delivery to be associated with poor birth outcome in PE. However, these results were observed when the pathology had progressed and may have been secondary to the effects of the disorder. To understand the role of antioxidant defense mechanisms in PE right from early pregnancy, in this prospective study, we measured malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH) concentrations in maternal blood at 3 time-points of gestation [16–20 weeks (T1), 26–30 weeks (T2), at delivery (T3)] and in cord blood. Gene expression of SOD and GPx and protein levels of endothelial nitric oxide synthase (eNOS) enzyme were also analyzed in the placenta. MDA levels were higher at T1 (p50.01) and T2 (p50.01) in women with PE as compared with control. GPx levels were higher at T3 (p50.05) while SOD levels were lower at T2 (p50.05), T3 (p50.01) and in cord (p50.01) in PE. GSH levels at T1 (p50.05) and expression of GPx in the placenta were lower in PE as compared with control. In conclusion, this study demonstrates that women who develop PE exhibit increased oxidative stress right from 16 to 20 weeks of gestation. This may alter placental development and lead to fetal programming of adult non-communicable disease in the offspring.
Antioxidants, early pregnancy, longitudinal study, oxidative stress, placenta, preeclampsia
Introduction It is well established that a certain amount of oxidative stress during pregnancy is necessary for embryonic and fetal growth (1). Oxidative stress is induced by reactive oxygen species (ROS) including superoxide anion (O 2 ), hydroxyl anion (OH) and hydrogen peroxide (H2O2) (2). A balance between ROS and antioxidants is necessary for fertilization, embryogenesis, embryonic implantation and placental growth and differentiation (3). However, excessive production of ROS damages the cell structures including lipids, proteins and DNA (4). An internal protection system comprised of both enzymatic and non-enzymatic antioxidant defenses such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH) are known to be helpful in combating excess ROS (5). Free ROS degrades polyunsaturated lipids to form malondialdehyde (MDA) which is used as a biomarker to measure the level of oxidative stress in an organism (6).
Correspondence: Dr. Sadhana Joshi, Scientist ‘G’ and Head, Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth Deemed University, Pune 411043, Maharashtra, India. Tel: 91-20-24366929/31. Fax: 91-20-24366931. E-mail: [email protected]
History Received 28 May 2015 Revised 23 July 2015 Accepted 27 July 2015 Published online 11 January 2016
In addition, there also exists an association between ROS and the enzyme endothelial nitric oxide synthase (eNOS) which produces nitric oxide in endothelial cells. Nitric oxide (NO) is a potent vasodilator and helps in maintaining vascular health and blood pressure (BP) (7) (Figure 1). Several studies have illustrated the effects of ROS on kidney function and BP in hypertensive animal models. ROS enhances vasoconstriction by reducing endothelium-dependent relaxation factor/NO (8). Preeclampsia (PE) is a pregnancy complication characterized by hypertension and proteinuria and has extensively been shown to be associated with elevated oxidative stress (9,10). Further, children born to mothers with PE are known to be at increased risk for endocrine, nutritional, and metabolic derangements during adolescence and early-adulthood (11). We have earlier reported an imbalance between oxidative stress and antioxidants in women with PE at delivery (12,13,14). However, these results were observed when the pathology had progressed and may have been secondary to the effects of the disorder. In addition to this, our earlier study in rats suggests that the antioxidant defense system of the brain may be programmed during pregnancy leading to impaired cognition in the offspring in later life (15). With this background, we hypothesize that an abnormal antioxidant
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
2
V. D’Souza et al.
Clin Exp Hypertens, Early Online: 1–8
Figure 1. Mechanism of oxidative stress leading to preeclampsia. SOD: Superoxide dismutase; GPx: Glutathione peroxidase; CAT: Catalase; GSH: Oxidised glutathione; GSSG: Reduced glutathione; H2O2: Hydrogen peroxide; H2O: Water; O2: Oxygen; O 2 : Superoxide anion; OH : hydroxyl ion; eNOS: Endothelial nitric oxide synthase; NO: Nitric oxide; ONOO: Peroxynitrite.
defense mechanism exists from early pregnancy in women who develop PE. There are few longitudinal studies which have examined changes in the oxidative stress parameters across gestation in women with PE. However, these studies are either on a small sample size, varying ethnic population or included women who smoke, all of which may confound the levels of oxidative stress (16,17,18,19,20). Further, there are limited studies associating the levels of maternal oxidative stress with birth outcome (21). Hence, this study was designed with an appropriate power and the data was analyzed after adjusting for potential confounders. This prospective study reports MDA, SOD, GPx and GSH levels at different gestational time-points in women who developed PE and in cord as compared with normotensive control (NC). The association of these biochemical markers with birth weight and maternal systolic and diastolic BP are also reported.
Materials and methods Study design Pregnant women were enrolled for this longitudinal and comparative study from two major hospitals, Bharati Hospital and Gupte Hospital in Pune, MH, India. Bharati Vidyapeeth Medical College Institutional Ethical Committee approved the research protocols and consent forms. This study is a part of a large ongoing departmental study which recruits all consecutive pregnant women at 16–20 weeks of gestation who are willing to participate and give a written consent. The detailed study design and recruitment criteria have been described by us earlier (22). Briefly, blood samples from pregnant women were collected at three different time-points across gestation, i.e. 16–20 weeks of gestation (T1), 26–30 weeks of gestation (T2) and at delivery (T3). All women were followed until delivery and categorized as PE if there was a presence of proteinuria (1 + protein on a dipstick test) and high BP (systolic BP4140 Hg and/or diastolic BP490 Hg). We calculated the power of the study based on our earlier cross sectional study where we reported MDA levels in mothers with PE. Power was calculated using the Power and Sample Size Calculation software (version 3.0.43) (14). For 3 controls per PE subject, we required 46 PE and 138 NC subjects to be able to reject the null hypothesis. The incidence
of PE in this population is about 8–10% of the total pregnancies. In view of this, during the period of our sample collection, 148 women for NC group were selected randomly among which 123 had complete data of all three gestational time-points with cord blood samples. In the case of PE, a total of 63 women were recruited out of which 34 women had complete data of all the gestational time-points including cord blood samples. Sample collection, processing and storing Ten milliliters of maternal venous blood was drawn into tubes containing EDTA at each gestational time points and cord blood was drawn at the time of delivery. Blood was processed as described by us earlier (23). Placental samples were obtained on a sub sample from NC and PE pregnancies right after delivery. The fetal membranes were trimmed off and small pieces were randomly cut out from the placental cotyledons from decidua plate. Tissue was rinsed in 1X phosphate-buffered saline to wash off the traces of blood. All the samples were stored at 80 C until assayed. Biochemical estimations Biochemical analysis was performed by blinding to PE classification and samples remained coded until analysis was completed. MDA levels were measured from maternal and cord plasma using the Oxis Research TM BioxytechÕ MDA-586TM Spectrophotometric Assay (Foster City, CA) and are expressed as mM/mL. Levels of GPx were measured from maternal and cord erythrocytes using Oxis Research TM BioxytechÕ GPx-340TM Colorimetric Assay for Cellular Glutathione Peroxidase (Foster City, CA) and are expressed as mU/ml. SOD levels were measured from maternal and cord erythrocytes using Cayman’s Superoxide Dismutase Assay Kit (Ann Arbor, MI). SOD activity was expressed as U/ml. GSH levels were measured in maternal and cord erythrocytes using Cayman’s Glutathione Assay Kit (Ann Arbor, MI) and was expressed as mM/mL and the method has been reported by us earlier (24). The eNOS levels were measured in placental homogenates using Quantikine Human eNOS Quantikine ELISA Kit by R&D Systems (Minneapolis, MN) and expressed as pg/ml.
Maternal oxidative stress in PE
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
DOI: 10.3109/10641963.2015.1081226
Measurement of placental mRNA levels
Table 1. Maternal and neonatal characteristics.
Isolation of total RNA followed by cDNA synthesis and estimation of mRNA levels of SOD, GPx, GAPDH genes from NC and PE placental samples were performed. Total RNA from placenta samples was isolated using Trizol method and quantified by the Eppendorf Biophotometer Plus (Hamburg, Germany) as described by us earlier (25). For each placental sample, 2 mg of RNA was reverse transcribed to cDNA using the High-Capacity cDNA reverse transcription Kit from Applied Biosystems (Foster City, CA). The qRTPCR for SOD, GPx and GAPDH were performed with the TaqMan Universal PCR Master Mix (Carlsbad, CA) using 7500 Standard Real Time PCR system from Applied Biosystems (Foster City, CA). The relative expression level of the gene of interest was examined with respect to GAPDH which has been standardized in our laboratory as housekeeping gene. Relative expression levels of genes were calculated and expressed as 2DCt. The following TaqManÕ primers were used in this study: GAPDH (Hs99999905_m1); SOD (Hs00829989_gH); GPx (Hs00166575_m1).
Maternal Characteristics
Statistical analysis SPSS/PC+ package software (Chicago, IL) was used to analyze the data (Version 20.0). Continuous variables are reported as mean ± SD and discrete variables as percent (%). Log10 transformation was used for skewed distributions. Mean values were compared at conventional levels of significance (p50.05 and p50.01) using the independent t- test and oneway analysis of variance (ANOVA). The data were also analyzed after adjusting for gestational age and socioeconomic status (SES). Association between different parameters with birth outcome and BP were done after adjusting for maternal age, BMI, gestational age and SES. In the current study, the variable sample numbers for different time-points and different parameters are due to either loss in the follow up of subjects at various time-points or insufficient sample volume available.
Results Maternal and neonatal demographic characteristics The maternal body mass index (BMI) at all gestational timepoints in women with PE were higher (p50.01 for all) as compared with the NC women. Systolic BP of women with PE were higher (p50.01 for all) at all time-points as compared with the NC women. Diastolic BP were higher at T2 and T3 (p50.01 for both) in women with PE as compared with the NC women. The percentage of nulliparous women was higher in the PE group compared with the NC group (70% vs. 45%, p50.01). Neonatal characteristics show that birth weight (p50.01), baby chest circumference (p50.05) and baby head circumference (p50.05) were lower in the PE group as compared with the NC group (Table 1). Maternal and cord MDA levels Maternal plasma MDA levels at T1 (p50.01), T2 (p50.01) and T3 (p50.05) were higher in women with PE than those in the NC women. No difference in cord
Age in years BMI (kg/m2) T1 T2 T3 Gestation (weeks) T1 T2 T3 Sys BP (mmHg) T1 T2 T3 Dias BP (mmHg) T1 T2 T3 Education (%) Lower Higher Graduation Post graduation Parity (%) Nulliparous Multiparous Neonatal Characteristics Birth weight (kg) Baby length (cm) Baby head circumference (cm) Baby chest circumference (cm)
NC (n ¼ 148)
3
PE (n ¼ 63)
26 ± 4
26 ± 8
22 ± 3 24 ± 4 25 ± 4
25 ± 5** 28 ± 6** 30 ± 6**
19 ± 2 29 ± 3 39 ± 1
18 ± 2** 30 ± 2 37 ± 3**
112 ± 8 113 ± 8 120 ± 9
117 ± 12** 122 ± 13** 142 ± 18**
73 ± 7 72 ± 7 78 ± 6
74 ± 8 76 ± 9** 94 ± 12**
35 12 36 18
22 14 43 21
45 55
70 30
2.9 ± 0.3 48.2 ± 2.8 33.7 ± 1.3 32.1 ± 1.6
2.8 ± 0.6** 47.4 ± 2.3 33.2 ± 1.3* 31.5 ± 2.5*
NC: Normotensive control; PE: Preeclampsia; BMI: Body mass index; Sys BP: Systolic blood pressure; Dias BP: Diastolic blood pressure; T1: 16–20 weeks of gestation; T2: 26–30 weeks of gestation; T3: At delivery; n: Number; *p50.05, **p50.01 as compared with NC; Type of analysis: Independent Student’s t-test, values given are mean ± SD.
plasma MDA levels were observed in the PE group as compared to NC group. However, after adjusting for age and SES the levels of MDA were lower at only T1 (p50.01) and T2 (p50.01) in women with PE as compared with NC (Figure 2–A1 and A2). In the NC group, the MDA levels at T3 were significantly higher (p50.01 for both) than T1 and T2. Similarly, cord levels were also higher (p50.01 for both) than T1 and T2. However, in women with PE, the levels of MDA did not vary across gestation. Maternal and cord GPx protein levels There was higher maternal erythrocyte GPx levels at T1 (p50.05) and T3 (p50.01) in women with PE as compared with NC women (Figure 2–B1). However, after adjustment for gestational age and SES, the levels of GPx were higher at only T3 (p50.05) in women with PE as compared with NC women (Figure 2–B2). In the NC group, the GPx levels were significantly lower (p50.01) at T3 than T2. Cord GPx levels were lowest (p50.01 for all) as compared to all the three time—points, i.e. T1, T2 and T3. However, in women with PE, the levels of GPx did not show any difference across gestation but was lowest (p50.01 for all) in the cord as compared to all the three time-points.
V. D’Souza et al.
Clin Exp Hypertens, Early Online: 1–8
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
4
Figure 2. Levels of MDA, SOD, GPx and GSH in maternal blood at different time-points and in cord blood in preeclampsia and normotensive control groups. T1: 16–20 weeks of gestation; T2: 26–30 weeks of gestation; T3: At delivery; C: Cord; PE: Preeclampsia; NC: Normotensive control; MDA: Malondialdehyde; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; GSH: Glutathione; (1) A1, B1, C1 and D1 are levels of MDA, SOD, GPx and GSH respectively without adjusting for gestational age and socio-economic status (SES); (2) A2, B2, C2 and D2 are levels of MDA, SOD, GPx and GSH respectively after adjusting for gestational age and SES; *p50.05; **p50.01 as compared to NC; Type of analysis: Independent Student’s t-test, values expressed as mean ± SD.
Maternal and cord SOD protein levels The levels of maternal erythrocyte SOD were lower at T2 (p50.05), T3 (p50.05) and in cord (p50.05) in the PE group as compared with NC group (Figure 2–C1). Similar to
this, after adjusting for gestational age and SES the SOD levels were lower at T2 (p50.05), T3 (p50.01) and in cord (p50.05) in PE group as compared with the NC group (Figure 2–C2). There were no longitudinal differences observed across gestation within both NC and PE groups.
Maternal oxidative stress in PE
DOI: 10.3109/10641963.2015.1081226
5
Figure 3. Placental SOD and GPx gene expressions in preeclampsia and normotensive control groups. PE: Preeclampsia; NC: Normotensive control; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; *p50.05 as compared to NC; Type of analysis: Independent Student’s t-test, values expressed as mean ± SD.
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
Maternal and cord GSH levels The maternal erythrocyte GSH levels were lower at T1 (p50.05) and T2 (p50.05) in women with PE as compared with NC women (Figure 2–D1). However, after adjusting for gestational age and SES, the GSH levels were lower only at T1 (p50.05) in women with PE as compared with NC women (Figure 2–D2). In the NC group, GSH levels showed reducing trend towards term where the levels at T3 was lower (p ¼ 0.05) than T2, although it was not statistically significant. However, in women with PE, the levels of GSH did not show any difference across gestation. Placental SOD and GPx gene expression In the PE group, placental GPx mRNA levels were lower (p50.05) as compared with NC. However, placental SOD mRNA levels were comparatively higher in PE as compared with NC, although it was not statistically significant (Figure 3). Placental eNOS protein levels Placental levels of eNOS were comparatively lower in PE as compared with NC group although it was not statistically significant (Figure 4). Association of MDA with blood pressure and birth weight Maternal MDA was positively (r ¼ 0.176, p ¼ 0.049, n ¼ 129) associated with diastolic BP at T2 in the NC group but not in the PE group. However, there were no associations of MDA with systolic BP and birth weight at any time-point. Association of SOD with blood pressure and birth weight Maternal SOD was positively (r ¼ 0.267, p ¼ 0.043, n ¼ 62) associated with diastolic BP at T1 in the PE group. However, there were no associations of SOD with systolic BP and birth weight at any time-point. Association of GPx with blood pressure and birth weight Maternal GPx was negatively (r ¼ 0.22, p ¼ 0.02, n ¼ 115) associated with diastolic BP at T2 in NC group. Maternal GPx
Figure 4. Placental levels of eNOS in preeclampsia and normotensive control groups. PE: Preeclampsia; NC: Normotensive control; eNOS: Endothelial nitric oxide synthase; Type of analysis: Independent Student’s t-test, values expressed as mean ± SD.
at T3 was negatively (r ¼ 0.195, p ¼ 0.035, n ¼ 122) associated with birth weight in the NC group while at T2 it was positively (r ¼ 0.386, p ¼ 0.026, n ¼ 37) associated with birth weight in PE group. However, there were no associations of GPx with systolic BP at any time-point.
Discussion This study demonstrates several important findings after adjusting for SES and gestation which are as follows. In the PE group the following changes were observed as compared to NC group, (a) higher plasma MDA levels at T1 and T2, (b) higher erythrocyte GPx levels at T3, (c) lower erythrocyte SOD levels at T2, T3 and in cord, (d) lower erythrocyte GSH levels at T1 and (e) lower expression of GPx in the placenta. Further, in the PE group there was a positive association of maternal SOD with diastolic BP at T1. In addition, in the NC group, there was a positive association between MDA and diastolic BP but a negative association between GPx and diastolic BP at T2. We also observed an association between GPx of T3 with birth weight at T3 in NC. In the current study, maternal plasma MDA levels were higher in women with PE at T1, and T2 as compared to NC. MDA is a byproduct of lipid peroxidation which has been shown to induce endothelial dysfunction, damage cell membranes directly and reduce endothelium-dependent vasorelaxation (28). It has been suggested that higher MDA levels
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
6
V. D’Souza et al.
parallel the severity of PE (29).MDA levels have been reported higher during 20–23 weeks of gestation in PE women (26,20).Further, studies have also shown higher MDA levels in serum, plasma and placental tissue samples in women with PE at delivery (30,31,32). Similar result was observed in our study when data was not adjusted for gestational age and SES. In the NC group, MDA levels increased at term and in cord blood. MDA level in umbilical cord blood is suggested to serve as an indication of perinatal oxidative stress (33). However, in women with PE the levels of MDA were high right from T1 and remained similar throughout suggesting that this may be affecting the early developmental processes. Reports indicate that oxidative stress is a cause of apoptosis in feto-placental unit which predates abnormalities in the fetus (34). In the NC group, MDA levels at T2 were positively associated with diastolic BP which supports the studies indicating oxidative stress increases mid gestation possibly elevating blood pressure in normal pregnancy(35). In this study, levels of SOD are lower at T2 and T3 and also in cord in PE as compared to control. SOD is the first barrier and antioxidant defense against ROS (36). Lower levels of SOD in women who develop PE has been previously reported at 10–14 and 20–24 weeks of gestation (17). Reports from cross-sectional studies also indicate lower maternal and cord SOD levels in the case of PE (37,38). Further, we also observed a positive association between maternal SOD and diastolic BP at T1 in the PE group. SOD is responsible for the production of H2O2 which if not converted to H2O results into the production of harmful radicals (39). It is reported that accumulation of H2O2 in kidney results in sustained hypertension and arterial BP (40). Treatment with SOD mimetic has been reported to reduce BP in rat models (41). In this study maternal erythrocyte GPx levels were higher at delivery in women with PE as compared with NC. There are several studies which report higher GPx levels in mothers with PE at term (42,43,44). In contrast, lower maternal serum levels of GPx have also been reported (37). GPx is directly involved in the elimination of ROS to inhibit lipid peroxidation especially in the membrane phospholipids. The elevated levels may be an adaptive response to elevated levels of oxidative stress in later stages of PE. The current study demonstrates reduced maternal GSH levels at T1 in PE. Developing embryos are sensitive to oxidative stress and mitochondria are known to provide ATP for GSH production to participate in the regeneration of NADPH (2). Reports indicate that glutathione and glutathione-related enzymes are involved in the metabolism and detoxification of cytotoxic compounds (45). However, no change in placental or serum GSH levels in women with PE has been reported (46). A study in animals reports that reduced GSH causes significant increase in BP (47). In the NC group, the GPx levels and GSH reduced at term. This supports other studies which report lower antioxidants activity at term (48,49). GPx levels were negatively while MDA levels were positively associated with BP at T2 in NC group which indicates that the level of oxidative stress during this period of pregnancy affects endothelial functioning of the mothers.
Clin Exp Hypertens, Early Online: 1–8
In this study, there was a lower expression of GPx in the placenta in PE. This is in accordance with other studies that show highly significant reductions in expression of GPx in placenta of women with PE (50,51). The placenta is known to synthesize extracellular glutathione peroxidase (27). Reduced expression in the placenta and increased levels in the maternal blood observed in this study may be a compensatory mechanism by the placenta in PE. In our study, the levels of eNOS were comparatively lower in PE than NC group but it was not statistically significant possibly due to small sample size. Our findings support earlier studies which report lower placental tissue of nitric oxide synthase in PE (52,53). Reduced placental nitric oxide synthesis by eNOS may adversely affect hemodynamic reducing the blood flow in PE placenta (52). This study reports results before and after adjusting for SES and gestation as confounders and suggests the need to identify confounders and use appropriate statistical tools while analyzing the data. Most of the studies reported in literature have not defined confounders while comparing means of the MDA and antioxidants. One limitation of this study is that we could not collect blood samples earlier than 16–20 weeks of gestation. In conclusion, women with PE demonstrate increased oxidative stress right from 16 to 20 weeks of gestation which leads to impaired fetal development. It is likely that the antioxidant enzymes are unable to cope with the increased oxidative stress levels in women with PE right from early pregnancy. This study contributes to a better understanding of the consequences of this disorder for the mother and the fetus. We have earlier demonstrated and discussed that increased levels of homocysteine is a consequence of altered one carbon cycle leading to increase in oxidative stress (54,55). It is likely that there may be epigenetic changes at the gene specific level for genes transcribing to antioxidant enzymes which can be studied further. Studies in our laboratory are currently focusing on the above mechanisms to explain the increased oxidative stress in women with PE. These changes can help explain the link between altered placental development and fetal programming of non-communicable diseases.
Acknowledgements The authors thank all the subjects who volunteered in this study and nurses of Bharati and Gupte Hospitals who helped in collection of the samples for this study.
Declaration of interest We declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding This research work was partially funded by Department of Biotechnology, Government of India.
References 1. Dennery PA. Oxidative stress in development: nature or nurture? Free Radical Biol Med 2010;49:1147–51.
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
DOI: 10.3109/10641963.2015.1081226
2. Dumollard R, Carroll J, Duchen MR, et al. Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol 2009; 20:346–53. 3. 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;42:1634–50. 4. Golbidi S, Laher I. Antioxidant therapy in human endocrine disorders. Med Sci Monitor 2010; 16:RA9–24. 5. Finkel T. Redox-dependent signal transduction. FEBS Lett 2000; 476:52–4. 6. Del Rio D, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis 2005;15:316–28. 7. Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Investigation 1997;100:2153–7. 8. Wilcox CS. Reactive oxygen species: roles in blood pressure and kidney function. Curr Hypertens Rep 2002;4:160–6. 9. Martinez-Varea A, Pellicer B, Perales-Marin A, Pellicer A. Relationship between maternal immunological response during pregnancy and onset of preeclampsia. J Immunol Res 2014;2014: 210241. 10. Poston L, Igosheva N, Mistry HD, et al. Role of oxidative stress and antioxidant supplementation in pregnancy disorders. Am J Clin Nutr 2011;94:1980S–5S. 11. Wu CS, Nohr EA, Bech BH, et al. Health of children born to mothers who had preeclampsia: a population-based cohort study. Am J Obstet Gynecol 2009;201:e261–9. 12. Dangat KD, Mehendale SS, Yadav HR, et al. Long-chain polyunsaturated fatty acid composition of breast milk in pre-eclamptic mothers. Neonatology 2010;97:190–4. 13. Kulkarni AV, Mehendale SS, Yadav HR, et al. Circulating angiogenic factors and their association with birth outcomes in preeclampsia. Hypertens Res 2010;33:561–7. 14. Mehendale S, Kilari A, Dangat K, et al. Fatty acids, antioxidants, and oxidative stress in pre-eclampsia. Int J Gynaecol Obstet 2008; 100:234–8. 15. Roy S, Sable P, Khaire A, et al. Effect of maternal micronutrients (folic acid and vitamin B) and omega 3 fatty acids on indices of brain oxidative stress in the offspring. Brain Dev 2013;36:219–27. 16. Atiba AS, Abbiyesuku FM, Adekanle DA, et al. Malondialdehyde and antioxidant enzymes in second and third trimesters of preeclamptic Nigerian women. Nigerian Postgrad Med J 2014;21: 150–4. 17. Genc H, Uzun H, Benian A, et al. Evaluation of oxidative stress markers in first trimester for assessment of preeclampsia risk. Arch Gynecol Obstet 2011;284:1367–73. 18. Loverro G, Greco P, Capuano F, et al. Lipoperoxidation and antioxidant enzymes activity in pregnancy complicated with hypertension. Eur J Obstet Gynecolo Reprod Biol 1996;70: 123–7. 19. Medrano Rodrı´guez JC, Mendoza PY, Bernal MP, Alvarado Acosta JL. Grado de estre´s oxidativo y cambios histolo´gicos placentarios durante la preeclampsia. Ginecol Obstet Mexico 2008;76:319–26. 20. Rudra CB, Qiu C, David RM, et al. A prospective study of earlypregnancy plasma malondialdehyde concentration and risk of preeclampsia. Clin Biochem 2006;39:722–6. 21. Min J, Park B, Kim YJ, et al. Effect of oxidative stress on birth sizes: consideration of window from mid pregnancy to delivery. Placenta 2009;30:418–23. 22. Sahay AS, Patil VV, Sundrani DP, et al. A longitudinal study of circulating angiogenic and antiangiogenic factors and AT1-AA levels in preeclampsia. Hypertens Res 2014;37:753–8. 23. Sundrani D, Khot V, Pisal H, et al. Gestation dependant changes in angiogenic factors and their associations with fetal growth measures in normotensive pregnancy. PloS One 2013;8:e54153. 24. Roy S, Dhobale M, Dangat K, et al. Differential oxidative stress levels in mothers with preeclampsia delivering male and female babies. J Maternal-Fetal Neonatal Med 2015;28:1973–80. 25. Sundrani DP, Reddy US, Joshi AA, et al. Differential placental methylation and expression of VEGF, FLT-1 and KDR genes in human term and preterm preeclampsia. Clin Epigenetics 2013;5:6. 26. Anastasakis E, Papantoniou N, Daskalakis G, et al. Screening for pre-eclampsia by oxidative stress markers and uteroplacental blood flow. J Obstet Gynaecol 2008;28:285–9.
Maternal oxidative stress in PE
7
27. Avissar N, Eisenmann C, Breen JG, et al. Human placenta makes extracellular glutathione peroxidase and secretes it into maternal circulation, Am J Physiol. 1994;267:E68–76. 28. Molyneux CA, Glyn MC, Ward BJ. Oxidative stress and cardiac microvascular structure in ischemia and reperfusion: the protective effect of antioxidant vitamins. Microvasc Res 2002;64: 265–77. 29. Wiktor H and Kankofer M. [Assessment of lipid peroxidation intensification in normal and preeclamptic placentas]. Ginekologia Polska 2001;72:1217–21. 30. Can M, Guven B, Bektas S, Arikan I. Oxidative stress and apoptosis in preeclampsia. Tissue Cell 2014;46:477–81. 31. Siddiqui IA, Jaleel A, Al’Kadri HM, et al. Biomarkers of oxidative stress in women with pre-eclampsia. Biomarkers Med 2013;7: 229–34. 32. Suhail M, Suhail S, Gupta BK, Bharat V. Malondialdehyde and antioxidant enzymes in maternal and cord blood, and their correlation in normotensive and preeclamptic women. J Clin Med Res 2009;1:150–7. 33. Gulbayzar S, Arica V, Hatipoglu S, et al. Malondialdehyde level in the cord blood of newborn infants. Iranian J Pediat 2011;21: 313–19. 34. Nakatsukasa Y, Tsukahara H, Tabuchi K, et al. Thioredoxin-1 and oxidative stress status in pregnant women at early third trimester of pregnancy: relation to maternal and neonatal characteristics. J Clin Biochem Nutr 2013;52:27–31. 35. Casanueva E, Viteri FE. Iron and oxidative stress in pregnancy. J Nutr 2003;133:1700S–8S. 36. Surh Y-J. Oxidative stress, inflammation, and health. Boca Raton, FL: Taylor & Francis, 2005. 37. Negi R, Pande D, Karki K, et al. Trace elements and antioxidant enzymes associated with oxidative stress in the pre-eclamptic/ eclamptic mothers during fetal circulation. Clin Nutr 2012;31: 946–50. 38. Patil SB, Kodliwadmath MV, Kodliwadmath M. Lipid peroxidation and antioxidant activity in complicated pregnancies. Clin Exp Obstet Gynecol 2009;36:110–12. 39. Ray G, Husain SA. Oxidants, antioxidants and carcinogenesis. Indian J Exp Biol 2002;40:1213–32. 40. Makino A, Skelton MM, Zou AP, Cowley Jr AW,. Increased renal medullary H2O2 leads to hypertension. Hypertension 2003;42: 25–30. 41. Rodriguez-Iturbe B, Zhan CD, Quiroz Y, et al. Antioxidant-rich diet relieves hypertension and reduces renal immune infiltration in spontaneously hypertensive rats. Hypertension 2003;41: 341–6. 42. Krishna Mohan S, Venkataramana G. Status of lipid peroxidation, glutathione, ascorbic acid, vitamin E and antioxidant enzymes in patients with pregnancy-induced hypertension. Indian J Physiol Pharmacol 2007;51:284–8. 43. Llurba E, Grataco´s E, Martı´n-Galla´n P, et al. A comprehensive study of oxidative stress and antioxidant status in preeclampsia and normal pregnancy. Free Radical Biol Med 2004;37:557–70. 44. Sharma JB, Sharma A, Bahadur A, et al. Oxidative stress markers and antioxidant levels in normal pregnancy and pre-eclampsia. Int J Gynecol Obstet 2006;94:23–7. 45. Knapen MF, Zusterzeel PL, Peters WH, Steegers EA. Glutathione and glutathione-related enzymes in reproduction: a review. Eur J Obstet Gynecol Reprod Biol 1999;82: 171–84. 46. Das B, Saha-Roy S, Das Gupta A, et al. Assessment of placental oxidative stress in pre-eclampsia. J Obstet Gynaecol India 2012;62: 39–42. 47. Vaziri ND, Wang XQ, Oveisi F, Rad B. Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats. Hypertension 2000;36:142–6. 48. Davidge ST, Hubel CA, Brayden RD, et al. Sera antioxidant activity in uncomplicated and preeclamptic pregnancies. Obstet Gynecol 1992;79:897–901. 49. Patil SB, Kodliwadmath M, Kodliwadmath SM. Study of oxidative stress and enzymatic antioxidants in normal pregnancy. Indian J Clin Biochem 2007;22:135–7. 50. Mistry HD, Kurlak LO, Williams PJ, et al. Differential expression and distribution of placental glutathione peroxidases 1, 3
8
V. D’Souza et al.
Downloaded by [University of California, San Diego] at 13:34 01 February 2016
and 4 in normal and preeclamptic pregnancy. Placenta 2010;31: 401–8. 51. Roland-Zejly L, Moisan V, St-Pierre I, Bilodeau JF. Altered placental glutathione peroxidase mRNA expression in preeclampsia according to the presence or absence of labor. Placenta 2011;32: 161–7. 52. Brennecke SP, Gude NM, Di Iulio JL, King RG. Reduction of placental nitric oxide synthase activity in pre-eclampsia. Clin Sci 1997; 93:51–5.
Clin Exp Hypertens, Early Online: 1–8
53. Morris NH, Sooranna SR, Learmont JG, et al. Nitric oxide synthase activities in placental tissue from normotensive, pre-eclamptic and growth retarded pregnancies. BJOG 1995;102:711–14. 54. D’Souza V, Chavan-Gautam P, Joshi S. Counteracting oxidative stress in pregnancy through modulation of maternal micronutrients and omega-3 fatty acids. Curr Med Chem 2013;20:4777–83. 55. Kulkarni A, Mehendale S, Pisal H, et al. Association of omega-3 fatty acids and homocysteine concentrations in pre-eclampsia. Clin Nutr 2011;30:60–4.
