Savita Mehendale

Fatty acids, antioxidants, and oxidative stress in pre-eclampsia

To investigate whether free radical‐mediated membrane lipid peroxidation may be implicated in the pathogenesis of pre‐eclampsia.

Global DNA Methylation Patterns in Placenta and Its Association with Maternal Hypertension in Pre-Eclampsia

Maternal nutrition is an important determinant of one-carbon metabolism that lies at the heart of intrauterine epigenetic programming. Exchange of nutrients and other vital molecules between the mother and fetus takes place across the placenta and hence may play direct role in fetal programming. Pre-eclampsia (PE) originates in the placenta and altered maternal nutrition may influence epigenetic patterns in the placenta, thereby affecting birth outcome. In the present study, we investigated the global DNA methylation levels in placentas of pre-eclampsia women (i.e., women delivering at term and those delivering preterm) and studied their associations with maternal blood pressure and birth outcome. Increased homocysteine and global DNA methylation levels were seen in the pre-eclampsia group (term and preterm PE) when compared with the normotensive group (p < 0.05). A positive association between global DNA methylation and systolic (p < 0.01) and diastolic (p < 0.05) blood pressure was seen in the term pre-eclampsia group, whereas there was no association with birth outcome. The study for the first time provides evidence for altered global DNA methylation patterns in pre-eclampsia placentas and its association with blood pressure. It is possible that increased homocysteine levels may be related to increased methylation in pre-eclampsia.

Differential placental methylation and expression of VEGF, FLT-1 and KDR genes in human term and preterm preeclampsia

BackgroundPreeclampsia, a pregnancy complication of placental origin is associated with altered expression of angiogenic factors and their receptors. Recently, there is considerable interest in understanding the role of adverse intrauterine conditions in placental dysfunction and adverse pregnancy outcomes. Since we have observed changes in placental global DNA methylation levels in preeclampsia, this study was undertaken to examine gene promoter CpG methylation and expression of several angiogenic genes.We recruited 139 women comprising, 46 normotensive women with term delivery (≥37 weeks), 45 women with preeclampsia delivering preterm (<37 weeks) and 48 women with preeclampsia delivering at term. Expression levels and promoter CpG methylation of VEGF, FLT-1 and KDR genes in placentae from respective groups were determined by Taqman-based quantitative real time PCR and by the Sequenom® EpiTYPER™ technology respectively.ResultsWe observed several differentially methylated CpG sites in the promoter regions of VEGF, FLT-1 and KDR between the normotensive and preeclampsia groups. We specifically observed hypomethylated CpGs in the promoter region and an increased expression of VEGF gene between term and preterm preeclampsia. However, mean promoter CpG methylation could not account for the higher expression of FLT-1 and KDR in preterm preeclampsia as compared to normotensive group.ConclusionsOur data indicates altered DNA methylation patterns in the VEGF, FLT-1 and KDR genes in preeclampsia as compared to the normotensive group, which could be involved in the pathophysiology of preeclampsia. Hypomethylation of VEGF promoter and consequent upregulation of VEGF mRNA levels could be a compensatory mechanism to restore normal angiogenesis and blood flow in preterm preeclampsia. This study suggests a role of altered DNA methylation in placental angiogenesis and in determining adverse pregnancy outcomes.

Circulating angiogenic factors and their association with birth outcomes in preeclampsia

This study was designed to test the hypothesis that altered angiogenic factors together with increased oxidative stress and reduced docosahexaenoic acid (DHA) levels may be associated with altered birth outcome parameters. To test this hypothesis, levels of plasma vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), the oxidative stress marker malondialdehyde (MDA) and fatty acids were estimated in women with preeclampsia and their cord samples and compared with those in normotensive women. The association of these parameters with birth outcome was also examined. Our results show that in preeclamptic women, maternal plasma VEGF and PlGF levels were lower, whereas sFlt-1 levels were higher (P<0.05 for all) than in normotensive women. In contrast, cord plasma VEGF levels were higher (P<0.05) in preeclamptic women, whereas there was no difference in sFlt-1 levels. Plasma DHA levels in both the mother and cord were lower (P<0.05) in the preeclamptic group compared with normotensive women. Maternal plasma sFlt-1 levels were positively (n=23, r=0.415, P=0.039) associated with MDA concentrations in preeclamptic women. Maternal plasma sFlt-1 levels showed a strong negative association with baby weight (n=37, r=−0.547, P=0.001), head circumference (n=37, r=−0.472, P=0.005) and baby chest circumference (n=37, r=−0.375, P=0.032) in the preeclamptic group. Cord plasma sFlt-1 concentrations were negatively associated with cord plasma DHA concentrations (n=28, r=−0.552, P=0.004). This study suggests that dysregulation of angiogenic factors may be associated with maternal oxidative stress. Increased oxidative stress may reduce cord DHA levels and increase sFlt-1 levels, leading to poor birth outcomes in preeclampsia.

Association of omega-3 fatty acids and homocysteine concentrations in pre-eclampsia.

BACKGROUND & AIMS The present study examines the associations of folic acid, vitamin B₁₂ and omega-3 fatty acids and increased homocysteine which are implicated in the pathology of pre-eclampsia. METHODS 49 Pre-eclamptic and 57 normotensive women were recruited at Bharati hospital, Pune, India. Plasma folate, vitamin B₁₂, homocysteine and erythrocyte omega-3 and omega-6 fatty acids were analyzed. RESULTS Homocysteine concentrations were higher in pre-eclamptic than in normotensive women (14.28±7.31 vs. 11.03±4.38 μmol/l, p<0.01) despite similar levels of folic acid and vitamin B₁₂. In the pre-eclamptic group, plasma folate levels were positively associated with erythrocyte omega-6 fatty acids (p<0.05) while erythrocyte docosahexaenoic acid levels were negatively associated with plasma homocysteine levels (p<0.01). CONCLUSIONS Our study provides evidence for the associations of altered omega-3 fatty acids especially docosahexaenoic acid and the resultant increased homocysteine concentrations in pre-eclampsia. Future studies need to examine if docosahexaenoic acid supplementation during pregnancy reduces homocysteine levels and ameliorates the risk of developing pre-eclampsia.

Reduced placental docosahexaenoic acid levels associated with increased levels of sFlt-1 in preeclampsia.

Our earlier studies, in preeclamptic women have shown altered levels of long chain polyunsaturated fatty acids (LCPUFA), essential constituents of the cell membrane lipids responsible for membrane stability as one of the key factors contributing to the pathophysiology of preeclampsia. We have also reported elevated levels of sFlt-1 in preeclampsia. The present study examines the levels of LCPUFA and their association with sFlt-1 levels in 69 pre-eclamptic women and 40 normotensive women. DHA and omega 3 fatty acid levels were lower (p<0.001) while arachidonic acid and omega 6 fatty acid levels were higher (p<0.05) in preeclamptic women as compared to normotensive women. Maternal plasma sFlt-1 levels were higher (p<0.05) in preeclamptic women and were negatively associated with DHA (p=0.008) and omega 3 fatty acids concentrations (p=0.031). Our results suggest that altered placental LCPUFA may result in altered membrane lipid fatty acid composition leading to increased release of sFlt-1 in circulation.

Long chain polyunsaturated fatty acids in mothers and term babies

Abstract Aim: To establish the levels of docosahexaenoic acid (DHA) and arachidonic acid (AA) in both plasma and erythrocytes of maternal and cord blood as well as in breast milk of mothers delivering babies at term. Methods: A total of 148 mothers delivering babies at term were recruited from Bharati Medical Hospital, Pune, India. Results: Levels of DHA and AA in both plasma and erythrocyte were higher in cord blood compared to levels in maternal blood (P<0.001). Maternal plasma and erythrocyte DHA levels had a positive association with the respective levels in cord blood (P<0.001). However, such an association was not seen for AA levels. Maternal plasma omega 3 and omega 6 fatty acids were positively associated with the respective milk fatty acids (P<0.01). Conclusions: Our results indicate that milk long-chain polyunsaturated fatty acids (LCPUFA) status reflects the concentrations of maternal LCPUFA in women delivering babies at term. Improving the maternal LCPUFA status throughout pregnancy and lactation may improve the milk LCPUFA status and ultimately benefit the infant.

Gestation‐dependent changes in human placental global DNA methylation levels

The placenta, which plays a critical structural and functional role duringpregnancy, is a key link in the chain of events that lead to intrauterine programming of adult health. Placental development involves spatio-temporally programmed epigenetic processes that, if altered, may affect the gene expression programs for different cell types leading to abnormal placentation, thereby affecting birth outcome. The present study, for the first time, examines the global DNA methylation levels as a function of gestation in placentas of normotensivewomendelivering at term and preterm who were matched for age and socioeconomic status. A total number of 98 normotensive pregnant women with singleton pregnancy recruited at the Department of Obstetrics and Gynecology, Bharati Hospital, Pune, during the years 2007--2009, were included in this study. Pregnant women with complications such as preeclampsia, gestational diabetes, anemia, chronic hypertension, and type I or type II diabetes mellitus were excluded from the study. Genomic DNA was isolated from placental tissues and global DNA methylation was measured using the Methylamp Global DNA Methylation Quantification Kit (EpigentekGroup, Inc., NewYork,NY) (Kulkarni et al., 2010). Our results show a positive association (r1⁄4 0.32; P< 0.01) between global DNA methylation and gestational age at birth in the study cohort (Fig. 1). In contrast to our previous study in preeclampsia (Kulkarni et al., 2011), the present study shows decreased DNAmethylation levels in preterm deliveries. The disturbed placental pathophysiology of preeclampsia could account for contrasting trends in DNA methylation levels in normotensive preterm and preeclamptic preterm placentas. Further the observed positive association with gestational age at birth suggests that the gestational age-dependent profile is important to evaluate and control, when considering any methylation change identified as a potential biomarker, particularly in preterm pregnancies. Placental development and function is coordinated by interactions among genetic, epigenetic, and physiological cues that are differentially interpreted as a function of gestational age (Oyama, 2000). It has been shown in animal models that after implantation, the bulk of the genome becomes hypermethylated through active de novo methylation. The dynamic nature ofmethylation patterns during development has also been demonstrated in humans. At every stage of fetal development there is a sequence of de novo methylation and chromatin remodeling that dictates the tissue structure and function through a finely tuned pattern involving the switching on and off of gene expression. There are critical spatiotemporal windows during which these programs must be completed; failure to complete these programs in time may be irrecoverable and have long-term consequences (Hales and Barker, 2001). Our results, therefore, have implications for the consequences of prematurity since premature birth may impede the normal spatio-temporal pattern of gene expression affecting later development of the infant after birth, and for fetal programming of adult diseases since preterm babies are known to be at increased risk of neurodevelopmental and metabolic disorders in later life. However, there is a need to study the methylation patterns of the relevant genes in placenta of women delivering preterm and at term to test this hypothesis.

High Maternal Plasma Antioxidant Concentrations Associated with Preterm Delivery

Background/Aims: Our earlier study has shown that increased maternal oxidative stress and reduced antioxidants like vitamin E and C play an important role in fetal growth in preeclampsia. However, the role of antioxidants and their effects on gestation and birth outcome in normotensive pregnancies are not conclusive. The present study examined plasma malondialdehyde as a marker of oxidative stress and antioxidant concentrations (vitamins E and C) in maternal as well as in cord blood samples in normotensive women who delivered both preterm and at term. Methods: 140 normotensive pregnant women were recruited at Bharati Medical Hospital, Pune, India, during the year 2007. Maternal and cord samples were examined for oxidative stress levels and vitamin C and E concentrations in women who delivered preterm (n = 40) and at term (n = 100). Mean values were compared with those of women delivering at term using the t test. Results: Increased (p < 0.05) oxidative stress was seen in preterm mothers as well as in cord samples. Preterm mothers had higher vitamin C concentrations (p < 0.05), and these were positively associated with oxidative stress (p = 0.02). Vitamin E levels were comparable between groups. Conclusions: Increased maternal circulating vitamin C concentrations and increased oxidative stress are associated with preterm delivery.

Gestation dependant changes in angiogenic factors and their associations with fetal growth measures in normotensive pregnancy.

BACKGROUND Earlier studies indicate that altered angiogenesis at birth is associated with poor birth outcome in women with preeclampsia. Now, we hypothesize that the progressive gestation dependant changes in markers of angiogenesis will be more useful to predict birth weight early even in a normotensive pregnancy. This study for the first time examines the association of gestation dependant changes in the levels of maternal angiogenic factors in addition to their levels in cord with birth weight. METHOD Ninety two pregnant women were followed at three different time points: 16-20 weeks, 26-30 weeks and at delivery during pregnancy. Plasma levels of angiogenic and anti angiogenic factors were determined by commercial enzyme-linked immunosorbent assay (ELISA) kits. RESULTS Maternal plasma VEGF levels increased (p<0.01) till the second time point and decreased (p<0.05) up to delivery while plasma sFlt-1 levels increased (p<0.01) at delivery. PlGF levels peaked (p<0.01) at second time point and decreased (p<0.01) at delivery. Cord plasma VEGF levels were higher (p<0.01) and sFlt-1 levels were lower (p<0.01) as compared to maternal values at all time points. Maternal plasma VEGF levels at first time point and PlGF levels at delivery were positively (p<0.05 and p<0.01 respectively), while sFlt-1/PlGF ratio at delivery was negatively associated (p<0.05) with birth weight. CONCLUSION Levels of pro- and anti-angiogenic factors may be differentially regulated across gestation. Maternal VEGF levels at early gestation (16-20 weeks) may be predictive of birth weight in healthy term pregnancies.