Hemlata Pisal

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.

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.

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.

Reduced Folate, Increased Vitamin B12 and Homocysteine Concentrations in Women Delivering Preterm

Background and Aim: Maternal nutrition is an important determinant of the duration of pregnancy and fetal growth, and thereby influences pregnancy outcome. Folic acid and vitamin B12 are involved in one-carbon metabolism and are reported to underlie intrauterine programming of adult diseases. Methods: In the present study, the levels of folate, vitamin B12 and homocysteine were measured in mothers delivering preterm (PT; gestation <37 weeks; n = 67), those delivering preterm due to preeclampsia (PT-PE; n = 49) and women delivering at term (control group; n = 76). Results: Increased vitamin B12 and homocysteine levels (p < 0.05 for both) were seen in the PT-PE and PT groups as compared to the controls. In addition, reduced folate levels (p < 0.05) were observed in the PT group. A negative association of maternal plasma homocysteine with birth weight was seen in the idiopathic preterm group. Conclusions: Altered maternal micronutrients and resultant increased homocysteine concentrations exist in women delivering preterm. These alterations may also be partly associated with other factors such as undiagnosed inflammatory conditions or inadequate placentation in some women. Since these micronutrients play an important role in epigenetic regulation of vital genes involved in the fetal programming of adult diseases, further studies need to be undertaken to understand their role in preterm deliveries.

Altered maternal proportions of long chain polyunsaturated fatty acids and their transport leads to disturbed fetal stores in preeclampsia

Our previous cross-sectional studies have shown altered proportions of long chain polyunsaturated fatty acids (LCPUFA) in preeclampsia (PE) at the end of pregnancy when the pathology has already progressed. The present longitudinal study for the first time reports fatty acid proportions from 16th week of gestation till delivery and placental transport in PE. This is a hospital based study where women were recruited in early pregnancy. Maternal blood was collected at 3 time points i.e. T1=16-20th week, T2=26-30th week and T3=at delivery. Cord blood and placenta were collected at delivery. This study reports data on 140 normotensive control (NC) and 54 PE women. In PE we report lower proportions of DHA in maternal plasma at T1, cord plasma and placenta (p<0.05 for all). The mRNA levels of placental ∆5 desaturase, fatty acid transport proteins -1, -4, were lower (p<0.05 for all) in PE. There was also a positive association between cord and maternal plasma DHA and total omega-3 fatty acids at T1. This study demonstrates that women with PE have lower fatty acids stores at 16-20th week of gestation and lower placental synthesis and transport. It is likely that supplementation of omega-3 fatty acids during the 16-20th week of gestation may help in improving fatty acid status in infants born to mothers with PE.

Reduced levels of placental long chain polyunsaturated fatty acids in preterm deliveries

Reports suggest that the placenta in preterm birth may provide clues to predicting the risk of individuals developing chronic diseases in later life. Placental delivery of long chain polyunsaturated fatty acids (LCPUFA) (constituents of the cell membrane and precursors of prostaglandins) is essential for the optimal development of the central nervous system of the fetus. The present study examines the levels of LCPUFA and their association with placental weight and birth outcome in 58 women delivering preterm and 44 women delivering at term. Docosahexaenoic acid (DHA) and arachidonic acid (ARA) levels were lower (p<0.01) in women delivering preterm. There was a positive association of placental DHA with placental weight (p=0.036) and nervonic acid with head circumference (p=0.040) in the preterm group. Altered placental LCPUFA status exists in Indian mothers delivering preterm, which may influence the birth outcome.

Increased oxidative stress from early pregnancy in women who develop preeclampsia.

Abstract 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 (p < 0.01) and T2 (p < 0.01) in women with PE as compared with control. GPx levels were higher at T3 (p < 0.05) while SOD levels were lower at T2 (p < 0.05), T3 (p < 0.01) and in cord (p < 0.01) in PE. GSH levels at T1 (p < 0.05) 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.

Differential expression of human placental neurotrophic factors in preterm and term deliveries

Neurotrophic factors such as brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are involved in development of the placenta and fetal brain. A series of human and animal studies in our department have shown that micronutrients (folic acid, vitamin B12) and omega 3 fatty acids like DHA are all interlinked in the one carbon cycle. Any alterations in one carbon components will lead to changes in methylation patterns that further affect the gene expression at critical periods of development resulting in complications during pregnancy. This may further contribute to risk for neurodevelopmental disorders in children born preterm. Therefore this study for the first time examines the mRNA levels from preterm and term placentae. A total number of 38 women delivering preterm (<37 weeks gestation) and 37 women delivering at term (=>37 weeks gestation) were recruited. The mRNA levels of BDNF and NGF were analyzed by real time quantitative polymerase chain reaction. Our results indicate that BDNF and NGF mRNA levels were lower in preterm group as compared to term group. There was a positive association of placental BDNF and NGF mRNA levels with cord plasma BDNF and NGF levels. The differential expression of BDNF and NGF gene in preterm placentae may also alter the vascular development in preterm deliveries. Our data suggests that the reduced mRNA levels of BDNF and NGF may possibly be a result of altered epigenetic mechanisms and may have an implication for altered fetal programming in children born preterm.

Matrix Metalloproteinase-1 and -9 in Human Placenta during Spontaneous Vaginal Delivery and Caesarean Sectioning in Preterm Pregnancy

Preterm birth is a major public health problem in terms of loss of life, long-term and short term disabilities worldwide. The process of parturition (both term and preterm) involves intensive remodelling of the extracellular matrix (ECM) in the placenta and fetal membranes by matrix metalloproteinases (MMPs). Our previous studies show reduced docosahexaenoic acid (DHA) in women delivering preterm. Further omega 3 fatty acids are reported to regulate MMP levels. This study was undertaken to examine the placental levels of MMPs and their association with placental DHA levels in women delivering preterm. The levels of MMP-1 and MMP-9 in 74 women delivering preterm (52 by spontaneous vaginal delivery and 22 by caesarean sectioning) and 75 women delivering at term (59 by spontaneous vaginal delivery and 16 by caesarean sectioning) were determined by enzyme-linked immunosorbent assay (ELISA) and their association with placental DHA was studied. Placental MMP-1 levels were higher (p<0.05) in women delivering preterm (both by spontaneous vaginal delivery and caesarean sectioning) as compared to those delivering at term. In contrast, placental MMP-9 levels in preterm pregnancies was higher (p<0.05) in women with spontaneous vaginal delivery while lower (p<0.05) in women delivering by caesarean sectioning. Low placental DHA was associated with higher placental MMP-9 levels. Our study suggests a differential effect of mode of delivery on the levels of MMPs from placenta. Further this study suggests a negative association of DHA and the levels of MMP-9 in human placenta although the mechanisms need further study.

Association of brain-derived neurotrophic factor and tyrosine kinase B receptor in pregnancy.

Abnormal brain development in a compromised prenatal and/or early postnatal environment is thought to be a risk factor for several neurobehavioural disorders. However, the mechanisms underlying these are not well understood. We have earlier reported reduced placental docosahexaenoic acid (DHA) levels in preterm deliveries. We have hypothesized that increased oxidative stress and reduced DHA levels may lead to changes in the circulating levels of maternal and cord brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (TrkB) levels. A total number of 96 women delivering preterm and 95 women delivering at term were recruited. Plasma BDNF levels were measured in both mother and cord blood plasma using the BDNF Immuno Assay kit. Placental TrkB levels were analysed using sandwich enzyme-linked immunosorbent assay (ELISA). Maternal plasma BDNF levels and placental TrkB levels were higher (p<0.05) while cord plasma BDNF levels were lower (p<0.01) in women delivering preterm as compared to term. There was a negative association between levels of placental TrkB and DHA (p=0.034). A negative association between maternal plasma BDNF levels and placental weight (p=0.001) was observed while a positive association was seen between cord plasma BDNF levels and gestation (p=0.025). The reduction in cord BDNF levels may have implications for altered neurodevelopment in childhood and later life. Studies need to be undertaken to follow up children born preterm for risk of neurobehavioural disorders like attention deficit hyperactivity disorder (ADHD) to understand the effect of altered BDNF at birth on neurodevelopment.