Responses of neurons in the rostral ventrolateral medulla of conscious cats to anticipated and passive movements.

The vestibular system contributes to regulating sympathetic nerve activity and blood pressure. Initial studies in decerebrate animals showed that neurons in the rostral ventrolateral medulla (RVLM) respond to small-amplitude (<10°) rotations of the body, as in other brain areas that process vestibular signals, although such movements do not affect blood distribution in the body. However, a subsequent experiment in conscious animals showed that few RVLM neurons respond to small-amplitude movements. This study tested the hypothesis that RVLM neurons in conscious animals respond to signals from the vestibular otolith organs elicited by large-amplitude static tilts. The activity of approximately one-third of RVLM neurons whose firing rate was related to the cardiac cycle, and thus likely received baroreceptor inputs, was modulated by vestibular inputs elicited by 40° head-up tilts in conscious cats, but not during 10° sinusoidal rotations in the pitch plane that affected the activity of neurons in brain regions providing inputs to the RVLM. These data suggest the existence of brain circuitry that suppresses vestibular influences on the activity of RVLM neurons and the sympathetic nervous system unless these inputs are physiologically warranted. We also determined that RVLM neurons failed to respond to a light cue signaling the movement, suggesting that feedforward cardiovascular responses do not occur before passive movements that require cardiovascular adjustments.

Uterine flushing with supernatant embryo culture medium in vitrified warmed blastocyst transfer cycles: a randomized controlled trial.

PURPOSE: Does transfer of supernatant embryo culture fluid (stimulation of endometrial embryo transfer - SEET) prior to vitrified warmed blastocyst transfer result in better clinical pregnancy and live birth rates than direct vitrified warmed blastocyst transfer? METHODS: This randomized controlled trial compared SEET group and direct transfer group (control) in 60 women undergoing vitrified warmed blastocyst transfers. The duration of the study was 3 years. The patients were undergoing vitrified warmed blastocyst transfer at university level infertility centre. Sixty women were randomized to SEET (n = 30) or control (n = 30). RESULTS: Data was available for analysis from all the 30 women in the SEET group and 30 women in the control group. There were no drop outs in the trial. The implantation rate was significantly lower in the SEET group compared to the control group (27 vs. 44 %, P = 0.018). The clinical pregnancy rates were similar in both the groups (47 vs. 53 %) but the live birth rate was also significantly lower in SEET group (23 vs. 50 %, P = 0.03). LIMITATIONS: The sample size based on clinical pregnancy rates was small and hence not adequately powered to detect differences in live birth rates. Lack of blinding leading to possible bias cannot be ruled out. CONCLUSION: There was no evidence of an improvement in clinical pregnancy rate following SEET in vitrified warmed blastocyst transfer compared to direct transfer.

Expression of genes encoding enzymes involved in the one carbon cycle in rat placenta is determined by maternal micronutrients (folic acid, vitamin B12) and omega-3 fatty acids.

We have reported that folic acid, vitamin B12, and omega-3 fatty acids are interlinked in the one carbon cycle and have implications for fetal programming. Our earlier studies demonstrate that an imbalance in maternal micronutrients influence long chain polyunsaturated fatty acid metabolism and global methylation in rat placenta. We hypothesize that these changes are mediated through micronutrient dependent regulation of enzymes in one carbon cycle. Pregnant dams were assigned to six dietary groups with varying folic acid and vitamin B12 levels. Vitamin B12 deficient groups were supplemented with omega-3 fatty acid. Placental mRNA levels of enzymes, levels of phospholipids, and glutathione were determined. Results suggest that maternal micronutrient imbalance (excess folic acid with vitamin B12 deficiency) leads to lower mRNA levels of methylene tetrahydrofolate reductase (MTHFR) and methionine synthase , but higher cystathionine b-synthase (CBS) and Phosphatidylethanolamine-N-methyltransferase (PEMT) as compared to control. Omega-3 supplementation normalized CBS and MTHFR mRNA levels. Increased placental phosphatidylethanolamine (PE), phosphatidylcholine (PC), in the same group was also observed. Our data suggests that adverse effects of a maternal micronutrient imbalanced diet may be due to differential regulation of key genes encoding enzymes in one carbon cycle and omega-3 supplementation may ameliorate most of these changes.

Levels of brain derived neurotrophic factors across gestation in women with preeclampsia.

Preeclampsia (PE) is a major pregnancy complication of placental origin which leads to adverse pregnancy outcome. Brain derived neurotrophic factor (BDNF) is suggested to promote trophoblast growth and regulate placental and fetal development. This study for the first time examines the levels of maternal plasma BDNF at various time points during gestation, cord plasma and placental BDNF levels and their association with birth outcome in women with PE. Normotensive control (NC) women (n=89) and women with PE (n=61) were followed at three different time points [16-20 weeks (T1), 26-30 weeks (T2) and at delivery (T3)]. Maternal blood at all time points and cord blood was collected. Results indicate that maternal BDNF levels at T1 (p=0.050) and T3 (p=0.025) were lower in women with PE than in NC women. Cord BDNF levels at delivery in women with PE were lower (p=0.032) than those in NC women. Placental BDNF gene expression was also lower (p=0.0082) in women with PE than in NC women. Our data suggests that BDNF plays an important role in the development of the materno-fetal-placental unit during pregnancy. Alteration in the levels of BDNF during pregnancy may be associated with an abnormal development of the placenta resulting in PE.

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.

A longitudinal study of circulating angiogenic and antiangiogenic factors and AT1-AA levels in preeclampsia.

Our earlier studies of preeclampsia (PE) at delivery have demonstrated the alteration of one carbon cycle, reduced placental omega 3 fatty acids, altered circulating levels of angiogenic factors and differential placental gene-specific methylation patterns of angiogenic factors. This study was undertaken to examine changes in the levels of angiogenic factors and angiotensin II type 1 receptor autoantibodies (AT1-AAs) throughout gestation, from early pregnancy until delivery, in women with PE and to examine their association with cord angiogenic factors, blood pressure and infant weight. A total of 81 pregnant women (46 normotensive and 35 with PE) were followed at three different time points during pregnancy: 16-20 weeks (T1), 26-30 weeks (T2) and at the time of delivery (T3). The plasma levels of angiogenic factors and AT1-AAs were determined in the maternal and cord plasma by commercial enzyme-linked immunosorbent assay kits. Maternal plasma levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) were lower (P<0.05 for both), whereas soluble fms-like tyrosine kinase-1 (sFlt-1; P<0.05) and the sFlt-1/PlGF ratio (P<0.01) were higher in early pregnancy in the PE group. Maternal plasma AT1-AA levels were higher (P<0.05) at T2 in women with PE. Cord plasma VEGF and soluble kinase insert domain receptor (sKDR) levels were lower (P<0.01 and P<0.05, respectively), whereas AT1-AA levels were higher (P<0.05) in the PE group. Maternal plasma VEGF levels in early pregnancy were positively associated with systolic blood pressure, whereas the sFlt-1/PlGF ratio at T2 was negatively associated with infant weight in the PE group. Low levels of proangiogenic factors (VEGF and PlGF) and high levels of AT1-AAs and antiangiogenic factors (sFlt-1 and sFlt-1/PlGF ratio) are present in the maternal circulation during early gestation in women with PE.

Maternal micronutrients, omega-3 fatty acids, and placental PPARγ expression.

An altered one-carbon cycle is known to influence placental and fetal development. We hypothesize that deficiency of maternal micronutrients such as folic acid and vitamin B12 will lead to increased oxidative stress, reduced long-chain polyunsaturated fatty acids, and altered expression of peroxisome proliferator activated receptor (PPARγ) in the placenta, and omega-3 fatty acid supplementation to these diets will increase the expression of PPARγ. Female rats were divided into 5 groups: control, folic acid deficient, vitamin B12 deficient, folic acid deficient + omega-3 fatty acid supplemented, and vitamin B12 deficient + omega-3 fatty acid supplemented. Dams were dissected on gestational day 20. Maternal micronutrient deficiency leads to lower (p < 0.05) levels of placental docosahexaenoic acid, arachidonic acid, PPARγ expression and higher (p < 0.05) levels of plasma malonidialdehyde, placental IL-6, and TNF-α. Omega-3 fatty acid supplementation to a vitamin B12 deficient diet normalized the expression of PPARγ and lowered the levels of placental TNF-α. In the case of supplementation to a folic acid deficient diet it lowered the levels of malonidialdehyde and placental IL-6 and TNF-α. This study has implications for fetal growth as oxidative stress, inflammation, and PPARγ are known to play a key role in the placental development.

Differential regulation of hepatic transcription factors in the Wistar rat offspring born to dams fed folic acid, vitamin B12 deficient diets and supplemented with omega-3 fatty acids.

Nutritional status of the mother is known to influence various metabolic adaptations required for optimal fetal development. These may be mediated by transcription factors like peroxisome proliferator activated receptors (PPARs), which are activated by long chain polyunsaturated fatty acids. The objective of the current study was to examine the expression of different hepatic transcription factors and the levels of global methylation in the liver of the offspring born to dams fed micronutrient deficient (folic acid and vitamin B12) diets and supplemented with omega-3 fatty acids. Female rats were divided into five groups (n = 8/group) as follows; control, folic acid deficient (FD), vitamin B12 deficient (BD) and omega-3 fatty acid supplemented groups (FDO and BDO). Diets were given starting from pre-conception and continued throughout pregnancy and lactation. Pups were dissected at the end of lactation. Liver tissues were removed; snap frozen and stored at -80°C. Maternal micronutrients deficiency resulted in lower (p<0.05) levels of pup liver docosahexaenoic acid (DHA) and arachidonic acid (ARA) as compared to the control group. Pup liver PPARα and PPARγ expression was lower (p<0.05) in the BD group although there were no differences in the expression of SREBP-1c, LXRα and RXRα expression. Omega-3 fatty acids supplementation to this group normalized (p<0.05) levels of both PPARα and PPARγ but reduced (p<0.05) SREBP-1c, LXRα and RXRα expression. There was no change in any of the transcription factors in the pup liver in the FD group. Omega-3 fatty acids supplementation to this group reduced (p<0.05) PPARα, SREBP-1c and RXRα expression. Pup liver global methylation levels were higher (p<0.01) in both the micronutrients deficient groups and could be normalized (p<0.05) by omega-3 fatty acid supplementation. Our novel findings suggest a role for omega-3 fatty acids in the one carbon cycle in influencing the hepatic expression of transcription factors in the offspring.

Maternal micronutrient imbalance alters gene expression of BDNF, NGF, TrkB and CREB in the offspring brain at an adult age.

Micronutrients like folate, vitamin B12, and fatty acids which are interlinked in the one carbon cycle play a vital role in mediating epigenetic processes leading to an increased risk for neurodevelopmental disorders in the offspring. Our earlier study demonstrates that a micronutrient imbalanced diet adversely affects docosahexaenoic acid (DHA) and protein levels of neurotrophins like brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain and cognition in the offspring by 3 months of age. In this study we attempt to analyze if these effects are a consequence of a change in gene expression of these molecules. Further, we also examined the effect of either a postnatal control diet or a prenatal omega-3 fatty acid supplementation on gene expression in the cortex of the offspring. Pregnant rats were divided into control and five treatment groups at two levels of folic acid (normal and excess folate) in the presence and absence of vitamin B12. Omega-3 fatty acid (eicosapentaenoic acid - EPA+DHA) supplementation was given to vitamin B12 deficient groups. Following delivery, 8 dams from each group were shifted to control diet and remaining continued on the same treatment diet. Our results demonstrate that the imbalanced diet caused a marked reduction in the mRNA levels of BDNF, NGF, TrkB, and cAMP response element-binding protein (CREB). Prenatal omega-3 fatty acid supplementation to the maternal imbalanced diet was able to normalize the mRNA levels of all the above genes. This study demonstrates that a maternal diet imbalanced in micronutrients (folic acid, vitamin B12) influences gene expression of neurotrophins and their signalling molecules and thereby adversely affects the brain of the offspring.

Differential regulation of brain-derived neurotrophic factor in term and preterm preeclampsia.

Our earlier studies in preeclampsia (PE) suggest a causal relationship between altered angiogenic factors and birth outcomes. Recent studies suggest that brain-derived neurotrophic factor (BDNF) can stimulate angiogenesis. The present study examines the levels of maternal and cord BDNF in women with PE (n = 106; full term [n = 60] and preterm [n = 46]) and normotensive women (n = 95; control) delivering at term. Maternal BDNF levels were lower (P < .05) in women with PE when compared to normotensive women. Cord BDNF levels were higher (P < .01) in women with PE delivering at term, while it was lower (P < .01) in women delivering preterm. Maternal BDNF levels were negatively associated with systolic and diastolic blood pressure (P < .01 for both). Our data for the first time suggest a possible role for BDNF in the pathophysiology of PE. Differential regulation of cord BDNF levels in preterm PE suggests a need to follow-up children to assess the neurodevelopmental effects in later life.

Management of severe ovarian hyperstimulation syndrome with thawed plasma.

Severe ovarian hyperstimulation syndrome remains one of the life threatening complication of assisted reproductive technology. In refractory cases of late ovarian hyperstimulation syndrome (OHSS), clinicians are left with limited therapeutic options. We report a case of refractory OHSS which was managed successfully using thawed plasma. Thawed plasma transfusion could be potential therapeutic option for managing patients with severe ovarian hyperstimulation not responding to conventional treatment.

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

BACKGROUND: Preeclampsia, 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Maternal micronutrients and omega 3 fatty acids affect placental fatty acid desaturases and transport proteins in Wistar rats.

Adequate supply of LCPUFA from maternal plasma is crucial for fetal normal growth and development. The present study examines the effect of maternal micronutrients (folic acid and vitamin B12) and omega 3 fatty acids on placental mRNA levels of fatty acid desaturases (Δ5 and Δ6) and transport proteins. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B12. Both the vitamin B12 deficient groups were supplemented with omega 3 fatty acid. Maternal vitamin B12 deficiency reduced placental mRNA and protein levels of Δ5 desaturase, mRNA levels of FATP1 and FATP4 (p<0.05 for all) as compared to control while omega 3 fatty acid supplementation normalized the levels. Our data for the first time indicates that altered maternal micronutrients and omega 3 fatty acids play a key role in regulating fatty acid desaturase and transport protein expression in placenta.

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.

Preconceptional omega-3 fatty acid supplementation on a micronutrient-deficient diet improves the reproductive cycle in Wistar rats.

Folic acid and vitamin B12 deficiencies are associated with high reproductive risks ranging from infertility to fetal structural defects. The aim of the present study was to examine the effects of preconceptional omega-3 fatty acid supplementation (eicosapentaenoic acid and docosahexaenoic acid) to a micronutrient-deficient diet on the reproductive cycle in Wistar rats. Female rats were divided into five groups from birth and throughout pregnancy: a control group, a folic acid-deficient (FD) group, a vitamin B12-deficient (BD) group, a folic acid-deficient + omega-3 fatty acid-supplemented (FDO) group and a vitamin B12 deficient + omega-3 fatty acid-supplemented (BDO) group. Dams were killed on gestation Day 20 and their ovaries and mammary glands were dissected out and subjected to histological examination. Maternal micronutrient deficiency (FD and BD groups) resulted in an abnormal oestrous cycle (P<0.001), whereas omega-3 fatty acid supplementation (FDO and BDO groups) restored the oestrous cycle to normal. There were fewer corpora lutea in the ovaries of FD rats compared with controls. In addition, rats in both the FD and BD groups exhibited an absence of lactating ducts in their mammary glands compared with controls. The findings of the present study indicate, for the first time, that maternal micronutrient deficiency affects the oestrous cycle and morphology of the ovary and mammary glands. Omega-3 fatty acid supplementation ameliorated these effects. This may have implications for infertility and pregnancy outcomes.