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During pregnancy, all major physiological systems undergo remarkable changes, driven largely by alterations in the maternal hormonal milieu. In healthy pregnancies, maternal cardiovascular and metabolic adaptation to pregnancy occurs to support fetal growth and maternal well-being. Impaired maternal adaptation to pregnancy is associated with a range of pregnancy complications, including gestational diabetes and preeclampsia. There is growing recognition of the importance of different maternal microbiota, including in the gut, vagina and oral cavity, in supporting normal maternal adaptations to pregnancy as well as evidence for microbial disturbances associating with pregnancy pathologies. Here, we aim to summarise emerging evidence demonstrating that differences in maternal microbiota associate with pregnancy outcomes and discuss potential therapeutic approaches under development that might restore an 'optimal' microbiome. In particular, we highlight recent work by ourselves and others exploring the role of the oral microbiome in pregnancy, given established links between poor oral health (e.g. periodontitis) and adverse pregnancy outcomes. Our research has focussed on specific nitrate-reducing oral bacteria which play a role in the generation of nitric oxide (NO) and other bioactive nitrogen oxides associated with cardiovascular health and maternal cardiovascular adaption to pregnancy. Ongoing research aims to define whether altered microbial profiles have clinical utility in the prediction of pregnancy pathologies, and whether interventions designed to optimise specific maternal microbiota could help prevent future complications. [ABSTRACT FROM AUTHOR]

Hypertension in pregnancy is prevalent, affecting around 10% of pregnancies worldwide, and significantly increases the risk of adverse outcomes for both mothers and their babies. Current treatment strategies for pregnant women with hypertension are limited, and new approaches for the management of hypertension in pregnancy are urgently needed. Substantial evidence from non‐pregnant subjects has demonstrated the potential for dietary nitrate supplementation to increase nitric oxide (NO) bioavailability and lower blood pressure, following bioactivation via the non‐canonical NO pathway. Emerging data suggest this approach may also be of benefit in pregnant women, although studies are limited. This review aims to summarise the current evidence from preclinical and clinical studies of nitrate supplementation in pregnancy, drawing on data from non‐pregnant populations where appropriate and highlighting key gaps in knowledge that remain to be addressed in future trials. [ABSTRACT FROM AUTHOR]

The generation of nitrite by the oral microbiota is believed to contribute to healthy cardiovascular function, with oral nitrate reduction to nitrite associated with systemic blood pressure regulation. There is the potential to manipulate the composition or activities of the oral microbiota to a higher nitrate-reducing state through nitrate supplementation. The current study examined microbial community composition and enzymatic responses to nitrate supplementation in sessile oral microbiota grown in continuous culture. Nitrate reductase (NaR) activity and nitrite concentrations were not significantly different to tongue-derived inocula in model biofilms. These were generally dominated by Streptococcus spp., initially, and a single nitrate supplementation resulted in the increased relative abundance of the nitrate-reducing genera Veillonella, Neisseria, and Proteus spp. Nitrite concentrations increased concomitantly and continued to increase throughout oral microbiota development. Continuous nitrate supplementation, over a 7-day period, was similarly associated with an elevated abundance of nitratereducing taxa and increased nitrite concentration in the perfusate. In experiments in which the models were established in continuous low or high nitrate environments, there was an initial elevation in nitrate reductase, and nitrite concentrations reached a relatively constant concentration over time similar to the acute nitrate challenge with a similar expansion of Veillonella and Neisseria. In summary, we have investigated nitrate metabolism in continuous culture oral biofilms, showing that nitrate addition increases nitrate reductase activity and nitrite concentrations in oral microbiota with the expansion of putatively NaR-producing taxa. IMPORTANCE Clinical evidence suggests that blood pressure regulation can be promoted by nitrite generated through the reduction of supplemental dietary nitrate by the oral microbiota. We have utilized oral microbiota models to investigate the mechanisms responsible, demonstrating that nitrate addition increases nitrate reductase activity and nitrite concentrations in oral microbiota with the expansion of nitrate-reducing taxa. [ABSTRACT FROM AUTHOR]

Complications of pregnancy represent a significant disease burden, with both immediate and lasting consequences for mother and baby. Two key pregnancy complications, fetal growth restriction (FGR) and preeclampsia (PE), together affect around 10%-15% of all pregnancies worldwide. Despite this high incidence, there are currently no therapies available to treat these pregnancy disorders. Early delivery remains the only intervention to reduce the risk of severe maternal complications and/or stillbirth of the baby; however early delivery itself is associated with increased risk of neonatal mortality and morbidity. As such, there is a pressing need to develop new and effective treatments that can prevent or treat FGR and PE. Animal models have been essential in identifying and screening potential new therapies in this field. In this review, we address recent progress that has been made in developing therapeutic strategies for pregnancy disorders, some of which are now entering clinical trials. [ABSTRACT FROM AUTHOR]

Fetal growth restriction associates with increased risk of adult cardiometabolic and neuropsychiatric disorders. Both maternal malnutrition [notably a low-protein (LP) diet] and stress/glucocorticoid exposure reduce fetal growth and cause persisting abnormalities (programming) in adult offspring. Deficiency of placental 11-hydroxysteroid dehydrogenase-2 (11β-HSD2), which inactivates glucocorticoids, is reduced by an LP diet and has been proposed as a unifying mechanism. Here, we explored the importance of glucocorticoids and placental 11β-HSD2 in dietary programming. Pregnant mice were fed a control or isocaloric LP diet throughout gestation. The LP diet first elevated fetal glucocorticoid levels, then reduced placental growth, and finally decreased fetal weight near term by 17%. Whereas the LP diet reduced placental 11β-HSD2 activity near term by ~25%, consistent with previous reports, activity was increased between 20 and 40% at earlier ages, implying that glucocorticoid overexposure in LP fetuses occurs via 11β-HSD2-indepen- dent mechanisms. Consistent with this, heterozygous 11β-+/- HSD2 crosses showed that although both LP and 11β-HSD2 deficiency reduced fetal growth, LP indeed acted independently of 11β-HSD2. Instead, the LP diet induced the fetal hypothalamic-pituitary-adrenal axis per se. Thus, maternal malnutrition and placental 11-HSD2 deficiency act via distinct processes to retard fetal growth, both involving fetoplacental overexposure to glucocorticoids but from distinct sources. [ABSTRACT FROM AUTHOR]

Oral supplementation with L-citrulline, which is sequentially converted to L-arginine then nitric oxide, improves vascular biomarkers and reduces blood pressure in non-pregnant, hypertensive human cohorts and pregnant mice with a pre-eclampsia-like syndrome. This early-phase randomised feasibility trial assessed the acceptability of L-citrulline supplementation to pregnant women with chronic hypertension and its effects on maternal BP and other vascular outcomes. Pregnant women with chronic hypertension were randomised at 12–16 weeks to receive 3-g L-citrulline twice daily (n = 24) or placebo (n = 12) for 8 weeks. Pregnant women reported high acceptability of oral L-citrulline. Treatment increased maternal plasma levels of citrulline, arginine and the arginine:asymmetric dimethylarginine ratio, particularly in women reporting good compliance. L-citrulline had no effect on diastolic BP (L-citrulline: − 1.82 95% CI (− 5.86, 2.22) vs placebo: − 5.00 95% CI (− 12.76, 2.76)), uterine artery Doppler or angiogenic biomarkers. Although there was no effect on BP, retrospectively, this study was underpowered to detect BP changes < 9 mmHg, limiting the conclusions about biological effects. The increase in arginine:asymmetric dimethylarginine ratio was less than in non-pregnant populations, which likely reflects altered pharmacokinetics of pregnancy, and further pharmacokinetic assessment of L-citrulline in pregnancy is advised. Trial Registration EudraCT 2015-005792-25 (2017-12-22) and ISRCTN12695929 (2018-09-20) In pregnant women with chronic hypertension, L-citrulline is an acceptable intervention which increased plasma L-citrulline bioavailability but did not affect BP, potentially due to altered pharmacokinetics of pregnancy. [ABSTRACT FROM AUTHOR]

Insufficient oxygen supply (hypoxia) during fetal development leads to cardiac remodeling and a predisposition to cardiovascular disease in later life. Previous work has shown hypoxia causes oxidative stress in the fetal heart and alters the activity and expression of mitochondrial proteins in a sex‐dependent manner. However, the functional effects of these modifications on mitochondrial respiration remain unknown. Furthermore, while maternal antioxidant treatments are emerging as a promising new strategy to protect the hypoxic fetus, whether these treatments convey similar protection to cardiac mitochondria in the male or female fetus has not been investigated. Therefore, using an established rat model, we measured the sex‐dependent effects of gestational hypoxia and maternal melatonin treatment on fetal cardiac mitochondrial respiration, reactive oxygen species (ROS) production, and lipid peroxidation. Pregnant Wistar rats were subjected to normoxia or hypoxia (13% oxygen) during gestational days (GDs) 6–20 (term ~22 days) with or without melatonin treatment (5 µg/ml in maternal drinking water). On GD 20, mitochondrial aerobic respiration and H2O2 production were measured in fetal heart tissue, together with lipid peroxidation and citrate synthase (CS) activity. Gestational hypoxia reduced maternal body weight gain (p <.01) and increased placental weight (p <.05) but had no effect on fetal weight or litter size. Cardiac mitochondria from male but not female fetuses of hypoxic pregnancy had reduced respiratory capacity at Complex II (CII) (p <.05), and an increase in H2O2 production/O2 consumption (p <.05) without any changes in lipid peroxidation. CS activity was also unchanged in both sexes. Despite maternal melatonin treatment increasing maternal and fetal plasma melatonin concentration (p <.001), melatonin treatment had no effect on any of the mitochondrial parameters investigated. To conclude, we show that gestational hypoxia leads to ROS generation from the mitochondrial electron transport chain and affects fetal cardiac mitochondrial respiration in a sex‐dependent manner. We also show that maternal melatonin treatment had no effect on these relationships, which has implications for the development of future therapies for hypoxic pregnancies. [ABSTRACT FROM AUTHOR]

Key points: Maternal hypertension is associated with increased rates of pregnancy pathologies, including fetal growth restriction, due at least in part to reductions in nitric oxide (NO) bioavailability and associated vascular dysfunction.Dietary nitrate supplementation, from beetroot juice (BRJ), has been shown to increase NO bioavailability and improve cardiovascular function in both preclinical and clinical studies.This study is the first to investigate effects of dietary nitrate supplementation in a pregnant animal model. Importantly, the effects of nitrate‐containing BRJ were compared with both 'placebo' (nitrate‐depleted) BRJ as well as water to control for potential nitrate‐independent effects.Our data show novel, nitrate‐independent effects of BRJ to lower blood pressure and improve vascular function in endothelial nitric oxide synthase knockout (eNOS−/−) mice.These findings suggest potential beneficial effects of BRJ supplementation in pregnancy, and emphasize the importance of accounting for nitrate‐independent effects of BRJ in study design and interpretation. Maternal hypertension is associated with adverse pregnancy outcomes, including fetal growth restriction (FGR), due in part to reductions in nitric oxide (NO) bioavailability. We hypothesized that maternal dietary nitrate administration would increase NO bioavailability to reduce systolic blood pressure (SBP), improve vascular function and increase fetal growth in pregnant endothelial NO synthase knockout (eNOS−/−) mice, which exhibit hypertension, endothelial dysfunction and FGR. Pregnant wildtype (WT) and eNOS−/− mice were supplemented with nitrate‐containing beetroot juice (BRJ+) from gestational day (GD) 12.5. Control mice received an equivalent dose of nitrate‐depleted BRJ (BRJ−) or normal drinking water. At GD17.5, maternal SBP was measured; at GD18.5, maternal nitrate/nitrite concentrations, uterine artery (UtA) blood flow and endothelial function were assessed, and pregnancy outcomes were determined. Plasma nitrate concentrations were increased in both WT and eNOS−/− mice supplemented with BRJ+ (P < 0.001), whereas nitrite concentrations were increased only in eNOS−/− mice (P < 0.001). BRJ− did not alter nitrate/nitrite concentrations. SBP was lowered and UtA endothelial function was enhanced in eNOS−/− mice supplemented with either BRJ+ or BRJ−, indicating nitrate‐independent effects of BRJ. Improvements in endothelial function in eNOS−/− mice were abrogated in the presence of 25 mm KCl, implicating enhanced EDH signalling in BRJ− treated animals. At GD18.5, eNOS−/− fetuses were significantly smaller than WT animals (P < 0.001), but BRJ supplementation did not affect fetal weight. BRJ may be a beneficial intervention in pregnancies associated with hypertension, endothelial dysfunction and reduced NO bioavailability. Our data showing biological effects of non‐nitrate components of BRJ have implications for both interpretation of previous findings and in the design of future clinical trials. Key points: Maternal hypertension is associated with increased rates of pregnancy pathologies, including fetal growth restriction, due at least in part to reductions in nitric oxide (NO) bioavailability and associated vascular dysfunction.Dietary nitrate supplementation, from beetroot juice (BRJ), has been shown to increase NO bioavailability and improve cardiovascular function in both preclinical and clinical studies.This study is the first to investigate effects of dietary nitrate supplementation in a pregnant animal model. Importantly, the effects of nitrate‐containing BRJ were compared with both 'placebo' (nitrate‐depleted) BRJ as well as water to control for potential nitrate‐independent effects.Our data show novel, nitrate‐independent effects of BRJ to lower blood pressure and improve vascular function in endothelial nitric oxide synthase knockout (eNOS−/−) mice.These findings suggest potential beneficial effects of BRJ supplementation in pregnancy, and emphasize the importance of accounting for nitrate‐independent effects of BRJ in study design and interpretation. [ABSTRACT FROM AUTHOR]

Abstract Chronic hypertension in pregnancy is associated with significant adverse pregnancy outcomes, increasing the risk of pre-eclampsia, fetal growth restriction and preterm birth. Dietary nitrate, abundant in green leafy vegetables and beetroot, is reduced in vivo to nitrite and subsequently nitric oxide, and has been demonstrated to lower blood pressure, improve vascular compliance and enhance blood flow in non-pregnant humans and animals. The primary aims of this study were to determine the acceptability and efficacy of dietary nitrate supplementation, in the form of beetroot juice, to lower blood pressure in hypertensive pregnant women. In this double-blind, placebo-controlled feasibility trial, 40 pregnant women received either daily nitrate supplementation (70 mL beetroot juice, n = 20) or placebo (70 mL nitrate-depleted beetroot juice, n = 20) for 8 days. Blood pressure, cardiovascular function and uteroplacental blood flow was assessed at baseline and following acute (3 h) and prolonged (8 days) supplementation. Plasma and salivary samples were collected for analysis of nitrate and nitrite concentrations and acceptability of this dietary intervention was assessed based on questionnaire feedback. Dietary nitrate significantly increased plasma and salivary nitrate/nitrite concentrations compared with placebo juice (p < 0.001), with marked variation between women. Compared with placebo, there was no overall reduction in blood pressure in the nitrate-treated group; however there was a highly significant correlation between changes in plasma nitrite concentrations and changes in diastolic blood pressure in the nitrate-treated arm only (r = −0.6481; p = 0.0042). Beetroot juice supplementation was an acceptable dietary intervention to 97% of women. This trial confirms acceptability and potential efficacy of dietary nitrate supplementation in pregnant women. Conversion of nitrate to nitrite critically involves oral bacterial nitrate reductase activities. We speculate that differences in efficacy of nitrate supplementation relate to differences in the oral microbiome, which will be investigated in future studies. Highlights • Dietary nitrate supplementation, via beetroot juice, has been shown to lower blood pressure in non-pregnant hypertensive adults. • This feasibility trial investigated whether nitrate supplementation via beetroot juice was acceptable to, and effective at lowering blood pressure in, hypertensive pregnant women. • Nitrate supplementation, via beetroot juice, was a highly acceptable dietary intervention to pregnant women. • In this feasibility trial, dietary nitrate supplementation did not significantly lower clinic BP compared with placebo. • A significant association between plasma nitrite concentrations and diastolic blood pressure lowering was seen, in the treatment arm only. [ABSTRACT FROM AUTHOR]

Abstract Adequate perfusion of the placental vasculature is essential to meet the metabolic demands of fetal growth and development. Lacking neural control, local tissue metabolites, circulating and physical factors contribute significantly to blood flow regulation. Nitric oxide (NO) is a key regulator of fetoplacental vascular tone. Nitrite, previously considered an inert end-product of NO oxidation, has been shown to provide an important source of NO. Reduction of nitrite to NO may be particularly relevant in tissue when the oxygen-dependent NO synthase (NOS) activity is compromised, e.g. in hypoxia. The contribution of this pathway in the placenta is currently unknown. We hypothesised that nitrite vasodilates human placental blood vessels, with enhanced efficacy under hypoxia. Placentas were collected from uncomplicated pregnancies and the vasorelaxant effect of nitrite (10−6–5x10−3 M) was assessed using wire myography on isolated pre-constricted chorionic plate arteries (CPAs) and veins (CPVs) under normoxic (p O 2 ∼5%) and hypoxic (p O 2 ∼1%) conditions. The dependency on the NO–sGC–cGMP pathway and known nitrite reductase (NiR) activities was also investigated. Nitrite caused concentration-dependent vasorelaxation in both arteries and veins, and this effect was enhanced by hypoxia, significantly in CPVs (P < 0.01) and with a trend in CPAs (P = 0.054). Pre-incubation with NO scavengers (cPTIO and oxyhemoglobin) attenuated (P < 0.01 and P < 0.0001, respectively), and the sGC inhibitor ODQ completely abolished nitrite-mediated vasorelaxation, confirming the involvement of NO and sGC. Inhibition of potential NiR enzymes xanthine oxidoreductase, mitochondrial aldehyde dehydrogenase and mitochondrial bc 1 complex did not attenuate vasorelaxation. This data suggests that nitrite may provide an important reservoir of NO bioactivity within the placenta to enhance blood flow when fetoplacental oxygenation is impaired, as occurring in pregnancy diseases such as pre-eclampsia and fetal growth restriction. Highlights • Nitrite has been shown to induce vasorelaxation in a number of vascular beds, with enhanced efficacy under hypoxia. • Here, we show that nitrite induces vasorelaxation also in isolated human placental blood vessels. • Our data suggest nitrite vasorelaxation in placental blood vessels is via nitrite->nitric oxide reduction. • The identity of the nitrite reductases responsible for the observed effects in this vascular bed remain unknown. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is essential in the control of fetoplacental vascular tone, maintaining a high flow−low resistance circulation that favors oxygen and nutrient delivery to the fetus. Reduced fetoplacental blood flow is associated with pregnancy complications and is one of the major causes of fetal growth restriction (FGR). The reduction of dietary nitrate to nitrite and subsequently NO may provide an alternative source of NO in vivo. We have previously shown that nitrite induces vasorelaxation in placental blood vessels from normal pregnancies, and that this effect is enhanced under conditions of hypoxia. Herein, we aimed to determine whether nitrite could also act as a vasodilator in FGR. Using wire myography, vasorelaxant effects of nitrite were assessed on pre-constricted chorionic plate arteries (CPAs) and veins (CPVs) from normal and FGR pregnancies under normoxic and hypoxic conditions. Responses to the NO donor, sodium nitroprusside (SNP), were assessed in parallel. Nitrate and nitrite concentrations were measured in fetal plasma. Hypoxia significantly enhanced vasorelaxation to nitrite in FGR CPAs (p < 0.001), and in both normal (p < 0.001) and FGR (p < 0.01) CPVs. Vasorelaxation to SNP was also potentiated by hypoxia in both normal (p < 0.0001) and FGR (p < 0.01) CPVs. However, compared to vessels from normal pregnancies, CPVs from FGR pregnancies showed significantly lower reactivity to SNP (p < 0.01). Fetal plasma concentrations of nitrate and nitrite were not different between normal and FGR pregnancies. Together, these data show that nitrite-mediated vasorelaxation is preserved in FGR, suggesting that interventions targeting this pathway have the potential to improve fetoplacental blood flow in FGR pregnancies. [ABSTRACT FROM AUTHOR]