Targeted Delivery of Epidermal Growth Factor to the Human Placenta to Treat Fetal Growth Restriction.

Placental dysfunction is the underlying cause of pregnancy complications such as fetal growth restriction (FGR) and pre-eclampsia. No therapies are available to treat a poorly functioning placenta, primarily due to the risks of adverse side effects in both the mother and the fetus resulting from systemic drug delivery. The use of targeted liposomes to selectively deliver payloads to the placenta has the potential to overcome these issues. In this study, we assessed the safety and efficacy of epidermal growth factor (EGF)-loaded, peptide-decorated liposomes to improve different aspects of placental function, using tissue from healthy control pregnancies at term, and pregnancies complicated by FGR. Phage screening identified a peptide sequence, CGPSARAPC (GPS), which selectively homed to mouse placentas in vivo, and bound to the outer syncytiotrophoblast layer of human placental explants ex vivo. GPS-decorated liposomes were prepared containing PBS or EGF (50-100 ng/mL), and placental explants were cultured with liposomes for up to 48 h. Undecorated and GPS-decorated liposomes containing PBS did not affect the basal rate of amino acid transport, human chorionic gonadotropin (hCG) release or cell turnover in placental explants from healthy controls. GPS-decorated liposomes containing EGF significantly increased amino acid transporter activity in healthy control explants, but not in placental explants from women with FGR. hCG secretion and cell turnover were unaffected by EGF delivery; however, differential activation of downstream protein kinases was observed when EGF was delivered via GPS-decorated vs. undecorated liposomes. These data indicate that targeted liposomes represent a safe and useful tool for the development of new therapies for placental dysfunction, recapitulating the effects of free EGF.

Enhanced Nitrite-Mediated Relaxation of Placental Blood Vessels Exposed to Hypoxia Is Preserved in Pregnancies Complicated by Fetal Growth Restriction.

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.

Kynurenine Relaxes Arteries of Normotensive Women and Those With Preeclampsia.

[Figure: see text].

Investigation of the outcome of pregnancies complicated by increased fetal movements and their relation to underlying causes - A prospective cohort study.

INTRODUCTION: Retrospective studies have reported an association between a single episode of significantly increased fetal movements (IFMs) and stillbirth after 28 weeks' gestation. This prospective study aimed to report the outcome of pregnancies associated with maternal perception of IFMs and determine whether this symptom is associated with adverse pregnancy outcome, a pathological intrauterine environment or placental dysfunction. MATERIAL AND METHODS: Women reporting IFMs after 28 weeks' gestation were recruited from St Mary's Hospital, Manchester and Liverpool Women's Hospital, UK, between 1 November 2017 and 1 May 2019. Demographic and clinical information were obtained and an ultrasound scan was performed to assess fetal biometry, liquor volume and umbilical artery Doppler. Maternal serum samples were collected for analysis of placentally derived biomarkers using ELISA. After delivery, maternal and fetal outcome data were collected and placentas and umbilical cord blood were obtained for analysis using immunohistochemistry and ELISA, respectively. Placental and serum samples were matched by gestation and maternal characteristics to participants with normal fetal activity. RESULTS: Seventy-seven women presented with IFM, representing 0.45% of the maternity population; 64 women consented to participate in the study, of which 7 (10.9%) experienced adverse pregnancy outcome: birthweight <3rd centile, 2 (3.1%); pH ≤7.10, 1 (1.6%); neonatal intensive care unit admission, 4 (6.3%). Women had IFM for varying lengths of time before presenting: 17.2% had IFM for less than 1 hour and 29.7% reported IFM lasting longer than 24 hours. Four women (6.3%) had abnormalities of the fetal heart rate trace on assessment. Women with IFM had similar modes of birth to women giving birth in participating maternity units. There was no evidence of macroscopic placental or umbilical cord abnormalities, alterations in microscopic placental structure, placental endocrine dysfunction or intrauterine hypoxia or infection in women with IFM compared with controls. CONCLUSIONS: This prospective study did not find evidence of an association between IFM and adverse pregnancy outcome. It also did not find evidence of underlying placental dysfunction, cord anomalies, intrauterine hypoxia or infection in pregnancies with IFM. Further work is required to determine the strength of association between IFM and adverse pregnancy outcome and its origins. At present, IFM cannot be used to identify fetuses at increased risk of adverse outcome.

Grape Seed Extract Polyphenols Improve Resistance Artery Function in Pregnant eNOS-/- Mice.

Hypertension during pregnancy is a leading cause of maternal and fetal morbidity and mortality worldwide, increasing the risk of complications including preeclampsia, intracerebral hemorrhage and fetal growth restriction. Increased oxidative stress is known to contribute to poor vascular function; however, trials of antioxidant supplementation have raised concerns about fetal outcomes, including risk of low birthweight. Grape seed extract polyphenols (GSEP) have been suggested to promote cardiovascular protection, at least in part through antioxidant actions. We tested the hypothesis that administration of GSEP during pregnancy would reduce oxidative stress and improve resistance artery function with no detrimental effects on fetal growth, in an established model of maternal hypertension associated with vascular dysfunction, the endothelial NO synthase knockout (eNOS-/-) mouse. Pregnant C57BL/6J (WT) and eNOS-/- mice received either GSEP (200 mg/kg/day) or drinking water, between gestational (GD) day 10.5 and GD18.5. At GD17.5, maternal systolic blood pressure (SBP) was measured; at GD18.5, maternal malondialdehyde (MDA) concentrations, vascular function of aortic, mesenteric, uterine and posterior cerebral arteries was assessed, and fetal outcome evaluated. GSEP reduced maternal SBP (P < 0.01) and plasma MDA concentrations (P < 0.01) in eNOS-/- mice. Whilst there was no effect of GSEP on vascular reactivity of aortas, GSEP improved endothelial-dependent relaxation in mesenteric and uterine arteries of eNOS-/- mice (P < 0.05 and P < 0.001, respectively) and normalized lumen diameters of pressurized posterior cerebral arteries in eNOS-/- mice (P < 0.001). Supplementation with GSEP had no effect in WT mice and did not affect fetal outcomes in either genotype. Our data suggest that GSEP improve resistance artery function, potentially through antioxidant actions, and provide a basis to further investigate these beneficial effects including in the prevention of intracerebral hemorrhage. Maternal supplementation with GSEP may be a safe intervention to improve outcomes in pregnancies associated with hypertension and vascular dysfunction.

Human placental uptake of glutamine and glutamate is reduced in fetal growth restriction.

Fetal growth restriction (FGR) is a significant risk factor for stillbirth, neonatal complications and adulthood morbidity. Compared with those of appropriate weight for gestational age (AGA), FGR babies have smaller placentas with reduced activity of amino acid transporter systems A and L, thought to contribute to poor fetal growth. The amino acids glutamine and glutamate are essential for normal placental function and fetal development; whether transport of these is altered in FGR is unknown. We hypothesised that FGR is associated with reduced placental glutamine and glutamate transporter activity and expression, and propose the mammalian target of rapamycin (mTOR) signaling pathway as a candidate mechanism. FGR infants [individualised birth weight ratio (IBR) < 5th centile] had lighter placentas, reduced initial rate uptake of 14C-glutamine and 14C-glutamate (per mg placental protein) but higher expression of key transporter proteins (glutamine: LAT1, LAT2, SNAT5, glutamate: EAAT1) versus AGA [IBR 20th-80th]. In further experiments, in vitro exposure to rapamycin inhibited placental glutamine and glutamate uptake (24 h, uncomplicated pregnancies) indicating a role of mTOR in regulating placental transport of these amino acids. These data support our hypothesis and suggest that abnormal glutamine and glutamate transporter activity is part of the spectrum of placental dysfunction in FGR.

Beetroot juice lowers blood pressure and improves endothelial function in pregnant eNOS-/- mice: importance of nitrate-independent effects.

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: 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.

Increased placental macrophages and a pro-inflammatory profile in placentas and maternal serum in infants with a decreased growth rate in the third trimester of pregnancy.

PROBLEM: There is growing evidence for the role of placental inflammation in the pathophysiology of pregnancy complications including fetal growth restriction (FGR). This study aimed to characterize the inflammatory profile in the maternal circulation and the placenta of infants who were growth restricted and those that were small for gestational age (SGA). METHOD OF STUDY: Placental villous tissue and maternal serum were obtained from pregnancies where infants were SGA at birth or who had a decreasing growth rate (≥25 centiles) across the third trimester. Immunohistochemical and histological analyses of placental samples were conducted for macrophage number, alongside vascular and cell turnover analysis. Inflammatory profile was analyzed in maternal and placental compartments via ELISAs and multiplex assays. RESULTS: There were significantly more CD163+ macrophages in placentas of infants with a decreased growth rate compared to controls, but not in SGA infants (median 8.6/ nuclei vs 3.8 and 2.9, P = .008 and P = .003, respectively). Uric acid (P = .0007) and IL-8 (P = .0008) were increased in placentas, and S100A8 (P < .0002) was increased in maternal serum of infants with decreased growth rate. No changes in the maternal serum or placental lysates of SGA infants were observed. CONCLUSION: The evidence of an altered inflammatory profile in infants with a decreasing growth rate, but not in those that were born SGA, provides further evidence that inflammation plays a role in true FGR. It remains unclear whether the increased placental macrophages occur as a direct result, or as a consequence of the pro-inflammatory environment observed in fetal growth restriction.

Evidence of adaptation of maternofetal transport of glutamine relative to placental size in normal mice, and in those with fetal growth restriction.

KEY POINTS: Fetal growth restriction (FGR) is a major risk factor for stillbirth and has significant impact upon lifelong health. A small, poorly functioning placenta, as evidenced by reduced transport of nutrients to the baby, underpins FGR. It remains unclear how a small but normal placenta differs from the small FGR placenta in terms of ability to transfer nutrients to the fetus. Placental transport of glutamine and glutamate, key amino acids for fetal growth, was assessed in normal mice and those with FGR. Glutamine and glutamate transport was greater in the lightest versus heaviest placenta in a litter of normally grown mice. Placentas of mice with FGR had increased transport capacity in mid-pregnancy, but this adaptation was insufficient in late pregnancy. Placental adaptations, in terms of increased nutrient transport (per gram) to compensate for small size, appear to achieve appropriate fetal growth in normal pregnancy. Failure of this adaptation might contribute to FGR. ABSTRACT: Fetal growth restriction (FGR), a major risk factor for stillbirth, and neonatal and adulthood morbidity, is associated with reduced placental size and decreased placental nutrient transport. In mice, a small, normal placenta increases its nutrient transport, thus compensating for its reduced size and maintaining normal fetal growth. Whether this adaptation occurs for glutamine and glutamate, two key amino acids for placental metabolism and fetal growth, is unknown. Additionally, an assessment of placental transport of glutamine and glutamate between FGR and normal pregnancy is currently lacking. We thus tested the hypothesis that the transport of glutamine and glutamate would be increased (per gram of tissue) in a small normal placenta [C57BL6/J (wild-type, WT) mice], but that this adaptation fails in the small dysfunctional placenta in FGR [insulin-like growth factor 2 knockout (P0) mouse model of FGR]. In WT mice, comparing the lightest versus heaviest placenta in a litter, unidirectional maternofetal clearance (Kmf ) of 14 C-glutamine and 14 C-glutamate (glutamine Kmf and glutamate Kmf ) was significantly higher at embryonic day (E) 18.5, in line with increased expression of LAT1, a glutamine transporter protein. In P0 mice, glutamine Kmf and glutamate Kmf were higher (P0 versus wild-type littermates, WTL) at E15.5. At E18.5, glutamine Kmf remained elevated whereas glutamate Kmf was similar between groups. In summary, we provide evidence that glutamine Kmf and glutamate Kmf adapt according to placental size in WT mice. The placenta of the growth-restricted P0 fetus also elevates transport capacity to compensate for size at E15.5, but this adaptation is insufficient at E18.5; this may contribute to decreased fetal growth.

Effects of hydroxychloroquine on the human placenta-Findings from in vitro experimental data and a systematic review.

Hydroxychloroquine (HCQ), a toll like receptor (TLR) 7 and 9 antagonist, is used during pregnancy for inflammatory conditions with limited understanding of its placental toxicology. We hypothesized that HCQ does not have toxic effects on the placenta and can modulate cytokine release in response to TLR7/9 activation. A systematic review was conducted and no studies of HCQ on multicellular human placental tissue were identified. Accordingly, placental villous explants were cultured for 7 days with HCQ +/- TLR7/9 agonists. HCQ did not affect cell turnover, nutrient transport or cytokine release but increased IL-10 (anti-inflammatory) secretion and promoted syncytiotrophoblast regeneration. Cytokine release stimulated by TLR7/9 agonists was unaffected by HCQ. In conclusion, HCQ did not adversely affect placental tissue and may have a protective anti-inflammatory function. Further research is needed to determine the mechanisms of HCQ actions on human placenta and whether they could be utilized to improve pregnancy outcomes.

The kynurenine pathway; A new target for treating maternal features of preeclampsia?

In preeclampsia, vasospasm, oxidative stress, endothelial dysfunction, and immune dysregulation are key mediators of maternal disease. A new time-of-disease treatment is needed with the potential to treat these areas of pathophysiology. A review of the literature has indicated that metabolites of the kynurenine pathway have the potential to; (i) induce vasorelaxation of resistance arteries and reduce blood pressure; (ii) exert antioxidant effects and reduce the effects of poly-ADP ribose polymerase activation (iii) prevent endothelial dysfunction and promote endothelial nitric oxide production; (iv) cause T cell differentiation into tolerogenic regulatory T cells and induce apoptosis of pro-inflammatory Th1 cells. This has led to the hypothesis that increasing Kynurenine pathway activity may offer a new treatment strategy for preeclampsia.

Mechanisms Underpinning Adaptations in Placental Calcium Transport in Normal Mice and Those With Fetal Growth Restriction.

Fetal delivery of calcium, via the placenta, is crucial for appropriate skeletal mineralization. We have previously demonstrated that maternofetal calcium transport, per gram placenta, is increased in the placental specific insulin-like growth factor 2 knockout mouse (P0) model of fetal growth restriction (FGR) compared to wild type littermates (WTL). This effect was mirrored in wild-type (WT) mice comparing lightest vs. heaviest (LvH) placentas in a litter. In both models increased placental calcium transport was associated with normalization of fetal calcium content. Despite this adaptation being observed in small normal (WT), and small dysfunctional (P0) placentas, mechanisms underpinning these changes remain unknown. Parathyroid hormone-related protein (PTHrP), elevated in cord blood in FGR and known to stimulate plasma membrane calcium ATPase, might be important. We hypothesized that PTHrP expression would be increased in LvH WT placentas, and in P0 vs. WTL. We used calcium pathway-focused PCR arrays to assess whether mechanisms underpinning these adaptations in LvH WT placentas, and in P0 vs. WTL, were similar. PTHrP protein expression was not different between LvH WT placentas at E18.5 but trended toward increased expression (139%; P = 0.06) in P0 vs. WTL. PCR arrays demonstrated that four genes were differentially expressed in LvH WT placentas including increased expression of the calcium-binding protein calmodulin 1 (1.6-fold; P < 0.05). Twenty-four genes were differentially expressed in placentas of P0 vs. WTL; significant reductions were observed in expression of S100 calcium binding protein G (2-fold; P < 0.01), parathyroid hormone 1 receptor (1.7-fold; P < 0.01) and PTHrP (2-fold; P < 0.05), whilst serum/glucocorticoid-regulated kinase 1 (SGK1), a regulator of nutrient transporters, was increased (1.4 fold; P < 0.05). Tartrate resistant acid phosphatase 5 (TRAP5 encoded by Acp5) was reduced in placentas of both LvH WT and P0 vs. WTL (1.6- and 1.7-fold, respectively; P < 0.05). Signaling events underpinning adaptations in calcium transport are distinct between LvH placentas of WT mice and those in P0 vs. WTL. Calcium binding proteins appear important in functional adaptations in the former whilst PTHrP and SGK1 are also implicated in the latter. These data facilitate understanding of mechanisms underpinning placental calcium transport adaptation in normal and growth restricted fetuses.

Nitrite mediated vasorelaxation in human chorionic plate vessels is enhanced by hypoxia and dependent on the NO-sGC-cGMP pathway.

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 (pO2 ∼5%) and hypoxic (pO2 ∼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 bc1 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.

Pomegranate Juice Supplementation Alters Utero-Placental Vascular Function and Fetal Growth in the eNOS-/- Mouse Model of Fetal Growth Restriction.

The eNOS-/- mouse provides a well-characterized model of fetal growth restriction (FGR) with altered uterine and umbilical artery function and reduced utero- and feto-placental blood flow. Pomegranate juice (PJ), which is rich in antioxidants and bioactive polyphenols, has been posited as a beneficial dietary supplement to promote cardiovascular health. We hypothesized that maternal supplementation with PJ will improve uterine and umbilical artery function and thereby enhance fetal growth in the eNOS-/- mouse model of FGR. Wild type (WT, C57Bl/6J) and eNOS-/- mice were supplemented from E12.5-18.5 with either PJ in their drinking water or water alone. At E18.5 uterine (UtA) and umbilical (UmbA) arteries were isolated for study of vascular function, fetuses and placentas were weighed and fetal biometric measurements taken. PJ supplementation significantly increased UtA basal tone (both genotypes) and enhanced phenylephrine-induced contraction in eNOS-/- but not WT mice. Conversely PJ significantly reduced UtA relaxation in response to both acetylcholine (Ach) and sodium nitroprusside (SNP), endothelium dependent and independent vasodilators respectively from WT but not eNOS-/- mice. UmbA sensitivity to U46619-mediated contraction was increased by PJ supplementation in WT mice; PJ enhanced contraction and relaxation of UmbA to Ach and SNP respectively in both genotypes. Contrary to our hypothesis, the changes in artery function induced by PJ were not associated with an increase in fetal weight. However, PJ supplementation reduced litter size and fetal abdominal and head circumference in both genotypes. Collectively the data do not support maternal PJ supplementation as a safe or effective treatment for FGR.

Equilibrative Nucleoside Transporter 1 (ENT1, SLC29A1) Facilitates Transfer of the Antiretroviral Drug Abacavir across the Placenta.

Abacavir is a preferred antiretroviral drug for preventing mother-to-child human immunodeficiency virus transmission; however, mechanisms of its placental transfer have not been satisfactorily described to date. Because abacavir is a nucleoside-derived drug, we hypothesized that the nucleoside transporters, equilibrative nucleoside transporters (ENTs, SLC29A) and/or Na+-dependent concentrative nucleoside transporters (CNTs, SLC28A), may play a role in its passage across the placenta. To test this hypothesis, we performed uptake experiments using the choriocarcinoma-derived BeWo cell line, human fresh villous fragments, and microvillous plasma membrane (MVM) vesicles. Using endogenous substrates of nucleoside transporters, [3H]-adenosine (ENTs, CNT2, and CNT3) and [3H]-thymidine (ENTs, CNT1, and CNT3), we showed significant activity of ENT1 and CNT2 in BeWo cells, whereas experiments in the villous fragments and MVM vesicles, representing a model of the apical membrane of a syncytiotrophoblast, revealed only ENT1 activity. When testing [3H]-abacavir uptakes, we showed that of the nucleoside transporters, ENT1 plays the dominant role in abacavir uptake into placental tissues, whereas contribution of Na+-dependent transport, most likely mediated by CNTs, was observed only in BeWo cells. Subsequent experiments with dually perfused rat term placentas showed that Ent1 contributes significantly to overall [3H]-abacavir placental transport. Finally, we quantified the expression of SLC29A in first- and third-trimester placentas, revealing that SLC29A1 is the dominant isoform. Neither SLC29A1 nor SLC29A2 expression changed over the course of placental development, but there was considerable interindividual variability in their expression. Therefore, drug-drug interactions and the effect of interindividual variability in placental ENT1 expression on abacavir disposition into fetal circulation should be further investigated to guarantee safe and effective abacavir-based combination therapies in pregnancy.

Effects of dietary nitrate supplementation, from beetroot juice, on blood pressure in hypertensive pregnant women: A randomised, double-blind, placebo-controlled feasibility trial.

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.

The problem with using the birthweight:placental weight ratio as a measure of placental efficiency.

INTRODUCTION: The ratio of birthweight to placental weight (BW:PW) is often used as a measure of placental efficiency in humans and animals. However, ratios have properties that are known to lead to spurious results. An alternative approach is the use of residuals from regression, which reflect whether birthweight is higher or lower than expected for a given placental weight, given the population pattern. We hypothesized that biologically meaningful measures of placental efficiency would differ between placentas with and without pathology, and between adverse and normal perinatal and postnatal outcomes. METHODS: We examined associations between measures of placental efficiency (BW:PW ratio or residuals) and placental pathology, Apgar scores and infant death using National Collaborative Perinatal Project data (4645 preterm births and 28497 term births). RESULTS: BW:PW ratios and residuals were significantly lower in placentas showing pathologies including signs of large infarcts or hemorrhage, although many of these differences were small. Low BW:PW ratios and residuals were also associated with low Apgar scores and increased risk of postnatal death. Whereas residuals were lower in term placentas that appeared immature by microscopic examination, the opposite was true for BW:PW ratios. CONCLUSION: The BW:PW ratio produced an artefact whereby histologically less mature placentas at term appeared to be more "efficient" than mature placentas, illustrating a known problem with the use of ratios. For other traits, residuals generally showed differences between placentas with and without pathology that were as great as those seen with BW:PW ratios, and often showed stronger associations with adverse outcomes.

Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction?

Cell free hemoglobin impairs vascular function and blood flow in adult cardiovascular disease. In this study, we investigated the hypothesis that free fetal hemoglobin (fHbF) compromises vascular integrity and function in the fetoplacental circulation, contributing to the increased vascular resistance associated with fetal growth restriction (FGR). Women with normal and FGR pregnancies were recruited and their placentas collected freshly postpartum. FGR fetal capillaries showed evidence of erythrocyte vascular packing and extravasation. Fetal cord blood fHbF levels were higher in FGR than in normal pregnancies ( P < 0.05) and the elevation of fHbF in relation to heme oxygenase-1 suggests a failure of expected catabolic compensation, which occurs in adults. During ex vivo placental perfusion, pathophysiological fHbF concentrations significantly increased fetal-side microcirculatory resistance ( P < 0.05). fHbF sequestered NO in acute and chronic exposure models ( P < 0.001), and fHbF-primed placental endothelial cells developed a proinflammatory phenotype, demonstrated by activation of NF-κB pathway, generation of IL-1α and TNF-α (both P < 0.05), uncontrolled angiogenesis, and disruption of endothelial cell flow alignment. Elevated fHbF contributes to increased fetoplacental vascular resistance and impaired endothelial protection. This unrecognized mechanism for fetal compromise offers a novel insight into FGR as well as a potential explanation for associated poor fetal outcomes such as fetal demise and stillbirth.-Brook, A., Hoaksey, A., Gurung, R., Yoong, E. E. C., Sneyd, R., Baynes, G. C., Bischof, H., Jones, S., Higgins, L. E., Jones, C., Greenwood, S. L., Jones, R. L., Gram, M., Lang, I., Desoye, G., Myers, J., Schneider, H., Hansson, S. R., Crocker, I. P., Brownbill, P. Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction?

Knowledge needed about the exchange physiology of the placenta.

There is now a basic understanding of the driving forces and mechanisms underlying rates of solute exchange across the placenta but there are still major gaps in knowledge. Here we summarise this basic understanding, whilst highlighting gaps in knowledge. We then focus on two particular areas where more knowledge is needed: (1) the electrical potential difference (PD) across the placenta and (2) the paracellular permeability of the placenta to hydrophilic solutes. In many species a PD has been recorded between a catheter in a maternal blood vessel and one in a fetal vessel. However, the key question is whether this PD is the same as that across the placental exchange barrier. We addressed this in the human placenta using microelectrodes to measure the PD in isolated villi in vitro; the transtrophoblast PD so measured had a median value of -3 mV (range 0-15 mV). There have been no subsequent studies to validate this measurement. The syncytiotrophoblast of haemochorial placentas lacks any obvious extracellular water filled paracellular space between the syncytial nuclei. However, in mouse, rat, guinea pig and human there is an inverse relationship between the rate of diffusion of inert hydrophilic solutes across the placenta and their molecular size. The simplest explanation is that a paracellular route exists but its morphological identity is still uncertain. Areas of syncytial denudation could provide a paracellular route but this has not been proven. Answers to these and similar questions are required to fully understand the exchange physiology of the normal placenta and how this is affected in pathology.