Murine models of advanced maternal age: a systematic review and meta-analysis.

In brief: Animal models have been developed to aid understanding of the increased incidence of adverse pregnancy complications observed in women of advanced maternal age (AMA). This systematic review of murine models of AMA demonstrates consistent effects of decreased litter size and fetal weight; this supports the future use of these models to determine pathophysiological mechanisms and test therapeutic strategies to improve poor pregnancy outcomes in AMA. Abstract: Advanced maternal age (AMA; ≥35 years of age) is associated with an increased risk of adverse pregnancy outcomes. To explore causes of adverse pregnancy outcomes in AMA, and to test candidate therapies, an increasing number of murine AMA models have been developed. The aim of this study was to systematically review the literature to assess whether murine AMA models demonstrate a reproducible effect on pregnancy outcomes. PubMed, Ovid, Web of Science and Google Scholar were searched. Studies that reported on pregnancy outcomes in AMA mice and rats were included; the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) tool evaluated the risk of bias. Eleven mouse and six rat studies were included. AMA mice and rats had reduced litter size (standardised mean difference (SMD): -1.59, 95% confidence interval (CI): -1.84, -1.34 for mice; SMD: -1.66, 95% (CI): -2.09, -1.23 for rats) and reduced fetal weight (SMD: -0.87, 95% CI: -1.24, -0.49 for mice; SMD: -1.05, 95% CI: -1.40, -0.69 for rats). Placental weight was increased in AMA mice (SMD: 0.62, 95% CI: 0.16, 1.08). Subgroup analysis indicated that C57Bl/6 mice had less heterogeneity than other, mostly outbred, mouse strains with regards to litter size (C57 strain I2 = 68.2% vs other strain types I2 = 85.7%). The risk of bias was high, mostly due to the lack of methodological detail and unclear reporting of findings. Murine models of AMA demonstrate similar adverse pregnancy outcomes to those observed in large human epidemiological studies. The reproducible phenotypes in AMA murine models allow the exploration of mechanisms underpinning poor pregnancy outcomes and the pursuit of therapeutic interventions.

Kynurenine Relaxes Arteries of Normotensive Women and Those With Preeclampsia.

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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.

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.

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.

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.

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?

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.

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.

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.

Dietary interventions for fetal growth restriction - therapeutic potential of dietary nitrate supplementation in pregnancy.

Fetal growth restriction (FGR) affects around 5% of pregnancies and is associated with significant short- and long-term adverse outcomes. A number of factors can increase the risk of FGR, one of which is poor maternal diet. In terms of pathology, both clinically and in many experimental models of FGR, impaired uteroplacental vascular function is implicated, leading to a reduction in the delivery of oxygen and nutrients to the developing fetus. Whilst mechanisms underpinning impaired uteroplacental vascular function are not fully understood, interventions aimed at enhancing nitric oxide (NO) bioavailability remain a key area of interest in obstetric research. In addition to endogenous NO production from the amino acid l-arginine, via nitric oxide synthase (NOS) enzymes, research in recent years has established that significant NO can be derived from dietary nitrate, via the 'alternative NO pathway'. Dietary nitrate, abundant in green leafy vegetables and beetroot, can increase NO bioactivity, conferring beneficial effects on cardiovascular function and blood flow. Given the beneficial effects of dietary nitrate supplementation to date in non-pregnant humans and animals, current investigations aim to assess the therapeutic potential of this approach in pregnancy to enhance NO bioactivity, improve uteroplacental vascular function and increase fetal growth.

Characterizing Villitis of Unknown Etiology and Inflammation in Stillbirth.

Villitis of unknown etiology (VUE) is an enigmatic inflammatory condition of the placenta associated with fetal growth restriction and stillbirth. Greater understanding of this condition is essential to understand its contribution to adverse outcomes. Our aim was to identify and quantify the cells in VUE in cases of stillbirth and to characterize immune responses specific to this condition. Immunohistochemistry was performed on placentas from stillborn infants whose cause of death was recorded as VUE to identify CD45(+) leukocytes, CD163(+) macrophages, CD4(+) and CD8(+) T cells, neutrophils, and proinflammatory and anti-inflammatory cytokines. Images were quantified with HistoQuest software. CD45(+) leukocytes comprised 25% of cells in VUE lesions: macrophages (12%) and CD4 T cells (11%) being predominant cell types; CD8 T cells were observed in all lesions. Leukocytes and macrophages were increased throughout the placenta in stillbirths; pan-placental CD4(+) and CD8(+) T cells outside VUE lesions were increased in stillbirth with VUE. There was increased IL-2 and IL-12 and reduced IL-4 immunostaining in VUE lesions. Our results suggest VUE in stillbirth has a similar immune cell profile to live birth. Pan-placental macrophages, CD4 and CD8 T cells indicate a wider inflammatory response unrestricted to VUE lesions. The cytokine profile observed suggests a skew towards inappropriate Th1 immune responses. Full characterisation VUE lesion phenotype confirms its immunological origins and provides foundations to develop novel investigations.

In vitro assessment of mouse fetal abdominal aortic vascular function.

Fetal growth restriction (FGR) affects 3-8% of human pregnancies. Mouse models have provided important etiological data on FGR; they permit the assessment of treatment strategies on the physiological function of both mother and her developing offspring. Our study aimed to 1) develop a method to assess vascular function in fetal mice and 2) as a proof of principle ascertain whether a high dose of sildenafil citrate (SC; Viagra) administered to the pregnant dam affected fetal vascular reactivity. We developed a wire myography methodology for evaluation of fetal vascular function in vitro using the placenta-specific insulin-like growth factor II (Igf2) knockout mouse (P0; a model of FGR). Vascular function was determined in abdominal aortas isolated from P0 and wild-type (WT) fetuses at embryonic day (E) 18.5 of gestation. A subset of dams received SC 0.8 mg/ml via drinking water from E12.5; data were compared with water-only controls. Using wire myography, we found that fetal aortic rings exhibited significant agonist-induced contraction, and endothelium-dependent and endothelium-independent relaxation. Sex-specific alterations in reactivity were noted in both strains. Maternal treatment with SC significantly attenuated endothelium-dependent and endothelium-independent relaxation of fetal aortic rings. Mouse fetal abdominal aortas reproducibly respond to vasoactive agents. Study of these vessels in mouse genetic models of pregnancy complications may 1) help to delineate early signs of abnormal vascular reactivity and 2) inform whether treatments given to the mother during pregnancy may impact upon fetal vascular function.

Reduced placental taurine transporter (TauT) activity in pregnancies complicated by pre-eclampsia and maternal obesity.

Taurine is an important nutrient in intrauterine life, being required for fetal organ development and cellular renewal of syncytiotrophoblast (STB), the nutrient transport epithelium of the placenta. As taurine is conditionally essential in human pregnancy, the fetal and placental demand for taurine is met by uptake from maternal blood into STB through the activity of TauT. Pre-eclampsia (PE) and maternal obesity are serious complications of pregnancy, associated with fetal growth restriction (FGR) and abnormal renewal of STB, and maternal obesity is a major risk factor for PE. Here we test the hypothesis that STB TauT activity is reduced in maternal obesity and PE compared to normal pregnancy.STB TauT activity, measured in fragments of placental tissue, was negatively related to maternal BMI over the range 18-46 kg/m(2) in both the first trimester (7-12 weeks gestation) and at term (p < 0.01; linear regression). Neither TauT activity nor expression in the first trimester differed to normal pregnancy at term. STB TauT activity was significantly lower in PE than normal pregnancy (p < 0.01). Neuropeptide Y (NPY), a protein kinase C (PKC) activator which is elevated in PE and obesity, reduced STB TauT activity by 20% (50 pM-50 nM: 2 h) (p < 0.03). Activation of PKC by phorbol 12-myristate-13-acetate (1 µM) reduced TauT activity by 18% (p < 0.05). As TauT activity is inhibited by phosphorylation, we propose that NPY activates PKC in the STB which phosphorylates TauT in PE and maternal obesity.Reduced TauT activity could contribute to dysregulated renewal of STB and FGR that are common to PE and maternal obesity.

Effect of maternal age and growth on placental nutrient transport: potential mechanisms for teenagers' predisposition to small-for-gestational-age birth?

Teenagers have an increased risk of delivering small-for-gestational-age (SGA) infants. Young maternal age and continued skeletal growth have been implicated as causal factors. In growing adolescent sheep, impaired placental development and nutrient transfer cause reduced birth weight. In human pregnancies, SGA is associated with reduced placental amino acid transport. Maternal growth has no effect on placental morphology or cell turnover, but growing teenagers have higher birth weight:placental weight ratios than nongrowing teenagers. We hypothesized that placental nutrient transporter activity would be affected by maternal age and/or growth status. Placentas from teenagers and adults were collected. Teenagers were defined as growing or nongrowing based on knee height measurements. System A amino acid transporter activity was quantified as sodium-dependent uptake of [(14)C]methylaminoisobutyric acid into placental fragments. Teenagers had lower placental system A activity than adults (P < 0.05). In adults, placental system A activity was lower in SGA infants than appropriate-for-gestational-age (AGA) infants (P < 0.05). In teenagers, AGA and SGA infants had lower placental system A activity than AGA infants born to adults (P < 0.05). Placental system A activity was higher in growing teenagers than in nongrowing teenagers (P < 0.001). Placental mRNA expression of system A transporter isoforms SLC38A1 and -2 was lower in teenagers than in adults (P < 0.05) but did not differ between growing and nongrowing teenagers. There was no difference in transporter protein expression/localization between cohorts. Teenagers have inherently reduced placental transport, which may underlie their susceptibility to delivering SGA infants. Growing teenagers appear to overcome this susceptibility by stimulating the activity, but not expression, of system A transporters.

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.

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.

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.