Model Of Fetal Growth Restriction Research Articles

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. 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 (K mf) of 14C‐glutamine and 14C‐glutamate (glutamine K mf and glutamate K mf) was significantly higher at embryonic day (E) 18.5, in line with increased expression of LAT1, a glutamine transporter protein. In P0 mice, glutamine K mf and glutamate K mf were higher (P0 versus wild‐type littermates, WTL) at E15.5. At E18.5, glutamine K mf remained elevated whereas glutamate K mf was similar between groups. In summary, we provide evidence that glutamine K mf and glutamate K mf 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 Maternal Sildenafil Treatment on Vascular Function in Growth-Restricted Fetal Sheep.

Objective- The objective of this study was to investigate the effect of intravenous maternal sildenafil citrate (SC) administration on vascular function in growth-restricted fetal sheep. Approach and Results- Fetal growth restriction (FGR) results in cardiovascular adaptations that redistribute cardiac output to optimize suboptimal intrauterine conditions. These adaptations result in structural and functional cardiovascular changes, which may underlie postnatal neurological and cardiovascular sequelae. Evidence suggests SC, a potent vasodilator, may improve FGR. In contrast, recent clinical evidence suggests potential for adverse fetal consequence. Currently, there is limited data on SC effects in the developing fetus. We hypothesized that SC in utero would improve vascular development and function in an ovine model of FGR. Preterm lambs (0.6 gestation) underwent sterile surgery for single umbilical artery ligation or sham (control, appropriately grown) surgery to replicate FGR. Ewes received continuous intravenous SC (36 mg/24 h) or saline from surgery until 0.83 gestation. Fetuses were delivered and immediately euthanized for collection of femoral and middle cerebral artery vessels. Vessel function was assessed via in vitro wire myography. SC exacerbated growth restriction in growth-restricted fetuses and resulted in endothelial dysfunction in the cerebral and femoral vasculature, irrespective of growth status. Dysfunction in the cerebral circulation is endothelial, whereas smooth muscle in the periphery is the origin of the deficit. Conclusions- SC crosses the placenta and alters key fetal vascular development. Extensive studies are required to investigate the effects of SC on fetal development to address safety before additional use of SC as a treatment.

Placental vascular abnormalities in the mouse alter umbilical artery wave reflections.

Current methods to detect placental vascular pathologies that monitor Doppler ultrasound changes in umbilical artery (UA) pulsatility have only moderate diagnostic utility, particularly in late gestation. In fetal mice, we recently demonstrated that reflected pressure waves propagate counter to the direction of flow in the UA and proposed the measurement of these reflections as a means to detect abnormalities in the placental circulation. In the present study, we used this approach in combination with microcomputed tomography to investigate the relationship between altered placental vascular architecture and changes in UA wave reflection metrics. Fetuses were assessed at embryonic day (E) 15.5 and E17.5 in control C57BL6/J mice and dams treated with combination antiretroviral therapy (cART), a known model of fetal growth restriction. Whereas the reflection coefficient was not different between groups at E15.5, it was 27% higher at E17.5 in cART-treated mice compared with control mice. This increase in reflection coefficient corresponded to a 36% increase in the total number of vessel segments, a measure of overall architectural complexity. Interestingly, there was no difference in UA pulsatility index between groups, suggesting that the wave reflections convey information about vascular architecture that is not captured by conventional ultrasound metrics. The wave reflection parameters were found to be associated with the morphology of the fetoplacental arterial tree, with the area ratio between the UA and first branch points correlating with the reflection coefficient. This study highlights the potential for wave reflection to aid in the noninvasive clinical assessment of placental vascular pathology. NEW & NOTEWORTHY We used a novel ultrasound methodology based on detecting pulse pressure waves that propagate along the umbilical artery to investigate the relationship between changes in wave reflection metrics and altered placental vascular architecture visualized by microcomputed tomography. Using pregnant mice treated with combination antiretroviral therapy, a model of fetal growth restriction, we demonstrated that reflections in the umbilical artery are sensitive to placental vascular abnormalities and associated with the geometry of the fetoplacental tree.

Assessment of prenatal cerebral and cardiac metabolic changes in a rabbit model of fetal growth restriction based on 13C-labelled substrate infusions and ex vivo multinuclear HRMAS.

BackgroundWe have used a previously reported rabbit model of fetal growth restriction (FGR), reproducing perinatal neurodevelopmental and cardiovascular impairments, to investigate the main relative changes in cerebral and cardiac metabolism of term FGR fetuses during nutrient infusion.MethodsFGR was induced in 9 pregnant New Zealand rabbits at 25 days of gestation: one horn used as FGR, by partial ligation of uteroplacental vessels, and the contralateral as control (appropriate for gestation age, AGA). At 30 days of gestation, fasted mothers under anesthesia were infused i.v. with 1-13C-glucose (4 mothers), 2-13C-acetate (3 mothers), or not infused (2 mothers). Fetal brain and heart samples were quickly harvested and frozen down. Brain cortex and heart apex regions from 30 fetuses were studied ex vivo by HRMAS at 4°C, acquiring multinuclear 1D and 2D spectra. The data were processed, quantified by peak deconvolution or integration, and normalized to sample weight.ResultsMost of the total 13C-labeling reaching the fetal brains/hearts (80–90%) was incorporated to alanine and lactate (cytosol), and to the glutamine-glutamate pool (mitochondria). Acetate-derived lactate (Lac C2C3) had a slower turnover in FGR brains (~ -20%). In FGR hearts, mitochondrial turnover of acetate-derived glutamine (Gln C4) was slower (-23%) and there was a stronger accumulation of phospholipid breakdown products (glycerophosphoethanolamine and glycerophosphocholine, +50%), resembling the profile of non-infused control hearts.ConclusionsOur results indicate specific functional changes in cerebral and cardiac metabolism of FGR fetuses under nutrient infusion, suggesting glial impairment and restricted mitochondrial metabolism concomitant with slower cell membrane turnover in cardiomyocytes, respectively. These prenatal metabolic changes underlie neurodevelopmental and cardiovascular problems observed in this FGR model and in clinical patients, paving the way for future studies aimed at evaluating metabolic function postnatally and in response to stress and/or treatment.

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.

Single-Cell Analysis Identifies Thymic Maturation Delay in Growth-Restricted Neonatal Mice.

Fetal growth restriction (FGR) causes a wide variety of defects in the neonate which can lead to increased risk of heart disease, diabetes, anxiety and other disorders later in life. However, the effect of FGR on the immune system, is poorly understood. We used a well-characterized mouse model of FGR in which placental Igf-2 production is lost due to deletion of the placental specific Igf-2 P0 promotor. The thymi in such animals were reduced in mass with a ~70% reduction in cellularity. We used single cell RNA sequencing (Drop-Seq) to analyze 7,264 thymus cells collected at postnatal day 6. We identified considerable heterogeneity among the Cd8/Cd4 double positive cells with one subcluster showing marked upregulation of transcripts encoding a sub-set of proteins that contribute to the surface of the ribosome. The cells from the FGR animals were underrepresented in this cluster. Furthermore, the distribution of cells from the FGR animals was skewed with a higher proportion of immature double negative cells and fewer mature T-cells. Cell cycle regulator transcripts also varied across clusters. The T-cell deficit in FGR mice persisted into adulthood, even when body and organ weights approached normal levels due to catch-up growth. This finding complements the altered immunity found in growth restricted human infants. This reduction in T-cellularity may have implications for adult immunity, adding to the list of adult conditions in which the in utero environment is a contributory factor.

Effect of sildenafil citrate treatment in the eNOS knockout mouse model of fetal growth restriction on long-term cardiometabolic outcomes in male offspring.

Fetal growth restriction (FGR) is associated with an increased risk of hypertension, insulin resistance, obesity and cardiovascular disease in adulthood. Currently there are no effective treatments to reverse the course of FGR. This study used the eNOS knockout mouse (eNOS-/-), a model of FGR, to determine the ability of sildenafil, a potential new treatment for FGR, to improve cardiovascular and metabolic outcomes in adult offspring following a complicated pregnancy. Pregnant eNOS-/- and C57BL/6J control dams were randomised to sildenafil treatment (0.2 mg/ml in drinking water) or placebo at day 12.5 of gestation until birth. After weaning, male offspring were randomised to either a high fat (HFD; 45% kcal from fat) or normal chow diet (ND), and raised to either postnatal day 90 or 150. Growth and body composition, glucose tolerance, insulin resistance, systolic blood pressure and vascular function were analysed at both time-points. eNOS-/- offspring were significantly smaller than their C57BL/6J controls at weaning and P90 (p < 0.01); at P150 they were a similar weight. Total adipose tissue deposition at P90 was significantly increased only in eNOS-/- mice fed a HFD (p < 0.001). At P150 both C57BL/6J and eNOS-/- offspring fed a HFD demonstrated significant adipose tissue deposition (p < 0.01), regardless of maternal treatment. Both diet and maternal sildenafil treatment had a significant effect on glucose tolerance. Glucose tolerance was significantly impaired in eNOS-/- mice fed a HFD (p < 0.01); this was significant in offspring from both sildenafil and vehicle treated mothers at P90 and P150. Glucose tolerance was also impaired in C57BL/6J mice fed a HFD at both P90 and P150 (p < 0.01), but only in those also exposed to sildenafil. In these C57BL/6J mice, sildenafil was associated with impaired insulin sensitivity at P90 (p = 0.020) but increased insulin resistance at P150 (p = 0.019). Exposure to sildenafil was associated with a significant increase in systolic blood pressure in eNOS-/- mice compared with their C57BL/6J diet controls at P150 (p < 0.05). Exposure to sildenafil had differing effects on vascular function in mesenteric arteries; it increased vasodilation in response to ACh in C57BL/6J mice, but was associated with a more constrictive phenotype in eNOS-/- mice. eNOS-/- mice demonstrate a number of impaired outcomes consistent with programmed cardiometabolic disease, particularly when faced with the 'second hit' of a HFD. Exposure to sildenafil treatment during pregnancy did not increase fetal growth or significantly improve adult metabolic or cardiac outcomes. Maternal sildenafil treatment was, however, associated with small impairments in glucose handling and an increase in blood pressure. This study highlights the importance of understanding the long-term effects of treatment during pregnancy in offspring from both complicated and healthy control pregnancies.

212. Effects of prenatal sildenafil treatment on long-term cardiovascular function in offspring from dahl salt-sensitive rats

Introduction Fetal growth restriction (FGR) is associated with increased risk for cardiovascular disorders in later life. Cardiovascular disease is the most common cause of death worldwide. Previous studies report that prenatal sildenafil improves pregnancy outcomes, such as birthweight, in FGR animal models; however, whether sildenafil treatment is protective against long-term cardiovascular disease in these offspring is unknown. Objective We hypothesize that prenatal sildenafil reduces blood pressure and endothelial dysfunction in FGR offspring from Dahl salt-sensitive (SS) rats on normal salt intake. Methods Sildenafil citrate (60 mg/kg/day) or control gel diet was administered from gestational day 10 until birth. Birthweight and litter size were measured (treated n = 10; untreated n = 8 dams). Telemetry devices (DSI) were implanted via the femoral artery to measure mean arterial pressure (MAP) from weeks 5–8 in the offspring (treated n = 12; untreated n = 4). Aortic rings were isolated from 10 week old offspring to assess vascular sensitivity ( logEC 50 ) to endothelial-dependent (acetylcholine) and -independent (sodium nitroprusside, SNP) vasorelaxation (treated n = 10; untreated n = 10). Data shown as mean ± S.E.M. Results No sex differences were observed in any variables; therefore, data were pooled between males and females. Sildenafil improved birthweight (treated 6.8 ± 0.2; untreated 6.2 ± 0.1 g; p = 0.02) without significantly changing viable litter size (treated 9.6 ± 0.9; untreated 7.9 ± 1.0; p = 0.23). While MAP at 5 weeks was similar between groups (treated 107 ± 1; untreated 108 ± 2 mmHg; p = 0.55), there was a trend towards lower MAP in prenatally treated offspring at 8 weeks (treated 116 ± 1; untreated 120 ± 2 mmHg; p = 0.09). Aortas from offspring of treated dams displayed enhanced sensitivity to acetylcholine ( logEC 50 : treated −7.4 ± 0.3; untreated −6.6 ± 0.3 M; p = 0.03), but not to SNP (treated −8.2 ± 0.3; untreated −7.9 ± 0.2 M; p = 0.43). Discussion Prenatal sildenafil treatment improves birthweight in a model of FGR. In young adult offspring, there was a trend towards a sex-independent lowering of blood pressure and increased endothelium-dependent relaxation.

Melatonin Increases Fetal Weight in Wild-Type Mice but Not in Mouse Models of Fetal Growth Restriction.

Fetal growth restriction (FGR) presents with an increased risk of stillbirth and childhood and adulthood morbidity. Melatonin, a neurohormone and antioxidant, has been suggested as having therapeutic benefit in FGR. We tested the hypothesis that melatonin would increase fetal growth in two mouse models of FGR which together represent a spectrum of the placental phenotypes in this complication: namely the endothelial nitric oxide synthase knockout mouse (eNOS-/-) which presents with abnormal uteroplacental blood flow, and the placental specific Igf2 knockout mouse (P0+/-) which demonstrates aberrant placental morphology akin to human FGR. Melatonin (5 μg/ml) was administered via drinking water from embryonic day (E)12.5 in C57Bl/6J wild-type (WT), eNOS-/-, and P0+/- mice. Melatonin supplementation significantly increased fetal weight in WT, but not eNOS-/- or P0+/- mice at E18.5. Melatonin did, however, significantly increase abdominal circumference in P0+/- mice. Melatonin had no effect on placental weight in any group. Uterine arteries from eNOS-/- mice demonstrated aberrant function compared with WT but melatonin treatment did not affect uterine artery vascular reactivity in either of these genotypes. Umbilical arteries from melatonin treated P0+/- mice demonstrated increased relaxation in response to the nitric oxide donor SNP compared with control. The increased fetal weight in WT mice and abdominal circumference in P0+/-, together with the lack of any effect in eNOS-/-, suggest that the presence of eNOS is required for the growth promoting effects of melatonin. This study supports further work on the possibility of melatonin as a treatment for FGR.

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.

Evaluation of Sildenafil and Tadalafil for Reversing Constriction of Fetal Arteries in a Human Placenta Perfusion Model.

Fetal growth restriction resulting from reduced placental blood perfusion is a major cause of neonatal morbidity and mortality. Aside from intense surveillance and early delivery, there is no treatment for fetal growth restriction. A potential treatment associated with placental vasoconstriction is the class of PDE5 (phosphodiesterase type 5) inhibitors such as sildenafil, which is known to cross the placenta. In contrast, tadalafil, a more potent and selective PDE5 inhibitor has not been studied in pregnancy or experimental models of fetal growth restriction. Therefore, we compared the efficacy of these 2 PDE5 inhibitors for reversing vasoconstriction in an ex vivo human placental model and evaluating molecular and physiological responses. Sildenafil and tadalafil were infused into the intervillous space in a preconstricted human placental dual cotyledon, dual perfusion assay for the comparison of arteriole pressures and molecular indicators of drug inhibition. Results indicate a decrease arterial pressure with sildenafil citrate compared with controls, whereas tadalafil showed no difference. PDE5 and endothelial nitric oxide synthase activity were altered with sildenafil but not tadalafil. Sildenafil citrate improved preconstricted placental arterial perfusion in a human placental model, whereas tadalafil showed no response. It is possible that tadalafil did not cross the human placental barrier or was degraded by trophoblasts. This study supports human clinical trials exploring sildenafil as a potential treatment for improving fetal blood flow in fetal growth restriction associated with vasoconstriction.

Nutritional intra-amniotic therapy increases survival in a rabbit model of fetal growth restriction.

ObjectiveTo evaluate the perinatal effects of a prenatal therapy based on intra-amniotic nutritional supplementation in a rabbit model of intrauterine growth restriction (IUGR).MethodsIUGR was surgically induced in pregnant rabbits at gestational day 25 by ligating 40–50% of uteroplacental vessels of each gestational sac. At the same time, modified-parenteral nutrition solution (containing glucose, amino acids and electrolytes) was injected into the amniotic sac of nearly half of the IUGR fetuses (IUGR-T group n = 106), whereas sham injections were performed in the rest of fetuses (IUGR group n = 118). A control group without IUGR induction but sham injection was also included (n = 115). Five days after the ligation procedure, a cesarean section was performed to evaluate fetal cardiac function, survival and birth weight.ResultsSurvival was significantly improved in the IUGR fetuses that were treated with intra-amniotic nutritional supplementation as compared to non-treated IUGR animals (survival rate: controls 71% vs. IUGR 44% p = 0.003 and IUGR-T 63% vs. IUGR 44% p = 0.02), whereas, birth weight (controls mean 43g ± SD 9 vs. IUGR 36g ± SD 9 vs. IUGR-T 35g ± SD 8, p = 0.001) and fetal cardiac function were similar among the IUGR groups.ConclusionIntra-amniotic injection of a modified-parenteral nutrient solution appears to be a promising therapy for reducing mortality among IUGR. These results provide an opportunity to develop new intra-amniotic nutritional strategies to reach the fetus by bypassing the placental insufficiency.

Effects of excess thromboxane A2 on placental development and nutrient transporters in a Mus musculus model of fetal growth restriction.

Hypertensive disease of pregnancy (HDP) with placental insufficiency is the most common cause of fetal growth restriction (FGR) in the developed world. Despite the known negative consequences of HDP both to the mother and fetus, little is known about the longitudinal placental changes that occur as HDP progresses in pregnancy. This is because longitudinal sampling of human placentae during each gestation is impossible. Therefore, using a mouse model of thromboxane A2-analog infusion to mimic human HDP in the last trimester, we calculated placental efficiencies based on fetal and placental weights; quantified spongiotrophoblast and labyrinth thicknesses and vascular density within these layers; examined whether hypoxia signaling pathway involving vascular endothelial growth factor A (VEGFA) and its receptors (VEGFR1, VEGFR2) and matrix metalloproteinases (MMPs) contributed to vascular change; and examined nutrient transporter abundance including glucose transporters 1 and 3 (GLUT1, GLUT3), neutral amino acid transporters 1, 2, and 4 (SNAT1, SNAT2, and SNAT4), fatty acid transporters 2 and 4 (FATP2, FATP4), and fatty acid translocase (CD36) from embryonic day 15.5 to 19 in a 20-day C57Bl/6J mouse gestation. We conclude that early-to-mid gestation hypertensive placentae show compensatory mechanisms to preserve fetal growth by increasing placental efficiencies and maintaining abundance of important nutrient transporters. As placental vascular network diminishes over late hypertension, placental efficiency diminishes and fetal growth fails. Neither hypoxia signaling pathway nor MMPs mediated the vascular diminution in this model. Hypertensive placentae surprisingly exhibit a sex-differential expression of nutrient transporters in late gestation despite showing fetal growth failure in both sexes.

Adaptations in Maternofetal Calcium Transport in Relation to Placental Size and Fetal Sex in Mice.

Appropriate placental transport of calcium is essential for normal fetal skeletal mineralization. In fetal growth restriction (FGR), the failure of a fetus to achieve its growth potential, a number of placental nutrient transport systems show reduced activity but, in the case of calcium, placental transport is increased. In a genetic mouse model of FGR this increase, or adaptation, maintains appropriate fetal calcium content, relative to the size of the fetus, despite a small, dysfunctional placenta. It is unknown whether such an adaptation is also apparent in small, but normally functioning placentas. We tested the hypothesis that calcium transfer would be up-regulated in the lightest vs. heaviest placentas in the same C57Bl/6J wild-type (WT) mouse litter. Since lightest placentas are often from females, we also assessed whether fetal sex influenced placental calcium transfer. Placentas and fetuses were collected at embryonic day (E)16.5 and 18.5; the lightest and heaviest placentas, and female and male fetuses, were identified. Unidirectional maternofetal calcium clearance (CaKmf) was assessed following 45Ca administration to the dam and subsequent radiolabel counts within the fetuses. Placental expression of calcium pathway components was measured by Western blot. Data (median) are lightest placenta expressed as percentage of the heaviest within a litter and analyzed by Wilcoxon signed-rank test. In WT mice having normally grown fetuses, CaKmf, per gram placenta near term, in the lightest placentas was increased (126%; P < 0.05) in association with reduced fetal calcium accretion earlier in gestation (92%; P < 0.05), that was subsequently normalized near term. Increased placental expression of calbindin-D9K, an important calcium binding protein, was observed in the lightest placentas near term (122%; P < 0.01). There was no difference in fetal calcium accretion between male and female littermates but a trend toward higher CaKmf in females (P = 0.055). These data suggest a small, normal placenta adapts calcium transfer according to its size, as previously demonstrated in a mouse model of FGR. Fetal sex had limited influence on this adaptive increase. These adaptations are potentially driven by fetal nutrient demand, as evidenced by the normalization of fetal calcium content. Understanding the regulatory mechanisms involved may provide novel avenues for treating placental dysfunction.

Taurine improves neuronal stem cell proliferation in fetal rats with growth restriction

Objective To detect the proliferation of neural stem cells (NSC) and analyze the effects of taurine on NSC proliferation in rats with fetal growth restriction (FGR). Methods For in vivo experiment, pregnant rats were randomly divided into three groups: control group (n=5), FGR group (n=7) and taurine group (n=6) [treated with taurine, 300 mg /(kg·d)]. Rats in the latter two groups were fed with 40% of food intake in control group throughout pregnancy to establish the FGR model. Fatty acid binding protein-7 (FABP-7) expression in brains was detected by immunohistochemistry. For in vitro experiment, pregnant rats were divided into two groups including control group and FGR group. FGR modeling was established in the same way as above. On the 14.5 day of pregnancy, NSC were isolated and cultured for five days to form neurospheres with different concentrations of taurine (control, 0, 5, 10, 15, 20, 30, 40 mmol/L). Immunofluorescence staining was used to detect the expression of NSC-specific marker FABP-7. CCK8 test was used to detect the cell proliferating index. Neurospheres were collected at the end of culture and disaggregated to get the total number of viable cells. One-way ANOVA, Kruskal-Wallis Test, SNK-test and Tamhane's test were used for statistical analysis. Results Compared with the control group (18 012±3 016), the number of FABP-7-positive cells in FGR model group (10 173±1 728) was decreased (P<0.05), but increased in taurine group (19 518±3 201) (F=200.8, P<0.05). Free-floating neurospheres could be formed in both control and FGR groups and the cells within neurospheres were mostly FABP-7-positive and were then considered as NSC. The proliferation indexes in control, FGR, FGR+5 mmol/L taurine, FGR+10 mmol/L taurine, FGR+15 mmol/L taurine, FGR+20 mmol/L taurine, FGR+30 mmol/L taurine, FGR+40 mmol/L taurine groups were 1, 0.80±0.07, 0.92±0.11, 1.33±0.24, 1.20±0.15, 1.11±0.14, 0.80±0.12 and 0.82±0.08, respectively. The proliferation indexes in FGR groups treated with 10, 15 or 20 mmol/L of taurine were significantly higher than that of FGR group without taurine interference (P<0.05). The total cell numbers in the eight groups were (24.5±0.6)×105, (20.23±1.09)×105, (22.80±0.86)×105, (27.53±1.22)×105, (25.30±1.05)×105, (22.70±0.72)×105, (21.80±1.80)×105 and (18.06±0.90)×105, respectively. Compared with the FGR group without taurine interference, those treated with 5, 10, 15 or 20 mmol/L of taurine showed higher total cell numbers ( all P<0.05 or <0.01), while that treated with 40 mmol/L of taurine was lower. Conclusions The number and proliferation of NSC in FGR fetal rat brains is significantly decreased, however, these could be reversed by antenatal supplementation of taurine. The best effect is shown when 10 mmol/L of taurine is given. Key words: Fetal growth retardation; Neural stem cells; Cell proliferation; Taurine

Antenatal taurine supplementation in fetal rats with growth restriction improves neural stem cell proliferation by inhibiting the activities of Rho family factors

Objective: To investigate whether antenatal taurine supplementation improves neural stem cell proliferation in rats with fetal growth restriction (FGR) through regulating the activity of Rho family factors.Methods: FGR models were established via food restriction throughout pregnancy. Pregnant rats were randomly divided into the control group, the FGR group (given 40% of the normal daily feeding in the control group), and the Taurine group (FGR model treated with 300 mg/kg·d taurine from gestational day seven). Expression of fatty acid binding protein-7 (FABP-7), Rho-associated coiled coil-forming protein kinase (ROCK2), Ras homolog gene family member A (RhoA), and rac in the brains of newborn rats was detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blotting (WB).Results: Relative FABP7 mRNA levels, the optical density (OD) values of FABP7-positive cells and the expression levels of the tested proteins all demonstrated that the number of neural stem cells (NSCs) in brain tissue was lower in the FGR group than in the control group but was significantly increased after antenatal taurine supplementation (p < .05). Compared with the control group, the mRNA and protein levels of RhoA and ROCK2 were higher in the FGR group but lower in the Taurine group (p < .05). In contrast, the rac mRNA level was lower in the FGR group than in the control group but was higher in the Taurine group (p < .05).Conclusions: Taurine prenatal supplementation improved neural stem cell proliferation in rats with FGR by inhibiting the activity of Rho family factors.

Effects of Antenatal Melatonin Treatment on the Cerebral Vasculature in an Ovine Model of Fetal Growth Restriction

Chronic moderate hypoxia, such as occurs in fetal growth restriction (FGR) during gestation, compromises the blood-brain barrier (BBB) and results in structural abnormalities of the cerebral vasculature. We have previously determined the neuroprotective and antioxidant effects of maternal administration of melatonin (MLT) on growth-restricted newborn lambs. The potential of maternal MLT therapy for the treatment of cerebrovascular dysfunction-associated developmental hypoxia has also been demonstrated in newborn lambs. We assessed whether MLT had an effect on the previously reported structural and cerebral vascular abnormalities in chronically hypoxic FGR lambs. Single umbilical-artery ligation surgery was performed in fetuses at approximately 105 days of gestation (term: 147 days) to induce placental insufficiency and FGR, and treatment with either saline or an MLT infusion (0.1 mg/kg) was started 4 h after surgery. Ewes delivered naturally at term and lambs were euthanased 24 h later. We found a significant reduction in the number of laminin-positive blood vessels within the subcortical and periventricular white matter (SCWM and PVWM) and the subventricular zone (SVZ) in FGR (p < 0.0005) and FGR + MLT brains (p < 0.0005 vs. controls), with no difference found between FGR and FGR + MLT animals. This was associated with a significant decrease in VEGF immunoreactivity in FGR and FGR + MLT brains versus controls (p < 0.0005; SCWM and PVWM) and in the SVZ in FGR brains versus controls (p < 0.005) and also with significantly lower levels of proliferating blood vessels versus controls (p < 0.0005). Glucose transporter-1 immunoreactivity (vascular endothelium) was decreased in FGR versus control lambs (p < 0.0005) in SCWM, PVWM, and the SVZ; it was significantly increased in FGR + MLT lambs compared with FGR lambs in SCWM and PVWM (p < 0.005) and even more markedly in the SVZ (p < 0.0005). FGR brains showed a 72% reduction in pericyte coverage versus control lambs and 68% versus FGR + MLT in PVWM. In SCWM, we found a 77 and 73% reduction compared with control and FGR + MLT lambs, respectively, while in the SVZ, we observed a 68% reduction versus controls and a 70% reduction in FGR versus FGR + MLT lambs. Astrocyte end-feet coverage in the SCWM showed a significant 24% reduction in FGR versus control levels, a 42% decrease within the PVWM, and a 35% decrease within the SVZ versus controls. MLT normalized astrocyte attachment to blood vessels, with no difference seen between controls and FGR + MLT animals in any of the brain regions examined. We also observed a decrease in albumin extravasation and microhemorrhage in controls and FGR + MLT brains versus FGR lambs. Our results demonstrate that umbilicoplacental insufficiency is associated with FGR-produced vascular changes in the white matter and SVZ of FGR newborn brains and that maternal MLT prevented disruption of the BBB by protecting perivascular cells essential for the maintenance of vascular homeostasis and stability.

Treating the dysfunctional placenta.

Placental dysfunction underlies major obstetric diseases such as pre-eclampsia and fetal growth restriction (FGR). Whilst there has been a little progress in prophylaxis, there are still no treatments for placental dysfunction in normal obstetric practice. However, a combination of increasingly well-described in vitro systems for studying the human placenta, together with the availability of more appropriate animal models of pre-eclampsia and FGR, has facilitated a recent surge in work aimed at repurposing drugs and therapies, developed for other conditions, as treatments for placental dysfunction. This review: (1) highlights potential candidate drug targets in the placenta – effectors of improved uteroplacental blood flow, anti-oxidants, heme oxygenase induction, inhibition of HIF, induction of cholesterol synthesis pathways, increasing insulin-like growth factor II availability; (2) proposes an experimental pathway for taking a potential drug or treatment for placental dysfunction from concept through to early phase clinical trials, utilizing techniques for studying the human placenta in vitro and small animal models, particularly the mouse, for in vivo studies; (3) describes the data underpinning sildenafil citrate and adenovirus expressing vascular endothelial growth as potential treatments for placental dysfunction and summarizes recent research on other potential treatments. The importance of sharing information from such studies even when no effect is found, or there is an adverse outcome, is highlighted. Finally, the use of adenoviral vectors or nanoparticle carriers coated with homing peptides to selectively target drugs to the placenta is highlighted: such delivery systems could improve efficacy and reduce the side effects of treating the dysfunctional placenta.

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.

Fetal origins of adult cardiac disease: a novel approach to prevent fetal growth restriction induced cardiac dysfunction using insulin like growth factor

Fetal growth restriction (FGR) is a risk factor for adult cardiovascular disease. Intraplacental gene transfer of human insulin-like growth factor-1 (IGF-1) corrects birth weight in our mouse model of FGR. This study addresses long term effects of FGR on cardiac function and the potential preventive effect of IGF-1. Laparotomy was performed on pregnant C57BL/6J mice at embryonic day 18 and pups were divided into three groups: Sham operated; FGR (induced by mesenteric uterine artery ligation); treatment (intraplacental injection of IGF-1 after uterine artery ligation). Pups were followed until 32 wk of life. Transthoracic echocardiography was performed starting at 12 wk. Systolic cardiac function was significantly impaired in the FGR group with reduced fractional shortening compared with sham and treatment group starting at week 12 of life (20 ± 4 vs. 31 ± 5 vs. 32 ± 5, respectively, n = 12 for each group; P < 0.001) with no difference between the sham and treatment groups. Intraplacental gene transfer of IGF-1 prevents FGR induced cardiac dysfunction. This suggests that in utero therapy may positively impact cardiac remodeling and prevent adult cardiovascular disease.