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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., (Copyright © 2018 IFPA, Elsevier Ltd. Published by Elsevier Ltd.. All rights reserved.)

The ANP knockout mouse is reported to exhibit pregnancy-associated hypertension, proteinuria and impaired placental trophoblast invasion and spiral artery remodeling, key features of pre-eclampsia (PE). We hypothesized that these mice may provide a relevant model of human PE with associated fetal growth restriction (FGR). Here, we investigated pregnancies of ANP wild type (ANP(+/+)), heterozygous (ANP(+/-)) and knockout (ANP(-/-)) mice. Maternal blood pressure did not differ between genotypes (E12.5, E17.5), and fetal weight (E18.5) was unaffected. Placental weight was greater in ANP(-/-) versus ANP(+/+) mice. Therefore, in our hands, the ANP model does not express phenotypic features of PE with FGR., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: To test the hypothesis that third trimester placental biometry and volume can be measured by two-dimensional (2D) and three-dimensional (3D) ultrasound in utero, determining which method of measurement was most strongly correlated with true placental size ex vivo., Methods: Singleton pregnancies underwent placental ultrasound within seven days of delivery (n = 87, 29(+3)-41(+5) weeks). Length and width (linear and curvilinear) and depth were estimated. Placental volume (PV) was estimated using 2D ellipse and shell techniques and 3D rotational (15⁰ and 30⁰ rotation angles) and multiplanar (5 and 10 mm slicing intervals) techniques. Measurements were compared to their true correlates following delivery. Intra- and inter-observer reliabilities of candidate placental size estimates were assessed by intraclass correlation coefficient (ICC)., Results: Curvilinear placental length (Rs = 0.24, p = 0.031), width (Rs = 0.27, p = 0.013) and depth (Rs = 0.31, p = 0.0056) correlated well with ex vivo measurements. All methods of PV estimation were related to ex vivo volume (Rs ≥ 0.32, p < 0.01) but not placental weight (p > 0.05); 30° rotational estimation demonstrated the strongest biological correlation (Rs = 0.40, p = 0.0004). Intra- and inter-observer placental size measurements intraclass correlation coefficients were suboptimal (0.59-0.70 and 0.10-0.58 respectively)., Discussion: We have demonstrated that it is possible to obtain information about the size of the third trimester placenta in utero using 2D and 3D ultrasound. However it is essential that the reliability (particularly interobserver reliability) of these estimates is improved prior to prospective studies to determine their predictive value., (Copyright © 2016. Published by Elsevier Ltd.)

Oxygen-enhanced MRI non-invasively monitors placental oxygenation in-vivo. This technique has been demonstrated at 1.5 Tesla (T) in healthy pregnancies. The aim of this study was to investigate whether findings are comparable at 3T. Nine pregnant volunteers underwent MRI at 3T. Scans obtained R1 (1/T1) measures from T1 maps under air, followed by a dynamic series breathing 100% oxygen. A statistically significant negative correlation was found between dR1 and gestation (P = 0.0008, r = -0.90, Pearson correlation test). The effect of the field strength was not significant within regression analysis. Placental Oxygen-Enhanced MRI at 3T gives comparable results to those previously obtained at 1.5T., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Syncytial nuclear aggregates (SNAs) are increased in pregnancy complications and include 'true' syncytial knots and inter-villous bridges. Apparent nuclear overlay caused by sectioning artefacts are frequently counted from single sections. Haematoxylin and eosin stained serial sections were assessed for frequency of SNA subtypes in placentas from normal, preeclamptic and fetal growth restricted (FGR) pregnancies. There were more sectioning artefacts and syncytial knots and fewer bridges in samples from preeclampsia compared to controls. There were no significant differences between FGR and control samples. This suggests the villous tree in preeclampsia has less inherent structural support and trophoblast cell dynamics are different., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: Syncytial nuclear aggregates (SNAs) are increased in pregnancy complications; however, little is known about their origin or function. This study aimed to characterise SNAs in more detail than has been reported previously., Methods: Immunohistochemistry and morphological examination at the light and ultrastructural level were used to determine the nature and structure of SNAs., Results: SNAs comprising bridges and syncytial knots had similar frequency with 974 per mm3 of villous tissue (IQR 717-1193) and 833 per mm3 (IQR 766-1190), respectively while there were approximately four times as many sectioning artefacts than knots and bridges combined. SNAs had increased proportions of condensed nuclei compared to the remaining syncytiotrophoblast (33.3% vs. 8.9%) and decreased proportions of euchromatic nuclei (0.0% vs. 16.2%), as assessed by examination of an electron micrograph archive. SNAs showed little evidence of apoptosis, with weak positivity for the apoptosis markers M30-neoepitope at 16.6% and TUNEL at 10.0%; strong staining was rarely seen for either marker. Immunofluorescence demonstrated rare association of actin (α, β or γ) with SNAs, whereas tubulin was in close proximity to SNAs and cytokeratin was seen within and surrounding SNAs., Discussion: M30-positive SNAs traced through serial sections were significantly more likely to be syncytial knots or sectioning artefacts than bridges. Nuclei within SNAs showed signs consistent with degeneration; however, this is unlikely to be an apoptotic process. There are few changes in configuration of cytoskeletal proteins around SNAs., Conclusions: These data suggest that the biogenesis and functional significance of SNAs still require resolution., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Since the advent of technologies to produce genetic knockout and transgenic mice, the number of mouse strains suggested to be useful as models for pregnancy-related complications in the human has risen substantially. Some of these share features in common with fetal growth restriction (FGR) and preeclampsia (PE) and could be useful for investigating aetiologies and for testing potential therapeutics to improve outcome in these diseases. However, since placental pathology is a major underlying factor in both FGR and PE, it is important to understand the similarities and differences in structure and function of the placenta between mice and women. The main aim of this review is to directly compare placental exchange physiology between human and mouse. The review will compare human and mouse in both normal and pathological circumstances, to attempt to answer the question of whether placental studies in the mouse can be translated to the human. The review includes descriptions of placental structure between the species, comparisons of nutrient transport, including amino acids, glucose and calcium, and evidence of how these transport systems are altered in both human FGR and mouse models of this disease. Finally, our review will conclude by examining studies in which mouse models of FGR/PE have been treated with drugs of potential therapeutic value in women and consider whether data obtained in mice can be a prelude for clinical trials in human., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: To characterise Chorionic Plate Artery (CPA) function in maternal obesity, and investigate whether leptin exposure reproduces the obese CPA phenotype in normal-BMI women., Study Design: CPA responses to the thromboxane-A(2) mimetic U46619 (pre/post leptin incubation), to the nitric oxide donor sodium nitroprusside (SNP) and the occurrence of tone oscillations (pre/post leptin incubation) were assessed in 46 term placentas from women of normal (18.5-24.9) or obese (>30) Body Mass Index (BMI)., Outcome Measures: Area Under the dose response Curve (AUC), maximum response (V(max)), sensitivity (EC(50)) to U46619 (pre/post leptin) and SNP; average vessel tone, oscillation amplitude and frequency (pre/post leptin)., Results: U46619 vasoconstriction was similar between BMI categories (p > 0.05), however vasodilatation to SNP was reduced in obesity (AUC p = 0.02, V(max)p = 0.04) compared to normal-BMI women. Leptin incubation altered responses to U46619 in both normal-BMI (EC(50) at 100 ng/ml leptin; p < 0.05) and obese women (AUC at 50 ng/ml; p < 0.05) but vasomotion was unaffected (p > 0.05)., Conclusions: Maternal obesity is associated with altered placental vascular function which may adversely affect placental oxygen and nutrient transport, placing the fetus at risk. Leptin incubation altered CPA vascular function but did not reproduce the obese phenotype., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Amino acid transfer to the fetus is dependent on several different factors. While these factors can be understood in isolation, it is still not possible to predict the function of the system as a whole. In order to do this an integrated approach is required which incorporates the interactions between the different determinants of amino acid transfer. Computational modelling of amino acid transfer in the term human placenta provides a mechanism by which this integrated approach can be delivered. Such a model would be invaluable for understanding amino acid transfer in both normal and pathological pregnancies. In order to develop a computational model it is necessary to determine all the biological factors which are important contributors to net amino acid transfer and the ways in which they interact. For instance, how different classes of amino acid transporter must interact to transfer amino acids across the placenta. Mathematically, the kinetics of each type of transporter can be represented by separate equations that describe their transfer rate as a non-linear function of amino acid concentrations. These equations can then be combined in the model to predict the overall system behaviour. Testing these predictions experimentally will demonstrate the strengths and weaknesses of the model, which can then be refined with increasing complexity and retested in an iterative fashion. In this way we hope to develop a functional computational model which will allow exploration of the factors that determine amino acid transfer across the placenta. This model may also allow the development of strategies to optimise placental transfer in pathologies associated with impaired amino acid transfer such as fetal growth restriction., (Copyright © 2012. Published by Elsevier Ltd.)

We tested the hypothesis that crossing two mouse models of fetal growth restriction (FGR) of differing phenotype would induce more severe FGR than either model alone. Female endothelial nitric oxide synthase knockout mice (eNOS(-/-)) were mated with placental-specific Igf2 knockout males (P0). Resultant fetuses were no more growth restricted than those with P0 deletion alone. However, P0 deletion attenuated the reduced placental system A amino acid transporter activity previously observed in eNOS(-/-) mice. Manipulating maternal and fetal genotypes provides a means to compare maternal and fetal regulation of fetal growth., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Unlabelled: Placental insufficiency is a major cause of fetal growth restriction (FGR) and accumulating evidence indicates several aspects of placental morphology are altered in this condition. MRI provides quantitative indices that may be used in non-invasive assessment of the human placenta, such as relaxation time measurements, T1 and T2. We hypothesised that placental relaxation times relate to alterations in placental tissue morphology and hence may be useful in identifying the changes associated with FGR. We report on the first phase of testing this hypothesis, in a study of women in normal pregnancy., Aims: To assess relaxation time measurements in the placenta in normal pregnancy and correlate these with gestational age and stereological analyses of placental morphology following delivery., Methods: 30 women underwent MRI examination (1.5 T) between 20 and 41 weeks gestation. Placental T1 and T2 measurements were acquired from a mid-depth placental region, co-localised to a structural scan. Fixed, wax-embedded sections of these placentas collected at delivery were stained with hematoxylin/eosin and subjected to stereological analysis., Results: Placental T1 and T2 show a significant negative correlation with gestation, (Pearson correlation p=0.01, 0.03 respectively). 17 placentas were analysed stereologically. In the group as a whole there was no significant correlation between T1 and T2 and morphological features. However, in a subset of 7 pregnancies scanned within a week of delivery, a significant positive correlation was observed between the fibrin volume density and the ratio of fibrin: villous volume densities and T2 (Spearman correlation p=0.02, 0.03 respectively)., Discussion: The correlations between placental T1 and T2 and gestation show that these variables are clearly influenced by changes in placental structure. Fibrin might be a key component but further work is needed to fully elucidate the major structural influences on placental T1 and T2., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: Teenagers are susceptible to delivering small-for-gestational-age infants. Previous studies implicate continued skeletal growth as a contributory factor, and impaired placental development was the primary cause of fetal growth restriction in growing adolescent sheep. The aims of this study were to examine the impact of young maternal age and growth on placental development., Study Design: Placentas were collected from 31 teenagers, of which 12 were growing and 17 non-growing based on knee height measurements. An adult control group (n = 12) was included., Main Outcome Measures: Placental weight and morphometric measurements of villous, syncytiotrophoblast, fibrin and vessel areas, as well as indices of proliferation and apoptosis, were analysed in relation to maternal growth and age., Results: Growing teenagers had a higher birthweight:placental weight ratio than non-growing teenagers (p < 0.05). Villous area, syncytial area, fibrin content, vascularisation and cell turnover did not differ between growing and non-growing teenagers. There were no differences in placental weight or morphometry between adult and teenage pregnancies. Maternal smoking, a potential confounding factor, did not exert a major influence on the placental parameters examined, except for a stimulatory effect on placental proliferation (p < 0.05) and syncytial knot formation (p < 0.05)., Conclusions: We were unable to detect any major differences in placental size or composition between growing and non-growing teenagers. Birthweight:placental weight ratio was higher in growing compared to non-growing teenagers. This suggests that maternal growth may affect placental function rather than development, and is consistent with our recent observations that maternal growth was not detrimental to fetal growth., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The increasing number of mouse models of fetal growth restriction (FGR) make it crucial to standardize the way FGR is defined. By constructing growth curves in the placental-specific Igf2 knockout mouse (P0) it was demonstrated that 93% of P0 fetuses fell below the 5th centile of wild-type weights at E18.5, up from 44% at E16.5. This analysis, coupled with anthropomorphic measurements showing evidence of head sparing in P0 fetuses, allows clinical comparisons of FGR in mice through the use of clinically relevant growth curves. We suggest this as a standardized approach to defining FGR in mouse, and other animal models., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: Deletion of the placental-specific P0 transcript of the insulin-like growth factor gene (Igf2) reduces placental growth from early pregnancy onwards. In Igf2 P0 knockout fetuses (P0), maternofetal flux of (14)C-methylaminoisobutyric acid ((14)C-MeAIB) mediated by system A amino acid transporter activity is increased at embryonic day 16 (E16), but this stimulation is not sustained, and by E19, fetal growth restriction (FGR) ensues. Here, we investigated whether upregulated (14)C-MeAIB transfer does occur concomitantly with a change in System A amino acid transporter activity and whether altered uteroplacental vascular function contributes to the FGR. We tested the hypothesis that FGR in P0 mice is attributable to altered nutrient transport rather than aberrant uteroplacental vascular function., Methods: Plasma membrane vesicles were isolated from placentas of P0 and wild-type (WT) fetuses at E16 and E19. System A amino acid transporter activity was measured as sodium-dependent (14)C-MeAIB uptake over 60s. Wire myography was performed on uterine artery branches supplying P0 or WT implantation sites and agonist-induced constriction and dilation measured., Results: Sodium-dependent uptake of (14)C-MeAIB (at 60s) was significantly (P < 0.05) higher in P0 compared to WT vesicles at E16; at E19 (14)C-MeAIB uptake was similar between P0 and WT. Uterine artery branch vascular reactivity was comparable between groups., Conclusions: System A activity in the maternal-facing plasma membrane of syncytiotrophoblast layer II underpins the adaptations observed in the transplacental MeAIB flux of P0 mice. Unaltered uterine artery vascular function suggests that the FGR phenotype of P0 fetuses is primarily due to deficient placental nutrient exchange capacity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The obesity epidemic, including childhood obesity, is rapidly gaining strength as one of the most significant challenges to the health of the global community in the 21st Century. The proportion of women who are obese at the beginning of pregnancy is also increasing. These women and their babies are at high risk of pregnancy complications, and of programming for metabolic disease in adult life. In particular, maternal obesity is associated with aberrant fetal growth, encompassing both growth restricted and large for gestational age, or macrosomic fetuses. This article considers the potential effect of obesity and adipose tissue on placental nutrient exchange mechanisms in relation to aberrant fetal growth. The review emphasizes the dearth of work on this topic to date despite its importance to current and future healthcare of the population., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Plasma corticotrophin-releasing factor (CRF) and urocortin are elevated in preterm labour and/or fetal growth restriction (FGR). FGR is associated with reduced placental system A amino acid transporter activity and in vitro data suggest altered endocrine status could be responsible. Here we test the hypothesis that CRF and urocortin inhibit placental system A activity. Chronic (48h) exposure of term placental villous explants to these hormones (10(-7)M) significantly reduced system A activity (Na(+)-dependent (14)C-methylaminoisobutyric acid uptake), whereas 1h exposure had no effect. We propose elevated CRF and urocortin contribute to FGR through negative regulation of placental system A activity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

During pregnancy, nutrient partitioning between the mother and fetus must balance promoting fetal survival and maintaining nutritional status of the mother for her health and future fertility. The nutritional status of the pregnant woman, reflected in her body composition, may affect placental function with consequences for fetal development. We investigated the relationship between maternal body composition and placental system A amino acid transporter activity in 103 term placentas from Southampton Women's Survey pregnancies. Placental system A activity was measured as Na(+)-dependent uptake of 10 mumol/L (14)C-methylaminoisobutyric acid (a system A specific amino acid analogue) in placental villous fragments. Maternal body composition was measured at enrollment pre-pregnancy; in 45 infants neonatal body composition was measured using dual-energy x-ray absorptiometry. Term placental system A activity was lower in women with smaller pre-pregnancy upper arm muscle area (r = 0.27, P = 0.007), but was not related to maternal fat mass. System A activity was lower in mothers who reported undertaking strenuous exercise (24.6 vs 29.7 pmol/mg/15 min in sedentary women, P = 0.03), but was not associated with other maternal lifestyle factors. Lower placental system A activity in women who reported strenuous exercise and had a lower arm muscle area may reflect an adaptation in placental function which protects maternal resources in those with lower nutrient reserves. This alteration may affect fetal development, altering fetal body composition, with long-term consequences., ((c) 2010 Elsevier Ltd. All rights reserved.)

Both syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments are used to characterize the placental uptake of maternal substrate and to investigate changes in uptake associated with pathological conditions. However, the two techniques have not been directly compared. In this study uptake of (14)C-L-serine was compared in placental villous fragments and in MVM prepared from the same placentas. (14)C-L-serine uptake into MVM vesicles was mediated by System L and System A and smaller unidentified Na(+)-dependent and Na(+)-independent components. In villous fragments an unidentified Na(+)-dependent component mediated the majority of (14)C-L-serine uptake followed by System A and System L. The unidentified Na(+)-independent component of L-serine uptake was not detected in villous fragments. The ratio of System A activity to System L activity was similar in villous fragments and MVM vesicles. However, the unidentified Na(+)-dependent component in villous fragments was significantly higher than that in MVM vesicles. This indicates that the main differences in serine uptake mechanisms identified using the two techniques were not due to differences in System A and System L activity but to differences in the unidentified Na(+)-dependent component. This study suggests that uptake of L-serine into MVM vesicles and villous fragments via Systems A and L is comparable, but that this is not true for all components of L-serine uptake., ((c) 2010 Elsevier Ltd. All rights reserved.)

Na(+)/H(+) exchanger (NHE) activity regulates intracellular pH (pH(i)) in the placental syncytiotrophoblast. In other tissues aldosterone and cortisol have been shown to up-regulate NHE activity via an acute, non-genomic effect. Here we tested the hypothesis that these corticosteroids stimulate NHE in the syncytiotrophoblast. Villous fragments from term placentas were loaded with 1 muM BCECF (pH sensitive fluorescent dye) and the syncytiotrophoblast acidified with a pre-pulse of 20 mM NH(4)Cl. The Na(+)-dependent recovery of pH(i) from this acid load was taken as a measure of NHE activity (pH units/sec, mean +/- SEM, n = number of placentas). In placental villi from female babies aldosterone significantly increased the rate of recovery of pH(i) from an acid load (0.0087 +/- 0.0005 versus 0.0056 +/- 0.0009 pH units/s, n = 8 p < 0.05 Paired Student's t-test) which was inhibited by the mineralocorticoid receptor antagonist, spironolactone (1 microM) but not the glucocorticoid antagonist mifepristone (1 microM). There was no effect on the rate of recovery from an acid load in villi from placenta from male babies. Alone, neither cortisol (1 microM, n = 5) nor carbenoxolone (100 microM, n = 9), an inhibitor of 11-beta-hydroxysteroid dehydrogenase-2 (11-beta-HSD-2), altered the rate of recovery from an acid load. However, simultaneous application of cortisol with carbenoxolone significantly increased the rate of recovery from an acid load but again only in placentas from female babies (0.0080 +/- 0.0017 versus control 0.0037 +/- 0.0005, p < 0.05 pH units/s, n = 9 Paired Student's t-test). Stimulation by cortisol in female tissue was inhibited by mifepristone but not spironolactone. In conclusion, syncytiotrophoblast NHE activity is increased acutely by aldosterone and, when 11-beta-HSD-2 is blocked, by cortisol. These non-genomic effects are only evident in placentas from female babies and are mediated by classical mineralocorticoid and/or glucocorticoid receptors., (Copyright 2010 Elsevier Ltd. All rights reserved.)

This review considers the hypothesis that adaptations in blood flow, exchange surface area and transporter activity enable placental supply capacity to meet fetal demand and cause alterations in fetal composition which result in life-long programming of homeostatic set points. We consider the components of placental supply capacity and describe the predominant changes each of these could impose on solute and water exchange across the placenta. We next consider the evidence that adaptations in placental nutrient supply to meet the demands of fetal growth and development do occur. Evidence from human and mouse studies suggests that adaptations occur in regulation of blood flow through the fetoplacental circulation, in exchange barrier surface area and in transporter-mediated processes for amino acids and calcium. Crucially there appear to be differences in the gestational timing of these adaptations. Finally we suggest that each of these adaptations could have separate effects on the composition of the fetus. These could affect physiological set points in different ways and so programme the lifetime responses of the individual., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Synthetic glucocorticoids (GCs) are given to women with threatened preterm labour but their administration has been linked to reduced infant birthweight. The underlying mechanisms are unknown, but impaired placental development and/or function has been implicated. The activity of the system A amino acid transporter is decreased in placentas from pregnancies complicated by fetal growth restriction. Whether GCs adversely affect placental amino acid transport is unknown. The objective of this study was to determine the regulatory effects of GCs on system A transport using a human in vitro placental explant model. Term explants (n=7) were treated with dexamethasone (DEX 10(-8)M or 10(-6)M) or vehicle for 48 h. System A activity was measured by the uptake of (14)C-N-methylated aminoisobutyric acid by explants. Explants were also processed for electron microscopy (EM), immunohistochemistry, and qRT-PCR. Lactate dehydrogenase (LDH), human chorionic gonadotropin (hCG) and human placental lactogen (hPL) release into the culture medium was measured. DEX (10(-6)M) stimulated system A activity compared to vehicle (p<0.05). System A transporter proteins were localized to the newly regenerating syncytiotrophoblast layer, but mRNA levels were unchanged with DEX treatment. DEX did not adversely affect explant viability as determined by reduced LDH release (p<0.05). DEX treatment was associated with morphological (accelerated apical microvilli formation, nuclear maturation, and increased cell organelle number) and functional (elevated hCG secretion, increased 11beta-HSD2 mRNA expression and reduced cytotrophoblast proliferation (p<0.05 for all)) markers of syncytiotrophoblast differentiation. These findings suggest that DEX stimulates system A activity and promotes syncytiotrophoblast differentiation and maturation., (2009 Elsevier Ltd. All rights reserved.)

Poor folate status during pregnancy can lead to elevated maternal plasma levels of homocysteine (Hcy) with associated pregnancy complications and adverse neonatal outcomes, suggesting placental metabolism of Hcy might be an important determinant in influencing fetal development. The metabolic pathways for Hcy in placenta are not well defined. In this study we examined the gene expression of key enzymes involved in Hcy metabolism in first trimester and term human placenta to determine which metabolic pathways prevail. Expression of mRNA for methionine synthase and 5,10-methylene tetrahydrofolate reductase, enzymes involved in the methionine cycle and responsible for the re-methylation of Hcy to methionine, were expressed at similar levels between first trimester and term and in comparison to human liver as positive control. In contrast, cystathionine beta-synthase mRNA expression was markedly lower than that in liver at both gestational periods. Betaine-homocysteine methyltransferase mRNA was undetectable at either gestational age. These data suggest that re-methylation of Hcy using methyl donation from 5-methyltetrahydrofolate is the prevalent pathway, indicating a marked reliance on folate availability. This led to further investigations examining the expression and localisation of folate transporters in first trimester and term placenta. Folate receptor alpha (FRalpha) was highly polarised to the microvillous plasma membrane (MVM) of the syncytiotrophoblast at both gestational periods, a distribution shared by the proton-coupled folate transporter which co-localised with FRalpha. Reduced folate carrier was distributed to both MVM and basal syncytiotrophoblast plasma membranes at term suggesting a role at both loci, and in first trimester was localised to MVM as well as cytotrophoblast plasma membranes. These data support the concept that placental folate transport is established early in pregnancy, providing folate for utilisation in placental Hcy metabolism., (2009 Elsevier Ltd. All rights reserved.)

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and beta, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl(2) precipitation and centrifugation. Vesicles were enriched 11.3+/-0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system beta activity in mouse placental vesicles, measured as Na(+)-dependent uptake of (14)C-methylaminoisobutyric acid (MeAIB) and (3)H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system beta activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Pre-eclampsia is associated with insufficient adaptations of spiral arteries which theoretically alter haemodynamics within the intervillous space. Such changes could damage the syncytiotrophoblast and release factors which instigate maternal endothelial dysfunction. We tested this hypothesis using an in vitro dual perfusion model of the human placenta, representing putative changes in flow arising from these spiral artery maladaptations. Whilst fetal-side flow rates remained constant (6 ml/min) perfusion rates on the maternal side were increased from 14 ml/min to 45 ml/min. As well as increasing placental derived intervillous hydrostatic pressures, and changes in flow dynamics observed by colour Doppler, these elevated flow rates resulted in morphologic damage, vacuolation and shedding of the syncytiotrophoblast, focal features previously defined in pre-eclampsia. The collected maternal perfusates recovered under high flow conditions also contained significantly elevated levels of biochemical markers of syncytial damage, including lactate dehydrogenase, alkaline phosphatase and human chorionic gonadotrophin. There were also significant elevations in chemokines GROalpha and RANTES, compared with the low flow perfusions. The soluble components of the maternal high flow rate perfusions decreased the number and proliferation of HUVECs after 24h exposure. These results could not be attributed to GROalpha or RANTES alone or in combination. This study provides evidence that alterations in intervillous flow have the potential to influence both the integrity of the syncytiotrophoblast and the liberation of potentially pathogenic soluble factors. This therefore offers a putative link between utero-placental maladaptations in pregnancy and the vascular endothelial complications of pre-eclampsia.

Vascular endothelial growth factor (VEGF) is a potent, physiologically relevant, vasodilator of the human term fetoplacental vasculature of placental lobules from normal pregnancy. There is evidence that VEGF and its receptors are dysregulated in preeclampsia (PE). Here, we used dual perfusion of the human placental lobule to test the hypothesis that the VEGF vasodilatory effect on the fetoplacental circulation is altered in PE and examined how vascular responsiveness relates to circulating levels of free VEGF in fetal sera in this disease. Umbilical cord sera and fetal venous perfusate concentrations of free VEGF from pregnancies complicated with PE were significantly lower compared to the normal group (P<0.05 and P<0.01, respectively). There was elevated in vitro placental release of the sequestrating soluble receptor, sVEGFR-1, into the fetal-side perfusate with PE compared to the normal group (P<0.05). The umbilical sera PlGF-1 level was higher by an order of magnitude in the fetal circulation in PE compared to normal pregnancy (P<0.0001), with the placenta appearing to contribute appreciably to these levels. Placental net contribution to maternal systemic free VEGF levels appeared to be negligible in both groups. sVEGFR-1 levels were elevated in the maternal-side venous perfusate with PE compared to the normal pregnancy (P<0.01). Perfused lobules from PE pregnancy exhibited an enhanced fetoplacental vasodilatory response to exogenous VEGF (P<0.001), with a longer recovery time (P<0.05), compared to the normal control group. Extrapolation of our combined functional and biochemical data suggests that a decrease in the in vivo circulating levels of free VEGF in PE is likely to contribute to compromised fetoplacental vascular patency in this disease.

The human fetus requires more glycine than any other amino acid but placental glycine transfer to the fetus is insufficient to meet fetal demand. L-Serine could represent a major metabolic source of glycine for the human fetus but little is known about the kinetics and physiology of L-serine uptake by the human placenta. We have characterised the amino acid transport systems involved in the uptake of L-serine by the microvillous membrane of the human placental syncytiotrophoblast and compared the uptake rates to those of glycine. L-Serine uptake into microvillous membrane (MVM) vesicles was primarily mediated by system A (MeAIB inhibitable) and system L (BCH inhibitable). Further characterisation using specific substrates of LAT1 and LAT2 found the pattern of L-serine uptake was consistent with that expected for uptake mediated by LAT2. Uptakes were performed with tracer levels of (14)C-L-serine, physiological levels of L-serine, or with physiological levels of amino acids. As amino acid concentrations rose, the proportion of uptake by System L decreased while uptake by uncharacterised Na(+)-independent systems increased. Uptake of Lserine into MVM vesicles had a V(max) of 2.1+/-0.4 nmol/mg protein/min, which was significantly higher than for glycine (V(max) 1.0+/-0.2 nmol/mg protein/min). This indicates that MVM vesicles have a higher uptake capacity for L-serine than glycine, despite a greater demand for glycine over serine for fetal protein synthesis. Further studies are now required to define the fate of L-serine taken up by the placenta and its importance for the fetus.

We used the in vitro dually perfused human placental lobule to test the hypothesis that known vasoconstrictors of the fetal placental circulation, angiotensin II and the thromboxane mimetic U46619 could induce fetomaternal water transfer. Secondly, we used a combination of vasoconstrictor and mechanically induced increases in fetal placental circulatory pressure to examine the role of the venous system in this context. Fetal-side administration of angiotensin II (A-II) and U46619 (n=6 and n=9, for A-II and U46619, respectively) induced dose dependent, recoverable elevations in fetal inflow hydrostatic pressure (HP; A-II: maximum contractility=83 mmHg, EC50=22.0 nM; U46619: maximum contractility was not achieved, but exceeded the A-II effect) and loss of perfusate from the fetal side (A-II: EC50=70.2 nM, maximum fetal-side solvent loss=1906 microl/min; U46619: maximum fetal-side solvent loss was not achieved, but exceeded the A-II effect). Fetal-side solvent loss, for both agonists, was correlated linearly with fetomaternal inflow HP (FMIHP) in a biphasic manner (between 0 and 30 mmHg the slopes (+/-S.E.) were 6.4+/-2.2 and 17.1+/-5.8 microl/(min mmHg) for A-II and U46619, respectively; between 30 and 70 mmHg the slopes (+/-S.E.) were 35.6+/-6.5 and 43.7+/-15.9 microl/(min mmHg) for A-II and U46619, respectively). Increasing fetal-side lumenal pressure (n=3) by raising the fetal outflow catheter caused a loss of perfusate from the fetal side which was reduced in the presence of U46619 (fetal solvent loss per unit increase in fetal-side inflow HP: slopes were 1.198+/-0.123 and 0.783+/-0.085 microl/(min mmHg mmHg), respectively). Notwithstanding the possibility of fetoplacental arterial constriction, we conclude that vasoconstrictive agonists in the fetoplacental circulation affect venous resistance, causing fetomaternal fluid loss. These observations could be relevant to the oligohydramnios associated with intrauterine growth restriction, a condition associated with increased resistance in the umbilical circulation.

This study investigates P-gp activity in placental villous fragments and the possibility of upregulating its expression and function by retroviral transduction. In fresh fragments, cyclosporin A caused a significant increase in 3H-vinblastine accumulation (187+/-48% at 180 min n=4), consistent with multi-drug resistance activity. After 7 days in culture, villous fragments showed a similar increase in 3H-vinblastine accumulation (143+/-10% at 180 min n=4), which was not significantly different from that in fresh tissue. Following transduction, immunohistochemistry revealed increased P-gp expression. However, the distribution of the protein differed from that in controls, with P-gp being located throughout the tissue as opposed to the normal specific location on the maternal facing plasma membrane. Transduced explants showed a significantly larger increase in 3H-vinblastine accumulation in the presence of cyclosporin A than control explants (245+/-15.5% at 180 min, n=4), suggesting reduced capacity to efflux vinblastine. This study demonstrates P-gp activity in intact placental tissue which is maintained in explant culture. Retroviral transduction of P-gp to such tissue leads to increased but undirected expression of the protein. The consequent increased activity at sites such as the basal, fetal facing, plasma membrane probably explains the increased substrate accumulation within the tissue.

Here we present methodology and validation (including measurement of unidirectional maternofetal clearance (Kmf) of (45)Ca and (14)C-mannitol) for in situ perfusion of the mouse placenta. On day 18 of gestation (term=19 days) mice were anaesthetised and the uterus delivered into a saline bath (40 degrees C). A fetus was selected, the umbilical artery and vein catheterised and perfused with Krebs Ringer (pH 7.4) at 60 microl/min. (45)Ca/(14)C-mannitol (2 microCi/5 microCi in 50 microl saline) was injected via maternal tail vein. Perfusate samples were collected every 5 min for 45 min. Maternal carotid artery pressure was monitored throughout perfusion. A terminal maternal cardiac blood sample was taken and analysed. Placentas were immersion fixed and processed for electron microscopy. Kmf for (45)Ca and (14)C-mannitol was calculated as perfusate [(45)Ca or (14)C-mannitol] x perfusion rate/maternal plasma [(45)Ca or (14)C-mannitol]xplacental weight. Maternal cardiac blood chemistry at termination (n=8-15, mean+/-SEM) was as follows: pH 7.153+/-0.016, PCO(2) 45.48+/-2.06 mmHg, PO(2) 66.47+/-7.10 mmHg, Na(+) 151.4+/-1.2 mmol/l, K(+) 5.54+/-0.17 mmol/l, Ca(2+) 1.15+/-0.03 mmol/l, glucose 7.2+/-0.5 mmol/l, and lactate 1.76+/-0.77 mmol/l. A successful 45 min perfusion in which perfusate recovery was >95% occurred in >50% of animals. Perfusion did not alter placental morphology or carotid pressure. Kmf (microl/min/g placenta) for (45)Ca (66.0+/-8.4 (n=7)) was significantly higher than Kmf for (14)C-mannitol (20.0+/-2.4 (n=5)) (p<0.01). These data demonstrate physiological perfusion of the mouse placenta in situ and its usefulness for measurement of solute transfer.

Dietary taurine is essential for cats and deficiency during pregnancy may lead to abortion, growth restriction or impaired neurological function of kittens. We previously described Na(+)- and Cl(-)-dependent taurine transport by system beta in fragments of freshly isolated cat placenta [Champion EE, Bailey SJ, Glazier JD, Jones CJP, Mann SJ, Rawlings JM, et al. Taurine uptake into cat placental tissue fragments. Placenta 2001;22:A.42]. Here we evaluate long term culture of cat placental explants as a model for the future study of chronic nutrient regulation of amino acid transport in this species. The cat placental explants displayed (i) Na(+)-dependent [(3)H]taurine uptake and (ii) taurine transporter protein on day 7 of culture, as observed in fresh cat placental fragments. The explants had preserved the ability to secrete PGF(2alpha) hormone until day 11 of culture and remained morphologically largely intact until day 7 of culture. This model of placental explant culture will provide an important in vitro method for the study of chronic regulation of amino acid transport in the cat.

Changes in tissue architecture and ultrastructure in the cat placenta during long-term explant culture have been examined over an 11-day period. Pieces of cat placenta, dissected from the lamellar region, were cultured in CMRL-1066 medium and tissue was fixed for electron microscopy at 2, 5, 7, and 11 days' culture, as well as before culture was initiated (day 0). Four samples were examined at each time point. After two days, the trophoblast basal lamina and attached cytotrophoblast cells had begun to separate from the syncytium and the cytotrophoblasts were spreading over the surface of the exposed fetal stromal compartment, and by five days were showing signs of growth. At seven days' culture, cytotrophoblast multilayering was common, and vascular and stromal components were also well preserved with collagen biosynthesis evident. By 11 days, the centre of the culture was compacted and degenerate with loss of tissue architecture, but on the outside polyp-like growths could be seen, with a well-developed covering of trophoblast containing fat and secretory droplets, lining a connective tissue matrix and stromal components. The cat placenta, therefore, like the human, has the capacity for regrowth in explant culture, including both trophoblast and stromal components.

Epidermal growth factor (EGF) reduces apoptosis in primary cytotrophoblast (CT) in culture through two separate pathways: the extracellular signal related kinase (ERK) 1/2 and phosphatidyl inositol 3-kinase (PI-3 kinase) paths. Whether other pathways are involved in survival signalling is unknown. We here show that the c-Jun NH2 terminal kinase (JNK) and the mitogen activated kinase (MAPK) p38 are also activated by EGF as seen by increases in JNK and p38 phosphorylation. However, inhibition of JNK phosphorylation with the specific inhibitor SP600125 increases apoptosis in a manner refractory to the addition of EGF but inhibition of p38 phosphorylation with its specific inhibitor SB 203580 does not increase apoptosis. EGF also activates sphingosine kinase-1 (SPHK-1), which converts sphingosine to sphingosine-1-phosphate, and its inhibition with dimethyl sphingosine (DMS) increased trophoblast death. Inhibition of SPHK-1 also did not affect EGF stimulated phosphorylation of PI-3 kinase, Akt, ERK1/2 or p38 but inhibition of PI-3 kinase with a specific inhibitor LY294002 partly (40%) inhibited the EGF-stimulated increase in SPHK-1 activity. We conclude that, in addition to the PI-3 kinase and ERK1/2 pathways, EGF acts through its receptor to stimulate JNK, p38 and SPHK-1 pathways, but that the JNK and SPHK-1, and not the p38, pathways are involved in suppressing apoptosis. This information provides evidence that EGF stimulates survival along multiple pathways that differ in trophoblast and other cell types.

This study aimed to determine whether there is a maternofetal potential difference (PD) in the mouse. The mean (+/- SEM) in vivo electrical potential difference (PD(sa)) between saline filled catheters in the maternal subcutaneous space (s) and the fetal amniotic sac (a) measured, according to strict criteria, in anaesthetised MF1 mice at a gestational age of 18-20 (term 20) days was 3.9+/-0.5 mV (significantly different from zero P<0.0001) in 16 conceptuses from 11 mice with the amniotic sac positive with respect to the maternal catheter. The PD(sv) between maternal tail vein (v) and maternal subcutaneous space was -0.8+/-0.4 mV (n=3: not significantly different from zero). Measurement of PD between two different maternal subcutaneous catheters (n=4) was < or =0.5 mV. This study shows that there is a maternofetal PD in the mouse and provides the foundation for studies addressing its mechanism of generation in this species.

The onset of maternal blood flow (10-12 weeks gestation) results in increased oxygenation of the placenta. We investigated whether the expressions of Na+/H+ exchanger (NHE) and Cl-/HCO3- anion exchanger (AE), thought to have an important role in maintaining intracellular pH of the syncytiotrophoblast and fetal pH homeostasis, are altered at the same time as this increase in blood flow. Real-time quantitative PCR was used to examine steady state levels of NHE (NHE1, 2, 3) and AE (AE1, 2) mRNA expression in early (6-9 weeks) and late (10-13 weeks) first trimester and full-term (38-40 weeks) placentas. beta-Actin, IF2B and GAPDH mRNA was also measured. None of the genes showed a significant difference in expression between the early and late first trimester groups. However, NHE2 (p < 0.001) and GAPDH (p < 0.05) mRNA expression significantly increased 18- and 3.7-fold between early first trimester and term. In conclusion, this study provides additional evidence that GAPDH is an unsuitable housekeeping gene for normalization of transcript levels in placenta. The expression of NHE and AE in the villous placenta is not altered concomitant with the onset of maternal blood flow. However, NHE2 transcripts appear to be gestationally regulated, which may contribute to changes in NHE activity.

We investigated the polarization of l-lactate transport in human syncytiotrophoblast by measuring uptake of [(14)C] l-lactate by both microvillous (maternal-facing; MVM) and basal (fetal-facing; BM) plasma membranes. [(14)C] l-lactate uptake by MVM and BM was stimulated in the presence of an inwardly directed H(+)gradient, with a significantly higher uptake in MVM than in BM at initial rate (15.4+/-2.3 vs 5.6+/-0.6 pmol/mg protein/20 sec). Stereospecific inhibition was observed in MVM, with a higher affinity for l-lactate compared with d-lactate. In BM, there was no difference in the inhibition by these two stereoisomers. Inhibition of lactate uptake in both MVM and BM by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of monocarboxylate transporter (MCT) activity, indicated MCT-mediated mechanisms across both membranes. Kinetic modelling supported a two-transporter model as the best fit for both MVM and BM, the K(m)of the major component being 6.21 mm and 25.01 mm in MVM and BM respectively. Western blotting and immunolocalization examining the distribution of MCT1 and MCT4, showed that MCT expression was polarized, MCT1 being predominantly localized to BM and MCT4 showing greater abundance on MVM. CD147, a chaperone protein for MCT1 and MCT4, was equally expressed by both membranes. These studies demonstrate that the opposing plasma membranes of human syncytiotrophoblast are polarized with respect to both MCT activity and expression.

We localized alkaline phosphatase and plasma membrane calcium-ATPase (PMCA) in the cat placental syncytiotrophoblast to address their polarized distribution and their potential as markers for specific plasma membrane purification. We used enzyme- (alkaline phosphatase) and immuno- (PMCA) histochemistry and, for alkaline phosphatase, compared data to observations on the human placenta. Alkaline phosphatase activity in the cat was localized to the decidual cell membranes, to within the associated interstitial space and on the subjacent apical (maternal facing) plasma membrane of the syncytiotrophoblast. Occasional maternal capillaries were positive on their basal surface and there was focal staining within the syncytiotrophoblast. This widespread distribution is less specific than in the human placenta where alkaline phosphatase was restricted to the apical and basal plasma syncytiotrophoblast membranes, with much greater density on the apical membrane. Expression of PMCA in the cat was restricted to the basal membrane of the syncytiotrophoblast only. This specific localization of PMCA is identical to the human placenta and all other species in which its placental localization has been studied. We conclude that the plasma membranes of the cat syncytiotrophoblast show a broadly similar functional polarization to the human and that PMCA would prove a useful marker in isolation of the cat syncytiotrophoblast basal plasma membrane.

This review article considers the purposes and mechanisms of regulation of placental transfer in general terms and then illustrates some key points with reference to the Na(+)/H(+) exchanger (NHE), a transport protein found in the syncytiotrophoblast. NHE probably has a role in the homeostasis of syncytiotrophoblast intracellular pH and may also be involved in syncytiotrophoblast cell volume regulation as well as H(+) loss from and Na(+) transfer to the fetus. The activity and expression of NHE in the microvillous plasma membrane of the syncytiotrophoblast is reduced in placentas from preterm, growth restricted babies as compared to their gestationally matched normally grown counterparts. There are differential effects of gestation in normal pregnancy on NHE mRNA, NHE protein and NHE activity. There is also evidence of acute modulation of NHE activity. Regulation of NHE in syncytiotrophoblast is therefore complex with control at transcription, post transcription and post translational loci., (Copyright 2002 IFPA and Elsevier Science Ltd.)

The purpose of this study was to determine whether there is any relationship between the activity of cationic amino acid transporters in the microvillous plasma membrane (MVM) of the syncytiotrophoblast and the oxygenation of the uteroplacental unit. Oxygenation data were obtained at the time of caesarean section from the uterine veins, the maternal radial artery and the umbilical vessels of 7 normal (AGA) and 13 intrauterine growth restricted (IUGR) pregnancies. Microvillous plasma membranes were isolated from the same placentas and the activity of the system y(+) and y(+)L cationic amino acid transporters determined by measuring (3)H- l -arginine uptake in the presence and absence of l -glutamine. In IUGR pregnancies uterine venous Po(2) was significantly higher (AGA=44.7+/-8.0 mmHg; IUGR=57.2+/-2.3 mmHg, P< 0.05) and umbilical venous Po(2) was significantly lower (AGA=33.4+/-3.0 mmHg; IUGR=25.1+/-2.0 mmHg, P< 0.05) than in AGA pregnancies. System y(+)L activity, but not system y(+) activity, was inversely correlated with uterine venous Po(2) (P< 0.01; r(2)=0.4) in AGA and IUGR pregnancies. In IUGR pregnancies without associated maternal pre-eclampsia, y(+)L activity, but not y(+) activity, was also directly related to the umbilical O(2) content difference (P< 0.01; r(2)=0.9). A significant negative correlation was found between system y(+) and the umbilical O(2) content difference in AGA pregnancies (P< 0.01; r(2)=0.9). Our data are consistent with the hypothesis that in IUGR fetuses uterine oxygenation is not reduced and can be increased. The inverse correlation between system y(+)L activity and uterine venous Po(2) and the correlations with umbilical venous-arterial O(2) content difference suggest a relationship between cationic amino acid transporter activity and oxygen tension in the uteroplacental unit., (Copyright 2002 IFPA and Elsevier Science Ltd.)

Ion transport is important for driving nutrient transport across the syncytiotrophoblast and yet is poorly understood. We have examined K(+)currents under basal conditions in cultured cytotrophoblast cells, at various stages of differentiation, using the whole cell patch clamp technique. Cytotrophoblast cells were isolated from human term placenta and maintained in culture for up to 3 days. Cells were studied at four stages of progressive morphological differentiation: (i) mononuclear cells, (ii) mononuclear cells in aggregates, (iii) small multinucleate cells and (iv) large multinucleate syncytiotrophoblast-like cells. In the conditions of whole cell recording the only K(+) selective current identified in all cell types was a strong inwardly rectifying current which was sensitive to Ba(2+) and Cs(+). This current was unaffected by intracellular ATP whereas intracellular GTPgammas caused either run down of the current or activated a linear current. The characteristics of the current described are consistent with those of the inwardly rectifying K(+) channel Kir2.1. The inwardly rectifying K(+) current was observed in three out of 19 (16 per cent ) mononuclear cells, seven out of 21 (33 per cent ) mononuclear aggregates, eight out of 21 (38 per cent ) small multinucleate cells and 16 out of 19 (84 per cent ) large multinucleate cells. This inwardly rectifying K(+) current is likely to have an important role in determining net K(+) diffusion across the syncytiotrophoblast cell membrane, perhaps increasing in importance as the cells terminally differentiate., (Copyright 2001 Harcourt Publishers Ltd.)

The aims of this study were to establish the sites of mRNA expression of both the cystic fibrosis (CF) and multidrug resistance (MDR1) genes in human placental sections from early pregnancy (first, early and mid-second trimesters). Riboprobes specific for each of these two genes were generated and used for in situ hybridization experiments. The results show parallel mRNA expression for the CF and MDR1 genes, with the signal detected in the syncytiotrophoblast and cytotrophoblast cells of the placental villi. Other cell types within the villous core were negative. Similar results were obtained at all stages of pregnancy studied., (Copyright 1999 W.B. Saunders Company Ltd.)

The expression of GLUT1 and GLUT3 mRNA and protein in human placental trophoblast-derived cells was investigated. A dividing choriocarcinoma derived cell line (JAr) was compared to differentiating cytotrophoblast cells, isolated from human term placenta, following 18 (mononucleate) and 66 h (multinucleate) in culture. JAr cells treated with 8-bromoadenosine, which inhibits growth and induces differentiation, were also studied. GLUT1 mRNA and protein expression were similar in the four groups of cells. However, GLUT3 mRNA expression was significantly higher (six- to sevenfold) in both control and 8-bromoadenosine-treated JAr cells compared to cytotrophoblast cells and was also significantly higher in untreated versus treated JAr cells. Western blotting showed that GLUT3 protein was undetectable in either cytotrophoblast cell groups, but was abundant in both groups of JAr cells. GLUT3 protein in JAr cells treated with 8-bromoadenosine was also significantly lower than in untreated JAr cells, in agreement with the mRNA data. We conclude that GLUT1 expression is unaffected by either growth or differentiation of trophoblast cells whereas GLUT3 expression is associated with dividing cells. We propose that in the placenta, GLUT3 may be involved in maintaining metabolic requirements of dividing trophoblast cells, rather than having a direct role in transport of glucose to the fetus.

In order to investigate mechanisms of fetal-maternal (F-M) transfer of Na+, clearance of 22Na+ and 51Cr-EDTA was measured simultaneously across the dually perfused placenta of the rat. In eight experiments clearance was measured successively in the F-M (Kfm) and in the maternal-fetal (M-F; Kmf) directions. Clearance of 22Na+ in the two directions was approximately equal (Kmf = 11.6 +/- 2.0 microliters/min; Kfm = 11.1 +/- 1.7 microliters/min: mean +/- s.d.) while Kfm of 51Cr-EDTA (4.4 +/- 0.7 microliters/min) was nearly double Kmf (2.4 +/- 0.8 microliters/min) for this tracer. Even greater asymmetry in the transfer of 51Cr-EDTA was found when measured across intact (non-perfused) placenta. It is suggested that this asymmetry is caused by volume flow in the F-M direction. In other experiments transfer was measured in the F-M direction only. Ouabain (0.1 mM) on the maternal side and reduced concentration of Na+ (25 mM) on the fetal side had no effect on the F-M transfer of the tracers. Reducing the temperature of the preparation by 5 degrees C significantly decreased transfer of 22Na+. The transfer of 22Na was inversely related to the concentration of K+ on the fetal side. These observations suggest that the F-M transfer of Na+ has three components: diffusion through paracellular routes; convective flow by filtration through wide placental pores, and transcellular transport by a mechanism which is uncertain at present.

The percentage of total placental water (%H2O(T)), maternal (%MBV) and fetal (%FBV) blood volumes, non-vascular extracellular (%EW) and intracellular (%IW) water, and villous histology were studied in placentas from 12 normal term pregnancies after a normal vaginal delivery, 19 caesarean sections at term after a normal pregnancy and history of a previous caesarean section and 47 caesarean sections at term or preterm due to pregnancy complications. Values were derived from change in placental dry weight, maternal and fetal haemoglobin content and 51CrEDTA space after incubation of placental fragments. Normal ranges (mean +/- SD) after term vaginal delivery were: H2O(T) 83.9 +/- 0.2%, MBV 10.9 +/- 0.2%, FBV 7.4 +/- 0.9%, EW 57.3 +/- 1.3% and IW 11.2 +/- 0.6%. %H2O(T) was higher after caesarean section; other measurements were not affected. There were no differences between placentas after 33-37 and after 38-42 weeks gestation. Three of eight placentas after rhesus incompatibility had %H2O(T) above the mean +2SD of term placentas and five of 17 IUGR placentas were below the mean -2SD. The remaining placentas following maternal pre-eclampsia, hypertension, or diabetes had no apparent alteration in %H2O(T). A blind histological diagnosis of 'true' oedema was associated with both a significantly high %IW and %H2O(T). Perhaps this is due to alteration in placental cell volume regulation in certain situations.