Computational modelling of placental amino acid transfer as an integrated system.

Placental amino acid transfer is essential for fetal development and its impairment is associated with poor fetal growth. Amino acid transfer is mediated by a broad array of specific plasma membrane transporters with overlapping substrate specificity. However, it is not fully understood how these different transporters work together to mediate net flux across the placenta. Therefore the aim of this study was to develop a new computational model to describe how human placental amino acid transfer functions as an integrated system. Amino acid transfer from mother to fetus requires transport across the two plasma membranes of the placental syncytiotrophoblast, each of which contains a distinct complement of transporter proteins. A compartmental modelling approach was combined with a carrier based modelling framework to represent the kinetics of the individual accumulative, exchange and facilitative classes of transporters on each plasma membrane. The model successfully captured the principal features of transplacental transfer. Modelling results clearly demonstrate how modulating transporter activity and conditions such as phenylketonuria, can increase the transfer of certain groups of amino acids, but that this comes at the cost of decreasing the transfer of others, which has implications for developing clinical treatment options in the placenta and other transporting epithelia.

Phenylalanine transfer across the isolated perfused human placenta: an experimental and modeling investigation.

Membrane transporters are considered essential for placental amino acid transfer, but the contribution of other factors, such as blood flow and metabolism, is poorly defined. In this study we combine experimental and modeling approaches to understand the determinants of [(14)C]phenylalanine transfer across the isolated perfused human placenta. Transfer of [(14)C]phenylalanine across the isolated perfused human placenta was determined at different maternal and fetal flow rates. Maternal flow rate was set at 10, 14, and 18 ml/min for 1 h each. At each maternal flow rate, fetal flow rates were set at 3, 6, and 9 ml/min for 20 min each. Appearance of [(14)C]phenylalanine was measured in the maternal and fetal venous exudates. Computational modeling of phenylalanine transfer was undertaken to allow comparison of the experimental data with predicted phenylalanine uptake and transfer under different initial assumptions. Placental uptake (mol/min) of [(14)C]phenylalanine increased with maternal, but not fetal, flow. Delivery (mol/min) of [(14)C]phenylalanine to the fetal circulation was not associated with fetal or maternal flow. The absence of a relationship between placental phenylalanine uptake and net flux of phenylalanine to the fetal circulation suggests that factors other than flow or transporter-mediated uptake are important determinants of phenylalanine transfer. These observations could be explained by tight regulation of free amino acid levels within the placenta or properties of the facilitated transporters mediating phenylalanine transport. We suggest that amino acid metabolism, primarily incorporation into protein, is controlling free amino acid levels and, thus, placental transfer.

Computational modelling of amino acid exchange and facilitated transport in placental membrane vesicles.

Placental amino acid transport is required for fetal development and impaired transport has been associated with poor fetal growth. It is well known that placental amino acid transport is mediated by a broad array of specific membrane transporters with overlapping substrate specificity. However, it is not fully understood how these transporters function, both individually and as an integrated system. We propose that mathematical modelling could help in further elucidating the underlying mechanisms of how these transporters mediate placental amino acid transport. The aim of this work is to model the sodium independent transport of serine, which has been assumed to follow an obligatory exchange mechanism. However, previous amino acid uptake experiments in human placental microvillous plasma membrane vesicles have persistently produced results that are seemingly incompatible with such a mechanism; i.e. transport has been observed under zero-trans conditions, in the absence of internal substrates inside the vesicles to drive exchange. This observation raises two alternative hypotheses; (i) either exchange is not fully obligatory, or (ii) exchange is indeed obligatory, but an unforeseen initial concentration of amino acid substrate is present within the vesicle which could drive exchange. To investigate these possibilities, a mathematical model for tracer uptake was developed based on carrier mediated transport, which can represent either facilitated diffusion or obligatory exchange (also referred to as uniport and antiport mechanisms, respectively). In vitro measurements of serine uptake by placental microvillous membrane vesicles were carried out and the model applied to interpret the results based on the measured apparent Michaelis-Menten parameters Km and Vmax. In addition, based on model predictions, a new time series experiment was implemented to distinguish the hypothesised transporter mechanisms. Analysis of the results indicated the presence of a facilitated transport component, while based on the model no evidence for substantial levels of endogenous amino acids within the vesicle was found.

In vitro dual perfusion of human placental lobules as a flow phantom to investigate the relationship between fetoplacental flow and quantitative 3D power doppler angiography.

Flow phantoms have been used to investigate and quantify three-dimensional power Doppler data but this is the first study to use the in vitro, dual perfused, placental perfusion model. We used this model to investigate and quantify the effect of variation in fetal-side flow rates and attenuation on 3D power Doppler angiography. Perfusion of a placental lobule was commenced within 30 min of delivery and experimentation was successful in 8 of the 18 placenta obtained. Fetal and maternal perfusate was modified Earle's bicarbonate buffer which, following equilibration, was supplemented on the fetal side with whole heparinised cord blood. Imaging was performed with a Voluson-i ultrasound machine. A 'vascular biopsy' the thickness of the placental lobule was defined and signal quantified within using VOCAL (GE Medical Systems, Zipf, Austria). Three vascular indices are generated: vascularisation index (VI) defined as the percentage of power Doppler data within a volume of interest; flow index (FI), the mean signal intensity of the power Doppler information; and vascularisation flow index (VFI), a combination of both factors derived through their multiplication. Attenuation was investigated in this model with the addition of tissue mimic blocks. Our results showed a predictable relationship between flow rates and the vascular indices VI and VFI. However the FI was a less reliable predictor of flow; thus it should be interpreted with caution. The power Doppler signal was markedly affected by attenuation leading to a complete loss of information at a depth of 6 cm in the model used. In conclusion this model can be adapted to provide a phantom to analyse and quantify 3D power Doppler signals and demonstrates that vascular indices within a tissue remain related to volume flow. This model provides further evidence that depth dependent attenuation of signal needs to be accounted for in any in vivo work where the probe is not in direct contact with the tissue of interest.

Live and let die - regulation of villous trophoblast apoptosis in normal and abnormal pregnancies.

Since 1995 the number of publications investigating apoptosis in villous trophoblast has increased exponentially. This scientific interest is in part due to observations that this specialised form of cell death is increased in pregnancy complications such as pre-eclampsia and intra-uterine growth restriction. In addition, apoptosis is described in normal villous trophoblast and elements of the apoptotic machinery are involved in the fusion between cytotrophoblast and the overlying multinucleate syncytiotrophoblast. The increase in descriptions of apoptotic cell death in villous trophoblast has been accompanied by investigations of regulators of apoptosis. It is anticipated that understanding the regulation of apoptosis in villous trophoblast may provide new insights into placental pathologies. This review describes current knowledge regarding the expression and function of these regulators in villous trophoblast, both in normal and complicated pregnancies.

Effects of oxygen on cell turnover and expression of regulators of apoptosis in human placental trophoblast.

Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are associated with aberrant cell turnover, including increased apoptosis, in placental villous trophoblast. The increased apoptosis is associated with exaggerated expression of p53, which promotes cell cycle arrest or apoptosis via downstream proteins such as p21 or Bax. These changes in apoptosis and p53 expression are purported to result from exposure to altered oxygen tension. Using a model of villous trophoblast turnover, we examined the effect of 20%, 6% and 1% ambient oxygen (O(2)) on apoptosis, necrosis, proliferation and expression of p53 and related regulators of cell turnover, compared to both fresh tissue. Altered O(2) tension exerted an effect on cell turnover in cultured term villous tissue: cytotrophoblast proliferation was increased by culture in 20% O(2) and reduced in 1% O(2) (median proliferative index: fresh tissue=0.32%, 20% O(2)=0.9%, 6% O(2)=0.28%, 1% O(2)=0.07%). Apoptosis was increased in all culture environments, but was significantly enhanced by culture in 1% O(2) (median apoptotic index: fresh tissue=0.64%, 20% O(2)=2.96%, 6% O(2)=3.81%, 1% O(2)=9.2%). Necrotic cell death was also increased by culture in 1% O(2) compared to 6% and 20% O(2). The expression of p53, p21 and Mdm2 in both cytotrophoblast and stromal cells was increased following culture in 1% O(2). There was no alteration in the expression of Bax or Bcl-2. This study provides evidence that p53 is elevated in trophoblast following exposure to hypoxia. The potential role of the p53-pathway in the control of cell turnover in villous trophoblast and the regulation of p53 by altered O(2) tension merits further investigation.

Analysis of the metabolic footprint and tissue metabolome of placental villous explants cultured at different oxygen tensions reveals novel redox biomarkers.

Pre-eclampsia (PE) is a multi-system disorder of pregnancy hypothesised to arise from circulating factors derived from an unhealthy placenta. Some changes in placental phenotype seen in PE can be reproduced by culture in altered oxygen (O2) tension. Currently, these circulating factors are unidentified, partly due to the complexity of maternal plasma. Investigation of factors released from placental tissue provides a potential method to identify bioactive compounds. Experimental strategies to study compounds present in a biological system have expanded greatly in recent years. Metabolomics can detect and identify endogenous and secreted metabolites. We aimed to determine whether metabolites could be identified in placental cultures with acceptable experimental variability and to determine whether altered O2 tension affects the composition of the placental metabolome. In this study we used gas-chromatography-mass spectroscopy to determine the presence of metabolites in conditioned culture medium (CCM) and tissue lysates of placental villous explants cultured in 1, 6 and 20% atmospheric O2 for 96h. This experimental strategy had an intra-assay variation of 6.1-11.6%. Intra and inter-placental variability were 15.7-35.8% and 44.8-46.2% respectively. Metabolic differences were identified between samples cultured in 1, 6 and 20% O2 in both CCM and tissue lysate. Differentially expressed metabolites included: 2-deoxyribose, threitol or erythritol and hexadecanoic acid. We conclude that metabolomic strategies offer a novel approach to investigate placental function. When conducted under carefully controlled conditions, with appropriate statistical analysis, metabolic differences can be identified in placental explants in response to altered O2 tension. Metabolomics could be used to identify changes in conditions associated with placental pathology.

Vasoactive and permeability effects of vascular endothelial growth factor-165 in the term in vitro dually perfused human placental lobule.

Vascular endothelial growth factor (VEGF) is an important vasodilator and effector of permeability in systemic blood vessels. Molecular and tissue culture techniques have provided evidence for its placental synthesis and release. Using an in vitro dual-perfusion model of the term placental lobule from normal pregnancy, we report here the relative secretion of total VEGF, soluble VEGF receptor (VEGFR)-1, and free VEGF into the maternal and fetoplacental circulations of the placenta. We tested the hypothesis that VEGF has vasomotor and permeability effects in the fetoplacental circulation of the human placenta, and we examined the broad intracellular pathways involved in the vasodilatory effect that we found. We show that total VEGF is released into the fetal and maternal circulations in a bipolar fashion, with a bias toward maternal side output. Soluble VEGFR-1 was also secreted into both circulations with bias toward the maternal side. Consequently, free VEGF (12.8 +/- 2.4 pg/ml, mean +/- se) was found only in the fetoplacental circulation. VEGF-165 was found to be a potent vasodilator of the fetoplacental circulation (maximum response: 77% of previous steady-state fetal-side inflow hydrostatic pressure after preconstriction with U46619; EC(50) = 71 pm). This vasodilatory effect was mediated by the VEGFR-2 receptor and nitric oxide in a manner-independent of the involvement of prostacyclin and the src-family tyrosine kinases. However, nitric oxide could explain only 50% of the vasodilatory effect. Finally, we measured the permeability of the perfused placenta to inert hydrophilic tracers and found no difference in the presence and absence of VEGF.

The mitotic manipulation of cytotrophoblast differentiation in vitro.

The placental syncytiotrophoblast is of paramount importance in optimising feto-maternal interactions. Syncytiotrophoblast is generated by the differentiation and fusion of underlying cytotrophoblasts. This process is aberrant in complicated pregnancies. We hypothesized that cell cycle withdrawal determines the phenotypic decision-making of cytotrophoblasts. We therefore investigated the effects of broad-spectrum mitotic inhibitors on cytotrophoblast differentiation. Villous tissue was dissected from term placentae of normal pregnancies and cultured on Netwell supports. Over 48 h, the original syncytiotrophoblast was detached and underlying cytotrophoblasts exposed. The resulting villi were treated with mitotic blockers (Ara-C, colcemid, cyclohexamide, doxorubicin hydrochloride, hydroxyurea, L-Mimosine, purvalanol A). The media was recovered and analysed for lactacte dehydrogenase (LDH) and human chorionic gondadotrophin (hCG), markers of tissue viability and cytotrophoblast differentiation, respectively. The resulting tissue was processed for proliferative activity thorough Ki-67 immunorecognition. Colcemid, cyclohexamide, hydroxyurea, and purvalanol A showed significant cytotoxicity over 48 h incubation. Villous tissue exposed to 0.01 mM and 0.1mM Ara-C, doxorubicin hydrochloride and L-Mimosine showed no increase in liberated LDH. hCG production increased exponentially with cytotrophoblast differentiation. Higher concentrations of Ara-C and L-Mimosine significantly encouraged hCG production. In addition, total cell and cytotrophoblast proliferation were reduced with Ara-C and L-Mimosine treatment. The inhibition of DNA synthesis and replication with Ara-C and L-Mimosine suppressed active proliferation of villus components and exaggerated the biochemical differentiation of cytotrophoblasts. Cell cycle disruption is therefore a basic trigger for cytotrophoblast differentiation. This approach provides a mechanism for encouraging syncytiotrophoblast formation and may hold benefits for conditions where syncytiotrophoblast cover is attenuated.

Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species.

The syncytiotrophoblast contains aggregates of nuclei termed syncytial knots. Increased numbers of syncytial knots have been reported in placentae of pregnancies complicated by pre-eclampsia and fetal growth restriction (FGR). As oxidative stress has been implicated in the pathophysiology of these disorders, we hypothesised that the formation of syncytial knots may be induced by exposure to hypoxia, hyperoxia or reactive oxygen species (ROS). We assessed both the number and morphology of syncytial knots induced by culture in hypoxia, hyperoxia and with ROS. We also investigated whether the presence of syncytial knots in normal tissue was associated with a down-regulation of anti-apoptotic proteins Bcl-2, Mdm2, XIAP and survivin. Using our measurement system we describe an increased number of syncytial knots when tissue is cultured in hypoxia, hyperoxia or in the presence of ROS. The morphology of these syncytial knots was similar to those seen in vitro, although the nuclei from cultured placental explants were morphologically more homogenous, had fewer nuclear pores, and a higher heterochromatin:euchromatin ratio. Despite the apoptotic appearances of nuclei we did not detect a loss of anti-apoptotic proteins in the region of syncytial knots. We conclude that the increased number of syncytial knots in placentae from pregnancies complicated by pre-eclampsia and FGR can be replicated in vitro by ROS or hypoxia, supporting their involvement in the pathogenesis of these conditions.

The in-vitro characterization of induced apoptosis in placental cytotrophoblasts and syncytiotrophoblasts.

Placental trophoblasts undergo apoptosis as part of normal epithelial turnover and placental ageing. Classically, the induction of apoptosis in in vitro preparations has utilized the cytokines TNFalpha and IFNgamma and has been measured using the TUNEL technique. The aim of this study was to compare apoptotic susceptibility of mononucleated and differentiated trophoblasts using a range of cytotoxic agents. To achieve this, an in vitro model of syncytialization was used, along with isolated placental cytotrophoblasts and an extravillous cytotrophoblast derived cell line (SGHPL-4). Cytotrophoblasts from term placentae (n=12), syncytiotrophoblasts (n=12) and SGHPL-4s (n=8) were cultured under reduced oxygen or with TNFalpha/IFNgamma, dexamethasone or staurosporine. Apoptosis assessments were made using TUNEL, Annexin V binding, fluorescence microscopy and ATP/ADP measurements. Each cytotoxic agent increased apoptosis in all three cell populations. For untreated cells, cytotrophoblasts showed the greatest levels of apoptosis in culture. With stimulation, these levels were significantly elevated using dexamethasone, TNFalpha/IFNgamma and staurosporine and further raised under hypoxic conditions. SGHPL-4 cells showed similar trends to those of cytotrophoblasts, however the syncytiotrophoblasts, although responsive to dexamethasone and TNFalpha/IFNgamma, showed lower levels of apoptosis with staurosporine and hypoxia. ADP : ATP measurements gave similar results to the other techniques and ratios of less than 1.0 were correlated with Annexin V measurements on the flow cytometer (P< 0.001). The typical morphological features of apoptosis i.e. chromatin margination, membrane blebbing and apoptotic body formation were detected in cytotrophoblasts and SGHPL-4 cells. However, only chromatin condensation could be recognized in syncytiotrophoblast preparations. Necrotic cell numbers were also increased under all cytotoxic conditions. Although elevated with dexamethasone, staurosporine and hypoxia, these levels were markedly raised in cytotrophoblasts and SGHPL-4 cells following incubations with TNFalpha/IFNgamma. These observations show variations in apoptosis between mononuclear trophoblasts and differentiated multinucleated syncytiotrophoblasts. Differential effects of stimuli may suggest disparate apoptotic pathways. These variations may reflect functional differences between placental cellular and syncytial components and may highlight the importance of exogenous stimulation in various stages of placental development.

Stereological investigation of placental morphology in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction.

The aim of this study was to quantify placental morphology in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction. Particular attention is given to the dimensions and composition of peripheral (intermediate+terminal) villi. Placentae from 9 control pregnancies, 5 cases of pre-eclampsia, 5 cases of intrauterine growth restriction and 5 cases of pre-eclampsia with intrauterine growth restriction were randomly sampled for location and position. Formalin-fixed, wax-embedded sections stained by the Masson trichrome method were subjected to stereological analysis in order to quantify the volumes of placental components and the surfaces and derived diameters for peripheral villi and fetal capillaries. Group comparisons were drawn using two-way analysis of variance. Fetal weights were reduced in all complicated pregnancies but only intrauterine growth restriction was accompanied by a significantly smaller placenta. Pre-eclampsia was not associated with main effects on placental morphology and (except for trophoblast thickness) there were no interaction effects involving pre-eclampsia. In contrast, intrauterine growth restriction was associated with a placenta which had reduced volumes of intervillous space and all types of villi (stem, intermediate, terminal). The impoverished growth of peripheral villi affected all tissues (trophoblast, stroma, capillaries) and was accompanied by smaller exchange surface areas and a thicker trophoblastic epithelium. The derived mean diameters of villi and capillaries were not affected. It is concluded that intrauterine growth restriction, but not pre-eclampsia, is associated with substantial changes in placental morphology including impoverished growth of villi and fetal vasculature. These changes are likely to reduce placental oxygen diffusive conductances and contribute to fetal hypoxic stress.

Characterization of small arteries isolated from the human placental chorionic plate.

Despite the essential role of the placenta in pregnancy, the control of the blood flow within the fetoplacental circulation is poorly understood. A handful of myography studies have directly assessed the role of vasoactive agonists in fetoplacental vasculature contractility but have used a range of steady-state conditions. Our aim, therefore, was to determine the optimal vessel diameter and oxygen tension to assess vascular function in small arteries isolated from the chorionic plate of normal term placentae. Biopsies were obtained from term placentae from uncomplicated pregnancies. Small arteries were dissected from the chorionic plate, mounted onto a wire myograph in HCO3(-) -buffered physiological salt solution at 37 degrees C and equilibrated for 20 min. Two methods for normalization of the optimal length/diameter for contractility of chorionic plate small arteries were assessed. Both classical normalization (CN) and length-tension curve (LTC) methods produced similar data. These data were agonist-independent. Data for CN and LTC were unaffected but maximal force generation (for U46619) was decreased in reduced oxygen tensions. Using conditions for optimal tension production in chorionic plate small arteries the thromboxane-mimetic U46619 produced the greatest and most reproducible constrictive effect. Relaxations were only achieved with endothelial-independent agonists (sodium nitroprusside and papaverine).

Syncytiotrophoblast degradation and the pathophysiology of the malaria-infected placenta.

Malaria is associated with excessive parasitic infection of the placenta and a reduction in neonatal birthweight. This study has investigated placental cell death in women with active and past malarial infection. Term placentae, with and without malarial pathology, were obtained from women in The Gambia. Active and past malaria infections were identified in placental sections and histological examination was used to determine the number of villi, the incidence of apoptosis, syncytial degradation, fibrinoid deposition and the frequency of syncytial knots. Placentae with active malaria infection showed erythrocyte adhesion of infected cells to syncytiotrophoblast, syncytial degradation, increased syncytial knotting and, in rare cases, localized destruction of the villi. Past malarial infection was characterized by syncytiotrophoblast disruption and fibrin-type fibrinoid (FTF) deposition. Perivillous FTF deposition was consistent with increased syncytial lesions and both increased lesions and syncytial knots were associated with birthweight reductions. Active malaria infection produced no alteration in placental apoptosis. The numbers of chorionic villi remained unchanged and infiltration of inflammatory cells, although not measured directly, appeared to be non-pervasive within the infected tissue. These observations establish a direct link between malaria parasitic infection and syncytiotrophoblast damage. The placental rejection of parasite-affected syncytia may invoke structural changes to compensate for inadequate placental exchange. Syncytial destruction could have serious implications; impairing fetal growth and in some rare cases, providing a previously unrecognized pathway to congenital infection.

Trophoblast invasion of spiral arteries: a novel in vitro model.

Extravillous trophoblasts invade the uterine wall (interstitial invasion) and the spiral arteries (endovascular invasion), replacing the cells of the vessel wall and creating a high-flow low-resistance vessel. We have developed a novel model to allow the interactions between the invading trophoblast cells and the cells of the spiral artery to be directly examined. Unmodified (non-placental bed) spiral arteries were obtained from uterine biopsies at caesarean section. Fluorescently labelled trophoblasts were seeded on top of artery segments embedded in fibrin gels (to study interstitial invasion) or perfused into the lumen of arteries mounted on a pressure myograph (to study endovascular invasion). Trophoblasts were incubated with the vessels for 3-5 days prior to cryo-sectioning. Both interstitial and endovascular interactions/invasion could clearly be detected and a comparison of the extravillous trophoblast cell line, SGHPL-4 and primary first trimester cytotrophoblasts showed both to be invasive in this model. This novel method will prove useful in an area where in vitro studies have been hampered by the lack of suitable models directly examining cellular interactions during invasion.

The anti-inflammatory effects of circulating fatty acids in obstructive jaundice: similarities with pregnancy-induced immunosuppression.

Rheumatoid arthritis (RA) is ameliorated during both obstructive jaundice and pregnancy. Previous studies of polymorphonuclear leukocyte (PMN) function during pregnancy have shown reductions in the stimulated release of arachidonic acid (AA) and leukotriene B4 (LTB4), and lower NADPH oxidase activity. These changes may account for the amelioration of RA. The cause of this reduction in PMN function appears to be a progressive change in circulating fatty acids (FA), with a reduction in polyunsaturated FA, predominantly AA. The NADPH oxidase responsible for the respiratory burst has a direct requirement for polyunsaturated FA, particularly AA. We investigated whether the same changes in PMN function and FA, occur during obstructive jaundice. Patients with biliary obstructions were investigated before and after surgical correction (n=14). Obstructive jaundice caused significant changes in the proportions of serum and cellular FA. There was a striking reduction in polyunsaturated FA, particularly AA (48% in serum, p<0.001; 42% in PMNs, p<0.001) and an increase in mono-unsaturated oleic acid (24% in serum, p<0.001; 15% in PMNs, p<0.005). Similar changes occurred in mononuclear cell FA. Jaundice also caused a significant reduction in PMN function. Respiratory burst activity was reduced by between 32% and 38% in response to physiological and non-physiological stimuli, and there were similar significant reductions in the release of AA and LTB4. These changes in stimulated PMN function were evident whether or not the cells were first primed with tumour necrosis factor alpha (TNFalpha). Incubation of PMNs from healthy donors in pooled serum from patients with obstructive jaundice caused a reduction of 32% in cellular AA and 38% in NADPH oxidase activity. These findings support the idea that circulating FA can regulate PMN inflammatory responsiveness. The FA-induced attenuation in PMN activity in both jaundice and pregnancy may explain their ameliorating effects upon RA.

Matrix metalloproteinase release from placental explants of pregnancies complicated by intrauterine growth restriction.

OBJECTIVE: There is evidence of impaired placental development in intrauterine growth restriction (IUGR). Matrix metalloproteinases (MMPs) are extracellular matrix-degrading enzymes that are released by placental cells during tissue remodeling processes. We hypothesized 1) that release of MMP-2 and -9 is decreased and/or release of tissue inhibitors of metalloproteinases (TIMPs) is increased from placental explants in pregnancies complicated by IUGR and 2) that oxygen levels affect such release. METHODS: Placental villous explants from normal (n = 7) and IUGR (n = 7) pregnancies were cultured at high (20%) and low (3%) oxygen levels for 24 hours. Supernatants were analyzed for MMP-2 and MMP-9 by zymography and for TIMP-1 and -2 by western blot analysis. RESULTS: : At 20% oxygen there was significantly reduced MMP-2 (P < .05) and TIMP-1 (P < .01) release and a trend for decreased MMP-9 release (P = .07) in explants from IUGR pregnancies compared with normal pregnancies; however, there were no differences at 3% oxygen. TIMP-2 was below detectable levels in all samples. Although MMP-2 and TIMP-1 release was significantly reduced at 3% compared with 20% oxygen in explants from both normal (P < .001; P < .05) and IUGR (P < .05) pregnancies, MMP-2 release changed less in IUGR compared with normal explant cultures. There were no significant effects of oxygen on MMP-9 release. CONCLUSION: Placental explants from IUGR pregnancies demonstrated reduced MMP-2, MMP-9, and TIMP-1 release compared with explants from normal pregnancies at high (20%) but not low (3%) oxygen.

Vascular endothelial growth factor but not placental growth factor promotes trophoblast syncytialization in vitro.

OBJECTIVE: The object of this study was to determine the effect of epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) on the differentiation of first-trimester and term cytotrophoblasts. METHODS: The first-trimester trophoblasts were isolated from villous tissue obtained at suction termination (n = 5), and the term trophoblasts were isolated from placentas (n = 6) at elective cesarean. Cultured cells were stimulated with EGF, VEGF, or PlGF at 0.5, 5, and 50 ng/mL, in the presence or absence of N(G)-nitro-L-arginine methyl ester hydrochloride (10(-4) M). Syncytialized trophoblasts were identified by immunostaining with antidesmosomal protein and anti-cytokeratin-7, whereas nuclei were counted in each syncytia using hematoxylin. RESULTS: Without treatment, background levels of syncytialization were significantly higher in term preparations than first-trimester cells. With VEGF and EGF, the number and size of syncytia increased significantly for the first-trimester cytotrophoblasts (P <.05). Neither VEGF nor EGF had any effect on the syncytialization of cultured cells at term. Nitric oxide showed no involvement in syncytial induction, and PlGF had no effect on syncytialization of cytotrophoblasts, from either the first or third trimester. CONCLUSION: Both EGF and VEGF appeared to enhance the in vitro syncytialization of first trimester cytotrophoblasts.

Review: Endothelial progenitor cells in pregnancy and obstetric pathologies.

Since their discovery, endothelial progenitor cells (EPCs) have generated considerable interest in vascular biology. They are a heterogeneous population of cells that exist in both the fetus and adult, and are mobilized to support de novo vessel formation or encourage vascular health. This review summarizes our understanding of these cells in pregnancy, paying particular attention to their physiological role in placental development and the uterus, alongside their involvement in related obstetric pathologies.

Review: Modelling placental amino acid transfer--from transporters to placental function.

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.