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.

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.

Contribution of PARP to endothelial dysfunction and hypertension in a rat model of pre-eclampsia.

BACKGROUND AND PURPOSE: Under conditions of increased oxidative stress, such as pre-eclampsia and diabetes, overstimulation of PARP leads to endothelial dysfunction. Inhibition of PARP has been demonstrated to reverse the vascular dysfunction associated with diabetes in vivo. The present study was carried out to investigate the role of PARP in mediating the endothelial dysfunction associated with pre-eclampsia. EXPERIMENTAL APPROACH: Uteroplacental perfusion was surgically reduced in pregnant rats to produce the reduced uterine perfusion pressure (RUPP) rat model of pre-eclampsia and the PARP inhibitor, PJ34, was administered either before or after surgery. Mean arterial BP and vascular function were measured in normal pregnant (NP) and both control and PJ34-treated RUPP rats. Mesenteric vessels from NP rats were incubated with either 3% RUPP or NP plasma alone or in combination with PJ34. Finally, immunohistochemical staining was carried out to measure nitrotyrosine (byproduct of peroxynitrite) immunoreactivity. KEY RESULTS: RUPP rats were characterized by hypertension, fetal growth restriction and endothelial dysfunction when compared with NP rats. PJ34 administered in vivo before, but not after, surgery prevented the development of both endothelial dysfunction and hypertension. RUPP plasma-induced impaired vasorelaxation was prevented following co-incubation with PJ34 in vitro. Furthermore, the protective effect of PARP inhibition in vivo was accompanied by a reduction in nitrotyrosine immunoreactivity. CONCLUSIONS AND IMPLICATIONS: PJ34 prevented the development of both endothelial dysfunction and hypertension and reduced vascular nitrotyrosine immunoreactivity, thus suggesting a role for oxidative-nitrosative stress/PARP activation in the aberration in both vascular and haemodynamic function in this rat model of pre-eclampsia.

IFPA Meeting 2011 workshop report II: Angiogenic signaling and regulation of fetal endothelial function; placental and fetal circulation and growth; spiral artery remodeling.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2011 there were twelve themed workshops, three of which are summarized in this report. These workshops related to vascular systems and circulation in the mother, placenta and fetus, and were divided in to 1) angiogenic signaling and regulation of fetal endothelial function; 2) placental and fetal circulation and growth; 3) spiral artery remodeling.

Magnetic resonance imaging relaxation time measurements of the placenta at 1.5 T.

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.

IFPA Meeting 2009 workshops report.

Workshops are an important part of the annual meeting of the International Federation of Placenta Associations (IFPA). At IFPA Meeting 2009 diverse topics were discussed in twelve themed workshops. Topics covered included: immune response to pregnancy; signaling between fetus and placenta; bioactive lipids in placenta; placenta in agricultural species; epigenetics and placentation; trophoblast deportation; glucocorticoids and placental function; endothelium; placental transport; genes and placenta; uteroplacental blood flow and placental stem cells. This report is a full summary of the various topics covered.

Endothelial progenitor cells: their potential in the placental vasculature and related complications.

Endothelial progenitor cells (EPCs) have received significant attention in recent times. A role for EPCs has been suggested in a range of pathologies and some recent studies of EPCs in pregnancy have been published. This review provides a guide to the confusing field of EPCs. Attention is paid to their phenotyping, as although elementary this remains a highly debated topic. The current understanding of different subtypes and physiological role of EPCs in the placenta, fetus and adult are also considered. An overview is given as to role of EPC's in the pathophysiology of different disease states and the possible therapeutic and diagnostic applications expected from EPC-related research in obstetrics.

Changes in the metabolic footprint of placental explant-conditioned culture medium identifies metabolic disturbances related to hypoxia and pre-eclampsia.

Pre-eclampsia (PE) is a multi-system disorder thought to be mediated by circulating factors released from damaged placental villous trophoblast. There is extensive evidence of changes in the villous tissue in PE, some of which may be replicated by culturing villous tissue in hypoxic conditions. Metabolic footprinting offers a hypothesis-generating strategy to investigate factors released from this tissue in vitro. This study investigated differences in the factors released from villous trophoblast from uncomplicated pregnancies (n=6) and those with PE (n=6). In both cases, explanted placental villous fragments were cultured for 96 h in 1% O(2) (hypoxia) or 6% O(2) (placental normoxia). Metabolites consumed from and released into serum-conditioned culture medium were analysed by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). The relative concentration of 154 features of the metabolic footprint were observed to change in culture medium from uncomplicated pregnancies cultured in normoxic and hypoxic conditions (p<0.00005). 21 and 80 features were also different in culture medium from PE versus uncomplicated pregnancies cultured in hypoxic and normoxic conditions, respectively (p<0.00005). When comparing all 4 groups, 47 metabolic features showed a similar relative concentration in PE-derived media cultured in normoxic conditions to conditioned media from normal villous tissue cultured in hypoxic conditions. These data suggest that hypoxia may have a role in the placental pathogenesis of PE. Three areas of metabolism were highlighted for systems biology investigation; glutamate and glutamine, tryptophan metabolism and leukotriene or prostaglandin metabolism.

A placental protective role for trophoblast-derived TNF-related apoptosis-inducing ligand (TRAIL).

Recent studies show that apoptosis, programmed cell death, plays an important role in the normal development of the human placenta and that an altered balance between proliferation and apoptosis of villous trophoblasts is associated with abnormal pregnancies. The TNF-related apoptosis-inducing ligand (TRAIL) is a molecule belonging to TNF superfamily. The role of TRAIL and its Death Receptor 5 (DR5) in regulating villous trophoblast cell turnover in normal and pathologic pregnancies remains to be explored. In order to elucidate the role of TRAIL in the regulation of placental growth, primary cytotrophoblast cells were isolated from normal term placentas (n=13) and cultured for 18 and 66h to generate mononucleate and multinucleate trophoblasts, respectively. The protein expression and localisation of TRAIL and DR5 were determined by Western blotting and immunofluorescence. Secreted sTRAIL was also measured by ELISA. Trophoblast apoptosis was measured by TUNEL in the presence of recombinant TRAIL (rTRAIL), and DR5 relocalisation was assessed by immunostaining after 18h exposure to TNFalpha. We demonstrated that TRAIL protein expression and the secretion of soluble TRAIL (sTRAIL) were down-regulated in syncytialised villous trophoblasts and that sTRAIL was independent of biochemical differentiation, as TRAIL-neutralizing antibody (2E5) failed to influence hCG production. TRAIL immunoreactivity was detected in mono- and multinucleated trophoblast cells and localised to the cytoplasm and cellular membranes -- more intense staining was associated with apoptotic nuclei. rTRAIL failed to induce apoptosis in trophoblasts cells owing to the nuclear localisation of DR5. However, TNFalpha treatment caused the redistribution of intracellular DR5 to the cell surface, potentiating apoptotic susceptibly to exogenously administered rTRAIL. These findings highlight a mechanism by which TRAIL and DR5 serve to protective trophoblasts in normal development, but may be activated in conditions of excessive TNFalpha.

Utero-placental haemodynamics in the pathogenesis of pre-eclampsia.

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.

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.

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.

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.

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.