Perinatal optimisation for periviable birth and outcomes: a 4-year network analysis (2018-2021) across a change in national guidance.

Introduction: The British Association of Perinatal Medicine (BAPM) released their revised framework for extremely preterm infant management in 2019. This revised framework promotes consideration of perinatal optimisation and survival-focused care from 22 weeks gestation onwards. This was a departure from the previous BAPM framework which recommended comfort care as the only recommended management for infants <23 + 0 weeks. Methods: Our study evaluates the clinical impact that this updated framework has had across the Northwest of England. We utilised anonymised network data from periviable infants delivered across the region to examine changes in perinatal optimisation practices and survival outcomes following the release of the latest BAPM framework. Results: Our data show that after the introduction of the updated framework there has been an increase in perinatal optimisation practices for periviable infants and an 80% increase in the number of infants born at 22 weeks receiving survival-focused care and admission to a neonatal unit. Discussion: There remain significant discrepancies in optimisation practices by gestational age, which may be contributing to the static survival rates that were observed in the lowest gestational ages.

A massively multi-scale approach to characterizing tissue architecture by synchrotron micro-CT applied to the human placenta.

Multi-scale structural assessment of biological soft tissue is challenging but essential to gain insight into structure-function relationships of tissue/organ. Using the human placenta as an example, this study brings together sophisticated sample preparation protocols, advanced imaging and robust, validated machine-learning segmentation techniques to provide the first massively multi-scale and multi-domain information that enables detailed morphological and functional analyses of both maternal and fetal placental domains. Finally, we quantify the scale-dependent error in morphological metrics of heterogeneous placental tissue, estimating the minimal tissue scale needed in extracting meaningful biological data. The developed protocol is beneficial for high-throughput investigation of structure-function relationships in both normal and diseased placentas, allowing us to optimize therapeutic approaches for pathological pregnancies. In addition, the methodology presented is applicable in the characterization of tissue architecture and physiological behaviours of other complex organs with similarity to the placenta, where an exchange barrier possesses circulating vascular and avascular fluid spaces.

Risk stratification for early-onset fetal growth restriction in women with abnormal serum biomarkers: a retrospective cohort study.

Abnormal maternal serum biomarkers (AMSB), identified through the aneuploidy screening programme, are frequent incidental findings in pregnancy. They are associated with fetal growth restriction (FGR), but previous studies have not examined whether this association is with early-onset (< 34 weeks) or late-onset (> 34 weeks) FGR; as a result there is no consensus on management. The aims of this study were to determine the prevalence and phenotype of FGR in women with AMSB and test the predictive value of placental sonographic screening to predict early-onset FGR. 1196 pregnant women with AMSB underwent a 21-24 week "placental screen" comprising fetal and placental size, and uterine artery Doppler. Multivariable regression was used to calculate a predictive model for early-onset FGR (birthweight centile < 3rd/< 10th with absent umbilical end-diastolic flow, < 34 weeks). FGR prevalence was high (10.3%), however early-onset FGR was uncommon (2.3%). Placental screening effectively identified early-onset (area under the curve (AUC) 0.93, 95% confidence interval (CI) 0.87-1.00), but not late-onset FGR (AUC 0.70, 95% CI 0.64-0.75). Internal validation demonstrated robust performance for detection/exclusion of early-onset FGR. In this cohort, utilisation of our proposed algorithm with targeted fetal growth and Doppler surveillance, compared with universal comprehensive surveillance would have avoided 1044 scans, potentiating significant cost-saving for maternity services.

Effect of sildenafil on maternal hemodynamics in pregnancies complicated by severe early-onset fetal growth restriction: planned subgroup analysis from a multicenter randomized placebo-controlled double-blind trial.

OBJECTIVES: Fetal growth restriction (FGR) is associated with maternal cardiovascular changes. Sildenafil, a phosphodiesterase type-5 inhibitor, potentiates the actions of nitric oxide, and it has been suggested that it alters maternal hemodynamics, potentially improving placental perfusion. Recently, the Dutch STRIDER trial was stopped prematurely owing to excess neonatal mortality secondary to pulmonary hypertension. The main aim of this study was to investigate the effect of sildenafil on maternal hemodynamics in pregnancies with severe early-onset FGR. METHODS: This was a cardiovascular substudy within a UK multicenter, placebo-controlled trial, in which 135 women with a singleton pregnancy and severe early-onset FGR (defined as a combination of estimated fetal weight or abdominal circumference below the 10th centile and absent/reversed end-diastolic flow in the umbilical artery on Doppler velocimetry, diagnosed between 22 + 0 and 29 + 6 weeks' gestation) were assigned randomly to receive either 25 mg sildenafil three times daily or placebo until 32 + 0 weeks' gestation or delivery. Maternal blood pressure (BP), heart rate (HR), augmentation index, pulse wave velocity (PWV), cardiac output, stroke volume (SV) and total peripheral resistance were recorded before randomization, 1-2 h and 48-72 h post-randomization, and 24-48 h postnatally. For continuous data, analysis was performed using repeated measures ANOVA methods including terms for timepoint, treatment allocation and their interaction. RESULTS: Included were 134 women assigned randomly to sildenafil (n = 69) or placebo (n = 65) who had maternal BP and HR recorded at baseline. At 1-2 h post-randomization, compared with baseline values, sildenafil increased maternal HR by 4 bpm more than did placebo (mean difference, 5.00 bpm (95% CI, 1.00-12.00 bpm) vs 1.25 bpm (95% CI, -5.38 to 7.88 bpm); P = 0.004) and reduced systolic BP by 1 mmHg more (mean difference, -4.13 mmHg (95% CI, -9.94 to 1.44 mmHg) vs -2.75 mmHg (95% CI, -7.50 to 5.25 mmHg); P = 0.048). Even after adjusting for maternal mean arterial pressure, sildenafil reduced aortic PWV by 0.60 m/s more than did placebo (mean difference, -0.90 m/s (95% CI, -1.31 to -0.51 m/s) vs -0.26 m/s (95% CI, -0.75 to 0.59 m/s); P = 0.001). Sildenafil was associated with a non-significantly greater decrease in SV index after 1-2 h post-randomization than was placebo (mean difference, -5.50 mL/m2 (95% CI, -11.00 to -0.50 mL/m2 ) vs 0.00 mL/m2 (95% CI, -5.00 to 4.00 mL/m2 ); P = 0.056). CONCLUSIONS: Sildenafil in a dose of 25 mg three times daily increases HR, reduces BP and reduces arterial stiffness in pregnancies complicated by severe early-onset FGR. These changes are short term, modest and consistent with the anticipated vasodilatory effect. They have no short- or long-term clinical impact on the mother. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

A clinical evaluation of placental growth factor in routine practice in high-risk women presenting with suspected pre-eclampsia and/or fetal growth restriction.

OBJECTIVE: To evaluate the use of plasma Placental Growth Factor (PlGF), recommended by the recent NICE guidance, in women with suspected pre-eclampsia (PE) and/or fetal growth restriction (FGR). STUDY DESIGN: Non-randomised prospective clinical evaluation study in high-risk antenatal clinics in a tertiary maternity unit. METHODS: PlGF testing was performed in addition to routine clinical assessment in 260 women >20 weeks' gestation with chronic disease (hypertension, renal disease ±â€¯diabetes) with a change in maternal condition or in women with suspected FGR to determine the impact on clinical management. Results were revealed and standardised care pathways followed. MAIN OUTCOME MEASURES: Outcome of pregnancies with a low PlGF (<12 pg/ml and 13-100 pg/ml), impact on clinical service and the diagnostic accuracy of alternative PlGF cut-offs. RESULTS: 206/260 (79.2%) women had an adverse outcome (PE/birthweight < 10th centile/preterm birth). In our cohort, a low PlGF (<12 pg/ml) was associated with a shorter test-birth interval and universally (100% PPV) with an adverse pregnancy outcome, although 29/61 (47.5%) of women with PlGF < 12 pg/ml continued their pregnancy >14 days. The PlGF result altered clinical management (surveillance or timing of birth) in 196/260 (75.4%) cases. Alternative PlGF thresholds did not significantly improve diagnostic performance. CONCLUSIONS: Our evaluation confirms the value of PlGF as a diagnostic tool for placental dysfunction. However, low PlGF in isolation should not trigger iatrogenic delivery. Further research linking placental pathology, maternal disease and maternal PlGF levels is urgently needed before this test can be implemented in routine clinical practice.

The effect of labetalol and nifedipine MR on blood pressure in women with chronic hypertension in pregnancy.

AIM: To compare the blood pressure (BP) lowering effects of labetalol and nifedipine modified release (MR) in hypertensive pregnant women. We also investigated the effect on the heart rate (HR) and determined the proportion of time spent in target. METHODS: This was an exploratory study. Women with chronic hypertension taking either labetalol or nifedipine were offered 24-h ambulatory blood pressure monitoring (ABPM). Sleep, wake and drug ingestion times were self-reported. An indirect response model was used to analyse the systolic BP (SBP), diastolic BP (DBP) and HR time-series; the effect of gestation and type of drug was evaluated. RESULTS: Forty-eight women were recruited: 24 in each group. There was no difference in clinical characteristics. In women taking nifedipine there was a positive association between the dose of nifedipine and pre-dose BP p = .002, this was not present in the labetalol group. There was a difference between the drug effects on both the SBP and DBP time-series (p = .014). In comparison to labetalol, there was less variation in day time BP in those women prescribed nifedipine. Women on labetalol spent a larger proportion of time with their DBP below target (<80 mmHg). The HR dynamics were qualitatively different, a stimulatory effect was found with nifedipine compared to an inhibitory effect with labetalol. CONCLUSION: There are significant and important differences between the BP lowering effects of nifedipine and labetalol. A large randomised control trial is required to investigate the relationship between BP variability and time in target on pregnancy outcomes.

Third trimester placental volume and biometry measurement: A method-development study.

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 (15° and 30° 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.

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.

Clinical evaluation of the IONA test: a non-invasive prenatal screening test for trisomies 21, 18 and 13.

OBJECTIVE: To evaluate the clinical accuracy of the IONA® test for aneuploidy screening. METHODS: This was a multicenter blinded study in which plasma samples from pregnant women at increased risk of trisomy 21 underwent cell-free DNA analysis utilizing the IONA test. For each sample, the IONA software generated a likelihood ratio and a maternal age-adjusted probability risk score for trisomies 21, 18 and 13. All results from the IONA test were compared against accepted diagnostic karyotyping. RESULTS: A total of 442 maternal samples were obtained, of which 437 had test results available for analysis and assessment of clinical accuracy. The IONA test had a detection rate of 100% for trisomies 21 (n = 43; 95% CI, 87.98-100%), 18 (n = 10; 95% CI, 58.72-100%) and 13 (n = 5; 95% CI, 35.88-100%) with cut-offs applied to likelihood ratio (cut-off > 1 considered high risk for trisomy) and probability risk score incorporating adjustment for maternal age (cut-off ≥ 1/150 considered high risk for trisomy). The false-positive rate (FPR) was 0% for trisomies 18 and 13 with both analysis outputs. For trisomy 21, a FPR of 0.3% was observed for the likelihood ratio, but became 0% with adjustment for maternal age. CONCLUSION: This study indicates that the IONA test is suitable for trisomy screening in a high-risk screening population. The result-interpretation feature of the IONA software should facilitate wider implementation, particularly in local laboratories, and should be a useful addition to the current screening methods for trisomies 21, 18 and 13.

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.

IFPA Gábor Than Award Lecture: Recognition of placental failure is key to saving babies' lives.

In high-income countries, placental failure is implicated in up to 65% of cases of stillbirth. Placental failure describes the situation where the placenta cannot meet the fetus' needs and may be the end-result of a variety of underlying pathological processes evident in the placental disc, membranes and umbilical cord. These include lesions with genetic, environmental, infectious, inflammatory, mechanical, metabolic, traumatic or vascular origin. Investigation of placental tissue from stillbirths and from pregnancies at an increased risk of stillbirth has demonstrated changes in macroscopic and microscopic structure which are themselves related to abnormal placental function. A better understanding and identification of placental failure may improve the management of pregnancy complications and of pregnancies after stillbirth (which have a 5-fold increased risk of stillbirth). The majority of current antenatal tests focus on the fetus and its response to the intrauterine environment; few of these investigations reduce stillbirths in low-risk pregnancies. However, some currently used investigations reflect placental development, structure and vascular function, while other investigations employed in clinical research settings such as the evaluation of placental structure and shape have a good predictive value for adverse fetal outcome. In addition, recent studies suggest that biomarkers of placental inflammation and deteriorating placental function can be detected in maternal blood suggesting that holistic evaluation of placental structure and function is possible. We anticipate that development of reliable tests of placental structure and function, coupled to assessment of fetal wellbeing offer a new opportunity to identify pregnancies at risk of stillbirth and to direct novel therapeutic strategies to prevent it.

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.

Fetoplacental vascular alterations associated with fetal growth restriction.

INTRODUCTION: Placental functional impairment in pregnancies with fetal growth restriction (FGR) can arise from fetoplacental vascular abnormalities. We aimed to compare the micro and macrovasculature of placentas from normal pregnancies with those showing late onset FGR. METHODS: Placental arterial casts (n = 12 normal, 6 FGR) were prepared. Chorionic arterial number and inter-branch length were examined. Microvascular features were quantified in CD34-stained tissue sections obtained by systematic (n = 12 normal, 12 FGR) and targeted (n = 6 normal, 6 FGR) sampling from the placental periphery and centre. RESULTS: Adjusted for the weight of the placenta or the surface area of the chorionic plate, the number of chorionic arteries was similar in normal and FGR arterial casts. Inter-branch length per unit placental weight was greater in the first generation of arterial branches in FGR (p < 0.05). Villi in FGR placentas were more poorly vascularised, particularly at the periphery and in grossly visible hypovascular regions. Intermediate and terminal FGR villi in these areas exhibited reduced vessel lumens, loss of CD34, and infilling with CD34-negative cells of what appeared to be previously existing vascular spaces. CONCLUSION: Differences in chorionic arterial branching patterns between normal and FGR placentas arise from differences in placental size. FGR placentas show microvascular regression and extreme hypovascularity in peripheral areas. These features may well limit the ability of the placenta to meet fetal nutrient requirements late in gestation. Targeted sampling is more effective than systematic random sampling in revealing vascular defects.

Absence of PO2 change in fetal brain despite PO2 increase in placenta in response to maternal oxygen challenge.

OBJECTIVE: Magnetic resonance imaging allows the noninvasive observation of PO2 changes between air breathing and oxygen breathing through quantification of the magnetic longitudinal relaxation time T1. Changes in PO2 are proportional to changes in the longitudinal relaxation rate ΔR1 (where ΔR1=1/T1oxygen-1/T1air). Knowledge of this response could inform clinical interventions using maternal oxygen administration antenatally to treat fetal growth restriction. We present in vivo measurements of the response of the fetal-placental unit to maternal hyperoxia. DESIGN: Prospective cohort. SETTING: Large tertiary maternity hospital. SAMPLE: Nine women undergoing low-risk pregnancy (21-33 weeks of gestation) and five nonpregnant adults. METHODS: During imaging the air supply to mothers was changed from medical air (21% oxygen) to medical oxygen (100% oxygen) and T1 was monitored over time in both the placenta and fetal brain using a periodically repeated magnetic resonance imaging sequence. To demonstrate that the method could detect a brain response, brain responses from five normal adult volunteers were measured using a similar imaging protocol. MAIN OUTCOME MEASURE: Changes in T1 following oxygen challenge. RESULTS: No significant ΔR1 (P=0.42, paired t-test) was observed in fetal brains. A significant placental ΔR1 (P=0.0002, paired t-test) of 0.02±0.01/s (mean±SD) was simultaneously observed in the same participants. In the brains of the nonpregnant adults, a significant ΔR1 (P=0.01, paired t-test) of 0.005±0.002/s was observed. CONCLUSION: Short-term maternal oxygen administration does not improve fetal brain oxygenation, in contrast to the response observed in the adult brain.

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.

IFPA Meeting 2011 workshop report I: Placenta: Predicting future health; roles of lipids in the growth and development of feto-placental unit; placental nutrient sensing; placental research to solve clinical problems--a translational approach.

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, four of which are summarized in this report. These workshops related to both basic science and clinical research into placental growth and nutrient sensing and were divided into 1) placenta: predicting future health; 2) roles of lipids in the growth and development of feto-placental unit; 3) placental nutrient sensing; 4) placental research to solve clinical problems: a translational approach.

Epidermal growth factor and sphingosine-1-phosphate stimulate Na+/H+ exchanger activity in the human placental syncytiotrophoblast.

The Na+/H+ exchanger (NHE) has a key role in intracellular pH ([pH]i) regulation of the syncytiotrophoblast in the human placenta and may have a role in the life cycle of this cell. In other cells the NHE (actually a family of up to 9 isoforms) is regulated by a variety of factors, but its regulation in the syncytiotrophoblast has not been studied. Here, we tested the hypotheses that EGF and sphingosine-1-phosphate (S1P), both of which affect trophoblast apoptosis and, in other cell types, NHE activity, stimulate syncytiotrophoblast NHE activity. Villous fragments from term human placentas were loaded with the pH-sensitive dye, BCECF. NHE activity was measured by following the recovery of syncytiotrophoblast [pH]i following an imposed acid load, in the presence and absence of EGF, S1P, and specific inhibitors of NHE activity. Both EGF and S1P caused a dose-dependent upregulation of NHE activity in the syncytiotrophoblast. These effects were blocked by amiloride 500 microM (a nonspecific NHE blocker) and HOE694 100 microM (NHE blocker with NHE1 and 2 isoform selectivity). Effects of EGF were also reduced by the NHE3 selective blocker S3226 (used at 1 microM). These data provide the first evidence that both EGF and S1P stimulate NHE activity in the syncytiotrophoblast; they appear to do so predominantly by activating the NHE1 isoform.

Multiple anti-apoptotic pathways stimulated by EGF in cytotrophoblasts.

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.