The association between first trimester placental biomarkers and placental lesions of maternal vascular malperfusion.

INTRODUCTION: Abnormal levels of first trimester placental biomarkers are associated with the development of placental syndrome (PS). However, prediction performance is moderate, possibly explained by the clinical heterogeneity of PS. Aim of this study is to investigate the association between first trimester biomarkers and the presence of maternal vascular malperfusion (MVM), as a marker for placental insufficiency. METHODS: This retrospective study included 195 women with available first trimester blood sample and placenta histological sections for examination at the Maastricht University Medical Centre. Women were divided into 4 groups, based on the presence of having MVM lesions and/or PS. Levels of PAPP-A, PlGF and sFlt-1 were measured and MVM lesions were classified according to the Amsterdam Placental Workshop Group Consensus Statement. RESULTS: MVM occurrence was observed in 32% of the uncomplicated pregnancies. Women with MVM (regardless of the PS) had lower levels of PAPP-A (p = 0.038) and sFLt-1 (p = 0.006), and a non-significant trend for lower PlGF and sFlt-1/PlGF ratio compared to women without MVM. Low PAPP-A levels individually and in combination with the presence of PS was significantly associated with MVM lesions (aOR = 3.0 and 6.1, respectively), as did the combination of low PlGF levels and PS (aOR = 4.6). In women with PS, having MVM increased the incidence of fetal growth restriction, small for gestational age neonates, lower birthweight and adverse neonatal outcome. DISCUSSION: Our findings suggest that MVM lesions were found to be associated with increased obstetric risks due to early placental dysfunction that can potentially be predicted by the use of first trimester biomarkers.

Feasibility of three dimensional power Doppler ultrasonography methods to assess placental perfusion.

OBJECTIVES: Given the crucial role of the placenta in establishing a healthy pregnancy, reliable non-invasive methods to measure placental perfusion are desirable. The aim of this study is to determine the reproducibility and potential bias in different three-dimensional power Doppler (3DPD) methods assessing placenta perfusion. METHODS: Ten singleton pregnancies around 16 weeks of gestation, with an anteriorly located placenta and centrally inserted umbilical cord were included in this study. Eight different combinations of a specific placental sweep and sonobiopsy method were used to evaluate placental perfusion. Vascularization index (VI), flow index (FI) and vascularization-flow index (VFI) were determined offline using the 4D-view program. Reproducibility and repeatability of the methods, expressed as correlation coefficients and Bland-Altman mean differences, were calculated. Differences between sampling methods were analyzed using t-test or Mann-Whitney U test. RESULTS: Intra- and inter-class correlation coefficient (CC) was highest when using a spherical centrally placed sonobiopsy of 2 cm3 in a whole placenta sweep (method 1; IntraCC VI 0.985, FI 0.769, VFI 0.993, InterCC VI 0.986, FI 0.784, VFI 0.987). Overall, intraCCs were higher compared to interCCs. Lowest mean differences in VI and FI were found comparing spherical to manual sonobiopsies, whereas the mean differences in VFI were lowest when comparing central versus peripheral located sonobiopsies. Comparing the three vascular indices, best median intra- and interCC and lowest mean differences were found for VFI. CONCLUSIONS: Three dimensional placental vascularization analysis showed best reproducibility using whole placental sweep volume and centrally located, spherical sonobiopsy of 2 cm3.

'Postnatal growth during the first five years of life in SGA and AGA neonates with reduced fetal growth'.

BACKGROUND: Even though a lot of research has been done on postnatal growth and the occurrence of catch-up growth in small-for-gestational age (SGA) neonates, this phenomenon has not been studied well in appropriate-for-gestational age (AGA) neonates. Postnatal catch-up growth may also occur in AGA neonates indicating a compensatory mechanism for undiagnosed intrauterine growth restriction, especially in AGA neonates with reduced fetal growth velocity. AIMS: To describe postnatal growth during the first 5 years of life in SGA and AGA neonates and evaluating the role of fetal growth velocity in catch-up growth. STUDY DESIGN: Retrospective study in a Dutch tertiary hospital. SUBJECTS: 740 singleton neonates, without congenital anomalies, with ultrasound fetal growth data from 20 weeks and 32 weeks of pregnancy. OUTCOME MEASURES: Postnatal growth measurements of height (cm) and weight (kg) from birth until five years of age. Postnatal catch-up growth defined as difference (delta) in both height and weight between 4 weeks and 3 years of age. RESULTS AND CONCLUSIONS: SGA neonates had a significantly lower height and weight compared to the AGA group for all available measurement moments till 3 years. The catch-up growth between the SGA and AGA groups from 4 weeks up to 3 years after birth was not different between the two groups. However, neonates with reduced fetal growth velocity had a significantly higher risk for catch-up growth in height during the first 3 years after birth. This suggests a role for fetal growth velocity measurement in predicting fetal and subsequent postnatal growth potential.

Understanding decidual vasculopathy and the link to preeclampsia: A review.

Preeclampsia is the archetype of a spectrum of clinical disorders related to abnormal placental development or function, characterized by placental histological lesions. Among those lesions, decidual vasculopathy is a term used to describe lesions of maternal spiral arteries, which are encountered on placental examination in about half of the women with preeclampsia. The morphological features of the lesions include perivascular lymphocytic infiltration, fibrinoid necrosis and foam cell incorporation within the vessel wall. Due to the resemblance of the latter characteristic to atherosclerosis, they are alternatively termed acute atherosis. Decidual vasculopathy correlates with worse maternal and neonatal outcomes, as well as placental pathology. In this article, we review the available literature on decidual vasculopathy and address the pitfalls in histological analysis of the lesions, including the varying definitions of the lesions and sample collection methods. We also discuss the current evidence on the etiology of the lesions and propose a novel hypothesis linking the three etiological pathways to the formation of decidual vasculopathy and, ultimately, the emergence of the heterogeneous group of placental dysfunction disorders, known as the great obstetric syndromes.

Universal screening versus risk-based protocols for antibiotic prophylaxis during childbirth to prevent early-onset group B streptococcal disease: a systematic review and meta-analysis.

BACKGROUND: Early-onset group B streptococcal (EOGBS) disease (including sepsis, meningitis, and pneumonia) causes significant morbidity and mortality in newborn infants worldwide. Antibiotic prophylaxis can prevent vertical streptococcal transmission, yet no uniform criteria exist to identify eligible women for prophylaxis. Some guidelines recommend universal GBS screening to pregnant women in their third trimester (screening-based protocol), whereas others employ risk-based protocols. OBJECTIVES: To compare the effectiveness of screening-based versus risk-based protocols in preventing EOGBS disease. SEARCH STRATEGY: Key words for the database searches included GBS, Streptococcus agalactiae, pregnancy, screening, culture-based, risk-based. SELECTION CRITERIA: Studies were included if they investigated EOGBS disease incidence in newborn infants and compared screening or risk-based protocols with each other or with controls. DATA COLLECTION AND ANALYSIS: Risk ratios (RR) and 95% confidence intervals (CI) were determined using Mantel-Haenszel analyses with random effects. MAIN RESULTS: Seventeen eligible studies were included. In this meta-analysis, screening was associated with a reduced risk for EOGBS disease compared either with risk-based protocols (ten studies, RR 0.43, 95% CI 0.32-0.56) or with no policy (four studies, RR 0.31, 95% CI 0.11-0.84). Meta-analysis could not demonstrate a significant effect of risk-based protocols versus no policy (seven studies, RR 0.86, 95% CI 0.61-1.20). In studies reporting on the use of antibiotics, screening was not associated with higher antibiotic administration rates (31 versus 29%). CONCLUSIONS: Screening-based protocols were associated with lower incidences of EOGBS disease compared with risk-based protocols, while not clearly overexposing women to antibiotics. This information is of relevance for future policymaking. TWEETABLE ABSTRACT: Meta-analysis: general screening is associated with lower rates of early-onset group B strep. neonatal sepsis compared with risk-based protocols.

Maternal vascular malformation in the placenta is an indicator for fetal growth restriction irrespective of neonatal birthweight.

INTRODUCTION: To study the association between placental pathology and neonatal birthweight and outcomes, and whether a combination of first trimester biomarkers and fetal growth velocity can predict placental lesions. METHODS: The presence of maternal vascular malperfusion (MVM) lesions (Amsterdam criteria) was recorded in a retrospective cohort of singleton pregnancies in the Maastricht University Medical Centre, 2011-2018. First trimester maternal characteristics and PAPP-A, PlGF and sFlt-1 levels were collected. Fetal growth velocities were calculated (mm/week) from 20 to 32 weeks for abdominal circumference, biparietal diameter, head circumference and femur length. Data were compared between neonates with 'small for gestational age' (SGA < p10) and different categories of 'appropriate for gestational age (AGA)': AGAp10-30, AGAp30-50 and AGA > p50 (reference), using one-way ANOVA and post hoc test. RESULTS: There were significantly more MVM lesions in the SGA group (94.6% p < .0001), but also in the AGAp10-30 (67.3% p < .0001) and AGAp30-50 (41.6% p = 0.002), compared to the reference AGA group (19.3%). The prediction of MVM for a 20% false-positive rate, with maternal characteristics was25.2%. The addition of birthweight percentile gave a prediction of 51.7% for MVM. However adding placental biomarkers and fetal growth velocities (instead of birthweight percentile) to the maternal characteristics, gave a prediction of 81.8% (PPV 49.5%, NPV 53.7%). DISCUSSION: Placental MVM lesions correlated inversely with birthweight even in AGA neonates, and was associated with slower fetal growth and more adverse outcome in SGA neonates. A combination of first trimester biomarkers and fetal growth velocity had good prediction of placental MVM lesions, as an indicator of fetal growth restriction irrespective of neonatal weight.

Reduced fetal growth velocities and the association with neonatal outcomes in appropriate-for-gestational-age neonates: a retrospective cohort study.

BACKGROUND: Fetal growth restriction is, despite advances in neonatal care and uptake of antenatal ultrasound scanning, still a major cause of perinatal morbidity. Neonates with birth weight > 10th percentile are assumed to be appropriate-for-gestational-age (AGA), although many are at increased risk of perinatal morbidity, because of undetected mild restriction of growth potential. We hypothesized that within AGA neonates, reduced fetal growth velocities are associated with adverse neonatal outcome. METHODS: A retrospective cohort study of singleton pregnancies, in the Maastricht University Medical Centre (MUMC) between 2010 and 2016. Women had two fetal biometry scans (18-22 weeks and 30-34 weeks of gestational age) and delivered a newborn with a birth weight between the 10th-80th percentile. Differences in growth velocities of the abdominal circumference (AC), biparietal diameter (BPD), head circumference (HC) and femur length (FL) were compared between the suboptimal AGA (sAGA) (birth weight centiles 10-50) and optimal AGA (oAGA) (birth weight centiles 50-80) group. We assessed the association between velocities and neonatal outcomes. RESULTS: We included 934 singleton pregnancies. In the suboptimal AGA group, fetal growth velocities were lower (in mm/week): AC 10.72 ± 1.00 vs 11.23 ± 1.00 (p < .001), HC 10.50 ± 0.80 vs 10.68 ± 0.77 (p = 0.001), BPD 3.01 ± 0.28 vs 3.08 ± 0.27 (p < .0001) and FL 2.47 ± 0.21 vs 2.50 ± 0.22 (p = 0.014), compared to the optimal AGA group. Neonates with an adverse neonatal outcome had significantly lower growth velocities (in mm/week) of: AC 10.57 vs 10.94 (p = 0.034), HC 10.28 vs 10.59 (p = 0.003) and BPD 2.97 vs 3.04 (p = 0.043) compared to those with normal outcome. An inverse association was observed between the AC velocity and a composite adverse neonatal outcome (OR) = 0.667 (95%CI 0.507-0.879, p = 0.004), and between the AC velocity and neonates with NICU stay (OR) = 0.733 (95%CI 0.570-0.942, p = 0.015). Neonates with a birthweight lower than expected (based on the abdominal circumference at 20 weeks) had significantly more composite adverse neonatal outcomes 8.5% vs 5.0% (p = 0.047), NICU stays 9.6% vs 3.8% (p < .0001) and hospital stays 44.4% vs 35.6% (p = 0.006). CONCLUSIONS: Appropriate-for-gestational-age neonates are a heterogeneous group with some showing suboptimal fetal growth. Abnormal fetal growth velocities, especially abdominal circumference velocity, are associated with adverse neonatal outcome and can potentially improve the detection of mild growth restriction when used in multivariate models.

Cardiac remodeling and pre-eclampsia: an overview of microRNA expression patterns.

Pre-eclampsia (PE) is strongly associated with heart failure (HF) later in life. During PE pregnancy, the left ventricle undergoes concentric remodeling which often persists after delivery. This aberrant remodeling can induce a molecular signature that can be evaluated in terms of microRNAs (miRNAs) and which may help to explain the associated increased risk of HF. For this review, we performed a literature search of PubMed (National Center for Biotechnology Information), identifying studies on miRNA expression in concentric remodeling and on miRNA expression in PE. The miRNA data were stratified based on origin (isolated from humans or animals and from tissue or the circulation) and both datasets compared in order to generate a list of miRNA expression patterns in concentric remodeling and in PE. The nine miRNAs identified in both concentric remodeling and PE-complicated pregnancy were: miR-1, miR-18, miR-21, miR-29b, miR-30, miR-125b, miR-181b, miR-195 and miR-499-5p. We found five of these miRNAs (miR-18, miR-21, miR-125b, miR-195 and miR-499-5p) to be upregulated in both PE pregnancy and cardiac remodeling and two (miR-1 and miR-30) to be downregulated in both; the remaining two miRNAs (miR-29b and miR-181b) showed upregulation during PE but downregulation in cardiac remodeling. This innovative approach may be a step towards finding relevant biomarkers for complicated pregnancy and elucidating their relationship with remote cardiovascular disease. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Prevalence of hypertensive disorders in women after preeclamptic pregnancy associated with decidual vasculopathy.

OBJECTIVE: A subgroup of preeclamptic women has spiral artery lesions termed decidual vasculopathy (DV) which relate to worse clinical outcome. We aimed to determine whether a history of preeclampsia (PE) with DV is associated with adverse overall and future pregnancy outcome, including increased recurrence risk of hypertensive diseases of pregnancy. METHODS: Via posted survey women with PE and DV (DV positive) in the index pregnancy were compared to those without the lesions (DV negative) on overall and future pregnancy outcome. RESULTS: DV positive cases showed a higher incidence of chronic hypertension both preconceptionally and at time of survey, adjusted odds ratio 4.8 (2.0-11.9). The DV positive group had a higher overall incidence of pregnancies with gestational hypertension (22% vs 13%, p = 0.04), preterm birth (59% vs 45%, p = 0.02) and a lower birth weight centile (30 vs 39, p = 0.02). There was no difference in outcome of future pregnancies, irrespective of the use of prophylactic aspirin. CONCLUSION: Women with DV-associated PE have a higher overall incidence of adverse obstetric outcome and of chronic hypertension, indicating an underlying vascular pathology, putting them at risk for pregnancy and cardiovascular complications. These women constitute a target group for counseling, monitoring and possibly lifestyle or pharmacological interventions.

Formerly eclamptic women have lower nonpregnant blood pressure compared with formerly pre-eclamptic women: a retrospective cohort study.

OBJECTIVE: To compare nonpregnant blood pressure and circulating metabolic factors between formerly pre-eclamptic women who did and did not deteriorate to eclampsia. DESIGN: Retrospective observational cohort study. SETTING: Tertiary referral centre. POPULATION: Formerly pre-eclamptic women with (n = 88) and without (n = 698) superimposed eclampsia. METHODS: Women who experienced pre-eclampsia with or without superimposed eclampsia during their pregnancy or puerperium were tested for possible underlying cardiovascular risk factors at least 6 months postpartum. We measured blood pressure and determined cardiovascular and metabolic risk markers in a fasting blood sample. Groups were compared using Mann-Whitney U test, Spearman's Rho test or Fisher's Exact test (odds ratios). MAIN OUTCOME MEASURES: Differences in postpartum blood pressures and features of the metabolic syndrome between formerly pre-eclamptic and formerly eclamptic women. RESULTS: Formerly pre-eclamptic women who developed eclampsia differed from their counterparts without eclampsia by a lower blood pressure (P < 0.01) with blood pressure correlating inversely with the likelihood of having experienced eclampsia (P < 0.001). In addition, formerly eclamptic women had higher circulating C-reactive protein levels than formerly pre-eclamptic women (P < 0.05). All other circulating metabolic factors were comparable. Finally, 40% of all eclamptic cases occurred in the puerperium. CONCLUSIONS: Formerly pre-eclamptic women with superimposed eclampsia have lower nonpregnant blood pressure compared with their counterparts without neurological sequelae with blood pressure negatively correlated to the occurrence of eclampsia. As about 40% of all eclamptic cases occur postpartum, routine blood pressure monitoring postpartum should be intensified.

Metabolic syndrome after pregnancies complicated by pre-eclampsia or small-for-gestational-age: a retrospective cohort.

OBJECTIVE: To study the prevalence of metabolic syndrome in women after a pregnancy complicated by pre-eclampsia or small-for-gestational-age (SGA), both epitomes of placental syndrome. DESIGN: A retrospective cohort study. SETTING: Single tertiary centre for maternal medicine in the Netherlands. POPULATION: Women with a history of pre-eclampsia in absence of SGA (n = 742) or pregnancy complicated by normotensive SGA (n = 147) between 1996 and 2010. METHODS: Women were routinely screened for underlying cardiometabolic and cardiovascular risk factors at least 6 months postpartum. Logistic regression analysis was used to calculate the odds ratio and adjusted odds ratio for each group. Adjustments were made for age, maternal height, smoking, parity, and interval between delivery and measurement. MAIN OUTCOME MEASURES: Prevalence of the metabolic syndrome. RESULTS: The prevalence of the metabolic syndrome in our population was two-fold higher for women with a history of pre-eclampsia (13.9%) compared with women with a history of SGA (7.6%). Calculated odds ratios for metabolic syndrome, fasting insulin, HOMA, and microalbuminuria were all higher for women with a history of pre-eclampsia compared with women with SGA. This difference persisted after adjustment for confounding factors: metabolic syndrome (adjusted odds ratio, aOR 2.11; 95% confidence interval, 95% CI 1.00-4.47) and hyperinsulinaemia (aOR 1.78; 95% CI 1.13-2.81) insulin resistance (HOMAIR ; aOR 1.80; 95% CI 1.14-2.86). Microalbuminuria (aOR 1.58; 95% CI 0.85-2.93) did not reach the level of significance after adjustment for confounding factors. CONCLUSIONS: A history of pre-eclampsia, rather than SGA, was associated with metabolic syndrome, suggesting that it relates to maternal rather than fetal etiology of placental syndrome.

Decidual vasculopathy in preeclampsia: lesion characteristics relate to disease severity and perinatal outcome.

OBJECTIVE: In a proportion of patients with preeclampsia, unremodeled spiral arteries develop additional pathological changes, termed decidual vasculopathy (DV), or acute atherosis. DV has been correlated to adverse clinical outcome and increased placental pathology. However, it was unclear whether these effects pertained to individual features of DV. METHODS: We performed a reanalysis of placental samples from preeclamptic pregnancies (n = 76), recording the number of vessels with DV, their location in the decidua and their morphological features. Results were correlated with clinical and placental parameters, using Spearman's rho test. P-value < 0.05 was considered significant. RESULTS: Total number of vessels with DV (totalDV) correlated with higher diastolic blood pressure, higher urine protein-to-creatinine ratio, shorter gestational age, lower birth weight, 5 min APGAR score and umbilical artery pH, and with increased accelerated villous maturity, infarction and hematoma formation, but not with HELLP syndrome markers. Additionally, there was a striking correlation of increased perinatal mortality with the number of vessels located in the decidua basalis (DVbas), and with vessels showing DV with thrombosis (DVthrom). Other morphological features, such as foam cell infiltration, did not increase correlation strength. DISCUSSION: In our study of preeclamptic placental samples, totalDV related to worse clinical outcome and increased placental pathology. Moreover, DVbas and DVthrom related to perinatal death. DV could be a manifestation of an underlying (vascular) pathology, increasing the risk of adverse pregnancy outcome. CONCLUSIONS: In preeclampsia, totalDV, DVbas and DVthrom correlated with increased placental pathology and adverse maternal and fetal outcome, most relevantly with perinatal mortality.

Decidual vasculopathy and adverse perinatal outcome in preeclamptic pregnancy.

OBJECTIVE: Decidual vasculopathy (DV) describes pathological findings seen in the spiral arteries in preeclampsia (PE). Morphologically, DV is characterized by fibrinoid necrosis and foamy macrophages within the vessel walls. The impact of the lesions on clinical outcome and placental pathology is unclear. We compared cases with DV to cases without these lesions on clinical outcome and placental histology in PE. STUDY DESIGN: Placental sections from 107 patients admitted with PE at the Radboud University Nijmegen Medical Centre, during the years 1995-2000, were analyzed. 25 cases were excluded due to incomplete records or multiple pregnancy. Cases with DV (n = 41) and without DV (n = 41) were compared for various clinical and placental histological parameters, using Mann-Whitney test. P-value < 0.05 was considered significant. RESULTS: Clinically, DV related to higher diastolic blood pressure, shorter gestational age, lower birth weight and lower umbilical artery pH. Histologically, DV related to more accelerated villous maturity and perivascular inflammatory cell infiltration. No differences were found in maternal biochemical variables (protein-to-creatinine ratio, constituents of HELLP syndrome), fetal-maternal interface parameters (placenta weight, infarctions, hematoma, calcifications), or neonatal outcome measures (birth weight centile, APGAR scores, and perinatal death). CONCLUSIONS: In PE, the presence of DV related to placental accelerated villous maturity, perivascular inflammatory cell infiltration, and adverse maternal and fetal outcome without affecting neonatal survival. Whether or not DV results from or raises the risk on severe preeclampsia remains to be elucidated.

Difficult prenatal diagnosis: fetal coarctation.

The prenatal diagnosis of fetal coarctation is still challenging. It is mainly suspected by ventricular disproportion (smaller left ventricle than right ventricle). The sensitivity of ventricular discrepancy is however moderate for the diagnosis of coarctation and there is a high false positive rate. Prenatal diagnosis of coarctation is important because the delivery can be arranged in a centre with a pediatric cardiac intensive careand this reduces postnatal complications and longterm morbidity. For many years the prenatal diagnosis of coarctation has been investigated to improve specificity and sensitivity by several of measurements. This article reviews all relevant articles from 2000 until 2011 searching pubmed and the reference list of interesting articles. An overview of specific measurements and techniques that can improve the diagnosis of coarctation has been made, such as the isthmus diameter, ductal diameter, isthmus/ductal ratio, z-scores derived from measurements of the distal aortic isthmus and arterial duct, the presence of a shelf andisthmal flow disturbance. Also 3-dimensional (3D) and 4-dimensional (4D) imaging with or without STIC has been -suggested to be used as newer techniques to improve diagnosis of coarctation in fetal life. Although more methods regarding prenatal diagnosis of coarctationare being investigated, the ultrasound specialist remains challenged to correctly diagnose this cardiac anomaly in prenatal life.

Interstitial trophoblastic cell fusion and E-cadherin immunostaining in the placental bed of normal and hypertensive pregnancies.

During their invasion of the placental bed, interstitial trophoblasts fuse to multinuclear giant cells which are thought to have lost their invasive properties. Trophoblast fusion is associated with downregulation of E-cadherin, and persistent E-cadherin expression has been linked to defective placentation in preeclampsia. Since a previous study suggested 'premature' giant cell formation in preeclampsia, we started with the working hypotheses that fusion is increased in hypertensive pregnancies, and that the intensity of fusion correlates with the severity of disease. Using double immunostaining for E-cadherin and cytokeratin 7/17, nuclei in interstitially invasive trophoblasts (IT) in the myometrial compartment of the placental bed from normotensive pregnancies (NT, n=8), gestational hypertension (GH, n=4), preeclampsia (PE, n=9), and HELLP syndrome (n=5) were categorised according to the E-cadherin staining of the cell and their occurrence in single, clustered or multinuclear cells. GH and PE patients showed a higher percentage of nuclei in clustered non-fused E-cadherin-positive cells (P<0.01 and P<0.05), and in smaller (bi- and trinuclear) placental bed giant cells (P<0.05) compared to NT pregnancies, suggesting defective IT fusion. In contrast, in HELLP syndrome no such failed fusion could be discerned, which may support the idea of a heterogeneous aetiology of different hypertensive diseases of pregnancy. Since we are still ignorant about the specific role of mononuclear and multinuclear trophoblasts in the placental bed, it is not yet possible to relate the present findings to the pathogenesis of different categories of hypertensive pregnancies.

The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells.

BACKGROUND: The current techniques for quantifying trophoblast viability, migration and invasion are mainly limited by the need to sacrifice the cells during the test procedure. In this study, the vital dye AB (AB) was used to quantify cell number and viability of BeWo and JEG-3 choriocarcinoma cells, as well as their migration and invasion through fibronectin-coated filters. METHODS: AB was directly added to culture medium of incubated test and control cells. At various time intervals, the redox reaction, in which AB is reduced by the cells, was measured by absorbance readings at 540 and 630 nm. For cell migration and invasion, cells were cultured onto uncoated or fibronectin-coated inserts, respectively. AB reduction of migrated cells was normalized to that of control cells to calculate percentages of migration. This model was also tested in the presence of a reported inhibitor, transforming growth factor (TGF) beta. RESULTS: The curve of %AB reduction versus cell number was linear, with intra- and inter-assay Coefficient of Variations of 1.88%and 2.94%, respectively. AB reduction increased with both seeding concentrations and incubation time with AB. TGFbeta treatment caused a modest decrease in AB reduction in both JEG-3 and BeWo cells. TGFbeta treatment also decreased migration in BeWo, but not in JEG-3, cells. CONCLUSIONS: AB assay is a simple and reliable method for quantifying trophoblast viability, migration and invasion.

Differential effects of inducers of syncytialization and apoptosis on BeWo and JEG-3 choriocarcinoma cells.

BACKGROUND: The interactions of trophoblasts with the cytokine network at the fetomaternal interface determine the pathway the cell undertakes, e.g. proliferation, differentiation and apoptosis. METHODS: We used cultures of fusigenic BeWo and non-fusigenic JEG-3 choriocarcinoma cells to study the effects of inducers of syncytialisation (forskolin) and apoptosis [tumour necrosis factor-alpha (TNFalpha)] on differentiation, viability, proliferation and apoptosis. RESULTS: E-cadherin immunostaining showed that syncytium formation was confined to BeWo and not JEG-3 cells, while secretion of hCG was promoted by forskolin in both cell types implying a 'dissociation' between morphological and biochemical differentiation. Forskolin also had differential effects on cell viability (MTT reduction test) and proliferation (Ki67 immunostaining with MIB-1 monoclonal antibody), both decreasing in BeWo and increasing in JEG-3 cells. TNFalpha increased apoptosis (cytokeratin neo-epitope immunostaining with M30 monoclonal antibody) in both cell types, an effect which was blocked by epidermal growth factor selectively in JEG-3 cells. CONCLUSION: Our results suggest that the differential responses of BeWo and JEG-3 cells to inducers of syncytialization and apoptosis might be related to their fusigenic capacity. Caution is needed when extrapolating results obtained by these models to normal trophoblast populations. However, we speculate that these models can help identify key factors involved in trophoblast differentiation at the placental bed.