Emerging role of SUMOylation in placental pathology.

INTRODUCTION: Small ubiquitin-like modifiers (SUMO) conjugate to target proteins in a dynamic, reversible manner to function as post-translational modifiers. SUMOylation of target proteins can impinge on their localization, in addition to their activity or stability. Differential expression of deSUMOylating enzymes (SENP 1 and 2) contributes to altered mammalian placental development and function in mice. Severe preeclampsia (sPE) is associated with abnormal placental development and chronic ischemic injury. Extra- and intracellular stimuli/stressors that include hypoxic-activated pathways are known modulators of SUMOylation. In this current study we hypothesized that placentas from sPE patients will display up regulation in the SUMO regulatory pathway. METHODS: Utilizing qRT-PCR, immuno-blotting and Western techniques, we determined the expression levels of SUMO pathway genes in healthy and diseased placentas. We also exposed placental explants to hypoxia to study the effect on the SUMOylation pathway. RESULTS: We observed steady-state expression of SUMO1-3, SUMO-conjugated enzyme-UBC9 and deSUMOylating enzymes - SENPs, throughout normal gestation. An elevated level of free SUMO1-3 and SUMO-protein conjugates was observed in sPE placentas. Furthermore, placental UBC9 levels were strikingly increased in the same sPE patients. Hypoxia-induced SUMOylation in first trimester placental explants. DISCUSSION: Our data demonstrate an elevated steady-state of SUMOylation in sPE placentas compared with gestational aged-matched controls. The observed hyper-SUMOylation in sPE placentas correlates with elevated expression of UBC9 rather than with reduced expression of SENPs Hypoxia may contribute to alterations in placental SUMOylation pathway. CONCLUSION: Increased placental SUMOylation may contribute to the pathogenesis of serious placental pathology that causes extreme preterm birth.

Placental hyperinflation and the risk of adverse perinatal outcome.

OBJECTIVES: To determine the pathological basis and clinical associations of excessively thick placentae observed at second-trimester ultrasound examination. METHODS: In a retrospective cohort of 19 singleton high-risk second-trimester pregnancies noted to have a placental length-to-maximum thickness ratio ≤ 2.0, maximum sonographic placental thickness was correlated with clinical outcome, maximum placental thickness after delivery and placental pathological findings. Results were compared with those of an intermediate group of 21 high-risk pregnancies with normal placental dimensions and a control group of 18 low-risk pregnancies also with normal placental dimensions. Increased maximum placental thickness (> 28 mm) and abnormal placental deflation following delivery (pathology - sonography difference in maximum placental thickness < -2 mm) were defined by the upper and lower quartile values, respectively, in the control group. RESULTS: The study group exhibited significantly more adverse outcomes and gross pathological placental features compared with both intermediate and control groups. Despite increased sonographic maximum placental thickness in the study group (median, 55 (range, 40-75) mm compared with both the intermediate group (median, 27 (range, 22-41) mm, P < 0.0001) and the control group (median 26 (range, 23-36) mm, P < 0.0001)), all three groups had similar maximal placental thickness following delivery (study group: median, 24 (range, 10-50) mm vs intermediate group: median, 27 (range, 15-40) mm, P = 0.82 and vs control group: median, 28.5 (range, 18-44), P = 0.42). Pathology-sonography difference in maximum placental thickness in the study group (median, -30 (range, -42 to 0) mm) was significantly greater than that in either the intermediate (median, -2 (range, -11 to 9) mm, P < 0.0001) or the control (median, 1.5 (range, -10 to 18) mm, P < 0.0001) group and was significantly associated with abnormal development of the gas-exchanging placental villi (distal villous hypoplasia) (P = 0.0001). CONCLUSIONS: Increased second-trimester sonographic maximum placental thickness represents a pathological finding associated with severe adverse perinatal outcome. This observation is due to overinflation of the intervillous space by maternal blood rather than to adaptive formation of functional placental tissue.

Umbilical cord diameter percentile curves and their correlation to birth weight and placental pathology.

OBJECTIVE: The aims of this study were to develop a nomogram of umbilical cord diameter (UCD) for pathologic examination of the placenta, to identify the umbilical cord components responsible for variations in UCD, and to examine the relationship between UCD and other placental pathologic features and perinatal outcome. STUDY DESIGN: We prospectively collected 497 umbilical cords between 18 and 41 weeks' gestation over a 1-year period. Fresh-tissue UCD were grouped according to gestational age and compared to sonographic and histological measurements. Associations between UCD percentile and placental pathologic findings or obstetrical outcomes were examined. RESULTS: Mean UCD increased with gestational age until a plateau at 1.0 cm in the third trimester, a value that was 0.56 cm less than sonographic measurements prior to delivery and 0.17 cm greater than UCD measured histologically. Umbilical cord components varied with UCD percentile, with umbilical vessel area increased in thick cords (p < 0.001) and Wharton's jelly area reduced in thin cords (p = 0.002). Thin umbilical cords were associated with at least one pathologic histological placental finding (p = 0.02), low placental weight (p < 0.001), single umbilical artery (p = 0.02), marginal cord insertion (p = 0.01), and low infant birth weight (p < 0.001). CONCLUSIONS: This study provides reference curves for post-delivery UCD from 18 to 41 weeks' gestation for use by perinatal pathologists. We show that increased UCD is a function of increased umbilical blood vessel volume and decreased UCD is a function of decreased Wharton's jelly volume. UCD shows a strong association with placental and infant birth weight.

Sex-specific basis of severe placental dysfunction leading to extreme preterm delivery.

OBJECTIVES: Since pregnancies with a male fetus have higher perinatal complications attributed to placental dysfunction, including severe pre-eclampsia and intrauterine growth restriction, the objective of our study was to formally evaluate placental pathology for a placental origin of these sex-specific differences. DESIGN: Retrospective study at Mount Sinai Hospital in Toronto, Canada. Identification of 262 singleton pregnancies affected by severe pre-eclampsia and/or intrauterine growth restriction who delivered between 22 and 32 weeks' gestation from 2000 to 2010. Detailed placental pathology was reviewed, and data from 140 pregnancies with male fetuses were compared with 122 pregnancies with female fetuses. A comparison group of 40 unaffected pregnancies who delivered in the same gestational range was used to determine baseline rates of placental pathology. MAIN OUTCOME MEASURED: Detailed placental pathology, including placental development/differentiation, velamentous umbilical cord insertion, maternal-fetal interface pathology, villous infarction, hemorrhagic lesions, villous development, and fetal vascular under-perfusion. RESULTS: Impaired placental development and differentiation was equally common amongst males (73/140, 52.1%) and females (69/122, 56.6%). Male placentas exhibited significantly higher rates of chronic deciduitis (17.9% vs. 9.0%; relative risk [RR] 1.98, 95% confidence interval [CI] 1.02-3.86) and velamentous umbilical cord insertion (9.5% vs. 1.7%; RR 5.66, 95% CI 1.30-24.6), and a significantly lower frequency of villous infarction (55.4% vs. 73.7%; RR 0.75, 95% CI 0.62-0.90) than female placentas. No significant differences were noted for other lesions. CONCLUSIONS: Fetal sex exerts a differential effect on the placental pathology that mediates severe pre-eclampsia and/or IUGR. Placental pathology at birth may provide insight into the mechanisms linking adverse in utero events with long-term adult disease since, for example, a male tendency to an inflammatory pathology at the maternal-fetal interface may be linked to the excess risk of coronary artery disease.

A distinct microvascular endothelial gene expression profile in severe IUGR placentas.

The placental microvasculature is essential for efficient transfer of gases, nutrients and waste between the mother and fetus. Microvascular hypoplasia of the terminal villi is a common pathology in severe Intra Uterine Growth Restriction (IUGR). We used novel methods to obtain placental micro-vascular endothelial cells (PlMEC) from preterm control placentas (n = 3) and placentas from pregnancies with severe IUGR (n = 6) with absent or reversed end-diastolic velocity in the umbilical artery. Distal placental villous tissue was collected to enrich for intermediate and terminal villi. Tissue was digested and PlMEC positively selected using tocosylated magnetic Dynabeads labeled with Human Endothelial Antigen lectin. The purity of the PlMEC (94 ± 2 SD %) was assessed by CD31 and vimentin immunocytochemistry. RNA was extracted from the PlMEC samples and subjected to Affymetrix microarray analysis (U133Plus2 array chips). Comparison of preterm and IUGR PlMEC gene expression profiles identified BTNL9 and NTRK2 transcripts to be upregulated and SAA1 and SLAMF1 transcripts to be downregulated in all 6 IUGR cases relative to preterm controls. A third downregulated gene GNAS was identified to be near significance. Changes were demonstrated to be significant at the mRNA level by Real Time PCR in the PlMEC samples. Changes in the IUGR endothelium were confirmed at the protein level by immunohistochemistry for the 3 with available antibodies. We used a tissue microarray constructed from an independent cohort of placental samples from severe IUGR (n = 7), preeclamptic (n = 7), preterm control (n = 6) and term control (n = 6) pregnancies. Results confirmed differential endothelial expression of BTNL9, NTRK2 and SLAMF1 in IUGR versus preterm and term samples. These studies are the first to characterize PlMEC gene expression profiles thus we have advanced our understanding of the molecular basis of placental micro-vascular pathophysiology in fetal growth restriction.

Heparin promotes soluble VEGF receptor expression in human placental villi to impair endothelial VEGF signaling.

BACKGROUND: Severe preeclampsia is characterized by hypertension, renal injury and placental dysfunction. Prothrombotic disorders are discovered in 10-20% of women with preeclampsia, providing the rationale for prescribing low-molecular-weight heparin (LMWH) in future pregnancies. Heparin has diverse molecular actions and appears to reduce the recurrence risk of preeclampsia in women without prothrombotic disorders. The placenta-derived anti-angiogenic splice-variant protein soluble vascular endothelial growth factor (VEGF) receptor-1 (sFLT1) is strongly implicated in the pathogenesis of the underlying endothelial dysfunction. As the placental syncytiotrophoblast is the principal source of sFLT1, we tested the hypothesis that heparin suppresses placental sFLT1 secretion. METHODS AND RESULTS: First trimester placental villi exposed to LMWH (0.25-25 IU mL(-1)) in an in vitro explant model significantly increased the expression and release of sFLT1 by the syncytiotrophoblast into culture media, reducing phosphorylation of FLT1 and KDR receptors in cultured human umbilical vein endothelial cells. This response was significantly diminished in placental villi from healthy term pregnancies. Placental villi from severely preeclamptic pregnancies had a higher baseline sFLT1 release, compared with first trimester placental villi and did not respond to LMWH treatment. LMWH promoted villous cytotrophoblast proliferation (BrdU incorporation) and impaired syncytial fusion-differentiation, causing syncytiotrophoblast apoptosis (by caspase 3&7 activity and TUNEL staining) and necrosis (ADP/ATP ratio). CONCLUSION: LMWH promotes sFLT1 synthesis and release from first trimester placental villi in a manner similar to that of severely preeclamptic placental villi, which antagonizes VEGF signaling in endothelial cells. These effects in part are mediated by an interaction between heparin and the cytotrophoblasts that regenerates the overlying syncytiotrophoblast responsible for sFLT1 secretion into the maternal blood.

Unfractionated heparin for second trimester placental insufficiency: a pilot randomized trial.

OBJECTIVE: To conduct a pilot randomized controlled trial of unfractionated heparin (UFH) in women considered at high risk of placental insufficiency in the second trimester. METHODS: Women with either false-positive first trimester (pregnancy-associated placental protein-A [PAPP-A] < 0.35 MoM) or second trimester (alpha-fetoprotein [AFP] > 2.0 MoM, inhibin > 3.0 MoM, human chorionic gonadotropin > 4.0 MoM) serum screening tests or medical/obstetric risk factors were screened for placental insufficiency by sonographic evaluation of the placenta and uterine artery Doppler between 18 and 22 weeks. Thrombophilia screen-negative women with two or three abnormal test categories were randomized by 23+6 weeks to self-administration of subcutaneous unfractionated heparin (UFH) 7500 IU twice daily until birth or 34 weeks, or to standard care. Maternal anxiety and other maternal-infant outcomes were determined. RESULTS: Thirty-two out of 41 eligible women consented, with 16 women randomized to UFH and 16 to standard care. There was no statistically significant difference identified between the two treatment groups (standard care vs. UFH) for the following: maternal anxiety score (mean [standard deviation]), 14.2 [± 1.6] vs. 14.0 [± 1.8]; birth weight (median [range]), 1795 [470-3295]g vs. 1860 [730-3050]g; perinatal death, 3 vs. 0; severe preeclampsia, 2 vs. 6; placental weight < 10th percentile, 7 vs. 4; or placental infarction, 4 vs. 3. CONCLUSION: Our study design identified women at high risk of adverse maternal-infant outcomes attributable to placental insufficiency. Women with evidence of placental insufficiency were willing to undergo randomization and self-administration of UFH without increased maternal anxiety.

Pathologic basis of echogenic cystic lesions in the human placenta: role of ultrasound-guided wire localization.

OBJECTIVE: The sonographic appearance of the placenta is normally homogenous throughout the second trimester. A variety of abnormalities in placental texture have been described, some of which may be pathologic and associated with adverse clinical outcomes. We characterized the pathologic basis of one lesion termed echogenic cystic lesions (ECLs) that may be a prognostic marker in intrauterine growth restriction (IUGR). STUDY DESIGN: We retrospectively correlated placental pathology in 50 pregnancies that had a total of 84 ECLs documented by ultrasound prior to delivery. Six additional women with placental ECLs prospectively underwent immediate post-delivery ultrasound-guided wire localization of 9 lesions followed by placental pathology. Obstetric outcome data were recorded. RESULTS: Severe pre-eclampsia (20%) and extreme IUGR (18%) were common outcomes. Of 93 ECLs identified, 46 (49%) gross lesions were found by placental pathology. Inter-villous thrombosis was the most significant lesion found (30/46, 65%) compared to all other lesions (35%; Z-Test, p = 0.007). Ultrasound guidance identified 8/9 (89%) lesions of which 6/8 (67%) were inter-villous thrombosis. Associated lesions (infarction, 36%; advanced villous maturation, 27%) and small placental weight (<10th centile, 38%) were present in 50%, but did not increase the risk of adverse perinatal outcome. CONCLUSIONS: ECLs are most commonly due to inter-villous thrombosis. The adverse clinical outcomes may be mediated by associated lesions not readily detectable by ultrasound. Ultrasound-guided wire localization is a promising research tool for future large-scale cohort studies needed to define the clinical utility of placental ultrasound findings.

Genetic polymorphisms in the fibrinolytic system of placentas with massive perivillous fibrin deposition.

Massive perivillous fibrin deposition (MPFD) and maternal floor infarction (MFI) of the placenta are rare related conditions associated with poor perinatal outcome including antepartum stillbirth. The diseases are characterized by pathologic accumulation of fibrinoid deposits that surround the placental villi (in the case of MFI predominantly in the basal regions adjacent to the decidual plate). These findings suggest either overproduction and/or defective clearance of fibrinoid within the intervillous space. Recently genetic polymorphisms of the plasminogen activator inhibitor-1 (PAI-1) gene have been found in association with impaired fibrinolysis in the pelvis predisposing to endometriosis. We hypothesized that polymorphisms in one or more of four genes that regulate fibrinolysis were associated with MPFD and MFI placentas. We retrospectively identified 20 consecutive cases of MPFD/MFI from our placental pathology database and generated 2 random gestational age-matched controls for each case. Clinical charts were reviewed. DNA was extracted from archived paraffin blocks of placental tissue from cases and controls. Single nucleotide repeat polymorphisms (SNPs) in loci within PAI-1 gene, thrombin activated fibrinolysis inhibitor (TAFI) gene, plasminogen activator urokinase (u-PA) gene and plasminogen activator tissue (t-PA) gene were studied using PCR methods. Outcomes in the study group included perinatal death (8), preterm IUGR (6), preeclampsia (4) and only 3 normal term deliveries. A spectrum of placental ultrasound abnormalities was observed. No SNP polymorphism was found to associate with MPFD/MFI. MPFD/MFI are associated with significant abnormal perinatal outcomes but have not been shown to be mediated by polymorphisms in candidate genes that are predicted to impair fibrinolysis in our study.

Placental size and the prediction of severe early-onset intrauterine growth restriction in women with low pregnancy-associated plasma protein-A.

OBJECTIVES: Screening studies for trisomy 21 demonstrate that low maternal serum pregnancy-associated plasma protein-A (PAPP-A) at 11-13 weeks' gestation is associated with stillbirth, intrauterine growth restriction (IUGR) and pre-eclampsia in chromosomally normal fetuses. However, the strength of these associations is too weak to justify screening for these placental insufficiency syndromes. Our objective was to evaluate placental size and uterine artery (UtA) Doppler imaging as second-stage screening tests for women with low PAPP-A. METHODS: We prospectively studied 90 normal singleton pregnancies with first-trimester PAPP-A

Double immuno-labelling of proliferating villous cytotrophoblasts in thick paraffin sections: integrating immuno-histochemistry and stereology in the human placenta.

In order to understand the pathological basis of abnormal villous trophoblast development in diseased placentas, the organ must be sampled by non-biased methods and subject to analysis by stereological tools. This approach permits quantification of cytotrophoblast density and syncytiotrophoblast structure including evidence of apoptotic shedding via syncytial knots. The stereological quantification of cells (or their) nuclei requires that each should be unambiguously identified and counted within a defined volume of tissue. A major limitation of such studies at present is the inability to accurately identify and phenotype subsets of villous cytotrophoblasts that either proliferate or are destined to fuse into the overlying syncytiotrophoblast. We describe the development of a novel double immuno-labelling protocol to selectively identify proliferating villous cytotrophoblast cells in human placental villi using thick (25microm) paraffin sections suitable for stereological quantification. Cytotrophoblast cells were selectively stained using a monoclonal anti-cytokeratin 7 (CK 7) antibody without antigen retrieval, followed by nuclear Ki-67 co-localisation. Both antibodies displayed full depth penetration with sharp, clearly defined staining precipitates and no cross-reactivity. This double immuno-labelling protocol is reproducible, cost effective and time efficient (8h). Use of a variety of antibodies following antigen retrieval will be a significant advancement in the ability to accurately quantify sub-populations of villous cytotrophoblast in normal and pathological placentas.

Rheological and physiological consequences of conversion of the maternal spiral arteries for uteroplacental blood flow during human pregnancy.

Physiological conversion of the maternal spiral arteries is key to a successful human pregnancy. It involves loss of smooth muscle and the elastic lamina from the vessel wall as far as the inner third of the myometrium, and is associated with a 5-10-fold dilation at the vessel mouth. Failure of conversion accompanies common complications of pregnancy, such as early-onset preeclampsia and fetal growth restriction. Here, we model the effects of terminal dilation on inflow of blood into the placental intervillous space at term, using dimensions in the literature derived from three-dimensional reconstructions. We observe that dilation slows the rate of flow from 2 to 3m/s in the non-dilated part of an artery of 0.4-0.5mm diameter to approximately 10 cm/s at the 2.5mm diameter mouth, depending on the exact radius and viscosity. This rate predicts a transit time through the intervillous space of approximately 25s, which matches observed times closely. The model shows that in the absence of conversion blood will enter the intervillous space as a turbulent jet at rates of 1-2m/s. We speculate that the high momentum will damage villous architecture, rupturing anchoring villi and creating echogenic cystic lesions as evidenced by ultrasound. The retention of smooth muscle will also increase the risk of spontaneous vasoconstriction and ischaemia-reperfusion injury, generating oxidative stress. Dilation has a surprisingly modest impact on total blood flow, and so we suggest the placental pathology associated with deficient conversion is dominated by rheological consequences rather than chronic hypoxia.

Early gene expression and morphogenesis of the murine chorioallantoic placenta in vivo and in vitro.

BACKGROUND: In mice the exchange of oxygen and nutrients between mother and fetus occurs in the chorioallantoic placenta where fetal capillaries come in close proximity with maternal blood perfusing trophoblast-lined sinusoids. Despite its critical importance, quantitative in vivo gene expression over the initial stages of chorioallantoic placental development has not been described, nor are there in vitro systems recapitulating the critical syncytiotrophoblast differentiation step in its formation. Here we describe molecular events that occur during the onset of chorioallantoic morphogenesis in mice in vivo, and in placental explant and whole conceptus cultures in vitro. RESULTS: Chorioallantoic morphogenesis began immediately following allantoic fusion with the chorion in vivo, and was associated with significant upregulation of syncytiotrophoblast associated mRNA (Gcm1 and Syncytin A). However mouse placentas with chorioallantoic point attachment cultured with the allantois or as whole conceptuses did not upregulate Gcm1 and/or Syncytin A, suggesting that syncytiotrophoblast differentiation did not occur in vitro. Failure of morphogenesis appeared to be due to failure to sustain in vitro the chorionic trophoblast cells from which the syncytiotrophoblast cells are derived. In vitro culture conditions did support the upregulation of ectoplacental cone marker Tpbpalpha, maintenance of giant cell marker Pl1, and maintenance of Fgfr2 expression; all of which mimicked in vivo events observed over this developmental interval. CONCLUSIONS: We conclude that chorionic trophoblast maintenance and the early events that occur in vivo between chorioallantoic point attachment and primary villous formation are dependent on undefined intrauterine factors that were not present in the in vitro culture system. Nevertheless, in vitro culture conditions were appropriate to reproduce in vivo expression levels of Fgfr2, Pl1, and Tpbpalpha in placental explants.

Screening for placental insufficiency in high-risk pregnancies: is earlier better?

OBJECTIVE: To compare a profile of placental function between the first and second trimesters in pregnancies at high risk of adverse perinatal outcomes attributable to placental insufficiency. STUDY DESIGN: Prospective cohort study in 61 singleton pregnancies. Uterine artery Doppler and placental morphology (shape and texture) were determined at 11-13(+6) weeks and at 18-23(+6) weeks. First trimester (pregnancy-associated placental protein-A [PAPP-A]) and second trimester (total hCG and alpha fetoprotein [AFP]) serum biochemistry were determined. The two screening periods were compared for the prediction of a range of severe adverse perinatal outcomes (intrauterine growth restriction [IUGR], abruption, severe pre-eclampsia/HELLP syndrome, delivery<32 weeks, or stillbirth). RESULTS: Adverse perinatal outcomes occurred in 14 (23%) women; 3 (4.9%) losses<20 weeks, 2 (3.3%) stillbirths>20 weeks, 4 (6.6%) IUGR, 7 (11.5%) severe pre-eclampsia/HELLP syndrome, and 10 (16.4%) deliveries<32 weeks. Abnormal second trimester placental morphology was significantly associated with adverse outcome [+LR: 3.6, 95% CI: 1.3-8.5; -LR: 0.63, 95% CI: 0.36-0.93; p=0.025], as was > or = 1 abnormal second trimester tests [+LR: 5.9, 95% CI: 1.6-24; -LR: 0.68, 95% CI: 0.59-0.89; p=0.005] or > or = 2 abnormal second trimester tests [+LR: 3.6, 95% CI: 1.3-7.7; -LR: 0.58, 95% CI: 0.27-0.94; p=0.035]. No combination of first trimester tests significantly predicted severe adverse perinatal outcomes. A study sample size of 822 women with similar high-risk characteristics would be needed in order to refute the conclusion that present methods of first trimester screening are not inferior to second trimester screening for severe placental insufficiency (p=0.05, power 80%, z-test). CONCLUSIONS: In clinically high-risk pregnancies, prediction of adverse perinatal outcomes using placental function testing is more effective in the second compared with the first trimester.

Assessment of fetal atrioventricular time intervals by tissue Doppler and pulse Doppler echocardiography: normal values and correlation with fetal electrocardiography.

OBJECTIVE: To establish gestational age-specific reference values of normal fetal atrioventricular (AV) time interval by spectral tissue Doppler imaging (TDI) and pulse-wave Doppler (PD) methods, and to assess their correlation with signal-averaged fetal PR intervals (ECG). DESIGN: Cohort study. SETTING: Tertiary centre for fetal cardiology. PATIENTS AND MEASURES: 131 pregnant women between 14 and 42 weeks' gestation underwent 196 fetal echocardiograms and 158 fetal ECG studies. TDI-derived AV intervals were measured as the intervals from atrial contraction (Aa) to isovolumic contraction (IV) and from Aa to ventricular systole (Sa) at the right ventricular free wall. PD-derived AV intervals were measured from simultaneous left ventricular inflow/outflow (in/out) and superior vena cava/aorta (V/AO) recordings. RESULTS: Measurements were possible by ECG in 61%, by TDI in 100%, by in/out in 100% and by V/AO in 97% of examinations. Aa-IV correlated significantly better with PR intervals (y = 0.67x + 38.29, R(2) = 0.15, p < 0.0001, mean bias 8.0 ms) than did in/out (R(2) = 0.10, p = 0.002, bias 18.7 ms) and V/AO (R(2) = 0.06, p = 0.02, bias 12.4 ms). Gestational age and AV intervals were positively correlated with all imaging modalities (R(2) = 0.19-0.31, p < 0.0001). CONCLUSION: This study showed the feasibility of fetal AV interval measurements by TDI, and established gestational age-specific reference data. TDI-derived Aa-IV intervals track ECG PR intervals more closely than PD-derived AV intervals and thus should be used as the ultrasound method of choice in assessing fetal AV conduction.

Placental morphology: from molecule to mother -- a dedication to Peter Kaufmann -- a review.

Theories on the development of placental pathologies and insufficiencies are based on the assumption that the development of the placental villous trees, the invasion of extravillous trophoblast or the differentiation of the villous trophoblast is somehow dysregulated. One of the pioneers trying to answer these questions is Peter Kaufmann, who started research on the placenta nearly 40 years ago. In this review, we try to shed light on various aspects of placental development, and on how important morphology is in combination with other disciplines to elucidate the differences between a normal and a pathological placenta.

EGF modulates trophoblast migration through regulation of Connexin 40.

In this study we show that decidua conditioned media (DCM) downregulate Connexin 40 (C x 40) expression in extravillous trophoblast (EVT) outgrowths and can promote EVT differentiation to the invasive phenotype resulting in switching of integrin and EGF receptor expression. This suggests that growth factors secreted by the decidua, such as EGF, mediate trophoblast migration/invasion and may do so by modulating C x 40 expression and function. To test this hypothesis we have utilized migration assays using cell lines expressing C x 40. Migration assays were performed with Jeg-3, Jeg-3 overexpressing C x 40 (JpUHD) and JAR cells seeded on fibronectin-coated inserts with 8 microm pores and incubated in the absence or presence of serum-starved decidual cells. Cell migration was only observed in the presence of DCM. Conversely overexpression of C x 40 in Jeg-3 cells resulted in inhibition of cell migration as compared to wild-type control. Addition of DCM to cultured JAR cells resulted in the downregulation of C x 40 protein. EGF is known to stimulate trophoblast migration/invasion and was detected in DCM; therefore, we investigated the action of EGF on C x 40. EGF (10 ng/mL) resulted in the downregulation of C x 40 in the JAR cell line. However, EGF had no effect on JAR cell migration. We conclude that decidual secretion of growth factors, such as EGF, may act to prime trophoblast for migration/invasion through modulation of connexin expression and function.

Bi-potential behaviour of cytotrophoblasts in first trimester chorionic villi.

Murine trophoblast stem (TS) cells express fibroblast growth factor receptor 2 (FGFR2) and are maintained in their proliferative state by fibroblast growth factor 4 (FGF4). We show in this report that in the first trimester human placenta FGFR2 expression is similarly found in a subset of villous cytotrophoblast and in proximal anchoring columns. Western analysis demonstrated declining FGFR2 protein expression as gestation advanced, suggesting a similar role for FGF in early human trophoblast proliferation. Mouse TS cell differentiation is known to occur along two distinct transcriptionally-regulated pathways; extravillous trophoblast (EVT) cells invade the uterine wall to promote maternal blood flow whilst syncytiotrophoblast lines chorionic villi in the labyrinth. Similar differentiation steps occur in the human placenta though the fate of human trophoblast stem cells is presently unknown. To investigate the mechanisms underlying human cytotrophoblast differentiation we have developed a novel cultured floating first trimester villous explant model in which denuded first trimester villi spontaneously regenerate syncytiotrophoblast following 48 h of culture. Addition of FGF4 and heparin inhibited syncytiotrophoblast regeneration in favor of forming clumps of cytotrophoblast. Proximal cells in these clumps were FGFR2 immuno-reactive and proliferative, intermediate parts expressed alpha5beta1-integrin, while the distal portion expressed HLA-G and the invasive integrin alpha1beta1 indicating differentiation to the EVT phenotype. In contrast, non-denuded villi exposed to FGF4 exhibited similar proliferation of the cytotrophoblast; however, these cells did not express any of the invasive EVT markers. We conclude that FGFR2-positive chorionic cytotrophoblasts exhibit bi-potential behaviour, being capable of forming either syncytiotrophoblast or EVT. We suggest bipotential trophoblast progenitor cells persist during first trimester human placental development.

Prognostic value of placental ultrasound in pregnancies complicated by absent end-diastolic flow velocity in the umbilical arteries.

Our objective was to evaluate the utility of gray-scale placental ultrasound for the detection of pathological lesions in the placentas of preterm pregnancies with abnormal fetoplacental blood flow (defined by absent or reversed end-diastolic flow velocities [ARED] in the umbilical arteries) before 32 weeks of gestation. Sixty consecutive structurally and chromosomally normal singleton pregnancies were evaluated. Pre-defined criteria were used to describe placental appearances using gray-scale real-time ultrasound. Proximal uterine artery Doppler waveforms were recorded using pulsed and color Doppler ultrasound. Each patient had a thrombophilia profile. Following delivery, a single perinatal pathologist reviewed each placenta at a gross and microscopic level blinded to the placental ultrasound findings. Placental shape or texture was abnormal on gray-scale ultrasound in 43/59 (73%) and echogenic cystic lesions (ECL) were found in 16 (27%). Uterine artery Doppler was abnormal in 47/60 (78%) cases. Thirty-eight pregnancies were subsequently delivered by planned Caesarean section in the fetal and/or maternal interest (birthweights 540-2300 g, mean gestational age 30.6 weeks) and 21 pregnancies resulted in the vaginal delivery of a stillborn fetus where fetal weight and/or gestational age did not justify Caesarean section (birthweights 85-600 g, mean gestational age 24.9 weeks). ECL had a low positive predictive value for both villous infarcts (63%) and for focal/massive perivillous fibrin deposition (40%). Nevertheless, the combination of abnormal uterine artery Doppler and abnormal gray-scale findings (abnormal placental morphology or ECL) was strongly predictive of stillbirth (17/21; sensitivity 81%, PPV 52%, p = 0.006 Fisher's exact test). Pregnancies with ARED in the umbilical arteries have a high perinatal mortality associated with pathology of the placental villi. Ultrasound examination of the placenta and its maternal blood supply may contribute to the perinatal management of these pregnancies.