Ettore Piccoli

Umbilical doppler waveforms and placental villous angiogenesis in pregnancies complicated by fetal growth restriction

OBJECTIVE To test the hypothesis that the characteristics of umbilical artery Doppler flow velocity waveforms in growth-restricted fetuses indicate angiogenesis within placental stem and gas-exchanging villi. METHODS We examined 18 placentas from singleton fetuses that were normal structurally and chromosomally but were growth-restricted, preterm, and complicated by preeclampsia. Ten cases with positive end-diastolic flow and eight with absent or reverse end-diastolic flow were compared with six gestational age-matched controls. Sections of villous placenta were examined to determine structural composition (percentage of fibrinoid, intervillous space, and villous tissue), relative proportion of villous types (stem, immature intermediate, and gas-exchanging villi), and the frequency distribution of stem arterial vessel calibers and their branching pattern. RESULTS Placentas with positive end-diastolic flow had a significantly (P < .05) higher percentage of gas-exchanging villi (median 69.6%, range 62.5-80.8%) than those with absent or reverse end-diastolic flow (58.3%, 29.9-71.9%) or controls (60.8%, 43.1-65.6%). The gas-exchanging villi from placentas with absent or reverse end-diastolic flow were slender, elongated, poorly branched, and poorly capillarized. There was a progressive trend toward reduced branching of the stem arteries from the controls (median 22%, range 2-38%), through the positive end-diastolic group (17%, 11-20%), to the absent or reverse end-diastolic group (13%, 4-23%). CONCLUSION Compared with absent or reverse end-diastolic flow, the placentas from growth-restricted fetuses with positive end-diastolic flow showed a normal pattern of stem artery development, accompanied by increased capillary angiogenesis and terminal villous development. These features suggest an adaptive pathway for the placenta in the face of uteroplacental ischemia.

Is three-dimensional power Doppler ultrasound useful in the assessment of placental perfusion in normal and growth-restricted pregnancies?

To investigate three‐dimensional (3D) power Doppler ultrasound indices in the assessment of placental perfusion and their relationship to gestational age (GA), placental position and umbilical artery Doppler flow velocity waveform (FVW) patterns in normal and intrauterine growth‐restricted (IUGR) pregnancies.

Macrophage Migration Inhibitory Factor in Human Pregnancy and Labor

PROBLEM: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in reproduction. Presently there is no information on the possible involvement of MIF in the onset of labor.

Pre-eclampsia is associated with Helicobacter pylori seropositivity in Italy.

Objectives Pre-eclampsia (PE) is characterized by an excess of inflammation and endothelial dysfunction. Helicobacter pylori (H. pylori) causes chronic inflammatory changes and endothelial damage. We investigated the prevalence of seropositivity for IgG against H. pylori and cytotoxin-associated antigen A (CagA) in PE patients and the presence of H. pylori DNA in their placentas. Methods We tested 47 pregnant women with PE and 47 with uneventful pregnancies for serum antibodies against H. pylori (enzyme immunoassays) and CagA protein (immunoblot assays). In 20 of them (10 normal and 10 PE) we assessed the presence, in the placenta, of H. pylori DNA by means of nested polymerase chain reaction (PCR). The odds ratios (OR) and 95% confidence intervals (CI), adjusted for parity, were calculated using logistic regression analysis to assess the risk of PE associated with H. pylori infection. Results Helicobacter pylori seropositivity frequency was higher in mothers with PE (51.1%) compared to women with uneventful pregnancy (31.9%) (OR, 2.668; 95% CI, 1.084–6.566; P = 0.033). The difference was even greater for CagA seropositivity (80.9 and 14.9%, respectively) (OR, 26.035; 95% CI, 8.193–82.729; P < 0.001). All placentas were negative for H. pylori DNA. Conclusions Helicobacter pylori, and especially strains carrying the CagA gene, may contribute to the inflammatory mechanisms involved in the pathogenesis of PE.

Pro-inflammatory profile of preeclamptic placental mesenchymal stromal cells: new insights into the etiopathogenesis of preeclampsia.

The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF), Vascular Endothelial Growth Factor (VEGF), soluble FMS-like tyrosine kinase-1 (sFlt-1) and free β-human Chorionic Gonadotropin (βhCG) by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia. Methods Chorionic villous PDMSCs were isolated from severe preeclamptic (n = 12) and physiological control term (n = 12) placentae. Control and PE-PDMSCs’s cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM) was collected. Physiological villous explants (n = 48) were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent. Results Cytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones. Conclusions Herein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs induced a PE-like phenotype in physiological villous explants. Our data clearly depict chorionic mesenchymal stromal cells as central players in placental physiopathology, thus opening to new intriguing perspectives for the treatment of human placental-related disorders as preeclampsia.

Macrophage Migration Inhibitory Factor in Fetoplacental Tissues from Preeclamptic Pregnancies with or without Fetal Growth Restriction

The proinflammatory cytokine MIF (macrophage migration inhibitory factor) is involved in physiological and pathological processes in pregnancy. MIF maternal serum levels are increased in preeclampsia (PE). We hypothesize that pregnancy tissues are the source of MIF overexpression in PE. MIF protein was studied in maternal sera, placental tissues, fetal membranes, and umbilical cord of 8 control and 20 PE pregnancies: 10 with normal fetal growth (PE-AGA) and 10 with fetal growth restriction (PE-FGR). MIF levels were significantly higher in PE-AGA membranes than in controls and PE-FGR. In PE-FGR, MIF cord concentrations were higher than in PE-AGA while MIF placental levels were lower than in controls. MIF maternal serum levels were higher in PE, compared to controls, and the difference was mainly due to PE-FGR samples. These data support MIF involvement in PE pathogenesis and suggest that different pregnancy tissues contribute to MIF production in PE with and without fetoplacental compromise.

Review: Feto-placental vascularization: A multifaceted approach

Doppler Ultrasound allows the in vivo study of feto-placental hemodynamics. Doppler flow velocity waveforms (FVWs) obtained from the umbilical arteries reflect downstream blood flow impedance, thus giving indirect evidence of vascular villous tree characteristics. Pulsatility Index, which quantifies FVWs, decreases throughout normal pregnancy, indicating decreasing impedance and is often higher in cases of fetal growth restriction (FGR). Different approaches (morphometrical, morphological, mathematical, immunohistochemical and molecular) have contributed to elucidation of which anomalies of the vascular villous tree underlie Doppler findings. 3D ultrasound may be useful in the study of feto-placental perfusion. However, the unsolved question is why developmental villous tree anomalies occur. Crucial to the success of future research is definition of the population studied based on the uniform and correct definition of FGR.

Investigation of Placental Stem Villi Arteries in Fetally Growth-Restricted Pregnancies: A Multivariate Analysis

Thirteen placentas were studied from 5 normal pregnancies and 8 from pregnancies complicated by fetal growth restriction (4 with present, 3 with absent, and 1 with reversed end-diastolic velocities at Doppler interrogation of the umbilical arteries). On immunohistochemically stained slides, the diameter (d) and the wall thickness (t) of the arterial vasculature of the stem villi were measured for a total of approximately 10,000 vessels. A multivariate ‘mixed effect model’ statistical analysis was performed using d and t as dependent variables and gestational age, delivery mode, fetal and placental weight, the degree of vascular collapse and Doppler blood flow patterns as independent variables. Gestational age, Doppler pattern and the degree of vascular collapse significantly affected both d and t, the mode of delivery influenced d while fetal and placental weights scarcely affected the dependent variables. The above parameters should therefore be taken into account when investigating placental stem vessel morphometry.

Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise

ABSTRACT Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16INK4A, p18INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16INK4A, p18INK4C, CDK4 and CDK6 expressions in physiological term villous explants. PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16INK4A, p18INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay. We reported significantly increased p16INK4A and p18INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16INK4 and p18INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast. We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise.