Congenital Diaphragmatic Hernia Fetuses Research Articles

Involvement of YAP and LATS1 in lung development in a rat model of nitrofen-induced congenital diaphragmatic hernia

Purpose: To investigate the role of Hippo pathway in lung development in congenital diaphragmatic hernia (CDH).
 Methods: One oral dose of nitrofen was maternally administered for induction of CDH on embryonic day 9.5 (E 9.5). Sildenafil was administered intragastrically at a dose of 100 mg/kg on E 11.5. Three rat groups were used: control, CDH, and CDH + sildenafil. Cesarean section was used for fetal delivery on E 21.5. Fetuses with left diaphragmatic hernia (except in control rats) were chosen for investigations. Fetal body weight and weight of lung tissue were recorded, and lung histological evaluation, western blot and PCR were carried out after lung processing.
 Results: There was markedly higher expression of YAP in CDH rats than in control rats was unaffected by antenatal sildenafil administration (p < 0.05). However, prenatal sildenafil intervention significantly increased LATS1 expression in the lung of CDH fetuses (p < 0.05).
 Conclusion: These results indicate that increased pulmonary YAP expression in CDH rat model might contribute to pulmonary vascular remodeling and suppression of lung development. Thus, antenatal sildenafil administration not only mitigates abnormal vascular remodeling but also promotes lung development, most likely via increased expression of LATS1.
 Keywords: Congenital diaphragmatic hernia (CDH), Hippo pathway, Lung development, Sildenafil

Prenatal prediction of postnatal survival in fetuses with congenital diaphragmatic hernia using MRI: lung volume measurement, signal intensity ratio, and effect of experience

Objective To evaluate various signal intensity ratios in isolated congenital diaphragmatic hernia (CDH) and to compare their potential in predicting survival with that of the observed-to-expected (O/E) ratio of total fetal lung volume (TFLV) using magnetic resonance imaging (MRI) measurements. Our second objective was to evaluate the impact of operator’s experience in comparing the prediction of postnatal survival by O/E-TFLV. Methods In 75 conservatively managed CDH fetuses and in 50 who underwent fetoscopic endoluminal tracheal occlusion (FETO), the fetal lung-to-amniotic fluid, lung-to-liver, lung-to-muscle, lung-to-spinal fluid signal intensity ratios, respectively LAFSIR, LLSIR, LMSIR, and LSFSIR, were measured, as was O/E-TFLV. Receiver operating characteristic (ROC) curves were constructed and used to compare the various signal intensity ratios with O/E-TFLV in the prediction of postnatal survival. In 72 MRI lung volumes assessed by the referring radiologists in Paris and Lille and secondarily by our expert radiologist in Brussels (M.M.C.) using the same MRI examinations, ROC curves were constructed and used to compare the value of O/E-TFLV determined by the two centers in the prediction of postnatal survival. Results In the total cohort of CDH fetuses, O/E-TFLV and LLSIR were predictive of postnatal survival whereas in the conservatively managed group O/E-TFLV, LLSIR, and LMSIR predicted postnatal survival. O/E-TFLV predicted postnatal survival far better than the signal intensity ratios: area under the ROC curve for prediction by O/E-TFLV in the total cohort was 0.866 (p < .001; standard error = 0.031). The area under the ROC curve for prediction of postnatal survival using O/E-TFLV by MRI evaluated at the referral centers was 0.640 (p = 102; standard error = 0.085), and with O/E-TFLV reevaluated by M.M.C., it was 0.872 (p < .001; standard error = 0.061). Pairwise comparison showed a significant difference between the areas under the ROC curves (difference = 0.187, p = .012; standard error = 0.075). Conclusion In fetuses with CDH with/without FETO, LLSIR was significantly correlated with the prediction of postnatal survival. However, measurement of O/E-TFLV was far better in predicting postnatal outcome. Operator experience in measurement of lung volumes using MRI seem to play a role in the predictive value of the technique.

Fetal MRI assessment of mediastinal shift angle in isolated left congenital diaphragmatic hernia: A new postnatal survival predictive tool?

To quantify the mediastinal shift angle (MSA) in fetuses with isolated left congenital diaphragmatic hernia (CDH) by magnetic resonance imaging and evaluate survival. Fetuses from singleton pregnancies with isolated left CDH were matched for gestational age with controls without thoracic malformations. For all fetuses the MSA was determined by two operators and inter-operator variability and differences between cases and controls were investigated. For all cases total fetal lung volume (TFLV) was calculated and the correlation between MSA and TFLV was assessed, and its predictive value towards survival was determined. Thirty-four fetuses were included as cases and 42 as controls. The mean gestational age for assessment of CDH fetuses was 32 weeks (range 27-38). Twenty-four fetuses survived until discharge and 10 did not. There was an excellent inter-operator reliability for measuring the MSA and a significant difference between MSA in cases and controls. There was an inverse correlation between MSA values and survival, a correlation between TFLV and survival and an inverse correlation between MSA and TFLV. The area under the ROC curve for MSA in predicting survival was 0.931 (95% CI 0.851-1.000). The MSA measured late in gestation correlates with postnatal survival in patients with isolated left CDH.

Calculating Observed-to-Expected Total Fetal Lung Volume in CDH Fetuses in Twin Gestation: Is There a Better Way?

Background: Congenital diaphragmatic hernia (CDH) is a potentially lethal birth defect, and identifying prenatal predictors of outcome is important. Observed-to-expected total fetal lung volume (o/e TFLV) has been shown to be a predictor of severity and useful in risk stratification but is variable due to different TFLV formulas. Objectives: To calculate o/e TFLV for CDH patients part of a twin gestation using the unaffected sibling as an internal control and comparing these values to those calculated using published formulas for TFLV. Methods: Seven twin gestations with one fetus affected by CDH were identified between 2006 and 2017. The lung volume for each twin was calculated using magnetic resonance imaging (MRI), and o/e TFLV was calculated using the unaffected twin’s TFLV. This percentage was then compared to the o/e TFLV calculated using published formulas. Results: Lung volumes in the unaffected twins were within normal ranges at the lower end of the spectrum. No single TFLV formula was found to correlate perfectly. Intraclass correlation coefficient estimate was most consistent for o/e TFLV calculated with the Meyers formula and supported by Bland-Altman plots. Conclusions: O/e TFLV measured in CDH/non-CDH twin gestations using the unaffected sibling demonstrated agreement with o/e TFLV calculated using the Meyers formula. We urge the fetal community to standardize the method, use, and interpretation of fetal MRI in the prenatal evaluation of CDH.

MIF inhibition enhances pulmonary angiogenesis and lung development in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a complex birth anomaly with significant mortality and morbidity. Lung hypoplasia and persistent pulmonary hypertension (PPHN) limit survival in CDH. Macrophage migration inhibitory factor (MIF), a key regulator of innate immunity, is involved in hypoxia-induced vascular remodeling and PPHN. We hypothesized that antenatal inhibition of MIF in CDH fetuses, would reduce vascular remodeling, and improve angiogenesis and lung development. Pregnant rats were randomized into three groups: Control, nitrofen, and nitrofen + ISO-92. Lung volumes of pups were measured by CT scanning. Right ventricular systolic pressure (RVSP) and vascular wall thickness (VWT) were measured together with MIF concentration, angiogenesis markers, lung morphometry, and histology. Prenatal treatment with ISO-92, an MIF inhibitor, improved normalization of static lung volume, lung volume-to-body weight ratio, decreased alveolar septal thickness, RVSP and VWT and improved radial alveolar count as compared to the non-treated group. Expression of MIF was unaffected by ISO-92; however, ISO-92 increased p-eNOS and VEGF activities and reduced arginase 1, 2 and Sflt-1. Prenatal inhibition of MIF activity in CDH rat model improves angiogenesis and lung development. This selective intervention may be a future therapeutic strategy to reduce the morbidity and mortality of this devastating condition.

The effects of tracheal occlusion on Wnt signaling in a rabbit model of congenital diaphragmatic hernia

PurposeTracheal occlusion (TO) reverses pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH), but its mechanism of action remains poorly understood. Wnt signaling plays a critical role in lung development, but few studies exist. The purpose of our study was to a) confirm that our CDH rabbit model produced PH which was reversed by TO and b) determine the effects of CDH +/− TO on Wnt signaling. MethodsCDH was created in fetal rabbits at 23 days, TO at 28 days, and lung collection at 31 days. Lung body weight ratio (LBWR) and mean terminal bronchiole density (MTBD) were determined. mRNA and miRNA expression was determined in the left lower lobe using RT-qPCR. ResultsFifteen CDH, 15 CDH + TO, 6 sham CDH, and 15 controls survived and were included in the study. LBWR was low in CDH, while CDH + TO was similar to controls (p = 0.003). MTBD was higher in CDH fetuses and restored to control levels in CDH + TO (p < 0.001). Reference genes TOP1, SDHA, and ACTB were consistently expressed within and between treatment groups. miR-33 and MKI67 were increased, and Lgl1 was decreased in CDH + TO. ConclusionTO reversed pulmonary hypoplasia and stimulated early Wnt signaling in CDH fetal rabbits. Type of studyBasic science, prospective. Level of evidenceII.

Longitudinal changes in lung size and intrapulmonary-artery Doppler during the second half of pregnancy in fetuses with congenital diaphragmatic hernia.

The objectives of the study are to evaluate longitudinal changes in lung size and intrapulmonary-artery (IPa) Doppler in fetuses with congenital diaphragmatic hernia (CDH) and assess their contribution in predicting neonatal survival. The observed/expected lung-to-head ratio (O/E-LHR) and IPa-pulsatility index (PI) and peak early diastolic reversed flow (PEDRF) were evaluated in a cohort of left-sided CDH fetuses managed expectantly during pregnancy. Longitudinal changes were analyzed by multilevel analysis, and their value to predict survival using the multiple logistic regression and decision-tree analysis was assessed. A total of 232 scans were performed on 69 CDH fetuses. The O/E-LHR values remained unchanged during fetal monitoring, whereas IPa-PI and PEDRF showed a progressive increase throughout follow-up, becoming abnormal on average at 30weeks of gestation. Absent/reversed end-diastolic velocity (EDV) in the IPa was observed in 20.3%. O/E-LHR and IPa Doppler indices were significantly associated with probability of survival (O/E-LHR≥26%, odds ratio [OR] 19.0; IPa-PI <+2.0 z score, OR 3.0; and positive EDV, OR 7.4). All cases with IPa-reversed EDV died after birth. While lung size remains stable during pregnancy, CDH fetuses show progressive deterioration in intrapulmonary blood flow. IPa Doppler evaluation may aid in predicting survival of CDH fetuses managed expectantly during pregnancy.

Prenatal treatment with rosiglitazone attenuates vascular remodeling and pulmonary monocyte influx in experimental congenital diaphragmatic hernia

IntroductionExtensive vascular remodeling causing pulmonary hypertension (PH) represents a major cause of mortality in patients with congenital diaphragmatic hernia (CDH). The chemokine monocyte chemoattractant protein-1 (MCP-1) is a biomarker for the severity of PH and its activation is accompanied by pulmonary influx of monocytes and extensive vascular remodeling. MCP-1 activation can be reversed by application of rosiglitazone (thiazolidinedione). We performed this study to evaluate the role of MCP-1 for the pathogenesis of PH in experimental CDH. We hypothesized that vascular remodeling and MCP-1 activation is accompanied by pulmonary influx of fetal monocytes and can be attenuated by prenatal treatment with rosiglitazone.MethodsIn a first set of experiments pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blot (WB), and immunohistochemistry (IHC) were used to evaluate MCP-1 expression, activation, and localization. Quantification and localization of pulmonary monocytes/macrophages were carried out by IHC.In a second set of experiments nitrofen-exposed dams were randomly assigned to prenatal treatment with rosiglitazone or placebo on D18+D19. Fetal lungs were harvested on D21, divided into control, CDH+rosiglitazone, and CDH+placebo and evaluated by WB as well as IHC.ResultsIncreased thickness of pulmonary arteries of CDH fetuses was accompanied by increased systemic and perivascular MCP-1 protein expression and significantly higher amounts of pulmonary monocytes/macrophages compared to controls (p<0.01). These effects were reversed by prenatal treatment with rosiglitazone (p<0.01 vs. CDH+P; control).ConclusionPrenatal treatment with rosiglitazone has the potential to attenuate activation of pulmonary MCP-1, pulmonary monocyte influx, and vascular remodeling in experimental CDH. These results provide a basis for future research on prenatal immunomodulation as a novel treatment strategy to decrease secondary effects of PH in CDH.

Characterizing cardiac dysfunction in fetuses with left congenital diaphragmatic hernia.

To evaluate cardiac function by conventional echocardiography and tissue Doppler imaging in fetuses with left congenital diaphragmatic hernia (CDH). Conventional echocardiography (myocardial performance index, ventricular filling velocities, and E/A ratios) and tissue Doppler imaging (annular myocardial peak velocities, E/E' and E'/A' ratios) in mitral, septal, and tricuspid annulus were evaluated in a cohort of 31 left-sided CDH fetuses and compared with 75 controls matched for gestational age 2:1. In comparison to controls, CDH fetuses had prolonged isovolumetric time periods (isovolumetric contraction time 35ms vs 28ms, P<.001), with higher myocardial performance index (0.49 vs 0.42, P<.001) and tricuspid E/A ratios (0.77 vs 0.72, P=.033). Longitudinal function assessed by tissue Doppler showed signs of impaired relaxation (mitral lateral A' 8.0 vs 10.1cm/s, P<.001 and an increased mitral lateral E'/A' ratio 0.93 vs 0.78, P<.001) in the CDH fetuses as compared with controls, with preserved systolic function. Left CDH fetuses show echocardiographic signs of diastolic dysfunction, probably secondary to fetal heart compression, maintaining a preserved systolic function.

Three-dimensional assessment of umbilical vein deviation angle for prediction of liver herniation in left-sided congenital diaphragmatic hernia.

To introduce a new sonographic marker of intrathoracic liver herniation in fetuses with left-sided congenital diaphragmatic hernia (CDH). In a consecutive series of fetuses with isolated CDH, an ultrasound volume of the fetal abdomen was acquired. On this volume, offline calculation of the angle formed by the midline of the abdomen (joining the center of the vertebral body to the abdominal insertion of the umbilical cord) and a second line joining the center of the vertebral body to the intra-abdominal convexity of the umbilical vein was carried out to give the umbilical vein deviation angle (UVDA). The UVDA was measured in a group of normal fetuses selected as controls. At follow-up, the presence of liver herniation was investigated in all cases of CDH. UVDA values were compared between the CDH group and controls, and between CDH 'liver-up' vs 'liver-down' cases. A receiver-operating characteristics (ROC) curve was constructed to identify a cut-off value of the UVDA with the highest accuracy in predicting liver herniation in the CDH group. Between 2009 and 2015, 22 cases of left-sided CDH were included in the study group, of which nine cases had liver herniation. Eighty-eight normal fetuses were recruited as controls. The UVDA was significantly higher in the cases vs controls (15.25 ± 7.91° vs 7.68 ± 1.55°; P < 0.0001). Moreover, the UVDA was significantly increased in CDH fetuses with liver-up vs liver-down (21.77 ± 8.79° vs 10.75 ± 2.10°; P < 0.0001). On ROC curve analysis the UVDA showed good prediction of liver herniation (area under the ROC curve, 0.94; P < 0.0001) with the best cut-off of 15.2°, yielding a sensitivity of 89% and a specificity of 100% (P < 0.0001). In fetuses with CDH, umbilical vein bowing may be quantified by measuring the UVDA using three-dimensional ultrasound. This sonographic marker seems to be an accurate predictor of liver herniation in left-sided CDH. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Current Strategy of Fetal Therapy II: Invasive Fetal Interventions

AbstractInvasive fetal intervention provides potential benefits to the fetus, but risks to the mother and the fetus are not negligible. Large congenital pulmonary airway malformation (CPAM) may cause fetal hydrops. Shunt placement in macrocystic type or steroids administration in microcystic type CPAM have been reported to reverse the hydrops in a small case series. In selected cases of fetal congenital diaphragmatic hernia (CDH), tracheal plugging may prevent egress of the lung fluid, promote lung proliferation, and maturation of pulmonary vasculature, which favor survival in isolated right-sided CDH fetuses or those with a lung-to-head ratio of ≤1.0. Fetoscopic cystoscopy with laser fulguration of the posterior urethral valve improves the 6-month survival and renal function. A randomized controlled trial to validate the real benefits of this procedure is still underway. Laser photocoagulation of anastomosing vessels is the standard of care for severe mid-trimester twin-to-twin transfusion syndrome. The principle of equipoise is likely to be reached on the issue of optimal treatment for Quintero stage I disease. Open fetal surgery has been offered in some cases of fetal tumor and myelomeningocele (MMC). Alternatively, endoscopic approach is being investigated to minimize procedure-related maternal morbidity. Only when procedure-related complications are properly prevented and managed, it is expected that invasive therapeutic innovation will then improve perinatal outcomes of selected fetal conditions.

Downregulation of KCNQ5 expression in the rat pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia

PurposePulmonary hypertension (PH) is a common complication of congenital diaphragmatic hernia (CDH). Voltage-gated potassium channels KCNQ1, KCNQ4, and KCNQ5 are expressed by rodent pulmonary artery smooth muscle cells, contributing to their vascular tone. We hypothesized that KCNQ1, KCNQ4, and KCNQ5 expression is altered in the pulmonary vasculature of nitrofen-induced CDH rats. MethodsAfter ethical approval (REC913b), time-pregnant rats received nitrofen or vehicle on gestational day (D)9. D21 fetuses were divided into CDH and control group (n=22). QRT-PCR and western blotting were performed to determine gene and protein expression of KCNQ1, KCNQ4, and KCNQ5. Confocal microscopy was used to detect these proteins in the pulmonary vasculature. ResultsRelative mRNA level of KCNQ5 (p=0.025) was significantly downregulated in CDH lungs compared to controls. KCNQ1 (p=0.052) and KCNQ4 (p=0.574) expression was not altered. Western blotting confirmed the decreased pulmonary KCNQ5 protein expression in CDH lungs. Confocal-microscopy detected a markedly diminished KCNQ5 expression in pulmonary vasculature of CDH fetuses. ConclusionsDownregulated pulmonary expression of KCNQ5 in CDH lungs suggests that this potassium channel may play an important role in the development of PH in this model. KCNQ5 channel activator drugs may be a potential therapeutic target for the treatment of PH in CDH. Level of evidence2b (Centre for Evidence-Based Medicine, Oxford)

Fetal Tracheal Occlusion for Severe Pulmonary Hypoplasia in Isolated Congenital Diaphragmatic Hernia: A Systematic Review and Meta-analysis of Survival.

To evaluate fetal survival after tracheal occlusion in fetuses with severe pulmonary hypoplasia and isolated congenital diaphragmatic hernia (CDH). Despite recent advances in neonatal intensive care, CDH still has a high mortality and morbidity. Fetoscopic endoluminal tracheal occlusion (FETO) stimulates lung growth and improves gas exchange in animal models of CDH, but the effects in humans are still under investigation. We searched Pubmed, Cochrane, EMBASE, and Scopus databases for clinical studies on tracheal occlusion and CDH. All studies comparing FETO and a contemporary control group were included. The primary outcome was survival, with the need for oxygen on discharge the secondary outcome. Meta-analysis of outcome measures was performed and odds ratios, relative risk ratios, and 95% confidence intervals were estimated with a fixed-effects model and were reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidance. Between 1997 and 2015, five eligible studies describing 211 patients were included (101 control and 110 FETO). All studies selected isolated severe CDH fetuses with a lung-to-head ratio 1.0 or less and liver herniation into the thoracic cavity. FETO favored survival outcome (odds ratio 13.32; 95% confidence interval, 5.40-32.87). Meta-analysis of the secondary outcome oxygen need at discharge could not be calculated, because it was not reported in all included studies. FETO improves survival in isolated CDH with severe pulmonary hypoplasia compared with the standard perinatal management.

Suppressed erythropoietin expression in a nitrofen-induced congenital diaphragmatic hernia.

Erythropoietin (EPO), an essential stimulator of erythropoiesis produced by the fetal liver, is important both in vascular remodeling and modulation of the endothelial response in the pulmonary vasculature. In addition, EPO guides alveolar development, along with retinoic acid (RA). EPO is a direct target of RA, and the retinoid pathway is altered in the nitrofen-induced congenital diaphragmatic hernia (CDH) model. In the present study, we tested the hypothesis that the synthesis of EPO is suppressed in a rat model of CDH. Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D19 and D21 and divided into control and CDH groups. Immunohistochemistry and quantitative real-time polymerase chain reaction (RT-PCR) were performed to determine the expression of EPO in the fetal liver and kidney. We also estimated the expression of EPO receptor in the fetal lung. The relative EPO mRNA expression in the liver on D19 and in the kidney on D21 were significantly lower in the CDH group than in the controls (P = 0.0008 and P = 0.0064, respectively). In addition, the results of immunohistochemistry supported the findings from the RT-PCR analysis. No significant changes were noted in the expression pattern or EPO receptor levels in the fetal lungs of the CDH group compared to the controls. Our results reveal the suppressed EPO synthesis in the CDH fetus, which may contribute to the pathogenesis of lung hypoplasia and modification of pulmonary vasculature in the CDH rat model. Pediatr Pulmonol. 2017;52:606-615. © 2016 Wiley Periodicals, Inc.

Why vessels do matter in pulmonary disease

In addition to structural abnormalities of the diaphragm, congenital diaphragmatic hernia (CDH) presents with severe hypoplasia of lung tissue involving both the pulmonary vasculature and the airways. These developmental defects...

Antenatal BAY 41-2272 reduces pulmonary hypertension in the rabbit model of congenital diaphragmatic hernia

Infants with congenital diaphragmatic hernia (CDH) fail to adapt at birth because of persistent pulmonary hypertension (PH), a condition characterized by excessive muscularization and abnormal vasoreactivity of pulmonary vessels. Activation of soluble guanylate cyclase by BAY 41-2272 prevents pulmonary vascular remodeling in neonatal rats with hypoxia-induced PH. By analogy, we hypothesized that prenatal administration of BAY 41-2272 would improve features of PH in the rabbit CDH model. Rabbit fetuses with surgically induced CDH at day 23 of gestation were randomized at day 28 for an intratracheal injection of BAY 41-2272 or vehicle. After term delivery (day 31), lung mechanics, right ventricular pressure, and serum NH2-terminal-pro-brain natriuretic peptide (NT-proBNP) levels were measured. After euthanasia, lungs were processed for biological or histological analyses. Compared with untouched fetuses, the surgical creation of CDH reduced the lung-to-body weight ratio, increased mean terminal bronchial density, and impaired lung mechanics. Typical characteristics of PH were found in the hypoplastic lungs, including increased right ventricular pressure, higher serum NT-proBNP levels, thickened adventitial and medial layers of pulmonary arteries, reduced capillary density, and lower levels of endothelial nitric oxide synthase. A single antenatal instillation of BAY 41-2272 reduced mean right ventricular pressure and medial thickness of small resistive arteries in CDH fetuses. Capillary density, endothelial cell proliferation, and transcripts of endothelial nitric oxide synthase increased, whereas airway morphometry, lung growth, and mechanics remained unchanged. These results suggest that pharmacological activation of soluble guanylate cyclase may provide a new approach to the prenatal treatment of PH associated with CDH.

Increased c-kit and stem cell factor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia

PurposePersistent pulmonary hypertension(PPH) in congenital diaphragmatic hernia (CDH) is caused by increased vascular cell proliferation and endothelial cell (EC) dysfunction, thus leading to obstructive changes in the pulmonary vasculature. C-Kit and its ligand, stem cell factor(SCF), are expressed by ECs in the developing lung mesenchyme, suggesting an important role during lung vascular formation. Conversely, absence of c-Kit expression has been demonstrated in ECs of dysplastic alveolar capillaries. We hypothesized that c-Kit and SCF expression is increased in the pulmonary vasculature of nitrofen-induced CDH. MethodsTimed-pregnant rats received nitrofen or vehicle on gestational day 9(D9). Fetuses were sacrificed on D15, D18, and D21, and divided into control and CDH group. Pulmonary gene expression levels of c-Kit and SCF were analyzed by qRT-PCR. Immunofluorescence double staining for c-Kit and SCF was combined with CD34 to evaluate protein expression in ECs of the pulmonary vasculature. ResultsRelative mRNA levels of c-Kit and SCF were significantly increased in lungs of CDH fetuses on D15, D18, and D21 compared to controls. Confocal laser scanning microscopy confirmed markedly increased vascular c-Kit and SCF expression in mesenchymal ECs of CDH lungs on D15, D18, and D21 compared to controls. ConclusionIncreased expression of c-Kit and SCF in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that increased c-Kit signaling during lung vascular formation may contribute to vascular remodeling and thus to PPH.

Learning Curve for Intrapulmonary Artery Doppler in Fetuses with Congenital Diaphragmatic Hernia

Objective: To assess the learning curve for intrapulmonary artery Doppler in fetuses with congenital diaphragmatic hernia (CDH). Methods: Three fetal medicine fellows with the theoretic knowledge, but without prior experience, in the evaluation of intrapulmonary artery Doppler in CDH fetuses were selected. Each trainee and 1 experienced explorer assessed the intrapulmonary artery in the contralateral lung to the side of the hernia for calculation of 2 Doppler parameters - pulsatility index (PI) and peak early diastolic reversed flow (PEDRF) - in a cohort of 90 consecutive CDH fetuses. The average difference between the 3 trainees and the expert was calculated. A difference below 15% was considered as accurate measurement. The average learning curve was delineated using the cumulative sum analysis (CUSUM). Results: Among the total 270 intrapulmonary artery Doppler measurements performed by the 3 trainees, the number of failed examinations was 14 (15.6%) and 16 (17.8%) for PI and PEDRF, respectively. The CUSUM plots demonstrate that the learning curve was achieved by 53 and 63 tests performed for calculations of the intrapulmonary artery PI and PEDRF, respectively. Conclusion: Competence in Doppler evaluation of the intrapulmonary artery in CDH fetuses is achieved only after intensive continuous training.

Expression of hepatic lipid droplets is decreased in the nitrofen model of congenital diaphragmatic hernia.

Prenatal mortality in newborn infants with congenital diaphragmatic hernia (CDH) has been attributed to increased amounts of liver hernia ion through the diaphragmatic defect. Antenatal studies in human and rodent fetus with CDH further demonstrated a contribution of the developing liver in the pathogenesis of CDH. The abnormal hepatic growth in experimental animal models, therefore, indicates a disruption of normal liver development in CDH. However, the underlying structural, histological and functional changes in the liver of animals with CDH remain unclear. We design this study to test the hypothesis that the morphological and cellular liver development is altered in the nitrogen-induced CDH model. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Livers and chest were harvested on D21 and divided into two groups: control (n=8), nitrofen with CDH (CDH, n=8). Haematoxylin-eosin (Straub et al. Histopathology 68:617-631, 2013) staining was performed to evaluate underlying morphological changes. Apoptosis was checked by using TUNEL staining and apoptotic cell number was counted on 16-16 slides in 25 fields by two independent viewers. Hepatic lipid droplet expressions were evaluated by hepatic adipose differentiation-related protein (ARDP) expression. Compared to controls markedly increased hypertrophy was seen in CDH group. Significantly increased apoptotic cell numbers were detected in CDH group compared to controls (5.1±1.5 vs 2.1±0.6) (p<0.05). The relative mRNA expression levels of ARDP were significantly reduced in CDH group compared to controls. Immunohistochemistry showed markedly decreased hepatic ADRP immunoreactivity in CDH fetuses compared to controls. Our findings provide strong evidence of hepatic hypertrophy and increased cell apoptosis in the liver of nitrofen-induced CDH. These morphological changes may affect liver lipid droplet expression function.

Prophylactic use of the Arabin cervical pessary in fetuses with severe congenital diaphragmatic hernia treated by fetoscopic endoluminal tracheal occlusion (FETO): preliminary experience.

The aim of this study was to describe whether the prophylactic use of a cervical pessary decreases the rate of premature birth in congenital diaphragmatic hernia (CDH) fetuses treated with fetoscopic tracheal occlusion (FETO). The study concerns a consecutive series of cases with CDH and FETO and a group of CDH without FETO. In a subgroup of the FETO group, a prophylactic cervical pessary was inserted the day following the procedure. Gestational age (GA) at birth was the primary outcome. Fifty-nine fetuses with FETO and 47 expectantly managed were included. The last 15 FETO had a cervical pessary inserted. The median GA at delivery in the FETO group with pessary was 35.1 weeks and was not different from that in the FETO group without a pessary (34.3 weeks; p = 0.28) but was below that in the expectantly managed group (38.3 weeks; p < 0.001). Early results suggest that prophylactic use of an Arabin cervical pessary does not prolong gestation of CDH fetuses treated with FETO. © 2015 John Wiley & Sons, Ltd.