Pbx1, Meis1, and Runx1 Expression Is Decreased in the Diaphragmatic and Pulmonary Mesenchyme of Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) are thought to originate from mesenchymal defects in pleuroperitoneal folds (PPFs) and primordial lungs. Pre-B-cell leukemia homeobox 1 (Pbx1), its binding partner myeloid ecotropic integration site 1 (Meis1), and runt-related transcription factor 1 (Runx1) are expressed in diaphragmatic and lung mesenchyme, functioning as transcription cofactors that modulate mesenchymal cell proliferation. Furthermore, Pbx1 -/- mice develop diaphragmatic defects and PH similar to human CDH. We hypothesized that diaphragmatic and pulmonary Pbx1, Meis1, and Runx1 expression is decreased in the nitrofen-induced CDH model. MATERIALS AND METHODS: Time-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on D13, D15, and D18, and were divided into control and nitrofen-exposed specimens. Diaphragmatic and pulmonary gene expression levels of Pbx1, Meis1, and Runx1 were analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence-double-staining for Pbx1, Meis1, and Runx1 was combined with mesenchymal/myogenic markers Gata4 and myogenin to evaluate protein expression. RESULTS: Relative mRNA expression of Pbx1, Meis1, and Runx1 was significantly decreased in PPFs (D13), developing diaphragms/lungs (D15), and muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning-microscopy revealed markedly diminished Pbx1, Meis1, and Runx1 immunofluorescence in diaphragmatic and pulmonary mesenchyme, associated with less proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls. CONCLUSION: Decreased Pbx1, Meis1, and Runx1 expression during diaphragmatic development and lung branching morphogenesis may reduce mesenchymal cell proliferation, causing malformed PPFs and disrupted airway branching, thus leading to diaphragmatic defects and PH in the nitrofen-induced CDH model.

The developmental biology of genetic Notch disorders.

Notch signaling regulates a vast array of crucial developmental processes. It is therefore not surprising that mutations in genes encoding Notch receptors or ligands lead to a variety of congenital disorders in humans. For example, loss of function of Notch results in Adams-Oliver syndrome, Alagille syndrome, spondylocostal dysostosis and congenital heart disorders, while Notch gain of function results in Hajdu-Cheney syndrome, serpentine fibula polycystic kidney syndrome, infantile myofibromatosis and lateral meningocele syndrome. Furthermore, structure-abrogating mutations in NOTCH3 result in CADASIL. Here, we discuss these human congenital disorders in the context of known roles for Notch signaling during development. Drawing on recent analyses by the exome aggregation consortium (EXAC) and on recent studies of Notch signaling in model organisms, we further highlight additional Notch receptors or ligands that are likely to be involved in human genetic diseases.

Case Report: Fetal Bilateral Diaphragmatic Agenesis, Ectopic Liver and Abnormal Pancreas.

Congenital bilateral diaphragm agenesis is a very rare condition. We describe limited (abdomen only) autopsy findings of a case of bilateral diaphragm agenesis in a 27-week male fetus with unusual findings of fibrosis of the pancreatic head and ectopic liver nodules in a mass at the upper abdomen that may represent a possible diaphragm anlage. We have correlated our observations with data from experimental and embryological studies to suggest possible mechanisms for the malformations that were present and their implications for our understanding of pancreas, liver and diaphragm development in the human fetus.

Antenatal maternally-administered phosphodiesterase type 5 inhibitors normalize eNOS expression in the fetal lamb model of congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypertension (pHTN), a main determinant of survival in congenital diaphragmatic hernia (CDH), results from in utero vascular remodeling. Phosphodiesterase type 5 (PDE5) inhibitors have never been used antenatally to treat pHTN. The purpose of this study is to determine if antenatal PDE5 inhibitors can prevent pHTN in the fetal lamb model of CDH. METHODS: CDH was created in pregnant ewes. Postoperatively, pregnant ewes received oral placebo or tadalafil, a PDE5 inhibitor, until delivery. Near term gestation, lambs underwent resuscitations, and lung tissue was snap frozen for protein analysis. RESULTS: Mean cGMP levels were 0.53±0.11 in placebo-treated fetal lambs and 1.73±0.21 in tadalafil-treated fetal lambs (p=0.002). Normalized expression of eNOS was 82%±12% in Normal-Placebo, 61%±5% in CDH-Placebo, 116%±6% in Normal-Tadalafil, and 86%±8% in CDH-Tadalafil lambs. Normalized expression of ß-sGC was 105%±15% in Normal-Placebo, 82%±3% in CDH-Placebo, 158%±16% in Normal-Tadalafil, and 86%±8% in CDH-Tadalafil lambs. Endothelial NOS and ß-sGC were significantly decreased in CDH (p=0.0007 and 0.01 for eNOS and ß-sGC, respectively), and tadalafil significantly increased eNOS expression (p=0.0002). CONCLUSIONS: PDE5 inhibitors can cross the placental barrier. ß-sGC and eNOS are downregulated in fetal lambs with CDH. Antenatal PDE5 inhibitors normalize eNOS and may prevent in utero vascular remodeling in CDH.

Repetitive MR measurements of lung volume in fetuses with congenital diaphragmatic hernia: individual development of pulmonary hypoplasia during pregnancy and calculation of weekly lung growth rates.

OBJECTIVE: To investigate individual changes in fetal lung volume (FLV) in fetuses with isolated congenital diaphragmatic hernia (CDH) and to calculate weekly growth rates of the FLV using serial MR examinations during pregnancy. METHODS: MR-FLV was measured in 89 fetuses with CDH. All fetuses received two MRIs. A mean weekly growth rate of the FLV was determined for each fetus and compared with the growth rate of healthy fetuses. RESULTS: Mean observed-to-expected MR-FLV (o/e MR-FLV) measured at the first MRI was 33.3 ± 12.2% and 29.5 ± 10.9% at the second MRI. In 61% of all fetuses (54/89) the o/e MR-FLV decreased during pregnancy, 26% (23/89) showed an increase in the o/e MR-FLV and 13 % (12/89) had stable values. First and last o/e MR-FLV values were significantly associated with mortality and neonatal extracorporeal membrane oxygenation (ECMO) requirement with a higher prognostic accuracy of MR-FLV measurements near delivery. Patients with CDH had lower weekly lung growth rates than healthy fetuses. There was a significant difference in the mean weekly growth rate between survivors and non-survivors and patients with and without ECMO requirement. CONCLUSION: Individual development of FLV in patients with CDH during pregnancy is extremely variable. Follow-up MR-FLV measurements are advisable before deciding upon pre- and postnatal therapeutic options. KEY POINTS: • Lung development in congenital diaphragmatic hernia (CDH) during pregnancy is extremely variable. • MRI demonstrates that lung growth rate is reduced in fetuses with CDH. • The final observed-to-expected fetal lung volume provides the best prognostic information. • Follow-up measurements are advisable before deciding upon therapeutic options.

Pulmonary FGF9 gene expression is downregulated during the pseudoglandular stage in nitrofen-induced hypoplastic lungs.

INTRODUCTION: The pathogenesis of pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) remains unclear. Fibroblast growth factor 9 (FGF9) is an essential component of the gene network that regulates lung development. FGF9 knockouts exhibit disrupted mesenchymal proliferation and reduced airway branching. The authors hypothesized that pulmonary FGF9 gene expression is downregulated during the pseudoglandular stage in nitrofen-induced hypoplastic lungs. MATERIAL AND METHODS: Pregnant rats received either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were dissected on D15 and D18, and were divided into controls, hypoplastic lungs with CDH (CDH+) and hypoplastic lungs without CDH (CDH-). Pulmonary FGF9 gene expression levels were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed to investigate FGF9 protein expression/distribution. RESULTS: Relative messenger RNA levels of FGF9 were significantly decreased on D15 in hypoplastic lungs compared with controls (p < 0.01), and on D18 in CDH+ and CDH- compared with controls (p< 0.05, respectively). Immunoreactivity of FGF9 was markedly diminished in mesothelium and distal airway epithelium on D15 and decreased in overall intensity on D18 in hypoplastic lungs compared with controls. CONCLUSIONS: Downregulation of FGF9 gene expression during the pseudoglandular stage may cause pulmonary hypoplasia in the nitrofen model by decreasing distal airway epithelial and mesenchymal proliferation throughout the branching morphogenesis.

Prenatal administration of retinoic acid increases the trophoblastic insulin-like growth factor 2 protein expression in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: The high mortality rate in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH). Insulin-like growth factor 2 (IGF2) is an important regulator of fetal growth. The highest levels of IGF2 expression are found in the placenta, which are negatively regulated by decidual retinoid acid receptor alpha (RARα). It has been demonstrated that prenatal administration of retinoic acid (RA) suppresses decidual RARα expression. Previous studies have further shown that prenatal administration of RA can reverse PH in nitrofen-induced CDH model. In IGF2 knockout animals, low levels of IGF2 are associated with decreased placental growth and PH. We therefore hypothesized that nitrofen decreases trophoblastic IGF2 expression and prenatal administration of RA increases it through decidual RARα in the nitrofen-induced CDH model. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). RA was given intraperitoneally on D18, D19 and D20. Fetuses were harvested on D21 and divided into three groups: control, CDH and nitrofen+RA. Immunohistochemistry was performed to evaluate decidual RARα and trophoblastic IGF2 expression. Protein levels of IGF2 in serum, intra-amniotic fluid and left lungs were measured by enzyme-linked immunosorbent assay. RESULTS: Significant growth retardation of placenta and left lungs was observed in the CDH group compared to control and nitrofen+RA group. Markedly increased decidual RARα and decreased IGF2 immunoreactivity were found in the CDH group compared to control and nitrofen+RA group. Significantly decreased IGF2 protein levels were detected in serum, intra-amniotic fluid and left lungs in the CDH group compared to control and nitrofen+RA group. CONCLUSION: Our findings suggest that nitrofen may disturb trophoblastic IGF2 expression through decidual RARα resulting in retarded placental growth and PH in the nitrofen-induced CDH. Prenatal administration of RA may promote lung and placental growth by increasing trophoblastic IGF2 expression.

Disruption of THY-1 signaling in alveolar lipofibroblasts in experimentally induced congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypoplasia (PH), characterized by alveolar immaturity, remains the main cause of neonatal mortality and long-term morbidity in infants with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) are critically important for normal alveolar development. Thymocyte antigen 1 (Thy-1) is a highly expressed cell-surface protein in this specific subset of lung fibroblasts, which plays a key role in fetal alveolarization by coordinating the differentiation and lipid homeostasis of alveolar LIFs. Thy-1 increases the lipid content of LIFs by upregulation of adipocyte differentiation-related protein (ADRP), a lipogenic molecular marker characterizing pulmonary LIFs. Thy-1 (-/-) mice further show impaired alveolar development with reduced proliferation of pulmonary LIFs, resulting in a PH-similar phenotype. We hypothesized that pulmonary Thy-1 signaling is disrupted in experimentally induced CDH, which may has an adverse effect on the lipid content of alveolar LIFs. METHODS: Timed-pregnant Sprague-Dawley rats were treated with either 100 mg nitrofen or vehicle on embryonic day 9.5 (E9.5). Fetuses were killed on E21.5, and lungs were divided into controls (n = 14) and CDH-associated PH (n = 14). Pulmonary gene expression levels of Thy-1 and ADRP were assessed by quantitative real-time PCR. ADRP immunohistochemistry and oil-red-O staining were used to localize alveolar LIF expression and lipid droplets. Immunofluorescence double staining for Thy-1 and oil-red-O was performed to evaluate Thy-1 expression and lipid content in alveolar LIFs. RESULTS: Radial alveolar count was significantly reduced in CDH-associated PH with significant downregulation of pulmonary Thy-1 and ADRP mRNA expression compared to controls. ADRP immunoreactivity and lipid droplets were markedly diminished in alveolar interstitial cells, which coincided with decreased alveolar LIF expression in CDH-associated PH compared to controls. Confocal laser scanning microscopy confirmed markedly decreased Thy-1 expression and lipid content in alveolar LIFs of CDH-associated PH compared to controls. CONCLUSION: Our study provides strong evidence that disruption of pulmonary Thy-1 signaling results in reduced lipid droplets in alveolar LIFs and may thus contribute to PH in the nitrofen-induced CDH model. Treatment modalities aimed at increasing lipid content in alveolar LIFs may therefore have a therapeutic potential in attenuating CDH-associated PH.

Spatiotemporal alterations in Sprouty-2 expression and tyrosine phosphorylation in nitrofen-induced pulmonary hypoplasia.

BACKGROUND/PURPOSE: Pulmonary hypoplasia (PH) is a life-threatening condition of newborns presenting with congenital diaphragmatic hernia (CDH). Sprouty-2 functions as a key regulator of fibroblast growth factor receptor (FGFR) signalling in developing foetal lungs. It has been reported that FGFR-mediated alveolarization is disrupted in nitrofen-induced PH. Sprouty-2 knockouts show severe defects in lung morphogenesis similar to nitrofen-induced PH. Upon FGFR stimulation, Sprouty-2 is tyrosine-phosphorylated, which is essential for its physiological function during foetal lung development. We hypothesized that Sprouty-2 expression and tyrosine phosphorylation are altered in nitrofen-induced PH. METHODS: Time-pregnant rats received either nitrofen or vehicle on gestation day 9 (D9). Foetal lungs were dissected on D18 and D21. Pulmonary Sprouty-2 gene and protein expression levels were analyzed by qRT-PCR, Western blotting and immunohistochemical staining. RESULTS: Relative mRNA expression of Sprouty-2 was significantly decreased in hypoplastic lungs without CDH (0.1050±0.01 vs. 0.3125±0.01; P<.0001) and with CDH (0.1671±0.01 vs. 0.3125±0.01; P<.0001) compared to controls on D18. Protein levels of Sprouty-2 were markedly decreased in hypoplastic lungs on D18 with decreased tyrosine phosphorylation levels on D18 and D21 detected at the molecular weight of Sprouty-2 consistent with Sprouty-2 tyrosine phosphorylation. Sprouty-2 immunoreactivity was markedly decreased in hypoplastic lungs on D18 and D21. CONCLUSION: Spatiotemporal alterations in pulmonary Sprouty-2 expression and tyrosine phosphorylation during the late stages of foetal lung development may interfere with FGFR-mediated alveolarization in nitrofen-induced PH.

Outcome of CDH infants following fetoscopic tracheal occlusion - influence of premature delivery.

PURPOSE: To evaluate the mortality and morbidity of infants with congenital diaphragmatic hernia who had undergone fetal endoscopic tracheal occlusion (FETO) and whether this was influenced by premature birth. METHODS: The gestational age at delivery, lung-head ratio (LHR) pre and post FETO, neonatal outcomes, and respiratory, gastro-intestinal, neurological, surgical, and musculoskeletal problems at follow up of consecutive infants who had undergone FETO were determined. Elective reversal of FETO was planned at 34 weeks of gestation. RESULTS: The survival rate of the 61 FETO infants was 48%, with 84% delivered prematurely. Thirty-one delivered <35 weeks of gestation. Their survival rate was 18%. Twenty-three of 24 infants who had emergency balloon removal were born <35 weeks of gestation. Survival was related to gestational age at delivery (OR 0.55, 95% CI 0.420, 0.77, p<0.001) and the duration of FETO (OR 0.73, 95% CI 0.59, 0.91, p<0.005). Infants born prior to 35 weeks of gestation compared to those born at ≥ 35 weeks required a longer duration of ventilation (median 45 days versus 12 days, p<0.001), and a greater proportion had surgery for gastro-oesophageal reflux (50% versus 9%, p=0.011). CONCLUSION: These results emphasize the need to reduce premature delivery following FETO.

The presence of a hernia sac in congenital diaphragmatic hernia is associated with better fetal lung growth and outcomes.

PURPOSE: The purpose of this study was to evaluate the relationship between the presence of a hernia sac and fetal lung growth and outcomes in infants with Congenital, Diaphragmatic Hernia (CDH). METHODS: The medical records of all neonates with CDH treated in our institution between 2004 and 2011 were reviewed. The presence of a hernia sac was confirmed at the time of surgical repair or at autopsy. Data were analyzed using parametric and non-parametric tests where appropriate. Multivariable regression and survival analyses were applied. RESULTS: Of 148 neonates treated for CDH, 107 (72%) had isolated CDH and 30 (20%) had a hernia sac. Infants with a hernia sac had significantly lower need for ECMO, patch repair, supplemental oxygen at 30 days of life, and shorter duration of mechanical ventilation and hospital stay. Ninety-three patients had prenatal imaging. The mean observed-to-expected total fetal lung volume in the sac group was higher throughout gestation. Although a greater percentage of sac patients had liver herniation as a dichotomous variable, the amount of herniated liver (%LH and LiTR) was significantly lower in the presence of a hernia sac. CONCLUSION: The presence of a hernia sac in Congenital Diaphragmatic Hernia is associated with less visceral herniation, greater fetal lung growth, and better post-natal outcomes.

Aberrant pulmonary lymphatic development in the nitrofen mouse model of congenital diaphragmatic hernia.

PURPOSE: Many infants develop a postsurgical chylothorax after diaphragmatic hernia repair. The pathogenesis remains elusive but may be owing to dysfunctional lymphatic development. This study characterizes pulmonary lymphatic development in the nitrofen mouse model of CDH. METHODS: CD1 pregnant mice were fed nitrofen/bisdiamine (N/B) or olive oil at E8.5. At E14.5 and E15.5, lung buds were categorized by phenotype: normal, N/B without CDH (N/B - CDH), or N/B with CDH (N/B+CDH). Anti-CD31 was used to localize all endothelial cells, while anti-LYVE-1 was used to identify lymphatic endothelial cells in lung buds using immunofluorescence. Differential protein expression of lymphatic-specific markers was analyzed. RESULTS: Lymphatic endothelial cells localized to the mesenchyme surrounding the airway epithelium at E15.5. CD31 and LYVE-1 colocalization identified lymphatic endothelial cells. LYVE-1 expression was upregulated in N/B+CDH lung buds in comparison to N/B - CDH and normal lung buds by immunofluorescence. Western blotting shows that VEGF-D, LYVE-1, Prox-1, and VEGFR-3 expression was upregulated in N/B+CDH lung buds in comparison to N/B - CDH or control lung buds at E14.5. CONCLUSIONS: Lung lymphatics are hyperplastic in N/B+CDH. Upregulation of lymphatic-specific genes suggests that lymphatic hyperplasia plays an important role in dysfunctional lung lymphatic development in the nitrofen mouse model of CDH.

Preferential streaming of the ductus venosus toward the right atrium is associated with a worse outcome despite a higher rate of invasive procedures in human fetuses with left diaphragmatic hernia.

PURPOSE: Preferential streaming of the ductus venosus (DV) toward the right atrium has been observed in fetuses with left diaphragmatic hernia (LDH). The purpose of this retrospective study was to compare survival rates to discharge between a group with preferential streaming of the DV toward the right heart and a group in which this abnormal flow pattern was not present. MATERIALS AND METHODS: We retrospectively searched our patient records for fetuses with LDH in whom liver position, DV streaming and postnatal outcome information was available. 55 cases were found and divided into two groups: Group I fetuses exhibited abnormal DV streaming toward the right side of the heart; group II fetuses did not. Various prognostic and outcome parameters were compared. RESULTS: 62 % of group I fetuses and 88 % of group II fetuses survived to discharge (p = 0.032). Fetoscopic tracheal balloon occlusion (FETO) was performed in 66 % of group I fetuses and 23 % of group II fetuses (p = 0.003). Postnatal ECMO therapy was performed in 55 % of group I fetuses and 23 % of group II infants (p = 0.025). Moderate to severe chronic lung disease in survivors was observed in 56 % of the survivors of group I and 9 % of the survivors of group II (p = 0.002). CONCLUSION: Preferential streaming of the DV toward the right heart in human fetuses with left-sided diaphragmatic hernia was associated with a poorer postnatal outcome despite a higher rate of invasive pre- and postnatal procedures compared to fetuses without this flow abnormality. Specifically, abnormal DV streaming was found to be an independent predictor for FETO.

Kif7 is required for the patterning and differentiation of the diaphragm in a model of syndromic congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a common birth defect that results in a high degree of neonatal morbidity and mortality, but its pathological mechanisms are largely unknown. Therefore, we performed a forward genetic screen in mice to identify unique genes, models, and mechanisms of abnormal diaphragm development. We identified a mutant allele of kinesin family member 7 (Kif7), the disorganized diaphragm (dd). Embryos homozygous for the dd allele possess communicating diaphragmatic hernias, central tendon patterning defects, and increased cell proliferation with diaphragmatic tissue hyperplasia. Because the patterning of the central tendon is undescribed, we analyzed the expression of genes regulating tendonogenesis in dd/dd mutant embryos, and we determined that retinoic acid (RA) signaling was misregulautted. To further investigate the role of Kif7 and RA signaling in the development of the embryonic diaphragm, we established primary mesenchymal cultures of WT embryonic day 13.5 diaphragmatic cells. We determined that RA signaling is necessary for the expression of tendon markers as well as the expression of other CDH-associated genes. Knockdown of Kif7, and retinoic acid receptors alpha (Rara), beta (Rarb), and gamma (Rarg) indicated that RA signaling is dependent on these genes to promote tendonogenesis within the embryonic diaphragm. Taken together, our results provide evidence for a model in which inhibition of RA receptor signaling promotes CDH pathogenesis through a complex gene network.

Use of ultrasound and MRI for evaluation of lung volumes in fetuses with isolated left congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is an anomaly that results in lung hypoplasia and pulmonary hypertension. The lungs of the CDH fetus have an abnormal architecture, with fewer bronchial branches and decreased number of arteries and veins, factors which result in pulmonary compromise postnatally. The goal of this review is to evaluate prenatal prognostic factors in the fetus with isolated left CDH, with particular emphasis on fetal MRI. These imaging indicators may be used to provide health professionals and the parents with the most accurate information about fetal prognosis.

An evidence-based review of the current treatment of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia is a rare but severe condition affecting 1 in 2000 to 3000 newborns with a survival rate of 67%. Although regular antenatal screening allows prenatal diagnosis in many cases, traditionally treatment has been based on postnatal surgical repair. Recent literature has pointed out the survival benefits of initial stabilization and the use of gentle ventilation strategies prior to definitive treatment, shifting the trend from immediate to delayed surgical repair. Advances in fetal intervention have allowed the introduction of fetal endoscopic tracheal occlusion as a method to hasten lung development before birth in order to minimize postnatal morbidity. Despite appropriate treatment, the long-term outcomes of these patients are plagued with numerous complications, associated with the primary pathology and also aggressive therapeutic measures. International centers of excellence have recently come together in an effort to standardize the care of such patients in hopes of maximizing their outcomes.

Quantitative lung index, contralateral lung area, or lung-to-head ratio to predict the neonatal outcome in isolated congenital diaphragmatic hernia?

OBJECTIVES: To estimate the accuracy of the quantitative lung index and contralateral lung area for prediction of the neonatal outcome in isolated congenital diaphragmatic hernia in comparison to other available prediction models. METHODS: Between January 2004 and December 2010, 108 fetuses with isolated (82 left-sided and 26 right-sided) congenital diaphragmatic hernia were prospectively evaluated. The quantitative lung index and observed-to-expected contralateral lung area were measured and compared to the neonatal survival rate and severe postnatal pulmonary arterial hypertension, along with the lung-to-head ratio, observed-to-expected lung-to-head ratio, and observed-to-expected total lung volume. RESULTS: Overall neonatal mortality was 64.8% (70 of 108). Severe pulmonary arterial hypertension was diagnosed in 68 (63.0%) of the cases, which was associated with neonatal death (P < .001). Both the quantitative lung index and observed-to-expected contralateral lung area were significantly associated with neonatal survival and pulmonary arterial hypertension (P < .001), with accuracy to predict survival of 70.9% and 70.0%, respectively, and accuracy to predict hypertension of 78.7% and 72.0%; however, they were both less accurate than the observed-to-expected total lung volume (83.3% and 86.1%; P < .01). The lung-to-head ratio (73.1% and 78.7%) and observed-to-expected lung-to-head ratio (75.9% and 72.2%; P > .05) had similar accuracy as the quantitative lung index and observed-to-expected contralateral lung area. CONCLUSIONS: The observed-to-expected total lung volume is the most accurate predictor of the neonatal outcome in cases of isolated congenital diaphragmatic hernia. Both the quantitative lung index and observed-to-expected contralateral lung area, albeit reasonably accurate, do not produce the same level of accuracy and render similar results as the lung-to-head ratio and observed-to-expected lung-to-head ratio.

Predictors of neonatal morbidity in fetuses with severe isolated congenital diaphragmatic hernia undergoing fetoscopic tracheal occlusion.

OBJECTIVES: To investigate neonatal morbidity in fetuses with severe congenital diaphragmatic hernia (CDH) treated with fetoscopic endoluminal tracheal occlusion (FETO) and compare it with historical controls with less severe forms of CDH that were managed expectantly. METHODS: This was a prospective, multicenter study on neonatal outcomes and prenatal predictors in 90 FETO survivors (78 left-sided, 12 right) and 41 controls from the antenatal CDH registry with either severe or moderate hypoplasia who were managed expectantly. We also investigated early neonatal morbidity indicators, including the need for patch repair, duration of mechanical ventilation and supplemental oxygen, age at full enteral feeding and incidence of pulmonary hypertension. RESULTS: Gestational age at delivery was predictive of duration of assisted ventilation (P = 0.046), days on supplemental oxygen (P = 0.019) and age at full enteral feeding (P = 0.020). When delivery took place after 34 weeks' gestation, neonatal morbidity of FETO cases was comparable with that of expectantly managed cases with moderate hypoplasia. CONCLUSIONS: Fetal intervention for severe CDH is associated with neonatal morbidity that is comparable with that of an expectantly managed group with less severe disease.

Effect of nitrofen in the final stages of development of the diaphragm muscle in rats.

PURPOSE: To evaluate the expression of myosin in muscle fibers of the diaphragm in experimental congenital diaphragmatic hernia (CDH). METHODS: Fetuses of pregnant rats were divided into four groups: External Control (EC), composed of non-manipulated rats; Nitrofen, composed of pregnant rats that received 100 mg of nitrofen (2,4-dichloro-4'nitrodiphenyl ether) diluted in olive oil on gestational day (GD) 9.5, whose fetuses developed CDH (N+) or not (N-), and Olive Oil Placebo (OO), composed of pregnant rats that received the oil on the same GD. The fetuses were collected on GD 18.5, 19.5, 20.5 and 21.5 (term = 22 days). We obtained body weight (BW) and photographed the diaphragm area (DA), hernia area (HA) and subsequent calculated the HA/DA ratio in N+ group. Samples of Diaphragm muscle were processed for histological staining with H/E and immunohistochemistry (IHQ) for myosin. RESULTS: The fetuses of N- and N+ groups had decreased BW and DA compared to EC and OO groups (p < 0.001). HA was decreased on GD 18.5 compared to 21.5 (p < 0.001) and the HA/DA ratio showed no difference. IHQ showed decreased expression of myosin in nitrofen groups. CONCLUSION: CDH induced by nitrofen model contributes to the understanding of muscularization in the formation of the diaphragm where the myosin expression is decreased.

Neuromuscular blockade and lung function during resuscitation of infants with congenital diaphragmatic hernia.

BACKGROUND: There is no consensus or evidence as to whether a neuromuscular blocking agent should be used during the initial resuscitation of infants with congenital diaphragmatic hernia (CDH) in the labour ward. OBJECTIVE: To determine if administration of a neuromuscular blocking agent affected the lung function of infants with CDH during their initial resuscitation in the labour ward. METHODS: Fifteen infants with CDH were studied (median gestational age 38 weeks, range 34-41; birth weight 2,790 g, range 1,780-3,976). Six infants had undergone feto-endotracheal occlusion (FETO). Flow, airway pressure, tidal volume and dynamic lung compliance changes were recorded using a respiratory function monitor (NM3, Respironics). Twenty inflations immediately before, immediately after and 5 min after administration of a neuromuscular blocking agent (pancuronium bromide) were analysed. RESULTS: The median dynamic lung compliance of the 15 infants was 0.22 ml/cm H2O/kg (range 0.1-0.4) before and 0.16 ml/cm H2O/kg (range 0.1-0.3) immediately after pancuronium bromide administration (p < 0.001) and remained at a similar low level 5 min after pancuronium bromide administration. The FETO compared to the non-FETO infants had a lower median dynamic compliance both before (p < 0.0001) and 5 min after pancuronium administration (p < 0.001) and required significantly longer durations of ventilation (p = 0.004), supplementary oxygen (p = 0.003) and hospitalisation (p = 0.007). CONCLUSIONS: Infants with CDH, particularly those who have undergone FETO, have a low lung compliance at birth, and this is further reduced by administration of a neuromuscular blocking agent.