Modulating effect of a selective endothelin A receptor antagonist on pulmonary endothelin system protein expression in experimental diaphragmatic hernia

Background/Purpose

Previously, we reported that perinatal administration of atrasentan, a selective endothelin A receptor (ET_A) antagonist, provided a beneficial effect on the cardiopulmonary profile under short-term conditions in newborn lambs with surgically induced congenital diaphragmatic hernia (CDH). We hypothesized that changes in the hemodynamic profile that we observed at birth in treated animals could be influenced by pulmonary modulation of the endothelin (ET) system.

Methods

The effect of atrasentan on protein expression levels of ETs and ET receptors (ET_A and ET_B receptor) was investigated by immunohistochemistry in lung tissues of untreated control (n = 3), treated control (n = 6), untreated CDH (n = 6), and treated CDH newborn lambs (n = 8).

Results

Right lung tissue of treated control lambs showed significantly higher ET_A protein expression levels in both vascular adventitia and airway epithelia when compared with that of untreated control lambs (P < .05). In contrast, protein expression levels of ET_A and ET _B receptor were significantly lower in the vascular smooth muscle cells among other tissue subcompartments of the right lung of treated CDH newborn lambs vs CDH lambs (P < .02 and P = .005, respectively).

Conclusions

We speculate that rapid pulmonary modulation of ET system protein expression levels by atrasentan results from an indirect effect possibly dependent on ventilation and/or perfusion. In CDH groups, this could contribute to the beneficial effect of the treatment.     

Lung function in lambs with diaphragmatic hernia after reversible fetal tracheal occlusion

Background/Purpose

Short-duration resuscitation (≤4 hours) of lambs with diaphragmatic hernia treated in utero with tracheal occlusion have shown improved lung function compared with untreated diaphragmatic hernia. This may be a transient phenomenon in the treated diaphragmatic hernia lambs because of surfactant deficiency. Our objective was to analyze the effect of fetal tracheal occlusion with or without release of the occlusion 1 week before delivery on pulmonary function during a longer period of resuscitation (8 hours) in the diaphragmatic hernia lamb model.

Methods

Four groups were compared: diaphragmatic hernia (n = 5), diaphragmatic hernia and tracheal occlusion until delivery (n = 5), diaphragmatic hernia and tracheal occlusion with release of the occlusion 1 week before delivery (n = 5), and normal controls (n = 4).

Results

Despite persistently decreased surfactant levels, diaphragmatic hernia lambs treated with tracheal occlusion had normal-sized lungs with marked improvement in lung function and gas exchange over 8 hours when compared with untreated lambs with diaphragmatic hernia. Release of the tracheal occlusion 1 week before delivery added no benefit.

Conclusions

It appears that surfactant-independent mechanisms such as pulmonary growth and structural changes are of foremost importance in relating to improved compliance, oxygenation, and ventilation of diaphragmatic hernia lambs treated with tracheal occlusion.     

Nitric oxide activity through guanylate cyclase and phosphodiesterase modulation is impaired in fetal lambs with congenital diaphragmatic hernia

Background

Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension and death. Administration of nitric oxide (NO) alone remains ineffective in CDH cases. We investigated in near full-term lambs with and without CDH the role of guanylate cyclase (GC), the enzyme activated by NO in increasing cyclic 3′-5′-guanylosine monophosphate, and the role of phosphodiesterase (PDE) 5, the enzyme-degrading cyclic 3′-5′-guanylosine monophosphate.

Methods

Congenital diaphragmatic hernia was surgically created in fetal lambs at 85 days of gestation. Pulmonary hemodynamics were assessed by means of pressure and blood flow catheters (135 days). In vitro, we tested drugs on rings of isolated pulmonary vessels.

Results

In vivo, sodium nitroprusside, a direct NO donor, and methyl-2(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032) and Zaprinast, both PDE 5 blockers, reduced pulmonary vascular resistance in CDH and non-CDH animals. The activation of GC by sodium nitroprusside and the inhibition of PDE 5 by T-1032 were less effective in CDH animals. In vitro, the stimulation of GC by 3(5′hydroxymethyl-2′furyl)-1-benzyl indazole (YC-1) (a benzyl indazole derivative) and the inhibition of PDE 5 by T-1032 were less effective in pulmonary vascular rings from CDH animals. The YC-1-induced vasodilation in rings from CDH animals was higher when associated with the PDE 5 inhibitor T-1032.

Conclusions

Guanylate cyclase and PDE 5 play a role in controlling pulmonary vascular tone in fetal lambs with or without CDH. Both enzymes seem to be impaired in fetal lambs with CDH.     

Main bronchus occlusion for treatment of congenital diaphragmatic hernia in fetal lambs

Purpose

The present study investigates a new surgical approach in the treatment of left diaphragmatic hernia in fetal sheep. We postulated that unlike tracheal occlusion where both lungs are occluded, selective left main bronchus occlusion (LMBO) would accelerate growth of only the left lung, reduce left visceral herniation, and recover space in the both thoraces necessary for lung development.

Methods

Left-sided congenital diaphragmatic hernia (CDH) was surgically created in 8 fetal lambs at approximately 65 days of gestation; in 4 of these animals, LMBO was performed at approximately 118 days. Four sham-operated animals served as controls. Lambs were delivered by hysterotomy at 137 days. We measured lung-to-body weight ratios, alveolar surface area, septal wall thickness, and AE2 cell density in the left and right lungs.

Results

Left main bronchus occlusion increased (P < .05) left lung growth causing severe right mediastinal shift but failed to reduce herniated abdominal viscera in 3 of 4 lambs. Wet lung-to-body weight ratio in LMBO group was similar to that of the control group; however, this was achieved by overgrowth of left lung, whereas the right wet lung-to-body weight ratio remained low. In the LMBO group, right lung AE2 cell density was higher than that of control group and not different to that of the CDH group.

Conclusions

Left main bronchus occlusion failed to restore normal pulmonary development in CDH.     

Is a vesicoamniotic shunt intrinsically bad? Shunting a normal fetal bladder

Introduction

We previously demonstrated that in utero vesicoamniotic shunting of obstructive uropathy in fetal lambs produces a shrunken noncompliant bladder. We hypothesized that the normal fetal bladder filling and emptying cycle in fetal life is critical to the development of normal bladder function.

Materials and Methods

We placed vesicoamniotic shunts in 4 normal fetal lambs at 74 days' gestation. The fetuses were delivered at term (145 days), and bladder volume and compliance were measured and compared with those measurements in 3 normal term fetuses. The lambs were then killed and the renal tracts and bladders removed submitted to histologic examination.

Results

All shunted lambs survived to term. Three normal control lambs were delivered at term. The mean bladder volume in shunted lambs was 4 ± 2.8 mL (n = 4) compared with 60 ± 17 mL (n = 3) in control lambs (P < .05). Bladders in the shunted lambs had very poor compliance compared with normal lambs' bladders. Histologic examination of the shunted bladders showed increased fibrosis and distortion of the muscle layers compared with control bladders.

Conclusion

Even in the absence of obstruction, preventing normal bladder filling and emptying in fetal life produces fibrotic bladders with poor compliance.     

Prenatal treatment with retinoic acid promotes pulmonary alveologenesis in the nitrofen model of congenital diaphragmatic hernia

Background/Purpose

Severe pulmonary hypoplasia remains the main cause of the high mortality in newborn infants with congenital diaphragmatic hernia (CDH). Retinoids are a family of molecules derived from vitamin A, which play an important role in lung development. We hypothesized that retinoids promote alveologenesis at the end of gestation and therefore designed this study to investigate the effects of retinoid acid on nitrofen-induced hypoplastic lungs in CDH.

Methods

Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9 of gestation. Retinoic acid 5 mg/kg was given intraperitoneally on days 18, 19, and 20 of gestation and fetuses were recovered on day 21. We had 4 study groups: control (n = 24), control + retinoic acid (n = 22), CDH (n = 24), and CDH + retinoic acid (n = 19). Lungs from the 4 study groups were fixed, and the following stereological measurements were performed on vertical random sections: total lung volume, volume density of airspaces, volume density of air walls, gas exchange surface area, alveolar volume, and total number of alveoli per lung. Total DNA content of each lung was measured using a spectrophotometer.

Results

Total lung volume increased in CDH lungs after the addition of retinoic acid but remained the same in the control group. Gas exchange surface area was larger in CDH lungs after the addition of retinoic acid but remained unchanged in the control group. The total number of alveoli per lung was higher after the addition of retinoic acid. Total DNA content as well as total DNA content-lung weight ratio of the left lung increased significantly in the CDH group after the addition of retinoic acid compared with CDH without retinoic acid.

Conclusions

Our results demonstrate that prenatal treatment with retinoic acid stimulates alveologenesis in hypoplastic lungs in CDH.     

Pressure-limited vesico-amniotic shunt tube for fetal obstructive uropathy

Introduction

In utero shunting (vesico-amniotic shunt) of obstructive uropathy in fetal lambs produces a shrunken, noncompliant bladder. We hypothesized that using a ventriculo-peritoneal shunt for the vesico-amniotic shunt may preserve the filling/emptying cycle and thus normal bladder development.

Materials and Methods

We created obstructive uropathy in 60-day gestation fetal lambs, ligating the urethra and urachus. Vesico-amniotic shunting was performed 21 days later using the valve end of a ventriculo-peritoneal shunt (valve shunt) or silastic tubing (nonvalve shunt). They were delivered at term (145 days), and the bladder volume was measured and compared to normal term fetuses. The lambs were sacrificed, and the kidneys and bladder removed for histology.

Results

Twenty-seven lambs were shunted. Of 14 valve shunts, 8 were effective. Of 13 nonvalve shunts, 11 were effective. The mean bladder volume was 57 ± 41 mL with a valve shunt and 8.8 ± 4.7 mL with a nonvalve shunt (P < .05) (normal term lambs, 65 ± 18 mL, n = 5). Histology of the shunted bladders showed increased fibrosis in the submucosal and muscle layers. This was less obvious in lambs with a valve shunt.

Conclusion

A pressure controlled shunt for fetal obstructive uropathy improves bladder volume but does not prevent bladder wall fibrosis.     

Nitrovasodilator responses in pulmonary arterioles from rats with nitrofen-induced congenital diaphragmatic hernia

Background/Purpose

Many infants with congenital diaphragmatic hernias (CDHs) experience persistent pulmonary hypertension that is refractory to treatment with inhaled nitric oxide (NO). We have examined the responses of isolated pulmonary arterioles from prenatal and postnatal rats with and without nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether)-induced CDH to a variety of activators of the NO-cyclic guanosine monophosphate (cGMP) pathway.

Methods

Right-sided CDH was induced in fetal rats by feeding nitrofen to pregnant rats on day 12 of gestation. Control rats were fed olive oil (vehicle). Third-generation pulmonary arterioles were isolated from the right lung of prenatal rats at term and from newborn rats within 8 hours after birth. Responses to increasing concentrations of sodium nitroprusside (SNP), atrial natriuretic peptide, or 8-bromo-cGMP were measured in pulmonary arterioles from control rats and from rats with nitrofen-induced CDH. Postnatal responses to 8-bromo-cGMP were also recorded in the presence of zaprinast, a type V phosphodiesterase inhibitor.

Results

Pulmonary arterioles from prenatal rats did not dilate in response to SNP, atrial natriuretic peptide, or 8-bromo-cGMP. Vasodilatory responses of postnatal pulmonary arterioles from control rats to SNP and 8-bromo-cGMP were significantly greater than for arterioles from rats with CDH. Zaprinast pretreatment resulted in similar responses for postnatal CDH and control arterioles to 8-bromo-cGMP.

Conclusions

Postnatal pulmonary arterioles from CDH rats exhibit altered nitrovasodilator responsiveness, which may be due to rapid degradation of cGMP.     

Effect of epidermal growth factor on pulmonary hypoplasia in experimental diaphragmatic hernia

Background/purpose

Currently, tracheal occlusion (TO) is a potent stimulus for fetal lung growth but also a rather invasive and high-risk procedure. The aim of this study was to investigate a new and much less invasive therapeutic strategy, namely the maternal intraperitoneal administration of epidermal growth factor (EGF) and its effect on pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) rat model, especially its effect on type II pneumocytes.

Methods

CDH was induced by maternal administration of a single oral dose (100 mg) of nitrofen on day 8.5 of pregnancy. Four groups of pregnant rats were designed on day 18.5: normal control (n = 4), CDH (n = 4), CDH plus Dex (n = 4), CDH plus EGF (n = 8). All fetuses were delivered by cesarean section on day 21. Accordingly, there were 4 groups of fetuses: normal controls (n = 33), nitrofen-induced CDH (n = 19), CDH plus Dex treatment (n = 15), and CDH plus EGF treatment (n = 24). Lung tissue weight (LW) and body weight (BW) of each fetus were recorded, lung histologic and morphometric evaluations were performed, and image analysis was combined after lung processing. Transmission electron microscopy was used for ultrastructural observation, especially type II pneumocytes.

Results

CDH was observed in 58 of the 94 rat fetuses (61.7%). Lw/Bw of CDH group was significantly lower than those of Dex and EGF (P < .05). The lungs of CDH fetuses showed marked hypoplasia, in contrast to improved mesenchymal differentiation in that of Dex and EGF fetuses. Statistical differences of these morphologic parameters (RAC, MTBD, interstitial%, and alveoli%) were found (P < .05). As to ultrastructural features, type II cells of CDH lungs had few if any lamellar bodies and cytoplasmic organelles, and showed evidence of abundant glycogen granules. The sparse type II cells also showed cytoplasmic degenerative changes. By contrast, type II cells of EGF lungs showed numerous mitochondria, abundant lamellar bodies (surfactant) and deficiency of glycogen granules, and displayed prominent microvillous projections and pitlike depressions. The density of type II pneumocyte were 65 ± 4.5, 31 ± 3.1, and 8 ± 1.5 for EGF, Dex, and CDH, respectively (EGF v Dex, P < .05; EGF v CDH, P < 0.01).

Conclusions

Compared with TO, prenatal EGF administration as a much less-invasive therapeutic strategy had shown marked improvement in pulmonary hypoplasia and promotion of type II pneumocyte differentiation in the nitrofen-induced CDH rat model. Thus, EGF could improve the prognosis of CDH by means of promoting pulmonary hypoplasia and improving the surfactant deficiency, which suggested a potential role in the clinical treatment of CDH.     

VEGF expression is downregulated in nitrofen-induced congenital diaphragmatic hernia

Background

Vascular endothelial growth factor (VEGF) is upregulated in pulmonary alveolarization. However, developmental expression of pulmonary VEGF and its possible role in the pathogenesis of CDH are not well described.

Methods

Timed-pregnant VEGF-LacZ mice, possessing a β-galactosidase reporter introduced into the 3′ region of the VEGF gene, were used to examine fetal lung gene expression in a model of nitrofen-induced CDH.

Results

VEGF gene expression increased from embryonic day 13 until its peak at embryonic day 16 and then decreased until term in all groups. This pattern was most apparent in the periphery with smaller differences noted in central lung locations. Expression of VEGF/β-gal in the lungs of nitrofen-treated mice was less than controls at all time-points (P < .0001) The type-II pneumocyte population did not significantly differ between the groups. Study concentrations of nitrofen showed no effect on vascular endothelial proliferation in vitro.

Conclusions

Nitrofen downregulates the production of VEGF during gestation and attenuates the peak seen at the onset of the canalicular stage, despite preservation of type-II pneumocytes. This effect was most pronounced in peripheral lung tissue. The authors speculate that altered VEGF expression may have a pivotal role in the pathogenesis of nitrofen-induced CDH.     

Fetal heart development in the nitrofen-induced CDH rat model: the role of mechanical and nonmechanical factors

Background/purpose

In congenital diaphragmatic hernia (CDH), it was recently shown that early and late gestational lung underdevelopment is caused by nonmechanical and mechanical factors, respectively. Heart underdevelopment, which might predict lung hypoplasia, is commonly attributed to mechanical factors. The authors analyzed whether nonmechanical and mechanical factors affect cardiac growth and correlations between lung and heart weights during gestation.

Methods

Left-sided CDH was induced in pregnant Wistar rats by administration of nitrofen on E9.5. At selected gestational ages (E18, E20, and E22), the lungs and heart were harvested, weighed, and analyzed for DNA and protein contents. Left lung and heart weights were correlated at those gestational ages. Two experimental groups: nitrofen without CDH (nitrofen), and nitrofen with CDH (CDH), were compared with normal controls.

Results

At E18, both nitrofen-exposed groups presented similar and significant left lung (LL) hypoplasia. As gestation progressed (E20 and E22), in the nitrofen group left lung (LL) hypoplasia decreased, whereas in the CDH group LL hypoplasia was exacerbated relative to normal controls. In contrast, at E18 and E20, heart-to-body weight ratios as well as cardiac DNA and protein contents were reduced significantly in all animals exposed to nitrofen, with no significant differences observed between nitrofen and CDH groups. As gestation progressed, the difference between cardiac parameters in nitrofen-exposed and normal control rats diminished, and at E22 no significant differences were documented. In the CDH group, significant correlations were seen between lung and heart weights at E18 (r = 0.65; P < .05) and E20 (r = 0.4; P < .05), whereas at term gestation (E22) no significant correlation was observed (r = 0.21, not significant).

Conclusions

Nonmechanical factors, which might be directed by nitrofen, play a role in the pathogenesis of lung and heart hypoplasia manifested precociously in fetal life, whereas mechanical compression might influence only lung growth during late gestation. Heart weight predicts lung weight only in early gestational ages.     

Administration of prenatal betamethasone suppresses the adrenal-hypophyseal axis in newborns with congenital diaphragmatic hernia

Background

Congenital diaphragmatic hernia (CDH) is a condition that is characterized by pulmonary hypoplasia and pulmonary hypertension. Prenatal betamethasone often is administered to fetuses with CDH to improve pulmonary function. In this study, the authors investigate the possible role of the adrenal-hypophyseal axis in CDH in an animal model and subsequently in human infants with CDH.

Methods

Twin fetal sheep underwent creation of DH or a sham thoracotomy, and levels of plasma and lung ACTH and plasma cortisol were compared. For the human studies, plasma levels of ACTH, cortisol, and DHEA were measured in cord blood samples collected from 9 CDH (5 that received prenatal betamethasone) and compared with those of 14 normal newborns. In both studies, ACTH and cortisol levels were determined by radioimmunoassay (RIA). Human (DHEA) levels were determined by ELISA.

Results

Plasma ACTH and cortisol levels were elevated in fetal DH sheep compared with sham-operated controls; however, levels of ACTH in lung tissues were not different. Human newborns with CDH who have been exposed to prenatal steroids have significantly lower plasma ACTH, cortisol, and DHEA levels than normal newborns and CDH newborns not exposed to prenatal betamethasone.

Conclusions

In an ovine model of CDH, the adrenal-hypophyseal axis appears up-regulated in DH fetuses compared with sham-operated animals. Conversely, the adrenal-hypophyseal axis in human CDH newborns appears normal but is suppressed by the administration of prenatal betamethasone.     

Diaphragmatic repair through fetal tissue engineering: a comparison between mesenchymal amniocyte- and myoblast-based constructs

Purpose

We have previously shown that fetal tissue engineering is a preferred alternative to diaphragmatic repair in a large animal model. This study was aimed at comparing diaphragmatic constructs seeded with mesenchymal amniocytes and fetal myoblasts in this model.

Methods

Neonatal lambs (n = 14) underwent repair of an experimental diaphragmatic defect with identical scaffolds, either seeded with labeled autologous cells (mesenchymal amniocytes in group 1 and fetal myoblasts in group 2) or as an acellular graft (group 3). At 1 to 12 months postoperatively, implants were harvested for multiple analyses.

Results

Repair failure (reherniation or eventration) was significantly higher in group 3 than in groups 1 and 2, with no difference between groups 1 and 2. Seeded fetal myoblasts quickly lost their myogenic phenotype in vivo. All grafts contained cells with a fibroblastic-myofibroblastic profile. Elastin concentrations and both modular and ultimate tensile strengths were significantly higher in group 1 than in groups 2 and 3. There were no differences in glycosaminoglycans and type I collagen levels among the groups.

Conclusions

Diaphragmatic repair with a mesenchymal amniocyte-based engineered tendon leads to improved structural outcomes when compared with equivalent fetal myoblast-based and acellular grafts. The amniotic fluid is a preferred cell source for tissue-engineered diaphragmatic reconstruction.     

Large fetal congenital cystic adenomatoid malformations: growth trends and patient survival

Purpose

The prognosis for fetuses with large congenital cystic adenomatoid malformations (CCAMs) remains uncertain. This study examined the natural history of large fetal CCAMs managed expectantly at a major referral center.

Methods

A 5-year retrospective review was conducted on fetuses diagnosed with a thoracic lesion (n = 59). Large CCAMs were identified on prenatal imaging and followed longitudinally. Perinatal outcomes were assessed.

Results

Twelve (20.3%) fetuses had large CCAMs in the absence of other congenital anomalies. Peak CCAM size occurred at 25.3 ± 3.6 weeks' gestation. Serial magnetic resonance volumetry demonstrated a trend toward decreasing CCAM mass volume relative to thoracic cavity volume over time. Overall, 6 patients, including 3 with signs of early hydrops, showed a marked regression of their lesions relative to estimated fetal weight. Five fetuses required an emergent intervention postnatally, including extracorporeal membrane oxygenation support (n = 1), cyst aspiration (n = 1), and lung resection (n = 5). Overall survival was 75%, with severe hydrops before 30 weeks seen in all 3 deaths.

Conclusion

Large fetal CCAMs tend to peak in size at 25 weeks' gestation and are characterized by in utero diminution relative to overall fetal growth. The prognosis for most fetuses with large CCAMs remains quite favorable under careful perinatal management.     

Abnormal intrinsic esophageal innervation in congenital diaphragmatic hernia: a likely cause of motor dysfunction

Purpose

Patients with congenital diaphragmatic hernia (CDH) often have dilated esophagus and gastroesophageal reflux. Sparse intrinsic esophageal innervation has been described in rats with CDH, but this issue has not been investigated in patients with CDH. The present study tests the hypothesis that innervatory anomalies could account for motor dysfunction in human CDH.

Methods

The esophagi of CDH (n = 6) and control babies dead of other causes (n = 6) were included in paraffin, transversally sectioned, and immunostained with antineurofilament and anti-S-100 antibodies. The proportion of the section surface occupied by neural structures, the ganglionar surface, and the number of neurons per ganglion were measured in 2 to 5 low-power fields from the proximal and distal esophagus with the assistance of image analysis software. Mann-Whitney tests were used for comparing the results using a threshold of significance of P < .05.

Results

The percentage of neural/muscle surface was similar in the upper esophagus in both groups, but it was significantly decreased in the lower esophagus of patients with CDH in comparison with controls. There was a relative scarcity of neural tissue in the intermuscular plexus of the lower esophagus. On the other hand, the ganglionar surface and the number of neurons per ganglion were identical in both groups. These results were similar with both immunostainings.

Conclusion

Intrinsic innervation of the lower esophagus in CDH is abnormal in terms of decreased density of neural structures in the intermuscular plexus. These neural crest-derived anomalies could explain in part the esophageal dysfunction in survivors of CDH.     

Intrapulmonary perfluorooctyl bromide instillation in fetal rabbits

Background

Instilling perfluorooctyl bromide (PFOB) into the fetal lung may lead to alveolar distension.

Objective

The aim of the study was to evaluate the safety of PFOB instillation into fetal lungs and to determine the radiographic distribution and tissue concentration of PFOB in New Zealand white rabbits.

Methods

Sibling fetuses of pregnant (day 27) New Zealand white rabbits were randomized to intratracheal instillation of 1 mL PFOB with tracheal ligation, instillation without ligation, and unmanipulated controls. The maternal animals were killed directly after instillation, at 3 or 6 hours (n = 10 each). For each study cohort, we determined fetal lung/body weight (FLBW) ratios, the radiographic distribution of PFOB, as well as pulmonary PFOB and water content by tissue distillation. PFOB concentrations in maternal and fetal tissues were assessed by gas chromatography.

Results

The relative amount of fetal lung PFOB recovered by fractional distillation was highest in ligated (25%) and lower in unligated lungs (9%). Extrapulmonary PFOB was found in the fetal brain (2.0 ± 0.7 ppm), but not in any other fetal or maternal tissues. Mean FLBW ratios were highest in ligated fetuses, followed by unligated fetuses and controls. PFOB partially displaced fetal lung water. PFOB was visible in the lungs of all treated fetuses. Fetal survival between manipulated and unmanipulated fetuses did not differ.

Conclusions

After prenatal intrapulmonary instillation, some PFOB remains in the lung, even if the trachea is not ligated, and may exert distending pressure on the alveoli.