AB and JB designed the study. OB, AB, BCH, MLFM, and JB performed the experiments. CE performed the statistical analyses. JM coordinated the autopsy organization and the collection of autopsy materials. OB, AB, BCH, CE, and JB contributed to writing the paper.
The authors have declared that no competing interests exist.
Pulmonary hypoplasia and persistent pulmonary hypertension account for significant mortality and morbidity in neonates with congenital diaphragmatic hernia (CDH). Global lung immaturity and studies in animal models suggest the presence of surfactant deficiency that may further complicate the pathophysiology of CDH. However, data about surfactant status in human fetuses with CDH at birth are contradictory. The lack of a chronological study of surfactant content in late pregnancy has been a significant limitation. The appropriateness of administering surfactant supplements to neonates with CDH is therefore a debated question.
We investigated surfactant content in human fetuses with CDH compared to age-matched fetuses with nonpulmonary diseases used as controls. Concentrations of disaturated phosphatidylcholine and surfactant proteins were found to be similar at a given stage of pregnancy, with both components showing a similar pattern of increase with progressing pregnancy in fetuses with CDH and in control fetuses. Thyroid transcription factor 1, a critical regulator of surfactant protein transcription, similarly displayed no difference in abundance. Finally, we examined the expression of three glucocorticoid-regulated diffusible mediators involved in lung epithelial maturation, namely: keratinocyte growth factor (KGF), leptin, and neuregulin 1 beta 1 (NRG1-β1). KGF expression decreased slightly with time in control fetuses, but remained unchanged in fetuses with CDH. Leptin and NRG1-β1 similarly increased in late pregnancy in control and CDH lungs. These maturation factors were also determined in the sheep fetus with surgical diaphragmatic hernia, in which surfactant deficiency has been reported previously. In contrast to the findings in humans, surgical diaphragmatic hernia in the sheep fetus was associated with decreased KGF and neuregulin expression. Fetoscopic endoluminal tracheal occlusion performed in the sheep model to correct lung hypoplasia increased leptin expression, partially restored KGF expression, and fully restored neuregulin expression.
Our results indicate that CDH does not impair surfactant storage in human fetuses. CDH lungs exhibited no trend toward a decrease in contents, or a delay in developmental changes for any of the studied surfactant components and surfactant maturation factors. Surfactant amounts are likely to be appropriate to lung size. These findings therefore do not support the use of surfactant therapy for infants with CDH. Moreover, they raise the question of the relevance of CDH animal models to explore lung biochemical maturity.
In an autopsy study of human fetuses, Jacques Bourbon and colleagues report that pulmonary surfactant content is not decreased in congenital diaphragmatic hernia.
Congenital diaphragmatic hernia (CDH), a fetal malformation in which the abdominal organs are displaced into the chest cavity, occurs in approximately one out of 3,000 live births and accounts for approximately 8% of major birth defects. The displaced lungs tend to be underdeveloped at birth, and decreased lung function is a major cause of sickness and death in affected babies.
Pulmonary surfactant, a substance naturally produced by cells in the lungs in the weeks before birth, is necessary for normal breathing. Surfactant acts to keep the walls of the lung's airspaces from collapsing onto each other, much as detergent can keep the walls of a moist plastic bag from sticking together.
The specific aspects of lung immaturity that cause decreased function in babies with CDH are not fully understood. In particular, it has been unclear whether abnormally low levels of pulmonary surfactant contribute to the lung problems caused by CDH. Some studies in sheep or rat models of CDH have found that surfactant levels are abnormally low, while others have found normal or even increased levels of surfactant. Studies in human CDH have also shown controversial results, and few have investigated surfactant production in lung tissue itself.
Doctors can artificially increase babies' pulmonary surfactant levels if necessary through the instillation of exogenous surfactant material, but doing so involves some risk. This is a routine treatment with demonstrated benefit in premature neonates with normal, although not fully developed, lungs. Pulmonary surfactant is sometimes given also to babies with CDH, but in this particular instance, the benefit of this practice is unclear. The authors of this study wanted to determine whether or not babies with CDH really have low levels of pulmonary surfactant.
The researchers performed autopsies to study lung tissue from human fetuses in pregnancies that ended in fetal death, were terminated for medical reasons, or resulted in death immediately after birth. They compared surfactant production in 16 fetuses with CDH to that in 33 fetuses with conditions not involving the lungs. They found that, taking fetal age into account, surfactant was present at similar levels in lung tissue from both groups, and that production of surfactant components and other factors involved in lung maturation was not delayed in CDH.
The researchers also studied sheep fetuses with surgically induced CDH, a model that has been used in the past to study lung development in CDH. In contrast with the findings in humans, they found that factors stimulating surfactant production were decreased in sheep with surgically induced CDH.
These findings suggest that surfactant deficiency is not a major contributor to lung problems in babies with CDH, and that further clinical research may be appropriate to determine whether administering surfactant to these babies is beneficial. The findings also suggest that commonly used animal models of CDH may be of limited relevance to human CDH.
Please access these Web sites via the online version of this summary at
Read the
Information from the MedlinePlus Encyclopedia (US National Library of Medicine) on
Article from eMedicine on
Wikipedia entry on
Congenital diaphragmatic hernia (CDH) is a developmental abnormality that affects one in 2,500–5,000 live births, depending on the studies, and accounts for 8% of all major congenital anomalies. CDH restricts fetal lung development. The resulting lung hypoplasia and hypertension have dramatic consequences at birth, and the disease continues to cause high rates of mortality and morbidity despite recent progress in neonatal care [
Pulmonary surfactant, which is a lipid–protein complex produced by alveolar type II (ATII) cells, prevents alveolar collapse during the breathing cycle by reducing surface tension at the air–liquid interface [
The prevailing opinion that the CDH lung is surfactant deficient is principally based on the immature morphological aspect of the parenchyma and on reports of diminished surfactant content in animal models of CDH. The surfactant system has been repeatedly reported to be deficient in two classical models of CDH, the surgically created diaphragmatic hernia (sDH) in the fetal sheep [
Controversy persists regarding whether or not human infants with CDH are surfactant deficient. Whereas some studies concluded that surfactant components failed to increase in amniotic fluid [
Clarifying this question is important insofar as decisions have to be made as to whether or not infants with CDH should be supplemented with surfactant at birth [
Postmortem lung tissue samples were obtained at autopsy after medical termination of pregnancy or neonatal death, with signed, informed consent from the parents. Terminations were performed according to the July 1994 French legislation, and the study was undertaken with the agreement of the institutional Ethical Committee. The prenatal diagnosis of CDH was made by echography, and was confirmed by postmortem examination. Reduced lung weight and consistent histological appearance confirmed the diagnosis of pulmonary hypoplasia (lung to body weight ratio < 0.015 before 28 wk of pregnancy, and < 0.012 thereafter [
Characteristics of CDH Fetuses
Characteristics of Fetuses with Nonpulmonary Diseases Used as Controls
All animal experiments were performed with the authorization of the French Ministry of Agriculture. Surgical procedures have been extensively described elsewhere [
The technique has been described in detail previously [
DNA was determined on residual pellet from delipidated homogenates by the colorimetric diphenylamine method [
Human lung tissues were homogenized in RIPA buffer containing protease inhibitors (Roche Diagnostics,
KGF and leptin concentrations in human fetal lung tissue were assessed with commercially sensitive and specific ELISA (R&D Systems), following the manufacturer's instructions, and normalized to total proteins. The intra-assay coefficient of variance was less than 5%.
Total RNA was extracted from fetal sheep lung tissue using Trizol reagent (Invitrogen,
cDNAs were reverse-transcribed from sheep lung total RNAs with Superscript II reverse transcriptase and random hexamer primers (Invitrogen) as above. Amplification of the partial cDNA sequence for NRG was performed using sense primer 5′-TCAGAACTTCGCATTAGCAAAGC-3′ (bovine NRG–specific sequence) and antisense primer 5′-GGGAGTGGACGTACTGTAGAAGCT-3′ (bovine/human NRG–specific sequence). Amplification was performed through 35 cycles (1 min at 94 °C, 1 min at 59 °C, and 1 min at 72 °C). The amplified sequence was purified with QIAquick PCR purification kit (Qiagen,
A 20-μl mix, containing 12.5 μl of Powerful SYBR Green PCR Master Mix (Applied Biosystems), 900 nM forward primer, and 900 nM reverse primer, was prepared for performing real-time PCR. Primers were designed to be intron-spanning to avoid co-amplification of genomic DNA, using Primer Express software (Applied Biosystems). Primer sequences are reported in
Primer Sequences for Real-Time PCR
Relationships between biological variables and fetal age were analyzed using linear-regression models. When appropriate, a quadratic term for time was introduced to fit an exponential increase with fetal age. Main effects and interaction terms were tested in order to compare biological values in human fetuses with CDH versus fetuses with nonpulmonary diseases used as controls (control fetuses). Sensitivity analyses were performed when outliers were present.
For NRG1-β1, SP-A, SP-B, proSP-C, and SP-D, comparison between human fetuses with CDH and control fetuses was made using the paired Mann-Whitney U-test. For these variables, except for NRG1-β1 in control lungs, regression models were not performed because of the restricted number of determinations.
For sheep data, multiple group comparisons were made using the Kruskal-Wallis test, and two-group comparisons were made using the Mann-Whitney U-test.
A
DSPC concentration was determined in lung tissue samples from ten fetuses with CDH, ranging from 24 to 33 wk of pregnancy, and in 14 age-matched control fetuses, and normalized for lung DNA concentration. Over the studied period, DSPC concentration displayed a significant exponential increase with fetal age in control lungs and in CDH lungs (
DSPC was normalized to lung wet weight (ww) (A) and lung DNA (B), and expressed as a ratio of DSPC to total PC (i.e., DSPC plus unsaturated PC) (C). A rise in DSPC concentration and in the ratio of DSPC to total PC, which is indicative of an enhanced rate of surfactant accumulation, was observed at fetal age 32–33 wk in CDH as well as in control fetal lungs. There were no differences in the data between the groups, whatever the considered fetal age. DSPC displayed the same exponential increase in control lungs and CDH lungs with fetal age (
Expression of SP-A, SP-B, SP-D, and proSP-C was evaluated at the post-translational level by Western blot analysis. Six pairs of CDH and age-matched control lungs ranging from 28 to 37 wk of pregnancy (i.e., all in saccular-alveolar stages) were studied comparatively. No obvious difference was observed for any SP between CDH and control lungs at any stage (
(A) Western blots: Samples were electrophoresed by SDS-PAGE, transferred, and successively incubated with specific anti-SP antibodies. (B) Densitometric analysis normalized by Ponceau S for gel loading (arbitrary units). SPs were detected at all stages. SP-A monomers were extremely faint before 36 wk, when they increased sharply (A) resulting in an increase in total amount (B). SP-B also increased markedly at 36–37 wk, ProSP-C did not display developmental changes, and SP-D showed a weak increase (A, B). There were no obvious differences between CDH and control lungs for any SP, with developmental changes occurring at the same fetal ages, the only exception being presentation of a high SP-B level in one CDH fetus, aged 28 wk.
In parallel with SP expression, we evaluated by Western blot the expression of TTF1, a transcription factor known to play a major role in various aspects of fetal lung development, including as a positive regulator of surfactant–protein gene-promoter activity [
Western blot was performed in lung samples of fetuses with CDH (lanes labeled H) and in fetuses with nonpulmonary diseases used as controls (lanes labeled C) at four fetal ages (Ponceau S stain was used as loading control). No differences were observed between CDH fetuses and age-matched control fetuses.
The expression of three growth factors involved in maturation control of ATII cells, namely KGF, leptin, and NRG1-β1 proteins, was studied in human lung samples at the post-translational level. In control lungs, KGF concentration decreased by about 50% between 14 and 38 wk (
ELISAs were performed on the lungs of 20 fetuses with nonpulmonary diseases used as controls, ranging from 14 to 37 wk of pregnancy, and on 11 fetuses with CDH ranging from 22 to 37 wk. Individual values are shown. Linear regression was performed for the period 22–37 wk when samples were available for both groups. KGF (A) displayed different profiles according to the groups concerned, with a negative slope for control lungs and a positive slope for CDH lungs (−1.17 versus 2.23, respectively,
(A) Developmental changes in control lungs were studied from 11 to 36 wk of pregnancy. Upper: representative Western blot. A 45-kDa band corresponds to NRG1-β1 (the 55-kDa band is nonspecific [
(B) Comparative study in seven pairs of age-matched CDH (lanes labeled H) and control (lanes labeled C) lungs. Upper: representative Western blot. Lower: densitometric analysis; there was no significant difference in expression level between CDH and control lungs (
The three growth factors were analyzed in fetal sheep lungs at the pre-translational level by real-time PCR. KGF mRNA was significantly decreased in the sDH group, and its expression level was partially restored by TO (
RT followed by real-time PCR analysis; mean ± standard error of the mean on six, five, and three individual samples, respectively, in sham-operated control fetuses (C), in a surgical model of CDH (sDH), and in sDH with subsequent tracheal occlusion (sDH + TO). The Kruskal-Wallis test was used for multiple-group comparison, with two-group comparisons made by Mann-Whitney U-test (*
(A) KGF mRNA level was reduced by half by sDH and was partially restored by sDH + TO.
(B) Leptin mRNA was not affected by sDH, whereas TO enhanced expression to 3.5 times the control level.
(C) NRG mRNA level was decreased by 60% by sDH and was restored by sDH + TO.
A persistent question regarding the care of neonates with CDH is whether these infants are effectively surfactant deficient. The question of whether to administer surfactant to infants born with CDH is not trivial, as this therapy can transiently compromise gas exchange. Information about surfactant status in humans is limited, has been obtained by indirect approaches, and has remained controversial. We reappraised the question through the ontogenetic study of surfactant directly in CDH lung tissue samples. We showed that, contrary to current opinion, surfactant accumulation occurs in CDH lungs at the normal time and rate.
The postmortem collection of tissue samples was a limitation of this study and prevented us from studying expression at the pre-translational level. Investigations in humans also raise the question of control appropriateness, since only lung samples from subjects with nonpulmonary diseases could be used as controls. A previous demonstration of the differences between CDH and control lungs, considering other parameters, nevertheless validated the approach [
Developmental changes in the concentration of DSPC, the major surfactant component, were identical in CDH and control lung tissue samples, with a marked increase in late pregnancy. Surfactant-storing lamellar bodies appear in the human fetus at around 20 wk [
Also indicative of alveolar cell maturation is the increase in both SP-A and SP-B that was observed at the same time in both groups. Although the available amount of tissue from the 37-wk CDH lung was not sufficient for studying DSPC concentration, our results indicate similar timing for DSPC and SP accumulation. ProSP-C is of particular interest because, contrary to other SPs that are also expressed in bronchiolar Clara cells, it is a specific marker of ATII cells [
Previous investigations about surfactant status in CDH have led to controversial data, which have left inconclusive the question of primary surfactant deficiency. Some studies that appear consistent with the present findings showed no change in surfactant content of amniotic fluid [
There are several possible explanations for the discrepancies highlighted by previous investigations. First, although lung concentrations of surfactant components are normal, the total lung surfactant content is decreased in the CDH lung as a consequence of lung hypoplasia. This may account for the decreased surfactant levels measured in amniotic or lavage fluids. Nevertheless, the amount of surfactant is probably appropriate for lung size and alveolar surface area. Second, ventilation may have influenced surfactant composition and/or concentration. It has indeed been reported that ventilation alters alveolar surfactant in rat pups [
In contrast with our findings in the human lung, surfactant deficiency has been repeatedly reported in the sheep [
Fully consistent with findings about surfactant components, we observed no decrease for either factor in CDH lungs as compared with control lungs. KGF concentrations were even slightly higher in CDH lungs at late-gestational stages, although this difference cannot readily be explained. These data suggest that the lung hypoplasia associated with CDH does not interfere with the mesenchymal–epithelial interactions that control alveolar cell maturation. On the whole, human CDH lungs did not exhibit a trend toward a decrease in contents, or a delay in developmental changes for any of the studied parameters, including surfactant components and surfactant maturation factors.
In contrast with human lungs, KGF and NRG transcripts were decreased in the ovine model of CDH. With regard to KGF, this is in agreement with previous reports in the same model [
Although they have provided considerable insight into the understanding of the disease, animal models of CDH have shown several limitations with respect to their relevance to the human condition [
Although exogenous surfactant was formerly reported to be effective in three high-risk newborns with CDH [
In conclusion, the present data challenge the paradigm that the human CDH lung is surfactant deficient as compared with the age-matched normal lung. CDH does not appear to interfere with surfactant accumulation, which occurs with normal timing and remains proportional to lung weight. Prophylactic administration of surfactant at birth in infants with CDH may therefore be useless. However, before surfactant administration can be ruled out as a potential therapy for CDH, a prospective randomized trial that also takes into account the severity of the underlying lung hypoplasia and the gestational age at delivery is necessary.
(30 KB DOC)
The GenBank (
OB was supported by a Doctoral Fellowship grant from the Fondation pour la Recherche Médicale.
alveolar type II
congenital diaphragmatic hernia
disaturated phosphatidylcholine
keratinocyte growth factor
neuregulin
neuregulin 1 beta 1
phosphatidylcholine
surgical diaphragmatic hernia
surfactant protein
tracheal occlusion
Tris-buffered saline containing 0.05% Tween-20
thyroid transcription factor 1