Antenatal and postnatal lung and vascular anatomic and functional studies in congenital diaphragmatic hernia Implications for clinical management.код для вставкиСкачать
American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 145C:184– 200 (2007) A R T I C L E Antenatal and Postnatal Lung and Vascular Anatomic and Functional Studies in Congenital Diaphragmatic Hernia: Implications for Clinical Management ROBERTA L. KELLER * Congenital diaphragmatic hernia is characterized by fetal and neonatal lung hypoplasia as well as vascular hypoplasia. Antenatal imaging studies have been performed that attempt to quantify the degree of hypoplasia and its impact on infant prognosis. Prenatal and perinatal growth of the lung and vasculature are interdependent and their continued coordinated growth is critical for survival after birth in this patient population. Lung protection strategies appear to improve survival in newborns with diaphragmatic hernia, but a subset of infants remain who demonstrate sufficiently severe lung hypoplasia that we are unable to provide support long-term after birth. Fetal intervention is a strategy designed to enhance fetal lung growth towards improving survival in this most severely affected group, though other therapies to enhance postnatal lung and vascular growth should be concurrently investigated. However, any of these interventions will require careful selection of those infants at risk for poor outcome and thorough follow up, since long-term morbidity is significant in children with diaphragmatic hernia. ß 2007 Wiley-Liss, Inc. KEY WORDS: lung hypoplasia; persistent pulmonary hypertension of the newborn; pulmonary function; tracheal occlusion How to cite this article: Keller RL. 2007. Antenatal and postnatal lung and vascular anatomic and functional studies in congenital diaphragmatic hernia: Implications for clinical management. Am J Med Genet Part C Semin Med Genet 145C:184–200. INTRODUCTION Congenital diaphragmatic hernia (CDH) is a condition characterized by fetal and neonatal pulmonary parenchymal and vascular hypoplasia. Pre-acinar airway and vascular structures develop together through the pseudoglandular stage of development (6–16 weeks’ gestational age, GA) [Bucher and Reid, 1961; Hall et al., 2000], while more peripheral intra-acinar airway and vessel develop- ment are also inter-dependent (reviewed by Hislop ). Respiratory surface area and the extent of the vascular bed, therefore, are closely related. Due to an early arrest of pre-acinar lung development, newborns with symptomatic CDH have respiratory failure with a delayed transition to postnatal circulation. Survival is limited by lung hypoplasia and postnatal lung injury. The use of gentle ventilation techniques has resulted in improved survival for new- Abbreviations: AP, anterior–posterior; CDH, congenital diaphragmatic hernia; ECMO, extracorporeal membrane oxygenation; EXIT procedure, ex utero intrapartum procedure; FTO, fetal tracheal occlusion; FRC, functional residual capacity; GA, gestational age; LD, lung diameter; LHR, lung-to-head ratio; L/T ratio, lung-to-thorax ratio; MRI, magnetic resonance imaging PA, pulmonary artery; PVR, pulmonary vascular resistance; ROC curve, receiver operator characteristic curve; SBP, systemic blood pressure; TC, thoracic circumference; 3D ultrasound, threedimensional ultrasound; 2D ultrasound, two-dimensional ultrasound. Roberta L. Keller, M.D. is an Assistant Professor of Clinical Pediatrics at the University of California San Francisco, an attending neonatologist in the William H. Tooley Intensive Care Nursery and the Medical Director of the neonatal Extracorporeal Membrane Oxygenation program at the UCSF Children’s Hospital in San Francisco, CA. Dr. Keller’s interests include lung and vascular growth and remodeling in congenital diaphragmatic hernia and other developmental disorders of the newborn lung. *Correspondence to: Roberta L. Keller, M.D., UCSF Box 0748, San Francisco, CA 94143. E-mail: email@example.com DOI 10.1002/ajmg.c.30130 ß 2007 Wiley-Liss, Inc. borns with symptomatic CDH in individual institutions, but overall mortality remains at 45–50% in population-based studies [Dott et al., 2003; Stege et al., 2003; Colvin et al., 2005]. This review will discuss the development of the lung in CDH, focusing on Bochdalek (posterolateral) hernias, and how antenatal and postnatal anatomical and physiological measurements of the lung and vasculature are related to morphometric observations. CDH AND PULMONARY HYPOPLASIA Lung Airway and Parenchymal Development Diaphragmatic defects in humans are associated with bilateral pulmonary hypoplasia. This pulmonary hypoplasia develops, in part, secondary to abdominal contents occupying the thoracic space with shift of mediastinal tissues into the contralateral hemithorax. Pul- ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c 185 monary hypoplasia may also have a primary developmental component, as animal models have confirmed that developmental regulation of the lung and diaphragm is controlled by some of the same genes [Guilbert et al., 2000; Diaphragmatic defects in humans are associated with bilateral pulmonary hypoplasia. This pulmonary hypoplasia develops, in part, secondary to abdominal contents occupying the thoracic space with shift of mediastinal tissues into the contralateral hemithorax. Pulmonary hypoplasia may also have a primary developmental component, as animal models have confirmed that developmental regulation of the lung and diaphragm is controlled by some of the same genes. Keijzer et al., 2000; Ackerman et al., 2005; You et al., 2005]. A typical severe diaphragmatic defect with herniation of bowel into the left chest is shown in Figure 1. This hypoplasia is an early fetal event occurring during the pseudoglandular stage of lung development in some cases, as evidenced by the arrest of pre-acinar airway branching at 10– 14 weeks’ GA. Pre-acinar airway branches are decreased in number to 1/3–2/3 of normal values, with the greatest reductions in the ipsilateral lung. Similarly, even the main stem bronchi are severely reduced in diameter, particularly in the ipsilateral lung [Areechon and Reid, 1963; Kitigawa et al., 1971; Thurlbeck et al., 1979; Geggel et al., 1985]. Although airway muscle is not significantly increased in those infants Figure 1. Infant with unrepaired left congenital diaphragmatic hernia. Note the presence of liver and intestines in the left thoracic cavity (white arrows). The heart and mediastinal structures are shifted to the right (H, heart). The stomach is usually intrathoracic and posterior in fetuses and infants with left diaphragmatic hernia and liver herniated into the thorax. The diaphragm under the heart is intact (marked with black arrow). Courtesy of KK Nobuhara. that die at less than 24 hr of age (compared to controls), increased musculature is demonstrated in infants that die later, and it is greatly increased in infants that die after receiving high ventilator assistance [Broughton et al., 1998]. Overall lung volume is reduced to 10–30% of normal volumes in those infants that die at less than 24 hr of age [Kitigawa et al., 1971; Thibeault and Haney, 1998]. Alveolar counts are relatively normal per acinus, but overall numbers of alveoli are severely reduced (6.6 106 compared to the normal average of approximately 50 106) and alveoli are generally smaller than those present in control lungs [Areechon and Reid, 1963; Kitigawa et al., 1971; Langston et al., 1984; Geggel et al., 1985]. The surface area available for gas exchange in these infants, therefore, is substantially decreased. However, the lung does have capacity for compensatory growth. One child with left CDH that later died from trauma at 64 months of age had 107 106 alveoli on the left side and 242 106 alveoli on the right side. Thus, the total alveolar numbers were normal for age, but the right lung demonstrated compensatory growth and had twice the number of alveoli expected [Thurlbeck et al., 1979]. Pulmonary Vascular Development The pulmonary vascular tree also demonstrates a developmental arrest of arterial branching at 12–14 weeks’ GA. Similarly, the diameter of the vessels is decreased, and related to lung volume [Kitigawa et al., 1971]. This results in a decreased cross-sectional area of the vascular bed, with a requisite increase in pulmonary vascular resistance (PVR). Pre-acinar and intra-acinar arteries are decreased in size in infants that die in the first week of life. In some infants, intraacinar arteries have a muscular wall, whereas other infants have no distal muscularization [Kitigawa et al., 1971; Geggel et al., 1985]. Muscular wall thickness and muscle mass are increased in arteries bilaterally, compared to normal controls, and this increase in muscle 186 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c mass is inversely proportional to the degree of lung hypoplasia [Kitigawa et al., 1971; Naeye et al., 1976]. influencing survival (e.g., ventilation strategy, GA at delivery and associated anomalies). ANTENATAL MEASUREMENTS OF PULMONARY HYPOPLASIA Measurement of Lung Hypoplasia to Predict Prognosis Pulmonary hypoplasia is a pathological diagnosis. Several anatomical measures of lung and vascular hypoplasia by fetal ultrasound have been proposed. In fetal CDH, these measurements have included thoracic, lung, and pulmonary artery (PA) sizes, as well as proxies for the mass of the herniated abdominal viscera (degree of mediastinal shift to the contralateral hemithorax, liver herniation into the hemithorax, decreased abdominal circumference and decreased left ventricular mass). However, most of these studies do not have pathological correlation to verify the accuracy of the diagnosis of lung hypoplasia. Instead, these measures have been evaluated as antenatal predictors of survival and other outcomes in CDH, to allow for counseling of families and to assess criteria for fetal intervention. Results from these prognostic studies have been inconsistent, which may be due to variability in the technique and gestational timing of measurement, the range of severity of lung hypoplasia in the population of fetuses and newborns being studied or the confounding effects of other factors There are various methods now available for quantification of in utero lung size. These include two-dimensional (2D) ultrasound to measure lung size in relation to transverse thoracic area (L/T ratio) or head circumference (LHR) and measurement of lung volume by magnetic resonance imaging (MRI) or three-dimensional (3D) ultrasound. Measurement of bilateral lung area by two-dimensional ultrasound (2D ultrasound), normalized to the transverse thoracic area (L/T ratio), was described in 1990 [Hasegawa et al., 1990]. This measurement was found to be decreased in CDH, with no differences in measurements between survivors and non-survivors with CDH in a small series; however, a lower L/T ratio was associated with worse indices of neonatal gas exchange and increased need for ECMO support [Kamata et al., 1992]. In subsequent publications from other centers, lung area measurements in CDH were confined to the contralateral lung, as the ipsilateral lung can often not be appreciated (Fig. 2A,B) [Guibaud et al., 1996; Lipshutz et al., ARTICLE 1997; Jani et al., 2006b]. In a recent study, the contralateral (right) lung area divided by the total thoracic area in seven fetuses with left CDH showed findings similar to Kamata and colleagues; specifically, the authors could not demonstrate a relationship of the ratio to survival in CDH, but they did demonstrate that lower ratios were associated with poorer initial oxygenation and need for ECMO support [Nakata et al., 2003]. Variations on these measurements have included the fetal right lung diameter (LD) at the level of the cardiac atria divide by the thoracic circumference (TC). In seven fetuses with CDH, when the LD/TC was less than 5th centile, lung hypoplasia was confirmed by pathological examination in all cases Variations on these measurements have included the fetal right LD at the level of the cardiac atria divide by the TC. In seven fetuses with CDH, when the LD/TC was less than 5th centile, lung hypoplasia was confirmed by pathological examination in all cases. Figure 2. Fetal ultrasound (A) and chest radiograph (B) from patients with severe left congenital diaphragmatic hernia. Ultrasound (A) is used to measure the right (contralateral) lung area (RL) using a cross-sectional view of the thorax at the level of the 4-chamber view of the heart (H). This fetus had liver herniated into the left hemithorax and the hypoplastic left lung cannot be appreciated (Ao, aorta and S, stomach). The chest radiograph (B) demonstrates findings in a newborn with severe left diaphragmatic hernia who is intubated and on mechanical ventilation. The extremely hypoplastic left lung is actually well-expanded (arrow). ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c [Merz et al., 1999]. A follow up study from this group demonstrated that 11/ 14 fetuses with ultrasound measurements <5th centile died (the nine infants with post-mortem examinations met pathological criteria for lung hypoplasia), whereas three fetuses with measures >5th centile survived. In 6/7 fetuses who had repeated fetal LD measures <5th centile, all infants expired [Bahlmann et al., 1999]. The one fetus with increasing LD centiles survived. A separate study, measuring contralateral lung area divided by hemithoracic area, demonstrated that a ratio less than 50% predicted was significantly associated with death (5/20 survivors vs. 6/7 survivors, P ¼ 0.009). There was excellent agreement between these measurements obtained by two different observers (kappa ¼ 0.87) [Guibaud et al., 1996]. The lung-to-head ratio (LHR), developed by our group at the University of California San Francisco (UCSF), is calculated by measuring the area of the contralateral lung at the level of the cardiac atria (obtained on a transverse section of the thorax (Fig. 2A)) divided by the biparietal head circumference. The lung-to-head ratio (LHR), developed by our group at the University of California San Francisco (UCSF), is calculated by measuring the area of the contralateral lung at the level of the cardiac atria (obtained on a transverse section of the thorax) divided by the biparietal head circumference. In a series of 55 fetuses with left CDH, the LHR was identified as the only independent predictor of survival (LHR 1.33 0.50 for survivors vs. 0.87 0.32 for non-survivors, P < 0.001). There were no survivors with LHR <0.6 (n ¼ 5) and 100% survival with LHR > 1.35 (n ¼ 14). The presence of liver herniated into the left hemithorax was also significantly associated with death (not independent of the LHR), and the severity of liver herniation was inversely proportional to the LHR [Metkus et al., 1996]. A subsequent small (n ¼ 15) UCSF series confined the analysis to fetuses with left CDH, liver herniated into the hemithorax and LHR measurement at 24–26 weeks’ GA. Again, the LHR was higher in survivors than in non-survivors; there were no survivors with LHR < 1.0 (n ¼ 3) and 100% survival with an LHR > 1.4 (n ¼ 4). We subsequently demonstrated that the LHR had good inter-observer agreement (among four blinded sonologists) with an intraclass correlation coefficient of 0.70 and excellent intra-observer agreement with an intraclass correlation coefficient of 0.80 [Keller et al., 2003]. However, the LHR was not found to be predictive of survival in fetuses with left CDH when the liver was not herniated on ultrasound at 26 weeks’ GA. Eight of nine fetuses with LHR 1.4 survived and 8/11 with LHR > 1.4 survived. In our experience, fetal liver herniation in left CDH is a significant predictor of neonatal morbidity [need for extracorporeal membrane oxygenation support (ECMO): 17/30 (57%) with liver herniation vs. 3/ 15 (20%) without liver herniation, P ¼ 0.04] and mortality [17/30 (57%) vs. 1/15 (7%), P ¼ 0.001] [Albanese et al., 1998]. Similar findings have been demonstrated in left CDH with fetal liver herniation detected by MRI (at 20–39 weeks’ GA); mortality was 60–70% with liver herniated versus 10– 20% without liver herniated [Walsh et al., 2000; Kitano et al., 2005]. Interestingly, the ultrasound LHR was significantly lower in fetuses with herniated liver (by MRI), compared to those without herniated liver (1.4 0.6 vs. 2.0 0.3, P ¼ 0.01) [Kitano et al., 2005]. Other single center studies reporting on the prognostic value of the LHR have had results that are not always consistent with ours. Flake et al.  reported that all fetuses with left CDH and LHR 1.0 (n ¼ 7) expired when 187 liver was herniated, and 20/24 fetuses with LHR > 1.4 survived. LHR (measured at 28–37 weeks’ GA) was higher in survivors than non-survivors in another series of 21 fetuses with left CDH. All fetuses with LHR < 1.1 expired, while all fetuses with LHR > 1.4 survived (n ¼ 8); the presence of liver herniation was not determined in this study [Laudy et al., 2003]. Heling et al. , however, did not find a relationship between LHR and survival, when fetuses with liver herniated and not herniated were included, as were fetuses with both left and right CDH. Similarly, in a series of 28 fetuses reported by Arkovitz et al., no relationship between LHR and survival was seen. However, only four infants subsequently died among this group of fetuses with both left and right CDH and with and without liver herniation [Arkovitz et al., 2007]. We participated in a recent large multicenter evaluation of LHR in fetal left CDH and liver herniation ascertained at 22–27 weeks’ GA. In this report of neonatal survival in 184 fetuses, the presence of liver herniation was associated with decreased odds of survival in a multivariate analysis (OR 0.48, P ¼ 0.035), as was a lower LHR. When confining the analyses to fetuses with or without liver herniation, the LHR relationship to mortality was stronger in those fetuses with liver herniation (OR 26.6 vs. OR 3.3). There were no survivors in this large group with liver herniation and LHR < 0.8, and only 3/27 infants (11%) with LHR < 1.0 survived [Jani et al., 2006a]. In comparison, 11/19 fetuses (58%) with LHR < 1.0 and without liver herniation survived. There are additional concerns regarding the interpretation of the LHR measurement, which may make extrapolation of studies from one center difficult to apply to findings at another center. One issue is the methodology of obtaining the measurement. Although all centers describe their measurement at the level of the cardiac atria obtained on a transverse section of the thorax, the lung area measurement can be obtained in two ways. One technique employs the aorta and lateral rib as landmarks for the 188 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c lateral measurement, with the orthogonal diameter the anterior–posterior (AP) measurement from the cardiac atria to the posterior rib (AP measurement, consistently performed and reported at UCSF, RA Filly, personal communication). Alternatively, the largest diameter of visible lung can be measured from a site lateral to the atria to the posterior rib with the resulting orthogonal measurement used to calculate the lung area (Long measurement) [Deprest et al., 2006]. As anticipated, LHR measures obtained by the Long technique are consistently higher. A blinded test series of 25 cases of left CDH with herniated liver yielded a mean LHR of 1.29 by AP measure compared to the LHR of 1.55 by Long measure, with excellent correlation (R2 ¼ 0.98) between the two measures [Keller et al., 2003; RA Filly, unpublished work]. A recent study of these two measurement techniques, which compared the resulting lung areas in control and CDH fetuses to lung volumes obtained by 3dimensional (3D) ultrasound, found that right lung areas measured by both techniques correlated equally well with lung volumes in control fetuses (R ¼ 0.95 for both methods). In CDH, lung area and LHR measures obtained by the AP method were consistently lower than those obtained by the Long method [Jani et al., 2006b]. Therefore, the measurement technique and its prognostic thresholds should be accounted for when counseling families with respect to prognosis. A second issue, regardless of technique, is which LHR measure is reported and used for counseling. There is variability in the measurement for any individual fetus. We previously reported the prognostic value for survival of the minimum, average, and maximum LHR measures. The minimum LHR measure demonstrated slightly better prognostication than the average LHR, based on the receiver operator characteristic curves (ROC curves) (area under the curve 0.73, 0.72, and 0.59 for minimum, average, and maximum LHR, respectively) [Keller et al., 2003]. All LHR reports from UCSF had used the minimum LHR, so we have continued to use the minimum LHR for counseling and reporting (RA Filly, personal communication). However, if maximum LHR is reported, the prognosis should be based on different thresholds. A third issue has to do with the timing of LHR measurement. The LHR was developed in an attempt to normalize the lung area to the size of the fetus, using the head circumference as a proxy for fetal size. In normal controls, the LHR increases over gestation (from 12 to 32 weeks’) when using measurements of either the right or left lung. The measurements have different quadratic relationships with respect to GA, with the head circumference increasing 4-fold and the lung area increasing 16-fold over the measurement period [Peralta et al., 2005]. Thus, timing of the LHR measurement may also have some relevance in prenatal counseling, and the implication of the timing of the measurement may be even greater since the rate of lung growth in severe CDH at 29–38 weeks’ GA may be decreased compared to lung growth in normal controls [Bahlmann et al., 1999]. The fourth issue is the site of delivery and neonatal care after a fetal diagnosis of severe CDH. From our fetal database, we have found that in the most severe CDH group (liver herniated with LHR 1.0), when LHR was measured at 20–29 weeks’ GA, infants were more likely to survive if their care was at UCSF compared to care given at a broad group of other institutions [17/30 (57%) vs. 8/ 37 (22%), P ¼ 0.01, unpublished work]. Therefore, prenatal counseling regarding prognosis in severe CDH should incorporate the planned site of delivery and postnatal care, with specific data as to survival in different fetal risk groups. Finally, data on LHR and other measures in right CDH is scarce. Right CDH is less frequent, and may be more difficult to diagnose antenatally, since mediastinal shift may not be appreciated. A single series of fetuses and newborns with right CDH demonstrated 3/4 fetuses with LHR 1.0 survived [Hedrick et al., 2004]. The right lung is consistently larger than the left lung in fetuses without lung hypoplasia [Peralta et al., 2005], although it is not clear how this differential would affect prognosti- ARTICLE cation in CDH where the problem is bilateral lung hypoplasia. Criteria for fetal intervention (such as fetal tracheal occlusion [FTO]) for severe right CDH at our center and others, however, are the same as those for severe left fetal CDH: the presence of liver herniated into the chest and LHR 1.0 [Deprest et al., 2006]. MRI and three-dimensional ultrasound (3D ultrasound) (which compare observed to predicted lung volume ratios) may be more promising tools to prognosticate survival throughout gestation. In unaffected fetuses, MRI lung volumes correlate well with standard biometric data obtained by fetal ultrasound [Coakley et al., 2000; MRI and 3D ultrasound (which compare observed to predicted lung volume ratios) may be more promising tools to prognosticate survival throughout gestation. In unaffected fetuses, MRI lung volumes correlate well with standard biometric data obtained by fetal ultrasound. Rypens et al., 2001]. Further, MRI lung volumes from control fetuses correlated well with autopsy lung volumes [Rypens et al., 2001]. A potential advantage of MRI and 3D ultrasound is that these techniques allow for quantification of bilateral lung volume in less severe CDH (compared to the unilateral measurements quantified by some 2D ratios). However, in more severe cases (where prognostication is most important), the ipsilateral lung can be difficult to measure or visualize by either of these newer techniques [Ruano et al., 2004b]. In one series, fetuses in whom the ipsilateral lung on MRI could not be identified had significantly higher mortality than those in whom the ipsilateral lung was visualized [Gorincour et al., 2005]. Other ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c limitations of these volumetric measurements include difficulties with reproducibility; studies have found both good agreement [Ruano et al., 2004b] and poor agreement [Peralta et al., 2006c] between MRI and 3D ultrasound lung volume measurements. Further, centers use different predictive equations for expected lung volume measurements, and prognostic ability within a center may be impacted by the relatively narrow ranges of normal lung volume predicted during mid-gestation, and the wide ranges of normal lung volume predicted later in gestation [Mahieu-Caputo et al., 2001; Rypens et al., 2001; Kasprian et al., 2006; Peralta et al., 2006a]. Walsh et al.  published a series of 41 fetuses with CDH and MRI lung volume measurements at 20–39 weeks’ GA. Seventeen infants expired, but there were no differences in lung volume between survivors and non-survivors, and also no differences in lung volume indexed for GA (ml/week GA). In a smaller series, the ratio of observed to expected lung volumes by MRI was evaluated in 13 fetuses with CDH at 28– 37 weeks’ GA. These ratios were significantly lower in fetuses with CDH than in controls, despite considerable variability in the measured lung volume of unaffected fetuses. Further, observed to expected lung volume ratios were lower among the nine CDH fetuses that expired as newborns, compared to the four that survived (median 0.26 vs. 0.46, P ¼ 0.03). In 5/6 infants undergoing autopsy, lung hypoplasia was confirmed by low lung to body weight ratios; all of these infants had an observed to expected lung volume ratio 0.35 [Mahieu-Caputo et al., 2001]. A series of 26 fetuses (20–28 weeks’ GA) from our center (using a different algorithm for predicting expected lung volume) demonstrated a strong correlation between observed to expected fetal lung volumes by MRI and LHR. Also, lower observed to expected fetal lung volumes correlated with worsening outcomes (e.g., death, need for ECMO support, prolonged duration of mechanical ventilation) in 11 infants with left CDH. This study further demonstrated excel- lent agreement in estimation of MR lung volume between two independent observers [Paek et al., 2001]. An additional recent series of 77 fetuses studied at 24–37 weeks’ GA (which again used a different predictive equation for expected lung volume) demonstrated lower ratios of observed to expected lung volume in non-survivors versus survivors (0.24 0.12 vs. 0.36 0.13, P < 0.001). Fetuses with a ratio below 0.25 had a survival rate lower than the 40% observed in the entire cohort, and those with a ratio above 0.25 had a survival rate greater than 40% (sensitivity 79%, specificity 64%) [Gorincour et al., 2005]. In a smaller study of 22 fetuses at 30–32 weeks’ GA using the same predictive equation, a threshold of 0.30 was the best fit for predicting mortality (sensitivity 83%, specificity 100%) [Bonfils et al., 2006] and in a study of 14 fetuses with CDH, all three infants that subsequently expired had an observed to expected lung volume ratio of 0.15 [Barnewolt et al., 2007]. Unfortunately, no reference is reported for the generation of the predictive algorithm in this study. Thus, there remains uncertainty in the prognostic ability of MRI measures in lung hypoplasia, similar to the concerns with 2D ultrasound measures. Three-dimensional ultrasound is proving useful for predicting lung size. Ipsilateral and contralateral lung volumes measured by 3D ultrasound are decreased in fetuses with CDH, although in some cases the contralateral lung is still within the normal range [Jani et al., 2006b; Peralta et al., 2006b]. Observed to expected lung volume ratios were found to be decreased in fetuses with CDH (compared to controls), and infants with CDH that expired had lower ratios than those that survived (median 0.16 vs. median 0.44, P ¼ 0.04). All infants with a ratio 0.32 expired and lung hypoplasia was confirmed on three of those infants that underwent post-mortem examination [Ruano et al., 2004a]. A subsequent study wherein absolute lung volumes ascertained by 3D ultrasound in CDH and controls were compared to postmortem lung volumes demonstrated 85% accuracy in measurements in fetuses 189 with CDH and 91% accuracy in controls [Ruano et al., 2005]. Fetal Measurement of Vascular Hypoplasia and Prognosis The data describing the impact of abnormal antenatal pulmonary vascular development on prognosis in CDH are limited. Main and branch PA diameter can be measured by fetal echocardiogram on a cross-section of the fetal thorax at the level of the main PA, ascending aorta and superior vena cava. In one series of fetuses with CDH, these branch PA measurements correlated well with individual lung weights obtained at autopsy. In both retrospective and prospective studies of these measurements (a total of 43 fetuses with left CDH), ipsilateral PA diameter was consistently smaller than contralateral PA diameter. On initial echocardiogram obtained during mid-gestation, ipsilateral PA diameter was at the low end of the normal range of values (usually <25th centile). Serial measures, however, demonstrated progressive hypoplasia of the ipsilateral PA, with the PA diameter percentile (corrected for GA) usually decreasing in studies after 30 weeks’ GA (consistent with the lack of lung growth observed by ultrasound in CDH [Bahlmann et al., 1999]). Fetal left PA measures were inversely correlated with the duration of mechanical ventilation, oxygen supplementation and length of hospital stay, and they were predictive of prolonged duration (>28 days) for all of these outcomes [Sokol et al., 2002, 2006]. Another method of quantification of the pulmonary vasculature in CDH is visualization of PA branches by Doppler in the contralateral lung on a cross-section of the thorax at the level of the largest image of the lung. In one study, the ability to visualize three branches of the PA was considered a normal vascular tree, two branches was considered a poor vascular tree, and one branch was considered an absent vascular tree. In this series of 42 fetuses with CDH, mortality was 25% (5/20) with normal vascular tree and 82% (18/22) with an abnormal vascular tree (P ¼ 0.0002) 190 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c [Mahieu-Caputo et al., 2004]. Finally, a more recent study proposed the use of power Doppler imaging by 3D ultrasound to ascertain vascularization index (percent of color signal in the lung volume as a measure of number of vessels detected), flow index (amplitude of color signal as a measure of pulmonary blood flow) and vascularization-flow index (a combination of vascularity and flow). These indices were all significantly correlated with the ratio of observed to expected lung volume. Among 21 fetuses with CDH, a lower vascularization index for either both lungs or the contralateral lung only was significantly associated with mortality. Lower pulmonary blood flow, however, was less strongly associated with mortality and there was no association if the contralateral lung alone was considered [Ruano et al., 2006]. These measures of pulmonary vascular development may enhance the prognostic abilities of the measures of fetal lung development described previously. There are also very limited data regarding fetal pulmonary vascular function in lung hypoplasia. In two fetuses with CDH and autopsy-proven pulmonary hypoplasia, pulsatility index (a Doppler measure of pulmonary vascular impedance) in the branch PA was elevated in utero [Chaoui et al., 1999]. A subsequent study evaluated reactivity of the pulmonary vasculature in response to maternal hyperoxygenation. A reactive test was defined as a decrease in branch PA pulsatility index of 20% (indicating a decrease in vascular impedance and an increase in pulmonary blood flow). Ten of the 29 fetuses evaluated in this study had a diagnosis of CDH. Eleven of 14 fetuses with a non-reactive test died from lung hypoplasia, while only 1/15 fetuses with a reactive test died from lung hypoplasia [Broth et al., 2002]. Another measurement that has been studied is the branch PA acceleration time/ejection time ratio. This parameter is low in adults with pulmonary arterial hypertension. In a series of fetuses with suspected lung hypoplasia, ten had CDH and four of these had low acceleration time/ejection time ratios in the contralateral branch PA. These fetuses either expired, or had lung hypoplasia confirmed by postmortem evaluation. The surviving newborns all had ratios that were within the normal range [Fuke et al., 2003]. EFFECTS OF FETAL TRACHEAL OCCLUSION (FTO) FTO was developed as a strategy to enhance fetal lung growth in CDH. Animal models of tracheal occlusion have provided an opportunity to start to understand the effects of stretch and distension on lung growth and maturation. These models of tracheal occlusion are discussed in detail in the review by Dr. Khan, Dr. Cloutier, and Dr. Piedboeuf. Tracheal occlusion was first performed on human fetuses only with severe CDH and was reported from our center in 1996 [Harrison et al., 1996]. The occlusion technique has evolved from the placement of an internal tracheal plug, to an external clip, and then to an endotracheal balloon [Harrison et al., 1996, 2001]. The surgical approach has also evolved from an open fetal surgical procedure with a maternal hysterotomy [Harrison et al., 1996], to an endoscopic procedure via maternal laparotomy [Harrison et al., 1998], and subsequently to a completely endoscopic procedure, with occlusion placed at 26–28 weeks’ GA and released prior to delivery at 33–36 weeks’ GA. The release prior to delivery has abrogated the need for a specialized Cesarean section delivery wherein the occlusion was released at delivery (ex utero intrapartum procedure [EXIT procedure]) [Deprest et al., 2004]. FTO and Survival in Severe CDH As FTO and predictive diagnostic techniques have evolved, so has the approach to FTO in humans. Initially, fetuses with CDH meeting inclusion criteria for FTO had to demonstrate herniated liver with an LHR 1.4 measured during mid-gestation. Fetuses also had to have a normal karyotype with no additional anomalies detected by ultrasound or echocardiography. The open procedure ARTICLE led to substantial fetal and neonatal morbidity [Harrison et al., 1996]. Once the endoscopic technique was accomplished, the early experience in this severely affected group was promising, with survival 75% (6/8) following FTO compared to survival of 38% (5/13) in a contemporary comparison group without FTO [Harrison et al., 1998]. These initial results led to our single-center randomized controlled trial of FTO for severe left CDH, with the procedure accomplished at 24–28 weeks’ GA. Interim analysis by the data safety monitoring board resulted in early trial termination due to the fact that we would be unlikely to detect a survival difference with completion of the study. These initial results led to our single-center randomized controlled trial of FTO for severe left CDH, with the procedure accomplished at 24–28 weeks’ GA. Interim analysis by the data safety monitoring board resulted in early trial termination due to the fact that we would be unlikely to detect a survival difference with completion of the study. We found survival at 90 days in both groups to be similar to the historical survival in the FTO group [10/13 (77%) vs. 8/11 (73%), control vs. FTO group, P ¼ 1.0]. Pre-term delivery remained a problem in the FTO group, with mean GA at delivery significantly higher in the control group (30.8 2.0 vs. 37.0 1.5 weeks, P < 0.001) [Harrison et al., 2003]. The data from this trial also suggested that an LHR threshold of 1.0 rather than 1.4 could be used for selection of candidates for FTO. Mortality was significantly increased when LHR was 0.90 (Hazard ratio 0.13, 95% ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c confidence interval 0.03, 0.64), with long-term survival 3/9 if LHR 0.90 and 13/15 if LHR > 0.90 [Harrison et al., 2003]. This was similar to the inclusion criteria for FTO previously used by Flake et al.  (liver herniated and LHR 1.0), and these criteria that have subsequently been adopted at our center and in Europe [Deprest et al., 2004]. Flake et al.  presented a survival of 33% (5/15) in a mixed group of left and right CDH following open FTO and the European FETO Task Group presented a survival of 48% in their first 21 endoscopic fetal procedures with both left and right CDH. Survival among fetuses that were evaluated by the same group but did not undergo FTO was 8% (1/12) [Deprest et al., 2004]. Contrary to the promising FTO data from Europe, a recent retrospective evaluation of the UCSF Fetal Treatment Center database, demonstrated that among fetuses with liver herniated and LHR 1.0 who were subsequently cared for in the neonatal period at UCSF, survival was comparable whether or not endoscopic FTO was performed [16/29 (55%) vs. 14/26 (54%), P ¼ 0.92, standard care vs. FTO, unpublished work]. Lung Growth After FTO Early data from our center on 16 infants undergoing endoscopic FTO with left CDH suggested that an initial LHR of 1.0 was a significant predictor of survival [Keller et al., 2003]. Similarly, the FETO Task Group has recently reported that initial LHR remains a significant predictor of survival [which was 57% (16/28) in this series for an LHR < 1.0 in left CDH] following FTO [Jani et al., 2006c]. Lung growth as assessed by ultrasound following FTO appears to be variable. It was thought to be occurring in the initial experience with FTO, with shift of the mediastinum back toward the midline beginning at 1 week following the procedure in 5/7 fetuses that survived FTO. Yet, only one of these fetuses (that had exhibited lung growth) ultimately survived the newborn period. In the remaining six infants, autopsy evidence of pulmonary hypoplasia was consistent with fetal ultrasound evidence of fetal lung growth [Harrison et al., 1996]. Flake et al.  demonstrated increasing LHR on serial ultrasounds in 8 of 13 fetuses and the FETO Task Group has also documented increased LHR after FTO, wherein the median LHR increased from 0.7 to 1.7 within 2 weeks of the procedure [Jani et al., 2005]. Data are not yet available investigating whether the appearance of lung growth is related to survival following FTO, and it may be that other imaging modalities (or functional studies) are needed to evaluate the intrauterine effects of FTO in humans. An autopsy series from UCSF demonstrated a mean lung-to-body weight ratio of 0.021 0.011 in fetuses and infants with fetal intervention (n ¼ 11) compared to a mean ratio of 0.011 0.004 in those without intervention (n ¼ 8, all subjects at 29 weeks post-conceptual age). A lung-to-body weight ratio less than or equal to 0.012 is considered to be evidence of lung hypoplasia [Askenazi and Perlman, 1979]. However, there was a tendency toward larger alveoli in the group of subjects with fetal intervention [Heerema et al., 2003]. In addition, four of 16 subjects with GAs of 29–30 weeks were classified in the alveolar stage of development following FTO, whereas no subjects without fetal intervention were in the alveolar stage prior to 34 weeks’ gestation (alveoli may be present as early as 32 weeks’ gestation in fetuses without CDH) [Langston et al., 1984; Heerema et al., 2003]. Morphometric data from the lungs of animals with CDH and FTO are discussed in the article by Dr. Khan et al. PULMONARY GROWTH AND FUNCTION IN CDH Short-Term Pulmonary Function Measurements A number of different investigators have studied lung function during the newborn hospitalization in CDH. Measurements include lung compliance, lung volume, and pulmonary resistance. Although timing of measurements and inclusion criteria for studies differ, some 191 general conclusions can be made. First, dynamic compliance of the respiratory system and/or the lung is decreased in CDH in this short-term period, although it tends to normalize over the first 6 months of life [Landau et al., 1977; Nakayama et al., 1991a; Koumbourlis et al., 2006]. Compliance usually improves (increases) over the first several days of life [Sakai et al., 1987; Nakayama et al., 1991a; Moffitt et al., 1995] although it transiently worsens (decreases) acutely in the post-operative period (Table I) [Sakai et al., 1987]. Thus, the delay of CDH repair until the patient is medically stabilized has most likely contributed to improved outcomes, as this delay allows for the development of improved lung and vascular function. Some studies have shown that higher lung compliance is observed in survivors versus non-survivors [Nakayama et al., 1991a; Tracy et al., 1994; Antunes et al., 1995], although results may depend on study selection criteria and timing of compliance measurements. Second, lung volume is decreased in the acute period in most infants with CDH, indicative of restrictive lung disease (Table II) [Landau et al., 1977; Nakayama et al., 1991b; Antunes et al., 1995; Kavvadia et al., 1997; Dinger et al., 2000]. However, over a varying time course (up to several months), it tends to normalize [Landau et al., 1977; Koumbourlis et al., 2006]. This time course may depend on the severity of the lung hypoplasia, as some infants have rapid increases in lung volume after surgery (with expansion of compressed lung) and others have no change for a period of weeks [Antunes et al., 1995; Kavvadia et al., 1997; Dinger et al., 2000]. In one series, all ten non-survivors had a pre-operative functional residual capacity (FRC) of <9 ml/kg (normal infant FRC 26– 31 ml/kg) [Chu et al., 1967; Schwartz et al., 1978], whereas among survivors, only 2/15 had FRC < 9 ml/kg [Antunes et al., 1995]. In some infants lung volumes are increased over normal values, indicating some degree of hyperinflation or emphysema [Landau et al., 1977; Koumbourlis et al., 2006]. Finally, many infants with CDH demonstrate 192 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c ARTICLE TABLE I. Respiratory System Compliance (Crs) in Infants With CDH at Various Time Points During Neonatal Hospitalization Respiratory system compliance (mL/cmH2O/kg) Study Sakai et al.  Nakayama et al. [1991a] Inclusion Respiratory distress <6 hr (n ¼ 9) Respiratory distress <24 hr (n ¼ 22) Respiratory distress <6 hr (n ¼ 8) Kavvadia et al. c Dinger et al. d Keller et al.  Post-op 8–92 hr (median 20 hr) 1.69 0.68 1.20 0.77a Immediate repair (10.6 1.5 hr) Survivors: 0.37 0.04 non-survivors: 0.18 0.04 Initial: 0.23 0.04 pre-op (137 29 hr): 0.46 0.04 7 days of age: Survivors: 0.38 0.03 nonsurvivors: 0.20 0.04 7 days of age: 0.37 0.03 Delayed repair (not stated) Nakayama et al. [1991b]b Pre-op Age at initial study 0.98 0.22 <30 days 1–9 days (median 2 days) 0.20 (range: 0.16–0.39) Prenatal diagnosis or present at birth (n ¼ 5) Severe fetal left CDH Standard care (n ¼ 9) 6–10 hr 0.25 0.10 <24 hr Initial: 0.20 0.06 pre-op (6.4 1.8 days): 0.17 0.04 Fetal tracheal occlusion (n ¼ 11) <24 hr Initial: 0.22 0.18 pre-op: (5.5 2.5 days): 0.28 0.12 1 day: 0.19 (0.13–0.40) 4 days: 0.27 (0.15–0.46) 24 hr: 0.34 0.13 7–9 days: 0.54 0.13 24 hr: 0.24 0.19 pre-extubation (30 10 days of age): 0.51 0.16 24 hr: 0.36 0.25 pre-extubation (34 20 days of age): 0.93 0.45 Pre-op indicates prior to CDH repair; post-op, following CDH repair (timing refers to post-operative day unless otherwise indicated). 7/9 subjects decreased Crs after surgery. b Data are mean SEM. c Subjects not studied if on high frequency ventilation. Data are median and range. d All measures fell immediately post-op, but improved by 24 hr. a some degree of increased expiratory airway resistance [Nakayama et al., 1991b; Boas et al., 1996; Koumbourlis et al., 2006]. There is variable response to bronchodilator challenge, but this may become more consistent over time, with studies at a later time point demonstrating some degree of airway reactivity [Boas et al., 1996]. This obstructive lung disease may be a reflection of the small caliber airways demonstrated in CDH, differences in airway muscle function and/or airway remodeling with increases in airway smooth muscle [Broughton et al., 1998] and/or increasing functional abnormalities fol- lowing periods of prolonged mechanical ventilation. Pulmonary Function After Tracheal Occlusion We have previously presented data on acute lung function following FTO, from the subjects enrolled in our randomized, controlled clinical trial [Keller et al., 2004]. Table I shows the differences in respiratory system compliance between the control group (n ¼ 9) and the FTO group (n ¼ 11) at four important time points in the neonatal course. Over the newborn hospitalization, compliance improved in all surviving infants, however, it improved more rapidly in the FTO group than in the control group. Peak expiratory flows were consistently higher in the TO group at all time points, possibly reflecting decreased airway muscle under the influence of FTO. However, there were no differences in indices of ventilation through the peri-operative period (with PaCO2 tightly controlled) and there were no differences in minute ventilation between the groups. Oxygenation from birth through the peri-operative period was also modestly better in the FTO group [Keller et al., 2004]. ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c 193 TABLE II. Lung Volume Measurements in Infants With CDH at Various Time Points During Neonatal Hospitalization Lung volume (ml/kg) Study Nakayama et al. [1991b] Antunes et al. b Kavvadia et al. c Dinger et al.  Thibeault et al.  Inclusion a Age at initial study Pre-op FVC: 20.8 3.3 Respiratory distress <6 hr (n ¼ 8) All admitted (n ¼ 25) <30 days All delivered at center (n ¼ 16) Prenatal diagnosis or distress at birth (n ¼ 5) Requiring ECMO support (n ¼ 6) 1–9 days (median 2 days) FRC: All CDH: 10.0 6.0 survivors: 14.0 2.0 nonsurvivors: 4.5 1.0 FRC: 9.4 (3.6–14.7) 6–10 hr FRC: 10.6 1.7 Not stated FRC: 13.3 3.3 2.3 1.5 days (8 hr–8 days) Post-op 3 and 7 days: survivors: increased nonsurvivors: no change 1 day: 8.4 (5.9–13.3) 4 days: 8.3 (6.9–19.1) 24 hr: 13.0 3.5 7–9 days: 22.7 3.5 22.4 5.6d Pre-op indicates prior to CDH repair; post-op, following CDH repair (timing refers to post-operative day), FVC, forced vital capacity; FRC, functional residual capacity. a Data are mean SEM. Control FVC was 39.8 3.3 ml/kg. b Normal infant FRC is 26–31 ml/kg [Chu et al., 1967; Schwartz et al., 1978]. c Subjects not studied if on high frequency ventilation. Data are median and range. d One infant expired prior to repeat measurement. This subject had the lowest pre-op lung volume of 8.5 ml/kg. We also performed pulmonary function measurements during spontaneous sleep at one year follow up visits. Thirteen children were studied (seven standard perinatal care, six FTO). There were no significant differences between groups in any of the parameters: respiratory rate, tidal volume, minute ventilation, or peak expiratory flow. In both groups, respiratory rates were elevated (42.7 11.4 breaths/min, normal 26 breaths/min) and tidal volumes were decreased (4.2 2.0 ml/kg, normal 8.9 ml/kg) [American Thoracic Society/ European Respiratory Society, 1993; Cortes et al., 2005]. Oxygen saturation on room air by pulse oximetry was not different between groups; the range for control subjects was 90–100% and for FTO subjects it was 86–99%. All subjects from this trial required a prosthetic patch or muscle flap repair due to severe hemidiaphragm hypoplasia. The findings from our 1 year evaluation are consistent with a number of possible explanations related to lung function, but ipsilateral diaphragmatic function is likely also an issue. At long-term follow up (5–26 years), diaphragmatic function is impaired even in subjects that underwent primary closure of the diaphragmatic defect [Wohl et al., 1977; Arena et al., 2005b]. Long-Term Pulmonary Function Measurements Pulmonary function studies in the CDH population have been performed in older children and adults. There is quite a bit of range in the age of these subjects (6–49 years), reflecting survivors over many years of neonatal intensive care with probable variability of diaphragmatic defect severity. In early childhood, chronic medications for respiratory indications are used by 35–50% of children with CDH [Muratore et al., 2001; Cortes et al., 2005]. Further, children and adults with CDH report respiratory symptoms more frequently than in age-matched control subjects [Falconer et al., 1990; Trachsel et al., 2005]. As previously noted, lung volume measurements normalize in the first weeks of life and most measurements of lung volume are normal at later follow up [Wohl et al., 1977; Trachsel et al., 2005]. However, a subset of subjects has increased residual volume, and this finding has been associated with prolonged mechanical ventilation in the newborn period and persistent chest wall deformity [Wohl et al., 1977; Ijsselstijn et al., 1997; Trachsel et al., 2005]. Morphometric evaluation of the lungs of two children with CDH dying of unrelated causes (8 and 64 months of age) revealed enlarged and emphysematous alveoli with alveolar destruction bilaterally (affecting the ipsilateral lung to a greater extent than the contralateral lung) [Thurlbeck et al., 1979]. These studies likely explain the functional findings, and they are consistent with hyperlucencies appreciated on follow up chest radiographs [Wohl et al., 1977; Arena et al., 2005a] and gas trapping noted on ventilation scans [Jeandot et al., 1989]. The most common lung function abnormality in survivors of CDH is expiratory obstruction, with up to 50% of these subjects demonstrating a pattern consistent with obstructive lung disease 194 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c The most common lung function abnormality in survivors of CDH is expiratory obstruction, with up to 50% of these subjects demonstrating a pattern consistent with obstructive lung disease. [Falconer et al., 1990; Vanamo et al., 1996; Ijsselstijn et al., 1997; Marven et al., 1998; Trachsel et al., 2005]. Further, the airways are hyperresponsive, with an abnormal response to methacholine challenge in 38% of children [Ijsselstijn et al., 1997]. Restrictive lung disease has also been appreciated in the minority of subjects (approximately 30%) in some series [Vanamo et al., Restrictive lung disease has also been appreciated in the minority of subjects (approximately 30%) in some series. 1996; Stefanutti et al., 2004]. However, diffusion capacity appears to be normal in other studies [Ijsselstijn et al., 1997; Trachsel et al., 2005]. Exercise capacity in children with CDH (measured by exercise stress testing) is impaired, compared to age-matched controls [Zaccara et al., 1996; Marven et al., 1998; Trachsel et al., 2006]. These children demonstrate shorter duration of exercise and decreased maximal oxygen consumption. In less active children with CDH, these abnormalities are more pronounced [Zaccara et al., 1996]. In one series, children with CDH had decreased tidal volumes and increased respiratory rates when exercising at maximal capacity [Marven et al., 1998]. In another series, children with CDH and decreased exercise capacity (<80% predicted) had previous evidence of obstructive lung disease on pulmonary function testing, whereas children with normal exercise capacity did not have obstructive lung disease [Trachsel et al., 2006]. However, since children with CDH also have abnormalities of the pulmonary vascular bed, impaired exercise capability may reflect abnormal vascular function, which cannot be independently studied by non-invasive means. VASCULAR GROWTH AND FUNCTION IN NEWBORNS WITH CDH Evaluation of the pulmonary circulation has been an important part of neonatal management of infants with CDH in recent years. Several studies have evaluated neonatal branch PA dimensions and related them to survival. In nine infants with left CDH, an analysis of the ratio of the left PA/right PA diameter at admission revealed that in the six infants with a ratio <0.80, four expired. Five of the six infants had evidence of persistent pulmonary hypertension of the newborn (i.e., failure to transition to postnatal circulation, evidenced by right-to-left or bidirectional shunting at the ductus arteriosus). Three of these infants expired and all had met criteria for lung hypoplasia on post-mortem examination [Hasegawa et al., 1994]. The modified McGoon index is the sum of the left PA and the right PA dimensions, divided by the size of the descending aorta. In a retrospective series of 40 subjects with CDH with an echocardiogram at admission, survivors had a larger McGoon index than non-survivors (1.7 0.2 vs. 1.2 0.2, P < 0.05) [Suda et al., 2000]. A subsequent series (n ¼ 34) found a significantly higher mortality with higher McGoon index; a cut-off of 1.25 had a sensitivity of 73% and specificity of 78% for survival [Casaccia et al., 2006]. Interestingly, another approach has evaluated the ratio between left and right heart cardiac output in CDH by measuring the area of the pulmonic valve divided by the area of the aortic valve. While healthy controls had a flow ratio centered at 1.0, newborns with CDH that subsequently required ECMO support had evidence of increased right heart output ARTICLE compared to left with a mean ratio of approximately 3.0 [Baumgart et al., 1998]. These findings are consistent with studies documenting decreased fetal left ventricular size (in comparison to right ventricular size) in CDH, potentially due to a compression effect of the hernia mass, or reflecting antenatal hemodynamic alterations in pulmonary hypoplasia [Thebaud et al., 1997]. These findings likely also reflect right-to-left shunting via the patent ductus arteriosus, with blood bypassing the pulmonary circulation and the left heart. The findings are not inconsistent with a higher McGoon index among survivors, since in that case it is the descending aorta dimension that is considered. Persistent pulmonary hypertension of the newborn is a common problem in newborns with CDH, contributing to their acute cardiopulmonary instability. Persistent pulmonary hypertension of the newborn is a common problem in newborns with CDH, contributing to their acute cardiopulmonary instability. Persistent supra systemic elevation of PVR frequently after delivery compromises systemic oxygenation and oxygen delivery due to extra-pulmonary shunting and impaired myocardial function. This high resistance may persist due to the anatomical problem of a small vascular bed with abnormal arterial muscular structure, but there also may be functional abnormalities of the pulmonary vasculature. A single center study of echocardiographic diagnosis of persistent pulmonary hypertension in newborns with CDH described an incidence of 46% (21/46) [Bos et al., 1993]. Serial measures of pulmonary arterial pressure by echocardiogram have also been proposed as a parameter to evaluate for stabilization prior to CDH repair. In a small series of eight subjects, either a fall in PA pressure or a reversal of ductus arteriosus blood flow were taken ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c as evidence of fall in PVR, which occurred at 3–20 days of age [Haugen et al., 1991]. Other investigators have attempted to quantify the ratio of right ventricular systolic pressure or PA pressure to systemic blood pressure (SBP), with decreases in this ratio reflecting decreases in PVR. In 34 infants with CDH that required ECMO support, pulmonary or right ventricular pressures equaled SBP (ratio of PA pressure to SBP ¼ 1.0) while in 16 of 20 survivors, there was a fall in the right-sided pressures to 50% SBP within 11 days of instituting ECMO support. The additional infants had intermediate ranges of pulmonary hypertension (PA pressure to SBP ratios between 0.50 and 0.69) when they were taken off of ECMO support. Among non-survivors, the right ventricle to systemic pressure ratio decreased to 0.50 after 14 days of ECMO support in 8/14 infants. In the remainder, the ratio remained >0.50 when they were removed from ECMO support. On autopsy, infants with CDH had increased thickness of the intraacinar arterial walls compared to controls, with the ipsilateral lung more affected than the contralateral lung. This pattern persisted even in infants that expired with a right ventricle to systemic pressure ratio of 0.50 [Thibeault and Haney, 1998]. Iocono et al., used the same definition of elevated PA pressure: a right ventricle to systemic ratio 0.50. At the time of hospital discharge (mean 71 days), 7/40 infants had persistent elevations in their estimated PA pressure despite the prolonged hospitalization in this group of infants. One infant subsequently died at 13 months with pulmonary hypertension and cor pulmonale [Iocono et al., 1999]. Later data from this center suggested a stratification of prognosis based on the timing of resolution of the pulmonary hypertension. Twenty-three of 47 infants resolved their pulmonary hypertension by 3 weeks of age and all survived. Sixteen of the 47 infants had persistently elevated estimates of PA pressure (a 0.50 but less than 1.0 ratio of PA to systemic pressure) and 12 survived. Median time to resolution of pulmonary hypertension in this group was 49 days. Eight infants had persistent elevations of PA pressure estimated at greater than or equal to systemic pressure; there were no survivors among this group despite ECMO therapy [Dillon et al., 2004]. Kinsella et al.  defined late pulmonary hypertension in CDH based on a threshold of estimated PA pressure 2/3 systemic pressure at the time of elective tracheal extubation (26 3 days); 10/42 infants met these criteria. These studies do not reveal information about pulmonary vascular function or pulmonary vascular tone. That is, does the vasculature have the ability to dilate to accommodate increases in cardiac output and does the vasculature respond to pulmonary vasodilator challenge? In patients who cannot cooperate with testing (or those with both cardiovascular and pulmonary disease) there is no definitive methodology for evaluating function. Therefore, we can only test the response to pulmonary vasodilator challenge. Early experiences with PA catheterization in the management of newborns with CDH demonstrated that, in some patients, the pulmonary circulation could vasodilate in response to sedation, high-inspired oxygen concentration and vasodilators. In these situations, pulmonary blood flow increased and oxygenation improved, although pulmonary arterial pressure did not always fall [German et al., 1977; Vacanti et al., 1984]. Bos et al., challenged newborns with CDH and persistent pulmonary hypertension of the newborn with two vasodilators: tolazoline and prostacyclin. There were no improvements in oxygenation with tolazoline (which, as a non-specific vasodilator also resulted in lower SBP). After prostacyclin challenge, oxygenation did improve and SBP was unchanged [Bos et al., 1993]. Subsequent studies of inhaled nitric oxide (a specific pulmonary vasodilator) during the acute management of newborns with CDH, have demonstrated sustained improvements in oxygenation in only 10–15% of subjects [Kinsella et al., 1997; The Neonatal Inhaled Nitric Oxide Study Group, 1997]. In using the change in PVR as the measure of response to inhaled nitric oxide, we 195 have documented significant pulmonary vasodilation during cardiac catheterization in three children with CDH at later time points (9 weeks to 11 years of age) [Keller et al., 2006]. Therefore, we suspect that abnormal pulmonary vascular tone is a component of pulmonary hypertension in a subset of patients with CDH and vascular hypoplasia. But, we do not know if and when specific treatment for pulmonary hypertension is beneficial in this patient population. Long-term follow up studies of abnormal vascular function in this patient population are difficult, due to compromise of both cardiovascular and pulmonary function. Schwartz et al., evaluated pulmonary hypertension in 21 children (3.2 1.4 years) that survived CDH and neonatal ECMO. They measured right ventricle systolic time intervals by echocardiogram and performed exercise testing in the children with abnormal echocardiograms. Eight children (38%) met criteria for pulmonary hypertension. Five of these children had abnormal exercise tests and seven were under treatment for chronic respiratory disease [Schwartz et al., 1999]. There is additional information available, however, as to the long-term development of the pulmonary vascular tree. Multiple groups of investigators have evaluated differential lung perfusion (ipsilateral vs. contralateral lung) or ventilation or volume to perfusion ratios in CDH. In late studies (6–18 years of age), the proportion of ventilation to the ipsilateral lung normalizes, whereas perfusion remains decreased, indicating failure of full development of the pulmonary vascular tree. Ipsilateral perfusion in these subjects was reduced, averaging 39% and 42% (with normal left lung perfusion approximately 46%) [Wohl et al., 1977; Falconer et al., 1990]. Children with CDH that were followed serially from the first several months of life to 6 years of age showed initial low perfusion and ventilation (or volume) in the ipsilateral lung. Although lung ventilation/volume tended to improve at follow up studies at age 1–6 years, perfusion remained low (or decreased), resulting in low perfusion to ventilation ratios. These findings were particularly 196 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c pronounced when the neonatal course and/or the anatomic findings were more severe (worse indices of oxygenation, prolonged mechanical ventilation and need for ECMO support or prosthetic patch repair of CDH) [Jeandot et al., 1989; Nagaya et al., 1996; Okuyama et al., 2006; Hayward et al., 2007]. Further, Okuyama and colleagues found an association between worse growth indices at one and two years of age and more asymmetric perfusion at initial and follow up studies [Okuyama et al., 2006]. We studied five children with severe left CDH (liver herniated into the chest and prosthetic patch repair of diaphragmatic defect). The mean perfusion to the left lung was 28% (22–37%, normal is approximately 46%) at 21– 38 days of age (unpublished work). Follow up studies in two children at 13 and 20 months showed no change (left lung perfusion 20% and 34%). This lack of growth of the pulmonary vascular tree may be more pronounced in severe CDH, consistent with a ‘‘pruning’’ of the pulmonary vascular tree and loss of microvasculature development seen by post-mortem angiogram in infants that die late in infancy, which does not appear in infants dying early in their course (Fig. 3). IMPLICATIONS OF ANATOMICAL AND FUNCTIONAL STUDIES OF THE LUNGS IN HUMAN CDH It is now clear that abnormal development of the diaphragm and lung in utero leads to chronic cardiopulmonary morbidity. In the acute neonatal period, there is a major focus on maintaining sufficient cardiopulmonary function for adequate oxygen delivery and survival, yet aggressive neonatal management practices that lead to lung injury have not served this group of patients well. Survival has increased with more gentle and permissive standards for both ventilation and oxygenation [Kays et al., 1999; Bohn, 2002]. It is likely longterm cardiopulmonary function in survivors also benefits from these practices, since later abnormalities in lung and vascular structure and function often follow a more severe neonatal clinical course, and reflect lung injury, airway and alveolar remodeling and impaired vascular growth. The implementation of these management standards is a challenge, as they require understanding and commitment from all intensive care staff members. ARTICLE It is still not clear if the potential lung growth effects of FTO will benefit the most severely affected fetuses. It is still not clear if the potential lung growth effects of FTO will benefit the most severely affected fetuses. The completion of our randomized trial [Harrison et al., 2003] was limited by at least two factors. First, survival in the control group was twice what was expected from our historical experience. This may have been the result of the consistent care delivered at our high volume center. Second, significant premature delivery likely affected the outcomes in the FTO group. Pre-term delivery has recently become less significant with less invasive complete endoscopic techniques enabled by the development of newer instruments [Deprest et al., 2004]. Thus, further studies employing these techniques in high volume experienced centers may be warranted, since survival in the highest risk fetal groups (based on liver herniation and LHR) remains below Figure 3. Post-mortem angiograms from infants with severe left diaphragmatic hernia obtained by perfusing the branch pulmonary arteries with barium gelatin under 100 cmH2O pressure. A: Demonstrates the right (R) lung of an infant dying at 15 days of age from hemorrhagic complications while receiving ECMO support. Note the well-developed peripheral pulmonary vasculature. B: Demonstrates the right (R) and left (L) lungs of an infant dying at 79 days of age with chronic lung disease, pulmonary hypertension, and cor pulmonale. Note severe ‘‘pruning’’ of the distal pulmonary vascular tree. ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c 60%. Additional fetal anatomical and functional studies may also be able to better define this high-risk group (enhancing selection criteria for fetal intervention) and long-term evaluation of survivors must occur. Alternatively, postnatal strategies to enhance lung growth should be studied in a controlled manner in severely affected infants. These may include trophic or anti-inflammatory agents administered directly to the lung (e.g., inhaled nitric oxide or perfluourocarbon [Hirschl et al., 2003]) or systemically (phosphodiesterase inhibitors or endothelin receptor antagonists). Again, timing of the intervention and appropriate selection criteria will be important, as many therapies also have the potential for harm in this vulnerable patient population. 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