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Fetal aortic stenosis and changes in amniotic fluid natriuretic peptides
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BASIC SCIENCE: OBSTETRICS
Fetal aortic stenosis and changes in amniotic fluid natriuretic peptides Walter C. Lubbers, BS; Pirooz Eghtesady, MD, PhD OBJECTIVE: Natriuretic peptides, especially brain natriuretic peptide (BNP), have demonstrated great usefulness in pediatric and adult cardiology. We studied their usefulness, based on amniotic fluid concentrations, in an ovine model of fetal aortic stenosis and in response to fetal cardiac intervention. STUDY DESIGN: After their natural history was established with gestation (n ⫽ 18 fetuses), natriuretic peptide levels were measured in a fetal model of aortic stenosis (50-60 days; term, 148 days; n ⫽ 9) and were correlated to the severity of fetal heart disease. Response to fetal cardiac intervention in 3 hydropic fetuses was also assessed. Significance was established with 2-sided paired t-tests at a probability value of ⬍.05.
RESULTS: Amniotic fluid BNP (but not atrial natriuretic peptide) concentrations were elevated significantly with aortic stenosis (181.9 ⫾ 109.9 pg/mL vs 50.0 ⫾ 40.5 pg/mL in control fetuses), especially if complicated with hydrops (283 ⫾ 74.4 pg/mL), and were correlated positively with the severity of stenosis and left ventricle hypertrophy. In the 1 animal surviving fetal intervention, BNP levels normalized. CONCLUSION: Amniotic fluid BNP concentrations correlate with the severity of fetal aortic stenosis.
Key words: fetal intervention, natriuretic peptide, congenital, ovine
Cite this article as: Lubbers WC, Eghtesady P. Amniotic fluid brain natriuretic peptide in an ovine model of fetal aortic stenosis. Am J Obstet Gynecol 2007; 196:253.e1-253.e6.
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ignificant knowledge has been gained regarding the natural history and outcome of congenital heart defects. Several risk factors that indicate poor prognosis have also been identified, such as the presence of chromosomal abnormalities, arrhythmias, or congestive heart failure as revealed by fetal hydrops.1-4
From the Department of Biomedical Engineering, University of Cincinnati (Mr Lubbers), and the Department of Surgery, University of Cincinnati College of Medicine, and the Division of Cardiothoracic Surgery, Cincinnati Children’s Hospital Medical Center (Dr Eghtesady), Cincinnati, OH. Presented at the 27th Annual Clinical Meeting of the Society for Maternal-Fetal Medicine, Feb. 5-10, 2007, San Francisco, CA. Received Dec. 8, 2006; revised Jan. 1, 2007; accepted Jan. 4, 2007. Reprints not available from the authors. Supported by the Thrasher Research Foundation and the Cincinnati Children’s Hospital Research Foundation Translational Research Initiative. 0002-9378/$32.00 © 2007 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2007.01.003
Despite these advances, the assessment of prognosis based on anatomy and presentation in utero remains limited, partly because signs of fetal heart failure, such as hydrops or valvular regurgitation that make such appraisals difficult. Further, given the differences in circulatory physiologic condition, some indicators of worsening cardiac function in postnatal life, such as flow and pressure, may not be measured easily in the fetus.5 Clinicians have developed various tools, such as the fetal cardiovascular score, to overcome these limits,5,6 yet assessment of fetal cardiovascular health remains a largely subjective and qualitative practice. A quantitative indicator of fetal cardiovascular health could complement data that is gained by fetal echocardiography and improve the effectiveness of fetal cardiovascular assessment and treatment. Such a prognostic tool may also be helpful for the advancement of fetal therapeutics.7,8 Natriuretic peptides, especially brain natriuretic peptide (BNP), have acquired this important complementary role in both pediatric and adult cardiology; point-of-care testing is now a routine part of clinical care. Significantly less is known about these peptides in fetal
and neonatal cardiac impairment. Besides their roles in regulating fluid balance and blood pressure,9-12 atrial natriuretic peptide (ANP) and BNP play an important part in the developing fetal circulatory system. Newborn infants in cardiac distress can have substantially increased umbilical plasma BNP concentrations.13,14 Similarly, increased plasma BNP concentrations have been linked with a number of congenital cardiovascular diseases in neonates and children.15,16 We have been interested in the potential usefulness of natriuretic peptides in fetuses who are affected by heart disease. We questioned whether amniotic fluid (AF) natriuretic peptide levels would correlate with severity of fetal cardiac distress, perhaps providing a complementary tool in the evaluation of fetal cardiac health. We previously had developed a model of fetal aortic stenosis in first-trimester fetal sheep, in which the severe pressure overload of the immature left ventricle (LV) led to a dichotomous phenotype of either compensatory LV hypertrophy or noncompensated LV dysfunction with associated fetal hydrops.17 To test our hypothesis, we measured ANP and BNP concentrations in
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FIGURE 1
Flow chart of animals in study Total number of fetuses = 38 (24 singletons; 7 sets of twins) Natural history Controls n=18
Aortic stenosis Experimental Sham-operated n=9 n=11 LV hypertrophy n=6
Fetal Hydrops n=3 Fetal intervention n=3
The number of fetuses in each experimental group.
the AF of animals from this experimental model. We also measured baseline concentrations of ANP and BNP in the blood and AF of the healthy ovine fetus and pregnant ewe to provide comprehensive data on the natural history of natriuretic peptides during gestation.
M ETHODS Animal care All animal procedures complied with the Cincinnati Children’s Hospital Research Foundation Institutional Animal Care and Use Committee standards. Timedated Suffolk-cross pregnant ewes (n ⫽ 31: 24 singletons, 7 sets of twins; total, 38 fetuses) were housed communally, had free access to food and water, and were allowed free movement. Animals were kept on a 12-hour light/dark cycle. The number of fetuses in each part of the study is shown in Figure 1.
Natural history of natriuretic peptide release To study the natural history of ANP and BNP release during normal ovine gestation, maternal and fetal blood and AF samples were obtained at 40% gestation (mean, 65 days; term, 145-148 days), 70% gestation (mean, 105 days), and at term (mean, 142 days) in a total of 18 animals (all singletons). Maternal blood 253.e2
samples were also taken from nonpregnant ewes.
Sample collection Pregnant ewes were anesthetized with ketamine and valium and maintained with isoflurane (2-2.5% minimal alveolar concentration) and fentanyl (10 g/ kg). Maternal blood samples were obtained through catheters that were placed in the jugular vein. A laparotomy and limited hysterotomy were performed, and AF (15 mL) was collected in polypropylene tubes that contained protease inhibitor (Complete, Mini; Roche Diagnostics, Mannheim, Germany). The fetus was exteriorized partially, and fetal blood samples (1 mL) were obtained from right internal jugular vein catheterization. Both fetal and maternal blood samples were collected in vacuum tubes that contained EDTA (BD Vacutainer, Franklin Lakes, NJ) and protease inhibitor. Both AF and blood samples were centrifuged at 1600 g at 4°C for 15 minutes. The supernatant was aliquoted in 1-mL samples and frozen until assayed.
Surgical preparation The surgical preparation of fetal lambs in our model of aortic stenosis has been described previously.17 Briefly, at 50-60 days of gestation (mean, 56 days), the fe-
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www.AJOG.org tus was exteriorized, and a left thoracotomy was performed after an AF sample was removed. A Dacron band was placed on the ascending aorta, then the thoracotomy was closed. The fetus was returned to the uterus, and lost AF in the uterus was replaced with warm saline solution that contained gentamycin and duacillin. During the remainder of gestation, we assessed fetal biophysical profiles and fetal hemodynamics with serial ultrasound measurements. Sham-operated control animals underwent the same surgical procedure, except that no band was placed about the aorta. At approximately 140 days estimated gestational age, a repeat laparotomy and hysterotomy were performed, and a second AF sample was collected. The fetus and ewe were then killed, and the fetus was removed for pathologic studies. In our animal model, the development of hydrops uniformly signals impending fetal death.17 For this reason, in the animals in which fetal hydrops developed (n ⫽ 3), fetal intervention was carried out within 2 weeks after onset of hydrops (approximately 100-110 days of gestation). Fetal intervention was successful in 1 case. Ultrasound examination revealed the development of fetal hydrops at 100 days of gestation in this animal; fetal intervention was carried out at 107 days of gestation. The other hydropic fetuses did not survive fetal intervention. To carry out our fetal surgical intervention, the ewe was anesthetized, the fetus was exposed, and a second thoracotomy was made through which the band was removed from the fetal aorta. An AF sample was obtained at this time. Incisions were closed, and the fetus was returned to uterus and allowed to grow to term; data collection was performed as mentioned earlier.
Natriuretic peptide measurement ANP and BNP concentrations were determined according to the manufacturer’s instructions with immunoassay kits (Peptide Enzyme Immunoassay ␣-ANP 1-28 [Human, Canine], and Peptide Enzyme Immunoassay BNP-26 [Porcine]; Bachem Bioscience Inc, King of Prussia, PA) and a microplate reader
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www.AJOG.org (Vmax Kinetic Microplate Reader; Molecular Devices, Sunnyvale, CA). Per the manufacturer’s specifications, these kits show 0% cross-reactivity for the nontarget natriuretic peptide (ie, 0% cross reactivity of the ANP kit for BNP and viceversa). All samples were assayed in duplicate. Interassay variation for both ANP and BNP was within the stated limits of the respective detection kit (⬍5%), with an average error of 10.2% for ANP and 8.4% for BNP.
FIGURE 2
Natriuretic peptide during gestation
Pathologic measurements Measurements of pathologic specimens were made after the animal was dead and included total fetal weight, weights of the fetal heart and LV (including septum), and circumferences of the mitral, aortic, tricuspid, and pulmonary valves along with other cardiac morphometry, as described previously.17 LV weight and valve diameters were normalized to total heart weight for each fetus. The LV hypertrophy was assessed with the use of a previously validated index,5,18 wherein the fraction of fetal heart weight comprised by the LV is calculated. Severity of LV hypertrophy is then indexed as any LV mass ⬎40% (⬎2 SD from mean control values).
Statistical analysis Statistical analyses were performed by the Director of the Statistical Consulting Laboratory, University of Cincinnati, with SAS software (SAS Institute Inc, Cary, NC). Values for groups are shown as mean ⫾ SD per group. Significance was established with a 2-sided unpaired t-test. Values were considered significant with a probability value of ⬍.05.
R ESULTS Fetal biophysical profile Basic biophysical measurements of fetuses (n ⫽ 18) were taken at 40%, 70%, and term gestation. Fetuses at 40% gestation had an average crown-to-rump length of 17.4 ⫾ 4.3 cm, a biparietal diameter of 4.7 ⫾ 1.1 cm, an anterior posterior (AP) chest diameter of 3.4 ⫾ 1.0 cm, and an average weight of 314 ⫾ 11.5 gm (the fetal weights were derived from fetuses that did not survive banding).
A, ANP and B, BNP concentration in AF and fetal and maternal blood throughout fetal gestation. Error bars show standard deviation. The closed squares represent the AF; the closed circles represent fetal blood; and the open triangles represent maternal blood.
Average fetal weight at 70% gestation was 1.94 ⫾ 0.75 kg. Average fetal weight at term was 4.47 ⫾ 1.47 kg.
Natural history of AF natriuretic peptides ANP concentration in AF. The average ANP concentration in AF samples at 40% gestation was 9.77 ⫾ 10.17 pg/mL, which increased to 51.25 ⫾ 8.54 pg/mL at 70% gestation (Figure 2A). The ANP levels then decreased to 45 ⫾ 33.9 pg/mL at term. ANP concentration in fetal and maternal plasma. ANP levels in fetal and maternal plasma remained relatively stable through the first 70% of gestation then significantly increased in the final 30% (Figure 2A). ANP levels in fetal plasma were unchanged between 40% (71.5 ⫾ 19.89 pg/mL) and 70% (70 ⫾ 28.98 pg/mL) gestation but increased significantly (P ⬍ .05) by term (228 ⫾ 36.6 pg/mL). Similarly, maternal plasma ANP levels were not significantly different among nonpregnant ewes (114 ⫾ 50.8 pg/mL), ewes at 40% gestation (126
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⫾ 40.5 pg/mL), and ewes at 70% gestation (93.33 ⫾ 15.27 pg/mL) but were significantly (P ⬍ .05) increased at term (176.6 ⫾ 23.4 pg/mL). BNP concentration in AF. The AF BNP concentration at 40% gestation was 9.28 ⫾ 13.98 pg/mL (Figure 2B). BNP levels increased significantly (P ⬍ .05) at 70% gestation to 46.7 ⫾ 25.03 pg/mL and remained at similar levels until term (50.0 ⫾ 40.5 pg/mL). BNP concentration in fetal and maternal plasma. BNP concentrations in fetal plasma were not significantly altered among 40% gestation (105 ⫾ 31.09 pg/mL), 70% gestation (118 ⫾ 75.9 pg/ mL), and at term (76.67 ⫾ 30.1 pg/mL; Figure 2B). Moreover, maternal blood BNP concentration at 40% gestation (186 ⫾ 82.3 pg/mL), 75% gestation (210 ⫾ 79.6 pg/mL), and at term (143 ⫾ 55 pg/mL) was not significantly different from concentrations in nonpregnant ewes (122 ⫾ 75.6 pg/mL).
Fetal aortic stenosis and AF natriuretic peptides Changes in AF natriuretic peptide levels with fetal aortic stenosis. In our model of aortic stenosis, 1 of 2 distinct phenotypes was induced: (1) survival to term with significant compensatory LV hypertrophy (n ⫽ 6) or (2) development of noncompensated heart failure, as evidenced by fetal hydrops at 100-110 days of gestation (n ⫽ 3), which is followed by fetal death within 1 or 2 weeks.17 Therefore, ANP and BNP levels were compared in animals that were divided into 1 of these 2 phenotype groups and in sham-operated control fetuses (n ⫽ 6) of similar gestational ages (Figure 3). In fetuses with aortic stenosis, ANP concentrations in AF at term (43.3 ⫾ 17.5 pg/mL) were not significantly different from those in control animals (45 ⫾ 33.9 pg/mL; Figure 3A). In contrast, in AF of fetuses with severe aortic stenosis, BNP concentrations (181. 9 ⫾ 109.9 pg/ mL) were nearly 4 times greater than concentrations in control animals (50 ⫾ 40.5 pg/mL; Figure 3B). This difference was statistically significant (P ⬍ .05). Animals with aortic stenosis experienced varying degrees of LV hypertro-
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FIGURE 3
FIGURE 4
FIGURE 5
Natriuretic peptides with fetal aortic stenosis
Correlation of LV hypertrophy with AF BNP levels
AF natriuretic peptide with hydrops
LV weight as a percentage of total fetal heart weight vs AF BNP concentration. Results from both banded and control animals are shown. LV hypertrophy was defined as LV weight being ⬎40% of total heart weight as shown by the dashed line. All points that corresponded to LV weights of ⬎40% correspond to banded animals, and all LV weights of ⱕ40% correspond to controls.
Average A, ANP and B, BNP concentrations in AF of experimental (banded) and control animals at term. Error bars show standard deviation. Differences in BNP concentration between groups were statistically significant (P ⬍ .05).
phy, as expected.17 None of the hydropic fetuses experienced LV hypertrophy; rather, they all experienced LV wall thinning, which was associated with severe LV dilation. AF BNP concentrations positively correlated (r2 ⫽ 0.6751) with the severity of LV hypertrophy (Figure 4). In contrast, AF ANP concentrations did not correlate with the developed LV hypertrophy (data not shown). No other physiologic parameters that were measured (valve diameters, fetal weight, organ weights) correlated with increased BNP or ANP concentrations. Of the 3 banded animals that experienced hydrops, 2 animals were from twin gestations, and 1 animal was a singleton gestation. Aortic stenosis in these animals led to the development of hydrops at approximately 100-110 days of gestation (mean, 45 days after banding). ANP concentrations in AF of hydropic fetuses (including the singleton hydropic twin that survived fetal intervention) were in253.e4
creased notably in comparison with their nonhydropic twins (99.0 ⫾ 37.5 pg/mL vs 25.0 ⫾ 21.0 pg/mL) and normal controls of the same gestational age (51.25 ⫾ 33.9 pg/mL; Figure 5A). These differences, however, did not reach statistical significance. In contrast, BNP concentrations significantly (P ⬍ .05) increased in the AF of hydropic animals, when compared with levels in nonhydropic twins (283 ⫾ 74.4 pg/mL vs 90 ⫾ 60.8 pg/mL; Figure 5B) and when compared with BNP concentrations in normal control animals of the same gestational age (46.7 ⫾ 40.5 pg/mL). Of note, the BNP values in these hydropic fetuses were significantly higher than the same measurements in animals with only compensatory LV hypertrophy (and no LV dilatation). Changes in AF natriuretic peptide levels in response to fetal intervention. Previous experiments with our model have revealed that fetal death follows the onset of fetal hydrops within 1 to 2 weeks. We therefore tested the effect of fetal intervention in these animals. We were able to prevent fetal death and restore normal cardiac function in 1 hydropic fetus by removing the band at 107 days gestation; 2 other hydropic animals did not survive the intervention (66% mortality rate). AF ANP concentrations in the surviving fetus were elevated at in-
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Average A, ANP and B, BNP concentrations in AF of hydropic banded fetuses, their nonhydropic control twins, and normal controls at 90-100 days of gestation. Error bars show standard deviation. ANP concentrations in hydropic fetuses and twins were not significantly different from control values. BNP concentrations in hydropic fetuses were statistically different from twin and control values, but twin values were not significantly different from controls.
tervention (125 pg/mL), similar to other hydropic fetuses (Figure 6A). The AF BNP concentration was also increased (205 pg/mL) in this animal at intervention, compared with control values (51.25 pg/mL), which is also similar to the other hydropic fetuses (Figure 6B). After fetal intervention, by the end of gestation, the BNP concentrations had decreased significantly but were still slightly increased (75 pg/mL; compared with control animals at term). Because these values represent data from only 1 animal, no statistical analysis was performed.
C OMMENT Our study, first, delineates the natural history of AF natriuretic peptides with
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FIGURE 6
AF natriuretic peptides with fetal intevention
A, ANP and B, BNP concentrations at the time of surgical intervention (removal of band from ascending aorta at 107 days gestation) and at term (140 days gestation) for a fetus with hydrops because of induced aortic stenosis.
gestation; second, demonstrates changes in concentrations of the peptides with induced fetal aortic stenosis; and, third, shows the fetal response and changes in the peptides with fetal cardiac intervention. These findings have potential implications with regard to fetal aortic stenosis and its potential management through fetal cardiac intervention. Data regarding amniotic natriuretic peptide concentrations, especially BNP, in either animal models or in humans are limited, although the results that were observed in our study parallel those seen in previous studies of humans and sheep.11,19,20 In our model of fetal aortic stenosis, significant pressure gradients developed across the LV outflow tract that correlate with increases in fetal left ventricular end-diastolic pressures. Similarly, an increase in LV mass is seen, which is directly proportional to the severity of stenosis.17 In this study, we found the
Basic Science: Obstetrics greater the degree of hypertrophy (ie, more severe the stenosis), the greater the rise in BNP concentrations. Further, we observed more dramatic increases in AF BNP levels in animals that experienced fetal hydrops, which is a sign of severe fetal cardiac distress and impending fetal death. In particular, BNP levels of hydropic fetuses were substantially higher than those with compensatory hypertrophy, especially when adjusted to gestational age (ie, the BNP levels are significantly elevated at a time in gestation when normally not much BNP is seen). These results suggest that AF BNP levels correlate with severity of fetal aortic stenosis. In adults with asymptomatic aortic stenosis, BNP levels correlate with outcome and, hence, have prognostic value in clinical decision making.21,22 Further studies are needed to determine whether AF BNP levels provide a similar value in the fetus with aortic stenosis or cardiac disease in general. For example, a “threshold” level may exist to foreshadow ensuing fetal hydrops and death. Such knowledge would be helpful for ongoing experimental fetal cardiac interventions, complementing data that are obtained from fetal echocardiography. An example of this potential use of amniotic BNP analysis may be found in our observation that the resolution of aortic stenosis and fetal cardiac distress (with fetal intervention) can lead to the restoration of normal amniotic BNP and ANP concentrations. Not only did fetal cardiac function recover, but also the elevated BNP concentrations and the fetal hydrops resolved through prenatal intervention in a fetus with induced aortic stenosis. Although this single example is not conclusive, we believe it suggests that BNP concentrations in AF may be useful as indicators of fetal cardiac health both before and after fetal intervention. If confirmed, this finding may have relevance to advancing experimental fetal cardiac interventions by indicating whether poor outcomes after “technically” successful interventions occur because of inadequate surgical correction or because the fetal heart is damaged already beyond repair.8
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Our observations correlate well with previously reported data, which were all obtained near term, for the healthy fetus, the pregnant ewe, and the fetus in distress.13,14,18,21,23 To our knowledge, our data represents the first natural history (and modified natural history) of fetal natriuretic peptide levels from early gestation onward. It would seem reasonable that AF natriuretic peptides may be of maternal origin; however, studies have now shown that ANP and BNP in fetal blood and AF are of fetal origin and, as seen here, do not correlate to maternal plasma concentrations.20,23 It is also remarkable that, in twin pairs in which 1 fetus experienced fetal hydrops, the nonhydropic twin may “sense” the distress of its sibling, as displayed by the rise in BNP levels, despite the fact that the twins do not share placentae. Additional experiments may help to clarify the involved mechanisms. A limitation of our study is that our animal model does not fully mimic fetal aortic stenosis as seen clinically. We currently are conducting a clinical trial to assess the potential relevance of AF BNP in humans. Another limitation of our natural history data is that it pertains to singleton gestation animals, although our limited data from control animals (all singletons) and sham-operated animals (twins and singletons) do not suggest substantial differences in the natriuretic peptide values at term. It is conceivable that, earlier in gestation than measured in our study, twin animals would have different AF BNP levels compared with singletons. Additionally, the origin of the AF BNP measured in the study is unknown; although it is assumed that most of AF BNP comes from fetal urine, the placenta and amniotic tissues cannot be ruled out as potential sources. Little published data are available regarding BNP levels in fetal urine, and, as such, this represents another avenue of research that we would like to explore. Along the same lines, it would be of interest to measure BNP concentrations in AF and fetal urine after the injection of BNP into fetal circulation, but currently only synthetic human BNP (Natrecor) is available for such an experiment.
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In summary, we have shown in an ovine model that the measurement of AF BNP concentrations has significant potential as a complementary tool for the assessment of fetal cardiac distress. If future experiments suggest BNP levels can be predictive of subsequent hydrops and if similar elevations can be demonstrated in affected human pregnancies, AF BNP levels may prove to be especially useful as quantifiable measures of fetal cardiac health and perhaps as added indicators of the need and timing for fetal intervention. f ACKNOWLEDGMENTS We thank Dr Anoop Brar and Scott Baker for insightful discussions and input in the preparation of this article.
REFERENCES 1. Sahn DJ, Shenker L, Reed KL, et al. Prenatal ultrasound diagnosis of hypoplastic left heart syndrome in utero associated with hydrops fetalis. Am Heart J 1982;104:1368-72. 2. Bronshtein M, Zimmer E, Gerlis LM, et al. Early ultrasound diagnosis of fetal congenital heart defects in high-risk and low-risk pregnancies. Obstet Gynecol 1993;82:225-9. 3. Harada T, Nagai R. The genetic disorders responsible for sudden cardiac death. Nippon Rinsho 2005;63:1273-83. 4. Soares G, Alvares S, Rocha C, et al. Congenital heart defects and chromosomal anomalies including 22q11 microdeletion (CATCH 22). Rev Port Cardiol 2005;24:349-71.
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5. Huhta JC. Guidelines for the evaluation of heart failure in the fetus with or without hydrops. Pediatr Cardiol 2004;25:274-86. 6. Huhta JC. Right ventricular function in the human fetus. J Perinat Med 2001;29:381-9. 7. Marshall AC, van der Velde ME, Tworetzky W, et al. Creation of an atrial septal defect in utero for fetuses with hypoplastic left heart syndrome and intact or highly restrictive atrial septum. Circulation 2004;110:253-8. 8. Tworetzky W, Wilkins-Haug L, Jennings RW, et al. Balloon dilation of severe aortic stenosis in the fetus: potential for prevention of hypoplastic left heart syndrome: candidate selection, technique, and results of successful intervention. Circulation 2004;110:2125-31. 9. Cameron VA, Ellmers LJ. Minireview: natriuretic peptides during development of the fetal heart and circulation. Endocrinology 2003; 144:2191-4. 10. Cameron VA, Aitken GD, Ellmers LJ, Kennedy MA, Espiner EA. The sites of gene expression of atrial, brain, and C-type natriuretic peptides in mouse fetal development: temporal changes in embryos and placenta. Endocrinology 1996;137:817-24. 11. Cheung CY, Gibbs DM, Brace RA. Atrial natriuretic factor in maternal and fetal sheep. Am J Physiol 1987;252:E279-82. 12. Walther T, Schultheiss HP, Tschope C, Stepan H. Natriuretic peptide system in fetal heart and circulation. J Hypertens 2002;20:785-91. 13. Itoh H, Sagawa N, Hasegawa M, et al. Brain natriuretic peptide levels in the umbilical venous plasma are elevated in fetal distress. Biol Neonate 1993;64:18-25. 14. Walther T, Stepan H, Faber R. Dual natriuretic peptide response to volume load in the fetal circulation. Cardiovasc Res 2001;49: 817-9.
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www.AJOG.org 15. Reynolds EW, Ellington JG, Vranicar M, Bada HS. Brain-type natriuretic peptide in the diagnosis and management of persistent pulmonary hypertension of the newborn. pediatrics. Pediatrics 2004;114:1297-304. 16. Kunii Y, Masahiro K, Ohtsuki S, et al. Plasma brain natriuretic peptide and the evaluation of volume overload in infants and children with congenital heart disease. Acta Med Okayama 2003;57:191-7. 17. Eghtesady P, Michelfelder E, Jones F, et al. Aortic stenosis in the ovine fetus: a model for fetal intervention? Pediatr Cardiol 2003;24:624. 18. Norman JE, Levy D. Improved electrocardiographic detection of echocardiographic left ventricular hypertrophy: results of a correlated data base approach. J Am Coll Cardiol 1995;26:1022-9. 19. Di Lieto A, Pollio F, Catalano D, et al. Atrial natriuretic factor in amniotic fluid and in maternal venous blood of pregnancies with fetal cardiac malformations and chromosomal abnormalities. J Matern Fetal Neonatal Med 2002;11:183-7. 20. Itoh H, Sagawa N, Hasegawa M, et al. Brain natriuretic peptide is present in the human amniotic fluid and is secreted from amnion cells. J Clin Endocrinol Metab 1993;76:907-11. 21. Bergler-Klein J, Klaar U, Heger M, et al. Natriuretic peptides predict symptom-free survival and postoperative outcome in severe aortic stenosis. Circulation 2004;109:2302-8. 22. Lim P, Monin JL, Monchi M, et al. Predictors of outcome in patients with severe aortic stenosis and normal left ventricular function: role of B-type natriuretic peptide. Eur Heart J 2004;25:2048-53. 23. Deloof S, VanCamp G, Chatelain A. Absence of transplacental transfer of atrial natriuretic peptide in the rat: direct experimental evidence. Med Sci Res 1995;23:347-50.
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Fetal aortic stenosis and changes in amniotic fluid natriuretic peptides Walter C. Lubbers, BS; Pirooz Eghtesady, MD, PhD OBJECTIVE: Natriuretic peptides, especially brain natriuretic peptide (BNP), have demonstrated great usefulness in pediatric and adult cardiology. We studied their usefulness, based on amniotic fluid concentrations, in an ovine model of fetal aortic stenosis and in response to fetal cardiac intervention. STUDY DESIGN: After their natural history was established with gestation (n ⫽ 18 fetuses), natriuretic peptide levels were measured in a fetal model of aortic stenosis (50-60 days; term, 148 days; n ⫽ 9) and were correlated to the severity of fetal heart disease. Response to fetal cardiac intervention in 3 hydropic fetuses was also assessed. Significance was established with 2-sided paired t-tests at a probability value of ⬍.05.
RESULTS: Amniotic fluid BNP (but not atrial natriuretic peptide) concentrations were elevated significantly with aortic stenosis (181.9 ⫾ 109.9 pg/mL vs 50.0 ⫾ 40.5 pg/mL in control fetuses), especially if complicated with hydrops (283 ⫾ 74.4 pg/mL), and were correlated positively with the severity of stenosis and left ventricle hypertrophy. In the 1 animal surviving fetal intervention, BNP levels normalized. CONCLUSION: Amniotic fluid BNP concentrations correlate with the severity of fetal aortic stenosis.
Key words: fetal intervention, natriuretic peptide, congenital, ovine
Cite this article as: Lubbers WC, Eghtesady P. Amniotic fluid brain natriuretic peptide in an ovine model of fetal aortic stenosis. Am J Obstet Gynecol 2007; 196:253.e1-253.e6.
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ignificant knowledge has been gained regarding the natural history and outcome of congenital heart defects. Several risk factors that indicate poor prognosis have also been identified, such as the presence of chromosomal abnormalities, arrhythmias, or congestive heart failure as revealed by fetal hydrops.1-4
From the Department of Biomedical Engineering, University of Cincinnati (Mr Lubbers), and the Department of Surgery, University of Cincinnati College of Medicine, and the Division of Cardiothoracic Surgery, Cincinnati Children’s Hospital Medical Center (Dr Eghtesady), Cincinnati, OH. Presented at the 27th Annual Clinical Meeting of the Society for Maternal-Fetal Medicine, Feb. 5-10, 2007, San Francisco, CA. Received Dec. 8, 2006; revised Jan. 1, 2007; accepted Jan. 4, 2007. Reprints not available from the authors. Supported by the Thrasher Research Foundation and the Cincinnati Children’s Hospital Research Foundation Translational Research Initiative. 0002-9378/$32.00 © 2007 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2007.01.003
Despite these advances, the assessment of prognosis based on anatomy and presentation in utero remains limited, partly because signs of fetal heart failure, such as hydrops or valvular regurgitation that make such appraisals difficult. Further, given the differences in circulatory physiologic condition, some indicators of worsening cardiac function in postnatal life, such as flow and pressure, may not be measured easily in the fetus.5 Clinicians have developed various tools, such as the fetal cardiovascular score, to overcome these limits,5,6 yet assessment of fetal cardiovascular health remains a largely subjective and qualitative practice. A quantitative indicator of fetal cardiovascular health could complement data that is gained by fetal echocardiography and improve the effectiveness of fetal cardiovascular assessment and treatment. Such a prognostic tool may also be helpful for the advancement of fetal therapeutics.7,8 Natriuretic peptides, especially brain natriuretic peptide (BNP), have acquired this important complementary role in both pediatric and adult cardiology; point-of-care testing is now a routine part of clinical care. Significantly less is known about these peptides in fetal
and neonatal cardiac impairment. Besides their roles in regulating fluid balance and blood pressure,9-12 atrial natriuretic peptide (ANP) and BNP play an important part in the developing fetal circulatory system. Newborn infants in cardiac distress can have substantially increased umbilical plasma BNP concentrations.13,14 Similarly, increased plasma BNP concentrations have been linked with a number of congenital cardiovascular diseases in neonates and children.15,16 We have been interested in the potential usefulness of natriuretic peptides in fetuses who are affected by heart disease. We questioned whether amniotic fluid (AF) natriuretic peptide levels would correlate with severity of fetal cardiac distress, perhaps providing a complementary tool in the evaluation of fetal cardiac health. We previously had developed a model of fetal aortic stenosis in first-trimester fetal sheep, in which the severe pressure overload of the immature left ventricle (LV) led to a dichotomous phenotype of either compensatory LV hypertrophy or noncompensated LV dysfunction with associated fetal hydrops.17 To test our hypothesis, we measured ANP and BNP concentrations in
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FIGURE 1
Flow chart of animals in study Total number of fetuses = 38 (24 singletons; 7 sets of twins) Natural history Controls n=18
Aortic stenosis Experimental Sham-operated n=9 n=11 LV hypertrophy n=6
Fetal Hydrops n=3 Fetal intervention n=3
The number of fetuses in each experimental group.
the AF of animals from this experimental model. We also measured baseline concentrations of ANP and BNP in the blood and AF of the healthy ovine fetus and pregnant ewe to provide comprehensive data on the natural history of natriuretic peptides during gestation.
M ETHODS Animal care All animal procedures complied with the Cincinnati Children’s Hospital Research Foundation Institutional Animal Care and Use Committee standards. Timedated Suffolk-cross pregnant ewes (n ⫽ 31: 24 singletons, 7 sets of twins; total, 38 fetuses) were housed communally, had free access to food and water, and were allowed free movement. Animals were kept on a 12-hour light/dark cycle. The number of fetuses in each part of the study is shown in Figure 1.
Natural history of natriuretic peptide release To study the natural history of ANP and BNP release during normal ovine gestation, maternal and fetal blood and AF samples were obtained at 40% gestation (mean, 65 days; term, 145-148 days), 70% gestation (mean, 105 days), and at term (mean, 142 days) in a total of 18 animals (all singletons). Maternal blood 253.e2
samples were also taken from nonpregnant ewes.
Sample collection Pregnant ewes were anesthetized with ketamine and valium and maintained with isoflurane (2-2.5% minimal alveolar concentration) and fentanyl (10 g/ kg). Maternal blood samples were obtained through catheters that were placed in the jugular vein. A laparotomy and limited hysterotomy were performed, and AF (15 mL) was collected in polypropylene tubes that contained protease inhibitor (Complete, Mini; Roche Diagnostics, Mannheim, Germany). The fetus was exteriorized partially, and fetal blood samples (1 mL) were obtained from right internal jugular vein catheterization. Both fetal and maternal blood samples were collected in vacuum tubes that contained EDTA (BD Vacutainer, Franklin Lakes, NJ) and protease inhibitor. Both AF and blood samples were centrifuged at 1600 g at 4°C for 15 minutes. The supernatant was aliquoted in 1-mL samples and frozen until assayed.
Surgical preparation The surgical preparation of fetal lambs in our model of aortic stenosis has been described previously.17 Briefly, at 50-60 days of gestation (mean, 56 days), the fe-
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www.AJOG.org tus was exteriorized, and a left thoracotomy was performed after an AF sample was removed. A Dacron band was placed on the ascending aorta, then the thoracotomy was closed. The fetus was returned to the uterus, and lost AF in the uterus was replaced with warm saline solution that contained gentamycin and duacillin. During the remainder of gestation, we assessed fetal biophysical profiles and fetal hemodynamics with serial ultrasound measurements. Sham-operated control animals underwent the same surgical procedure, except that no band was placed about the aorta. At approximately 140 days estimated gestational age, a repeat laparotomy and hysterotomy were performed, and a second AF sample was collected. The fetus and ewe were then killed, and the fetus was removed for pathologic studies. In our animal model, the development of hydrops uniformly signals impending fetal death.17 For this reason, in the animals in which fetal hydrops developed (n ⫽ 3), fetal intervention was carried out within 2 weeks after onset of hydrops (approximately 100-110 days of gestation). Fetal intervention was successful in 1 case. Ultrasound examination revealed the development of fetal hydrops at 100 days of gestation in this animal; fetal intervention was carried out at 107 days of gestation. The other hydropic fetuses did not survive fetal intervention. To carry out our fetal surgical intervention, the ewe was anesthetized, the fetus was exposed, and a second thoracotomy was made through which the band was removed from the fetal aorta. An AF sample was obtained at this time. Incisions were closed, and the fetus was returned to uterus and allowed to grow to term; data collection was performed as mentioned earlier.
Natriuretic peptide measurement ANP and BNP concentrations were determined according to the manufacturer’s instructions with immunoassay kits (Peptide Enzyme Immunoassay ␣-ANP 1-28 [Human, Canine], and Peptide Enzyme Immunoassay BNP-26 [Porcine]; Bachem Bioscience Inc, King of Prussia, PA) and a microplate reader
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www.AJOG.org (Vmax Kinetic Microplate Reader; Molecular Devices, Sunnyvale, CA). Per the manufacturer’s specifications, these kits show 0% cross-reactivity for the nontarget natriuretic peptide (ie, 0% cross reactivity of the ANP kit for BNP and viceversa). All samples were assayed in duplicate. Interassay variation for both ANP and BNP was within the stated limits of the respective detection kit (⬍5%), with an average error of 10.2% for ANP and 8.4% for BNP.
FIGURE 2
Natriuretic peptide during gestation
Pathologic measurements Measurements of pathologic specimens were made after the animal was dead and included total fetal weight, weights of the fetal heart and LV (including septum), and circumferences of the mitral, aortic, tricuspid, and pulmonary valves along with other cardiac morphometry, as described previously.17 LV weight and valve diameters were normalized to total heart weight for each fetus. The LV hypertrophy was assessed with the use of a previously validated index,5,18 wherein the fraction of fetal heart weight comprised by the LV is calculated. Severity of LV hypertrophy is then indexed as any LV mass ⬎40% (⬎2 SD from mean control values).
Statistical analysis Statistical analyses were performed by the Director of the Statistical Consulting Laboratory, University of Cincinnati, with SAS software (SAS Institute Inc, Cary, NC). Values for groups are shown as mean ⫾ SD per group. Significance was established with a 2-sided unpaired t-test. Values were considered significant with a probability value of ⬍.05.
R ESULTS Fetal biophysical profile Basic biophysical measurements of fetuses (n ⫽ 18) were taken at 40%, 70%, and term gestation. Fetuses at 40% gestation had an average crown-to-rump length of 17.4 ⫾ 4.3 cm, a biparietal diameter of 4.7 ⫾ 1.1 cm, an anterior posterior (AP) chest diameter of 3.4 ⫾ 1.0 cm, and an average weight of 314 ⫾ 11.5 gm (the fetal weights were derived from fetuses that did not survive banding).
A, ANP and B, BNP concentration in AF and fetal and maternal blood throughout fetal gestation. Error bars show standard deviation. The closed squares represent the AF; the closed circles represent fetal blood; and the open triangles represent maternal blood.
Average fetal weight at 70% gestation was 1.94 ⫾ 0.75 kg. Average fetal weight at term was 4.47 ⫾ 1.47 kg.
Natural history of AF natriuretic peptides ANP concentration in AF. The average ANP concentration in AF samples at 40% gestation was 9.77 ⫾ 10.17 pg/mL, which increased to 51.25 ⫾ 8.54 pg/mL at 70% gestation (Figure 2A). The ANP levels then decreased to 45 ⫾ 33.9 pg/mL at term. ANP concentration in fetal and maternal plasma. ANP levels in fetal and maternal plasma remained relatively stable through the first 70% of gestation then significantly increased in the final 30% (Figure 2A). ANP levels in fetal plasma were unchanged between 40% (71.5 ⫾ 19.89 pg/mL) and 70% (70 ⫾ 28.98 pg/mL) gestation but increased significantly (P ⬍ .05) by term (228 ⫾ 36.6 pg/mL). Similarly, maternal plasma ANP levels were not significantly different among nonpregnant ewes (114 ⫾ 50.8 pg/mL), ewes at 40% gestation (126
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⫾ 40.5 pg/mL), and ewes at 70% gestation (93.33 ⫾ 15.27 pg/mL) but were significantly (P ⬍ .05) increased at term (176.6 ⫾ 23.4 pg/mL). BNP concentration in AF. The AF BNP concentration at 40% gestation was 9.28 ⫾ 13.98 pg/mL (Figure 2B). BNP levels increased significantly (P ⬍ .05) at 70% gestation to 46.7 ⫾ 25.03 pg/mL and remained at similar levels until term (50.0 ⫾ 40.5 pg/mL). BNP concentration in fetal and maternal plasma. BNP concentrations in fetal plasma were not significantly altered among 40% gestation (105 ⫾ 31.09 pg/mL), 70% gestation (118 ⫾ 75.9 pg/ mL), and at term (76.67 ⫾ 30.1 pg/mL; Figure 2B). Moreover, maternal blood BNP concentration at 40% gestation (186 ⫾ 82.3 pg/mL), 75% gestation (210 ⫾ 79.6 pg/mL), and at term (143 ⫾ 55 pg/mL) was not significantly different from concentrations in nonpregnant ewes (122 ⫾ 75.6 pg/mL).
Fetal aortic stenosis and AF natriuretic peptides Changes in AF natriuretic peptide levels with fetal aortic stenosis. In our model of aortic stenosis, 1 of 2 distinct phenotypes was induced: (1) survival to term with significant compensatory LV hypertrophy (n ⫽ 6) or (2) development of noncompensated heart failure, as evidenced by fetal hydrops at 100-110 days of gestation (n ⫽ 3), which is followed by fetal death within 1 or 2 weeks.17 Therefore, ANP and BNP levels were compared in animals that were divided into 1 of these 2 phenotype groups and in sham-operated control fetuses (n ⫽ 6) of similar gestational ages (Figure 3). In fetuses with aortic stenosis, ANP concentrations in AF at term (43.3 ⫾ 17.5 pg/mL) were not significantly different from those in control animals (45 ⫾ 33.9 pg/mL; Figure 3A). In contrast, in AF of fetuses with severe aortic stenosis, BNP concentrations (181. 9 ⫾ 109.9 pg/ mL) were nearly 4 times greater than concentrations in control animals (50 ⫾ 40.5 pg/mL; Figure 3B). This difference was statistically significant (P ⬍ .05). Animals with aortic stenosis experienced varying degrees of LV hypertro-
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FIGURE 3
FIGURE 4
FIGURE 5
Natriuretic peptides with fetal aortic stenosis
Correlation of LV hypertrophy with AF BNP levels
AF natriuretic peptide with hydrops
LV weight as a percentage of total fetal heart weight vs AF BNP concentration. Results from both banded and control animals are shown. LV hypertrophy was defined as LV weight being ⬎40% of total heart weight as shown by the dashed line. All points that corresponded to LV weights of ⬎40% correspond to banded animals, and all LV weights of ⱕ40% correspond to controls.
Average A, ANP and B, BNP concentrations in AF of experimental (banded) and control animals at term. Error bars show standard deviation. Differences in BNP concentration between groups were statistically significant (P ⬍ .05).
phy, as expected.17 None of the hydropic fetuses experienced LV hypertrophy; rather, they all experienced LV wall thinning, which was associated with severe LV dilation. AF BNP concentrations positively correlated (r2 ⫽ 0.6751) with the severity of LV hypertrophy (Figure 4). In contrast, AF ANP concentrations did not correlate with the developed LV hypertrophy (data not shown). No other physiologic parameters that were measured (valve diameters, fetal weight, organ weights) correlated with increased BNP or ANP concentrations. Of the 3 banded animals that experienced hydrops, 2 animals were from twin gestations, and 1 animal was a singleton gestation. Aortic stenosis in these animals led to the development of hydrops at approximately 100-110 days of gestation (mean, 45 days after banding). ANP concentrations in AF of hydropic fetuses (including the singleton hydropic twin that survived fetal intervention) were in253.e4
creased notably in comparison with their nonhydropic twins (99.0 ⫾ 37.5 pg/mL vs 25.0 ⫾ 21.0 pg/mL) and normal controls of the same gestational age (51.25 ⫾ 33.9 pg/mL; Figure 5A). These differences, however, did not reach statistical significance. In contrast, BNP concentrations significantly (P ⬍ .05) increased in the AF of hydropic animals, when compared with levels in nonhydropic twins (283 ⫾ 74.4 pg/mL vs 90 ⫾ 60.8 pg/mL; Figure 5B) and when compared with BNP concentrations in normal control animals of the same gestational age (46.7 ⫾ 40.5 pg/mL). Of note, the BNP values in these hydropic fetuses were significantly higher than the same measurements in animals with only compensatory LV hypertrophy (and no LV dilatation). Changes in AF natriuretic peptide levels in response to fetal intervention. Previous experiments with our model have revealed that fetal death follows the onset of fetal hydrops within 1 to 2 weeks. We therefore tested the effect of fetal intervention in these animals. We were able to prevent fetal death and restore normal cardiac function in 1 hydropic fetus by removing the band at 107 days gestation; 2 other hydropic animals did not survive the intervention (66% mortality rate). AF ANP concentrations in the surviving fetus were elevated at in-
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Average A, ANP and B, BNP concentrations in AF of hydropic banded fetuses, their nonhydropic control twins, and normal controls at 90-100 days of gestation. Error bars show standard deviation. ANP concentrations in hydropic fetuses and twins were not significantly different from control values. BNP concentrations in hydropic fetuses were statistically different from twin and control values, but twin values were not significantly different from controls.
tervention (125 pg/mL), similar to other hydropic fetuses (Figure 6A). The AF BNP concentration was also increased (205 pg/mL) in this animal at intervention, compared with control values (51.25 pg/mL), which is also similar to the other hydropic fetuses (Figure 6B). After fetal intervention, by the end of gestation, the BNP concentrations had decreased significantly but were still slightly increased (75 pg/mL; compared with control animals at term). Because these values represent data from only 1 animal, no statistical analysis was performed.
C OMMENT Our study, first, delineates the natural history of AF natriuretic peptides with
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FIGURE 6
AF natriuretic peptides with fetal intevention
A, ANP and B, BNP concentrations at the time of surgical intervention (removal of band from ascending aorta at 107 days gestation) and at term (140 days gestation) for a fetus with hydrops because of induced aortic stenosis.
gestation; second, demonstrates changes in concentrations of the peptides with induced fetal aortic stenosis; and, third, shows the fetal response and changes in the peptides with fetal cardiac intervention. These findings have potential implications with regard to fetal aortic stenosis and its potential management through fetal cardiac intervention. Data regarding amniotic natriuretic peptide concentrations, especially BNP, in either animal models or in humans are limited, although the results that were observed in our study parallel those seen in previous studies of humans and sheep.11,19,20 In our model of fetal aortic stenosis, significant pressure gradients developed across the LV outflow tract that correlate with increases in fetal left ventricular end-diastolic pressures. Similarly, an increase in LV mass is seen, which is directly proportional to the severity of stenosis.17 In this study, we found the
Basic Science: Obstetrics greater the degree of hypertrophy (ie, more severe the stenosis), the greater the rise in BNP concentrations. Further, we observed more dramatic increases in AF BNP levels in animals that experienced fetal hydrops, which is a sign of severe fetal cardiac distress and impending fetal death. In particular, BNP levels of hydropic fetuses were substantially higher than those with compensatory hypertrophy, especially when adjusted to gestational age (ie, the BNP levels are significantly elevated at a time in gestation when normally not much BNP is seen). These results suggest that AF BNP levels correlate with severity of fetal aortic stenosis. In adults with asymptomatic aortic stenosis, BNP levels correlate with outcome and, hence, have prognostic value in clinical decision making.21,22 Further studies are needed to determine whether AF BNP levels provide a similar value in the fetus with aortic stenosis or cardiac disease in general. For example, a “threshold” level may exist to foreshadow ensuing fetal hydrops and death. Such knowledge would be helpful for ongoing experimental fetal cardiac interventions, complementing data that are obtained from fetal echocardiography. An example of this potential use of amniotic BNP analysis may be found in our observation that the resolution of aortic stenosis and fetal cardiac distress (with fetal intervention) can lead to the restoration of normal amniotic BNP and ANP concentrations. Not only did fetal cardiac function recover, but also the elevated BNP concentrations and the fetal hydrops resolved through prenatal intervention in a fetus with induced aortic stenosis. Although this single example is not conclusive, we believe it suggests that BNP concentrations in AF may be useful as indicators of fetal cardiac health both before and after fetal intervention. If confirmed, this finding may have relevance to advancing experimental fetal cardiac interventions by indicating whether poor outcomes after “technically” successful interventions occur because of inadequate surgical correction or because the fetal heart is damaged already beyond repair.8
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Our observations correlate well with previously reported data, which were all obtained near term, for the healthy fetus, the pregnant ewe, and the fetus in distress.13,14,18,21,23 To our knowledge, our data represents the first natural history (and modified natural history) of fetal natriuretic peptide levels from early gestation onward. It would seem reasonable that AF natriuretic peptides may be of maternal origin; however, studies have now shown that ANP and BNP in fetal blood and AF are of fetal origin and, as seen here, do not correlate to maternal plasma concentrations.20,23 It is also remarkable that, in twin pairs in which 1 fetus experienced fetal hydrops, the nonhydropic twin may “sense” the distress of its sibling, as displayed by the rise in BNP levels, despite the fact that the twins do not share placentae. Additional experiments may help to clarify the involved mechanisms. A limitation of our study is that our animal model does not fully mimic fetal aortic stenosis as seen clinically. We currently are conducting a clinical trial to assess the potential relevance of AF BNP in humans. Another limitation of our natural history data is that it pertains to singleton gestation animals, although our limited data from control animals (all singletons) and sham-operated animals (twins and singletons) do not suggest substantial differences in the natriuretic peptide values at term. It is conceivable that, earlier in gestation than measured in our study, twin animals would have different AF BNP levels compared with singletons. Additionally, the origin of the AF BNP measured in the study is unknown; although it is assumed that most of AF BNP comes from fetal urine, the placenta and amniotic tissues cannot be ruled out as potential sources. Little published data are available regarding BNP levels in fetal urine, and, as such, this represents another avenue of research that we would like to explore. Along the same lines, it would be of interest to measure BNP concentrations in AF and fetal urine after the injection of BNP into fetal circulation, but currently only synthetic human BNP (Natrecor) is available for such an experiment.
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In summary, we have shown in an ovine model that the measurement of AF BNP concentrations has significant potential as a complementary tool for the assessment of fetal cardiac distress. If future experiments suggest BNP levels can be predictive of subsequent hydrops and if similar elevations can be demonstrated in affected human pregnancies, AF BNP levels may prove to be especially useful as quantifiable measures of fetal cardiac health and perhaps as added indicators of the need and timing for fetal intervention. f ACKNOWLEDGMENTS We thank Dr Anoop Brar and Scott Baker for insightful discussions and input in the preparation of this article.
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5. Huhta JC. Guidelines for the evaluation of heart failure in the fetus with or without hydrops. Pediatr Cardiol 2004;25:274-86. 6. Huhta JC. Right ventricular function in the human fetus. J Perinat Med 2001;29:381-9. 7. Marshall AC, van der Velde ME, Tworetzky W, et al. Creation of an atrial septal defect in utero for fetuses with hypoplastic left heart syndrome and intact or highly restrictive atrial septum. Circulation 2004;110:253-8. 8. Tworetzky W, Wilkins-Haug L, Jennings RW, et al. Balloon dilation of severe aortic stenosis in the fetus: potential for prevention of hypoplastic left heart syndrome: candidate selection, technique, and results of successful intervention. Circulation 2004;110:2125-31. 9. Cameron VA, Ellmers LJ. Minireview: natriuretic peptides during development of the fetal heart and circulation. Endocrinology 2003; 144:2191-4. 10. Cameron VA, Aitken GD, Ellmers LJ, Kennedy MA, Espiner EA. The sites of gene expression of atrial, brain, and C-type natriuretic peptides in mouse fetal development: temporal changes in embryos and placenta. Endocrinology 1996;137:817-24. 11. Cheung CY, Gibbs DM, Brace RA. Atrial natriuretic factor in maternal and fetal sheep. Am J Physiol 1987;252:E279-82. 12. Walther T, Schultheiss HP, Tschope C, Stepan H. Natriuretic peptide system in fetal heart and circulation. J Hypertens 2002;20:785-91. 13. Itoh H, Sagawa N, Hasegawa M, et al. Brain natriuretic peptide levels in the umbilical venous plasma are elevated in fetal distress. Biol Neonate 1993;64:18-25. 14. Walther T, Stepan H, Faber R. Dual natriuretic peptide response to volume load in the fetal circulation. Cardiovasc Res 2001;49: 817-9.
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www.AJOG.org 15. Reynolds EW, Ellington JG, Vranicar M, Bada HS. Brain-type natriuretic peptide in the diagnosis and management of persistent pulmonary hypertension of the newborn. pediatrics. Pediatrics 2004;114:1297-304. 16. Kunii Y, Masahiro K, Ohtsuki S, et al. Plasma brain natriuretic peptide and the evaluation of volume overload in infants and children with congenital heart disease. Acta Med Okayama 2003;57:191-7. 17. Eghtesady P, Michelfelder E, Jones F, et al. Aortic stenosis in the ovine fetus: a model for fetal intervention? Pediatr Cardiol 2003;24:624. 18. Norman JE, Levy D. Improved electrocardiographic detection of echocardiographic left ventricular hypertrophy: results of a correlated data base approach. J Am Coll Cardiol 1995;26:1022-9. 19. Di Lieto A, Pollio F, Catalano D, et al. Atrial natriuretic factor in amniotic fluid and in maternal venous blood of pregnancies with fetal cardiac malformations and chromosomal abnormalities. J Matern Fetal Neonatal Med 2002;11:183-7. 20. Itoh H, Sagawa N, Hasegawa M, et al. Brain natriuretic peptide is present in the human amniotic fluid and is secreted from amnion cells. J Clin Endocrinol Metab 1993;76:907-11. 21. Bergler-Klein J, Klaar U, Heger M, et al. Natriuretic peptides predict symptom-free survival and postoperative outcome in severe aortic stenosis. Circulation 2004;109:2302-8. 22. Lim P, Monin JL, Monchi M, et al. Predictors of outcome in patients with severe aortic stenosis and normal left ventricular function: role of B-type natriuretic peptide. Eur Heart J 2004;25:2048-53. 23. Deloof S, VanCamp G, Chatelain A. Absence of transplacental transfer of atrial natriuretic peptide in the rat: direct experimental evidence. Med Sci Res 1995;23:347-50.