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DOI: 1 0 . 1 1 1 1 / j . 1 4 7 1 - 0 5 2 8 . 2 0 0 4 . 0 0 0 7 0 . x

Amniotic fluid inflammatory proteins and digestive compounds profile in fetuses with gastroschisis undergoing amnioexchange$
Laurence Burc1,a,b,c, Jean-Luc Volumenie1,a,c, Pascal de Lagausiea,c, Jean Guibourdenchea,b,c, Jean-Franc ois Ourya,c, Edith Vuillarda,c, Olivier Sibonya,c, Philippe Blota,c, Carole Saizoua,c, Dominique Luton*,a,c
Objective In gastroschisis, an inflammatory process related to the presence of digestive compounds may be involved in intestinal damage. We measured the amniotic fluid concentrations of total protein, ferritin and amylase, lipase, g-glutamyl transferase and bile acids before each amnioexchange performed in women whose infants had gastroschisis. We estimated the correlation among total proteins, ferritin and digestive compounds and postnatal outcome. Design All women whose infants had gastroschisis in our fetal medicine unit are offered repeated amnioexchange during the third trimester of pregnancy to improve the quality of the exteriorised bowel at birth. Amniotic fluid was sampled at the beginning of each amnioexchange and total proteins, ferritin and digestive compounds were assayed. Setting This study was conducted in the Department of Perinatology of the University Hospital Robert Debre in Paris. Population Thirty pregnant women with a gastroschisis affected fetus diagnosed antenatally. Methods The biological results were expressed as multiples of the median with respect to a control population. Main outcome measure Gestational age at delivery and the outcome of the infants were recorded and correlated with amniotic fluid total proteins, ferritin and digestive compounds. Results There was a positive correlation ( P < 0.01) between digestive compounds (except amylase at the final amnioexchange) and ferritin on the one hand, and all digestive compounds and total proteins concentration at the final amnioexchange on the other. In addition, among total proteins amylase and lipase, lipase concentrations were related with parameters of short term outcome ( P < 0.05). Conclusion Amniotic total proteins and ferritin are elevated in fetuses presenting with gastroschisis as a consequence of an inflammatory process. Inflammation may be induced by the presence of digestive compounds in the amniotic fluid. The concentrations of which may constitute a marker of short term outcome of the newborn infant. INTRODUCTION Gastroschisis is a defect of the anterior abdominal wall with extrusion of fetal intestine into the amniotic cavity. The prognosis of this congenital anomaly is mainly influenced by the degree of inflammation of the intestine at birth. Identification of prognostic markers relies mainly on ultrasound with maximal dilatation of the exteriorised bowel appearing to be the most important variable.1 6 As dilatation of the intestine has poor specificity and sensitivity, other tests are desirable to assess intestinal alteration and inflammation changes. Intestinal damage at birth includes a fibrous coating of bowel loops with smooth muscle thickening and mucosal blunting.7 As prolonged exposure to amniotic fluid may be responsible for these alterations in the intestine, amniotic fluid exchange has been attempted, in experimental8 and in clinical settings.9 11 Amnioexchange is supposed to act by relieving intestinal compression due to oligohydramnios, which is frequently encountered in gastroschisis, by eliminating deleterious compounds present in amniotic fluid such as digestive enzymes and, perhaps, through improved supply of blood to the exteriorised intestine.12
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pital Robert Debre , Paris, Department of Perinatology, Ho France b pital Robert Laboratory of Hormonal Biochemistry, Ho , Paris, France Debre c veloppement du tube Maladies Inflammatoires et du de Paris VII-Denis Diderot, digestif chez lenfant, Universite EA3102, Paris, France
cologie-obste trique, * Correspondence: Dr D. Luton, Service de gyne pital Robert Debre , 48 blvd Se rurier, 75019 Paris, France. Ho $ Part of this work was presented at the International Fetal Medicine Surgery Society Congress 2000. 1 These two authors contributed equally to this work. D RCOG 2004 BJOG: an International Journal of Obstetrics and Gynaecology

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It has been previously shown that an inflammatory reaction exists in the amniotic fluid of fetuses with gastroschisis in humans and in animals. There is an increase in the number of activated neutrophils and in the concentrations of cytokines and ferritin. Ferritin reflects iron stores, but is also a marker of chronic inflammation, being an inflammatory protein secreted by monocyte and macrophages. Its measurement is more convenient than cytokines.13,14 We sought to determine whether in gastroschisis there was a correlation between inflammatory markers in amniotic fluid, such as ferritin, total proteins and digestive compounds, and if these biochemical data recorded during repeated amnioexchanges could yield prognostic information.

METHODS The study took place in a tertiary referral fetal medicine unit from 1997 to 2000. Thirty infants with gastroschisis diagnosed antenatally were treated during this period. In all cases, the diagnosis of gastroschisis was confirmed by a single operator (EV). With the approval of our local ethics committee, all the women were offered amnioexchange starting at 30 weeks of gestation and repeated every two or three weeks. The decision to start amnioexchange during the third trimester was justified by the fact that most intestinal damage occurs during this period.15 The procedure consisted in a transabdominal amniotic drainage followed by an infusion of a saline solution through a 20-gauge needle under ultrasound guidance (Hitachi Katana-5, Hitachi, France with a 3.5 MHz sector transducer). Amniotic fluid was replaced in 300 mL fractions at each procedure, to a total of 600 900 mL. Karyotyping was always performed at the first amnioexchange if not done before. Where oligohydramnios was present, the procedure corrected the volume of amniotic fluid. Normal amniotic fluid volume was restored in all cases. Samples were collected from the first 300 mL for assay of total proteins, ferritin, amylase, lipase, g-glutamyl transferase and bile acids. A cardiotocogram was performed after the procedure and the women were allowed to return home if no uterine activity or fetal heart rate abnormality was detected. When necessary, the women were admitted and tocolysis was prescribed. Ultrasound scans were performed every two weeks from the beginning of the third trimester until delivery, noting the maximal diameter of exteriorised bowel, the thickness of intestinal wall, the volume of amniotic fluid, the echogenicity of the amniotic fluid and peristaltic movements of the exteriorised fetal gut. Gastroschisis did not constitute a sufficient motive for caesarean delivery in our unit. Thus, vaginal delivery was allowed and caesarean section was only performed if there were classical obstetric indications. Induction of labour was performed for obstetric indications. After delivery, a sterile bag was wrapped around the abdomen of the newborn, who
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was then transferred to the paediatric surgical unit in the same hospital. Abdominal closure was generally performed in one stage in the first hours following birth. In some cases, the extent of the fibrous coating necessitated a two-stage procedure, with secondary closure. The outcomes were gestational age at delivery, the extent of the fibrous coating of the intestine classified according to Debeugny et al.,16 the length of stay in the neonatal intensive care unit, the length of ventilatory assistance, the need for and length of curarisation, the need for and length of haemodynamic support (administration of vasoactive drugs), the length of stay in hospital, the duration of parenteral nutrition, and the length of time before full oral feeding. These variables were correlated with the concentrations of total proteins, ferritin, amylase, lipase, g-glutamyl transferase and bile acids measured at the first and the last amnioexchange, and with maximal bowel dilatation. To establish normal ranges, concentrations of ferritin, total proteins, amylase, lipase, g-glutamyl transferase and bile acids were first assayed in amniotic fluid sampled for karyotyping in 898 fetuses. The indications for karyotyping were various (short femur, congenital heart disease, maternal age above 38 years, positive maternal Downs Syndrome screening test). Only amniotic fluid samples from fetuses whose karyotyping proved normal were considered for analysis. All samples were harvested from pregnant women free of inflammatory or infectious diseases. Any stained samples were discarded. Written informed consent was obtained from all women. The amniotic fluid was sampled by transabdominal amniocentesis between 14 and 38 weeks of gestation. The samples were centrifuged for 10 minutes at 2000g to remove cellular material and stored at 20jC until assay. Assays were performed using a Hitachi 911 instrument (Roche Diagnostics, Mannheim, Germany) at 37jC with spectrophotometric and turbidity methods developed for serum as follows: g-glutamyl transferase (Roche Diagnostics, ref. 1489496); amylase (Biotrol, toile, France); lipase (Sigma, St. Louis, ref. A03051, Marcy lE Missouri); ferritin (Tinaquant Roche Diagnostics, Mannheim, Germany); bile acids (Randox, ref. BI1689, Antrim, UK); total proteins (Biotrol, ref. AO1217). Medians and 5th and 95th centiles were calculated using StatView (SAS, Berkeley, California) software for PC version 4.5. Some of these data have been published in Clinical Biochemistry17 and are reproduced with permission on Table 1, other nonpublished data are reproduced on Table 2. All data were entered in the StatView 4.5 software. For normal pregnancy, Wilcoxons signed rank test was used for analysis of variation of biochemical markers during pregnancy with a P value <0.05 considered as statistically significant. Then Mann Whitney U test was used to compare group to group with a P value <0.01. Weeks of gestation were grouped for a P > 0.01. For gastroschisis, Spearmans rank correlation coefficient test was estimated to establish association among the biological markers with one

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Table 1. Reference ranges for g-glutamyl transferase, 5V nucleotidase, total alkaline phosphatase, amylase and aspartate amino transferase from 14 to 35 weeks of gestation. Results are expressed as medians and 5th and 95th centiles (iu/L) for each enzyme.

GGT, g-glutamyl transferase; 5V nuc, 5V nucleotidase; t-ALP, total alkaline phosphatase; ALPp, placental alkaline phosphatase. Reproduced with permission L Burc et al. Clin Biochem 2001;34:317 322. * Placental alkaline phosphatase is expressed as a percentage of total alkaline phosphatase activity.

another and the association of the biological markers with neonatal outcome. A P value <0.05 and a r V value >0.38 at initial exchange and r V value >0.43 at final exchange (with a  5%) were considered as statistically significant. A m2 test was used to assess a difference between the median multiple of median (MoM) of biological markers in gastroschisis and the control population.

RESULTS Thirty infants with gastroschisis were managed between 1997 and 2000. They were all singletons. Karyotyping was always normal and no associated malformation was diagnosed. Pregnancy was allowed to go to term in all cases.

The mean maternal age was 24 years (range 18 34 years). Gastroschisis was discovered at a mean gestational age of 19.5 weeks (range 12 34.5 weeks) and the mean gestational age at delivery was 36.5 weeks (range 30 39.5 weeks). Labour was induced in 10 women, was spontaneous in 11 and a caesarean section before labour was judged necessary in nine women. Three infants delivered preterm; one was born at 33 weeks of gestation by caesarean section for an abnormal non-stress test; one was born spontaneously at 30 weeks of gestation; and one was born by caesarean section at 30 weeks of gestation for intrauterine growth restriction and an abnormal non-stress test. The mean gestational age at the first amnioexchange was 32 weeks (range 26 38 weeks). The procedure was repeated up to six times (mean 2.5).

Table 2. Normal values of ferritin (n 898), lipase (n 499), bile acids (n 150) and proteins (n 91) in control fetuses. Weeks of gestation 14 Ferritin (Mmol/L) n 57 Median 99 5th 95th 24 290 Lipase (iu/L) n 30 Median 18 5th 95th 3 54 Bile acids (Mmol/L) n Median Proteins (g/L) n Median 5th 95th 15 16 17 18 19 20 21 22 23 24 25 30 31 38

155 144 59 540

151 172 42 513

112 180 81 583

90 202 75 590

63 235 78 651

36 168 49 162

22 178 86 396

25 184 41 482

27 163 58 540

20 141 69 274

96 95 21 245

44 24 5 65

97 14 3 40

82 8 1 33

49 7 1 28

49 7 1 25

192 5 1 17

150 2

91 5.0 2.8 8.3

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FETUSES WITH GASTROSCHISIS UNDERGOING AMNIOEXCHANGE Table 3. Amniotic fluid concentrations of ferritin, amylase, lipase, gglutamyl transferase, bile acid and total proteins in gastroschisis-affected fetuses at initial and final amnioexchange. Values are expressed as median of MoM matched for gestational age and compared with reference population by mean of m2 test. Initial amnioexchange Ferritin Median MoM Extreme ranges P Amylase Median MoM Extreme ranges P Lipase Median MoM Extreme ranges P ;-Glutamyl transferase Median MoM Extreme ranges P Bile acids Median MoM Extreme ranges P Total proteins Median MoM Extreme ranges P Final amnioexchange Lipase (initial amnioexchange) 3.7 0.5 56.9 <0.01 8.3 0.4 233.5 <0.0001 Lipase (final amnioexchange)

295

Table 5. Correlation between amniotic lipase and short term outcome. Results were obtained with non-parametric Spearmans rank correlation coefficient test providing r V( P) values. Duration of haemodynamic support n 26 0.43 (0.02) n 26 NS Duration of curarisation n 26 NS n 26 0.47 (0.02)

1.7 0.6 3.9 <0.05

2.4 0.6 7.7 0.002

7.8 0.2 808 <0.01

6.6 0.2 992 <0.001

1.3 0.3 53 NS

0.9 0.5 155 NS

1.5 0.5 11 NS

3.0 1 98 NS

2.4 1 7.5 <0.0001

2.6 1.2 8.1 <0.0001

Reference intervals for g-glutamyl transferase and amylase are published in Clinical Biochemistry17 and values are available for the range of gestation involved in our gastroschisis. Reference ranges for ferritin, lipase and bile acid were established in amniotic fluid samples collected at 14 38. Each parameter showed a change in concentrations or enzymatic activities between 14 and 35 weeks of gestation ( P < 0.0001) except for bile acid and total protein. Ferritin peaked at 19 weeks of gestation at 235 Amol/L and then fell to 24 Amol/L by the end of gestation. Lipase activity decreased significantly throughout gestation, from 18 to 5 iu/L. Bile acids did not fluctuate during gestation and their

concentration was 2 Amol/L (the lower threshold of measurement is 0.5 Amol/L) (see Tables 1 and 2). In pregnancies with gastroschisis, total protein, ferritin, g-glutamyl transferase, amylase, lipase and bile acids were measured and expressed as median of MoM using this reference range (Table 3). The concentration of total protein, ferritin, amylase and lipase in amniotic fluid were significantly higher with gastroschisis. At initial amnioexchange (n 30), ferritin was positively correlated with all digestive compounds and total proteins concentration was only positively correlated with g-glutamyl transferase. At final amnioexchange (n 21), ferritin concentration was positively correlated with gglutamyl transferase, lipase and bile acids and total proteins was positively correlated with all digestive compounds (Table 4). We also measured maximal bowel dilatation. A mean value of 22 mm (from 6 to 44 mm) was noted for exteriorised gut. No correlation could be established between this parameter and outcome data, nor between bowel dilatation and any total protein or enzymatic value. Conversely, we did find a positive correlation between the initial ferritin concentration and the maximal bowel dilatation. We sought to determine whether the biological markers measured in amniotic fluid during the initial and final amnioexchanges could yield information about the neonatal outcome. Although we could establish a relation among total protein, amylase or lipase and some of the short term outcome, this relation seemed more relevant for lipase (Table 5). No relation could be established with any of the long term outcome parameters. The pattern of changes in the different amniotic concentrations during the series of amnioexchanges was variable and no correlation was observed between the increase or decrease in the markers analysed and the outcome data.

Table 4. Correlation between inflammatory markers (total proteins and ferritin) and digestives enzymes at initial (n 30) and final amnioexchange (n 21). Results were obtained with non-parametric Spearmans rank correlation coefficient test providing r V ( P) values. g-Glutamyl transferase Total proteins initial Ferritin initial Total proteins final Ferritin final 0.61 0.43 0.87 0.66 (0.003) (0.02) (0.0001) (0.003) Lipase NS 0.64 (0.0009) 0.83 (0.0002) 0.64 (0.004) Bile acids NS 0.58 (0.003) 0.85 (0.0002) 0.71 (0.002) Amylase NS 0.61 (0.001) 0.56 (0.01) NS

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DISCUSSION Gastroschisis carries a good prognosis, with a survival rate exceeding 90%.18,19 Nonetheless, intestinal function may be impaired in some cases, leading to death or to resection with the short bowel syndrome. Antenatal criteria predicting these high risk situations lack sensitivity and specificity, although many authors have attempted to evaluate ultrasonographic signs of bowel complications. The degree of dilatation of the exteriorised intestine was the most commonly reported ultrasonographic prognostic factor,1 6 with outcome worsening with growing bowel diameter. Nevertheless, opinions of this criterion varied, and we now interpret it with caution.1 6,20 23 Identification of early signs of intestinal deterioration could help the obstetrician to induce delivery in spite of prematurity to avoid irreversible lesions.1,2 Thus, preterm delivery and even caesarean section could prevent progressive bowel impairment. This approach is controversial,22 preterm delivery can increase the risk of complications, such as sepsis.19 We observed no correlation between maximal bowel dilatation and prognosis, whereas many authors consider this ultrasonographic sign as the most important.1 6 It is noteworthy that none of these authors studied the relation between bowel dilatation and outcome in pregnancies treated antenatally by serial amnioexchanges. We suggest that intestinal damage is mainly attributable to prolonged exposure to amniotic fluid5 12,15 21 and progressive mechanical constriction of the eviscerated intestine.2 5 From 1995, amnioexchange and amnioinfusion amnioexchange was proposed to improve the quality of extruded fetal intestine at birth.24 Amnioexchange and amnioinfusion has proved successful in animal8,24 models and in human.9 11 It has also been shown in ewes that amnioexchange resulted not only in improvement of the bowel at delivery but also in clearance of various digestive compounds and inflammatory markers.25 The rationale for amnioexchange is that inflammatory processes are likely to induce changes in the fetal bowel, particularly fibrous coating.15 Other mechanisms, such as chemical aggression by meconium rather than urine,26 perhaps attributable to the presence of digestive compounds, are probably implicated in intestinal damage. The positive correlation we observed among ferritin, protein and digestive compounds in initial and final amnioexchange may support the hypothesis that inflammation is at least partly attributable to digestive compounds. Inflammatory cells and molecules have been identified in amniotic fluid from pregnancies with gastroschisis,9 12,15 27 whereas they are absent from amniotic fluid in healthy pregnancies.9 In a previous study, we found that total protein and ferritin were particularly elevated in pregnancies with gastroschisis.9 These biochemical substances can be routinely and easily measured with high reproducibility. Carrol et al.28 found a high level of protein in the amniotic fluid of infants with gastroschisis to be associated with low levels of

protein in umbilical cord blood at delivery. These authors state that this protein loss could partly explain the morbidity associated with gastroschisis, but they do not link this result to an inflammatory reaction. Nonetheless, these results are consistent with ours. Our results also indicate that digestive compounds, such as lipase and total proteins, may be antenatal prognostic factors of short term neonatal morbidity in gastroschisis. The need for longer neonatal intensive care may be due to increased tension in the anterior abdominal wall after surgical closure of the abdominal defect, which is one of the main problems in the treatment of gastroschisis. When abdominal pressure is elevated, prolonged ventilatory assistance and curarisation may be necessary, and so the length of stay in the neonatal intensive care unit increases accordingly. Nevertheless, we failed to demonstrate a relationship between any of the biochemical variables we measured and the appearance of the intestine at birth, such as the extent of fibrous coating of the bowel. This criterion, although graded according to well-defined data,16 is subjective. The surgical team was not blinded to the antenatal investigations and treatment and may have been influenced by knowledge the amnioexchanges. The fibrous coating on the surface of the intestine was classified as severe in only four cases, and we therefore probably lacked the power to reach statistical significance. The long term outcome of infants born with gastroschisis is influenced by several factors, some of them appearing after birth.19,29 The antenatal condition of the infant may be less important than the early neonatal period. Gestational age at birth, infectious complications and pulmonary bronchodysplasia are important determinant of outcome. This may partly explain why there was no correlation between the concentrations of biochemical substances in the amniotic fluid and the longer term outcome for the infants, measured by the time to establish oral feeding, the length of parenteral nutrition, and the length of time spent in hospital. Although a causal relationship between the increase of amylase, lipase or total proteins and neonatal outcome cannot be ascertained from our data, the correlations we observed encourage us to explore the involvement of these compounds in the pathophysiology of alteration in the intestine of gastroschisis. Digestive compounds may either act either through an alteration of the bowel function or an increase of parietal inflammatory reaction. Alternatively, increase of digestive compounds may be a consequence of in utero bowel dysfunction, which can be used to disclose it. We hypothesise that digestive compounds result from meconium emission or vomiting due to periods of subocclusion. The result is then similar to what can be observed in biliary peritonitis, leading in this special setting to a predominant aseptic inflammation. The concentrations of lipase and possibly total proteins, ferritin and other digestive compounds may be markers of a
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poor initial outcome in infants whose mothers were treated by repeated amnioexchanges during pregnancy. Total protein, ferritin and digestive compounds enable us to gain more insight into the pathophysiology of gastroschisis, particularly the mechanisms responsible for alteration in the function of the intestine. Digestive enzymes may be the cause or consequence of intestinal alteration, and the correlations observed between digestive compounds (gglutamyl transferase, lipase, amylase, bile acid) and markers of inflammation (total proteins and ferritin) are probably significant. Further investigations are required before these biochemical markers can be considered prognostic factors for early neonatal outcome.

13.

14. 15.

16.

17.

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Accepted 14 November 2003

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