The Nutrition of the Fetus With Intestinal Atresia: Studies in the Chick Embryo Model By B. Lopez de Torre, J.A. Tovar, S. Uriarte, and P. Aldazabal San Sebastiiin, Spain 0 This article examines the effects of experimental tal intestinal

obstruction

prena-

on the growth and blood composi-

tion of chick embryos. Intestinal atresia (IA) was produced by bipolar bowel electrocoagulation in fertile eggs on the 14th day of incubation. The chicks killed on the 19th day were measured, weighed, and blood-sampled. Twenty-three control,

10 sham-operated,

and

11 IA chicks were

studied.

Animals with IA were severely undernourished by weight (43.4 f 4.7 v 70.3 f 7.6% of egg weight, P < .OOl) and length (15.3 -c 1.1 v 18.1 +: 0.9 mm tibia1 length, P c .OOl) in comparison with sham-operated

ones. Their hematocrit

was slightly

lower, and total protein increased. Prealbumin was absent in their sera and albumin, a and 6 globulins were significantly decreased, whereas y-globulin was greatly increased. Sodium, potassium chloride, urea, and glucose remained within normal

limits.

precludes

The lack of placenta

any supply

of nutrients

in the avian embryo by this route

and the

ingestion of amniotic fluid, which is protein-rich after the 13th day of incubation, when the opening of the seroamniotic connection allows albumen to be mixed with it, becomes the main source of nutrients until hatching. Obstruction of the main incoming avenue by IA induces severe malnutrition

in

this model which relies on this route to a greater extent than the human fetus. In spite of the obvious biological differences between the avian embryo and the human fetus, the present evidence supports the hypothesis that prenatal interruption of the amniotic fluid transit contributes

to fetal undergrowth

in IA. Copyright

o 1992 by W.B. Saunders Company

INDEX WORDS: fetal nutrition.

Intestinal

atresia;

intestinal

MATERIALS obstruction,

B

ABIES BORN with intestinal atresia (IA) are frequently small for their gestational age and although associated malformations may account for this intrauterine undernourishment in some cases, the gut anomaly itself should be incriminated in most of them. Jolleys pointed out the contribution of the interruption of prenatal amniotic fluid transit to this underdevelopment and emphasized that diseases like esophageal atresia, malrotation, and anorectal malformations, in which such transit is only partially disturbed, interfere slightly with fetal growth, whereas duodenal atresia or high jejunal atresia are frequently associated with considerable underweight.’ Pierro et al have produced additional evidence that, regardless of associated malformations, complete and high gastrointestinal (GI) tract congenital obstructions are more often accompanied by polyhydramnios and intrauterine malnutrition than incomplete and low ones.2 lending further support to this interpretation JournalofPediafricSurgery,

Vol27,No

and recalling the relevance of the nutritional role of amniotic fluid at the end of gestation. In the last few years a relatively simple chick embryo experimental model has revealed itself as a valuable tool for research on IA”_5 in spite of the obvious biological differences between the avian embryo and the human fetus. Among these differences one of the more relevant is the isolation of the chick within its shell where it is devoid of any transplacental supply of nutrients from the mother. Precisely because of this, amniotic fluid deglutition becomes the main nutritional avenue for the chick embryo in the last days of incubation, when yolk and albumen foodstuff reserves are nearly exhausted. In fact, the remaining albumen is incorporated into the amniotic fluid during this period to make enteral nutrition possible. This paper examines the effects of surgical obstruction of the GI tract on body growth and blood composition of chick embryos in which this manipulation leads to the interruption of the main nutritional income avenue creating a situation in which the risk of subsequent undergrowth is very exaggerated in comparison with that in the human IA.

lO(O.ctober),1992: pp 1325-1328

AND METHODS

We used domestic hen (Gal/us domesticus) fertile eggs incubated in an appropriate incubator at 37.X, 80% humidity and 2-hour automatic tilting. On the 14th day a l-cm window was opened in the shell and the allantoic cavity was entered. Using microsurgical sterile instruments and a binocular operating microscope (OM-650; Wild, Herrburgg, Switzerland) we mobilized and opened the amniotic membrane, located the umbilical stalk until we had intestinal loops in view by transparence and opened the umbilical wall. A midgut loop was carefully grasped with tine bipolar coagulating forceps (Esculap GK-4. Tuttlingen, Germany) and a length of 3 mm of intestine was coagulated. The bowel was then reintegrated into the abdomen, the egg was sealed with a plastic sterile dressing and the incubation was resumed. This technique is basically the same as the one previously described by Tibboel et

From the Pediattic Surgery and Immunology Services, Universidad de1 Pais Vasco, Hospital NO.!+’ de Aranzazu, San Sebastian, Spain. Presented at the 23rd Annual Meeting of the Canadian Association of Paediatric Surgeons, Quebec City, Quebec, September 19-22, 1991. Supported in part by FIS grants 8711606, 891390, and 911363 and by the Depatiment of Health of the Basque Government. Address reprint requests to Professor Dr J.A. Tovar, Hospital Infantil La Paz, P de la Castellana 261, 28046 Madrid, Spain, Copyright Q 1992 by W.B. Saunders Company 0022.3468l9212710-0020$03.0010 1325

1326

TORRE ET AL

a13s4and by ourselves.s By daily inspection through the plastic dressing we checked the survival of the operated embryos and discarded those which died. On the 19th day, 24 to 48 hours before hatching, we opened the shell with scissors, exteriorized the chick with its membranes and sacrificed it by puncturing an allantoic vessel with a 26-gauge needle and drawing as much blood as possible (usually 1 mL). After sacrifice, animals were carefully examined, weighed in a precision balance, and had their right tibia measured with a precision caliper. An abdominal autopsy was then performed and tissue samples were taken for histological studies. These were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E and Masson trichromic. We measured hasmatocrit, serum Na, Cl, K, urea, glucose, and total protein by standard laboratory micromethods. In order to minimize variations in animals with different hzematocrits we recalculated the values for all these substances by multiplying them by (100-hematocrit) and dividing the result by 100. We expressed all values in units per unit of serum volume (instead of units of blood volume). Protein electrophoresis led to the separation of seven fractions that were identified as prealbumin, albumin, a-l-globulin, o-2-globulin, B-l-globulin, S-2-globulin, and y-globulin based on data published by RomanofL6 Weller and Bowdon,’ and Slade and Mi1ne.s We added p fractions because we did not find their separate consideration interesting enough. Finally, in several unmanipulated eggs we studied the protein composition of amniotic fluid on days 14, 16, and 18 of incubation by conventional and polyacrylamide gel electrophoresis (PAGE-SAS) using molecular weight standards for comparison. The eggs for these experiments were randomly allocated to three groups: (1) control group (n = 23) in which the embryos were not manipulated before sacrifice on day 19 of incubation (hatching at 21st); (2) sham group (n = lo), in which a l-cm window was opened on the 14th day, the umbilical stalk mobilized but without further manipulation, and the incubation resumed after plastic dressing application until the 19th day; (3) IA group (n = 11) in which the operations were completed as described above in order to reproduce the malformation. Before manipulations and after them all eggs were treated identically and experiments in the three groups were simultaneously carried out. We described each variable in every group by standard statistical methods (mean and standard deviation). Given the size of the samples we compared variables between groups by non-parametric tests (Mann-Whitney U test). A level of P < .05 was accepted as significant throughout. RESULTS

Postoperative embryonal mortality was 0% for the control group, 45% for the sham group, and 84% for the Atresia group. In fact, we needed 35 surviving IA embryos to gather complete blood samples in 11. Egg weights were similar in the three groups at the beginning of incubation but chick weight in the sham group was significantly reduced (70.37% 2 7.6% of egg weight, P < .Ol) and it was even more so in the IA group chicks (43.47% + 4.8% of egg weight, P < .Ol), which weighed almost 50% less than the control ones (80.8% + 4.2% of egg weight). There were also significant reductions of tibia1 length in sham (1.81 + 0.09 mm, P < .Ol) and IA (1.53 + 0.11 mm, P < .Ol) groups in comparison with controls (2.15 ? 0.26 mm). These data are graphicahy displayed in Fig 1.

CHICK WEIGHT

TIBIAL LENGTH

Fig 1. (A) Chick weight on the 19th day as a percentage of original egg weight. Animals in the sham group were significantly undernourished, but those in the IA group were much more so. (6) The same deviations were present in length as judged by tibia1 length. Values are means f SD. l*P < .Ol.

Intestinal atresia lesions were identical to those described in a previous paper using the same techniques.5 In short, the small bowel was either obstructed or interrupted at the point where coagulation was performed, the proximal end was very dilated with bilious contents and the distal end was narrow and unused. Histologically there was some evidence of “meconium” peritonitis and the wall lesions were as formerly described. Both gross and microscopic anatomy were strikingly similar to those of the human malformation. Blood electrolytes, urea and glucose were not significantly different in the sham and IA groups (Figs 2 and 3), showing that, as far as these solutes are concerned, the internal environment was not substantially modified by prenatal intestinal obstruction. On the other hand, contrary to our expectations, total serum proteins in IA chicks (both in raw values and after correction for hematocrit, which was slightly lower) was significantly increased. Changes in protein fractions account probably for hyperproteinemia in this group: prealbumin was absent, there was a significant decrease of albumin, ol-l-globulin, a-2SODIUM

HAEMATOCRIT

mEq/L serum

% 601

$+jfl fi Control Sham Atresia

CHLORIDE 801

mEq/L serum

100 50 0 II3-D-D Control Sham Atresia

POTASSIUM mEq/L serum ^I

Control Sham

Atrssia

Fig 2. Hematocrit was minimally decreased in IA animals. No modifications were found in serum Na, Cl, and K in comparison with sham animals, although Na was minimally increased. Potassium was significantly increased in all operated chicks. Values are means r SD. lP < .05.

1327

FETAL NUTRITION IN INTESTINAL ATRESIA

UREA

GLUCOSE

Control Sham Atresis Fig 3. Glucose and urea were also unmodified in IA animals in comparison with sham ones. Values are means f SD.

globulin, and P-globulin, and a marked hyper-yglobulinemia. These results are summarized in Table 1 with those in the control and sham groups. The figures corresponding to the composition of the normal amniotic fluid revealed that its protein content was richer than that of the human on day 14 of gestation, peaked at the 17th day, and decreased thereafter. PAGE-SAS assays demonstrated that the great majority of amniotic protein was egg-albumin (molecular weight, 45,000) with some transferrin (molecular weight, 78,000). DISCUSSION

Amniotic fluid deglutition by the human fetus was first studied in 1966 by Pritchard who, by injecting 51Cr-tagged erithrocytes into the amniotic cavity of pregnant women on the last gestational month, could demonstrate that up to 750 mL of the 850 to 1,000 mL present at that moment were swallowed daily.Y Given the low protein content of this liquid (less than 1 g/dL), its role as a nutritional avenue is apparently modest, but in fact it amounts to 10% to 13% of daily nitrogen requirements. Lev and OrliclO demonstrated the absorption of amniotic components by the fetal gut by intrauterine injection of horseradish peroxidase in pregnant rats, and Pitkin and Reynolds” confirmed that exogenously injected S3,-methionintagged proteins were absorbed by the primate fetus. Carbohydrates can also be absorbed by the fetal lamb when administered intragastrically.‘* Studies of protein absorption by the human fetal gut were performed by Gitlin et al by injecting radionuclidetagged amniotic and exogenous proteins into the uterus of pregnant women in the last month of gestation.‘” They could demonstrate that 63% of them were absorbed daily, confirming, at the same time, that 200 to 57.5 mL of fluid was swallowed daily.

The recovery in maternal blood of a fraction of the radionuclides injected in these studies showed that there was not only deglutition and absorption of proteins, but also metabolic utilization by the fetus. In their remarkable study, these authors confirmed that 10% to 15% of daily nitrogen requirements were supplied by this route and that the proteins involved were not only fetal (lanugo, desquamated cells) but also low-molecular weight maternal proteins transferred through the placenta and passed into the amniotic fluid by filtration through the fetal kidney which excretes as much as 400 mL of urine a day. Further evidence of the nutritional role of amniotic fluid deglutition was provided by demonstration of undergrowth after experimental esophageal ligation in the rabbit fetus,14J5 and delivery of nutrients by intraamniotic infusion has confirmed the possibility of growth manipulation through this route.‘6-‘a The chick embryo, which has all nutrients contained within the egg, right from the beginning of incubation, is nourished first by imbibition, afterward by vascular extraction of the nutrients contained into the yolk sac and the albumen, and, finally, when its gastrointestinal tract is sufficiently developed, by deglutition. Amniotic fluid, which except for the absence of urinary contribution to its formation (urine is deversed into the allantoic cavity in the avian embryo), remains rather similar to the human one until day 13 of incubation,” incorporates albumen at that time through the opening of the sero-amniotic connection and becomes very rich in proteins until the 18th day.?’ Its volume decreases constantly during these days as a consequence of deglutition and, although this process has not been extensively studied, it is obvious that enteral nutrition might be even more relevant in the chick embryo than in the human fetus, and that its interference by intestinal obstruction should lead to malnutrition. Our results largely confirm this hypothesis. Chicks with IA had their Iength (as expressed by tibia1 length) significantly reduced in comparison with sham ones and weighed almost 50% less. The high mortality in these experiments, which was lower when the chicks were recovered earlier, further proves the severe nutritional deficiencies suffered by the obstructed chicks. Although we do i-lot have a fully

Table 1. Serum Proteins and Electrophoretic Total Protein

Prealbumin

Fractions

Albumin

u-1-G

&Z-G

W)

(“4

(%I

35 f 5.2

17.6 + 2.5

13.7 k 3.5

28.2 + 4.6

3.6 + 0.9

31.1 r 5.2

16.8 -e 2.3

16.3 c 3.8

31.6 2 4.4

4.1 _c 0.9

Group

(g/dL serum)

1%)

Control (n = 23)

1.26 2 0.3

1.1 2 0.8

Sham (n = 10)

1.30 k 0.4

1 * 0.3

IA(n = 11)

2.21 + 0.7*

0*

23.3 + 9.5*

8.6 t- 5*

11.9

t-

P-G I%)

3.9t

21.4 +- 5.1”

Y-G i%l

34.5 2 9*

NOTE. Values expressed as mean c standard deviation. lP < .Ol and tP < .05 against control 19th day. There were no significant differences for any fraction when comparing control v sham groups.

TOME

1328

satisfactory explanation for the increased proteinemia in this group, the pattern of protein fractions allows us to maintain that there was real undernourishment since prealbumin was absent and albumin and cxand P-globulins were markedly decreased. The important increase of y-globulin can account for hyperproteinemia, but there are some other alternative explanations: the tendency (albeit nonsignificant) to higher values of serum Na suggest some degree of dehydration and, although this is apparently contradicted by a mildly decreased hematocrit, the existence of anemia may mask intravascular depletion of water and electrolytes. Unfortunately, we have not studied hematologic status in detail. These protein alterations are in some way specific to this experimental setting because they were absent in very similar

ET AL

experiments directed to assess the nutritional status of the chick embryo with gastroschisis.22 In conclusion, our experiments lend support to the hypothesis that small weight for gestational age in babies with IA is in part due to the interference with the nutritional supply represented by the swallowed amniotic fluid in the last period of prenatal life. The use of the chick embryo as an experimental model for this purpose is particularly interesting given its probably greater reliance on this nutritional source and the absence of any transplacental maternal correction. Once again the chick embryo model has shown itself to be an interesting tool for studies of “fetal” biology in spite of the considerable zoological distance between birds and humans.

REFERENCES 1. Jolleys A: An examination of the birthweights of babies with some abnormalities of the alimentary tract. J Pediatr Surg 16:160163,198l 2. Pierro A, Cozzi F, Colarossi G, et al: Does fetal gut obstruction cause hydramnios and growth retardation? J Pediatr Surg 22:454-457,1987 3. Tibboel D. Van der Kamp AWM, Molenaar JC: The effect of experimentally induced intestinal perforation at an early developmental stage. J Pediatr Surg 16:1017-1020,198l 4. Tibboel D, Van der Kamp AWM, Molenaar JC: An experimental study on the effect of an intestinal perforation at various developmental stages. Z Kinderchir 37:62-66,1982 5. Tovar JA, Sun01 M, Lopez de Torre B. et al: Mucosal morphology in experimental intestinal atresia: Studies in the chick embryo. J Pediatr Surg 26:184-189,199l 6. Romanoff AL: Biochemistry of the Avian Embryo: A Quantitative Analysis of Prenatal Development. New York, NY, Interscience Publishers-John Wiley & Sons, 1967 7. Weller EM, Bowdon HR: Ontogeny of avian serum proteins. Ala J Med Sci 11:6-13,1974 8. Slade B, Milne J: The ontogeny of cu-foetoprotein in the chicken. Experientia 34:520-522, 1977 9. Pritchard JA: Fetal swallowing and amniotic fluid volume. Obstet Gynecol28:606-610, 1966 10. Lev R, Orlic D: Protein absorption by the intestine of the fetal rat in utero. Science 177:522-524, 1972 11. Pitkin RM, Reynolds WA: Fetal ingestion and metabolism of amniotic fluid protein. Am J Obstet Gynecol 123:356-363, 1975 12. Charlton-Char V, Rudolph AM: Digestion and absorption of carbohydrates by the fetal lamb in utero. Pediatr Res 13:10181023.1979

13. Gitlin D, Kumate J, Morales C, et al: The turnover of amniotic fluid protein in the human conceptus. Am J Obstet Gynecoll13:632-645.1975 14. Wesson D, Muraji T, Kent G, et al: The effect of intrauterine esophageal ligation on growth of fetal rabbits. J Pediatr Surg 19:398-399,1984 15. Mulvihill SJ. Stone MM, Debas HT, et al: The role of amniotic fluid in fetal nutrition. J Pediatr Surg 20:668-672, 1985 16. Heller L: Intrauterine aminoacid feeding of the fetus, in Bode H, Warshaw JB (eds): Parenteral Nutrition in Infancy and Childhood. New York, NY, Plenum, 1974, pp 206-213 17. Harrison MR, Villa RL: Trans-amniotic fetal feeding, I: Development of an animal model: Continuous amniotic infusion in rabbits. J Pediatr Surg 17:376-380,1982 18. Mulvihill SJ, Albert A, Synn A, et al: In utero supplemental fetal feeding in an animal model: Effects on fetal growth and development. Surgery 98:500-505,1985 19. Flake AW, Villa-Troyer RL, Adzick NS: Transamniotic fetal feeding, III: The effect of nutrient infusion on fetal growth retardation. J Pediatr Surg 21:481-484, 1986 20. Phillips JD, Fonkalsrud EW, Mirzayan A, et al: Uptake and distribution of continuously infused intraamniotic nutrients in the fetal rabbit. J Pediatr Surg 25:374-380, 1991 21. Romanoff AL: The Avian Embryo: Structural and Functional Development (ed 1). New York, NY, Macmillan, 1960, p 1304 22. Lopez de Torre B, Tovar JA, Uriarte S, et al: Transperitoneal exchanges of water and solutes in the fetus with gastroschisis. Experimental study in the chick embryo. Eur J Pediatr Surg 1:346-352, 1991