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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, Pediatrics, Physiology, and the California Regional Research Primate Center, University of California, Davis, California; and Division of Pulmonary Biology and Neonatology Children’s Hospital, Cincinnati, Ohio
Address reprint requests to: William M. Gilbert, MD, Department of Obstetrics and Gynecology, University of California, Davis, 4860 Y Street, Suite 2500, Sacramento, CA 95817; E-mail: wmgilbert{at}ucdavis.edu.
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ABSTRACT |
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METHODS: Twelve pregnant monkeys (Macaca mulatta) on gestational day 125 (term 165 ± 10 days) had surfactant protein A and B concentrations measured in amniotic fluid. In four controls, normal saline was injected into the amniotic fluid; four others (intra-amniotic) received intra-amniotic betamethasone (1 mg) and T4 (60 µg); and in four others (maternal), the dam was given betamethasone (12 mg) intramuscularly, repeated in 24 hours, plus TRH (400 µg) intravenously, repeated every 6 hours for 24 hours. Seventy-two hours after the initial amniocentesis, a hysterotomy was performed and fetal tissue and amniotic fluid harvested for determination of surfactant protein A and B concentrations and immunohistochemical staining for surfactant protein A.
RESULTS: Amniotic fluid surfactant protein A was higher with intra-amniotic injection than with maternal treatment (P < .04) or controls (P = .07). Amniotic fluid surfactant protein B was higher in the intra-amniotic group than in controls (P = .06). Immunohistochemical staining for surfactant protein A in the lung tissue was increased in the intra-amniotic group compared with controls (0.145 ± 0.01 versus 0.097 ± 0.001, percent positive staining for surfactant protein A cells per lung tissue cells; P < .03). Birth weight was greater in the intra-amniotic group compared with the maternal group (P < .03) although not different from the controls. Finally, gut motility and the presence of formed meconium were increased in the intra-amniotic group compared with the other groups (P < .05).
CONCLUSION: Intra-amniotic injection of betamethasone and T4 enhanced lung (and possibly intestinal) maturation of the preterm rhesus fetal monkey compared with maternal injections.
The primary neonatal morbidity associated with preterm delivery is respiratory distress syndrome (RDS).1 Betamethasone, a synthetic glucocorticoid, administered to the mother over 24 hours has been shown to decrease the incidence of RDS in premature fetuses if delivery is delayed for a minimum of 48 hours.1–3 TRH administered to the mother over 24 hours has been shown to further decrease RDS when given in conjunction with betamethasone in several studies4–6 but not in others.7 Thyroxine (T4), administered into the amniotic fluid (because it does not cross the placenta), has been shown to accelerate the maturation of preterm human fetal lungs as demonstrated by a more rapid rise in the lecithin–sphingomyelin (L/S) ratio compared with a control population.8 Frequently, when pregnant women present in premature labor, amniocentesis is often performed to rule out infection and to determine if the fetal lungs are mature.9,10 One potential route of therapy is to inject medications into the amniotic fluid at the time of amniocentesis. This approach could overcome problems associated with maternal administration of these medications including choice of appropriate dosages and time required for efficacy.
In prior studies, we observed that furosemide, injected into the amniotic cavity in sheep, was rapidly absorbed by the fetus through blood vessels, which perfuse the fetal surface of the placenta and the fetal membranes resulting in a significant diuresis.11 This diuresis occurred despite the ligation of the fetal esophagus (preventing fetal swallowing). The absorption from the amniotic fluid into the fetal blood vessels on the fetal surface of the placenta, or intramembranous absorption, is believed to play an important role in amniotic fluid volume regulation and composition.11,12 Subsequent work has demonstrated the presence of the intramembranous pathway in the rhesus monkey and the importance this pathway may play in this nonhuman primate model.13 Thus, intra-amniotic administration of therapies may be a more efficient method of treatment for the fetus than maternal administration.
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METHODS |
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Amniocentesis was performed on approximately gestational day 125 as previously described with the removal of 3 mL of amniotic fluid for examination.15 Animals were assigned to three groups of four each as follows: controls had 3 mL of saline injected intra-amniotically; intra-amniotic had 1 mg betamethasone and 60 µg of T4 (total volume 3 mL) injected intra-amniotically; and maternal received maternal administration of two doses, 24 hours apart, of 12 mg betamethasone intramuscularly and 400 µg TRH intravenously every 6 hours for 24 hours.
Seventy-two hours after amniocentesis and initiating treatment, each animal underwent a hysterotomy and the gestational sac removed intact for the collection of amniotic fluid and fetal tissues using standardized methods.16 Included in the gross evaluation of the fetus were measurements (body weight; crown-rump, humerus, femur, hand, and foot lengths; biparietal and occipitofrontal diameters; head, chest, arm circumferences) and organ weights (brain, thymus, spleen, liver, right and left kidneys, right and left adrenals). The small and large intestines were examined for overall length, motility, and for the presence or absence of formed meconium. Representative sections of tissues were collected and specimens immersed in 10% buffered formalin and O.C.T. (Tissue-Tek O.C.T., VanWaters and Rogers, San Francisco, CA) embedding compound and frozen over liquid nitrogen for histopathology.
The lungs were removed and were further dissected from the bronchus down into right and left sections and weighed. Lung morphology and morphometry were determined using both light and electron microscopy. The right cranial lung lobe was canalized and fixed by airway perfusion (30 cm of fixative pressure) of glutaraldehyde-paraformaldehyde in 0.2 M Cacodylate buffer (adjusted to 330 mOsm and 7.4 pH).17 For immunohistochemistry, the right intermediate and accessory lobes were immersed in O.C.T. embedding compound and frozen in super cooled Freon. The blocks of tissue were sectioned using a Reichert Jung 2040N cryostat, and the sections were incubated overnight with surfactant protein A specific antibody (1:30,000 dilution) containing 10% monkey serum in phosphate buffered saline.17 The lung tissue slides for surfactant protein A immunohistochemical staining were examined, and quantification of surfactant protein A cells was performed as previously reported.17 Staining was performed on a subset of animals: control (N = 3), intra-amniotic (N = 3), and maternal (N = 3) with one control, one maternal, and one intra-amniotic animal lung tissues unavailable.
The amniotic fluid samples collected before treatment and at hysterotomy were analyzed for surfactant protein A and surfactant protein B concentrations. The amniotic fluid was screened with an assay using a goat antihuman surfactant protein A antibody and a rabbit antihuman surfactant protein A antibody in a capture assay previously described.18 Human surfactant protein A was used as the standard. Surfactant protein B was detected by an assay using a rabbit antihuman surfactant protein B antibody by competitive assay using bovine surfactant protein B as standard.19
Groups were compared by analysis of variance for repeated measures for the amniotic fluid surfactant protein A and B comparisons. For the other nonrepetitive comparisons, Student’s t-test was used with all data expressed as mean ± standard error (SE) unless otherwise stated. Statistical significance was presumed to be P < .05 unless otherwise stated.
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RESULTS |
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. There were no differences in maternal weight at the time of surgery among the three groups. Mean fetal weight in the maternal group was less than the intra-amniotic group (P < .04) but not the controls (P = .09). Individual organ weights, including lung, adrenal, and placenta, when normalized to the fetal body weights, were not different between groups, suggesting that none of the routes of therapy were detrimental to the overall organ size as determined by wet weight. The change in concentration of surfactant protein A in amniotic fluid from the initial amniocentesis until delivery 72 hours later was significantly increased (Table 1) in the intra-amniotic group when compared with the maternal group (P < .04) and nonsignificantly increased compared with controls (P = .07, Table 1). The change in concentration of surfactant protein B measured in the amniotic fluid increased in the intra-amniotic group compared with the controls (P = .06, Table 1).
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DISCUSSION |
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Surfactant protein A is the most abundant surfactant protein found in the rhesus monkey and first appears in the early second trimester (approximately gestational day 62).20 Surfactant protein A concentrations correlate with increased L/S ratios and lung function indicating pulmonary maturity.20,21 Our findings of increased surfactant protein A in the intra-amniotic group would suggest that direct amniotic therapy may provide a better method for the premature maturation of fetal lungs. In addition, surfactant protein A is an important host defense protein that is involved in fighting infection, which may further benefit premature newborns.22
Several investigators have previously reported the results of in utero therapy. Jobe et al directly injected the sheep fetus with betamethasone or saline and found a more rapid improvement in fetal pulmonary maturation when compared with control injections, as demonstrated by neonatal pulmonary function tests.22 In other work, when Jobe et al compared a single treatment of betamethasone given to the pregnant ewe with a single injection directly into the sheep fetus, the maternal treatment demonstrated better outcomes whereas the single fetal treatment showed no benefit.23 In the baboon, Ervin et al demonstrated a nonsignificant improvement in premature neonatal pulmonary function and significant improvement in renal function with direct fetal injections of betamethasone compared with control injection.24 In all of these cases, the medications were given by direct fetal injection under ultrasound guidance. We have previously demonstrated that injected substances into the amniotic cavity are rapidly absorbed into the fetal circulation via the intramembranous pathway.11–13 Because of this rapid absorption into the fetal circulation, we believe that direct injection into the fetus or umbilical cord is not necessary.
In our study, a significant decrease in fetal body weight was shown in the maternal group when compared with the intra-amniotic group (Table 1
), which was seen with a single course of therapy. Because of the numbers of animals in our study, we cannot state with certainty that the difference in birth weight resulted from the effect of treatment. In addition, when organ to body weight ratios were examined, there were no differences between groups for any organ system, suggesting that no obvious effect on organ growth was seen with any treatment regimen.
Maternal antenatal steroids have been used primarily for their effect on lung maturation because RDS is the major cause of perinatal morbidity and mortality. However, glucocorticoids effects on gastrointestinal maturation were first noted in the 1960s. Necrotizing enterocolitis remains a significant clinical problem of premature infants, and antenatal steroids have long been known to decrease necrotizing enterocolitis when compared with controls.2,3 Our subjective finding of an increase in intestinal motility and the presence of solid meconium in the intra-amniotic treatment group as compared with the other group is, therefore, of interest. Direct intra-amniotic injection of the hormones betamethasone and T4 could have resulted in a more rapid movement of these medications into the fetal intestine via fetal swallowing. This may explain the increase in motility and formed meconium in the intra-amniotic group as compared with the other groups.
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Footnotes |
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Received November 2, 2000. Received in revised form March 28, 2001. Accepted March 30, 2001.
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REFERENCES |
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2. Liggins GC, Howie RN. A controlled trial of antepartum glucorticoid treatment for prevention of the respiratory distress syndrome in premature infants. Pediatrics 1972; 50:515–25.
3. Crowley P, Chalmers I, Keirse MJN. The effects of corticosteroid administration before preterm delivery: An overview of the evidence from controlled trials. Br J Obstet Gynaecol 1990;97:11–6.[Medline]
4. Morales WJ, O’Brien WF, Angel JL, Knuppel RA, Sawai S. Fetal lung maturation: The combined use of corticosteroids and thyrotropin-releasing hormone. Obstet Gynecol 1989;73:111–6.
5. Ballard RA, Ballard PL, Creasy RK, Padbury J, Polk DH, Bracken M, et al. Respiratory disease in very-low-birth-weight infants after prenatal thyrotropin-releasing hormone and glucocorticord. Lancet 1992;339:510–5.[Medline]
6. Knight DB, Liggins GC, Wealthall SR. A randomized controlled trial of antepartum thyrotropin-releasing hormone and betamethasone in the prevention of respiratory disease in preterm infants. Am J Obstet Gynecol 1994;171:11–6.[Medline]
7. Ballard RA, Ballard PL, Cnaan A, Pinto-Martin J, Davis DJ, Padbury JF, et al. Antenatal thyrotropin-releasing hormone to prevent lung disease in preterm infants. North American Thyrotropin-releasing Hormone Study Group. NEJM 1998;338:493–8.
8. Mashiach S, Barkai G, Sack J, Stern E, Goldman B, Brish M, et al. Enhancement of fetal lung maturity by intra-amniotic administration of thyroid hormone. Am J Obstet Gynecol 1978;130:289–93.[Medline]
9. Gibbs RS, Romero R, Hillier SL, Eschenbach DA, Sweet RL. A review of premature birth and subclinical infection. Am J Obstet Gynecol 1992;166:1515–28.[Medline]
10. Romero R, Jimenez C, Lohda AK, Nores J, Hanaoka S, Avila C, et al. Amniotic fluid glucose concentration: A rapid and simple method for the detection of intraamniotic infection in preterm labor. Obstet Gynecol 1990;163:968–74.
11. Gilbert WM, Newman PS, Brace RA. Potential route for fetal therapy: Intramembranous absorption of intraamniotically injected furosemide. Am J Obstet Gynecol 1995; 172:1471–6.[Medline]
12. Gilbert WM, Brace RA. The missing link in amniotic fluid volume regulation: Intramembranous absorption. Obstet Gynecol 1989;74:748–54.
13. Gilbert WM, Eby-Wilkins E, Tarantal AF. The missing link in Rhesus monkey amniotic fluid volume regulation: Intramembranous absorption. Obstet Gynecol 1997;89: 462–5.[Abstract]
14. Tarantal AF, Hendrickx AG. Prenatal growth in the cynomolgus and rhesus macaque (Macaca fascicularis and Maccaca mulatta)—A comparison by ultrasonography. Am J Primatol 1988;15:309–23.
15. Tarantal AF. Interventional ultrasound in pregnant macaques: Embryonic/fetal applications. J Med Primatol 1990;19:47–58.[Medline]
16. Tarantal AF, Marthas ML, Gargosky SE, Otsyula M, McChesney MB, Miller CJ, et al. Effects of viral virulence on intrauterine growth in SIV-infected fetal rhesus macaques (Macaca mulatta). J AIDS and Hum Retrovirol 1995;10:129–38.
17. Plopper CG, St. George JA, Read LC, Nishio SJ, Weir AJ, Edwards L, et al. Acceleration of alveolar type II cell differentiation in fetal rhesus monkey lung by administration of EGF. Am J Physiol 1992;262:L313–21.
18. Whitsett JA, Weaver TE, Lieberman MA, Clark JC, Daughter C. Differential effects of epidermal growth factor and transforming factor-beta synthesis of Mr = 35,000 surfactant-associated protein in fetal lung. J Biol Chem 1987;262:7908–13.
19. Pryhuber GS, Hull WM, Fink I, McMahan MJ, Whitsett JA. Ontogeny of surfactant proteins A and B in human amniotic fluid as indices of fetal lung maturation. Pediatr Res 1991;30:597–605.[Medline]
20. Have-Opbroek AAWT, Plopper CG. Morphogenic and functional activity of type II cells in early fetal rhesus monkey lungs. The Anatom Rec 1992;234:93–114.
21. Rooney SA, Young SL, Mendelson CR. Molecular and cellular processing of lung surfactant. FASEB J 1994;8:957–67.[Abstract]
22. Jobe AH, Polk D, Ikegami M, Newnham J, Kohen R, Kelly R. Lung responses to ultrasound-guided fetal treatments with corticosteroids in preterm lambs. J Appl Physiol 1993;75:2099–205.
23. Jobe AH, Newnham J, Willet K, Sly P, Ikegami M. Fetal versus maternal and gestational age effects of repetitive antenatal glucocorticoids. Pediatrics 1998;102:1116–25.
24. Ervin MG, Seidner SR, Leland MM, Ikegami M, Jobe AH. Direct fetal glucocorticoid treatment alters postnatal adaptation in premature newborn baboons. Am J Physiol 1998; 274:R1169–76.
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