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.
Normally cycling adult female rhesus monkeys (Macaca mulatta; N = 12) with a history of prior pregnancies were bred and identified as pregnant using established methods.14 All procedures employed within the study conformed to the requirements of the Animal Welfare Act, and study protocols were approved before implementation by the Institutional Animal Use and Care Administrative Advisory Committee at the University of California at Davis. Pregnancy in the rhesus monkey is divided into trimesters with gestational days 0–55 representing the first trimester, gestational days 56–110 representing the second trimester, and gestational days 111–165 representing the third trimester (term gestational 165 ± 10 days).15 All pregnancies were sonographically assessed to confirm normal growth and development before assigning to the study.14 The dams were administered ketamine hydrochloride (10 mg/kg) for these and subsequent ultrasound examinations.
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.
The characteristics of the three groups are shown in Table 1. 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).
Immunohistochemical staining for surfactant protein A was increased (P < .03) in the intra-amniotic group lung tissue (0.145 ± 0.011, mean ± SE percentage of mature cells staining positive for surfactant protein A per total lung tissue cells) as compared with the control group (0.097 ± 0.001) but not the maternal group (0.14 ± 0.03). Intestinal motility was noted by direct observation in all animals in the intra-amniotic group at necropsy. None of the animals in the other two groups had spontaneous intestinal contractions noted either sonographically or grossly (P < .05), suggesting that the intra-amniotic animals had more mature intestinal development. Formed meconium was found only in the colons of the intra-amniotic group (P < .05).
This study demonstrates that injection of betamethasone and T4 directly into the amniotic fluid increased the primary fetal surfactant, surfactant protein A, in preterm rhesus monkey amniotic fluid when compared with maternal treatment with betamethasone and TRH. In addition, immunohistochemical staining for surfactant protein A was greater in fetal lung tissue in the intra-amniotic group when compared with the control group. This significant increase in surfactant protein A concentrations in the amniotic fluid and similar levels in the lung tissue sections suggests increased synthesis and excretion of surfactant protein A in the intra-amniotic group as compared with the maternal group. Increased gut motility and the presence of solid meconium also suggest that the intra-amniotic route of therapy may accelerate intestinal development in the third trimester rhesus fetus when compared with controls and maternal therapy.
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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