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Mothers and Babies Research Centre (V.E.M., T.Z., R.S., W.B.G., V.L.C.), University of Newcastle; and Department of Respiratory and Sleep Medicine (P.G.G.), John Hunter Hospital, Newcastle, New South Wales 2310, Australia
Address all correspondence and requests for reprints to: Dr.Vicki Clifton, Mothers and Babies Research Centre, Endocrine Unit, John Hunter Hospital, Locked Bag 1, Hunter Region Mail Center, Newcastle, New South Wales, 2310, Australia. E-mail: . vclifton{at}mail.newcastle.edu.au
Abstract
Pregnancies complicated by asthma are associated with an increased risk of low birth weight. Currently, the mechanisms causing this outcome are unknown. To investigate whether impaired placental function may be a determinant, we measured placental 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) activity, protein and mRNA, placental CRH mRNA, fetal cortisol, and fetal estriol concentrations at delivery.
Asthmatic subjects were classified according to inhaled glucocorticoid intake during pregnancy and compared with a control nonasthmatic group. There was a 25% reduction in neonatal birth weight centile in asthmatic women who did not use inhaled glucocorticoid treatment. This was accompanied by significantly reduced placental 11ß-HSD2 activity, significantly increased fetal cortisol, and a trend toward increased placental CRH mRNA and reduced fetal estriol concentrations. The use of inhaled glucocorticoids for treatment was associated with birth weight centile, 11ß-HSD2 activity, CRH mRNA, fetal cortisol, and estriol concentrations similar to control levels. There was a significant inverse correlation between fetal cortisol and fetal estriol concentrations across all groups.
These studies demonstrate that inhaled glucocorticoid intake for the treatment of asthma is associated with improved placental function and fetal outcome, suggesting that inflammatory factors associated with asthma may be detrimental to fetal growth and development in these pregnancies.
ASTHMA DURING PREGNANCY is a common problem in Australia, complicating approximately 12% of pregnancies (1), a rate much greater than that observed in other parts of the world such as North America (2). Several epidemiological studies have indicated that asthmatic pregnancies are associated with a higher incidence of maternal and fetal complications. These include a greater risk of having a low birth weight neonate (3, 4, 5, 6, 7), an increased incidence of preterm labor or delivery (3, 4, 6, 7, 8, 9) or delivery by cesarean section (5, 7, 10), and a greater risk of perinatal mortality (6, 11). Although some studies suggest that asthma is not associated with poor outcomes (8, 9, 12, 13), most reports state that poor control of asthma and severe asthma itself are the greatest contributors to adverse perinatal outcomes, including low birth weight. The mechanisms that alter fetal growth in pregnant asthmatic women are currently unknown.
The enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD) plays a crucial role in mediating the availability of glucocorticoids for both the GR and the MR by interconverting glucocorticoids with their 11-keto metabolites. In the placenta, the high affinity 11ß-HSD2 enzyme converts active cortisol to inactive cortisone, thus acting as a barrier to protect the fetus from the relatively high concentrations of glucocorticoids found in the mother (14). 11ß-HSD2 immunoreactivity and mRNA are localized to the syncytiotrophoblast and villous cytotrophoblast (15, 16, 17), and there is a progressive increase in placental 11ß-HSD2 activity as gestation progresses (18).
Reductions in 11ß-HSD2 activity have been associated with reduced human fetal growth. Shams et al. (18) demonstrated that there was a significant reduction in enzyme activity in placentae from pregnancies complicated by intrauterine growth restriction (IUGR), and further work demonstrated that there were also reductions in 11ß-HSD2 mRNA levels (18, 19). Furthermore, one of the clinical features of patients suffering from the syndrome of apparent mineralocorticoid excess, which results from mutations of the 11ß-HSD2 gene, is moderate IUGR (20).
We have questioned whether there are alterations in placental 11ß-HSD2 in pregnancies complicated by asthma. The aims of our study were to determine whether there are any changes in mRNA, protein, or activity of 11ß-HSD2 in these pregnancies, and further, to assess whether these changes are associated with rises in fetal cortisol and alterations in placental pathways regulated by cortisol (CRH mRNA) or the fetal hypothalamic-pituitary-adrenal axis (fetal estriol concentrations). In addition, we determined whether 11ß-HSD2 activity was associated with neonatal birth weight centile in pregnancies complicated by asthma.
Subjects and Methods
The following experiments were formally approved by the Hunter Area Health Service Research Ethics Committee and the University of Newcastle Human Research Ethics Committee. Both asthmatic women and control nonasthmatic women were recruited to the study from the antenatal clinic at the John Hunter Hospital during the first trimester of their pregnancy. Written, informed consent was obtained from women for participation in the study.
Assessment of asthma severity
Clinical asthma severity was rated as mild, moderate, or severe using the integrated severity score described in the Australian Asthma Management Guidelines (21), which closely approximate the National Heart, Lungs and Blood Institute Guidelines (22). Measurements of current daytime symptoms, nocturnal and morning symptoms due to asthma, bronchodilator use, forced expiratory volume at 1 sec (FEV1), peak expiratory flow (PEF), and hospitalizations were used to assign the patient a severity rating of either mild, moderate, or severe. The patient was assigned to the most severe grade in which any feature occurred during her pregnancy. Subjects with mild persistent asthma exhibited any of the following characteristics: FEV1 greater than 80% predicted, no night-time asthma symptoms, no asthma symptoms on awakening, infrequent short-acting bronchodilator use, no severe attacks in the past year, and only occasional daytime symptoms (less than four times per week). Moderate asthma was defined by symptoms on most days, short-acting ß2 agonist requirements most days, night symptoms up to once per week, FEV1 greater than 60% and less than 80% predicted, or PEF variability less than 25%. In severe asthma, there were daily symptoms, limited physical activity, frequent night-time symptoms (more than once per week) and asthma on awakening, FEV1 less than 60% predicted, or PEF variability greater than 25%. Subjects were managed according to a standard treatment protocol (23) in a combined antenatal/asthma management clinic. Control subjects had spirometry monitored at 18 wk gestation, whereas asthmatic women received two to eight visits at the asthma management service, depending on severity of asthma and individual needs. At the first visit, a history of asthma control was recorded, including number of emergency presentations, hospital admissions, and oral glucocorticoid use in the previous 2 yr before pregnancy. Each asthmatic study subject received instruction with an asthma educator in asthma control, management skills, and a crisis plan. All subjects were asked to perform self-monitoring peak flows and to record these in a diary for 2 wk after each asthma management service visit or after an exacerbation. Follow-up visits were guided by asthma severity, with subjects being reviewed at ultrasound appointments at 18 and 30 wk gestation. During these visits, there was reinforcement of asthma knowledge, management skills, and inhaler technique, and compliance with medications and individual issues were discussed. Inhaled glucocorticoid therapy and oral prednisolone intake were recorded for each trimester during the pregnancy. Subjects with uncontrolled asthma or experiencing an exacerbation were reviewed by a respiratory physician.
Assessment of glucocorticoid use
Cumulative, inhaled glucocorticoid dose was calculated for each trimester and summarized as the mean daily dose of beclomethasone dipropionate or equivalent used during the pregnancy, where 1 µg beclomethasone dipropionate was considered equal to 1 µg budesonide and to 0.5 µg fluticasone propionate. Subjects were then grouped into the following categories, based on glucocorticoid dosage: nil, no glucocorticoid use; low, low dose glucocorticoid, where the average daily dose during pregnancy was less than 400 µg; moderate, moderate use where the average daily dose during pregnancy was between 400–1,500 µg; and high, high dose where the average daily dose during pregnancy was greater than 1,500 µg. Asthmatic women in the nil group used a ß2 agonist (ventolin) when required. Oral glucocorticoid intake was noted for each woman, and the percentage of women in each group requiring oral medication has been reported.
11ß-HSD2 activity study
Chemicals were obtained from Sigma (St. Louis, MO), unless specified. Placental samples were collected within 45 min of delivery (vaginal or cesarean section). Tissues were snap-frozen in liquid nitrogen and stored at -80 C until further use.
Placental tissue was homogenized in 10 vol 0.1 M sodium phosphate buffer (pH 7.4) containing 2 mM EDTA; protease inhibitor cocktail tablets, 20 per liter (Roche Diagnostics, Mannheim, Germany); Trasylol, 50,000 kIU/liter (Bayer Corp., Leverkusen, Germany); dithiothreitol, 100 µM; pepstatin A, 1 µM; benzamidine, 1 mM; bacitracin, 65,000 U/liter; and sucrose, 0.25 M, using a polytron homogenizer (Kinematica AG, Lucerne, Switzerland). This homogenate was centrifuged at 1,000 x g for 10 min to remove cellular debris. The supernatant was centrifuged at 105,000 x g, and the pellet that contained microsomes was rehomogenized in 1 ml sodium phosphate buffer containing protease inhibitors, but no sucrose. The protein concentration was determined by Bradford Assay (Bio-Rad Laboratories, Inc., Hercules, CA) (24) against a standard curve of BSA (1 mg/ml).
11ß-HSD2 activity was determined using a radiometric conversion assay, adapted from the work of Sun et al. (25). The protein fraction prepared above was incubated in triplicate at three protein concentrations (usually 100, 150, and 200 µg/ml) to ensure that the experiment was carried out in the linear range of enzyme activity. Incubated with the protein was a saturating concentration of cofactor (NAD+, 1 mM), cold substrate (cortisol, 5 µM), and approximately 200,000 cpm 3H-cortisol (Amersham Pharmacia Biotech, Buckinghamshire, UK) made up to 1 ml with sodium phosphate buffer containing protease inhibitors. The incubation was performed at 37 C for 15 min in a shaking water bath. The reaction was stopped by removing the solution from the water bath into a tube containing 2 ml of ice-cold ethyl acetate (BDH Ltd., Dorset, UK). The solution was thoroughly mixed, and the organic phase containing the steroids was removed and dried overnight under high-speed vacuum. The steroids were reconstituted in 100 µl ethyl acetate, and 10 µl each of 10 mM cold cortisol and cortisone were added as markers. This solution was spotted onto a glass-backed TLC plate (Alltech, Deerfield, IL) and chromatographed with 95:5 chloroform:ethanol (BDH Ltd.) as the mobile phase. The bands were visualized using UV light, and the silica band was scraped into a vial containing 10 ml of scintillation fluid (Amersham Pharmacia Biotech). Steroids were quantified using a liquid scintillation counter (1217 Rackbeta, LKB Wallac, Turku, Finland). Enzyme activity was expressed as nanomoles of cortisone formed per milligram of protein per hour.
Initial studies using the method outlined above were performed using at least two placental samples each in triplicate to optimize the assay procedure. These included a time optimization in which the incubation was carried out for 2, 10, 15, 30, 60, and 120 min. A cortisol saturation curve was obtained using a range of concentrations of cortisol (10, 25, 50, 100, 250, and 500 nM and 1, 5, and 10 µM). The data obtained were transformed into a Lineweaver-Burk plot to determine enzyme kinetics (Microsoft Excel 97 was used for line fitting). An internal standard of three pooled placentae was included in each assay of the asthmatic samples to allow direct comparison of results. The intra-assay variation was 19.4%, and the interassay variation was 20.1%.
Determination of protein levels using Western blot
Placental samples were homogenized, and protein was extracted as described above. Placental samples (10 µg protein) were suspended in reduced SDS sample buffer, heated for 3 min at 80 C, and loaded onto precast 12% Tris-glycine gels (Novex, San Diego, CA). Placental proteins were separated by electrophoresis (26) and transferred to a polyvinylidene fluoride membrane (NEN Dupont). Blots were stained for immunoreactive 11ß-HSD2 using specific sheep antihuman antibodies (The Binding Site, Birmingham, UK) and alkaline phosphatase conjugated second antibody (antisheep IgG). Specific immunostaining was then visualized using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Nalgene, Boston, MA). A band to which the second antibody bound in proportion to the total protein was used to adjust for loading. Blots were analyzed using computer-assisted densitometry (Scion Image, Release ß 4.0.2, NIH, Bethesda, MD).
RNA extraction and quantitative real-time RT-PCR
Total RNA was extracted from frozen placental samples using the trizol method (Life Technologies, Inc., Frederick, MD). Briefly, 1 ml trizol/0.1 g tissue was added to crushed placental samples before homogenizing (Polytron homogenizer, Kinematica AG). The homogenate was centrifuged at 7,800 x g for 10 min, and 0.2 ml chloroform/1 ml trizol was added to the supernatant. The mixture was shaken vigorously by hand for 15 sec, then centrifuged again at 7,800 x g for 15 min. The supernatant was removed, and 0.5 ml isopropanol (BDH Ltd.)/ 1 ml trizol was added. After standing at room temperature for 10 min, the solution was centrifuged at 7,800 x g for 10 min, the supernatant was removed, and 1 ml 75% ethanol/1 ml trizol was added. The OD (Cary50 UV-Visible Spectrophotometer, Varian, Palo Alto, CA) was used to determine RNA concentration. RNA (60 µg) was loaded onto RNeasy mini kit columns (QIAGEN, Clifton Hill, Victoria, Australia), and the purification was performed according to the manufacturer’s instructions. The RNA was eluted from the column with 60 µl RNase free water. RNA (1 µg) was reverse-transcribed using the Taqman RT kit (Perkin-Elmer Corp., Branchburg, NJ) and a 9600 GeneAmp PCR machine (Perkin-Elmer Applied Biosystems). The following cycles were used: 10 min at 25 C, 30 min at 48 C, and 5 min at 95 C; the samples were held at 4 C, then stored at -80 C until further use. RNA (1 µg) was run on a 1.5% agarose gel to verify the presence of 18S and 28S RNA bands, indicating that the extracted RNA was intact.
Quantitative real-time RT-PCR was used to determine mRNA abundance of 11ß-HSD1, 11ß-HSD2, and CRH in placental samples, which were compared with levels of a constitutively expressed gene, ß-actin. Primers were designed using Primer Express version 1.0b6 (Perkin-Elmer Applied Biosystems) and published sequences for the above genes found on the NCBI Entrez Nucleotide database (accession no. AH006349 for 11ß-HSD1, U27317 for 11ß-HSD2, and V00571 for CRH). ß-actin primers were supplied by Dr. Sam Mesiano (Mothers and Babies Research Centre, Newcastle, Australia). Sequences for the primers used were as follows: 11ß-HSD1, forward primer 5'-GAATTCAGACCAGAGATGCTCCA-3' and reverse primer 5'-GCCCCTGTGACAATCACTTT-3'; 11ß-HSD2, forward primer 5'-TCAAGACAGAGTCAGTGAGAAACG-3' and reverse primer 5'-GGAACTGCCCATGCAAGTG-3'; CRH, forward primer 5'-AGAAAGGCGGTCCGAGGA-3' and reverse primer 5'-GGAACTGCCCATGCAAGTG-3'; ß-actin, forward primer 5'-GGCCGCGGTGTACGCCAACACAGTGCTG-3' and reverse primer 5'-CCCGGGGCCGTCATACTCCTGCTTGCTG-3'. Primers (Life Technologies, Inc., Frederick, MD) were made up to a concentration of 100 pmol/µl with RNase free water. The PCR contained 40 ng reverse transcribed sample, 10 pmol of the appropriate primer mix, and 12.5 µl SybrGreen master mix (Perkin-Elmer Applied Biosystems) made up to a total volume of 25 µl with RNase free water in a MicroAmp Optical 96-well plate (PE Applied Biosystems, Foster City, CA). PCR analysis was performed on triplicate samples using the ABI Prism 7700 sequence detector (Perkin-Elmer Applied Biosystems). Reaction conditions were 2 min at 50 C, 10 min at 95 C, and 40 cycles of 15 sec at 95 C and 1 min at 60 C. A duplicate sample that had not been reverse-transcribed was included as a control for each sample, along with duplicate no-template controls for each primer. The Comparative CT method (where CT is the threshold cycle) was used to derive a relative quantitative measure of the test gene expression compared with ß-actin expression.
Determination of fetal cortisol and estriol concentrations
A sample of blood was taken from the umbilical vein immediately after delivery. Plasma was separated by centrifugation at 2,000 x g (4 C) for 15 min and stored at -20 C until use. Concentrations were determined using commercial RIA kits for cortisol (Orion Diagnostica, Espoo, Finland) and unconjugated estriol (Diagnostics Systems Laboratories, Inc., Webster, TX), following the manufacturer’s instructions. The sensitivity of the cortisol assay was 4.7 nmol/liter, and the sensitivity of the estriol assay was 0.03 ng/ml.
Statistical analyses
All results are presented as means ± SEM. Statistical analysis was performed using the GraphPad InStat program (version 2.04a, GraphPad Software, Inc., San Diego, CA) and the t test, one-way ANOVA, Kruskal-Wallis nonparametric ANOVA test, Bartlett’s test for homogeneity of variances, the Tukey-Kramer multiple comparisons test, and the Mann-Whitney U test. A P value of <0.05 was considered significant.
Results
Tables 1–4
show the maternal characteristics, glucocorticoid intake, fetal and neonatal characteristics, and asthma classifications for all women recruited to our study (n = 127). Not all women had their placenta or cord blood collected. The maternal characteristics of age, height, weight in early pregnancy, weight gain during pregnancy, gravidity, and parity were not significantly different between the groups (one-way ANOVA; P > 0.05). However, FEV1 was significantly decreased in the severe asthmatics compared with control subjects (t test; P < 0.02) and in the high glucocorticoid group compared with control subjects (t test; P < 0.05). Placental and cord blood samples taken for 11ß-HSD2, CRH, cortisol, and estriol measurements were representative of the whole group in terms of maternal and fetal characteristics and inhaled glucocorticoid dose during pregnancy.
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). It was significantly higher in the moderate (P < 0.05) and severe (P < 0.01) asthmatic groups compared with the mild asthmatic group (Tukey-Kramer multiple comparisons test) and in the moderate (P < 0.001) and high (P < 0.001) glucocorticoid groups compared with the low glucocorticoid group (Tukey-Kramer multiple comparisons test) both before pregnancy and in the first, second, and third trimesters. Inhaled glucocorticoid dose increased throughout pregnancy in all groups (low, moderate, and high). This was a significant increase for the moderate glucocorticoid group from the first to second trimester (Tukey-Kramer multiple comparisons test; P < 0.05), and from the first to third trimester (Tukey-Kramer multiple comparisons test; P < 0.01).
The fetal characteristics of abdominal circumference at 18 and 30 wk and the neonatal characteristics of birth weight, length, head circumference, and gestational age at delivery (Table 3) were not significantly different between the groups (one-way ANOVA; P > 0.05). When birth weight centile data were analyzed according to inhaled glucocorticoid intake, we found that neonates from the nil group had significantly lower birth weight centiles than those from the control nonasthmatic group and the low glucocorticoid group (t test; P < 0.05). This corresponded to a 25% reduction in birth weight centile compared with control infants and a 17% reduction compared with infants from mothers who used inhaled glucocorticoids for asthma treatment during pregnancy. Birth-weight centiles in the moderate and high glucocorticoid groups were not significantly different from the control group (t test; P > 0.05).
Table 4 shows the number of patients with mild, moderate, and severe asthma in the nil, low, moderate, and high dose glucocorticoid groups.
Optimization of 11ß-HSD2 enzyme activity assay
Time optimization indicated that the reaction was under conditions of initial velocity up to 15 min. The cortisol saturation curve indicated that the enzyme was saturated at 5 µM cortisol. The Km, determined by a Lineweaver-Burk plot, was found to be 249 ± 57 nM. A Lineweaver-Burk plot from a representative enzyme preparation is shown in Fig. 1. A protein concentration dependence study indicated that the region where 11ß-HSD2 activity increases linearly with enzyme concentration was between 100–200 µg/ml protein.
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Results from the study of enzyme activity in asthmatic and nonasthmatic placentae were analyzed in two ways. First, mean enzyme activity values were analyzed according to asthma severity (control, mild, moderate, and severe). As depicted in Fig. 2A, there were no significant differences in 11ß-HSD2 activity between any of these groups (control, 4.36 ± 0.65 nmol/mg·h; mild, 4.93 ± 0.71 nmol/mg·h; moderate, 5.32 ± 0.75 nmol/mg·h; and severe, 4.99 ± 0.63 nmol/mg·h; one-way ANOVA; P > 0.05). The data were also analyzed according to inhaled glucocorticoid intake (control, nil, low, moderate, and high). There was a significant reduction in 11ß-HSD2 activity (2.77 ± 0.30 nmol/mg·h) in the nil group of asthmatics not taking inhaled glucocorticoids to control their asthma (Fig. 2B), compared with both the nonasthmatic controls (4.36 ± 0.65 nmol/mg·h; t test; P < 0.02) and the other groups in which daily inhaled glucocorticoids were used for asthma treatment (low, 5.62 ± 1.09 nmol/mg·h, t test, P < 0.02; moderate, 5.77 ± 0.80 nmol/mg·h, t test, P < 0.01; and high, 5.99 ± 0.69 nmol/mg·h, t test, P < 0.01). Labor, mode of delivery, and smoking had no significant effect on placental 11ß-HSD2 activity (t test; P > 0.05; data not shown).
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; t test; P < 0.05) and in the nil and high glucocorticoid groups compared with control (Fig. 3B; t test; P < 0.05).
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; one-way ANOVA; P > 0.05). 11ß-HSD1 mRNA abundance was also measured in the placenta and was approximately 25-fold lower than 11ß-HSD2 mRNA abundance. There were no significant differences in 11ß-HSD1 mRNA levels between the asthma severity groups (one-way ANOVA; P > 0.05; data not shown) or glucocorticoid intake groups (Kruskal-Wallis nonparametric ANOVA; P > 0.05; data not shown).
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). These levels were inversely correlated with 11ß-HSD2 activity (Fig. 5B; r = 0.183; n = 49). However, this correlation was not statistically significant.
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). However, this was not statistically significant compared with the control group (t test; P > 0.05). Furthermore, there was no change in CRH mRNA abundance in placentae from women taking any dose of synthetic glucocorticoids, compared with the nonasthmatic control subjects (Kruskal- Wallis ANOVA; P > 0.05).
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; one-way ANOVA; P > 0.05). Across all groups, there was a significant inverse correlation with fetal cortisol concentration (Fig. 7B; r = 0.394; n = 50; P < 0.005).
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This is the first study to show an association between reduced placental 11ß-HSD2 activity and decreased birth weight in asthmatic pregnancies. 11ß-HSD2 activity is significantly reduced in asthmatic women who do not take inhaled glucocorticoids for asthma treatment. This reduction in activity is accompanied by a decrease in birth weight centile, increased fetal cortisol concentration, and decreased fetal estriol concentration. These data indicate that there are alterations in placental function in pregnancies complicated by asthma that may contribute to low birth weight neonates. We speculate that these alterations may be mediated by the inflammatory factors associated with asthma rather than the glucocorticoid treatment.
Our results support work from previous studies that suggest that not only are the effects of asthma during pregnancy most pronounced when the asthma is untreated, or poorly controlled, but that asthma medication itself is not harmful to the fetus (27). The changes in placental function that we observed were in the group of women not taking inhaled steroids for treatment. Placental function and neonatal birth weight centile were comparable in the asthmatic women using daily inhaled and periodic oral steroids to the control nonasthmatic group. The degree of asthma control may be assessed by examining lung function (FEV1) or monitoring hospitalizations for asthma treatment. Schatz et al. (28) demonstrated that lower maternal mean FEV1 during pregnancy is associated with increased incidences of birth weight in the lower quartile and ponderal indices less than 2.2, suggestive of asymmetric growth retardation. These associations were found to be independent of the effects of smoking, age, asthmatic episodes during pregnancy, and medication (28). In addition, some studies indicate that lower birth weights were more likely to occur when mothers had been hospitalized for their asthma during pregnancy (29, 30). Inhaled glucocorticoid treatment significantly reduces the number of exacerbations of asthma (31) and is not associated with adverse maternal or fetal outcomes (27). Interestingly, our data show that most women with decreased 11ß-HSD2 activity were mild asthmatics (76% of nil group) with a normal FEV1, who were recommended by their physician to use only a ß2 agonist when required. The effect of asthma on placental function appears to be restored to control levels when glucocorticoid treatment is used. Our data suggest, therefore, that the inflammatory effects of asthma may alter placental function and consequently fetal growth.
Inflammation may be an important factor in regulating the activity of 11ß-HSD2 in the placenta. Because glucocorticoids act specifically to reduce the effects of inflammatory mediators, it is possible that the inflammatory factors present in the asthmatic women not using inhaled glucocorticoids are inhibiting 11ß-HSD2 activity. A recent study from Cooper et al. (32) demonstrated that IL-1ß and TNF-
dose dependently inhibited 11ß-HSD2 activity and mRNA levels in a human osteosarcoma cell line. In addition, Hardy et al. (33) showed that the prostaglandins PGE2 and PGF2 could inhibit 11ß-HSD2 activity without changing mRNA levels in JEG-3 choriocarcinoma cells. Leukotriene B4, a product of the 5-lipoxygenase pathway, also inhibited 11ß-HSD2 enzyme activity without a change in gene expression (33). Therefore, it is possible that pro-inflammatory mediators may alter placental 11ß-HSD2 activity, but not mRNA abundance in asthmatic women, to favor the passage of active cortisol to the fetal compartment.
In pregnant women with malaria, altered cytokine levels in the placenta have been shown to be associated with poor birth weight outcomes. Fried et al. (34) showed that higher levels of IFN-
and TNF- in the placenta of malaria sufferers were associated with low birth weight infants. Moorman et al. (35) showed that increased placental gene expression of IL-8 and TNF- in malaria was associated with IUGR. Other inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosis, are also known to be associated with adverse pregnancy outcomes, including small for gestational age neonates (36, 37, 38), IUGR (39), and preterm delivery (36, 40).
Previous studies in animals and humans have indicated that systemic administration of synthetic glucocorticoids antenatally is detrimental to fetal growth and development. Multiple doses of betamethasone in the human result in a 9% reduction in birth weight and a 4% reduction in head circumference (41). Betamethasone administration to sheep results in a birth weight reduction of 19% at term (42). Our data indicate that in asthmatic pregnancies in which women used daily inhaled and/or periodic oral glucocorticoids, there were no changes in birth weight centile compared with a control nonasthmatic group. This difference with previous studies could be due to the different route of administration of synthetic glucocorticoids (primarily by inhalation). Paradoxically, it appears that a lack of glucocorticoid treatment in the asthmatic mother is associated with an impaired placental 11ß-HSD2 barrier, which allows endogenous maternal glucocorticoids to pass to the fetus and contribute to reduced fetal growth. This reduction in birth weight centile observed in untreated asthmatics appears to be overcome by glucocorticoid treatment, indicating the importance of continued inhaled glucocorticoid medication for asthma treatment during pregnancy.
Synthetic glucocorticoids are known to up-regulate 11ß-HSD2 in a number of tissues, and this may explain the trend toward increased placental 11ß-HSD2 activity in the asthmatics taking high doses of inhaled glucocorticoids for treatment during pregnancy. Increased 11ß-HSD2 activity after dexamethasone treatment has been shown previously in both human endometrial cells (43) and rat renal cells (44). In the baboon, administration of betamethasone to the mother significantly increased both protein and mRNA levels of 11ß-HSD2 in the placenta (45). This supports our observation of increased 11ß-HSD2 protein levels in the nil group, where there were increased levels of endogenous glucocorticoid, and in the high group, where there were high doses of synthetic glucocorticoids used. Our study did not, however, find any alterations in 11ß-HSD2 mRNA abundance with inhaled glucocorticoid intake.
Estriol is the major estrogen of pregnancy, produced exclusively from dehydroepiandrosterone sulfate derived from the fetal adrenal gland (46). Our results show a decrease in fetal estriol concentrations in the untreated asthmatics. This group also had higher levels of fetal cortisol and reduced 11ß-HSD2 activity in the placenta. These data suggest that cortisol of maternal origin was able to exert negative feedback on the fetal adrenal, resulting in a reduction of estriol output. This indicates not only that placental function is altered in asthmatic women who do not take preventative glucocorticoid medication, but also that fetal hypothalamic-pituitary-adrenal axis function may be suppressed.
Our study has indicated that alterations in placental 11ß-HSD2 activity are associated with changes in fetal growth. We have found that birth weight centile is reduced in asthmatics with reduced 11ß-HSD2 activity and that cortisol levels in the fetus inversely correlate with placental 11ß-HSD2 activity. Despite the fact that smoking is known to contribute to low birth weight outcomes (47), we found that the effect of untreated asthma on birth weight centile was greater than the effect of smoking alone. Overall, 11ß-HSD2 activity was not altered by smoking, and within the nil group itself, smokers and nonsmokers had similar 11ß-HSD2 activity and birth weight centile. Reduced 11ß-HSD2 activity has previously been associated with low birth weight outcomes. Shams et al. (18) showed that 11ß-HSD2 activity was reduced in the placentae from IUGR births compared with normal term placentae, and a recent study from this group indicates that mRNA levels of 11ß-HSD2 were also reduced in IUGR placentae, but that this was not due to any gene mutations (19). In apparent mineralocorticoid excess, in which patients have one or more genetic mutations of 11ß-HSD2, moderate IUGR is often observed (20). Our data do not demonstrate a corresponding reduction in 11ß-HSD2 mRNA abundance in the placentae from the nil group. This suggests that alterations in 11ß-HSD2 in asthmatic pregnancies are the result of posttranslational events. Studies in rats have suggested a relationship between 11ß-HSD2 activity and birth weight (48). One human study found a positive correlation between placental 11ß-HSD2 activity and birth weight (49). However, further work from this group was unable to confirm this result with larger study numbers (50). In the latter report, all but one baby weighed more than 2,500 g, suggesting that the correlation of activity and birth weight may not be apparent within the normal weight range, but may become more obvious when studying low birth weight infants (18). In the present study, we observed a 25% reduction in birth weight centile in the nil group compared with control subjects. This did not translate into a significant reduction in birth weight itself, and large epidemiological studies would be required to define birth weight outcome in relation to asthma severity and inhaled glucocorticoid intake. However, this study identifies possible mechanisms for changes in fetal growth in asthmatic pregnancies. Further work into the effects of inflammatory mediators on placental 11ß-HSD2 activity will aid our understanding of what contributes to changes in the placenta that lead to a lower birth weight outcome in these and other pregnancies complicated by inflammatory disease.
In summary, we found that pregnancies complicated by untreated asthma were associated with a decreased birth weight centile, reduced activity of placental 11ß-HSD2, increased fetal cortisol, and decreased fetal estriol. These changes were restored to control levels by the use of daily inhaled glucocorticoids for asthma treatment, suggesting an effect of inflammation on placental function in the untreated asthmatic group. This is the first study to show an association between reduced placental 11ß-HSD2 activity and decreased birth weight in pregnancies complicated by asthma, and it highlights the potential importance of inflammatory mediators in determining fetal well-being. The activity of placental 11ß-HSD2 is a crucial factor involved in fetal development in asthmatic pregnancies.
Acknowledgments
We thank Sr. Carolyn Kessell for helping recruit patients into this study and the staff of the Department of Obstetrics and Gynaecology for their assistance. We thank Dr. Sam Mesiano for the use of ß-actin primers.
Footnotes
This work was supported by The Asthma Foundation of New South Wales and the National Health and Medical Research Council.
Abbreviations: 11ß-HSD, 11ß-Hydroxysteroid dehydrogenase; FEV1, forced expiratory volume at 1 sec; IUGR, intrauterine growth restriction; PEF, peak expiratory flow.
Received June 28, 2001.
Accepted December 13, 2001.
References
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 V. E. Murphy, R. Smith, W. B. Giles, and V. L. Clifton Endocrine Regulation of Human Fetal Growth: The Role of the Mother, Placenta, and Fetus Endocr. Rev., April 1, 2006; 27(2): 141 - 169. [Abstract] [Full Text] [PDF]
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 K. Yang, L. Julan, F. Rubio, A. Sharma, and H. Guan Cadmium reduces 11{beta}-hydroxysteroid dehydrogenase type 2 activity and expression in human placental trophoblast cells Am J Physiol Endocrinol Metab, January 1, 2006; 290(1): E135 - E142. [Abstract] [Full Text] [PDF]
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 V. E. Murphy, P. Gibson, P. I. Talbot, and V. L. Clifton Severe Asthma Exacerbations During Pregnancy Obstet. Gynecol., November 1, 2005; 106(5): 1046 - 1054. [Abstract] [Full Text] [PDF]
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 V. E. Murphy, P. G. Gibson, P. I. Talbot, C. G. Kessell, and V. L. Clifton Asthma self-management skills and the use of asthma education during pregnancy Eur. Respir. J., September 1, 2005; 26(3): 435 - 441. [Abstract] [Full Text] [PDF]
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 V. E. Murphy, R. F. Johnson, Y.-C. Wang, K. Akinsanya, P. G. Gibson, R. Smith, and V. L. Clifton The Effect of Maternal Asthma on Placental and Cord Blood Protein Profiles Reproductive Sciences, July 1, 2005; 12(5): 349 - 355. [Abstract] [PDF]
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V. E. Murphy, P. G. Gibson, R. Smith, and V. L. Clifton Asthma during pregnancy: mechanisms and treatment implications Eur. Respir. J., April 1, 2005; 25(4): 731 - 750. [Abstract] [Full Text] [PDF]
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J. R. SECKL and M. J. MEANEY Glucocorticoid Programming Ann. N.Y. Acad. Sci., December 1, 2004; 1032(1): 63 - 84. [Abstract] [Full Text] [PDF]
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 V. E. Murphy, P. G. Gibson, W. B. Giles, T. Zakar, R. Smith, A. M. Bisits, C. G. Kessell, and V. L. Clifton Maternal Asthma Is Associated with Reduced Female Fetal Growth Am. J. Respir. Crit. Care Med., December 1, 2003; 168(11): 1317 - 1323. [Abstract] [Full Text] [PDF]
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