Fetal Glucocorticoid-regulated Pathways Are Not Affected by Inhaled Corticosteroid Use for Asthma during Pregnancy

Transcription

1 Fetal Glucocorticoid-regulated Pathways Are Not Affected by Inhaled Corticosteroid Use for Asthma during Pregnancy Nicolette A. Hodyl 1, Michael J. Stark 1, Annette Osei-Kumah 1, Maria Bowman 2, Peter Gibson 3, and Vicki L. Clifton 1,2 1 The Robinson Institute, University of Adelaide, South Australia, Australia; and 2 Mothers and Babies Research Centre, and 3 Department of Respiratory and Sleep Medicine, Hunter Medical Research Institute, University of Newcastle, Newcastle, New South Wales, Australia Rationale: Inhaled corticosteroids (ICS) are currently advised for the control of asthma during pregnancy, despite the lack of evidence regarding potential systemic effects on maternal, placental, and fetal systems. Objectives: To determine maternal plasma concentrations of cortisol, estriol, osteocalcin, and corticotropin-releasing hormone in pregnant women with asthma (n 5 156) and without asthma (n 5 51). Methods: During each trimester of pregnancy, the use and dose of ICS was recorded and blood samples were collected. Ultrasound was performed at 18 and 30 weeks gestation, and birth weight and fetal sex were recorded at delivery. Measurements and Main Results: Maternal hormone concentrations were not affected by the presence of asthma; however, they were inhibited by ICS use in a dose-dependent manner. This was dependent on fetal sex: in pregnancies with a female, ICS was inversely associated with maternal cortisol in first trimester and inversely associated with maternal osteocalcin in second and third trimester. When pregnant with a male, no effect of ICS dose was observed on maternal cortisol, estriol, or osteocalcin levels, whereas corticotropinreleasing hormone levels were increased with ICS use only in the first trimester. Conclusions: Maternal glucocorticoid-regulated systems appeared susceptible to ICS only when pregnant with a female. Fetal adrenal functionappearedunaffectedbyics in pregnanciesofbothmales and females. This provides clinically important information suggesting that ICS do not exert effects on glucocorticoid-regulated pathways in thefetus, and thereforeareunlikelytocontributeto adverseeffects on fetal growth and development. Keywords: inhaled corticosteroids; asthma; pregnancy; cortisol; corticotropin-releasing hormone Asthma is one of the most prevalent chronic conditions to affect pregnancy, with incidence rates varying among populations. In the United States, asthma affects up to 8.4% of pregnant women (1), whereas in Australia up to 14% of women are affected (2). During pregnancy, untreated or severe asthma (as indicated by the use of multiple asthma medications) has been associated with a number of adverse outcomes for both the mother and the fetus, including preeclampsia, preterm birth, congenital malformations, (Received in original form July 30, 2010; accepted in final form October 7, 2010) Supported by The Asthma Foundation of New South Wales, Department of Health and Ageing Grant, and the National Health and Medical Research Council of Australia. Correspondence and requests for reprints should be addressed to Vicki L. Clifton, Ph.D., Robinson Institute, University of Adelaide, Lyell McEwin Hospital, DX , Haydown Road, Elizabeth Vale, Adelaide 5112, South Australia. Vicki. clifton@adelaide.edu.au This article has an online supplement, which is accessible from this issue s table of contents at Am J Respir Crit Care Med Vol 183. pp , 2011 Originally Published in Press as DOI: /rccm OC on October 8, 2010 Internet address: AT A GLANCE COMMENTARY Scientific Knowledge on the Subject The impact of inhaled corticosteroids for asthma management in pregnancy on maternal hypothalamic-pituitaryadrenal axis function and glucocorticoid-mediated pathways in the placenta and fetus are not well understood. What this Study Adds to the Field We demonstrate that inhaled corticosteroid use for asthma does not affect fetal adrenal function during pregnancy. Inhibition of maternal glucocorticoid responsive pathways was dependent on both the sex of the fetus and the pregnancy trimester. and low birth weight (3 15). Appropriate doses and compliance with inhaled corticosteroids (ICS) for asthma treatment have been shown to be efficacious in decreasing both the incidence of these adverse outcomes (16, 17) and the number of asthmarelated physician and emergency department visits made by pregnant women (18). This has led to national recommendations for the continued use of ICS during pregnancy (19, 20). However, controversy remains, with questions remaining regarding the potential for ICS to exert adverse effects on the developing fetus. Furthermore, some evidence exists that suggests the use of high doses of ICS (. 1,000 ug/d) increases the risk of congenital malformations (14). This has led to cautious consideration by the medical community to recommend ICS use by pregnant women with asthma. In addition, up to 40% of women with asthma choose to discontinue ICS use without medical consultation when becoming pregnant (21, 22). Concerns exist that ICS exposure during pregnancy may have detrimental effects on the ontogeny and function of fetal systems, particularly the fetal adrenal (for review, see Reference 23). This stems from evidence relating fetal adrenal suppression to administration of other exogenous glucocorticoids in pregnancy (for example, betamethasone or prednisolone) (24, 25). However, the effect of ICS on the mother, placenta, and fetus throughout pregnancy has not been addressed. In the current study we have examined whether markers of maternal, placental, and fetal origin, which are known to be regulated by glucocorticoids, are altered by ICS treatment for maternal asthma during pregnancy. Maternal estriol concentrations are a reliable measure of fetal adrenal activity, as dehydroepiandrosterone (DHEA) produced by the fetal adrenal becomes sulfated in the fetal liver to dehydroepiandrosterone sulfate (DHEAS) and then aromatized by the placenta to form estriol, which is released into the maternal circulation (26). Previous studies showing that maternal dexamethasone administration during pregnancy significantly inhibits

2 Hodyl, Stark, Osei-Kumah, et al.: Inhaled Corticosteroid Effects during Pregnancy 717 estriol concentrations (27) suggest the fetal adrenal is suppressed by this glucocorticoid. Placental corticotropin-releasing hormone (CRH) also plays a critical role in both fetal adrenal maturation and steroid output (for review, see Reference 28), and its production can be increased by glucocorticoid administration (29). We measured CRH levels in maternal plasma in relation to ICS treatment as a marker of placental function. In healthy adults and nonpregnant women with asthma, ICS treatment exerts systemic effects on the hypothalamic-pituitary-adrenal (HPA) axis (30). It is unknown whether ICS treatment during pregnancy has a similar effect; therefore, maternal cortisol levels were also measured. Because cortisol exhibits marked diurnal variation, we chose to also measure osteocalcin, a marker of bone turnover (31 33) that is also suppressed by exogenous glucocorticoids and, unlike cortisol, does not vary diurnally (34). As we have previously demonstrated an increase in ICS requirements during pregnancy in women with asthma that is dependent on the sex of the fetus, as well as differential fetal growth and placental responses to glucocorticoids in pregnancies complicated by asthma that are dependent on fetal sex (11, 35 40), we assessed the effects of ICS on maternal hormone concentrations in each trimester separately according to fetal sex. METHODS Subjects Pregnant women were prospectively recruited at their first visit to the John Hunter Hospital antenatal clinic (NSW, Australia) after giving written informed consent. All experiments were approved by the Hunter Area Health Service Research Ethics Committee and The University of Newcastle Human Research Ethics Committee. Assessment of Asthma and Treatment Asthma severity was rated as previously described (21). Detail on the method for grading severity is provided in the online data supplement. Women were assigned to the most severe grade of asthma experienced during pregnancy. A total of 207 women were recruited in to the study: 51 control subjects, 78 with mild asthma, and 78 with moderate-severe asthma. Control subjects had spirometry monitored at 18 weeks gestation, whereas women with asthma received two to eight visits at the asthma management service (AMS) depending on severity of asthma and individual needs. Details of the service provided by the AMS can be found on the online data supplement. Dose of ICS was recorded at these visits. Cumulative, inhaled glucocorticoid dose was then calculated for each trimester and summarized as the mean daily dose of beclomethasone dipropionate or equivalent used during the pregnancy, where 1 mg beclomethasone dipropionate was considered equal to 1 mg budesonide and to 0.5 mg fluticasone propionate. As high-dose glucocorticoids are known to result in maternal (41) and fetal adrenal suppression (24), women who used oral glucocorticoids during pregnancy were excluded from this study. When required, women with asthma used b 2 -agonists (salbutamol) for the relief of symptoms. Our previous studies of women using the AMS demonstrate an 80% compliance rate with medication (21). Blood Collection and Cortisol, Osteocalcin, and Estriol Radioimmunoassay Plasma samples were collected at antenatal visits between 11 and 38 weeks gestation. All samples were collected between 2:00 and 5:00 P.M. Total cortisol was assayed using a commercial kit from Orion Diagnostica (Espoo, Finland). Assay sensitivity was 5 nm and interassay variability was less than 5%. Total maternal estriol was measured using a radioimmunoassay kit from Abbott Laboratories (Abbott Park, IL). Sensitivity of the assay was 6.6 ng/ml and interassay variability was less than 8%. Osteocalcin was measured using a commercial kit from Diagnostic Systems Laboratories (Webster, TX). Assay sensitivity was nmol/l and interassay variability was 4.24%. CRH Radioimmunoassay Immunoreactive CRH was assayed in maternal plasma using an inhouse assay described previously (42). Plasma samples (1 ml) were extracted using Vycor glass and the sample reconstituted in 1 ml of radioimmunoassay buffer (0.1 M sodium phosphate, 0.25% bovine serum albumin, 0.1% b-mercaptoethanol, and phenol red, ph 7.4). Samples were then diluted and assayed for immunoreactive CRH using an ovine CRH antibody, synthetic human CRH (Peninsula, CA) as standard, and [ 125 I]-Tyr-CRH purified in propan-1-ol gradient in 1% trifluoroacetic acid over a C18 Sep-Pak and then purified by highpressure liquid chromatography. The assay sensitivity was 10 pg/ml of extracted plasma and the interassay coefficient was 21.5%. Assessment of Fetal Growth Ultrasound measurements of abdominal circumference and umbilical systolic-to-diastolic (S:D) ratio were obtained twice during pregnancy at approximately 18 and 30 weeks gestation. Birth weight and fetal sex were recorded at birth. Birth weight centiles were calculated using (Perinatal Institute, Birmingham, UK). Infants with a birth weight less than the tenth centile were defined as small for gestational age. Statistical Analyses The data were analyzed using SPSSv17 (Statistical Package for the Social Sciences), using P values of 0.05 or less to indicate significance. Maternal CRH, estriol, cortisol, and osteocalcin levels were log (natural) transformed to normalize the data. Data were analyzed using repeated measures analysis of variance to assess asthma and gestational timing effects. Post hoc comparisons were performed using t tests with the Bonferroni correction. Planned a priori comparisons were made using analysis of variance to assess asthma severity and treatment effects on maternal hormone concentrations in each trimester. For analysis of these treatment effects, women with asthma were grouped according to whether they used ICS for asthma treatment. Given that a high dose of ICS (. 1,000 mg/d) has been associated with adverse fetal outcomes (14), we also analyzed the maternal hormone data comparing the use of lowmoderate doses of ICS (, 1,000 mg/d) to high-dose ICS (> 1,000 mg/d) in each trimester. Chi-square analyses were used to assess frequency data. Spearman correlations were used to identify associations between ICS dose and untransformed maternal hormone concentrations. RESULTS Overall, 75% of women attended two or more visits during their pregnancy, and data were only included when a minimum of two samples were collected. These data were grouped into three gestational periods corresponding to each trimester: first trimester (11 15 wk): control subjects, n 5 31; no ICS, n 5 40; and ICS, n 5 30; second trimester (16 24 wk): control subjects, n 5 47; no ICS, n 5 80; and ICS, n 5 47; third trimester (25 34 wk): control subjects, n 5 51; no ICS, n 5 80; and ICS, n No patient provided more than one sample during any individual time period. Clinical Characteristics Maternal data are reported in relation to asthma treatment used during the third trimester of pregnancy (Table 1). Significantly more women with mild asthma used No ICS treatment for asthma during pregnancy compared with ICS (67.5% vs. 25%), whereas the use of ICS was most frequently associated with severe asthma (40.8%; Table 1). The daily dose of ICS used during each trimester increased significantly over pregnancy (x , P, 0.001), as did the proportion of women who used high-dose ICS (> 1,000 mg/d). FEV 1 adjusted for height and weight was significantly reduced in women with asthma who used ICS compared with control subjects (Table 1; F[3, 209] , P ). Significantly more women in the No ICS group smoked cigarettes in pregnancy compared with the control and

3 718 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL TABLE 1. MATERNAL CHARACTERISTICS ACCORDING TO ASTHMA TREATMENT USED DURING THE THIRD TRIMESTER Maternal Characteristics Control No ICS ICS P Value n Age, yr 27.7 (0.7) 25.6 (0.6) 26.8 (0.6) 0.08 Height, cm (0.8) (0.7) (0.7) 0.28 Weight at 12 wk, kg 73.8 (2.8) 73.4 (2.2) 74.3 (2.3) 0.96 Gravidity, median (min-max) 2 (1 6) 2 (1 6) 2 (1 5) 0.77 Parity, median (min-max) 1 (0 4) 1 (0 4) 1 (0 4) 0.96 Asthma severity, n (%) Mild 54 (67.5) 24 (31.6),0.001 Moderate 6 (7.5) 21 (27.6) Severe 20 (25) 31 (40.8) ICS daily dose, mg/d, median, (min-max) First trimester 800 (100 2,000),0.001 Second trimester 800 (100 3,200) Third trimester 900 (100 3,200) ICS. 1,000 mg/d, n (%) First trimester 18 (25),0.001 Second trimester 31 (43) Third trimester 38 (53) FEV 1 (% predicted) 99.7 (2.2) 95.0 (1.8) 90.1 (1.6) 0.002* VC, L 3.89 (0.09) 3.76 (0.06) 3.73 (0.07) 0.38 Smoker, n (%) 6 (12) 28 (35) 14 (18) Definition of abbreviations: No ICS 5 no inhaled corticosteroid treatment; ICS 5 inhaled corticosteroids; max 5 maximum; min 5 minimum. Data represented as mean (6 SEM) unless otherwise indicated. Reported P values relate to main effect of asthma treatment (analysis of variance) or chi-square analysis for frequency data. * P, 0.05, ICS compared to Control and No ICS. ICS groups (x , P ). Control women and women with asthma were not significantly different in maternal age, height, weight, gravidity, or parity (Table 1). Fetal and neonatal data are presented in Table 2 according to fetal sex. Birth weight and birth weight centiles were significantly higher in males compared with females (P, in each case), but were unaffected by asthma treatment (Table 2) or severity (data not shown). Comparable rates of small for gestational age and preterm births were observed in the asthma and control groups. At 18 and 30 weeks gestation, no effect of asthma or treatment was demonstrated on abdominal circumference measures or SD ratios (Table 2). The Effect of Asthma on Maternal Hormone Concentrations Circulating levels of CRH and estriol increased significantly from first to second trimester, and again from second to third trimester (CRH, F[2, 114] , P, 0.001, post hoc comparisons P, 0.01 in each instance; estriol, F[2, 94] , P, 0.001, post hoc comparisons P, 0.01 in each instance; Table 3). Maternal cortisol levels increased from first to second trimester (F[2, 114] , P, 0.001, post hoc comparison P, 0.01) and then remained constant for the remainder of pregnancy (Table 3). Maternal osteocalcin levels were comparable in first and second trimester, and increased significantly from second to third trimester (F[2, 94] , P , post hoc comparison P, 0.01). Asthma alone (Table 3) and asthma severity (data not shown) had no overall effect on maternal hormone levels over gestation. The Effect of ICS Treatment during Pregnancy on Maternal Hormone Concentrations When pregnant with a male, CRH levels were significantly elevated in first trimester with ICS use compared with the control group (F[2, 45] , P ; Table 4). However, CRH was unaltered by ICS treatment in the second and third trimesters. Within the ICS group, CRH concentrations were unaffected by high compared with low-moderate doses of ICS and no correlation between ICS dose and CRH was observed. No significant associations were observed between ICS dose and maternal cortisol, estriol, or osteocalcin levels in any trimester in women pregnant with a male. When pregnant with a female, ICS dose was inversely associated with maternal cortisol levels in the first trimester (r , P ) but not in the second or third trimester. In the second trimester and third trimester, ICS dose was inversely associated with maternal osteocalcin levels (second trimester r , P ; third trimester r , P ). Osteocalcin concentrations were lower overall in the women treated with ICS compared with the No ICS group (F[2, 65] , P ; Table 4). There was no association between ICS dose and estriol or CRH at any measured time point. Maternal hormone levels in each trimester were unaffected by the use of high as compared with low-moderate doses of ICS (data not shown). The Relationship between ICS and Fetal Growth In women who used ICS for asthma treatment, the association between ICS dose and fetal ultrasound measures (abdominal circumference and umbilical S:D ratio) as well as ICS dose and birth weight centiles were assessed for each sex. No association between ICS dose and abdominal circumference, S:D ratios, and birth weight centiles were observed in pregnancies with males or females. DISCUSSION Our findings indicate that treatment of asthma with ICS during pregnancy does not affect fetal adrenal function, as demonstrated by unchanging measurements of estriol with increasing doses of ICS used during pregnancy. These data indicate that ICS use for asthma does not influence fetal glucocorticoidregulated pathways. In pregnancies with a female fetus, maternal glucocorticoid-regulated pathways were responsive to ICS treatment for asthma. ICS suppressed maternal cortisol in the first trimester and osteocalcin in the final two trimesters of

4 Hodyl, Stark, Osei-Kumah, et al.: Inhaled Corticosteroid Effects during Pregnancy 719 TABLE 2. FETAL AND NEONATAL CHARACTERISTICS ACCORDING TO ASTHMA TREATMENT Control No ICS ICS P Value Female infants n 5 28 n 5 43 n 5 38 Abdominal circumference, mm 18 wk (3.1) (2.6) (2.8) wk (6.8) (4.9) (4.7) 0.88 Umbilical systolic:diastolic ratio 18 wk 4.45 (0.4) 5.06 (0.30) 4.66 (0.35) wk 2.97 (0.11) 2.96 (0.08) 2.97 (0.08) 0.99 Birth weight, g 3,358 (110) 3,336 (89) 3,295 (94) 0.90 Birth weight centile 45.9 (5.3) 45.4 (4.3) 43.0 (4.6) 0.90 Head circumference, cm 34.1 (0.3) 34.1 (0.2) 34.0 (0.3) 0.96 Length, cm 51.4 (0.5) 51.0 (0.4) 50.2 (0.4) 0.16 SGA, no. (%) 3 (10.7) 4 (9.3) 6 (15.7) 0.49 Preterm delivery, no. (%) 1 (3.6) 3 (7.0) 2 (5.3) 0.95 Placental weight, g (32.0) (27.9) (34.2) 0.73 Cord blood cortisol, nmol/l median (95% CI) 371 ( ) 310 ( ) 235 ( ) 0.31 Male infants n 5 24 n 5 38 n 5 38 Abdominal circumference, mm 18 wk (3.5) (3.1) (3.0) wk (4.2) (2.9) (3.2) 0.69 Umbilical systolic:diastolic ratio 18 wk 4.35 (0.30) 3.79 (0.23) 4.24 (0.22) wk 2.86 (0.10) 2.79 (0.07) 2.83 (0.08) 0.84 Birth weight, g 3,541 (133) 3,776 (105) 3,465 (105) 0.10 Birth weight centile 62.2 (5.9) 63.8 (4.7) 57.9 (4.7) 0.65 Head circumference, cm 34.6 (0.4) 35.3 (0.3) 34.8 (0.3) 0.31 Length, cm 52.3 (0.6) 53.1 (0.4) 52.3 (0.4) 0.42 SGA, no. (%) 1 (3.6) 1 (2.3) 2 (5.3) 0.77 Preterm delivery, no. (%) 2 (7.1) 0 (0) 2 (5.3) 0.99 Placental weight, g (45.9) (42.5) (36.5) 0.75 Cord blood cortisol, nmol/l 200 (7 392) 314 ( ) 315 ( ) 0.54 Definition of abbreviations: CI 5 confidence interval; ICS 5 inhaled corticosteroids; No ICS 5 no inhaled corticosteroid treatment; SGA 5 small for gestational age. Data reported as means (6 SEM) unless otherwise indicated. pregnancy in a dose-dependent manner. This was not evident in pregnancies with a male fetus despite the use of comparable doses of ICS by mothers in both groups. The suppression of cortisol and osteocalcin by ICS was dose dependent and not specifically due to high-dose steroid use, as defined by a 1,000 mg/d cutoff. Suppression of these hormones by ICS did not appear to affect fetal growth. These are clinically important findings, as this study indicates the continued use of ICS during pregnancy does not exert adverse effects on fetal growth and development. In nonpregnant women with asthma, high-dose ICS and oral prednisolone are both associated with adrenal suppression (31, 43 46). We questioned whether the use of daily ICS for the treatment of asthma during pregnancy would affect maternal HPA function, as indicated by cortisol suppression. Inhibition of cortisol by ICS was observed in the first trimester alone, and only in those women pregnant with a female fetus. This suggests that women pregnant with a female fetus exhibit greater sensitivity to ICS, with negative feedback observed on the maternal HPA axis. Furthermore, this comparative increased sensitivity to ICS treatment was only evident in the early stages of pregnancy. This finding cannot be explained by increased size of ICS dose in women pregnant with a female compared with a male fetus, nor can it be explained by increased doses in first trimester compared with later in pregnancy. Indeed, the opposite was true, as an increased requirement and use of higher doses of ICS were observed in second and third trimester compared with first trimester. These findings may suggest that the maternal HPA institutes a state of increased sensitivity to inhaled glucocorticoids in early pregnancy, which is dependent on the sex of the fetus. The collection of samples in this study was constrained by timing of antenatal clinic appointments. The observed dosedependent inhibition of cortisol evident in the first trimester may therefore be a result of timing of blood collection, due to circadian fluctuations and variation between individuals (47). Because this could not be circumvented, we also measured osteocalcin, which is known to be highly sensitive to ICS treatment and does not fluctuate diurnally (34). In the second and third trimesters, a dose-dependent suppression of osteocalcin was observed, indicating that the maternal system is sensitive to ICS throughout pregnancy. Again, this effect was only observed TABLE 3. MATERNAL HORMONE CONCENTRATIONS IN EACH TRIMESTER IN CONTROL WOMEN AND WOMEN WITH ASTHMA First Trimester Second Trimester Third Trimester P Value* CRH Control 0.87 (0.16) 1.18 (0.18) 3.40 (0.18),0.001 Asthma 1.04 (0.11) 1.48 (0.14) 3.29 (0.13) Cortisol Control 5.80 (0.10) 6.07 (0.10) 6.23 (0.08) Asthma 5.62 (0.07) 6.04 (0.07) 6.26 (0.05),0.001 Estriol Control 1.19 (0.38) 4.53 (0.20) 5.60 (0.09),0.001 Asthma 0.49 (0.29) 4.25 (0.15) 5.42 (0.07) Osteocalcin Control 0.48 (0.14) 0.61 (0.15) 0.59 (0.17) x Asthma 0.67 (0.17) 0.53 (0.19) 0.90 (0.21) CRH 5 corticotropin-releasing hormone. Data are reported as means of transformed data (6 SEM). * P value reflects significance for time effect (comparing values across each trimester). No effects of asthma were observed. Third trimester significantly greater than second, second trimester significantly greater than first trimester. Second and third trimester significantly greater than first trimester. x Third trimester significantly greater than second trimester.

5 720 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL TABLE 4. MATERNAL HORMONE CONCENTRATIONS IN EACH TRIMESTER OF PREGNANCY ACCORDING TO ASTHMA TREATMENT Control No ICS ICS P Value Pregnancies with a female fetus First trimester CRH 0.89 (0.20) 1.10 (0.17) 1.11 (0.19) NS Cortisol 5.84 (0.13) 5.67 (0.11) 5.51 (0.13) NS Estriol 0.58 (0.36) 0.49 (0.32) 0.53 (0.38) NS Osteocalcin 0.57 (0.21) 0.86 (0.19) 0.99 (0.27) NS Second trimester CRH 1.52 (0.15) 1.50 (0.12) 1.49 (0.15) NS Cortisol 6.29 (0.12) 6.40 (0.09) 6.06 (0.12) NS Estriol 4.59 (0.30) 4.12 (0.24) 4.33 (0.24) NS Osteocalcin 0.98 (0.18) 1.30 (0.13) 0.67 (0.19)* Third trimester CRH 3.26 (0.19) 3.32 (0.16) 3.16 (0.16) NS Cortisol 6.15 (0.13) 6.21 (0.11) 6.18 (0.11) NS Estriol 5.55 (0.14) 5.40 (0.14) 5.48 (0.10) NS Osteocalcin 1.40 (0.23) 1.94 (0.23) 1.10 (0.23)* Pregnancies with a male fetus First trimester CRH 0.84 (0.20) 1.16 (0.16) 1.57 (0.18) Cortisol 5.76 (0.12) 5.87 (0.12) 5.68 (0.14) NS Estriol 1.20 (0.52) 1.74 (0.60) 0.21 (0.60) NS Osteocalcin 0.67 (0.25) 1.25 (0.36) 1.09 (0.36) NS Second trimester CRH 1.36 (0.15) 1.75 (0.11) 1.66 (0.15) NS Cortisol 6.41 (0.13) 6.24 (0.10) 6.08 (0.12) NS Estriol 4.67 (0.34) 4.50 (0.27) 4.11 (0.31) NS Osteocalcin 0.90 (0.18) 1.27 (0.18) 1.02 (0.24) NS Third trimester CRH 3.29 (0.24) 3.43 (0.20) 3.24 (0.21) NS Cortisol 6.16 (0.10) 6.32 (0.08) 6.21 (0.08) NS Estriol 5.65 (0.17) 5.43 (0.18) 5.16 (0.14) NS Osteocalcin 1.12 (0.25) 1.76 (0.22) 1.57 (0.25) NS Definition of abbreviations: CRH 5 corticotropin-releasing hormone; ICS 5 inhaled corticosteroids; NS 5 not significant. Data are reported as means (6 SEM) of log (natural) transformed data. Reported P values reflect significance level of main effect of treatment (analysis of variance) using transformed data. * P, 0.05 compared to No ICS, post hoc comparison. P, 0.05 compared to Control group, post hoc comparison. in the presence of a female fetus. Limitations in sample collection cannot explain these sex-specific effects. Why this alteration in maternal function is dependent on the sex of the fetus is unclear. However we have previously reported differential alterations in the function of maternal and placental systems in the presence of asthma with respect to fetal sex. Specifically, maternal inflammatory pathways are up-regulated in the presence of a female fetus, but remain unaltered when the fetus is male (35). Furthermore, placental glucocorticoid receptor and metabolic pathways also appear responsive to endogenous corticosteroids only in the presence of a female fetus (37, 39). The current findings add to this body of work, further supporting the presence of differential mechanisms regulating glucocorticoid and inflammatory pathways that are dependent on fetal sex. A major concern of asthma treatment during pregnancy is the potential for teratogenic side effects of ICS use. Evidence for ICS effects on the fetus can be demonstrated through glucocorticoid suppression of the fetal HPA axis, measurable through maternal estriol levels, which directly reflect fetal adrenal steroidogenic activity (26). Previous studies have shown that dexamethasone or betamethasone administration for fetal lung maturation to women at risk of preterm labor results in suppression of maternal estriol levels (24, 25). Our study found that maternal plasma estriol concentrations were not reduced in women with asthma who used ICS, nor were any dose-dependent effects evident within this group. These data suggest that the fetal adrenal is not susceptible to glucocorticoid inhibition by ICS. This may be due to protection of the fetus from glucocorticoid exposure conferred by the placental glucocorticoid barrier. This barrier is formed primarily by the enzyme 11b hydroxysteroid dehydrogenase type 2 (11bHSD2), which inactivates glucocorticoids from the maternal system into their 11 keto-metabolites (48), together with placental glucocorticoid efflux proteins, such as P-glycoprotein. We have previously demonstrated that 11bHSD2 mrna and activity are not altered by ICS use for asthma in pregnancy (39) and do not appear to metabolize the inhaled synthetic steroid preparations used for asthma treatment (49). However, increased expression of other glucocorticoid barrier proteins may explain why ICS does not affect the fetal adrenal, despite altering maternal glucocorticoid-regulated pathways. Whether glucocorticoid efflux proteins present in the placenta are altered by ICS use is currently unknown. Potential limitations should be considered in this study. Rhinitis is frequently associated with asthma, and the first line of treatment for both conditions is corticosteroids. We do not have information relating to the prevalence or treatment of rhinitis in women participating in this study. This should be considered when interpreting the current data, as the impact of ICS on maternal hormones may be due to a cumulative effect. In addition, the effects of ICS use before and at the beginning of pregnancy were not examined, as women were recruited into the study at their first antenatal visit; for most women this coincided with the end of the first trimester. It is known that HPA axis function is attenuated with asthma in both children and nonpregnant adults, and it is therefore possible that subsequent effects of ICS treatment on maternal cortisol production may be masked by long-term adjustments to HPA function. However, through comparison of osteocalcin concentrations in control women and women with asthma, it is clear that ICS use through pregnancy can affect maternal systemic function. Further limitations to this study include the potential for recall bias, as women are being asked to recall the use of ICS during pregnancy. Although questions relating to asthma medications and adherence were designed to obtain an accurate assessment of these factors, this information is subject to recall and therefore the use of ICS may be overestimated. This should be considered in the interpretation of the data. There are a number of studies that confirm that adequate control of asthma during pregnancy is beneficial to both mother and fetus. Adequate control of asthma with ICS significantly reduces the incidence of acute exacerbations (4), decreases the number of hospital admissions (50, 51), and reduces the need for regular use of oral steroids, which are independently associated with low birth weight (12). Careful multidisciplinary management of asthma by both obstetricians and respiratory physicians to provide adequate ICS treatment reduces the rate of preterm delivery, perinatal death, and low birth weight to the rates observed in nonasthmatic pregnancies (4). Although maternal indices of systemic function appeared susceptible to ICS in the current study, no effects were observed on markers of fetal adrenal function. Our study provides evidence that ICS do not exert effects on glucocorticoid-regulated pathways in the placenta and fetus, and therefore are unlikely to contribute to adverse effects on fetal growth and development. Author Disclosure: N.A.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.O.-K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.G. received lecture fees from GlaxoSmithKline, AstraZeneca, Novartis, and Boehringer Ingelheim (up to

6 Hodyl, Stark, Osei-Kumah, et al.: Inhaled Corticosteroid Effects during Pregnancy 721 $1,000). V.L.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgment: The authors thank the numerous collaborators involved in this project who contributed to the collection of these data, including Prof. Warwick Giles, Prof. Roger Smith, Ms. Carolyn Kessell, Ms. Philippa Talbot, and Dr. Vanessa Murphy. They also thank all the women who offered to participate in this study and acknowledge the support of John Hunter Hospital Antenatal Clinic and Delivery Suite staff who assisted with the recruitment and care of the study subjects. References 1. Kwon HL, Belanger K, Bracken MB. Asthma prevalence among pregnant and childbearing-aged women in the United States: estimates from national health surveys. Ann Epidemiol 2003;13: Australian Centre for Asthma Monitoring. Asthma in Australia 2008, in AIHW Asthma Series, AIHW, editor. Canberra: Australian Institute for Health and Welfare; Bahna SL, Bjerkedal T. The course and outcome of pregnancy in women with bronchial asthma. Acta Allergol 1972;27: Stenius-Aarniala B, Piirila P, Teramo K. Asthma and pregnancy: a prospective study of 198 pregnancies. Thorax 1988;43: Demissie K, Breckenridge MB, Rhoads GG. Infant and maternal outcomes in the pregnancies of asthmatic women. Am J Respir Crit Care Med 1998;158: Breton MC, Beauchesne MF, Lemiere C, Rey E, Forget A, Blais L. Riskof perinatal mortality associated with asthma during pregnancy. Thorax 2009;64: Kallen B, Rydhstroem H, Aberg A. Asthma during pregnancy a population based study. Eur J Epidemiol 2000;16: Kallen B, Otterblad Olausson P. Use of anti-asthmatic drugs during pregnancy. 2. Infant characteristics excluding congenital malformations. Eur J Clin Pharmacol 2007;63: Enriquez R, Griffin MR, Carroll KN, Wu P, Cooper WO, Gebretsadik T, Dupont WD, Mitchel EF, Hartert TV. Effect of maternal asthma and asthma control on pregnancy and perinatal outcomes. J Allergy Clin Immunol 2007;120: Wen SW, Demissie K, Liu S. Adverse outcomes in pregnancies of asthmatic women: results from a Canadian population. Ann Epidemiol 2001;11: Murphy VE, Gibson PG, Giles WB, Zakar T, Smith R, Bisits AM, Kessell CG, Clifton VL. Maternal asthma is associated with reduced female fetal growth. Am J Respir Crit Care Med 2003;168: Perlow JH, Montgomery D, Morgan MA, Towers CV, Porto M. Severity of asthma and perinatal outcome. Am J Obstet Gynecol 1992;167: Kallen B, Otterblad Olausson P. Use of anti-asthmatic drugs during pregnancy. 1. Maternal characteristics, pregnancy and delivery complications. Eur J Clin Pharmacol 2007;63: Blais, L., Beauchesne MF, Lemiere C, Elftouh N. High doses of inhaled corticosteroids during the first trimester of pregnancy and congenital malformations. J Allergy Clin Immunol 2009;124: e Clifton VL, Engel P, Smith R, Gibson P, Brinsmead M, Giles WB. Maternal and neonatal outcomes of pregnancies complicated by asthma in an Australian population. Aust N Z J Obstet Gynaecol 2009;49: Bakhireva LN, Schatz M, Jones KL, Chambers CD. Asthma control during pregnancy and the risk of preterm delivery or impaired fetal growth. Ann Allergy Asthma Immunol 2008;101: Murphy VE, Gibson PG, Smith R, Clifton VL. Asthma during pregnancy: mechanisms and treatment implications. Eur Respir J 2005;25: Schatz M, Leibman C. Inhaled corticosteroid use and outcomes in pregnancy. Ann Allergy Asthma Immunol 2005;95: National Asthma Council Australia. Asthma management handbook (revised and updated). South Melbourne: Asthma Management Council of Australia Ltd National Asthma Education and Prevention Program. Quick reference from the Working Group Report on asthma during pregnancy: recommendations for pharmacological treatment. Update In: NIH Publication No Bethesda, MD: US Department of Health and Human Services; National Institutes of Health; National Heart, Lung and Blood Institute Murphy VE, Gibson PG, Talbot PI, Kessell CG, Clifton VL. Asthma self-management skills and the use of asthma education during pregnancy. Eur Respir J 2005;26: Chambers K. Asthma education and outcomes for women of childbearing age. Case Manager 2003;14: Mesquita AR, Wegerich Y, Patchev AV, Oliveira M, Leao P, Sousa N, Almeida OF. Glucocorticoids and neuro- and behavioural development. Semin Fetal Neonatal Med 2009;14: Haning RV Jr, Curet LB, Poole WK, Boehnlein LM, Kuzma DL, Meier SM. Effects of fetal sex and dexamethasone on preterm maternal serum concentrations of human chorionic gonadotropin, progesterone, estrone, estradiol, and estriol. Am J Obstet Gynecol 1989;161: Hendershott CM, Dullien V, Goodwin TM. Serial betamethasone administration: effect on maternal salivary estriol levels. Am J Obstet Gynecol 1999;180:S219 S Patrick J, Challis J, Natale R, Richardson B. Circadian rhythms in maternal plasma cortisol, estrone, estradiol, and estriol at 34 to 35 weeks gestation. Am J Obstet Gynecol 1979;135: Maltau JM, Stokke KT, Moe N. Effects of betamethasone on plasma levels of estriol, cortisol and HCS in late pregnancy. Acta Obstet Gynecol Scand 1979;58: Grammatopoulos DK. Placental corticotrophin-releasing hormone and its receptors in human pregnancy and labour: still a scientific enigma. J Neuroendocrinol 2008;20: Korebrits C, Yu DH, Ramirez MM, Marinoni E, Bocking AD, Challis JR. Antenatal glucocorticoid administration increases corticotrophinreleasing hormone in maternal plasma. Br J Obstet Gynaecol 1998; 105: Wlodarczyk JH, Gibson PG, Caeser M. Impact of inhaled corticosteroids on cortisol suppression in adults with asthma: a quantitative review. Ann Allergy Asthma Immunol 2008;100: Martin RJ, Szefler SJ, Chinchilli VM, Kraft M, Dolovich M, Boushey HA, Cherniack RM, Craig TJ, Drazen JM, Fagan JK, et al. Systemic effect comparisons of six inhaled corticosteroid preparations. Am J Respir Crit Care Med 2002;165: Richy F, Bousquet J, Ehrlich GE, Meunier PJ, Israel E, Morii H, Devogelaer JP, Peel N, Haim M, Bruyere O, et al. Inhaled corticosteroids effects on bone in asthmatic and COPD patients: a quantitative systematic review. Osteoporos Int 2003;14: Toogood JH, Jennings B, Hodsman AB, Baskerville J, Fraher LJ. Effects of dose and dosing schedule of inhaled budesonide on bone turnover. J Allergy Clin Immunol 1991;88: Toogood JH. Inhaled steroids for chronic asthma. Hosp Pract (Off Ed) 1991;26:15 18, 23, Clifton VL, Murphy VE. Maternal asthma as a model for examining fetal sex-specific effects on maternal physiology and placental mechanisms that regulate human fetal growth. Placenta 2004;25: S45 S Clifton VL, Hodyl NA, Murphy VE, Giles WB, Baxter RC, Smith R. Effect of maternal asthma, inhaled glucocorticoids and cigarette use during pregnancy on the newborn insulin-like growth factor axis. Growth Horm IGF Res 2010;20: Hodyl N, Wyper H, Osei-Kumah A, Scott N, Murphy VE, Gibson P, Smith R, Clifton VL. Sex-specific associations between cortisol and birth weight in pregnancies complicated by asthma are not due to differential glucocorticoid receptor expression. Thorax 2010;65: Murphy VE, Clifton VL. Alterations in human placental 11Bhydroxysteroid dehydrogenase type 1 and 2 with gestational age and labour. Placenta 2003;24: Murphy VE, Zakar T, Smith R. Reduced 11B-hydroxysteroid dehydrogenase type 2 activity is associated with decreased birth weight centile in pregnancies complicated by asthma. J Clin Endocrinol Metab 2002; 87: Scott NM, Hodyl NA, Murphy VE, Osei-Kumah A, Wyper H, Hodgson DM, Smith R, Clifton VL. Placental cytokine expression covaries with maternal asthma severity and fetal sex. J Immunol 2009;182: Sorkness CA, LaForce C, Storms W, Lincourt WR, Edwards L, Rogenes PR. Effects of the inhaled corticosteroids fluticasone propionate, triamcinolone acetonide, and flunisolide and oral prednisone on the hypothalamic-pituitary-adrenal axis in adult patients with asthma. Clin Ther 1999;21: Chan EC, Smith R, Lewin T, Brinsmead MW, Zhang HP, Cubis J, Thornton K, Hurt D. Plasma corticotropin-releasing hormone, betaendorphin and cortisol inter-relationships during human pregnancy. Acta Endocrinol (Copenh) 1993;128: Brus R. Effects of high-dose inhaled corticosteroids on plasma cortisol concentrations in healthy adults. Arch Intern Med 1999;159:

7 722 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Grebe SK, Feek CM, Durham JA, Kljakovic M, Cooke RR. Inhaled beclomethasone dipropionate suppresses the hypothalamo-pituitaryadrenal axis in a dose dependent manner. Clin Endocrinol (Oxf) 1997; 47: Clark DJ, Lipworth BJ. Evaluation of corticotropin releasing factor stimulation and basal markers of hypothalamic-pituitary-adrenal axis suppression in asthmatic patients. Chest 1997;112: Clark DJ, Lipworth BJ. Adrenal suppression with chronic dosing of fluticasone propionate compared with budesonide in adult asthmatic patients. Thorax 1997;52: Peters SP. Safety of inhaled corticosteroids in the treatment of persistent asthma. J Natl Med Assoc 2006;98: Edwards CR, Benediktsson R, Lindsay RS, Seckl JR. Dysfunction of placental glucocorticoid barrier: link between fetal environment and adult hypertension? Lancet 1993;341: Murphy VE, Fittock RJ, Zarzycki PK, Delahunty MM, Smith R, Clifton VL. Metabolism of synthetic steroids by the human placenta. Placenta 2007;28: Wendel PJ, Ramin SM, Barnett-Hamm C, Rowe TF, Cunningham FG. Asthma treatment in pregnancy: a randomized controlled study. Am J Obstet Gynecol 1996;175: Suissa S, Ernst P, Kezouh A. Regular use of inhaled corticosteroids and the long term prevention of hospitalisation for asthma. Thorax 2002; 57: