Differential Effects of Maternal Dexamethasone Treatment on ...

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Endocrinology 148(2):800 – 805 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1194

Differential Effects of Maternal Dexamethasone Treatment on Circulating Thyroid Hormone Concentrations and Tissue Deiodinase Activity in the Pregnant Ewe and Fetus Alison J. Forhead, Juanita K. Jellyman, David S. Gardner, Dino A. Giussani, Ellen Kaptein, Theo J. Visser, and Abigail L. Fowden Department of Physiology, Development and Neuroscience (A.J.F., J.K.J., D.S.G., D.A.G., A.L.F.), University of Cambridge, Cambridge CB2 3EG, United Kingdom; and Department of Internal Medicine (E.K., T.J.V.), Erasmus Medical Center, Rotterdam, The Netherlands and renal and placental D3 activities were lower, than in the saline-exposed fetuses. In the ewes, plasma concentrations of T3 and T4 were reduced, and rT3 increased, by dexamethasone treatment without any change in tissue deiodinase activity. Therefore, maternal dexamethasone treatment has different effects on the thyroid hormone axis of the pregnant ewe and fetus. In the fetus, the dexamethasone-induced rise in circulating T3 may be due to both increased hepatic production of T3 from T4, and reduced clearance of T3 by the kidney and placenta. Changes in T3 bioavailability may mediate some of the maturational effects of antenatal glucocorticoid treatment in the preterm fetus. (Endocrinology 148: 800 – 805, 2007)

Clinically, treatment of pregnant women at risk of preterm delivery with synthetic glucocorticoids accelerates fetal maturation. This study investigated the effect of maternal dexamethasone treatment, in clinically relevant doses, on plasma thyroid hormone concentrations and tissue deiodinase activities (D1, D2, and D3) in ewes and their fetuses. From 125 d of gestation (term 145 ⴞ 2 d), pregnant ewes were injected twice im with either saline (2 ml of 0.9% NaCl, n ⴝ 11) or dexamethasone (2 ⴛ 12 mg in 2 ml of saline, n ⴝ 10) at 24-h intervals. Maternal dexamethasone treatment increased plasma T3 and reverse T3 (rT3), but not T4, concentrations in the fetuses. In the dexamethasone-exposed fetuses, hepatic D1 activity was higher,

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N CLINICAL PRACTICE, synthetic glucocorticoids such as dexamethasone are used to accelerate fetal maturation in pregnant women at risk of preterm delivery (1). Antenatal glucocorticoids mimic the actions of the endogenous rise in plasma cortisol normally seen in the fetus close to term (2). In man and other species, the prepartum cortisol surge induces structural and functional changes in fetal tissues that prepare the fetus for delivery and extrauterine life (2). Therefore, antenatal glucocorticoid treatment has markedly improved the survival and health of the preterm neonate (3, 4). Some of the maturational effects of endogenous and exogenous glucocorticoids on fetal tissues may be mediated by other endocrine systems, such as the thyroid hormones. In sheep fetuses near term, the rise in plasma cortisol causes tissue-specific changes in iodothyronine deiodinase activity that lead to a coincident rise in plasma T3 concentration (5). Deiodinase enzymes have an important role in determining the bioavailability of thyroid hormones: type I and II deiodinases (outer ring, D1 and D2) convert T4 to T3, whereas type III deiodinase (inner ring, D3) is responsible for T3 clearance and production of biologically inactive reverse T3 (rT3) from T4. In the sheep fetus, the prepartum cortisol surge increases T3 production by activation of D1 in hepatic, renal,

and perirenal adipose tissues, and reduces T3 clearance by suppression of D3 activity in the fetal kidney and placenta (5). The rise in plasma T3 near term has been shown to be essential for the normal maturation of fetal tissues (6 – 8). For instance, in the sheep fetus, thyroidectomy impairs normal maturation of gluconeogenic enzymes in the liver and kidney near term (8). Preterm infants often have low serum T4 and T3 concentrations (9) which may account, in part, for the dysmaturity of their tissues and physiological systems. Therefore, like endogenous glucocorticoids near term, exogenous glucocorticoid treatment may improve perinatal maturation and survival by stimulation of thyroid hormone activity in the fetus. However, the consequence of exogenous glucocorticoids on thyroid hormone metabolism in fetal and maternal tissues is unknown. Therefore, this study investigated the effects of maternal dexamethasone treatment, in clinically relevant doses, on plasma thyroid hormone concentrations and tissue deiodinase activities in ewes and their fetuses. The sheep fetus is an ideal animal model for the study of thyroid hormone physiology in utero. The sheep fetus closely resembles the human fetus in the temporal development of the thyroid hormone system, more so than the rodent species (10). The onset of thyroid hormone activity occurs from around mid-gestation in the human and ovine fetus, and the thyroid hormone axis is fully mature soon after delivery, whereas in rats, maturation of thyroid hormone activity is relatively delayed and extends up to 4 wk of postnatal life. In addition, the sheep fetus is large enough to

First Published Online November 16, 2006 Abbreviations: PAT, Perirenal adipose tissue; rT3, reverse T3. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

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Forhead et al. • Dexamethasone and Thyroid Hormones in Utero

undergo experimental manipulation in utero and to provide adequate blood and tissue samples for analysis. Materials and Methods Animals Twenty-one Welsh Mountain sheep fetuses of known gestational age were used in this study; 16 were singletons, and five were twin fetuses. The ewes were maintained on 200 g kg⫺1 concentrates with free access to hay, water, and a salt-lick block. Food, but not water, was withheld for 18 –24 h before surgery. All surgical and experimental procedures were in accordance with the UK Animals (Scientific Procedures) Act 1986.

Surgical and experimental procedures Study 1: chronically catheterized animals. Fetuses of eleven pregnant ewes were chronically catheterized to obtain blood samples during maternal saline and dexamethasone treatment. There were six singleton and five twin fetuses used in the study; only one of the twin fetuses in a single pregnancy was instrumented. Two and three of the twin fetuses were in the saline and dexamethasone-treated groups, respectively. Between 116 and 119 d of gestation (term 145 ⫾ 2 d), iv catheters were inserted into the femoral artery and vein of eleven fetuses using surgical techniques described previously (11) and carried out under halothane anesthesia (1.5% in O2-N2O) with positive pressure ventilation. All catheters were exteriorized through the flank of the ewe and secured in a plastic bag sutured to the skin. The catheters were flushed daily with heparinized saline solution (100 IU heparin ml⫺1 0.9% saline wt/vol) from the day after surgery. At surgery, all fetuses were administered 100 mg ampicillin iv (Penbritin; Beecham Animal Health, Brentford, UK) and 2 mg gentamycin iv (Frangen-100; Biovet, Mullingar, Ireland). The ewes were given antibiotics im (procaine penicillin, Depocillin; Mycofarm, Cambridge, UK) on the day of surgery and for 3 d thereafter. From 125 d of gestation, the ewes were injected twice im with either saline (2 ml of 0.9% NaCl, n ⫽ 6) or dexamethasone (2 ⫻ 12 mg in 2 ml 0.9% NaCl, n ⫽ 5) at 24 h intervals. Arterial blood samples (2 ml) were collected the day before and immediately before the first injection (d ⫺1 and 0), at 24 h after each injection (d 1 and 2) and at 48 h after the last injection (d 3). Saline and dexamethasone injections were administered at immediately after blood sampling at 0 and 24 h.

Endocrinology, February 2007, 148(2):800 – 805

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Tissue D1 [liver, kidney, perirenal adipose tissue (PAT)], D2 (placenta), and D3 (liver, kidney, placenta) activities were determined by radiometric enzyme assays as described previously (5). Previous analyses using ovine placental and fetal tissues have shown negligible activities of D1 in the placenta, of D2 in PAT (relative to D1), and of D3 in the liver and PAT at 130 d of gestation (5).

Statistical analyses All values are presented as mean ⫾ sem, and were log10-transformed for statistical analysis where appropriate. There were no differences in the values obtained from the single and twin fetuses in each treatment group and, therefore, data were pooled from these animals. Plasma hormone concentrations were compared by two-way ANOVA with repeated measures followed by the Tukey post hoc test for the chronically catheterized fetuses in Study 1, and by Student’s unpaired t test for the noninstrumented ewes and fetuses in Study 2. Significant differences in tissue deiodinase activity between the treatment groups were assessed by Student’s unpaired t test. Differences where P ⬍ 0.05 were regarded as significant.

Results Study 1: chronically catheterized animals

Plasma hormone concentrations. Maternal dexamethasone treatment caused a significant increase in plasma T3 concentration in the chronically catheterized fetuses (P ⬍ 0.05; Fig. 1A). On d 1, 2, and 3 of the study, plasma T3 concentrations in the dexamethasone-exposed fetuses were significantly greater than the pretreatment baseline values, and the values observed in the saline-exposed fetuses (P ⬍ 0.05; Fig. 1A). In the chronically catheterized fetuses, the maximum plasma T3

Study 2: noninstrumented animals. Ten pregnant ewes and their singleton fetuses were used in a study to collect maternal and fetal tissues after maternal saline or dexamethasone treatment. From 125 d of gestation, all of the ewes were injected twice im with either saline (2 ml of 0.9% NaCl, n ⫽ 5) or dexamethasone (2 ⫻ 12 mg in 2 ml 0.9% NaCl, n ⫽ 5) at 24-h intervals. The fetuses were delivered by Caesarean section under general anesthesia (20 mg kg⫺1 sodium pentobarbitone iv) at 10 h after the second injection. Before anesthesia, a 10-ml blood sample was obtained from the ewe by jugular venepuncture. At delivery, a 10-ml blood sample was taken from the umbilical artery, and a number of tissues were collected from the ewe and fetus after the administration of a lethal dose of barbiturate (200 mg kg⫺1 sodium pentobarbitone). All tissue samples were immediately frozen in liquid nitrogen and stored at ⫺80 C until analysis.

Biochemical analyses All blood samples obtained from the two studies were immediately placed into EDTA-containing tubes and centrifuged for 5 min at 1000 ⫻ g and 4 C. The plasma aliquots were stored at ⫺20 C until analysis. Plasma cortisol concentration was measured by RIA validated for use with ovine plasma as described previously (12). The lower limit of detection was 3.5 nm, and the interassay coefficient of variation was 12%. Plasma T3 and T4 concentrations were also measured by RIA using a commercial kit validated for ovine plasma (13) (ICN Biomedicals, Thame, UK). The lower limits of detection were 0.11 nm for T3 and 9.8 nm for T4. The interassay coefficients of variation were 10% for both assays. Plasma rT3 concentrations were measured by RIA (14); the lower limit of detection was 0.01 nm, and the samples were measured in a single assay where the intraassay coefficient of variation was 3– 4%.

FIG. 1. Mean (⫾SEM) plasma concentrations of (A) T3 and (B) cortisol in saline (n ⫽ 6) and dexamethasone-exposed (n ⫽ 5) fetuses of Study 1. Arrows indicate time of maternal dexamethasone injection (dex) at immediately after blood sampling at 0 and 1 d (24 h) of the study. *, Significant difference from saline-exposed fetuses at same time point; P ⬍ 0.05. Within each group of catheterized fetuses, columns with different letters are significantly different from each other, P ⬍ 0.05.

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Fig. 2. Mean (⫾SEM) plasma concentrations of cortisol, T4, T3, and rT3 in the fetuses of the saline (n ⫽ 5) and dexamethasone-treated (n ⫽ 5) ewes of Study 2 sampled at 10 h after the second daily injection. *, Significant difference from saline-exposed fetuses; P ⬍ 0.05.

concentration was observed at 24 h after the second maternal dexamethasone injection (Fig. 1A). No significant difference in plasma T4 was observed between the fetuses of the two treatment groups on any day of the study (data not shown). On d 1 and 2, plasma cortisol concentration in the dexamethasone-exposed fetuses was significantly lower than that seen in the saline-exposed fetuses (P ⬍ 0.05; Fig. 1B). Study 2: noninstrumented animals

Plasma hormone concentrations. Maternal dexamethasone treatment caused a significant increase in plasma T3 and rT3 concentrations in the fetuses at delivery (P ⬍ 0.001; Fig. 2). Plasma T4 concentrations were similar between the saline and dexamethasone-exposed fetuses (Fig. 2). At delivery, plasma cortisol concentration was significantly lower in the dexamethasone-exposed fetuses compared with the fetuses of the ewes injected with saline (P ⬍ 0.05; Fig. 2). Maternal plasma concentrations of T3, T4, and cortisol were all significantly lower in the dexamethasone-injected ewes compared with the saline-injected ewes at delivery (P ⬍ 0.05; Fig. 3). A significant increase in plasma rT3 concentration was observed in the ewes treated with dexamethasone (P ⬍ 0.05; Fig. 3). Tissue deiodinase activities

Maternal dexamethasone treatment caused a significant increase in D1 activity in the liver of the sheep fetus (P ⬍

Fig. 3. Mean (⫾SEM) plasma concentrations of cortisol, T4, T3, and rT3 in the saline (n ⫽ 5) and dexamethasone-treated (n ⫽ 5) ewes of Study 2 sampled at 10 h after the second daily injection. *, Significant difference from saline-treated ewes; P ⬍ 0.05.

0.001; Fig. 4). Compared with hepatic D1 activity, D1 activities were low in the fetal kidney and PAT (Table 1). There were no significant differences in D1 activity in the fetal kidneys or PAT, or maternal liver, kidneys, and PAT, between the saline and dexamethasone-injected animals (Table 1). Similar D2 activity was observed in the placentae from the two treatment groups (Table 1); D1, D2, and D3 activities were negligible in PAT of the pregnant sheep (data not shown). In the dexamethasone-treated ewes, D3 activities in the placenta and fetal kidney were reduced markedly compared with the saline-treated animals (P ⬍ 0.001; Fig. 4). Hepatic D3 activity in the pregnant ewe was relatively low and no difference was observed between the two treatment groups (Table 1). Renal D3 activity in the saline and dexamethasonetreated ewes was negligible (data not shown). Discussion

In the present study, maternal dexamethasone treatment increased plasma T3 in the immature sheep fetus to the concentration seen in mature fetuses near term (5) by tissuespecific changes in deiodinase enzyme activities. In the fetus, the rise in circulating T3 appeared to be due to both increased T3 production by stimulation of hepatic D1 activity and decreased T3 clearance by suppression of D3 enzymes in the kidney and placenta. The changes in plasma thyroid hormone concentrations and tissue deiodinase activities in the



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Fig. 4. Mean (⫾SEM) hepatic D1, and renal and placental D3, activities in saline (n ⫽ 5) and dexamethasone-exposed (n ⫽ 5) fetuses of Study 2 sampled at 10 h after the second daily injection. *, Significant difference from saline-exposed fetuses; P ⬍ 0.001.

fetus induced by clinically relevant doses of exogenous synthetic glucocorticoid were similar to those seen previously in response to endogenous glucocorticoids (5). Furthermore, the effects of dexamethasone on circulating thyroid hormone concentrations in the ovine fetus resembled those seen in umbilical cord blood of human infants delivered within 48 h of antenatal dexamethasone treatment at a comparable dose (15). Therefore, maternal dexamethasone treatment largely mimics the effect of endogenous glucocorticoids on thyroid hormone metabolism near term, and may promote fetal maturation by stimulation of thyroid hormone activity in utero. Not all ovine fetal tissues influenced by the prepartum cortisol surge, however, were affected by maternal dexamethasone treatment in the present study. Cortisol stimulated D1 activity in the fetal kidney and PAT (5), whereas dexamethasone had no effect on deiodinase activities in these tissues. Likewise, in the crocodile embryo, the rise in plasma T3 concentration induced by dexamethasone was associated with increased hepatic but not renal D1 activity (16). Differences in glucocorticoid action may be due to the duration of exposure and relative mineralocorticoid receptor activity. The lack of effect of dexamethasone on D1 activity in fetal PAT may mean that local tissue concentrations of T3 are not as high as in mature fetuses near term. This may have implications for the degree of tissue-specific maturation, such as nonshivering thermogenesis in PAT, induced by dexamethasone in the immature fetus. However, all fetal tissues would be exposed to elevated circulating concentrations of T3. In addition, the findings suggest that glucocorticoid-induced changes in hepatic D1, and renal and placental D3, enzyme activities are primarily responsible for the rise in the circulating concentration of T3 in utero, and that changes in

the relatively low levels of renal and PAT D1 activities are less important for the endocrine production of T3. Endogenous and exogenous glucocorticoids are likely to have effects on gene expression of the deiodinase enzymes in utero, although the presence of a glucocorticoid response element has not yet been reported for any of the deiodinase genes. Previous studies have shown that dexamethasone up-regulates hepatic D1 mRNA and enzyme activity in chick and crocodile embryos (16, 17) and in adult rat liver cells in vitro (18). Furthermore, the glucocorticoid-induced rise in circulating and local concentrations of T3 may contribute to some of the changes in deiodinase enzyme activity by a positive feedback mechanism. Dexamethasone and T3 have been shown to have synergistic effects on D1 mRNA abundance in adult rat hepatocytes in vitro (19, 20). Indeed, T3 is known to have positive feedback effects on hepatic D1 activity in adult rats, possibly via a thyroid hormone response element identified on the D1 gene (21, 22). In addition, in immature fetal sheep, raising plasma concentrations of T3 alone by exogenous hormone infusion stimulates hepatic D1 and reduces renal D3 activities (5). Therefore, the rise in plasma T3 induced by dexamethasone may help to maintain thyroid hormone bioavailability by promoting production and reducing clearance of T3 in the fetus. In contrast to the effects on thyroid hormone activity in the fetus, maternal dexamethasone treatment suppressed plasma T3 and T4 concentrations in the ewe without any change in tissue deiodinase activity. The fall in biologically active thyroid hormones in the maternal circulation may be due to suppression of hypothalamic-pituitary-thyroid activity. Indeed, in nonpregnant adult human subjects, dexamethasone has been shown previously to decrease circulating

TABLE 1. Mean (⫾SEM) tissue deiodinase activities in saline and dexamethasone-treated animals of Study 2 sampled at 10 h after the second daily injection Tissue deiodinase activity

Fetus Kidney D1 (pmol min⫺1 mg protein⫺1) PAT D1 (pmol min⫺1 mg protein⫺1) Placental D2 (fmol min⫺1 mg protein⫺1) Ewe Liver D1 (pmol min⫺1 mg protein⫺1) Liver D3 (fmol min⫺1 mg protein⫺1) Kidney D1 (pmol min⫺1 mg protein⫺1)

Saline (n ⫽ 5)

Dexamethasone (n ⫽ 5)

0.48 ⫾ 0.06 2.35 ⫾ 0.65 0.39 ⫾ 0.17

0.59 ⫾ 0.07 1.76 ⫾ 0.31 0.45 ⫾ 0.21

11.13 ⫾ 2.89 0.41 ⫾ 0.15 7.90 ⫾ 1.28

8.92 ⫾ 1.37 0.60 ⫾ 0.16 7.17 ⫾ 0.87

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concentrations of T3 and T4 via a reduction in pituitary sensitivity to TSH-releasing hormone and secretion of TSH (23, 24). In adult animals, this may be an important endocrine adaptation to reduce metabolic rate in response to stressful stimuli. Therefore, glucocorticoids have different effects on thyroid hormone activity before and after birth. Indeed, dexamethasone has been shown to increase hepatic D1 activity and plasma T3 in the chick embryo at 18 d of gestation (term 21 d), and yet reduce hepatic D1 activity, and plasma T4 and T3 concentrations, in the chick on postnatal d 8 (17, 25). The differences in dexamethasone action in adult and fetal life may be due, in part, to differences in the sensitivity of the developing hypothalamic-pituitary-thyroid axis to glucocorticoids. In addition, it remains to be determined whether there are different transcripts for the deiodinase genes in preand postnatal life with different glucocorticoid-responsive elements in their promoter regions. In both the pregnant ewe and fetus, maternal dexamethasone treatment caused an increase in plasma rT3 concentration. This may have been due to increased conversion of T4 to rT3 by inner ring deiodination. However, there were no significant changes in D3 activity in the liver or kidney of the ewe, and D3 enzyme activities were reduced in the fetal kidney and placenta. Instead, there may have been glucocorticoid-induced changes in metabolism of the sulfated thyroid hormones. These hormones are predominant in the circulation of the sheep fetus and can undergo placental transfer to the ewe (26 –28) but were not measured in the present study. The rise in plasma T3 induced in utero by maternal dexamethasone treatment is likely to have important consequences for fetal maturation. Both endocrine and local production of T3 may contribute to the maturation of fetal tissues in the preparation for extrauterine life. For example, thyroid hormones are known to be important for the activation of hepatic gluconeogenic enzymes (8), for the reabsorption of lung liquid (6) and for the stimulation of nonshivering thermogenesis in brown adipose tissue (7). Therefore, up-regulation of plasma T3 concentration in the preterm fetus exposed to dexamethasone may improve the likelihood of survival over the immediate neonatal period. Indeed, the use of antenatal glucocorticoid treatment has reduced the incidence of postnatal complications, such as respiratory distress syndrome, which are common in premature infants with low circulating concentrations of thyroid hormones (1, 4, 9, 29). Although the timing of dexamethasone treatment in clinical practice (0.80 of gestation) would be somewhat earlier than in the present study (0.86 of gestation), glucocorticoid receptors are present in a variety of human and ovine fetal tissues from early in gestation (30, 31). Therefore, similar changes in thyroid hormone metabolism may be induced by glucocorticoids at other points in gestation. Therefore, in the present study, maternal dexamethasone treatment had different effects on thyroid hormone activity in the pregnant ewe and fetus. In the fetus, the exogenous glucocorticoid induced an increase in circulating T3 concentration by tissue-specific changes in deiodinase enzyme activities, whereas in the adult animal, thyroid hormone activity was suppressed by dexamethasone without any alteration in tissue deiodinases. Overall, glucocorticoid-induced changes in T3 bioavailability in the fetal circulation


and tissues may mediate some of the maturational effects of dexamethasone in the preterm infant. Acknowledgments The authors are grateful to the members of the Department of Physiology, Development and Neuroscience, University of Cambridge, who have assisted with this study. Received August 31, 2006. Accepted November 6, 2006. Address all correspondence and requests for reprints to: Dr. Alison J. Forhead, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom. E-mail: [email protected]. The study was supported by the Biotechnology and Biological Sciences Research Council, the Isaac Newton Trust, and Tommy’s, the baby charity. Disclosure Statement: The authors have nothing to disclose.

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Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.