Abstract
A randomized, placebo-controlled, masked study was conducted of the responses of thyroid parameters, cortisol, and the cardiovascular system to a single dose of triiodothyronine (T 3 ) 24 h after birth, followed by a daily dose of thyroxine (T 4 ) during 6 wk to infants Ͻ28 wk gestational age. Thirty-one infants were assigned to three groups: 1) group A: T 3 24 h after birth plus daily T 4 during 6 wk; 2) group B: placebo T 3 and T 4 during 6 wk; and 3) group C: placebo T 3 and placebo T 4 . T 4 , free T 4 , T 3 , free T 3 , reverse T 3 , thyroid-stimulating hormone, and cortisol were measured in cord blood and on days 1, 3, 7, 14, 21, 42, and 56. Data on pulse rate, blood pressure, and cumulative dose of inotropic agents were collected. T 3 (0.5 g/kg) resulted in a plasma increase until day 3. Thereafter, plasma T 3 levels were comparable between the groups. T 4 , free T 4 , and reverse T 3 were increased in groups A and B during the period of T 4 administration. Thyroid-stimulating hormone suppression was of shorter duration in group A. T 3 and T 4 administration did not have any effect on cortisol levels. We did not find any effects of T 3 or of T 4 administration on the cardiovascular system. A single injection of T 3 (0.5 g/kg) given 22-26 h after birth only leads to a 2-d increase of T 3 levels and does not have effects on the cardiovascular system. This study does not support the use of T 3 according to our regimen in preterm infants. Thyroid hormones play an important role in normal growth and development of the CNS (1, 2). In term infants, thyroxine (T 4 ) and triiodothyronine (T 3 ) increases shortly after birth. In premature infants, transient hypothyroxinemia after birth is a common finding (3-7). This hypothyroxinemia is characterized by low levels of T 4 and T 3 , which do not cause elevations in thyroid-stimulating hormone (TSH) above 20 mU/L. The degree of hypothyroxinemia is related to gestational age (GA) and the severity of neonatal disease. Low T 3 and T 4 levels in premature infants have been found to be associated with poor neurologic and developmental outcome (8 -11).
Abstract
Transient hypothyroxinemia is a multicausal phenomenon in which immaturity of thyroid hormone metabolism with high activity of deiodinase type 3 together with factors such as immaturity of the hypothalamo-pituitary-thyroid axis and the sudden interruption of the maternal contribution to fetal thyroid hormone pools are involved. Thyroid hormone supplementation in preterm infants has been the subject of several clinical studies with clinical and neurodevelopmental parameters as primary outcome (12) (13) (14) . To date, no clear effect of supplemental T 4 treatment on neurodevelopmental outcome has been demonstrated. In an earlier study, however, we observed that a subgroup of T 4 -treated infants of Ͻ29 wk GA tended to have a better developmental outcome than infants of similar GA in the placebo group (13, 15) .
Abstract
T 4 administration to preterm infants of Ͻ30 wk GA did not result in increased plasma T 3 levels; in fact, it resulted in decreased plasma T 3 levels and increased plasma reverse T 3 (rT 3 ) levels (16). It was suggested that this T 3 decrease was caused by lower endogenous thyroidal T 3 production as a result of TSH suppression and that, because of low thyroidal and hepatic type 1 deiodinase activity and high deiodinase type 3 activity, an increase in plasma T 4 did not result in an increase in plasma T 3 . 

effect on cortisol levels. We did not find any effects of T 3 or of T 4 administration on the cardiovascular system. A single injection of T 3 (0.5 g/kg) given 22-26 h after birth only leads to a 2-d increase of T 3 levels and does not have effects on the cardiovascular system. This study does not support the use of T 3 according to our regimen in preterm infants. Thyroid hormones play an important role in normal growth and development of the CNS (1, 2) . In term infants, thyroxine (T 4 ) and triiodothyronine (T 3 ) increases shortly after birth. In premature infants, transient hypothyroxinemia after birth is a common finding (3) (4) (5) (6) (7) . This hypothyroxinemia is characterized by low levels of T 4 and T 3 , which do not cause elevations in thyroid-stimulating hormone (TSH) above 20 mU/L. The degree of hypothyroxinemia is related to gestational age (GA) and the severity of neonatal disease. Low T 3 and T 4 levels in premature infants have been found to be associated with poor neurologic and developmental outcome (8 -11) .

Transient hypothyroxinemia is a multicausal phenomenon in which immaturity of thyroid hormone metabolism with high activity of deiodinase type 3 together with factors such as immaturity of the hypothalamo-pituitary-thyroid axis and the sudden interruption of the maternal contribution to fetal thyroid hormone pools are involved. Thyroid hormone supplementation in preterm infants has been the subject of several clinical studies with clinical and neurodevelopmental parameters as primary outcome (12) (13) (14) . To date, no clear effect of supplemental T 4 treatment on neurodevelopmental outcome has been demonstrated. In an earlier study, however, we observed that a subgroup of T 4 -treated infants of Ͻ29 wk GA tended to have a better developmental outcome than infants of similar GA in the placebo group (13, 15) .

T 4 administration to preterm infants of Ͻ30 wk GA did not result in increased plasma T 3 levels; in fact, it resulted in decreased plasma T 3 levels and increased plasma reverse T 3 (rT 3 ) levels (16) . It was suggested that this T 3 decrease was caused by lower endogenous thyroidal T 3 production as a result of TSH suppression and that, because of low thyroidal and hepatic type 1 deiodinase activity and high deiodinase type 3 activity, an increase in plasma T 4 did not result in an increase in plasma T 3 .

Although the T 3 decrease after T 4 administration did not result in worse clinical outcome, a T 3 increase shortly after birth could very well improve clinical outcome. We hypothesized that a single dose of T 3 is able to switch on deiodinating enzymes and thus simulate the rapid postnatal increase of T 3 levels as is especially seen in more mature infants. This increase in T 3 levels is thought to be necessary for metabolic adaptation after birth.

In an earlier study using historical controls, we described that a single dose of T 3 12 h after birth resulted in a sustained increase of plasma T 3 both if given as a single treatment and in combination with 6 wk of T 4 supplementation. Because this study was done with a historical control group, these results need to be reproduced in a randomized trial (17) .

The aim of the current study was to investigate the hormonal effects of three supplementation schemes (T 4 plus T 3 , T 4 plus placebo, and placebo) in a randomized, masked way in infants Ͻ28 wk GA. In addition, we measured plasma cortisol levels concurrently with thyroid hormone levels. This was done because, to our knowledge, until now, there are no reports on the influence of transient hypothyroxinemia in preterm infants on adrenal cortex function, whereas glucocorticoid hormones and thyroid hormones are known to be functionally interrelated with respect both to maturational effects and to influencing each other's regulatory pathways (18, 19) .

In several pediatric studies, it has been suggested that T 3 administration improves myocardial function, especially after cardiac surgery (20 -22) . In an earlier study, we found higher heart rates in children who received only T 4 (23) . For studying the effects of T 3 plus T 4 administration on the cardiovascular system, intra-arterial blood pressure, heart rates, and cumulative doses of inotropic agents were measured in all infants included in the current study.
METHODS
Eligibility and randomization. This study was approved by the Committee of Medical Ethics of the Academic Medical Center in Amsterdam (The Netherlands) and by the National Committee on Human Research. Infants who were admitted to our neonatal intensive care unit and were born between September 2000 and September 2001 with a GA Ͻ28 wk were eligible for the study. Infants were excluded when they had a severe congenital anomaly or when there was maternal endocrine disease. When informed consent had been obtained from at least one of the parents within 24 h after birth, the infant was included in the study.
METHODS
After inclusion, the infants were randomized to receive T 3 plus T 4 (group A), placebo-T 3 plus T 4 (group B), or placebo-T 3 plus placebo-T 4 (group C). Randomization took place with the use of a computerized randomization program in blocks of five. Randomization was stratified according to GA (27 wk and Ͻ27 wk). All investigators, medical staff, and parents remained unaware of the infants' study-group assignment throughout the study.
METHODS
Intervention. For each infant who entered the study, a numbered set of 1 ampoule containing T 3 or placebo and 50 ampoules containing T 4 or placebo was prepared. T 3 was given in a single i.v. dose of 0.5 g/kg 22-26 h after birth. T 4 was given in a daily i.v. dose of 8 g/kg during 42 d starting immediately after the single dose of T 3 . As in previous studies of our group (13, 17) , T 4 was given by enteral route when all feeding was given enterally.
METHODS
Thyroid hormone and cortisol determinations. The primary outcome was thyroid function measured as plasma levels of T 4 , free T 4 (FT 4 ), T 3 , free T 3 (FT 3 ), rT 3 , TSH, and plasma cortisol levels. Blood samples (1 mL) were taken from cord blood 24 h after birth (just before T 3 administration) and again on days 3, 7, 14, 21, 42, and 56. Furthermore, 0.3-mL blood samples were taken 30, 120, and 360 min after T 3 administration for determination of T 3 levels.
METHODS
T 4 , T 3 , and rT 3 were measured by in-house RIA methods. FT 3 , FT 4 , and TSH were measured by time-resolved fluoroimmunoassay (Delfia FT 4 , respectively, Delfia FT 3 and Delfia hTSH Ultra; Wallac Oy, Turku, Finland). Cortisol was measured by luminescence enzyme immunoassay (Immulite; Diagnostic Products Corporation, Los Angeles, CA, U.S.A.)
METHODS
Detection limits were as follows:
METHODS
.01 mU/L; and cortisol, 30 nmol/L. Intra-assay variations were as follows: T 4 , 2-4%; T 3 , 3-4%; rT 3 , 4-5%; FT 3 , Ϯ6%; FT 4 , 4-6%; TSH, 1-2%; cortisol, 3.6 -6.4%. Interassay variations were as follows: T 4 , 3-6%; T 3 , 7-8%; rT 3 , 5-9%; FT 3 , Ϯ9%; FT 4 , 5-8%; TSH, 3-4%; cortisol, 4.7-9.0%. Cardiovascular measurements. Secondary outcomes were daily mean heart rate, daily mean intra-arterial blood pressure during the first week of life, and cumulative dose of inotropic agents. These data all were collected prospectively. Daily mean heart rate and intra-arterial blood pressure were calculated as the mean of four values at predefined time points (0400, 1000, 1600, and 2200 h). Cumulative doses of inotropic agents were calculated over the total study period and each of the first 4 d after T 3 administration separately.
METHODS
Statistical analysis. Data analysis was done using SPSS version 10.0.7 (SPSS Inc., Chicago, IL, U.S.A.). The one-way ANOVA with Bonferroni post hoc analysis was used to compare continuous variables in the three study groups. For analysis of categorical data, the 2 -test (3 ϫ 2 tables) was used. Statistical significance was set at p ϭ 0.05.
RESULTS

Patients.
Between September 1, 2000, and September 1, 2001, 42 infants were eligible for the study. Five infants died before consent could be asked. For three infants, consent was not given. Two infants were excluded because of severe congenital malformations and one because of thyroid illness of the mother.
Patients.
Population characteristics of the remaining 31 infants enrolled in the study are shown in Table 1 . In group A, mean birth weight was lowest and percentage of small-for-gestational-age infants was highest (NS). Clinical characteristics of the infants who were enrolled in the study are shown in Table 2 . Infants in group A seemed to be sicker because they had a significantly higher incidence of intraventricular hemorrhage grades 3 and 4. They also needed higher cumulative doses of inotropic 249 T 3 PLUS T 4 ADMINISTRATION IN PRETERMS agents and had more septicemia than infants in groups B and C, but these differences were not significant. All 31 children included received the intervention as described above until day 42 or, if earlier, until death.
Patients.
Hormonal outcome. Figure 1 shows the time course of the mean plasma concentrations of T 3 , FT 3 , T 4 , FT 4 , TSH, rT 3 , and cortisol. Mean T 3 levels in group C (placebo group) remained low at approximately 0.7 nmol/L until approximately day 7; thereafter, mean T 3 levels rose gradually to approximately 3.0 nmol/L on day 56.
Patients.
In group B, T 3 levels were similar to the placebo group until approximately day 3. From day 7 onward, however, T 3 levels remained lower compared with the placebo group without reaching statistical significance.
Patients.
In group A, after T 3 administration, there was a 200% increase compared with groups B and C with a maximum at 30 min after injection. At 5.5 h after this maximum, T 3 levels were already decreased by 30%. On day 3, T 3 levels were comparable to levels in group B. In group A, T 3 concentrations were somewhat higher on day 21 and day 42 compared with group B but not significantly.
Patients.
Mean FT 3 levels remained comparable among the three groups until day 21. On day 42, the mean FT 3 level in group B was lower but not significantly lower than the mean FT 3 levels in groups A and C. On day 56, mean FT 3 levels were comparable among the groups.
Patients.
Mean FT 4 levels in group C remained between 10.3 and 12.9 pmol/L during the first 14 d and increased slowly to approximately 15 pmol/L on day 56. In groups A and B, mean FT 4 levels showed a clear elevation in the first 21 d of T 4 administration. Thereafter, the differences with the placebo group gradually disappeared. Two weeks after discontinuation of T 4 administration, FT 4 levels were similar in the three groups. T 3 administration did not have any effect on mean FT 4 levels.
Patients.
Mean T 4 levels showed the same pattern as the mean FT 4 levels, but significance was not reached. Mean TSH levels in all groups initially showed the highest values (approximately 7 mU/L) in cord blood. In group C, the mean TSH levels dropped to approximately 3 mU/L at 24 h after birth and showed an increase at approximately day 14. Thereafter, mean TSH levels gradually decreased again to approximately 3 mU/L.
Patients.
In groups A and B, mean TSH levels fell deeper to Ͻ1 mU/L on day 3. Mean TSH levels were equally suppressed in groups A and B during the first 21 d. After day 21, mean TSH levels in group A seemed to be less suppressed than in group B, and on day 42, mean TSH levels were decreased only in the children who received only T 4 . After cessation of T 4 administration, TSH levels were comparable in all three groups. 
T 3 PLUS T 4 ADMINISTRATION IN PRETERMS
Mean rT 3 levels in all groups were highest in cord blood (group A, 5.4 Ϯ 0.8 nmol/L; group B, 3.0 Ϯ 0.5 nmol/L; group C, 3.9 Ϯ 0.4 nmol/L). During the whole period of T 4 administration, mean rT 3 levels were higher in groups A and B compared with those in group C, although significance was not reached at all points. T 3 administration did not influence the time course of mean rT 3 levels.
T 3 PLUS T 4 ADMINISTRATION IN PRETERMS
Mean cortisol levels showed a peak at 24 h after birth (before T 3 and T 4 administration was started) and a lower peak 14 d after birth in all groups and stayed comparable between the groups. T 3 and T 4 administration did not influence the time course of cortisol levels.
T 3 PLUS T 4 ADMINISTRATION IN PRETERMS
Cardiovascular outcome. The cumulative doses of inotropic agents in the three groups are shown in Table 2 . Mean heart rates, mean arterial blood pressure, and cumulative dose of inotropic agents showed no statistically significant differences among the three study groups, although the cumulative dose of inotropic agents was somewhat higher in group A compared with groups B and C. Even during the period of higher T 3 levels (days 1-3) in group A, there were no significant differences in mean heart rate, mean daily blood pressure, or daily use of inotropic agents.
DISCUSSION
In this randomized, placebo-controlled trial, we have studied the effect of T 3 plus T 4 administration in comparison with T 4 supplementation alone and with a placebo-treated control group on thyroid hormone and cortisol levels and on the cardiovascular condition in preterm infants. We found a significant, short-lasting increase in T 3 levels after the T 3 bolus without any effect of T 3 administration on concentrations of T 4 , FT 4 , and rT 3 . TSH levels in the group that received combined T 3 and T 4 were less suppressed on day 42 than in the group that received T 4 alone.
DISCUSSION
In an earlier study from our group (17), we described a longitudinal T 3 increase after a single T 3 dose, given 12 h after birth, in comparison with historical controls. We hypothesized that this T 3 increase was caused by a T 3 -mediated induction of type 1 deiodinase expression (24) , most probably of thyroidal origin, causing increased levels of T 3 as a result of increased conversion of T 4 to T 3 . In this study, our findings are less strong.
DISCUSSION
T 3 is known to be decreased as a result of T 4 administration (16). Indeed, also in this study, T 3 concentrations were in the lowest range in the T 4 group. In the T 4 plus T 3 group, T 3 concentrations were somewhat higher on day 21 and day 42 compared with the T 4 group, but not significantly.
DISCUSSION
TSH seemed to be less suppressed in the T 4 plus T 3 group, compared with the T 4 group on day 42. A possible explanation for this higher TSH concentration could be the more severe illness in this group with a more pronounced rebound in TSH secretion when nonthyroid illness resolved.
DISCUSSION
In an earlier study, we found a clear nadir in the mean FT 4 concentration at approximately day 7 in a placebo group of preterm infants (25) . It was also found that disease decreased FT 4 concentrations, especially during the first week of life. In a recent and larger study from the United Kingdom (26), an FT 4 nadir was also found in infants of comparable GAs. In the present study, we did not find this nadir in the mean FT 4 concentrations in the placebo group, although our lowest FT 4 concentrations between day 3 and day 14 were well comparable with those from our earlier study (25) . Although we cannot readily explain the different course in time in the placebo group, it could be due to the somewhat better clinical condition in this small study group.
DISCUSSION
Cortisol levels in all three study groups showed a longitudinal pattern similar to that described by others in preterm infants (27, 28) . Although glucocorticoid hormones and thyroid hormones are known to influence each other's regulation in animal studies (18), we could not demonstrate any effect of T 3 and/or T 4 supplementation on cortisol levels. This is an important observation, as hypocortisolism is often suspected in sick preterm infants, and the need for cortisol supplementation in these infants is under ongoing debate (29) . As cortisol levels did not differ among the study groups at time points on which low thyroid hormone levels were measured in one or two study groups, our study suggests that hypothyroxinemia is not involved in the development of hypocortisolism. In a recent study published by Biswas et al. (26) , no effect of simultaneous administration of T 3 and hydrocortisone on pulmonary outcome was found, whereas in agreement with our results, no relationship between free thyroid hormone concentrations and plasma cortisol levels was found within the study group.
DISCUSSION
Several studies have suggested that T 3 and T 4 administration influences the cardiovascular system (20 -23). We did not find any effect of T 3 or T 4 administration on the cardiovascular parameters measured. A possible explanation for this negative finding is the worse clinical condition of the infants in the group that received T 3 plus T 4 requiring relatively more inotropic agents for low blood pressure. It could also be that only a regimen of daily T 3 administration results in measurable cardiovascular effects.
DISCUSSION
Indeed, that our group A had a lower mean birth weight and a less favorable clinical course is a limitation in the interpretation of the results of this study. In this group, there were more deaths (NS) and significantly more infants with grades 3 and 4 intracranial bleeding. We do not believe that there is a causal relationship between T 3 plus T 4 administration and the clinical course, because of the unchanged cardiovascular parameters and the far too low power of this study to pick up such an effect, but of course, we cannot rule it out either.
DISCUSSION
The lack of both positive clinical findings and pronounced changes in thyroid hormone levels, especially T 3 levels, does not support the use of T 3 in preterm infants according to our regimen. For future studies in this field this could indicate that other treatment regimens, for example, with a daily or higher dose of T 3 , might be more successful in improvement of both hormonal and clinical outcome.

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