Collection time of Thyroid hormones and TSH in ...

HealthMED - Volume 5 / Number 3 / 2011

Collection time of Thyroid hormones and TSH in preterm newborns Simone Holzer de Moraes1, Silvia Espiridião2, Fernando A. Fonseca2, Luiz Carlos de Abreu2, Vitor E. Valenti2, 3, Ricardo Peres do Souto1 1 2 3

Disciplina de Bioquímica, Faculdade de Medicina do ABC, Brasil, Departamento de Morfologia e Fisiologia, Faculdade de Medicina do ABC, Brasil, Departamento de Medicina, Disciplina de Cardiologia, Universidade Federal de São Paulo, Brasil.

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observed in preterm neonates; this is generally referred to as hypothyroxinemia of prematurity [4]. %DFNJURXQG In this study we aimed to evalu- In contrast to typical congenital hypothyroidism, a ate the collection time of T3, free T4 and TSH in normal TSH level upon initial screening followed preterm newborns. by delayed TSH elevation is observed in some 0HWKRGV Prospective study, composed of 85 preterm infants [5]. preterm newborns (PTI) as the gestational age 7KHPDLQIDFWRUVWKDWLQÀXHQFHWK\URLGIXQFWLVWUDWL¿HGLQWRIRXUJURXSV*URXS$Q OHVV on in preterm infants are immaturity of the hypotthan 30 weeks); Group B: n = 13 (30 + 1 / 7-32 halamic-pituitary-thyroid axis, immature thyroid weeks); Group C: n = 19 (32 + 1 / 7 to 34 weeks) hormone synthesis, immature thyroid hormone and; Group D: n = 35 (34 + 1 / 7 to 36 weeks). PHWDEROLVP DQG V\VWHPLF GLVHDVHV ,QVXI¿FLHQW The collections of T3, T4 and thyroid stimulating RUH[FHVVLYHLRGLQHLQWDNHVDOVRLQÀXHQFHSUHWHUP hormone (TSH) were collected by chemilumines- thyroid function [6]. cence in six stages: 1 - umbilical cord; 2 - 3 days Although the survival rate of very low birth old; 3 - 7 days old; 4 - 14 days old; 5 - 30 days old weight infants has increased in recent years [6], and; 6 – 60 days old. guidelines for thyroid function monitoring have 5HVXOWV :H REVHUYHG VLJQL¿FDQW GLIIHUHQFHV not been established for preterm infants. Therefountil the dosage of 30 days, for free T4 between re, we aimed to evaluate the collection time of T3, groups A and D; for the T3 between A and B free T4 and TSH in preterm newborns. groups with C and D groups. In the comparison EHWZHHQWKHWLPHVWKHWLPHZDVVLJQL¿FDQWO\GLfferent for TSH and T3. 0HWKRG &RQFOXVLRQ The hormone levels are gradually lower the lower the gestational age and the miniStudy Population mum ideal time for the collection of tests is from the 3rd day of life for TSH and the 7th day to the T4 We investigated 85 preterm newborns, they and T3. were born from June 2004 to December 2004. .H\ZRUGV Thyroid Gland; Hormones; TSH They were separated into four groups based on (Thyroid Stimulating Hormone); Infant, Newborn. gestational age: Group A (GA, n = 18) – newborns aged < 30 weeks old, Group B (GB, n = 13) – newborns aged between 30 weeks + 1 day old and Introduction 32 weeks old, Group C (GC, n = 19) – newborns aged between 32 weeks + 1 day old and 34 weeks Endocrine dysfunctions are an important event old and Group D (GD, n = 35) – newborns aged to be studied [1-3]. Postnatal thyroid function of between 34 weeks + 1 day old and 36 weeks old. preterm newborns is different compared to term All procedures were approved by the Ethical Conewborns. Impaired postnatal thyrotropin (TSH) mmittee in Research of our University. surges and low serum T4 levels are frequently Journal of Society for development in new net environment in B&H

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,QFOXVLRQ&ULWHULD We included newborns younger than 36 weeks old after the responsible for each infant sign an agreement of Consent. Gestational age was determined by maternal data: date of the last menstruation and/or ultrasound, and/or by Capurro [7] and New Ballard [8] methods. ([FOXVLRQ&ULWHULD We excluded newborns with severe congenital anomalies, chromosomal abnormalities, those in which there was some indication in the maternal history of thyroid disease or medication with iodine and non-compliance of family participation in the research. /DERUDWRU\([DPLQDWLRQ The levels of TSH (3rd generation), free T4 and total T3 were determined by chemiluminescence of blood samples that ranged between 1 and 2 ml of whole blood. The reference values of these tests for the equipments and Immulite® kit of the DPC Medlab manufacturer used in the study are, respectively, 0.4 – 4 mUI/mL for TSH, 70 – 170 ng/dL for T3 and for free T4 values are 0.65 – 2.3 ng/dL for children aged from one to 12 years old and 0.8 to 1.9 for adults. The values of TSH and T3 do not change in relation to age and to the three tests for differentiation for premature newborns. The samples were collected in six stages: 1st time

– umbilical cord; 2nd time – 3 days old; 3rd time – 7 days old; 4th time – 15 days old; 5th time –30 days old and; 6th time – 60 days old. 6WDWLVWLFDO$QDO\VLV We applied Levene and Kolmogorov-Smirnov tests in order to check homogeneity of variances and adherence to the normal curve, respectively. For comparison between the groups we applied the one way ANOVA for parametric distributions and the Kruskal-Wallis for nonparametric variables. :KHQZHGHWHFWHGVLJQL¿FDQWGLIIHUHQFHVZHDSSOLHGWKH7XNH\+RQHVW6LJQL¿FDQW'LIIHUHQFHV (HSD) posttest in order to identify in which group there was difference. We used the ANOVA test for repeated measures to compare groups and times at the same time and the multiple comparison test of Newman-Keuls to determine at which time there were difference and the Tukey HSD posttest to verify differences between groups. Differences ZHUHFRQVLGHUHGVLJQL¿FDQWZKHQWKHSUREDELOLW\ of a Type I error was less than 5% (p < 0.05). 5HVXOWV We evaluated 85 preterm newborns, with a balance between genders and with predominance of appropriate newborns for gestational age. The assessment of vitality at birth performed using the Apgar score of 1 and 5 minutes was similar between the four groups (Table 1).

Table 1. Characteristics of newborns regarding Apgar score at 1 and 5 minutes and gestational age (weeks) in each group 3DUDPHWHUV

Group A ZHHNV Q

Group B ZHHNVQ

*URXS& ZHHNV Q

Group D ZHHNV Q

$SJDUPLQXWH P ± SD Min - Max $SJDUPLQXWH P ± SD Min – Max *HVWDWLRQDODJH P ± SD Min - Max

n=17 6.29 ±2.33 2-9 n=17 8.29± 1.40 6- 10 n=18 27.92±2.08 24-30

n=13 5.07±3.09 1-9 n=13 7.92±1.65 4- 9 n=13 30.9±0.44 30.4- 31.86

n=19 7.26±1.85 1- 9 n=19 8.7±1.48 3-10 n=19 33.19±0.56 32.43- 34

n=34 7.08±2.17 1-9 n=34 8.8±1.19 5-10 n=35 35.25±0.63 34- 36.29

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Journal of Society for development in new net environment in B&H


We compared the results of hormone levels according to gestational age at the time of collection and the reference values of the supplier of the kit used. In relation to TSH we observed that it progressively increased according to gestational age advancing and in all groups the higher levels were found in umbilical cord dosage where there ZDVVLJQL¿FDQWGLIIHUHQFHEHWZHHQJURXSV$DQG& Moreover, at this time of collection the values in all groups were above the benchmarks set by the manufacturer (0.4 – 4 mIU/ml) and the remaining doses were very close to those limits (Figure 1).

Figure 1. Variations of serum TSH according to the progress of pregnancy and sampling period. Group A (GA, n = 18) - newborns aged less than 30 weeks of gestational age, Group B (GB, n = 13) - newborns between 30 weeks + 1 day and 32 weeks of gestational age, Group C (GC, n = 19) - newborns between 32 weeks + 1 day and 34 weeks of gestational age and Group D (GD, n = 35) - newborns between 34 weeks + 1 day and 36 weeks of gestational age 5HJDUGLQJWKHIUHH7ZHIRXQGVLJQL¿FDQWGLIferences between gestational age groups, from the collection of umbilical cord until 60 days, always with newborns younger than 30 weeks old with lower values for 32 weeks old. No difference was observed between the collection times and all values were within the reference set (0.65 – 2.3 ng/ dl) (Figure 2). We observed difference of T3 at doses of 3, 7, 14 and 30 days old, where the newborns with gestational age younger than 30 weeks old presented lower values than newborns with more than 34 weeks of gestational age. In relation to the collection times, WKHGRVDJHRIFRUGYDOXHVZDVVLJQL¿FDQWO\ORZHU than other times. When we performed the compa-

rison with the reference values (70 – 170 ng/dl), we observed that the two smaller groups presented lower levels compared to those established until the collection of the 7th day of life. In the two higher groups the averages were found above the determined at collections of 30 and 60 days old (Figure 3).

Figure 2. Variations of serum free T4 according to the progress of pregnancy and sampling period. Group A (GA, n = 18) - newborns aged less than 30 weeks of gestational age, Group B (GB, n = 13) - newborns between 30 weeks + 1 day and 32 weeks of gestational age, Group C (GC, n = 19) - newborns between 32 weeks + 1 day and 34 weeks of gestational age and Group D (GD, n = 35) - newborns between 34 weeks + 1 day and 36 weeks of gestational age

Figure 3. Variations of serum T3 according to the progress of pregnancy and sampling period. Group A (GA, n = 18) - newborns aged less than 30 weeks of gestational age, Group B (GB, n = 13) - newborns between 30 weeks + 1 day and 32 weeks of gestational age, Group C (GC, n = 19) - newborns between 32 weeks + 1 day and 34 weeks of gestational age and Group D (GD, n = 35) - newborns between 34 weeks + 1 day and 36 weeks of gestational age


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VDJH7KLVSHDNLQWKH¿UVWPLQXWHVRIOLIHZDV GHVFULEHGE\VHYHUDODXWKRUVDQGUHÀHFWVWKHUHV2XU ¿QGLQJV VKRZ WKDW WKH YDOXHV RI WK\URLG ponse to cooling. After this initial peak there was hormones changed in preterm newborns, in infants a fall in TSH levels that remain similar to the last below 30 weeks of gestational age it was directly GRVHZLWKQRVLJQL¿FDQWGLIIHUHQFHVEHWZHHQJURproportional to gestational age. Moreover, the ide- ups. Additionally, Murphy et al [18] reported sial time of collection by this method for preterm milar results to ours, they found no statistical difnewborns was from the 3rd day of life for TSH and ference of TSH values between preterm and term free T4 and from the 7th day of life for T3. Thyroid newborns. Williams et al [19] also observed no hormones are essential for adaptation to extraute- differences in gestational age, however, the values rine life, including lung development and control of cord dosage were higher compared to other coof liquids, thermogenesis and metabolic processes llection times, which in this study was conducted [9-12]. An adequate level of serum T3 and T4 is im- at 7, 14 and 28 days of life. Williams et al [20] portant for the maturation and functions to stimula- evaluated the hormones only in the dosage of the te the growth and development of various tissues, cord and also found no statistical differences in including skeletal, heart, gastrointestinal tract, and gestational age by checking only a trend to lower especially the central nervous system [13]. Des- levels at the extremes of gestation (below 27 and pite the knowledge about the formation, function above 42 weeks). and thyroid dysfunction in the fetus and newborn, Analyzing the values of free T4, we found difLWLVVWLOOGLI¿FXOWWRGLVWLQJXLVKSK\VLRORJLFDOIURP ferences related to gestational age from the collecpathological conditions due to the peculiarities of tion of cord until 30 days. The group A (below 30 thyroid function during this period [14]. weeks) always presented lower values compared It is already well-established the differences of to those of groups C and/or D (over 32 weeks). By hormone levels between preterm newborns, term observing the curves we found that for newborns newborns and adult, however, the increase in sur- with less than 32 weeks of gestational age, there vival of smaller infants with extremely low birth was decrease of free T4 in dosages of 72 hours weight and lower gestational age limits has made and 7 days compared to the dosage of cord, while new reference values for this population. Given this the opposite occurred with the two higher groups, QHZ UHDOLW\ ZH VWUDWL¿HG WKH SUHPDWXUH LQIDQWV LQ FRQ¿UPLQJWKHUHYLHZOLWHUDWXUHDQGFRQWUDGLFWLQJ four gestational age groups and analyzed the levels Van Wassenaer and Kok [15] who described hyof TSH, free T4 and T3 from birth to the collection pothyroxinemia of prematurity as the period in of umbilical cord blood within 60 days. This co- which the levels of T3 and total and free T4 are llection period was determined from the analysis of lower, particularly among those with gestational the literature which shows that even for extremely age less than 30 weeks. On the other hand, Cuestas premature infants stabilization of hormonal levels [21], comparing premature infants 30 – 37 weeks come after 45 days of life [14, 15]. of gestational age with term newborns found in We found levels of T3, free T4 and TSH gradu- both groups higher values of free T4 at doses of 12 ally lower the lower the gestational age. A similar and 72 hours compared to those in the dosage of ¿QGLQJZDVYHUL¿HGE\%LVZDVHWDO>@WKH\UH- FRUGKRZHYHUZLWKYDOXHVVLJQL¿FDQWO\ORZHULQ SRUWHGVLJQL¿FDQWO\ORZHUOHYHOVLQQHZERUQVEH- SUHWHUPLQIDQWV&RQ¿UPLQJRXU¿QGLQJV)UDQNHW low 30 weeks of gestational age, especially T3 and al [22], using population screening of 1989 – 1993 free T4 compared to term newborn. Furthermore, in Massachusetts found lower levels of free T4 a recent study showed that infants born with ge- dosage from second to third day until 14 days of stational age lower than 32 weeks old present high life in newborns weighing less than 1500g, howeincidence of hypothyroidism [17]. YHULQWKHLUVWXG\WKHFODVVL¿FDWLRQRIWKHQHZERUQ When we analyzed the TSH, we observed that was made from the weight and not gestational age. their values increased progressively with advan- 5RRPDQHWDO>@DOVRIRXQGVLJQL¿FDQWO\ORZHU cing gestational age in all groups and the highest levels of free T4 when collecting in 14 days of life, level of this hormone was found in the cord do- FRPSDUHGWRWKH¿UVWGD\RQJHVWDWLRQDODJHEHORZ 630



31 weeks, while the opposite occurred, i.e. higher values with 14 days in infants with 32 weeks of gestational age. In the analysis between groups, the authors found differences between the values RIIUHH7LQWKH¿UVWGD\VLQWKHJURXSZLWKJHVWDtional age below 29 weeks, with all the others with higher gestational age. Williams et al [19] observed a gradually decrease of postnatal free T4 levels in newborns aged 31 – 34 weeks and 28 – 30 weeks and markedly lower in infants aged 23-27 ZHHNVEXWGLGQRW¿QGORZHUYDOXHVWKDQXPELOLFDO cord in either group. In our study T3 showed parallel curves, accompanying the increase of gestational age. In our analysis we suggest that for T3, gestational age EHORZZHHNVUHVXOWVLQVLJQL¿FDQWO\ORZHUYDOXHVD¿QGLQJQRWVKDUHGE\DSUHYLRXVVWXG\>@ which found no differences between preterm and term newborns. Williams et al [19] also found a progressive increase in T3 levels from the measurement of umbilical cord and the magnitude of this increase was related to gestational age. The authors compared the umbilical cords levels of T3 with 7, 14 and 28 days of life of the preterm QHZERUQVDQGYHUL¿HGVWDWLVWLFDOGLIIHUHQFHLQDOO collection times. However, unlike our study, they did not evaluate the differences between groups and within each collection time. According to our results, comparing them to the benchmark we observed that TSH values in the dosage of the umbilical cord in all groups are far above it, keeping the other times very close to the standard. With these data we may consider that the ideal time of collection is from 72 hours of life. The free T4 has practically all averages within the reference range, except in Group A that samples of 72 hours and seven days presented values below this standard, supporting the hypothesis of hypothyroxinemia, since this condition is related to newborns below 30 weeks of gestational age and our group presented an average of 28 weeks. Based on levels of free T4 the collection of screening congenital hypothyroxinemia should occur after the 7th day in order to minimize the margin of error. In relation to T3 the ideal time of collection in which gestational age does not change the result LVDIWHUWKH¿UVWZHHNRIOLIH Adams et al [24] aimed to establish benchmarks for the technique of equilibrium dialysis

straight to the T4 and TSH immunometric test in premature infants and reported results similar to RXUV7KHVHDXWKRUVGH¿QHGWZRGHIDXOWYDOXHVIRU T4, one for infants 25 – 30 weeks of gestational age and another for 31 – 36 weeks of gestational age they found correlation for TSH, however, they determined only a benchmark for all premature of 25 – 36 weeks. 2XUVWXG\IRUWKHPRVWSDUWFRQ¿UPVWKH¿Qdings of other authors on the evolution of thyroid hormones in preterm newborns and the presence of transient hypothyroxinemia in these infants. Nevertheless, it would be required a larger number of subjects and a collection time of at least one year to prepare a reference curve for premature newborns, since seasonal changes may affect reVXOWV 1HYHUWKHOHVV PRUH WKDQ WKLV FRQ¿UPDWLRQ this study reveals to the team of neonatal care how thyroid function has been “forgotten”, especially in very premature newborns, allowing from this moment on another way to evaluate this function with rapid results and assurance if it is collected at the ideal time. In conclusion, the values of T3, free T4 and TSH ranged in preterm newborns, they were directly proportional to gestational age in newborns under 30 weeks of gestational age. Clinical maQLIHVWDWLRQ ZLWK FRQ¿UPDWLRQ RI WUDQVLHQW K\SRWhyroxinemia in preterm newborns characterized E\ORZIUHH7DQG76+ZDVQRUPDOLQWKH¿UVW 30 days of life. The ideal time of collection by this method for premature newborn is from the 3rd day of life on for TSH and free T4 and from the 7th day on for T3. $FNQRZOHGJHPHQWV 7KLV UHVHDUFK UHFHLYHG ¿QDQFLDO VXSSRUW IURP Núcleo de Estudos de Pesquisa da Faculdade de Medicina do ABC (NEPAS-FMABC). 5HIHUHQFHV 1. Khan DA, Cheema AN, Anwar M, Khan FA. Endocrine Dysfunction in Beta-Thalassaemic Major Patients at Rawalpindi, Pakistan. HealthMED J 2010;3:580-585.


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