ORI­GI­NAL AR­TIC­LE DO­I: 10.4274/jcrpe.galenos.2022.2022-2-1 J Clin Res Pediatr Endocrinol 2022;14(4):453-462 Thyroid Function in 509 Premature Newborns Below 31 Weeks of Gestational Age: Evaluation and Follow-up Ariadna Campos-Martorell1, Alicia Montaner Ramon2, Karla Narváez Barros3, Jose Luis Marin Soria4, Rosa Maria López Galera4, Diego Yeste Fernández5, María Clemente León5 1Autonomous University of Barcelona, Vall d’Hebron University Hospital, Clinic of Pediatric Endocrinology, Barcelona, Spain 2Vall d’Hebron University Hospital, Clinic of Neonatal, Barcelona, Spain 3Hospital del Mar, Clinic of Pediatric Endocrinology, Barcelona, Spain 4University of Barcelona, Spain School of Medicine, Neonatal Screening Program of Catalonia, Inborn Errors Metabolism Unit Biochemistry and Molecular Genetics Department Center for Biomedical Diagnosis (CDB) Hospital Clinic, Barcelona, Spain 5Autonomous University of Barcelona, Vall d’Hebron University Hospital, Clinic of Pediatric Endocrinology; Vall d’Hebron Research Institute, Clinic of Paediatric Endocrinology, Barcelona, Spain What is already known on this topic? Preterm and low birth weight (LBW) newborns are at risk of thyroid dysfunction during a critical period for neurodevelopment and this dysfunction can be missed in the congenital hypothyroidism screening program performed in whole-blood. The utility of a second screening, its optimal timing and the need of levothyroxine (LT4) still remain subjects for debate. What this study adds? This study included a large number of preterms and their follow up. This protocol was able to detect thyroid dysfunction in neonates who were not identified by the current program based on thyroid stimulating hormone determination in whole-blood. Most cases of thyroid dysfunction resolve spontaneously in a few months, but in some cases LT4 could be needed. Abstract Objective: Preterm and low birth weight (LBW) neonates may present with thyroid dysfunction during a critical period for neurodevelopment. These alterations can be missed on routine congenital hypothyroidism (CH) screening which only measures thyroid stimulating hormone (TSH). The objective of this study was to evaluate a protocol for thyroid function screening (TFS) six years after national implementation. Methods: Serum TSH and free thyroxine (fT4) were measured during the second week of life in neonates below 31 weeks. Patients with abnormal TFS (fT4 <0.8 ng/dL and/or TSH >5 mU/L) were followed up with repeated tests until normal levels were reported. Patients who were still on levothyroxine (LT4) at three years of age were re-evaluated. Results: Five-hundred and nine neonates were included. Thyroid dysfunction was detected in 170 neonates (33%); CH n=20 (3.9%) including typical CH n=1; delayed TSH elevation CH n=19; hypothyroxinemia of prematurity (HOP) n=15 (2.9%); and transient hyperthyrotropinemia n=135 (26.5%). Twenty-one neonates (4.1%) were treated (20 for CH and 1 for HOP). At 3-year follow-up only three patients were diagnosed with permanent CH and still need treatment. LBW infants tended to have TSH levels higher than those with adequate weight. Conclusion: This protocol was able to detect thyroid dysfunction in preterm neonates who were not identified by the current program based on TSH determination in whole-blood. This thyroid dysfunction seems to resolve spontaneously in a few months in the great majority of neonates, but in some cases LT4 could be needed. There is a critical need for specific guidelines regarding the follow-up and re-evaluation of transient CH in preterm neonates. Keywords: Preterm newborn, low birth weight, congenital hypothyroidism, hypothyroxinemia of prematurity, delayed TSH rise Address for Correspondence: Ariadna Campos-Martorell MD, Autonomous University of Barcelona, Vall Conflict of interest: None declared d’Hebron University Hospital, Clinic of Pediatric Endocrinology, Barcelona, Spain Received: 21.02.2022 E-mail: arcampos@vhebron.net ORCID: orcid.org/0000-0002-8134-4934 Accepted: 18.07.2022 ©Copyright 2022 by Turkish Society for Pediatric Endocrinology and Diabetes The Journal of Clinical Research in Pediatric Endocrinology published by Galenos Publishing House. 453 Campos-Martorell A et al. J Clin Res Pediatr Endocrinol Thyroid Function Evaluation in Preterm Neonates 2022;14(4):453-462 Introduction currently require a routine second sample on preterm neonates or LBW neonates. Therefore, thyroid function Thyroid hormones are essential for the growth and screening (TFS) based on measurement of serum TSH and development of the central nervous system, as well as fT4 has been implemented in our tertiary hospital NICU for bone, pulmonary and cardiac maturation throughout for preterm neonates born below 31 weeks of gestational foetal and neonatal life (1,2,3). Preterm neonates usually age (GA). This protocol has been conducted in addition to exhibit lower thyroid hormones levels compared to term routine CH screening in dried blood spot samples. neonates, in proportion to their degree of prematurity (4). The immaturity of the hypothalamic-pituitary-thyroid axis The aims of the present study were: first, to determine the and the influence of pathologies, such as respiratory distress incidence of thyroid dysfunction detected by the application syndrome, sepsis, and intraventricular hemorrhage, and the of this protocol in preterm neonates below 31 weeks of GA; therapeutic measures (dopamine, corticosteroids) make and second, to describe the follow-up of the treated patients these infants prone to thyroid dysfunction (5,6). at reassessment. In addition, thyroid function of preterm neonates with LBW for GA was evaluated separately. Hypothyroxinemia of prematurity (HOP) refers to low levels of free thyroxine (fT4) generally without elevation of thyroid stimulating hormone (TSH) (4,7,8). This condition Methods is difficult to differentiate from central hypothyroidism and This was a prospective, observational and descriptive study. from non-thyroidal illness syndrome. Although in preterms TFS based on measurement of serum TSH and fT4 during with HOP low thyroid hormone levels have been related to the second week of life was performed on all preterm worse neurodevelopmental outcome, a causal relationship neonates below 31 weeks GA admitted in a tertiary hospital has not been clearly established as it is difficult to adjust for NICU from January 2011 to March 2017. Patients who died other co-morbidities present in this population (9,10,11,12). before 14 days of age, those who were transferred from However, some preterm neonates, especially those with other centers after 14 days, those born to mothers with low birth weight (LBW), have congenital hypothyroidism thyroid disorders and those who had no TFS performed for (CH) with delayed elevation in serum TSH levels (5,13,14). any other reason were excluded. The immaturity of the hypothalamic-pituitary axis, iodine TFS was conducted at the same time as routine blood tests overload due to any procedure involving iodine-contining scheduled during the second week after birth for preterm antiseptics, drugs, and acute nonthyroidal illness can neonates and did not involve an extra blood test or a larger contribute to the elevation of TSH and its later occurrence volume of blood to be drawn. TSH and fT4 were measured in time (5,15,16). This CH will be transient in the majority of by immunochemoluminescence in an automated analyzer infants but it could be permanent in some cases. Moreover, (Immulite 2000; Diagnostic Products Corporation, Los it is unclear whether treatment with levothyroxine (LT4) is Angeles, CA, USA) using commercially-available kits. necessary for milder elevations of TSH (13). HOP was defined by low fT4 (lower than 0.8 ng/dL) Although the incidences of permanent CH and central and normal TSH (lower than 5 mU/L) according to the hypothyroidism are similar in preterm and term newborns, literature reviewed and a previous study published by our these disorders can be missed in CH screening performed group (4,7,19). In those preterm neonates with increased using only TSH determination in a dried blood spot test TSH levels, the differential diagnosis between CH and taken at 48-72 hours of life. Accordingly, guidelines of the hyperthyrotropinemia was based on if they were treated or European Society for Paediatric Endocrinology and the not (13). A TSH serum cut-off value of 12 mU/L to start LT4 European Society for Endocrinology strongly recommend was used, based on consensus guideline recommendations a second screening for preterm neonates, low or very LBW to treat patients if TSH was between 10-20 mU/L and on neonates, and sick neonates admitted to the neonatal the experience of previous cases, and with the intention of intensive care unit (NICU) (17). The utility of the second evaluating whether this cut-off point could be raised (17,20). screening, its optimal timing, whether it measures TSH CH was diagnosed in neonates with increased serum TSH alone in dried blood spot or TSH and fT4 in a serum sample, (>12 mU/L) and normal or low fT4 who were treated the TSH cutoffs to be used, and the need to start replacement with LT4. It was categorized as transient or permanent therapy still remain subjects of active debate (18). CH, depending on whether treatment could be withdrawn The Neonatal Screening Program of Catalonia centralizes all before or at 3 years of age. CH was classified as typical when the birth centers throughout Catalonia and only mandates it was detected by routine CH screening on whole-blood or a TSH determination in dried blood spot and does not with delayed TSH rise (TSH levels increased from the first 454 J Clin Res Pediatr Endocrinol Campos-Martorell A et al. 2022;14(4):453-462 Thyroid Function Evaluation in Preterm Neonates or more TFS). Hyperthyrotropinemia was defined as the all treated patients. Those who were still on replacement presence of increased TSH (but always below 20 mU/L) with therapy at discharge from the NICU were followed up in fT4 in the normal range in neonates in whom LT4 was not the pediatric endocrinology outpatient clinic. All patients started either because this TSH elevation was transient or who were still on replacement therapy at three years of lower than 12 mU/L. age were re-evaluated, and those who still showed thyroid dysfunction after three weeks without LT4 were assessed by LBW for GA was defined as birth weight below -2 standard genetic screening for thyroid dyshormonogenesis with Next deviation (SD) score according to the Spanish newborn Generation Sequencing. This consisted of PCR amplification reference population (21,22). of coding exonic sequences and flanking intronic regions Maternal and neonatal data were collected from the NICU of DUOX2, DUOXA2, IYD, TPO, SLC26A4, SLC5A5, TG, TSHR, database including maternal thyroid dysfunction, type of and PAX8 genes using the GeneRead (Quiagen) methodology pregnancy (single or twin), sex, cause of preterm birth, and sequencing using the Illumina MiSeqd sequencer. Data administration of prenatal corticosteroids and magnesium analysis was performed with the following platforms MiSeq sulfate, GA, anthropometry at birth, Apgar test, type of Control Software (MCS), MiSeq Reporter (Illumina Inc, San resuscitation, endotracheal surfactant administration, Diego CA, USA) and GeneRead SeqVariant Analysis software neonatal evolution variables (respiratory and hemodynamic (Quiagen). support, intraventricular hemorrhage, early and late sepsis, All neonates were tested for CH within the Catalonia death), genetic diseases of the neonate, and iodine overload Neonatal Screening Program. This program uses a primary procedures. TSH test strategy from whole-blood sample on filter paper The TFS flow-chart is shown in Figure 1. Patients with following a heel-prick at 48-72 h of life. Whole-blood TSH abnormal TFS (fT4 <0.8 ng/dL and/or TSH >5 mU/L) were concentration is measured by fluorescence immunoassay followed up with repeated thyroid function test until normal in an autoanalyzer (AutoDELFIA Neonatal hTSH kit, Perkin levels of thyroid hormones were reported. Patients with TSH Elmer, Turku, Finland), using commercially-available kits. If level >12 mU/L, persistently elevated TSH 5-12 mU/L and/or the TSH level is higher than 20 mU/L, the patient is referred persistently low fT4 below 0.8 ng/dL were started on LT4 after to our endocrinology unit for confirmation and treatment. confirmation and taking into account the patient’s clinical If TSH is between 10-20 mU/L a second dried blood spot condition and the preceding TFS. Oral LT4 replacement sample is requested, and if TSH persists above 10 mU/L the patient is also referred to our endocrinology unit. was administered at 4-6 mcg/kg/day for HOP and 10-15 mcg/kg/day for CH (7,17,20). In those preterm infants with Serial thyroid hormone determinations (date of analysis, normal TFS at first sample, TFS was repeated if iodine TSH and fT4 levels), as well as the indication for treatment overload occurred. A thyroid ultrasound was performed on with LT4, its start date and, if it occurred, end date, were Figure 1. Thyroid function screening protocol TFS: thyroid function screening, fT4: free thyroxine, TSH: thyroid stimulating hormone, LT4: levothyroxine 455 Campos-Martorell A et al. J Clin Res Pediatr Endocrinol Thyroid Function Evaluation in Preterm Neonates 2022;14(4):453-462 collected from the NICU database. TFS data from routine after TFS was performed at a median of 30 days. A total CH screening were provided from the Neonatal Screening of 687 TFS were performed (Table 1). The total number of Program of Catalonia. Treated patients follow up data were repeat analyzes were 104, for abnormal values (n=58) or collected from the Pediatric Endocrinology Unit outpatient insufficient sample (n=46). clinic medical reports. Thyroid dysfunction was detected in 170 patients (33.3%); The study was conducted in compliance with the terms of CH was diagnosed in 20 (3.9%); and hyperthyrotropinemia the Helsinki II Declaration and was approved by the Drug in 135 neonates (26%) [TSH between 13-20 mU/L n= 9; TSH Research Committee and the Research Project Committee between 5-12 mU/L n=126 patients]; HOP was diagnosed of Vall d’Hebron University Hospital [PR (AMI)271/2018]. in 15 neonates (2.9%). Results are shown in Figure 2. Informed consent was obtained from all patients. Twenty-one neonates (4.1%) were treated with LT4 (20 for CH and one for HOP). Characteristics of neonates treated Statistical Analysis are shown in Table 2. Statistical analysis was performed using Statistical Package Congenital Hypothyroidism for the Social Sciences, version 20.0 (IBM Inc., Armonk, NY, USA). Normal distribution was assessed by the Kolmogorov- Twenty preterm neonates were diagnosed with CH of whom one was typical (patient 16) and 19 with delayed TSH rise) Smirnov test. Comparisons between groups were performed with median (IQR) TSH levels of 25.9 (16.8,42) mU/L and using Student’s t-test or the Mann-Whitney U test, as mean±SD fT4 levels of 1.0±0.3 ng/dL. Remarkably, 11 appropriate. The Pearson test was used in the correlation (55%) of the 20 patients received an iodine overload due analysis in normally distributed data. The x2 test was used to routine procedures (intestinal surgery, lumbar puncture to test for differences in patient group distribution. Data and surgical closure of ductus arteriosus). Seven patients are expressed as frequency, mean±SD and median and were LBW. To highlight, all patients with delayed TSH rise interquartile range (IQR), whenever appropriate. In some presented with TSH levels at the first TFS above 5 mU/L variables, range (minimum-maximum) was also shown. (14 patients between 13-20 mU/L and five patients between Statistical significance was assumed when p<0.05. 5-12 mU/L). In relation to the Neonatal Screening Program, all neonates Results were tested at a median (IQR) of 4 (3,13) days of life. Three A total of 550 preterm neonates below 31 weeks GA were patients had a TSH above 10 mU/L in the first dried spot born in the observation period. Forty-one neonates were sample. Patient 16 was diagnosed with typical CH because excluded: 30 died within two weeks of birth, nine did not he had a TSH level of 18.6 mU/L in dried spot sample and have TFS performed and two had mothers with thyroid TSH levels of 66 mU/L in the serum sample at fourteenth pathology. Thus, 509 preterm neonates were enrolled. day of life. Patients 15 and 17 had TSH levels in the first Median (IQR) of GA at birth was 28 weeks (26.4,29.4) and sample of 13.6 and 13.2 mU/L, but showed normal TSH the birth weight was 1000 g (800,1230). The distribution levels in the second blood spot sample. by weeks of GA was: 23 (n=3), 24 (n=30), 25 (n=54), 26 LT4 was started at a median (IQR) of 27.9 (18,33) days of (n=59), 27 (n=94), 28 (n=91), 29 (n=91) and 30 (n=87). life and stopped at median (IQR) of 12.5 (2,36) months. Fifty-six neonates (11%) were LBW. Nineteen neonates died Indication for treatment was done according to the protocol Table 1. Number of TFS performed Number of TFS Number of patients Days of life (min-max) Number of patients with TSH >12 mU/L (TSH min-max) 1st TFS 509 15 (3-61) 21 (12.4-66) 2 TFS nd 104 28 (5-106) 10 (12.5-58.3) 3rd TFS 46 38 (13-278) 1 (17.9) 4th TFS 11 42 (23-76) 1 (19.6) 5 TFS th 11 51 (34-64) 1 (13) 6th TFS 4 60.5 (42-80) 0 7th TFS 1 98 0 8 TFS th 1 118 0 687 TFS: thyroid function screening, min-max: minimum-maximum, TSH: thyroid stimulating hormone 456 J Clin Res Pediatr Endocrinol Campos-Martorell A et al. 2022;14(4):453-462 Thyroid Function Evaluation in Preterm Neonates shown in Figure 1, except for patient 9 who was treated the other one died 15 days after birth. Six patients (28.6%) due to persistent elevated TSH levels (9.37 mU/L) at 66 were lost to follow-up because they returned to their country days of life on the third TFS. All the patients had normal or region of origin. thyroid ultrasound. At re-evaluation after more than three years of follow up, three patients were finally diagnosed Low Birth Weight for Gestational Age Neonates with permanent CH. One patient was diagnosed with Fifty-six patients (11%) were LBW. These neonates were Williams syndrome, another one with Down syndrome at around two weeks of GA older than those preterm neonates follow up and the last one was diagnosed with probable born with adequate birth weight for GA (ABW) (p<0.001). thyroid dyshormonogenesis. A heterozygous variant of LBW neonates presented with TSH levels at first and unknown significance (VUS), c.2654G>A, (p.Arg885Gln), second TFS higher than those of ABW neonates. However, was found in exon 20 of the DUOX2 gene. This variant no statistically significant differences were found for fT4 has been previously described in the bibliography and in levels between those groups. Results are shown in Table 3. the HGMD database as a variant associated with transient Remarkably, there was a statistically significant relationship hypothyroidism resulting from a single compromised allele, between being LBW and having a TSH >12 mU/L at first TFS but at re-evaluation TSH levels had increased up to 8.6 mU/L (p<0.0001) and receiving treatment with LT4 (p=0.006). (after three weeks without LT4) (23). This patient is currently Of the LBW neonates, only one was finally diagnosed with 9 years old and is still receiving 2 mcg/kg of LT4. In contrast, permanent CH corresponding to the newborn with Williams 15 patients were diagnosed with transient CH because LT4 syndrome. replacement was withdrawn before 6 years of age (before 3 years of age in 13 patients, and between 4 and 6 years Hypothyroxinemia of Prematurity of age in another two). Of the remaining two patients, one Fifteen neonates presented with HOP during the second is currently 2 years old and has not been re-evaluated and week of life [median (IQR) 15 (9,17) days] with TSH Figure 2. Flowchart of thyroid function screening results of premature neonates GA: gestational age, TSH: thyroid stimulating hormone, fT4: free thyroxine, TFS: thyroid function screening, TSH: thyroid stimulating hormone, CH: congenital hypothyroidism 457 Campos-Martorell A et al. J Clin Res Pediatr Endocrinol Thyroid Function Evaluation in Preterm Neonates 2022;14(4):453-462 Table 2. Characteristics of premature newborns treated with levothyroxine Weeks of TSH (mU/L)/ Days of life / TSH Surgical or Levothyroxine Thyroid Transient/ Follow up: GA/ weight fT4 TFS number (mU/L) medical therapy dysfunction permanent Current age (years)/ (g)/ (ng/dL) when when at CH procedure duration CH dyshormonogenesis LBW or levothyroxine levothyroxine screening with iodine (months) genetic test ABW was started was started (whole- overload/days blood)/ of life days of life (2nd sample) 1 28/700/ 19.6/1.02 33/4 1.4/20 Intestinal 48 Delayed TSH Transient, 4y ABW surgery/4 rise although Dyshormonogenesis therapy until test negative 4 years old 2 24/750/ 25/0.76 19/2 4.8/30 PDA surgery/12 30 Delayed TSH Transient 3y ABW rise 3 28/820/ 26.8/1.05 21/2 7.5/3 LP/10 72 Delayed TSH Transient, 6y ABW rise although Dyshormonogenesis therapy until test negative 6 years old 4 25/630/ 15.6/1.18 25/2 3.2/3 PDA surgery/20 1 Delayed TSH Transient 3y ABW rise LOFU 5 29/900/ 94/0.65 18/2 1.7/3 - Still on LT4 Delayed TSH Permanent 3y LBW rise Williams Sd 6 24/495/ 13/1.2 60/5 4.63/40 PDA surgery/8 4 Delayed TSH Tansient 4y ABW rise 7 28/1220/ 58/0.55 33/2 1.12/8 - 12 Delayed TSH Transient 5y ABW rise LOFU 8 30/685/ 12.5/1.62 24/2 1.7/4 - 6 Delayed TSH Transient 3y LBW rise 9 30/645/ 9.37/1.19 66/3 3.61/19 - 36 Delayed TSH Transient 3y LBW rise 10 27/600/ 32.4/1.23 30/2 1.9/3 - 6 Delayed TSH Transient 5y LBW rise 11 27/730/ 18/1.6 36/2 4/5 Inguinal hernia 42 Delayed TSH Transient, 5y ABW surgery/33 rise although Dyshormonogenesis therapy until test negative 3.5 years old 12 29/1600/ 20.8/1.2 18/2 4.2/5 - Still on LT4 Delayed TSH Permanent 2y ABW rise Down Sd 13 29/750/ 39.8/0.94 20/2 3/4 Intestinal 36 Delayed TSH Transient 3y LBW surgery/7 rise LOFU 14 28/1070/ 15/1.05 51/2 3.02/4 Intestinal 13 Delayed TSH Transient 2y LBW surgery/60 rise 15 30/1450/ 41/0.48 25/1 13.6/13 - 2 Delayed TSH Transient 6y LBW 0.6 (2nd rise LOFU sample) 16 26/790/ 66/0.6 14/1 18.6/4 Intestinal 1.5 Typical CH Transient 2y ABW surgery/7 LOFU 17 28/815/ 44.4/0.85 17/1 13.2/4 Intestinal - Delayed TSH ? Died at 15 days of life ABW 1 (2nd surgery/2 rise sample) 18 30/1410/ 46/0.79 23/1 3.5/13 - Still on LT4 Delayed TSH Permanent 6y ABW rise Dyshormonogenesis test: Mut in DUOX2 19 30/825/ 37/0.94 11/1 5.3/4 - Still on LT4 Delayed TSH ? 2y LBW rise 20 29/600/ 22.9/1.29 14/1 10/13 LP/1 13 Delayed TSH Transient 2y LBW rise 21 24/720/ 1.27/0.41 15/1 0.24/60 Hemodynamic 0.5 HPO Transient 6y ABW instability, LOFU dopamine/2 ABW: adequate bierth weight for gestational age, LBW: low birth weight for gestational age, NA: not available, PDA: surgical closure of patent ductus arteriosus, LP: lumbar puncture, TG: tyroglobulin: LOFU: lost of follow up: US: ultrasound, HPO: hypothyroxinemia of prematurity, CH: congenital hypothyroidism, LT4: levothyroxine, Sd: syndrome 458 J Clin Res Pediatr Endocrinol Campos-Martorell A et al. 2022;14(4):453-462 Thyroid Function Evaluation in Preterm Neonates median (IQR) levels of 2 (1.3-3.6) mU/L and fT4 mean The diagnosis of CH in the preterm population is challenging, levels of 0.68±0.1 ng/dL. Two neonates with HOP were especially in the more immature infants because TSH may LBW. Only one patient (26 weeks of GA, TSH 2.1 mU/L, not be elevated in initial samples. Remarkably, in this study fT4 0.41 ng/dL) was treated with LT4 at 15 days of life only one patient was diagnosed by the routine CH screening while suffering a septic shock; his thyroid ultrasound was program in a dried blood spot sample, highlighting the normal and LT4 and hydrocortisone were stopped after need for specific protocols for this population. Whether two weeks. On follow up, 10 patients had TFS with normal repeat screenings should be done at the state level or the levels, two patients had died and in two patients TFS was individual NICU level remains a matter of debate (14). Some not repeated. authors have emphasized the need for a standard repeat of whole-blood TSH samples in very preterm infants as taking Correlation and Association Analysis only a second sample could miss a significant proportion FT4 levels at first TFS correlated positively with birth of neonates with delayed TSH rise (48% in the study of weight (r=0.193, p<0.001) and GA (r=0.343, p<0.001), McGrath) (14,18). Catalonia’s CH screening program does although no association was found with the other variables not currently require a routine second sample on preterm (LBW, prenatal corticoid administration, ventilatory or LBW neonates, and, given this, it seems more efficient support, oxygen therapy, inotropic support, intraventricular and practical that each NICU undertakes the TFS (in venous hemorrhage and neonatal sepsis). sample and with TSH and fT4 determination) in a consistent way integrated with any other screening of co-morbidities TSH levels at first TFS correlated negatively with birth weight related to prematurity (intraventricular haemorrhage, (r=-0.146, p=0.002), although no correlation was found anaemia or retinopathy). Furthermore, the results can be with GA. In association analysis, neonates who received obtained faster and hypothyroxinemic states with low TSH dopamine had TSH levels [3.98 mU/L (0.6,22.9)] slightly values can be treated earlier. higher than neonates who had not received dopamine [3.16 mU/L (0.2,37); p=0.019], but no association was found In the present NICU-based protocol a single serum with the other variables. determination of TSH and fT4 is performed, unlike those described in other studies in which serial determinations of whole-blood TSH were required. However, through the Discussion requisition of repeating all serum TSH above 5 mU/L, we Our study focuses on the incidence of thyroid dysfunction consider that cases of CH will not be missed, and are not in a cohort of 509 preterm neonates below 31 weeks of GA aware of any infant who was diagnosed late with CH, to evaluated with a protocol of TFS in a venous sample during date. The second week of life appears to be an optimal the second week of life, in addition to routine CH screening moment to detect affected neonates in order to start in a dried blood spot (13,16). Thyroid dysfunction was treatment as soon as possible and preferably no later identified in 170 neonates (33%), although only 21 of them than the second week after birth, as European Guidelines (4.1%) were finally treated, 20 for CH and one for HOP. recommend (17). Table 3. Comparison of TSH and fT4 levels at first and second TFS between LBW for gestational age and ABW for gestational age LBW ABW p value 1 TFS st N 56 393 Gestational age median (IQR) 29.1 (28,30) 27.6 (26.2,29.2) 0.001 Birth weight median (IQR) 735 (630,735) 1045 (830,1251.2) <0.001 Days of life when TFS was performed median (IQR) 14 (13,16) 15 (13,17) NS fT4 (ng/dL) mean±SD and range 1.2±0.2 1.18±0.2 NS 0.84-1.76 0.8-2.05 TSH (mU/L) median (IQR) 5.3 (3.6,9) 3.3 (2.1,5.2) <0.001 2nd TFS N 21 94 Days of life when TFS was performed median (IQR) 28 (22,32.5) 28 (21,34) NS fT4 (ng/dL) mean±SD and range 1.31±0.2 1.22±0.2 NS 0.96-1.84 0.83-1.76 TSH (mU/L) median (IQR) 6.9 (5.1,9.4) 3.8 (2.6,6) <0.001 TFS: thyroid function screening, LBW: low birth weight for gestational age, ABW: adequate birth weight for gestational age, NS: non-significant, SD: standard deviation, IQR: interquartile range, fT4: free thyroxine, TSH: thyroid stimulating hormone 459 Campos-Martorell A et al. J Clin Res Pediatr Endocrinol Thyroid Function Evaluation in Preterm Neonates 2022;14(4):453-462 Several longitudinal studies have shown that some preterm weeks of GA. In addition to the GA effect, this may be due neonates have a characteristic fluctuation in thyroid to the fact that our NICU has a higher number of complex hormone levels during the first few weeks of postnatal pregnancies (fetal pathologies such as genetic and life, consisting of transient mild lower levels of fT4, chromosomal syndromes) and also to the smaller size of followed by a mild and transient elevation in serum TSH the study cohort compared to the general population. Our levels. Some preterm neonates present with delayed TSH data are similar to other studies with extremely preterm elevations greater than this mild compensatory rise and neonates: Kaluarachchi et al. (5) reported an incidence of the etiology of this greater delayed TSH elevation remains 1:143 and 1:64 in <32 weeks of GA neonates and Woo unclear (4). It may reflect true primary hypothyroidism et al. (26) reported 14-fold higher incidence of CH in very or the recovery of illness-induced suppression of the LBW infants. hypothalamic-pituitary-thyroid axis. In addition, iodine deficiency or excess iodine levels could also be associated The TSH cut-off point for starting treatment is unclear, but with the development of delayed TSH elevation in this given the evolution of our patients, perhaps an increase in population (13,24). Although TSH elevation usually this cut-off from 12 to 15 mU/L could be considered, as long resolves spontaneously in a few months, some of these as fT4 levels remain within normal range. A recent study by neonates have low fT4 concentrations at diagnosis and Kaluarachchi et al. (27) suggested using age-related TSH cut- in a small but significant proportion of neonates, thyroid offs from birth to term equivalent gestation to avoid missed dysfunction is permanent (13). Infants with extremely diagnoses. LBW, have greater and more persistent increase in TSH In preterm neonates with low levels of fT4 and normal levels (4,6). In general, the strategy is to start replacement levels of TSH it is difficult to distinguish between HOP, treatment with LT4 (see protocol) and attempt to withdraw central hypothyroidism and non-thyroidal illness syndrome. it at 3 years of age when neurodevelopment is completed. Central hypothyroidism is an extremely rare condition and In our study some neonates with mild and delayed other pituitary hormone deficiencies and midline brain TSH elevations were re-evaluated earlier during these defects could suggest this diagnosis. The incidence of HOP 36 months post-birth because of the low doses of LT4 is difficult to compare in the studies published because related to their weight. New, recently published guidelines recommend re-evaluating a child with no permanent CH of different definitions (free T4 or total T4) and different diagnosis and a gland in situ if thyroxine dose required cut-off levels. It is reported from 7 to 50% of preterm is less than 3 mcg/kg per day at the age of 6 months neonates below 28 weeks of GA (7,28,29). In our study (17,25). Another important factor to be considered is the the incidence of HOP was lower (3%), but is comparable susceptibility of preterm neonates to iodine overload, with that reported in a previous study (7). In the present despite the very restrictive protocols for iodine use in study fT4 levels correlated positively with GA and levels NICU. In our sample, 55% of neonates diagnosed with rise throughout the first postnatal weeks (7,28,29). Only CH received an iodine overload and in 60% of these cases one newborn received LT4 for HOP while suffering a septic replacement therapy was withdrawn before 3 years of age. shock. This data confirms that not all preterm infants are Our study highlights the importance of repeating TFS and hypothyroxinemic and there is no reason for indiscriminate close monitoring when iodine overload occurs, as was also treatment with LT4 in this population (4). reported by Mcgrath et al. (18) who found an incidence of 25% of iodine overload in infants with delayed TSH rise Study Limitations in their cohort. Limitations of our study are that some neonates might have After 6 years of follow up only three of the treated been acutely ill at the time of TFS. However, this limitation neonates were diagnosed with permanent CH, one with an may have been mitigated with repeated abnormal heterozygous DUOX2 mutation of unknown significance findings. Another issue is that starting LT4 treatment was and the other two were syndromic, Down and Williams decided on an individual basis and not from a randomized syndrome, respectively. To highlight, none of them were control trial, denoting the lack of consensus on the start diagnosed using routine dried blood spot TSH-based of treatment in this group of neonates. It should also be screening. The global incidence of primary CH in our added that the choice of the serum TSH cut-off point of community is 1:2305 and in our study the incidence of 12 mU/L to start treatment with LT4 was used based on CH is 1:509 (patients with Down and Williams syndrome consensus guidelines recommendation to treat patients were excluded because these syndromes already if TSH is between 10-20 mU/L and on the experience of predispose to thyroid disorders) in preterm newborn <31 previous cases (17,20). 460 J Clin Res Pediatr Endocrinol Campos-Martorell A et al. 2022;14(4):453-462 Thyroid Function Evaluation in Preterm Neonates Conclusion References 1. Calvo RM, Jauniaux E, Gulbis B, Asunción M, Gervy C, Contempré B, In conclusion, this protocol was able to detect thyroid Morreale de Escobar G. Fetal tissues are exposed to biologically relevant dysfunction in preterm neonates that were not detected free thyroxine concentrations during early phases of development. 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