Extremely premature infants are at high risk of disrupted brain development with consequent acquired damage, especially to the white matter because of multiple factors in an adverse environment. Adverse conditions are thought to include hypoxic ischaemia, free radical injury i.e. factors related to their premature birth because of low concentrations of antioxidants, undernutrition and sepsis 1 . At untimely extreme premature delivery (24 – 28 weeks of gestation), brain development is at a critical stage because neuronal migration has only just completed and synaptogenesis is occurring: therefore the cellular milieu is critical 2 .

Brain magnetic resonance imaging (MRI) studies of survivors of low birth weight who have subtle cognitive abnormalities have shown diminished volumes of the caudate nucleus and hippocampus. Diminished volumes of the caudate nucleus and hippocampus were associated with lower IQ, learning disorder and attention deficit 3 4 . Recent brain MRI studies of preterm infants also show that quantitative cerebral structural abnormalities are related to the degree of immaturity at birth and are followed by abnormal neurodevelopmental outcome 5 . Preterm infants assessed at term show reduced myelination when compared to infants born at term 5 6

Only 70% of infants born before 26 weeks' gestation survive until expected date of delivery 7 . Of the survivors at least half have significant neurocognitive disabilities 8 9 . Many factors are associated with an increased risk of neurocognitive impairment. Brain growth following birth is a major determinant of neurocognitive outcome in these infants and poor brain growth is recognised as a dominant determinant of neurocognitive outcome 10 . Brain growth at the expected date of delivery in preterm infants are key predictors of long-term brain growth and intelligence quotient (IQ) score 10 . This implies that postnatal head growth is a more important determinant of IQ than intrauterine growth.

Observational studies have found that low thyroid hormone levels in the first few weeks of life in preterm infants are associated with poor neurodevelopmental outcome. (see Table 1). These associations persist after adjustment for confounders and suggest that hypothyroxinemia during the critical window period for brain development which occurs following birth at extreme prematurity has important effects on neurodevelopment. However, at present it is unclear whether poor neurodevelopment is caused by hypothyroxinaemia or whether the hypothyroxinaemia is a consequence of other factors that also cause poor neurodevelopmental outcome and simply an associated abnormality.

There is prime facie evidence that thyroid hormones directly contribute to brain development which leads us to suspect that hypothyroxinaemia among preterm infants has adverse effects on brain development. Animal studies have shown that a deficiency of thyroxine results in impaired neurological pathways during early neurogenesis which results in abnormalities of the cytoarchitecture of specific brain structures such as the hippocampus, temporal and sensorimotor cortex 11 12 13 14 15 . Eayrs and colleagues have shown in early studies 16 17 that perinatal hypothyroidism in rats alter the density and size of neurones and fibre density in specific brain regions and the orientation of cortical layers. Berbel et al 18 19 also described effects on spine density of pyramidal neurones in the cerebral cortex, organisation of callosal connection and maturation of dendrite connections as a result of hypothyroidism. Thyroid hormones are also demonstrated to increase proliferation of cerebellar granule cells as well as affect apoptosis which is important in suppression of proliferation and downgrading of these cells 20 21 . Thyroid hormones are essential for normal axonal myelination and for normal interhemispheric commisure development 22 23 . Thyroid hormones are also important in regulating transcription of specific genes expressed in the developing brain 24 . This suggests an important role for thyroid hormone in the complex developing brain throughout gestation.

While neurocognitive outcome in childhood is the long-term concern, previous work has demonstrated that the size of key brain structures at expected date of delivery is a predictor of subsequent development and is a more immediate way to evaluate strategies that aim to enhance brain growth 9 33 . This is because the size of brain structures reflect the extent of myelination. Myelination is the process by which nerve fibres are coated with thin layers of lipid called myelin. Myelin acts as electrical insulation and increases the efficiency of nerve fibres. In children with a congenital lack of thyroid hormone (congenital hypothyroidism) there is a lack of cerebral myelination 25 26 . Thyroid hormones are therefore a factor in the regulation of the extent of myelination during gestation and after birth. Furthermore, TSH receptors have been found in early human foetal brain and human astrocytes in culture, and they are thought to mediate extrathyroidal cyclic AMP independent biological effects of TSH and stimulation of the type 2 deiodinases in astroglial cells. 27

At present, there is a paucity of published data concerning the relationship between thyroid hormone status and brain development in extreme preterm infants. Previous work has demonstrated that the size of key brain structures and brain volume at term is a key predictor of subsequent development 2 6 9 28 . Current evidence also shows that hypothyroxinemia is common in extreme prematurity 29 30 31 32 33 . Derangements of critical thyroid function during the sensitive window in prematurity when early development occurs could have a range of long term effects for brain development.

A randomised controlled trial of levothyroxine supplementation by van Wassanaer 34 35 enrolled 200 infants under 30 weeks gestation showed no overall effect of levothyroxine supplementation on medium- or long-term outcomes. However, post hoc subgroup analysis showed an improved Bayley MDI and PDI at 2 years and 5.5 years of follow up among infants born below 27 weeks' gestation and a better motor outcome in those born below 28 weeks' gestation in the treatment arm. Paradoxically, the reverse was true for those infants born at 29 weeks' gestation and above which had a worse Bayley MDI and PDI developmental score. However, the sample size for this sub-set of infants with gestational age under 27 weeks who benefited from the intervention was small with 19 infants in the treatment arm and 27 in the placebo arm. Several reasons may explain this gestation dependent positive effect of thyroxine in infants born below 28 weeks' gestation. Firstly, FT4 concentration in the very immature infants may be too low to ensure normal brain development, of which thyroid dependent maturation in the brain takes place in the window period before 30 weeks. It is also possible that thyroid hormones are important in its role of repairing ischaemic damage of the brain which commonly occurs in extreme prematurity 36 .

This study is an explanatory randomised controlled trial of levothyroxine vs placebo in infants born below 28 weeks' gestation to determine the effects on brain volume, and development, the hypothalamic-pituitary-adrenal (HPA) axis and somatic growth (see TIPIT protocol 37 ).

This trial will provide the opportunity to assess whether supplementation with a thyroid hormone during development up until 32 weeks gestation affects the extent of myelination because we are undertaking detailed cranial MRI in a subset of trial participants. An MRI technique called Diffusion Tensor Imaging will assess the structural integrity of the white matter 38 39 . The anisotropy of water diffusion will be measured which is dependent on the degree of myelination. In addition, we aim to qualitatively and quantitatively evaluate the effect of thyroid hormones on cerebro-vascular morphology, including vessel size (using measures of diameter), architecture (using measures of tortuosity) and vessel density in extremely premature infants and to understand more about the interdependence of vessel and brain development and their relationship to prematurity. In addition, this trial will determine whether the effects of levothyroxine on the regulation of brain development are clinically relevant.

Important outcomes that will be assessed with cranial MRI are the extent of white matter myelination and integrity, vessel morphology and the volumes of key brain structures at term equivalent which will be detailed in this protocol.

All infants recruited to the TIPIT study 37 will be consented separately to have cranial MRI scans at term equivalent. This protocol describes the study we will perform on the infants that have cranial MRI scans.

The authors declare that they have no competing interests.

SMN conceived the TIPIT/cranial MRI study, participated in the design and management of the TIPIT trial, the design and coordination of the cranial MRI study and drafted the manuscript. AMW and MAT conceived the TIPIT/cranial MRI study, and participated in its design and management of the TIPIT trial. MD and SV participated in the design of the TIPIT trial and its management and the design and coordination of the cranial MRI study. CG participated in the design of the study and the statistical analysis. LMP, VS and LA conceived the cranial MRI study and participated in its design and coordination. AT, CM and LG participated in design sequence on the MRI protocol. All authors read and approved the final manuscript.