Analysis of Factors Related To Head MRI Changes In Neonatal Hyperbilirubinemia And Effect On Neurodevelopmental Outcomes.

Background and Objective This study aims to detect how hyperbilirubinemia affects cerebral structures by imagery (MRI) to prevent irreversible future brain damage. Research Design and Methods In this retrospective study, 50 infants of 35 weeks gestation or more with hyperbilirubinemia were assigned to two groups based on abnormal or normal head imaging (MRI). The primary outcomes included peak bilirubin levels according to socio-demographic factors and how these levels inuence MRI. The secondary outcome was neurodevelopment at 1, 3, 6, 9 and 12 months for the imaging changes versus the normal imaging groups assessed by a combined formulaire based on the CDC’s developmental milestones, the Denver II Developmental Screening Test (DDST), the Hammersmith infant neurological exam, and auditory brainstem response (ABR) by investigators. Findings from our study showed that neonates with hyperbilirubinemia who have abnormal MR imaging had an association with their gestational ages, birth weights, albumin levels, peak TSB levels, maternal pathology which may have an effect on the neurodevelopment outcome of these neonates. In the present retrospective cohort, it was observed that the mean gestational age of infants with hyperbilirubinemia which presented with high signal intensity on MR imaging was lower compared to those without changes in imaging. A lengthier change process in dynamic properties and mitochondrial changes has been observed in preterm infants, especially extremely premature ones and this may explain the abnormal image scans preceding the normal ones 22 . In addition, myelination may be a factor causing high signal intensity in preterm populations which may be seen falsely as abnormal imaging due to high bilirubin levels 23 . Our study also supports a higher risk of MRI changes in newborns with low gestational age.


Introduction
Neonatal hyperbilirubinemia (NHB) is a common occurrence affecting about 60% of term and 80% of preterm infants 1,2 . In 1904, Schmorl described yellow staining of the basal ganglia in postmortem ndings of infants with neonatal jaundice 3,4 . It refers to increased bilirubin levels seen in neonates with a variable clinical outcome from reversible acute bilirubin encephalopathy (ABE) to irreversible kernicterus 5 .
10% of term and 25% of late preterm deliveries will undergo phototherapy to avoid acute and chronic bilirubin encephalopathy 6 . The cost of caring for a patient with kernicterus over a lifetime is estimated at around $USD 900000 while the estimated cost to prevent a single case of kernicterus with universal total serum bilirubin and/or transcutaneous bilirubin screening is between $USD 5.7 and 9.2 million 7 , making neonatal jaundice a health concern.
Magnetic resonance imaging (MRI) is used as the imaging of choice for examining structural changes at the level of the basal ganglia and subthalamus, the two most affected cortical areas in encephalopathy 8 .
The most widely accepted changes show abnormally increased signal intensity on T1-weighted imaging in these areas. Few studies have been used to show the structural changes observed using DWI in neonatal hyperbilirubinemia 9 .
Can early detection of central nervous system damage in children with hyperbilirubinemia be detected to guide early intervention and reduce permanent damage and sequelae? Can Magnetic resonance imaging (MRI), be used as an indicator for predicting brain damage and late neurodevelopment? To answer these questions, we conducted retrospective cohort studies in 50 children with hyperbilirubinemia.

Patients And Methods
Overall 50 participants (21 males, 29 females) with TSB ≥ 210µmol/L were included in the study after being born or referred at the rst a liated hospital of Dalian Medical University between December 2019 and February 2021. A gestational age (GA) of at least 35 weeks, a postnatal age less than 2 weeks were criteria for inclusion. Others were clinically observable jaundice with TSB ≥ 210µmol/L on the rst day of birth or on subsequent days. They were divided according to their TSB levels as follows : low ≤ 256µmol/L, moderate 256-340µmol/L and high > 340µmol/L. Infants were divided into preterm (< 37 weeks gestational age, GA), early-term (37-38 weeks GA), and full-term (> 38 weeks GA). In addition, participants with signs of acute or chronic bilirubin encephalopathy (ABE,CBE) manifested as lethargy, shill cry, hypo/hypertonia, seizures, auditory or occular abnormalities were also included. Congenital anomaly, con rmed infections,or metabolic derangement, intracranial hemorrhage or injury, and those who did not give their accord to do MRI, DWI sequencing or ABR were excluded. All participants were treated with phototherapy.
Anthropometric measurements including gender, birth weight, head circumference of each participant was recorded as predictors. Birth weight was classi ed using AAP criteria as low birth weight LBW < 2500g, normal birth weight NBW 2500-4000g and high birth weight HBW > 4000g. In addition, the method of delivery, gestational age at delivery, feeding methods were equally recorded. Etiologies for hyperbilirubinemia were obtained if found.
MRI was performed with a 1.5 Tesla (T) clinical MR unit (Magnetom Sonata; Siemens, Erlangen, Germany), with a standard infant head coil after administration of 5% chloral hydrate 0.5ml/kg per os for sedation. Transverse and axial planes were used to obtained with T1-weighted spin echo images. Fluid Attenuation recovery (FLAIR) and diffusion-weighted imaging (DWI) were equally obtained. Apparent diffusion coe cients (ADC) were rcorreletaed with DWI to determine uid restriction, thereby hyperintensity. MRI scanning documented the following ndings as abnormal: a) High signal intensity SI in the GP and basal ganglia b) white matter SI with extent and location of lesion and comparing with SI of the basal ganglia.
Universal screening for hearing loss or dysfunction was done in all participants who had their audition tested using auditory brainstem evoked response (ABER) and audiometry (in situ and autoacoustic emission), with abnormal results recorded.
A questionnaire based on attainment of milestones at 1, 3, 6, 9 and 12 months respectively was designed for the purpose of this study, based on Hammersmith infant neurologic exam 10 , the CDC (Center of Disease Control) developmental milestones 11 , and the Denver II Developmental Screening Test (DDST) 12 and was used for follow-up to access development. DDST includes a series of age speci c questions and an evaluation chart to characterize development 4 outcomes in terms of social (emotional), language (communication), and both gross and motor skills.On this chart, questions and observation were recorded as percentages, according to the patients age in months. According to the guideline, a patient was classi ed as having a delay when failing on an item matching 90% the age. This delay was then assessed for one of each of the four subgroups. If there are two or more delays, the result is considered abnormal. After obtaining a nal score, patients results in subgroups were classi ed as normal or abnormal. Baseline characteristics in were recorded in Table 1 below. Abbreviations: LBW-low birth weight, NBW -normal birth weight, HBW -high birth weight, GA -gestational age, HC -Head circumference, Tc -Transcutaneous bilirubin, P-phototherapy, P+A -phototherapy and antibiotics, P+Alb -phototherapy and albumin, P+Alb+IVIg -phototherapy, albumin and intravenous immunoglobulins, P+M+Ph -phototherapy, mannitol and phenobarbital, others include HELLP syndrome, APS, thyroiditis and vaginitis.
Informed parental consent was obtained from the guardians of all participants in the study. Our methodology was carried out in accordance with relevant guidelines and regulations. Our study was approved by the ethical committee of the First A liated Hopital of Dalian Medical University.
Partaking to statistical analysis, mean, standard deviation, and minimum and maximum values were referred to as continuous variables, and the categorical variables were expressed as numbers and percentages. The Mann-Whitney U test was used in the comparison of continuous variables. Moreover, the student t-test was used in the comparison of MRI changes and normal groups.In addition, binary logistic regression analysis was performed to separately access hyperbilirubinemia, changes in imaging, and their relationship with milestone attainment. The statistical analysis was performed with SPSS software (version 25.0). The probability level of less than 0.05 was considered signi cant.

Neonatal and maternal ndings.
Our study comprised of 50 neonates; 21 boys and 29 girls, admitted with a mean age of 3.3± 2.1 days and who presented in our department with a mean time of 2.3± 1.1 days from birth to clinical observation of jaundice from guardians. The mean weight of admission was 3360.5±489.9 g with a mean gestational age of 38.2± 1.2 weeks, and a head circumference of 33.8± 1.4 cm. Baseline characteristics of participants were recorded and TSB values were recorded in Table 2. None of the infants on admission demonstrated abnormal neurological ndings of ABE (lethargy, drowsiness, high-pitched cry or de cient motor tones) nor CBE.
Maternal factors such as age, gravida number, blood group, and antepartum pathologies were collected and compared between groups. There was a statistically signi cant difference between antenatal pathologies in the group of infants presenting with MRI imaging compared to those without. Infants with mothers with both gestational hypertension (eclampsia, others) and gestational diabetes had higher bilirubin levels (322.9±48.5μmol/L) compared to those with other pathologies. In addition, the mean maternal age of infants presenting with changes on imaging is higher than those without changes.  Table 3 below. Using the evaluation forms based on DDST and CDC milestone attainment, neurodevelopment was evaluated in terms of language, social, ne and gross motor skills. There was no signi cant difference between MRI changes and normal MRI groups when evaluating milestones (p=0.237). 3 participants (2 males, 1 female) had abnormal outcomes during follow-up as seen in Table 4. 1 male showed delayed milestones at 3 months and the last male showed a delay at 6 months. In addition, a cross-tab comparison between MRI and milestones depicts MRI as having a 66.7% 70.2% sensitivity and speci city while 12.5% 97.1% PPV (positive predictive value) and NPV (negative predictive values) respectively for milestone attainment. It is interesting to note that a 2-day old female infant who had MRI changes (bilateral symmetrical lentiform increased intensity) in Figure 1, had the second lowest albumin level among the participants (31.8 g/L), with moderate peak TSB levels (305.5μmol/L), showed a left ear abnormal ABR and showed delayed milestone attainment at 9 months of age, whereas she showed normal attainment at 1, 3 and 6 months of age. Compared to her age group at 9 months, she scored she showed delays in 3 out of 4 subcategories.
All neonates participating in the study were tested for audition after admission in the neonatology ward.
Mean peak TSB values for normal and abnormal milestone attainment were 304.1±54.6μmol/L and 284.8±40.3μmol/L respectively while peak mean TSB values for normal ABR and abnormal ABR were 302.9±50.0μmol/L and 303.7±88.9μmol/L. 3 neonates (2 males, 1 female) showed abnormalities on follow-up.
In addition, a cross-tab comparison between MRI and milestones depicts MRI as having a 66.7% and 33.3% sensitivity and speci city In addition, a cross-tab comparison between MRI and milestones depicts MRI as having a 66.7% 33.3% sensitivity and speci city while 12.5% 97.1% PPV and NPV respectively for milestone attainment.
Direct logistic regression was employed to assess the impact of several demographic and clinical factors on the likelihood that infants with hyperbilirubinemia will present with abnormal changes on imaging.
From our results obtained in Table 5 below, univariate analysis depicted peak TSB accounting for the greatest variance in abnormal MRI at 26.3%, followed by days in observing jaundice in participants (9.5%), age of participants (8.4%), and maternal gestational pathology (7.4%). In our study, we were able to show a consistent relationship between hyperbilirubinemia and MRI changes manifested as bilateral hyperintensity of the GP on T1WI, but a signi cant correlation between these changes and neurodevelopmental outcomes could not be established with certainty.
Neonatal jaundice is a chronic worldwide problem and a major cause of morbidity especially in developing countries [16][17][18] . Bilirubin encephalopathy is not uncommon in China, accounting for about 4.8% of neonatal hyperlirubinemia. Neonatal hyperbilirubinemia has been found to have an association with development outcomes as it is well known that chronic bilirubin encephalopathy (kernicterus) may lead to long-term neurological and physical de cits 8, [19][20][21] .
Findings from our study showed that neonates with hyperbilirubinemia who have abnormal MR imaging had an association with their gestational ages, birth weights, albumin levels, peak TSB levels, maternal pathology which may have an effect on the neurodevelopment outcome of these neonates.
In the present retrospective cohort, it was observed that the mean gestational age of infants with hyperbilirubinemia which presented with high signal intensity on MR imaging was lower compared to those without changes in imaging. A lengthier change process in dynamic properties and mitochondrial changes has been observed in preterm infants, especially extremely premature ones and this may explain the abnormal image scans preceding the normal ones 22 . In addition, myelination may be a factor causing high signal intensity in preterm populations which may be seen falsely as abnormal imaging due to high bilirubin levels 23 . Our study also supports a higher risk of MRI changes in newborns with low gestational age.
Birth weight is another risk factor for hyperbilirubinemia. Birth weight and prematurity work in tandem as a low gestational age neonate will most likely have a low birth weight after delivery. A study by Woodward demonstrated that low-weight preterm infants with mild to moderate white matter abnormality on MRI had delays by 4 years of age 24 . These studies were done in preterm infants hence the delay in language development could not be ascertained to be due to prematurity, hyperbilirubinemia, or both.
Findings from our study showed that neonates who presented with jaundice had lower mean weights in the group with MRI changes compared to those without MRI changes. The difference though was not statistically signi cant (p=0.332). Contrary to their ndings, our cohort showed a signi cant difference in peak TSB levels between groups, with the TSB levels of participants with MRI changes signi cantly higher than those without (p=0.000).
It is known that hyperbilirubinemia affects more boys than girls 25 . A study showed that male neonates with a TSB>340μmol/L had an increased risk of having an IQ<85 on follow-up at 17 years with affected cognition and physical performance 26 . In our study, gender was shown to be statistically different in both groups in term of peak bilirubin levels and MRI changes (p=0.09). Although there were 21 males compared to 28 females, males had signi cantly higher TSB levels than females (325.8±43.9μmol/L vs 286.4±54.8μmol/L) respectively.
Auditory neural pathways are highly sensitive to bilirubin-induced toxicity especially in preterm infants who are at a higher risk of this toxicity 6,27 . ABR may predict sensory neural hearing loss with rising TSB levels, but predictive value of ABR to determine neurodevelopmental outcome is not clear. Infants born at ≤ 36 weeks gestational age with a mean peak TSB of 12 mg/dL was a signi cant predictor for hearing loss 28 . In our study, 3 out of 16 patients with abnormal MR imaging had an abnormal audio brainstem response (ABR). It is worth mentioning that among these 3 participants, only one went on to show a delayed attainment of milestones at 9 months of age after previously having normal milestones at 1, 3, and 6 months of age ( Figure 1). Audiologic follow-up of patients showed normal ABR examinations. This concords with previous ndings which show that auditory impairment due to hyperbilirubinemia is reversible. In addition none of the participants with abnormal ABR was preterm nor had birth weights less than 3500g. We can therefore ascertain that abnormal ABR results was primarily due to hyperbilirubinemia.
Maternal factors such as delivery methods, age, and antepartum pathology can be risk factors for neonatal hyperbilirubinemia. In our study, it was shown that mothers with hypertension and diabetes with other factors had infants with higher mean bilirubin levels and had signi cant changes on imaging compared to mothers of infants without MRI changes (p=0.047). Our study showed there were more mothers of A+ blood groups in the MRI change group compared to the group without change. Multipara deliveries were associated with infants having higher TSB levels than primipara deliveries although the difference was not signi cant (p=0.638). Cesarean delivery is a well-known protective factor in infants with neonatal jaundice and decreased chances of readmission because of hyperbilirubinemia due to prolonged hospital stay, hence improved monitoring, early formula supplementation, less placental transfusion and stress prior to delivery prompting induction of conjugating enzymes or metabolism of bilirubin as an oxidant 29  Among all the participants involved, 3 participants had abnormal DDST scores during the study, with 1 participant having a delay at 9 months during follow-up. Johnson observed cognitive de cits in neonates as they develop regardless of the cause of neonatal jaundice 37 . Some aspects of nervous system functioning and cognition in infants and children may not be apparent until much later than 3 years 38 .Therefore, the effect of imaging changes in MRI on the long-term prognosis of children with hyperbilirubinemia may require longer follow-up times and increased sample size for further study.
Limitations of our retrospective cohort involve a small sample size and loss of participants during followup for various reasons. Moreover the time for follow-up was short as most neurological de cits are observed at around 2 years of age after maturation of frontal cortices and neuropsychiatric symptoms are observed much later. For example athetoid cerebral palsy or other signs of kernicterus may remain undetected by year 1. A strength of our study was we evaluated MRI scans including DWI sequences, ABR tests in all participants and showed a cut-off TSB level for increasing sensitivity and speci ty of MRI scanning.

Conclusion
1. In children with hyperbilirubinemia, with the increase of total serum bilirubin levels, the probability of abnormalities in the head magnetic resonance imaging (MRI) increased signi cantly≥342μmol/L; 2. Although early MRI imaging changes cannot independently predict the outcome of early neurodevelopment in infants with hyperbilirubinemia, it is necessary to extend the follow-up regarding growth and development in children with MRI abnormalities.