Hyperbilirubinemia is a prevalent newborn condition and a major factor in neonatal morbidity and mortality, especially among preterm infants. Nearly all neonates exhibit a total serum or plasma bilirubin (TSB) concentration over 1 mg/dL (17 µmol/L), with clinical signs of neonatal jaundice generally emerging when TSB levels surpass 5 mg/dL.1 Newborn jaundice, marked by yellow pigmentation of the skin and sclera resulting from hyperbilirubinemia, is the most common newborn disease, especially in Southeast Asia, impacting around 50% of term and 80% of preterm infants during the initial week of life.2

Excess bilirubin accumulation is a recognized neurological risk factor in both preterm and term neonates, in conjunction with other perinatal complications including prematurity, low birth weight, intrauterine growth restriction, perinatal asphyxia, sepsis, bronchopulmonary dysplasia, neonatal hypoglycemia, periventricular leukomalacia, and intraventricular hemorrhage.3,4

Acute bilirubin encephalopathy (ABE) is a critical consequence of hyperbilirubinemia, arising when bilirubin penetrates the blood-brain barrier, attaches to neural tissue, and causes neurotoxicity.5,6 Clinically, ABE manifests as lethargy, atypical behavior, neonatal encephalopathy, opisthotonus, and seizures, with surviving children frequently experiencing enduring neurodevelopmental consequences such as cerebral palsy, sensorineural hearing loss, cognitive deficits, and significant developmental delays.7. Severe neonatal hyperbilirubinemia is a recognized etiology of kernicterus, or chronic bilirubin encephalopathy (CBE), which is marked by choreoathetoid cerebral palsy, impaired upward vision, and sensorineural hearing loss, while cognitive function is generally intact.8 Furthermore, extended exposure to neurotoxic bilirubin concentrations may result in auditory impairments and linguistic difficulties.9,10 A research by Murki et al. in India revealed a 21.8% incidence of kernicterus in neonates with non-hemolytic hyperbilirubinemia.11 A bilirubin level beyond 20 mg/dL correlates with a significant risk of kernicterus, with pathology data indicating that kernicterus occurs in roughly 30% of newborns with bilirubin levels reaching 25–30 mg/dL. Recent research suggest that diminished bilirubin levels may be associated with neurodevelopmental impairments, cerebral palsy, and auditory abnormalities, especially in preterm infants.12Bilirubin-induced neurologic dysfunction (BIND) is a less precisely defined condition that encompasses a wider range of neurological abnormalities caused by mild hyperbilirubinemia, excluding the severe characteristics of kernicterus.13 BIND may manifest clinically beyond the neonatal phase as developmentaldelays, cognitive impairments, executive dysfunction, and behavioral or psychiatric disorders.14 Advancements in perinatal care, such as phototherapy and exchange transfusion, have markedly diminished the prevalence of severehyperbilirubinemia and kernicterus.15

The proportionalimpact of neonatal hyperbilirubinemia compared to other perinatal risk factors in the emergence of long-term neurodevelopmental problems remains contentious. Moreover, information regarding the predictors of neurological outcomes in hyperbilirubinemia neonates in our nation is still scarce.

This study aimed to investigate the association betweenhyperbilirubinemiaand neurodevelopmental outcomes in term and late preterm neonates. Furthermore, it evaluated the correlation between hearing assessment and neurodevelopmental delay and assess MRI changes in hyperbilirubinemia and their relationship with adverse neurodevelopmental outcomes.

This hospital-based prospective observational study was conducted in the Neonatal Intensive Care Unit (NICU) of the Department of Pediatrics, Chacha Nehru Bal Chikitsalaya, Geeta Colony, Delhi. Data collection commenced in April 2014 following approval from the Institutional Ethics Committee of Maulana Azad Medical College and associated hospitals, New Delhiand was completed in March 2015, with an additional two months allocated for data analysis.

The study population comprised term (37 to 42 weeks of gestation) and late preterm neonates (>34 weeks of gestation) admitted to the NICU during the study period. Using convenience sampling, the first 60 neonates meeting the inclusion and exclusion criteria were enrolled. Eligible participants were hemodynamically stable term and late preterm neonates, admitted primarily for neonatal jaundice requiring phototherapy or exchange transfusion, whose parents provided written informed consent.

Neonates with major congenital anomalies, genetic or metabolic disorders, perinatal asphyxia with hypoxic-ischemic encephalopathy (HIE), seizures, hypoglycemia, meningitis, or intracranial hemorrhage were excluded. Additionally, those with a complicated neonatal course (e.g., respiratory distress syndrome, shock, severe hypothermia) or head circumference outside the 10th–90th percentile for gestational age were not included.

Methodology

Informed consent was obtained from the parents. A detailed antenatal, perinatal, and postnatal history was recorded using a predesigned proforma, followed by a thorough clinical examination. Under aseptic conditions, a 3.5 mL venous blood sample was collected and processed accordingly. The clotted bloodsample was centrifuged at 3000 rpm for 10 minutes, and the supernatant serum was collected for routine analysis of total and direct bilirubin using the Jendrassik-Grof method. The following biochemical investigations were performed: total and indirect serum bilirubin, blood group and Rh typing (mother and baby), Direct Coombs test, complete blood count (CBC) with peripheral smear and reticulocyte count, thyroid profile, and glucose-6-phosphate dehydrogenase (G6PD) levels. In neonates presenting with clinical signs of sepsis, the diagnosis was confirmed through a sepsis screen and blood culture. Management of hyperbilirubinemia was carried out as per the AAP 2004 guidelines.

Neurodevelopmental and Hearing Assessment

A detailed neurological examination was conducted using the Developmental Assessment Scales for Indian Infants (DASII) at admission, discharge, and follow-up visits (3 and 6 months of age). DASII assesses motor and mental development through 230 items (67 motor and 163 mental items). A Development Quotient (DQ) ≤ 70 (≤2 SD below the mean) in either domain was considered indicative of developmental delay. Hearing assessment was performed at discharge using behavioral methods and/or otoacoustic emissions (OAE). Neonates with neurological abnormalities or no response to sound at 3 months underwent Brainstem Evoked Response Audiometry (BERA) for further evaluation.

Statistical Analysis

Datawas entered into Microsoft Excel Spreadsheet. Discrete data was expressed in form of proportion and continuous data was expressed as mean and standard deviations. The difference in proportion was analyzed using Chi-Square test and the difference in mean among the groups was analyzed using the student-t Test, 0.5% probability level was considered as statistically significant.

Two thirds of newbornwere male and rest were female. 85% of the newborns were full-term, while 15% were pre-term. Three fourth (75%, 45/60) of the newborns were appropriate for gestational age, whereas rest one fourth were small for gestational age. Majority of newborns (91.7%) were through vaginal delivery, while rest 8.3% via caesarean section. The mean age, weight at admission, period of gestation was 131.58±74.74 hours, 2424.00±409.95 grams, and 37.77±1.51 weeks. The mean serum bilirubin was 21.57±4.76mg/dL, and the low birth weight was 23(47.9%). And the mean duration of phototherapy was 41.13±10.18 hours [Table 1].

The most common neonatal condition observed in this study was ABO incompatibility in 30% of neonates, followed by sepsisin 28.3% neonates, Rhesus (Rh) incompatibility in 8.3% of neonates, both ABO and Rh incompatibility in 5% of cases. Other less common causes included breastfeeding jaundice in 3.3%, Polycythemia in 1.7% of cases, and 18.3% of neonates, the cause of their condition remained unknown. Out of 60 neonates, eight (13.3%) were diagnosed with acute bilirubin encephalopathy (ABE) at the time of admission. Among these, three neonates (37.5%) had sepsis, two (25%) had ABO incompatibility, and another two (25%) had Rhesus incompatibility. In one case, the underlying cause remained unidentified [Figure 1].

In our study, 8(13.3%) out of 60had abnormal neurological findings at discharge. At discharge, six(10%) neonates had active tone abnormalities, which decreased to five(8.3%) neonates by three months and reduced to four (6.7%) neonates by six months. Similarly, passive tone abnormalities were observed in eight (13.3%) neonates at discharge, which declined to six(10%) neonates at three months and five(8.3%) neonates at six months.

Neurobehavioral abnormalities were present in four (6.7%) neonates at discharge, with no change observed at three months, and by six months, three (5%) neonates had neurobehavioral abnormalities. Abnormal neonatal reflexes were noted in six (10%) neonates at discharge, in five (8.3%) neonatesat three months and in four(6.7%) neonates at six months.

At discharge, all neonates underwent hearing screening using behavioral methods and/or OAE. Nine(15%) neonates who failed OAE were further evaluated with BERA, which was abnormal in four(6.7%) cases, with the most common finding being a raised threshold of wave V.  For neurodevelopmental assessment, MRI brain was performed in six (10%)neonates with abnormal neurological exams or DQ ≤ 70 at 3 months. Abnormal MRI findings were observed in five (8.3%) cases, with four (6.7%) showing bilateral symmetric high signal areas in the globus pallidus on T2-weighted images and one displaying hyperintense signals in the cerebral white matter.

In our study, most 53(83.3%) neonates received phototherapy, and rest seven(16.7%) had exchange transfusion. Among the 53 patients who received Phototherapy alone, two (3.8%) had an abnormal neurological examination, whereas 51(96.2%) had a normal neurological examination. And among the seven patients who received exchange transfusion, 4 (57.1%) had an abnormal neurological examination, while 3 (42.9%) had a normal neurological examination [Table 2]. The association between treatment type and neurological outcome at 3 months was statistically significant (p<0.05).

Patients with serum bilirubin levels ≤25 mg/dL had a mean motor development quotient of 91.8 ± 10.2, whereas those with >25 mg/dL had a significantly lower mean motor development quotient of 73.6 ± 25.0 (p<0.001), and similarly, the mean mental development quotient was 91.8 ± 9.8 in the lower bilirubin group and 77.5 ± 23.7 in the higher bilirubin group, with a statistically significant difference (p<0.001) [Table 3].

One (2.9%) neonate with 20–25 mg/dL bilirubin level, 5(62.5%) neonates in the >25 mg/dL group, none of the neonates with bilirubin <20 mg/dL had an abnormal neurological examination at 3 months. At 6 months, neurological abnormalities observed in 1(2.9%) neonate in the 20–25 mg/dL group and 4 (50%) in the >25 mg/dL group. No neonates in the <20 mg/dL group had a DASII score ≤70, whereas 1 (2.9%) in the 20–25 mg/dL group and 4 (50%) in the >25 mg/dL group had impaired developmental scores. Abnormal BERA findings were observed in 1 (2.9%) neonate in the 20–25 mg/dL group and 3 (37.5%) in the >25 mg/dL group. 1 (2.9%) in the 20–25 mg/dL group and 4 (50%) in the >25 mg/dL group showed abnormalities in MRI brain. The association of serum bilirubin with neurological, developmental, and auditory outcomes was statistically significant (p<0.05) [Table 4].

Table 1-Demographic and Perinatal Characteristics of the Newborn

Table 2- Association between Treatment Type and Neurological Outcome at 3 Months

Table 3-Comparison of Development Quotient (Motor and Mental) Based on Serum Bilirubin Levels

Table 4- Association of Serum Bilirubin Levels with Neurological, Developmental, and Auditory Outcomes

In our study, the most common neonatal condition observedwas ABO incompatibility in 30%, followed by sepsisin 28.3%, Rh incompatibility in 8.3%, both ABO and Rh incompatibility in 5% of neonates. These findings align with previous research on neonatal jaundice and its varied etiologies. Monika Sharma et al16 reported that 51.04% of hyperbilirubinemia cases were due to hemolysis (ABO, Rh, ABO+Rh incompatibility, and DCT positivity), while 48.96% were non-hemolytic. Among affected neonates, 79.17% required phototherapy, and 20.83% underwent exchange transfusion.

A study from the West Indies attributed jaundice to ABO incompatibility (35%), infection (18%), prematurity (11%), and Rh incompatibility (3.5%), while 9% had an unidentified cause.17 Similarly, Kulkarni et al18, in a study of 120 neonates with serum bilirubin >10 mg/dl, reported idiopathic jaundice in 35%, physiological jaundice in 30%, ABO incompatibility in 15%, septicemia in 8.33%, Rh incompatibility in 6.66%, cephalhematoma in 2.5%, and G6PD deficiency in 0.83%.These studies emphasize the various etiologies of neonatal jaundice, reinforcing the need for comprehensive diagnostic evaluation and timely intervention to prevent complications.

In our study, neonates who underwent exchange transfusion were more likely to show abnormal neurodevelopment compared to those treated with phototherapy. This is consistent with findings by Monika Sharma et al16, who reported similar association of exchange transfusion with neurodevelopment. This indicated that severe hyperbilirubinemia requiring exchange transfusion may provide a higher risk of neurological impairment due to the condition's underlying severity.

Our study found a positive association between serum bilirubin levels and neurodevelopmental outcomes, with higher bilirubin levels correlating with motor and mental deterioration. This aligns with findings by P. Dubey et al19 who reported significantly higher total serum bilirubin (TSB) levels in neonates with delayed neurodevelopment compared to those with normal development. Similarly, Yilmaz Y et al20 observed a strong correlation between elevated bilirubin levels and adverse neurological outcomes. However, studies by Newman et al21 and Croen et al22 suggested reversibility of neurodevelopmental effects after treatment or found no significant long-term impact. This contrasts with our findings, this may be due to difference in inclusion criteria of neonates, or difference in tools for neurodevelopment assessment.

Despite these variations, our findings support that severe hyperbilirubinemia (>25 mg/dL) poses a risk for neurotoxicity, highlighting the importance of early intervention to prevent long-term impairments.

Monika S. et al16 reported that the prevalence of abnormal neurodevelopmental outcomes (DQ ≤70) using the DASII method was 10.42% at three months, decreasing to 6.25% at 12 months, suggesting that some neurodevelopmental impairments due to hyperbilirubinemia may be transient or reversible. Neonates with peak serum bilirubin (PSB) >25 mg/dL had a higher prevalence of neurodevelopmental abnormalities—37.5% at three months and 25% at 12 months—compared to those with PSB <25 mg/dL, where the prevalence was significantly lower (7.95% at three months and 4.54% at 12 months). These findings support our study, reinforcing the association between severe hyperbilirubinemia and neurodevelopmental risk while also indicating the potential for partial recovery over time. Abnormal BERA findings were observed in 1 (2.9%) neonate in the 20–25 mg/dL group and 3 (37.5%) in the >25 mg/dL group. A large number of studies have supported the transient nature of BERA abnormalities in infants with hyperbilirubinemia or bilirubin encephalopathy, which improves after phototherapy and/or exchange transfusion.23-25

Even a moderate concentration of bilirubin considerably impacts developmental outcomes, it is imperative to prioritize early detection and prevention of serum bilirubin elevation before it attains hazardous levels. Antenatal screening for high-risk moms, cord blood bilirubin assessment in high-risk newborns, and the administration of anti-D both antenatally and perinatally are interventions that may avert the onset of substantial hyperbilirubinemia and its consequences at primary and secondary levels.26

Limitations

This study is limited by its small sample size, which may affect the generalizability of the findings. And, the six-month follow-up period may not be sufficient to assess long-term outcomes, particularly neurodevelopmental effects of severe hyperbilirubinemia. A larger sample size and extended follow-up is warranted to assess the long-term impact of neonatal hyperbilirubinemia.

This study demonstrated a significant association between hyperbilirubinemia and neurodevelopmental outcomes in term and late preterm neonates. Findings indicate that neonates with serum bilirubin levels >25 mg/dL are at a higher risk of neurological abnormalities, developmental delays, hearing impairment, and structural brain changes. At both 3 and 6 months, neonates with severe hyperbilirubinemia showed significantly higher rates of abnormal neurological examinations, and those with elevated bilirubin levels had lower DASII scores, indicating motor and mental developmental delays. Hearing assessment revealed an association between hyperbilirubinemia and auditory dysfunction. MRI brain abnormalities were also prevalent in neonates with abnormal neurological findings or low developmental scores, with the most common finding being bilateral symmetric high signal areas in the globus pallidus on T2-weighted images, reinforcing the impact of bilirubin toxicity on brain structures. Additionally, neonates who required exchange transfusion had significantly poorer neurodevelopmental outcomes compared to those managed with phototherapy alone.

These findings underscore the neurotoxic effects of severe hyperbilirubinemia, emphasizing the need for early identification, monitoring, and intervention to prevent long-term neurological and developmental impairments.

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