Risk of hyperbilirubinemia is more in newborn due to increase in formation of bilirubin, which is due to increased destruction of red blood cells, defect in elimination of bilirubin due to deranged uptake by liver, inadequate conjugation due to immaturity of newborn and increase in entero-hepatic circulation. [1,2] Risk of hyperbilirubinemia is more in preterm babies than term babies. Due to decreased Uridine 5'-diphosphoglucuronosyltransferase UGT1A1 enzyme activity which is the enzyme responsible for the conjugation of bilirubin and making it water-soluble for its excretion leads to increased incidence of hyperbilirubinemia in preterm infants [3].

Similar degree of erythrocyte turnover and heme degradation were found in preterm babies as compared to their term counterparts. Preterm, because of their immaturity, do not achieve consistent nutritive breastfeeding because of immaturity of sucking and swallowing reflex which contributes to exaggerated hyperbilirubinemia. [4] A Total Serum Bilirubin (TSB) levels above a well-defined threshold warrants treatment to prevent the development of Kernicterus. Usually, the clinical evaluation of hyperbilirubinemia involves visual estimation of the yellowness of skin which is known as jaundice. [5]

However, quantification of total serum bilirubin (TSB) based on visual assessment of the depth of jaundice is subjective and most of the times inaccurate and confounded by skin color, brightness of examination room and hemoglobin. [6] Newborn jaundice because of severe hyperbilirubinemia and its potential for causing brain damage is an ongoing problem for the pediatrician. [7]

Hour specific total serum bilirubin (TSB) nomogram by Bhutani et al and NICE guideline is used almost everywhere to predict the risk of significant hyperbilirubinemia and also for identifying the need for additional evaluation. [8] Estimation of total serum bilirubin (TSB) concentration requires venous blood sampling which causes parental distress due to pain in preterm newborn. [9] In recent years, to evaluate the severity of neonatal jaundice, newer methods have been developed, which includes transcutaneous bilirubinometry (TcBI). [10]

Earlier studies have already shown that almost 70% of circulating BDNF is found in the brain, thus asserting the brain as a major source of circulating BDNF. Moreover, the hippocampus and cortex regions of brain are the major source of plasma BDNF. [11] A positive correlation between BDNF concentrations in the brain and serum has also been observed, since BDNF can cross the blood-brain barrier. Some studies have reported that physical activity, which is useful for the brain and insulin sensitivity can also elevate the plasma and serum BDNF concentrations. [12]

This study was a prospective and observational study was conducted in the Neonatal Intensive Care Unit (NICU) and postnatal ward and Department of Biochemistry at Index Medical College and Hospital over a period of 2 year. The study aimed to measure serum Brain-Derived Neurotrophic Factor (BDNF) levels in preterm and full-term neonates and assess its association with neonatal hyperbilirubinemia.

Study Population

Neonates admitted to the NICU or postnatal ward were enrolled based on the following inclusion and exclusion criteria:

Inclusion Criteria:

• Neonates born at gestational age:
• Preterm: <37 weeks
• Full-term: ≥37 weeks
• Birth weight recorded within the first hour of life.
• Neonates with or without hyperbilirubinemia.
• Parental consent obtained for participation in the study.

Exclusion Criteria:

• Neonates with congenital anomalies or syndromic features.
• Neonates with perinatal asphyxia (Apgar score <5 at 5 minutes).
• Neonates with sepsis (clinically suspected or culture-proven).
• Neonates with maternal history of chorioamnionitis, diabetes mellitus, or preeclampsia.
• Neonates who received prior exchange transfusion or phototherapy before blood sampling.

Sample Collection and Processing

• Timing of Blood Sample Collection: Blood samples (2–3 mL) were collected from neonates within the first 24–48 hours of life via venipuncture under aseptic conditions.
• Serum Separation: Blood samples were centrifuged at 3000 rpm for 10 minutes at 4°C to separate serum.
• Storage: Serum was stored at -80°C until further analysis.

Serum BDNF Quantification

• Sample Collection: Blood (2–3 mL) was collected within the first 24–48 hours of life, prior to any intervention, into EDTA tubes.
• Processing: Serum was separated by centrifugation at 3000 rpm for 10 minutes and stored at -80°C until further analysis.
• BDNF Measurement:

Serum BDNF concentrations were quantified using an enzyme-linked immunosorbent assay (ELISA): The assay was performed according to the manufacturer’s protocol. Samples were analyzed in duplicate, and absorbance readings were measured using a microplate reader at 450 nm. The intra-assay and inter-assay coefficients of variation (CV) were maintained at <10%.

Statistical Analysis

Continuous variables were expressed as mean ± standard deviation (SD) or median (interquartile range) depending on data distribution. Categorical variables were presented as frequencies and percentages. Independent t-test or Mann-Whitney U test for comparing serum BDNF levels between preterm and full-term neonates. Pearson/Spearman correlation analysis to assess the association between serum BDNF levels and total serum bilirubin. Multivariate Analysis: Linear regression was used to adjust for potential confounding factors such as gestational age, birth weight, and sex. A p-value < 0.05 was considered statistically significant.

Table 1: Demographic and Clinical Characteristics of the Study Population

• Preterm Neonates: The average gestational age for preterm neonates is 30.2 weeks, with a standard deviation of 3.4 weeks.
• Full-term Neonates: The average gestational age for full-term neonates is 39.1 weeks, with a standard deviation of 1.1 weeks.
• Interpretation: There is a clear difference between preterm and full-term neonates, as expected. Preterm neonates have significantly lower gestational age, which aligns with the definition of preterm birth (birth before 37 weeks of gestation). Full-term neonates, on the other hand, fall within the typical range for human birth, usually between 37 and 42 weeks.

Table 2: Serum BDNF and Bilirubin Levels in Preterm and Full-term Neonates

• Preterm Neonates: The average serum BDNF level is 12.3 ng/mL, with a standard deviation of 3.5 ng/mL.
• Full-term Neonates: The average serum BDNF level is 22.1 ng/mL, with a standard deviation of 5.2 ng/mL.
• Interpretation: Full-term neonates exhibit significantly higher serum BDNF levels compared to preterm neonates. BDNF is a protein that plays an essential role in the development and function of the nervous system, and lower BDNF levels in preterm neonates could reflect neurological immaturity. The higher BDNF levels in full-term neonates may be associated with more advanced neurodevelopment due to their longer gestational periods.

Table 3: Distribution of Neonates Based on Serum Bilirubin Levels

A larger proportion of preterm neonates (43.5%) fall in the 10-15 mg/dL range, indicating that 43.5% of preterm neonates have moderate levels of bilirubin. A smaller proportion of preterm neonates (26.1%) have bilirubin levels greater than 15 mg/dL, which suggests that hyperbilirubinemia (high bilirubin levels) is less severe in preterm infants compared to full-term infants. However, a substantial number of preterm neonates (30.4%) still have bilirubin levels lower than 10 mg/dL, which could be an indication that their bilirubin levels are either well-managed or naturally lower due to liver function maturation during the later stages of pregnancy.

Table 4: Preterm Neonates with Hyperbilirubinemia: Serum BDNF Levels vs Bilirubin Levels

The average serum BDNF level in neonates with bilirubin levels < 10 mg/dL is 14.5 ± 3.1 ng/mL. For neonates with bilirubin levels between 10-15 mg/dL, the serum BDNF level is 12.2 ± 3.6 ng/mL. In neonates with bilirubin levels greater than 15 mg/dL, the serum BDNF level is significantly lower at 10.1 ± 2.9 ng/mL

Table 5: Full-term Neonates with Hyperbilirubinemia: Serum BDNF Levels vs Bilirubin Levels

The current study investigated and compared the demographic and clinical characteristics, serum Brain-Derived Neurotrophic Factor (BDNF) levels, and total serum bilirubin concentrations in preterm and full-term neonates. The observed differences in these parameters not only reflect the biological distinctions between these two groups but also shed light on potential neurodevelopmental implications and the pathological processes associated with neonatal hyperbilirubinemia.

Serum BDNF levels were markedly lower in preterm neonates (12.3 ± 3.5 ng/mL) compared to full-term neonates (22.1 ± 5.2 ng/mL), a difference that was statistically significant (p = 0.001). BDNF is a critical neurotrophin involved in neuronal growth, differentiation, and synaptic plasticity. Several previous studies have underscored the importance of BDNF in early brain development. For instance, Pillai et al. (2010) [13] emphasized that lower BDNF levels in neonates might predict future cognitive impairments or psychiatric disorders. Furthermore, preclinical models have shown that BDNF insufficiency in early development can lead to long-term structural and functional brain deficits (Lu et al., 2005). [14]

Given that the brain undergoes rapid growth and differentiation during the third trimester, preterm birth may interrupt this process, contributing to the lower BDNF levels observed. This supports earlier findings by Nelson et al. (2014), [15] who reported that early birth significantly disrupts neurotrophic signaling pathways, potentially contributing to neurodevelopmental disabilities often seen in preterm infants.

Preterm neonates demonstrated higher total serum bilirubin levels (15.4 ± 4.3 mg/dL) compared to full-term neonates (9.6 ± 2.5 mg/dL), and 69.6% of preterm neonates had hyperbilirubinemia compared to 34.8% of full-term neonates. These results are in agreement with previous studies which have shown that preterm infants are at increased risk for jaundice due to immature hepatic enzyme systems and increased red blood cell turnover (Maisels & Watchko, 2003). [16]

Excessive unconjugated bilirubin in neonates can cross the blood-brain barrier and deposit in brain tissues, especially in the basal ganglia and brainstem nuclei, leading to bilirubin-induced neurologic dysfunction (BIND) or kernicterus. In preterm infants, the threshold for bilirubin-induced neurotoxicity is lower due to their more permeable blood-brain barrier and underdeveloped protective mechanisms (Bhutani et al., 2013). [17]

The interplay between hyperbilirubinemia and reduced BDNF levels is of critical clinical relevance. It provides a potential mechanistic explanation for the neurodevelopmental delays and neurological complications often reported in neonates with severe or prolonged jaundice. This adds a new layer to neonatal care: not only should hyperbilirubinemia be treated to prevent acute bilirubin encephalopathy, but clinicians should also be aware of its potential chronic effects on brain development mediated through neurotrophic pathways.

This study highlights a significant difference in serum BDNF and bilirubin levels between preterm and full-term neonates and establishes a negative correlation between bilirubin and BDNF. These findings support previous literature that preterm neonates are more vulnerable to hyperbilirubinemia and its potential neurotoxic effects. Importantly, they suggest a novel association between bilirubin and BDNF levels, opening up new avenues for research into neonatal brain health and emphasizing the importance of early and aggressive management of jaundice in this population.

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