Neonatal hyperbilirubinemia or Neonatal jaundice is a common problem encountered in the newborn period. Nearly 85% of all the term and most of the preterm neonates become visibly jaundiced in the first week of their life. As per the Indian scenario, almost 60% of term and 80% of preterm neonates develop neonatal jaundice or hyperbilirubinemia. [1] The word jaundice was taken from the French word which means yellow. Similarly, the word hyperbilirubinemia was derived from term, taken from Greek literature. [2]

These infants, who develop kernicterus and an increased expert concern that -Induced Neurological Dysfunction (BIND) has altered our perception. [3] Estimation of bilirubin levels are vital for the effective treatment of neonatal jaundice, which is usually done clinically by visual assessment or by cutaneous, and serum evaluations. [4] Although clinical or visual assessment is simple, it has two major shortcomings: firstly, it is dependent on the physician's experience with no accurate and reliable criteria; secondly, possible estimations in this process is based on the cephalocaudal trend of jaundice. Moreover, lighting of the room, color of the skin and clothes affect this visual estimation. [5]

The biochemical analysis of blood for estimation of total serum bilirubin (TSB) involve invasive extraction of venous blood from premature neonates, making the frequent use of painful stimuli which is a significant issue marked in neonates over the past decade. Position statement by the American Academy of Pediatrics recognizes this epidemic and calls for other purposeful methods to minimize this painful stimulus among neonates. The prevention of this pain can reduce the risk of long-term adverse sequelae in preterm neonates related to repetitive painful procedures. [6]

TcB is considered to be an easy method as it is non-invasive and minimizes blood loss of an infant through more invasive venous blood assays. The first clinically appropriate and portable transcutaneous bilirubinometer was introduced by Yamanouchi and associates. [7] They worked with the Minolta Camera Company and developed a hand-held instrument which was used to measure the yellow color intensity in the skin. Transcutaneous bilirubinometery (TcBI) a non-invasive, safe, simple, quick, cost-effective method was used to evaluate transcutaneous bilirubin (TcB) to find out the correlation of TcB with total serum bilirubin (TSB) levels in preterm babies. [8]

Brain-Derived Neurotrophic Factor (BDNF) is a neurotrophic, immunotrophic, epitheliotrophic and metabotrophic factors, that shows its high expression in the central and peripheral nervous system, influencing the dopaminergic, serotonergic, glutamatergic, cholinergic and noradrenergic signaling pathways. [9] BDNF performs the function of proliferation, differentiation, activity dependent plasticity and survival of neurons in CNS. BDNF influences the paraventricular, arcuate and ventromedial nuclei brain regions for regulating the feeding mechanisms, energy expenditure and homeostasis. [10]

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.

Clinical Data Collection

Demographic and clinical details were recorded, including:

• Maternal history: Age, parity, mode of delivery, history of gestational diabetes, hypertension.
• Neonatal characteristics: Gestational age, birth weight, Apgar scores, sex, and mode of feeding.
• Laboratory parameters: Hemoglobin, hematocrit, reticulocyte count, and serum bilirubin levels.

Demographic and Clinical Data Collection

Neonatal and maternal data were recorded at the time of enrollment, including:

• Neonatal Parameters:
  • Gestational age (weeks), birth weight (grams), sex, Apgar scores at 1 and 5 minutes, mode of delivery (vaginal vs. cesarean section), and feeding method (breastfeeding vs. formula).
  • Presence of comorbidities such as respiratory distress syndrome (RDS), sepsis, or necrotizing enterocolitis (NEC).
  • Jaundice onset (time of appearance) and severity.
• Maternal Parameters:
  • Maternal age, parity, medical history (e.g., diabetes, hypertension), history of gestational diabetes or pre-eclampsia, and antenatal care received.

2. 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

Table 2: Serum BDNF Levels in Preterm vs. Full-term Neonates (Group Comparison)

The serum BDNF level is significantly lower in preterm neonates compared to full-term neonates. The p-value of 0.001 indicates strong statistical evidence that this difference is not due to chance. Since BDNF is crucial for brain development, the lower levels observed in preterm neonates suggest delayed neurodevelopment or neurological immaturity in these infants. Preterm infants are born before the brain has fully developed, which likely contributes to lower BDNF levels.

Table 3: Serum Bilirubin Levels in Preterm vs Full-term Neonates (Group Comparison)

The total serum bilirubin level is significantly higher in preterm neonates compared to full-term neonates. The p-value of 0.001 indicates strong statistical evidence that this difference is not due to random chance, and the difference is highly significant. Bilirubin is a byproduct of the breakdown of red blood cells, and its processing is primarily carried out by the liver. Preterm neonates often have immature liver function, which leads to an inability to efficiently metabolize and eliminate bilirubin from their system. This results in higher bilirubin levels, which can lead to jaundice. Full-term neonates, on the other hand, have more mature liver function, which allows them to process and clear bilirubin more efficiently, resulting in lower serum bilirubin levels.

Table 4: Correlation between Serum BDNF Levels and Total Serum Bilirubin Levels in Preterm Neonates

Negative Correlation (r = -0.45): The correlation coefficient of -0.45 indicates a moderate negative correlation between serum BDNF levels and total serum bilirubin levels in preterm neonates. This suggests that as total serum bilirubin increases, serum BDNF levels tend to decrease, and vice versa. Statistical Significance (p = 0.02): The p-value of 0.02 indicates that the observed negative correlation is statistically significant, meaning that the relationship between BDNF and bilirubin levels in preterm neonates is unlikely to have occurred by chance.

Table 5: Correlation Between Serum BDNF Levels and Total Serum Bilirubin Levels in Full-term Neonates

Negative Correlation (r = -0.30): The correlation coefficient of -0.30 indicates a weak to moderate negative correlation between serum BDNF levels and total serum bilirubin levels in full-term neonates. This suggests that as total serum bilirubin increases, serum BDNF levels tend to decrease, but the relationship is weaker than in preterm neonates.

Table 6: Distribution of Hyperbilirubinemia in Preterm and Full-term Neonates

A significant proportion of preterm neonates (69.6%) were diagnosed with hyperbilirubinemia, which is more common in preterm infants due to their immature liver function and lower ability to process and excrete bilirubin effectively. Only 30.4% of preterm neonates did not experience hyperbilirubinemia, indicating that the majority of preterm infants in this cohort required some form of intervention, such as phototherapy.

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.

Apgar scores at both 1 and 5 minutes were significantly lower in preterm neonates, indicating reduced immediate postnatal health status and adaptation to extrauterine life. These scores improved by 5 minutes, suggesting that early resuscitation or adaptation mechanisms are often effective, though less optimal in preterm infants. Consistent with studies by Ehrenstein (2009), lower Apgar scores in preterm infants often reflect respiratory distress and immature cardiovascular adaptation. [11]

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). [12]

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). [13]

A moderate negative correlation (r = -0.45, p = 0.02) was observed between BDNF and total bilirubin levels in preterm neonates, and a weaker but significant negative correlation (r = -0.30, p = 0.04) was found in full-term neonates. This implies that higher bilirubin levels are associated with lower BDNF levels in both groups. These findings are novel and highly relevant. They suggest that hyperbilirubinemia may negatively impact neurodevelopment not only through direct neurotoxicity but potentially through suppression of neurotrophic factors such as BDNF.

Earlier animal studies have indicated that hyperbilirubinemia can reduce BDNF expression in brain tissues (Chen et al., 2011), and our findings support the possibility of a similar effect in human neonates. [14] It is conceivable that elevated bilirubin levels downregulate BDNF synthesis or its availability, thereby compounding the risk of impaired neurodevelopment in affected infants.

Stratified analysis showed that preterm neonates with bilirubin levels > 15 mg/dL had significantly lower BDNF (10.1 ± 2.9 ng/mL) compared to those with levels < 10 mg/dL (14.5 ± 3.1 ng/mL; p = 0.001). A similar trend was seen in full-term neonates. This gradient further reinforces the dose-dependent detrimental association of bilirubin on BDNF concentration. These results underscore the clinical importance of timely management of hyperbilirubinemia to possibly preserve neurotrophic support in neonates.

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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