Unconjugated Hyperbilirubinaemia and Its Effect on Neonatal Distortion Product Otoacoustic Emission and Auditory Brainstem Response- An Observational Study

Unconjugated Hyperbilirubinaemia and Its Effect on Neonatal Distortion Product Otoacoustic Emission and Auditory Brainstem Response- An Observational Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unconjugated Hyperbilirubinaemia and Its Effect on Neonatal Distortion Product Otoacoustic Emission and Auditory Brainstem Response- An Observational Study SAKSHAM DHAWAN, Vijendra Shenoy S, Neehal Zuturu, ANANYA SINHA, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7473326/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract AIM To evaluate the association between neonatal hyperbilirubinemia, particularly elevated unconjugated bilirubin, and early hearing dysfunction using Distortion Product Otoacoustic Emissions (DPOAE) and Auditory Brainstem Response (ABR). MATERIALS AND METHODS A prospective study of 2-year duration at a tertiary care centre was performed wherein hyperbilirubinaemic neonates were cases and controls had normal bilirubin levels. Groups 1 and 2 had cases with total bilirubin levels below and above the average (5.17mg/dl) while Groups 3 and 4 included cases with unconjugated bilirubin levels below and above the average (4.18 mg/dl) respectively. All neonates underwent DPOAE on day 3 of life. ABR and repeat DPOAE were done on 30 th day of life. Neonates with abnormal ABR were followed up at 3 rd and 12 th month, with repeat ABR. RESULTS 30 th day ABR responses between Group 3 and Group 4 showed statistical significance for the right and left ear (p = 0.037, 0.007), respectively. Repeat DPOAE on day 30 was significant between preterm and term neonates (p = 0.047). There was no association noted between hyperbilirubinaemia and birthweight or gestational age. DPOAE responses showed no statistical significance between cases and controls or any specific DPOAE frequency. All cases of hearing loss were transient and resolved within the first year of life. CONCLUSION Unconjugated hyperbilirubinemia poses a notable risk for early auditory dysfunction, even in the absence of kernicterus. DPOAE and serial ABR are valuable tools for early detection and monitoring. Neonates with elevated unconjugated bilirubin should be prioritized for repeated audiological assessments. Early identification and timely intervention can prevent long-term speech and language delays and improve developmental outcomes. Hyperbilirubinaemia Neonate Hearing loss ABR DPOAE Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION One child out of every thousand is born profoundly deaf, while four times as many are born with bilateral hearing loss, either moderate or severe. Congenital hearing loss is more common in newborns than the total prevalence of all metabolic disorders that are now detected through blood tests ( 1 ). Infants in Neonatal Intensive Care Units are 10–20 times more likely to have substantial hearing loss as compared to the general population. Hyperbilirubinemia affects a substantial number of newborns, up to 84% of term and late preterm infants in the first week of life ( 2 ). While neonatal jaundice is often benign, high levels of unconjugated bilirubin can lead to kernicterus and irreversible neurological damage. Bilirubin induced neurological damage leading to impairment of the hearing mechanism is one of the prime concerns in these hyperbilirubinaemic infants. Hearing loss is among the earliest and most subtle manifestations of bilirubin neurotoxicity ( 2 ). The auditory system, particularly the brainstem and cochlear nuclei, is particularly sensitive to excessive bilirubin levels. The major cause of damage is when lipid-soluble unconjugated bilirubin (UB) breaches the blood brain barrier (BBB). Organic anion efflux cannot remove the UB, which then builds up in the cytoplasm and is ultimately fatal to the cells. Presence of adequate and correct auditory stimuli in the first year of life is necessary not only for the audiological, but also for the overall development of the child. As hearing outcomes in newborns can be bettered with prompt surveillance and identification, and proper rehabilitation, the National Institute of Health advised universal neonatal hearing testing by three months of age, using Otoacoustic Emissions (OAE) in 1993. Current guidelines in various countries recommend universal neonatal hearing screening using OAE and confirmatory testing with Auditory Brainstem Response (ABR). With early detection and intervention, infants with auditory impairment can now achieve near-normal language and cognitive milestones. This study aims to evaluate the association between neonatal hyperbilirubinemia, particularly elevated unconjugated bilirubin, and early hearing dysfunction using DPOAE and ABR. Furthermore, the study also aims to determine the prognostic utility of serial ABR assessments over the first year of life. 2. METHODS OF DATA COLLECTION This was a prospective study conducted over a period of 2 years at a tertiary care Neonatal Intensive Care Unit (NICU). After obtaining consent from the family, all live births requiring NICU admission (n = 106), both preterm (< 37 weeks period of gestation) and term (37 to 42 weeks period of gestation), with and without hyperbilirubinemia were included into the study. Neonates with a family history of sensorineural hearing loss, in-utero TORCH infection, craniofacial abnormalities and anomalies incompatible with life, APGAR score < 5 in 1st minute or 5 days were excluded from the study. Neonates for whom the consent of the family could not be obtained were excluded from the study (n = 12) as well as those which were lost to follow up were excluded from the study (n = 10). Neonates with hyperbilirubinaemia were deemed as cases (n = 44) and the ones with normal bilirubin were deemed as controls (n = 40). The case group was further subdivided into subgroups. Group 1 (n = 12) and Group 2 (n = 32) included the cases with total bilirubin levels below and above the average value of total bilirubin in all the cases, respectively. Similarly, Group 3 (n = 16) and Group 4 (n = 28) included the cases with unconjugated bilirubin levels below and above the average value of unconjugated bilirubin in all the cases, respectively. After clinical examination, all neonates underwent istortion Product Otoacoustic Emissions (DPOAE) examination on the 3rd day of life under universal neonatal screening for hearing loss. A repeat DPOAE on the 30th day of life was performed to rule out any temporary causes of conductive hearing loss and to substantiate whether hyperbilirubinaemia, particularly elevated unconjugated bilirubin, is a cause for development of hearing impairment in neonates. This was followed by evaluation using ABR. The ones with abnormal ABR findings were followed up at 3 months and an ABR was repeated. Infants with abnormal ABR at 3 months were re-evaluated at 12 months, by ABR, to check whether the abnormalities in ABR due to hyperbilirubinemia were transient or permanent. 3. RESULTS 3.1 Demographics This study evaluated the association between neonatal hyperbilirubinemia and auditory function across 84 neonates, 44 with hyperbilirubinemia (cases) and 40 without (controls). Both term and preterm infants were included. Birth weight and gestational age were comparable between groups. There was no significant correlation between serum bilirubin levels and birth weight. Although males are biologically more prone to neonatal jaundice ( 3 ), our study did not show any significant difference in gender-based distribution between the groups. 3.2 OAE Comparisons 3.2.1 First screening : In the initial DPOAE screening, a considerable number of absent responses were observed in both cases and controls. Among the 88 ears in the case group, 41 ears (46.6%) showed absent DPOAE responses, compared to 26 ears (32.5%) out of the 80 in the control group. Results across various frequencies were not statistically significant (p = 0.062) (FIGURE 1 ) . There was no significant difference in DPOAE responses between preterm cases and preterm controls. The majority of absent DPOAE readings occurred in the lower frequencies, namely 1kHz and 2kHz, though no statistically significant differences were noted for any specific frequency. Across both low and high frequencies, a higher number of ears in the case group demonstrated absent readings as compared to the controls, although this too lacked statistical significance. While higher bilirubin levels tended to be associated with more absent DPOAE responses, this did not show statistical significance. Similarly, there was no significant association noted between the absence of different DPOAE frequencies in the ears of preterm and term neonates either. However, both preterm and term neonates exhibited more absent DPOAE responses in lower frequencies (1kHz and 2kHz). 3.2.2 Comparison of OAE responses within two subgroups of cases based on total bilirubin levels The mean total bilirubin value in the case group was found to be 5.17 mg/dl. Out of the total 88 ears in the cases, 24 ears belonged to neonates with total bilirubin values below the average of the cases group (Group 1) and 64 ears belonged to those with their total bilirubin values above the average (Group 2). Higher bilirubin levels trended toward more absent DPOAE responses, i.e., absent responses were found in 8 ears (33.3%) in Group 1 and 33 ears (52.6%) in Group 2 ( FIGURE 2 ) . This difference was found to be statistically insignificant (p = 0.127). However, a significant association was found between left ear DPOAE absence and hyperbilirubinemia (p = 0.018), suggesting a possible asymmetric vulnerability. 3.2.3 Comparison of OAE responses within two subgroups of cases based on unconjugated bilirubin levels. The mean unconjugated bilirubin level in the case group was found to be 4.18mg/dl. Out of the 88 ears, there were 32 ears (Group 3) for which the unconjugated bilirubin values were below the average of the cases group and 56 ears (Group 4) with their unconjugated bilirubin values above the average of the total cases. Absent DPOAE responses were found in 10 ears (31.2%) of Group 3, compared to 31 ears (55.4%) in Group 4 ( FIGURE 3 ). This difference was statistically significant (p = 0.029), suggesting a stronger relationship between elevated unconjugated bilirubin and DPOAE absence. This finding strengthens the hypothesis that elevated unconjugated bilirubin, more than total bilirubin, has a stronger predictive value for early cochlear dysfunction as captured by DPOAE. 3.2.4 Repeat DPOAE analysis DPOAE testing was repeated on the 30th day of life for all neonates with initially absent responses. Among the case group, 22 out of 41 ears (53.6%) continued to show absent responses. In the control group, 10 out of 26 ears (38.4%) remained absent (FIGURE 4) . Numerically, fewer neonates in both the case and the control group showed an absent DPOAE response on the repeat DPOAE as compared to the initial screening ( Table 1 ) . These results highlight a reduction in absent responses in both groups over time, suggesting some degree of reversibility in the auditory effects of bilirubin. Table 1 SHOWING THE PROGRESSIVE DECREASE IN THE NUMBER OF NEONATES WITH ABSENT DPOAE RESPONSES NUMBER OF EARS WITH ABSENT RESPONSE ON 1 ST DPOAE (3 RD DAY OF LIFE) NUMBER OF EARS WITH ABSENT DPOAE RESPONSE ON REPEAT DPOAE (30 TH DAY OF LIFE) CASES CONTROLS CASES CONTROLS 41 22 26 10 3.3 ABR Comparisons: On the 30th day of life, ABR testing was performed on both the cases and the controls (Table 2 ). Although a higher number of neonates in the case group showed absent responses to ABR than those in the control group, the association between the ABR responses of both was found to be statistically insignificant (p = 0.090). Table 2 DEPICTING THE NUMBER AND PERCENTAGES OF THE ABSENT AND PRESENT ABR RESPONSES THAT WERE NOTED AMONGST CASES(n = 44) AND CONTROLS (n = 40) Case (88 ears) Controls (80 ears) ᵡ 2 P Value ABR, n (%) 2.873 0.090 Absent 17(19.3%) 08(10.0%) Present 71(80.7%) 72(90.0%) ABR results were compared based on bilirubin levels in all 4 groups. In Group 1, none of the 24 ears had absent ABR response. In contrast, 17 out of 64 ears in Group 2 showed absent ABR response. This was found to be statistically significant (p = 0.005) (Table 3 ). The stronger relationship emerged when comparing groups based on unconjugated bilirubin. None of the 32 ears in Group 3 had absent ABR responses. However, in Group 4, 17 out of the 56 ears exhibited absent ABR responses (Table 4 ). This was highly statistically significant (p = 0.001), underlining the neurotoxic potential of elevated unconjugated bilirubin over total bilirubin. Table 3 NUMBER OF NEONATES IN GROUP 1 AND GROUP 2 OF CASES SHOWING ABSENT ABR RESPONSES. ABR ON THE BASIS OF TOTAL BILIRUBIN LEVELS P value GROUP 1 GROUP 2 0.005 ABSENT 0 17(26.6) PRESENT 24(100) 47(73.4) Table 4 NUMBER OF NEONATES IN GROUP 3 AND GROUP 4 OF CASES SHOWING ABSENT ABR RESPONSES ABR ON THE BASIS OF UNCONJUGATED BILIRUBIN LEVELS P Value BELOW AVERAGE; n(%) ABOVE AVERAGE; n(%) 0.001 ABSENT 0 17(30.4%) PRESENT 32(100%) 39(69.6%) Repeat ABRs were done at 3 months for both the cases and controls who previously showed absent ABR responses during the first screening at 30 days. Only one case and one control had persistently absent ABR waveforms, both of which became normal at the 12-month evaluation, indicating recovery. 4. DISCUSSION One of the most frequent problems concerning newborns is hyperbilirubinaemia. Even though majority of the infants with jaundice are healthy, excessive serum levels of bilirubin, especially unconjugated bilirubin, can induce kernicterus which can lead to sensorineural hearing loss. The major cause of damage is UB. Being lipid-soluble, it breaches the cell membranes and the BBB. Organic anion efflux cannot remove the UB, which then accumulates in the cytoplasm of neural cells, leading to cytotoxicity. This build-up is fatal to the cells. Early detection and evaluation of jaundice are crucial to avoid the consequences such as bilirubin encephalopathy, which can cause hearing loss. OAE and ABR are routine and reliable non-invasive screening tools that can be employed to achieve such early detection to enable timely intervention ( 4 ). 4.1 HYPERBILIRUBINAEMIA AND HEARING LOSS Several studies reinforce the link between high bilirubin levels and auditory dysfunction. For instance, Boskabadi et al. evaluated risk factors for SNHL in 200 icteric newborns and found abnormal ABRs in 4.8% of them. They also stated that with regard to infant hearing health, total bilirubin level had the highest prediction and in infants with severe jaundice, hearing impairment develops about 10–50 times more commonly ( 5 ). Similarly, Ahmed et al. reported hearing impairment in 13 out of 234 neonates with hyperbilirubinemia, linking hyperbilirubinemia and hearing loss in newborns ( 6 ). As per Chavan et al’s study, hyperbilirubinemia was found to be a significant factor (p = 0.01) for REFER responses in OAE in infants with normal bilirubin levels and those with hyperbilirubinaemia ( 7 ). Our study found that higher unconjugated bilirubin levels significantly predicted absent ABR responses. This supports the hypothesis that unconjugated bilirubin at high levels is particularly neurotoxic, affecting auditory pathways by delaying neural transmission, leading to delayed or absent ABR responses. Specifically, 17 ears (19.3%) out 88 in the case group (hyperbilirubinaemic neonates) showed absent ABRs, compared to 8 out of 80 ears (10%) in the control group (non-hyperbilirubinaemic neonates). This corroborates that the presence of hyperbilirubinaemia may be a causative factor for hearing loss, although it was statistically insignificant. While higher total bilirubin levels showed trends of association with increased absence of DPOAE responses (statistically insignificant), the stronger and statistically significant associations were consistently observed with elevated unconjugated bilirubin. Its dose-dependent ability to interfere directly with cochlear structures may explain its clearer impact on ABR outcomes. Even moderate elevations may disrupt outer hair cell function and neural transmission, detectable through frequency-specific DPOAE testing and ABR. Surprisingly, left ear vulnerability was observed in our study (p = 0.018) possibly suggesting asymmetry in early subclinical cochlear damage that precedes measurable hearing loss in neonates. However, the mechanisms remain speculative and warrant further neurophysiological exploration. In addition, the higher rate of absent responses at low frequencies (1kHz, 2kHz) across groups, though not statistically significant, also emerged as a curious trend, needing further research into frequency-specific cochlear sensitivity to bilirubin toxicity. 4.2 GESTATIONAL AGE AND HYPERBILIRUBINAEMIA Studies have linked prematurity with auditory dysfunction. According to a study by Gulati et al., diagnostic Brainstem Evoked Response Audiometry (BERA) in conjunction with OAE should be performed in all high-risk newborns who are preterm neonates (</= 34 weeks) in order to detect cases of auditory neuropathy spectrum disorders ( 8 ). Jiang et al. found BERA abnormalities in late-preterm neonates, indicating brainstem auditory pathway impairment, suggesting that perinatal complications have a negative impact on the late preterm auditory brainstem when compared to low-risk late preterm newborns ( 9 ). Previous studies, such as those by Gulati et al. and Jiang et al. emphasize the vulnerability of preterm neonates to auditory damage. However, our study did not find a statistically significant correlation between gestational age and auditory dysfunction. There were 7 neonates who were </= 34 weeks preterm out of which 5 were cases and 2 were controls. Of the 5 cases, 4 (80%) had absent DPOAE responses initially and 3 of them continued to have absent responses at day 30. Out of the 3 cases with absent DPOAE, 2 had an absent ABR at 30 days. 1 of the case continued to have an absent ABR which was repeated at 3 months of age, which later showed normal response to repeat ABR at 12 months. Out of the 2 controls, both had absent DPOAE at the first assessment and on repeat DPOAE on day 30, 1 control had normal DPOAE and 1 had absent DPOAE, in which the latter had absent ABR at 30 days but a normal ABR at 3 months of age. Despite these findings, statistical significance was not reached linking prematurity alone to auditory loss as preterm neonates did not show significantly different DPOAE results compared to term neonates, implying that bilirubin levels may be a more critical factor than gestational age when other variables like birth weight are controlled. Our findings are also consistent with Chavan et al. (p = 0.16)( 7 ) and Ahmed Kaenat et al. ( 6 ), where gestational age had no statistically significant influence on initial DPOAE outcomes. These findings suggest that bilirubin toxicity by elevated unconjugated bilirubin may exert a more central influence on auditory function than gestational age alone. 4.3 REVERSIBILITY OR PERMANENCE OF THE HEARING LOSS INDUCED BY BILIRUBIN Emerging evidence suggests bilirubin-induced auditory dysfunction can be transient and hence bilirubin-induced hearing loss may often be reversible. With the administration of albumin infusions, there is a chance that bilirubin-induced neurological damage can be reversed, and there is emerging proof that ABR abnormalities can be corrected in animal models ( 10 – 13 ). Furthermore, certain minor ABR abnormalities in babies can be treated with phototherapy and exchange transfusions. For example, Nakamura et al. observed that delayed ABR wave latencies (peaks I and V) normalized after exchange transfusion in 56 neonates with TB ≥ 15 mg/dL (compared to 24 infants with normal TB levels) ( 14 ). Similarly, our study found that only 2 out of 84 neonates had persistent abnormal ABR responses at 3 months. They were monitored further in our study and at the end of the 12 months follow up, there were no neonates in either the case or the control group who had abnormal responses on ABR, signifying that transient nature of bilirubin-induced auditory dysfunction may not translate into broader neurodevelopmental impairment. This indicates that the auditory dysfunction linked to unconjugated bilirubin may be transient, especially with early intervention. Maturation of neural pathways due to the neonates developing neuroplasticity may also mitigate the transient hearing loss caused due to hyperbilirubinaemia. Our results align with findings from Abdollahi et al. who reported that hyperbilirubinaemic neonates with initial ABR abnormalities often improved by the second evaluation and hence such neonates should have their hearing tested again after receiving therapy before any aggressive hearing interventions are made. In that study, 7 infants who had ABR threshold values between 35 and 65 dBnHL at the first examination displayed a threshold of less than 30 dBnHL during the second evaluation. However, infants who had an ABR threshold of 70 dBnHL at the first evaluation still had the same threshold during the subsequent evaluation ( 15 ). This was also noted in a study conducted by Nam et al. where ABR abnormalities resolved in a subset of affected neonates. 13 out of these 30 preterm infants recovered completely while the remaining 17 had either worse responses on ABR or had not changed. Out of these 17, 2 recovered to normal hearing as shown by the follow up ABRs ( 16 ). Similarly, Ahmed et al. ( 6 ) reported significant improvement in hearing over time using repeat OAE and BERA assessments. They found that among 57 (out of 234) enrolled neonates referred after initial OAE, 38 passed on re-evaluation at 3 months. With a p value of 0.0001, it was determined that the improvement in hearing impairment seen on the OAE after 3 months was significant. In the same study, BERA done for 51 subjects at the 3rd month showed normal findings in 38 (75.64%) neonates and abnormal findings in 13 (25.5%) neonates and both these groups had the same finding on repeat BERA at 6 months. This was found to be dissimilar to our study, as at the end of 3 months, repeat ABR of only 2 out of the 15 neonates (who had initial abnormal ABR results) was found to be abnormal. Even for these 2 neonates, ABR results were found to be normal at 12 months. Additionally, 10–15% of ears in controls showed absent ABR at day 30 in our study. Contribution by other perinatal factors like hypoxia, medications, or delivery mode need to be studied to see if they are confounding factors. In our study, DPOAE referrals dropped from 46.6% to 32.5% in cases and from 25% to 12.5% in controls after 30 days. This finding underscores the importance of repeated testing and continuous monitoring (Table 1 ). The fact that none of the neonates in our study showed persistent ABR abnormalities at 12 months reinforces the possibility of spontaneous recovery. Serial testing remains critical for differentiating transient and permanent damage, aiding timely intervention and also preventing unnecessary treatments in neonates whose hearing normalizes over time. 4.4. IMPLICATIONS IN PUBLIC HEALTH DPOAE and ABR serve as valuable, non-invasive tools in detecting early auditory dysfunction in neonates and distinguishing reversible dysfunction from permanent damage. Together, they offer a reliable framework for monitoring auditory risk in jaundiced neonates. These tests minimize neonatal stress and do not require sedation, while providing reliable results, making them ideal for widespread screening, even in resource-limited settings. Our study supports public health initiatives for universal newborn hearing screening using these accessible methods. Given the neurotoxic risk of elevated unconjugated bilirubin, universal screening for jaundiced neonates becomes even more important. Early identification enables timely interventions, reducing the risk of permanent damage. In addition, repeat testing should be the standard protocol, as early ABR failures may not reflect permanent damage. Our study’s 12-month follow-up showed complete recovery in all affected neonates. Given the transient nature of many abnormalities, clinicians may be able to reassure guardians that hearing loss, even in cases of moderate hyperbilirubinemia, is often reversible, particularly with prompt treatment and follow-up. With vigilant monitoring and timely interventions, long-term auditory outcomes in hyperbilirubinemic neonates can be favourable. Continued follow-up into early childhood, particularly assessing speech and cognitive development could help identify subtle effects that may not be apparent in the first year. 4.5 LIMITATIONS As the study was conducted in a single tertiary care hospital, consistent protocols were utilised while studying neonates for a period of 12 months. Although there is internal validity, generalisability across various populations may be studied by multicentric studies. A larger cohort in future studies could help validate the findings and discover more subtle effects of unconjugated hyperbilirubinaemia on hearing. Further research can also be carried out compare outcomes across different types of treatment like phototherapy and transfusion, to explore their specific auditory effects. Longer-term studies could explore how early auditory changes due to neonatal hyperbilirubinaemia could influence language development and academic performance. While our study prioritized non-invasive, accessible screening tools like ABR and DPOAE, incorporation of genetic profiling or neuroimaging in future research may provide more in-depth understanding of individual susceptibility. 5. CONCLUSION & KEY MESSAGES Our study shows that elevated unconjugated bilirubin, more than total bilirubin, is significantly associated with early auditory dysfunction. While comparisons between cases and controls did not reach statistical significance, neonates with higher unconjugated bilirubin consistently showed more absent DPOAE and ABR responses. Importantly, most hearing loss observed was transient and resolved within the first year of life. We strongly recommend routine DPOAE and ABR screening in all neonates with hyperbilirubinemia regardless of gender or gestational age, priority evaluation for those with elevated unconjugated bilirubin, serial testing to track recovery or progression of hearing loss and early rehabilitation for those with persistent auditory deficits. Early identification enables timely interventions, reducing the risk of permanent damage and supporting speech development, leading to positive long-term outcomes. Declarations Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki. All the study participants have provided written informed consent for participation in the study. The study protocol was approved by Institutional Ethics Committee vide letter IEC KMC MLR- 09/2020/257. Clinical trial number Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding This research was not funded by any organization Authors' contributions Saksham Dhawan, Vijendra Shenoy S –contributed significantly to conception and design of the work Saksham Dhawan, Vijendra Shenoy S, Neehal Zuturu-contributed significantly to drafting the work, Saksham Dhawan, Vijendra Shenoy S, Neehal Zuturu, Pooja Varshini Raja, Ananya Sinha, Divya Subramanian- All the authors made a major contribution to data collection, study, and interpretation. All authors have finally approved the version to be published and are responsible for all aspects of the work to ensure that problems relating to the quality or credibility of any section of the work are adequately reviewed and resolved. 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Hearing Assessment in Infants with Otoacoustic Emission and Auditory Brainstem Response: A Retrospective Study. Gulati A, Sakthivel P, Singh I, Ramji S. The Hearing Status of Preterm Infant’s ≤ 34 Weeks as Revealed by Otoacoustic Emissions (OAE) Screening and Diagnostic Brainstem Evoked Response Audiometry (BERA): A Tertiary Center Experience. Indian Journal of Otolaryngology and Head & Neck Surgery. 2022 Aug 8;74(S1):178–83. Jiang ZD, Zhou Y, Ping LL, Wilkinson AR. Brainstem auditory response findings in late preterm infants in neonatal intensive care unit. Acta Paediatr. 2011 Aug;100(8):e51–4. Amin SB. Clinical assessment of bilirubin-induced neurotoxicity in premature infants. Semin Perinatol. 2004 Oct;28(5):340–7. Amin SB, Ahlfors C, Orlando MS, Dalzell LE, Merle KS, Guillet R. Bilirubin and Serial Auditory Brainstem Responses in Premature Infants. Pediatrics. 2001 Apr 1;107(4):664–70. Agrawal VK, Shukla R, Misra PK, Kapoor RK, Malik GK. Brainstem auditory evoked response in newborns with hyperbilirubinemia. Indian Pediatr. 1998 Jun;35(6):513–8. Vinodh M, Ambikapathy P, Aravind MA, Ganesh J. Reversibility of brainstem evoked response audiometry abnormalities at 3 months in term newborns with hyperbilirubinemia. Indian Pediatr. 2014 Feb 5;51(2):134–5. Nakamura H, Takada S, Shimabuku R, Matsuo M, Matsuo T, Negishi H. Auditory nerve and brainstem responses in newborn infants with hyperbilirubinemia. Pediatrics. 1985 Apr;75(4):703–8. Abdollahi FZ, Ahmadi T, Manchaiah V, Lotfi Y. Auditory Brainstem Response Improvements in Hyperbillirubinemic Infants. J Audiol Otol. 2016;20(1):13. Nam GS, Kwak SH, Bae SH, Kim SH, Jung J, Choi JY. Hyperbilirubinemia and Follow-up Auditory Brainstem Responses in Preterm Infants. Clin Exp Otorhinolaryngol. 2019 May 1;12(2):163–8. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7473326","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":522245114,"identity":"bee2bd58-d9f7-4ed2-adf1-6d31067a6205","order_by":0,"name":"SAKSHAM DHAWAN","email":"","orcid":"","institution":"Kasturba Medical College Mangalore, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"SAKSHAM","middleName":"","lastName":"DHAWAN","suffix":""},{"id":522245115,"identity":"712c7ec7-1a2d-4c81-8517-db8569bb3df0","order_by":1,"name":"Vijendra Shenoy 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Education","correspondingAuthor":false,"prefix":"","firstName":"Neehal","middleName":"","lastName":"Zuturu","suffix":""},{"id":522245119,"identity":"aac64a53-35ff-44da-ba83-7ae34bf1546b","order_by":3,"name":"ANANYA SINHA","email":"","orcid":"","institution":"Kasturba Medical College Mangalore, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"ANANYA","middleName":"","lastName":"SINHA","suffix":""},{"id":522245121,"identity":"966a7052-accc-407e-b1bd-81a9c5d9574f","order_by":4,"name":"Pooja Raja Varshini","email":"","orcid":"","institution":"Kasturba Medical College Mangalore, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Pooja","middleName":"Raja","lastName":"Varshini","suffix":""},{"id":522245123,"identity":"04d5b0b5-1347-42ec-87be-1b20c2780e4a","order_by":5,"name":"DIVYA SUBRAMANIAN","email":"","orcid":"","institution":"Kasturba Medical College Mangalore, Manipal Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"DIVYA","middleName":"","lastName":"SUBRAMANIAN","suffix":""}],"badges":[],"createdAt":"2025-08-27 15:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7473326/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7473326/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92575217,"identity":"ec28ffd1-6cfc-42d3-b5e4-84e846d1e8ec","added_by":"auto","created_at":"2025-10-01 08:20:08","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110604,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7473326/v1/beee504b536ab9bfb92f02ea.docx"},{"id":92576312,"identity":"66b7c758-1d7c-4c33-836f-2e924a830762","added_by":"auto","created_at":"2025-10-01 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1","display":"","copyAsset":false,"role":"figure","size":78816,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7473326/v1/a1df6c2685c31133213456f1.jpg"},{"id":92575209,"identity":"35f82d80-44d8-4cb8-842c-4b76538e3c0a","added_by":"auto","created_at":"2025-10-01 08:20:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54229,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7473326/v1/3ae11f87effe55ef31ce1c50.jpg"},{"id":92575212,"identity":"fc762d92-0759-47b5-9c5c-bae6a1643eaa","added_by":"auto","created_at":"2025-10-01 08:20:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58947,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7473326/v1/97a3ecdd6672067793483787.jpg"},{"id":92832107,"identity":"437424a7-7756-4d01-95d5-673660848bb4","added_by":"auto","created_at":"2025-10-06 06:39:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":952353,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7473326/v1/6e938174-ec9d-4a21-85e9-1c587713cb17.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unconjugated Hyperbilirubinaemia and Its Effect on Neonatal Distortion Product Otoacoustic Emission and Auditory Brainstem Response- An Observational Study","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eOne child out of every thousand is born profoundly deaf, while four times as many are born with bilateral hearing loss, either moderate or severe. Congenital hearing loss is more common in newborns than the total prevalence of all metabolic disorders that are now detected through blood tests (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Infants in Neonatal Intensive Care Units are 10\u0026ndash;20 times more likely to have substantial hearing loss as compared to the general population.\u003c/p\u003e\u003cp\u003eHyperbilirubinemia affects a substantial number of newborns, up to 84% of term and late preterm infants in the first week of life (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). While neonatal jaundice is often benign, high levels of unconjugated bilirubin can lead to kernicterus and irreversible neurological damage. Bilirubin induced neurological damage leading to impairment of the hearing mechanism is one of the prime concerns in these hyperbilirubinaemic infants.\u003c/p\u003e\u003cp\u003eHearing loss is among the earliest and most subtle manifestations of bilirubin neurotoxicity (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The auditory system, particularly the brainstem and cochlear nuclei, is particularly sensitive to excessive bilirubin levels. The major cause of damage is when lipid-soluble unconjugated bilirubin (UB) breaches the blood brain barrier (BBB). Organic anion efflux cannot remove the UB, which then builds up in the cytoplasm and is ultimately fatal to the cells.\u003c/p\u003e\u003cp\u003ePresence of adequate and correct auditory stimuli in the first year of life is necessary not only for the audiological, but also for the overall development of the child. As hearing outcomes in newborns can be bettered with prompt surveillance and identification, and proper rehabilitation, the National Institute of Health advised universal neonatal hearing testing by three months of age, using Otoacoustic Emissions (OAE) in 1993. Current guidelines in various countries recommend universal neonatal hearing screening using OAE and confirmatory testing with Auditory Brainstem Response (ABR). With early detection and intervention, infants with auditory impairment can now achieve near-normal language and cognitive milestones.\u003c/p\u003e\u003cp\u003eThis study aims to evaluate the association between neonatal hyperbilirubinemia, particularly elevated unconjugated bilirubin, and early hearing dysfunction using DPOAE and ABR. Furthermore, the study also aims to determine the prognostic utility of serial ABR assessments over the first year of life.\u003c/p\u003e"},{"header":"2. METHODS OF DATA COLLECTION","content":"\u003cp\u003eThis was a prospective study conducted over a period of 2 years at a tertiary care Neonatal Intensive Care Unit (NICU). After obtaining consent from the family, all live births requiring NICU admission (n\u0026thinsp;=\u0026thinsp;106), both preterm (\u0026lt;\u0026thinsp;37 weeks period of gestation) and term (37 to 42 weeks period of gestation), with and without hyperbilirubinemia were included into the study. Neonates with a family history of sensorineural hearing loss, in-utero TORCH infection, craniofacial abnormalities and anomalies incompatible with life, APGAR score\u0026thinsp;\u0026lt;\u0026thinsp;5 in 1st minute or \u0026lt;\u0026thinsp;7 in 5th minute and infants on ventilators for \u0026gt;\u0026thinsp;5 days were excluded from the study. Neonates for whom the consent of the family could not be obtained were excluded from the study (n\u0026thinsp;=\u0026thinsp;12) as well as those which were lost to follow up were excluded from the study (n\u0026thinsp;=\u0026thinsp;10). Neonates with hyperbilirubinaemia were deemed as cases (n\u0026thinsp;=\u0026thinsp;44) and the ones with normal bilirubin were deemed as controls (n\u0026thinsp;=\u0026thinsp;40).\u003c/p\u003e\u003cp\u003eThe case group was further subdivided into subgroups. Group 1 (n\u0026thinsp;=\u0026thinsp;12) and Group 2 (n\u0026thinsp;=\u0026thinsp;32) included the cases with total bilirubin levels below and above the average value of total bilirubin in all the cases, respectively. Similarly, Group 3 (n\u0026thinsp;=\u0026thinsp;16) and Group 4 (n\u0026thinsp;=\u0026thinsp;28) included the cases with unconjugated bilirubin levels below and above the average value of unconjugated bilirubin in all the cases, respectively.\u003c/p\u003e\u003cp\u003eAfter clinical examination, all neonates underwent istortion Product Otoacoustic Emissions (DPOAE) examination on the 3rd day of life under universal neonatal screening for hearing loss. A repeat DPOAE on the 30th day of life was performed to rule out any temporary causes of conductive hearing loss and to substantiate whether hyperbilirubinaemia, particularly elevated unconjugated bilirubin, is a cause for development of hearing impairment in neonates. This was followed by evaluation using ABR. The ones with abnormal ABR findings were followed up at 3 months and an ABR was repeated. Infants with abnormal ABR at 3 months were re-evaluated at 12 months, by ABR, to check whether the abnormalities in ABR due to hyperbilirubinemia were transient or permanent.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Demographics\u003c/h2\u003e\u003cp\u003eThis study evaluated the association between neonatal hyperbilirubinemia and auditory function across 84 neonates, 44 with hyperbilirubinemia (cases) and 40 without (controls). Both term and preterm infants were included. Birth weight and gestational age were comparable between groups. There was no significant correlation between serum bilirubin levels and birth weight. Although males are biologically more prone to neonatal jaundice (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), our study did not show any significant difference in gender-based distribution between the groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e3.2 OAE Comparisons\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3.2.1 First screening\u003c/span\u003e:\u003c/h2\u003e\u003cp\u003eIn the initial DPOAE screening, a considerable number of absent responses were observed in both cases and controls. Among the 88 ears in the case group, 41 ears (46.6%) showed absent DPOAE responses, compared to 26 ears (32.5%) out of the 80 in the control group. Results across various frequencies were not statistically significant (p\u0026thinsp;=\u0026thinsp;0.062) (FIGURE \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. There was no significant difference in DPOAE responses between preterm cases and preterm controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe majority of absent DPOAE readings occurred in the lower frequencies, namely 1kHz and 2kHz, though no statistically significant differences were noted for any specific frequency. Across both low and high frequencies, a higher number of ears in the case group demonstrated absent readings as compared to the controls, although this too lacked statistical significance. While higher bilirubin levels tended to be associated with more absent DPOAE responses, this did not show statistical significance. Similarly, there was no significant association noted between the absence of different DPOAE frequencies in the ears of preterm and term neonates either. However, both preterm and term neonates exhibited more absent DPOAE responses in lower frequencies (1kHz and 2kHz).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3.2.2 Comparison of OAE responses within two subgroups of cases based on total bilirubin levels\u003c/span\u003e\u003c/h2\u003e\u003cp\u003eThe mean total bilirubin value in the case group was found to be 5.17 mg/dl. Out of the total 88 ears in the cases, 24 ears belonged to neonates with total bilirubin values below the average of the cases group (Group 1) and 64 ears belonged to those with their total bilirubin values above the average (Group 2). Higher bilirubin levels trended toward more absent DPOAE responses, i.e., absent responses were found in 8 ears (33.3%) in Group 1 and 33 ears (52.6%) in Group 2 \u003cb\u003e(\u003c/b\u003eFIGURE \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. This difference was found to be statistically insignificant (p\u0026thinsp;=\u0026thinsp;0.127). However, a significant association was found between left ear DPOAE absence and hyperbilirubinemia (p\u0026thinsp;=\u0026thinsp;0.018), suggesting a possible asymmetric vulnerability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3.2.3 Comparison of OAE responses within two subgroups of cases based on unconjugated bilirubin levels.\u003c/span\u003e\u003c/h2\u003e\u003cp\u003eThe mean unconjugated bilirubin level in the case group was found to be 4.18mg/dl. Out of the 88 ears, there were 32 ears (Group 3) for which the unconjugated bilirubin values were below the average of the cases group and 56 ears (Group 4) with their unconjugated bilirubin values above the average of the total cases. Absent DPOAE responses were found in 10 ears (31.2%) of Group 3, compared to 31 ears (55.4%) in Group 4 \u003cb\u003e(\u003c/b\u003eFIGURE \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This difference was statistically significant (p\u0026thinsp;=\u0026thinsp;0.029), suggesting a stronger relationship between elevated unconjugated bilirubin and DPOAE absence. This finding strengthens the hypothesis that elevated unconjugated bilirubin, more than total bilirubin, has a stronger predictive value for early cochlear dysfunction as captured by DPOAE.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3.2.4 Repeat DPOAE analysis\u003c/span\u003e\u003c/h2\u003e\u003cp\u003eDPOAE testing was repeated on the 30th day of life for all neonates with initially absent responses. Among the case group, 22 out of 41 ears (53.6%) continued to show absent responses. In the control group, 10 out of 26 ears (38.4%) remained absent \u003cb\u003e(FIGURE 4)\u003c/b\u003e. Numerically, fewer neonates in both the case and the control group showed an absent DPOAE response on the repeat DPOAE as compared to the initial screening \u003cb\u003e(\u003c/b\u003eTable \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These results highlight a reduction in absent responses in both groups over time, suggesting some degree of reversibility in the auditory effects of bilirubin.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSHOWING THE PROGRESSIVE DECREASE IN THE NUMBER OF NEONATES WITH ABSENT DPOAE RESPONSES\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eNUMBER OF EARS WITH ABSENT RESPONSE ON 1\u003csup\u003eST\u003c/sup\u003e DPOAE (3\u003csup\u003eRD\u003c/sup\u003e DAY OF LIFE)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eNUMBER OF EARS WITH ABSENT DPOAE RESPONSE ON REPEAT DPOAE (30\u003csup\u003eTH\u003c/sup\u003e DAY OF LIFE)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCASES\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCONTROLS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCASES\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCONTROLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.3 ABR Comparisons:\u003c/h2\u003e\u003cp\u003eOn the 30th day of life, ABR testing was performed on both the cases and the controls (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although a higher number of neonates in the case group showed absent responses to ABR than those in the control group, the association between the ABR responses of both was found to be statistically insignificant (p\u0026thinsp;=\u0026thinsp;0.090).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDEPICTING THE NUMBER AND PERCENTAGES OF THE ABSENT AND PRESENT ABR RESPONSES THAT WERE NOTED AMONGST CASES(n\u0026thinsp;=\u0026thinsp;44) AND CONTROLS (n\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCase (88 ears)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControls (80 ears)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eᵡ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP Value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eABR, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e2.873\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.090\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbsent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17(19.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e08(10.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e71(80.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e72(90.0%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eABR results were compared based on bilirubin levels in all 4 groups. In Group 1, none of the 24 ears had absent ABR response. In contrast, 17 out of 64 ears in Group 2 showed absent ABR response. This was found to be statistically significant (p\u0026thinsp;=\u0026thinsp;0.005) (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The stronger relationship emerged when comparing groups based on unconjugated bilirubin. None of the 32 ears in Group 3 had absent ABR responses. However, in Group 4, 17 out of the 56 ears exhibited absent ABR responses (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This was highly statistically significant (p\u0026thinsp;=\u0026thinsp;0.001), underlining the neurotoxic potential of elevated unconjugated bilirubin over total bilirubin.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eNUMBER OF NEONATES IN GROUP 1 AND GROUP 2 OF CASES SHOWING ABSENT ABR RESPONSES.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eABR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eON THE BASIS OF TOTAL BILIRUBIN LEVELS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGROUP 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGROUP 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eABSENT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17(26.6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePRESENT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24(100)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47(73.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eNUMBER OF NEONATES IN GROUP 3 AND GROUP 4 OF CASES SHOWING ABSENT ABR RESPONSES\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eABR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eON THE BASIS OF UNCONJUGATED BILIRUBIN LEVELS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP Value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBELOW AVERAGE; n(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eABOVE AVERAGE; n(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eABSENT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17(30.4%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePRESENT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e32(100%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39(69.6%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eRepeat ABRs were done at 3 months for both the cases and controls who previously showed absent ABR responses during the first screening at 30 days. Only one case and one control had persistently absent ABR waveforms, both of which became normal at the 12-month evaluation, indicating recovery.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eOne of the most frequent problems concerning newborns is hyperbilirubinaemia. Even though majority of the infants with jaundice are healthy, excessive serum levels of bilirubin, especially unconjugated bilirubin, can induce kernicterus which can lead to sensorineural hearing loss. The major cause of damage is UB. Being lipid-soluble, it breaches the cell membranes and the BBB. Organic anion efflux cannot remove the UB, which then accumulates in the cytoplasm of neural cells, leading to cytotoxicity. This build-up is fatal to the cells. Early detection and evaluation of jaundice are crucial to avoid the consequences such as bilirubin encephalopathy, which can cause hearing loss. OAE and ABR are routine and reliable non-invasive screening tools that can be employed to achieve such early detection to enable timely intervention (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4.1 HYPERBILIRUBINAEMIA AND HEARING LOSS\u003c/h2\u003e\u003cp\u003eSeveral studies reinforce the link between high bilirubin levels and auditory dysfunction. For instance, Boskabadi et al. evaluated risk factors for SNHL in 200 icteric newborns and found abnormal ABRs in 4.8% of them. They also stated that with regard to infant hearing health, total bilirubin level had the highest prediction and in infants with severe jaundice, hearing impairment develops about 10\u0026ndash;50 times more commonly (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Similarly, Ahmed et al. reported hearing impairment in 13 out of 234 neonates with hyperbilirubinemia, linking hyperbilirubinemia and hearing loss in newborns (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). As per Chavan et al\u0026rsquo;s study, hyperbilirubinemia was found to be a significant factor (p\u0026thinsp;=\u0026thinsp;0.01) for REFER responses in OAE in infants with normal bilirubin levels and those with hyperbilirubinaemia (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur study found that higher unconjugated bilirubin levels significantly predicted absent ABR responses. This supports the hypothesis that unconjugated bilirubin at high levels is particularly neurotoxic, affecting auditory pathways by delaying neural transmission, leading to delayed or absent ABR responses. Specifically, 17 ears (19.3%) out 88 in the case group (hyperbilirubinaemic neonates) showed absent ABRs, compared to 8 out of 80 ears (10%) in the control group (non-hyperbilirubinaemic neonates). This corroborates that the presence of hyperbilirubinaemia may be a causative factor for hearing loss, although it was statistically insignificant.\u003c/p\u003e\u003cp\u003eWhile higher total bilirubin levels showed trends of association with increased absence of DPOAE responses (statistically insignificant), the stronger and statistically significant associations were consistently observed with elevated unconjugated bilirubin. Its dose-dependent ability to interfere directly with cochlear structures may explain its clearer impact on ABR outcomes. Even moderate elevations may disrupt outer hair cell function and neural transmission, detectable through frequency-specific DPOAE testing and ABR.\u003c/p\u003e\u003cp\u003eSurprisingly, left ear vulnerability was observed in our study (p\u0026thinsp;=\u0026thinsp;0.018) possibly suggesting asymmetry in early subclinical cochlear damage that precedes measurable hearing loss in neonates. However, the mechanisms remain speculative and warrant further neurophysiological exploration. In addition, the higher rate of absent responses at low frequencies (1kHz, 2kHz) across groups, though not statistically significant, also emerged as a curious trend, needing further research into frequency-specific cochlear sensitivity to bilirubin toxicity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.2 GESTATIONAL AGE AND HYPERBILIRUBINAEMIA\u003c/h2\u003e\u003cp\u003eStudies have linked prematurity with auditory dysfunction. According to a study by Gulati et al., diagnostic Brainstem Evoked Response Audiometry (BERA) in conjunction with OAE should be performed in all high-risk newborns who are preterm neonates (\u0026lt;/= 34 weeks) in order to detect cases of auditory neuropathy spectrum disorders (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Jiang et al. found BERA abnormalities in late-preterm neonates, indicating brainstem auditory pathway impairment, suggesting that perinatal complications have a negative impact on the late preterm auditory brainstem when compared to low-risk late preterm newborns (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious studies, such as those by Gulati et al. and Jiang et al. emphasize the vulnerability of preterm neonates to auditory damage. However, our study did not find a statistically significant correlation between gestational age and auditory dysfunction. There were 7 neonates who were \u0026lt;/= 34 weeks preterm out of which 5 were cases and 2 were controls. Of the 5 cases, 4 (80%) had absent DPOAE responses initially and 3 of them continued to have absent responses at day 30. Out of the 3 cases with absent DPOAE, 2 had an absent ABR at 30 days. 1 of the case continued to have an absent ABR which was repeated at 3 months of age, which later showed normal response to repeat ABR at 12 months. Out of the 2 controls, both had absent DPOAE at the first assessment and on repeat DPOAE on day 30, 1 control had normal DPOAE and 1 had absent DPOAE, in which the latter had absent ABR at 30 days but a normal ABR at 3 months of age. Despite these findings, statistical significance was not reached linking prematurity alone to auditory loss as preterm neonates did not show significantly different DPOAE results compared to term neonates, implying that bilirubin levels may be a more critical factor than gestational age when other variables like birth weight are controlled.\u003c/p\u003e\u003cp\u003eOur findings are also consistent with Chavan et al. (p\u0026thinsp;=\u0026thinsp;0.16)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and Ahmed Kaenat et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), where gestational age had no statistically significant influence on initial DPOAE outcomes. These findings suggest that bilirubin toxicity by elevated unconjugated bilirubin may exert a more central influence on auditory function than gestational age alone.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.3 REVERSIBILITY OR PERMANENCE OF THE HEARING LOSS INDUCED BY BILIRUBIN\u003c/h2\u003e\u003cp\u003eEmerging evidence suggests bilirubin-induced auditory dysfunction can be transient and hence bilirubin-induced hearing loss may often be reversible. With the administration of albumin infusions, there is a chance that bilirubin-induced neurological damage can be reversed, and there is emerging proof that ABR abnormalities can be corrected in animal models (\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Furthermore, certain minor ABR abnormalities in babies can be treated with phototherapy and exchange transfusions. For example, Nakamura et al. observed that delayed ABR wave latencies (peaks I and V) normalized after exchange transfusion in 56 neonates with TB\u0026thinsp;\u0026ge;\u0026thinsp;15 mg/dL (compared to 24 infants with normal TB levels) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSimilarly, our study found that only 2 out of 84 neonates had persistent abnormal ABR responses at 3 months. They were monitored further in our study and at the end of the 12 months follow up, there were no neonates in either the case or the control group who had abnormal responses on ABR, signifying that transient nature of bilirubin-induced auditory dysfunction may not translate into broader neurodevelopmental impairment. This indicates that the auditory dysfunction linked to unconjugated bilirubin may be transient, especially with early intervention. Maturation of neural pathways due to the neonates developing neuroplasticity may also mitigate the transient hearing loss caused due to hyperbilirubinaemia.\u003c/p\u003e\u003cp\u003eOur results align with findings from Abdollahi et al. who reported that hyperbilirubinaemic neonates with initial ABR abnormalities often improved by the second evaluation and hence such neonates should have their hearing tested again after receiving therapy before any aggressive hearing interventions are made. In that study, 7 infants who had ABR threshold values between 35 and 65 dBnHL at the first examination displayed a threshold of less than 30 dBnHL during the second evaluation. However, infants who had an ABR threshold of 70 dBnHL at the first evaluation still had the same threshold during the subsequent evaluation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). This was also noted in a study conducted by Nam et al. where ABR abnormalities resolved in a subset of affected neonates. 13 out of these 30 preterm infants recovered completely while the remaining 17 had either worse responses on ABR or had not changed. Out of these 17, 2 recovered to normal hearing as shown by the follow up ABRs (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSimilarly, Ahmed et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) reported significant improvement in hearing over time using repeat OAE and BERA assessments. They found that among 57 (out of 234) enrolled neonates referred after initial OAE, 38 passed on re-evaluation at 3 months. With a p value of 0.0001, it was determined that the improvement in hearing impairment seen on the OAE after 3 months was significant. In the same study, BERA done for 51 subjects at the 3rd month showed normal findings in 38 (75.64%) neonates and abnormal findings in 13 (25.5%) neonates and both these groups had the same finding on repeat BERA at 6 months. This was found to be dissimilar to our study, as at the end of 3 months, repeat ABR of only 2 out of the 15 neonates (who had initial abnormal ABR results) was found to be abnormal. Even for these 2 neonates, ABR results were found to be normal at 12 months. Additionally, 10\u0026ndash;15% of ears in controls showed absent ABR at day 30 in our study. Contribution by other perinatal factors like hypoxia, medications, or delivery mode need to be studied to see if they are confounding factors.\u003c/p\u003e\u003cp\u003eIn our study, DPOAE referrals dropped from 46.6% to 32.5% in cases and from 25% to 12.5% in controls after 30 days. This finding underscores the importance of repeated testing and continuous monitoring (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The fact that none of the neonates in our study showed persistent ABR abnormalities at 12 months reinforces the possibility of spontaneous recovery. Serial testing remains critical for differentiating transient and permanent damage, aiding timely intervention and also preventing unnecessary treatments in neonates whose hearing normalizes over time.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.4. IMPLICATIONS IN PUBLIC HEALTH\u003c/h2\u003e\u003cp\u003eDPOAE and ABR serve as valuable, non-invasive tools in detecting early auditory dysfunction in neonates and distinguishing reversible dysfunction from permanent damage. Together, they offer a reliable framework for monitoring auditory risk in jaundiced neonates. These tests minimize neonatal stress and do not require sedation, while providing reliable results, making them ideal for widespread screening, even in resource-limited settings. Our study supports public health initiatives for universal newborn hearing screening using these accessible methods.\u003c/p\u003e\u003cp\u003eGiven the neurotoxic risk of elevated unconjugated bilirubin, universal screening for jaundiced neonates becomes even more important. Early identification enables timely interventions, reducing the risk of permanent damage. In addition, repeat testing should be the standard protocol, as early ABR failures may not reflect permanent damage. Our study\u0026rsquo;s 12-month follow-up showed complete recovery in all affected neonates.\u003c/p\u003e\u003cp\u003eGiven the transient nature of many abnormalities, clinicians may be able to reassure guardians that hearing loss, even in cases of moderate hyperbilirubinemia, is often reversible, particularly with prompt treatment and follow-up. With vigilant monitoring and timely interventions, long-term auditory outcomes in hyperbilirubinemic neonates can be favourable. Continued follow-up into early childhood, particularly assessing speech and cognitive development could help identify subtle effects that may not be apparent in the first year.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.5 LIMITATIONS\u003c/h2\u003e\u003cp\u003eAs the study was conducted in a single tertiary care hospital, consistent protocols were utilised while studying neonates for a period of 12 months. Although there is internal validity, generalisability across various populations may be studied by multicentric studies. A larger cohort in future studies could help validate the findings and discover more subtle effects of unconjugated hyperbilirubinaemia on hearing. Further research can also be carried out compare outcomes across different types of treatment like phototherapy and transfusion, to explore their specific auditory effects. Longer-term studies could explore how early auditory changes due to neonatal hyperbilirubinaemia could influence language development and academic performance. While our study prioritized non-invasive, accessible screening tools like ABR and DPOAE, incorporation of genetic profiling or neuroimaging in future research may provide more in-depth understanding of individual susceptibility.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. CONCLUSION \u0026 KEY MESSAGES","content":"\u003cp\u003eOur study shows that elevated unconjugated bilirubin, more than total bilirubin, is significantly associated with early auditory dysfunction. While comparisons between cases and controls did not reach statistical significance, neonates with higher unconjugated bilirubin consistently showed more absent DPOAE and ABR responses. Importantly, most hearing loss observed was transient and resolved within the first year of life.\u003c/p\u003e\u003cp\u003eWe strongly recommend routine DPOAE and ABR screening in all neonates with hyperbilirubinemia regardless of gender or gestational age, priority evaluation for those with elevated unconjugated bilirubin, serial testing to track recovery or progression of hearing loss and early rehabilitation for those with persistent auditory deficits. Early identification enables timely interventions, reducing the risk of permanent damage and supporting speech development, leading to positive long-term outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki. All the study participants have provided written informed consent for participation in the study. The study protocol was approved by Institutional Ethics Committee vide letter IEC KMC MLR- 09/2020/257.\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis research was not funded by any organization\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eSaksham Dhawan, Vijendra Shenoy S \u0026ndash;contributed significantly to conception and design of the work\u003c/p\u003e\n\u003cp\u003eSaksham Dhawan, Vijendra Shenoy S, Neehal Zuturu-contributed significantly to drafting the work,\u003c/p\u003e\n\u003cp\u003eSaksham Dhawan, Vijendra Shenoy S, Neehal Zuturu, Pooja Varshini Raja, Ananya Sinha, Divya Subramanian- All the authors made a major contribution to data collection, study, and interpretation. All authors have finally approved the version to be published and are responsible for all aspects of the work to ensure that problems relating to the quality or credibility of any section of the work are adequately reviewed and resolved.\u0026nbsp;\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCoates H, Gifkins K. Newborn hearing screening. Aust Prescr. 2003 Aug 1;26(4):82\u0026ndash;4.\u003c/li\u003e\n \u003cli\u003eOlds C, Oghalai JS. Audiologic impairment associated with bilirubin-induced neurologic damage. Semin Fetal Neonatal Med. 2015 Feb;20(1):42\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003eZaman BU, Lone PA, Irtika, Mir NY. Clinico-etiological profile of neonates with jaundice in a tertiary care hospital of Northernmost India. Int J Contemp Pediatrics. 2024 Apr 25;11(5):566\u0026ndash;70.\u003c/li\u003e\n \u003cli\u003ePoornima Shankar and V.C. Manjunath. No Title. J Evol Med Dent Sci. 2014;3(10).\u003c/li\u003e\n \u003cli\u003eBoskabadi H, Zakerihamidi M, Moradi A, Bakhshaee M. Risk Factors for Sensorineural Hearing Loss in Neonatal Hyperbilirubinemia. Iran J Otorhinolaryngol. 2018 Jul;30(99):195\u0026ndash;202.\u003c/li\u003e\n \u003cli\u003eAhmed K, Havle A, Sravya YL, Ahmeds S. Assessment of hearing in newborns with hyperbilirubinemia using otoacoustic emmisions and brainstem evoked response audiometry. Int J Health Sci (Qassim). 2022 Apr 25;3564\u0026ndash;73.\u003c/li\u003e\n \u003cli\u003eChavan RP, Ingole SM, Damodhar AP et al. Hearing Assessment in Infants with Otoacoustic Emission and Auditory Brainstem Response: A Retrospective Study.\u003c/li\u003e\n \u003cli\u003eGulati A, Sakthivel P, Singh I, Ramji S. The Hearing Status of Preterm Infant\u0026rsquo;s \u0026le; 34 Weeks as Revealed by Otoacoustic Emissions (OAE) Screening and Diagnostic Brainstem Evoked Response Audiometry (BERA): A Tertiary Center Experience. Indian Journal of Otolaryngology and Head \u0026amp; Neck Surgery. 2022 Aug 8;74(S1):178\u0026ndash;83.\u003c/li\u003e\n \u003cli\u003eJiang ZD, Zhou Y, Ping LL, Wilkinson AR. Brainstem auditory response findings in late preterm infants in neonatal intensive care unit. Acta Paediatr. 2011 Aug;100(8):e51\u0026ndash;4.\u003c/li\u003e\n \u003cli\u003eAmin SB. Clinical assessment of bilirubin-induced neurotoxicity in premature infants. Semin Perinatol. 2004 Oct;28(5):340\u0026ndash;7.\u003c/li\u003e\n \u003cli\u003eAmin SB, Ahlfors C, Orlando MS, Dalzell LE, Merle KS, Guillet R. Bilirubin and Serial Auditory Brainstem Responses in Premature Infants. Pediatrics. 2001 Apr 1;107(4):664\u0026ndash;70.\u003c/li\u003e\n \u003cli\u003eAgrawal VK, Shukla R, Misra PK, Kapoor RK, Malik GK. Brainstem auditory evoked response in newborns with hyperbilirubinemia. Indian Pediatr. 1998 Jun;35(6):513\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eVinodh M, Ambikapathy P, Aravind MA, Ganesh J. Reversibility of brainstem evoked response audiometry abnormalities at 3 months in term newborns with hyperbilirubinemia. Indian Pediatr. 2014 Feb 5;51(2):134\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eNakamura H, Takada S, Shimabuku R, Matsuo M, Matsuo T, Negishi H. Auditory nerve and brainstem responses in newborn infants with hyperbilirubinemia. Pediatrics. 1985 Apr;75(4):703\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eAbdollahi FZ, Ahmadi T, Manchaiah V, Lotfi Y. Auditory Brainstem Response Improvements in Hyperbillirubinemic Infants. J Audiol Otol. 2016;20(1):13.\u003c/li\u003e\n \u003cli\u003eNam GS, Kwak SH, Bae SH, Kim SH, Jung J, Choi JY. Hyperbilirubinemia and Follow-up Auditory Brainstem Responses in Preterm Infants. Clin Exp Otorhinolaryngol. 2019 May 1;12(2):163\u0026ndash;8.\u003cstrong\u003e\u003cu\u003e\u003c/u\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hyperbilirubinaemia, Neonate, Hearing loss, ABR, DPOAE","lastPublishedDoi":"10.21203/rs.3.rs-7473326/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7473326/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAIM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the association between neonatal hyperbilirubinemia, particularly elevated unconjugated bilirubin, and early hearing dysfunction using Distortion Product Otoacoustic Emissions (DPOAE) and Auditory Brainstem Response (ABR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMATERIALS AND METHODS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA prospective study of 2-year duration at a tertiary care centre was performed wherein hyperbilirubinaemic neonates were cases and controls had normal bilirubin levels. Groups 1 and 2 had cases with total bilirubin levels below and above the average (5.17mg/dl) while Groups 3 and 4 included cases with unconjugated bilirubin levels below and above the average (4.18 mg/dl) respectively. All neonates underwent DPOAE on day 3 of life. ABR and repeat DPOAE were done on 30\u003csup\u003eth\u003c/sup\u003e day of life. Neonates with abnormal ABR were followed up at 3\u003csup\u003erd\u003c/sup\u003e and 12\u003csup\u003eth\u003c/sup\u003e month, with repeat ABR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e30\u003csup\u003eth\u003c/sup\u003e day ABR responses between Group 3 and Group 4 showed statistical significance for the right and left ear (p = 0.037, 0.007), respectively. Repeat DPOAE on day 30 was significant between preterm and term neonates (p = 0.047). There was no association noted between hyperbilirubinaemia and birthweight or gestational age. DPOAE responses showed no statistical significance between cases and controls or any specific DPOAE frequency. All cases of hearing loss were transient and resolved within the first year of life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnconjugated hyperbilirubinemia poses a notable risk for early auditory dysfunction, even in the absence of kernicterus. DPOAE and serial ABR are valuable tools for early detection and monitoring. Neonates with elevated unconjugated bilirubin should be prioritized for repeated audiological assessments. Early identification and timely intervention can prevent long-term speech and language delays and improve developmental outcomes.\u003c/p\u003e","manuscriptTitle":"Unconjugated Hyperbilirubinaemia and Its Effect on Neonatal Distortion Product Otoacoustic Emission and Auditory Brainstem Response- An Observational Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 08:20:03","doi":"10.21203/rs.3.rs-7473326/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b1ed0765-e58b-446f-807a-7345dc60deac","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T06:39:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-01 08:20:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7473326","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7473326","identity":"rs-7473326","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}