Comparative Effectiveness of Non-Pharmacological Pain Management Interventions in Neonates: A Cross- Sectional Observational Study

Comparative Effectiveness of Non-Pharmacological Pain Management Interventions in Neonates: A Cross- Sectional 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 Comparative Effectiveness of Non-Pharmacological Pain Management Interventions in Neonates: A Cross- Sectional Observational Study Anjali Gupta, Ramesh Choudhary, Chetan Meena, Vijendra Garg, Sourabh Agrawal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9112827/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 Background: Neonates in intensive care units undergo numerous painful procedures. Non-pharmacological interventions offer safe approaches to reduce procedural pain, but comparative studies evaluating their relative effectiveness are limited, particularly in low-resource settings. Methods: We enrolled 240 neonates requiring heel lancing for blood glucose monitoring. Neonates were allocated to three intervention groups: breastfeeding, kangaroo mother care (KMC), and oral dextrose. Pain was assessed using the Neonatal Infant Pain Scale (NIPS) at baseline and at 2, 10, and 120 seconds post-procedure. Results: All three interventions effectively reduced procedural pain. Mean NIPS scores at 2 seconds were 2.17±1.76 for breastfeeding, 2.29±1.69 for KMC, and 2.61±1.72 for oral dextrose group (p=0.25). Complete pain relief (NIPS=0 at 10 seconds) differed significantly across groups: 83.8% for breastfeeding, 78.8% for KMC, and 65.0% for oral dextrose (p=0.016). However, after adjusting for age and birth weight, KMC showed lower pain scores than breastfeeding, though the difference was not significant. Conclusion: All three non-pharmacological methods were effective in reducing procedural pain. Between-group differences in mean pain scores were not statistically significant. These methods offer safe, accessible options for procedural pain management in neonates. neonatal pain non-pharmacological pain relief breastfeeding kangaroo mother care skin-to-skin contact oral dextrose neonatal infant pain scale procedural pain Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Neonates admitted to intensive care units undergo numerous painful procedures daily. Studies report an average of 7–17 painful procedures per day, with some neonates experiencing over 300 procedures during their NICU stay. 1,2,3 Common procedures include heel lancing, venipuncture, endotracheal suctioning, and intravenous cannulation. Although clinically necessary, procedural pain has important implications for both immediate well-being and long-term neurodevelopmental outcomes of the infant. The neurobiological basis of pain perception is established early in fetal life. By the second trimester, nociceptors, sensory neurons, and cortical pain-processing centers are functional. Ionotropic glutamate receptors involved in nociceptive transmission (AMPA and kainate) are expressed early in the fetal life. 4 Therefore, neonates possess the capability to detect and transmit painful stimuli. 5 However, neonatal pain differs from adult pain. Immature pain pathways increase the vulnerability to painful stimuli. Structural and functional differences in neonatal spinal cord sensory connections can enhance and prolong the effects of both noxious and innocuous sensory inputs. Normally, pain signals use AMPA receptors, but with repeated painful stimulation, NMDA receptors activate and cause central sensitization and hypersensitivity. This immature but hyperreactive NMDA system, combined with underdeveloped inhibitory control, causes the neonate to experience exaggerated pain. 5,6 At the spinal level, dorsal horn neurons occupy a larger receptive field area compared to adults. 7,8 As a result, a given stimulus activates a broader neuronal population, producing a more diffuse sensory response. In addition, the descending inhibitory pathways are immature at birth, limiting endogenous analgesic attenuation of persistent noxious stimuli. 9,10 Because neonates can detect painful stimuli but have limited capacity to modulate them, painful procedures activate their stress response. This manifests as surges in catecholamines and cortisol; increases in heart rate, blood pressure, and respiratory rate; oxygen desaturation; and disrupted glucose metabolism. 11,12 These changes increase the risk of complications such as intraventricular hemorrhage and hypoxia and may contribute to poor weight gain and delayed healing, particularly in preterm and critically ill neonates. Such sensitization may persist beyond the NICU stay. Neonates exposed to repeated heel lances in the first 24 to 36 hours exhibit more intense responses during subsequent venipuncture. 13 In preterm and extremely low birth weight infants, greater exposure to invasive procedures is associated with less mature pain responses and subtle differences in autonomic regulation and behavioral recovery. 14,15 Infants who underwent painful circumcision showed amplified pain responses to vaccination at 4 and 6 months compared to uncircumcised infants. 16 Children with neonatal pain exposure and poor maternal sensitivity were found to have higher somatization at 4.5 years, suggesting that early pain's long-term effects may be influenced by the quality of parent-child interaction. 17 Animal models further support these findings, demonstrating decreased pain thresholds and altered stress-related behavior after repetitive neonatal pain. 18 Pharmacological pain management in neonates remains limited because immature hepatic and renal function can make drug metabolism unpredictable and increase accumulation risk, 19 and because opioids such as morphine and fentanyl carry risks of respiratory depression, hypotension, and potential neurotoxicity. 20,21 These limitations have driven interest in non-pharmacological alternatives such as breastfeeding, kangaroo mother care (KMC), and oral dextrose. These interventions act through complementary mechanisms: endogenous opioid pathway activation, stress regulation with vital-sign stabilization, and comforting sensory input that competes with pain signaling. 22 Analgesic effects may be mediated via endogenous endorphin release at the spinal level 23 and via neuropeptide systems that potentiate opioid activity. 24 These approaches are physiologically compatible with the developing nervous system, feasible without specialized equipment, and carry minimal risk of cumulative toxicity. 25,26 Breastfeeding and kangaroo mother care may also promote calming through skin-to-skin contact, maternal scent, and bonding, strengthening parent–infant relationships. 27,28 The Neonatal Infant Pain Scale (NIPS) provides a validated, practical tool for bedside pain assessment. 29 It evaluates six parameters—facial expression, crying, breathing pattern, arm movement, leg movement, and state of arousal—yielding scores from 0 to 7, with scores ≥3 indicating significant pain requiring intervention. Its simplicity and rapid scoring time make it particularly suitable for busy clinical environments and low-resource settings. While individual non-pharmacological interventions have demonstrated efficacy in reducing procedural pain, direct comparative studies evaluating their relative effectiveness remain limited. Most evidence derives from placebo-controlled trials of single interventions. This gap is particularly pronounced in low- and middle-income settings, where implementation feasibility is a primary consideration. We compared breastfeeding, kangaroo mother care, and oral dextrose for heel-lance pain relief in neonates. The primary objective of our study was to compare NIPS scores across groups at standardized time points; secondary objectives were recovery time to baseline, and the proportion of neonates achieving effective and complete pain relief. Methods Study Design and Setting This was a cross-sectional observational study conducted at a tertiary care referral center in the state of Rajasthan from August 2023 to December 2024. The facility is a public care center with high patient turnover and nearly 100% bed occupancy. The center has 378 neonatal beds across 9 neonatal care units, with a patient-to-nurse ratio of 5.5:1 per shift. Study Participants All neonates admitted to the neonatal care units, requiring heel lancing for blood glucose monitoring were screened for eligibility. Inclusion: Term and preterm neonates aged 0–28 days, delivered under spinal anesthesia if cesarean, and hemodynamically stable, were included in the study. Exclusion: Neonates with perinatal asphyxia or intracranial bleeding; those who received sedatives, muscle relaxants, analgesics, or antiepileptics within the preceding 24 hours; those with hypoglycemia, defined as blood glucose levels of ≤ 45 mg/dl, 34 diagnosed during heel prick; those with acute conditions requiring critical care, surgery, or continuous sedation; those exposed to maternal opioid use during pregnancy or labor; those with congenital malformations affecting pain perception or expression; and those whose parents refused informed consent were excluded. Sample size was calculated for comparing mean NIPS scores between two groups using the formula: n = (Z₁₋α/₂ + Z₁₋β)² × 2σ² / d². Based on Efe and Savaşer, 35 we assumed a standard deviation (σ) of 1.9 and a minimum clinically meaningful difference (d) of 1.0 point: n = (1.96 + 0.84)² × 2 × 1.9² / 1.0² = 7.84 × 7.22 = 57 per group. The sample size was increased to 80 per group to enhance statistical power for the three-group comparison and to account for potential dropouts. Sampling and group allocation: We used convenience sampling to enroll eligible neonates during routine clinical rounds. The pain relief intervention was selected by the treating pediatric consultant as part of standard clinical practice. Neonates receiving breastfeeding were included in the breastfeeding group (n=80), those in the KMC nursery were included in the KMC group (n=80), and those for whom maternal interventions were not feasible—mother not present, breastfeeding not allowed due to prematurity, or feeding restricted for medical reasons—received oral dextrose as per unit protocol (n=80). Procedures Breastfeeding protocol: Mother positioned comfortably holding her baby in cradle hold; breastfeeding initiated at least 1 minute before the procedure; good latch confirmed before heel lance; breastfeeding continued throughout and after the procedure. KMC protocol: Neonate placed prone on mother’s bare chest between breasts, wearing only a diaper and covered with KMC gown and cotton sheet; skin-to-skin contact initiated at least 15 minutes before the procedure and maintained throughout. Oral dextrose protocol: 2 mL of 10% dextrose was administered using a pacifier made from autoclaved cotton rolled in sterile autoclaved bandage, given at least 1 minute before heel lance; neonate positioned supine in radiant warmer. A standardized painful stimulus was given—heel lancing using a spring-loaded lancet—for clinically indicated blood glucose monitoring. The baby’s foot was held stable, heel cleansed with surgical spirit and allowed to dry. The lateral aspect of the baby’s heel was punctured using an automatic spring-loaded lancet. The prick was instantaneous; blood was then collected onto a test strip for bedside glucometer measurement (2–5 seconds), followed by gentle pressure with sterile gauze for hemostasis. The researcher documented NIPS scores at standardized time points without influencing care decisions. Pain Assessment NIPS assessment was performed by a single trained pediatric resident doctor who observed the neonate throughout the procedure. The resident’s role was limited to pain assessment; the heel lancing procedure was performed by trained nursing staff responsible for routine neonatal procedures. Neither the resident assessing NIPS nor the nursing staff performing the procedure were blinded to the pain relief intervention, as the nature of the interventions (breastfeeding, KMC, oral dextrose) precluded blinding in this study design. Training of assessor: Prior to data collection, the pediatric resident received hands-on training sessions of approximately 30 minutes per day for six consecutive days, from pediatric consultants, including scoring of neonates during routine procedures. Training continued until the resident achieved consistent NIPS scores, verified by two independent pediatric consultants. Training of nursing staff: Nursing staff responsible for routine neonatal procedures received separate hands-on training in lancet-based heel prick technique. Data Collection Baseline data collected included demographic characteristics (age, sex, birth weight category), clinical condition, and pre-procedure NIPS assessment. Outcome Measures The primary outcome was NIPS score measured at four time points: baseline (120 seconds before heel prick), immediate response (2 seconds post-procedure), early recovery (10 seconds post-procedure), and late recovery (120 seconds post-procedure). Secondary outcomes were recovery time, defined as duration for NIPS to return to baseline, for which the examiner continuously assessed NIPS after the heel prick until the scores returned to 0, and recorded the time of return to baseline score; proportion of neonates achieving effective pain relief (NIPS≤3 at 10 seconds); proportion of neonates achieving complete pain relief (NIPS=0 at 10 seconds). Statistical Analysis Data were compiled using Microsoft Excel. Categorical variables were expressed as frequencies and percentages; continuous variables as mean ± standard deviation, or median and range where appropriate. Chi-square tests were used for categorical comparisons, one-way ANOVA for continuous variables across groups, and independent t-tests for pairwise comparisons. Because of statistically significant differences in age and birth weight categories across all groups, analysis of covariance (ANCOVA) was performed with intervention group as independent variable, NIPS score as dependent variable, and age (days) and birth weight category (LBW vs. normal) as covariates; adjusted means were calculated using estimated marginal means. All statistical analysis was done using R version 4.5.1 (R Core Team, 2025), 31 with the Companion to Applied Regression (car) package for ANCOVA 32 and Estimated Marginal Means (emmeans) package for adjusted means. 33 The full reproducible code is available in supplementary materials. P-value of <0.05 was considered statistically significant. Ethics Written informed consent was obtained from all mothers before enrollment. The study protocol was approved by the Institutional Ethics Committee (Reference no. MC/EC/2023/368; Date: 18/01/2024) and conducted in accordance with the Declaration of Helsinki. Heel prick procedures done in this study were clinically indicated, and no additional heel pricks were done for study purposes. Results A total of 312 neonates were screened for eligibility during the study period. Of these, 72 were excluded and 240 were enrolled and allocated to three intervention groups. All 240 completed the study with no post-enrollment exclusions or missing data (figure 1). The study groups differed significantly in age distribution (p<0.001), and birth weight categories (p<0.001). Clinical condition profiles also varied across the intervention groups (Table 1). Table 1: Baseline demographic characteristics Characteristic Breastfeeding (n=80) KMC (n=80) Oral Dextrose (n=80) Total (N=240) p-value Age (days), Mean±SD 5.69±6.26 15.09±7.94 8.70±8.43 9.82±8.53 — Age group <0.001 <7 days 62 (77.5%) 16 (20.0%) 47 (58.8%) 125 (52.1%) ≥7 days 18 (22.5%) 64 (80.0%) 33 (41.3%) 115 (47.9%) Sex 0.58 Male 46 (57.5%) 47 (58.8%) 52 (65.0%) 145 (60.4%) Female 34 (42.5%) 33 (41.3%) 28 (35.0%) 95 (39.6%) Birth weight <0.001 ≥2500 g 69 (86.3%) 6 (7.5%) 39 (48.8%) 114 (47.5%) <2500 g 11 (13.7%) 74 (92.5%) 41 (51.2%) 126 (52.5%) Clinical conditions Respiratory distress 13 (16.2%) 64 (80.0%) 52 (65.0%) 129 (53.8%) <0.001 Neonatal jaundice 7 (8.8%) 8 (10.0%) 16 (20.0%) 31 (12.9%) 0.067 Refusal to feed 1 (1.2%) 8 (10.0%) 10 (12.5%) 19 (7.9%) 0.022 Infant of diabetic mother 1 (1.2%) 0 (0.0%) 3 (3.8%) 4 (1.7%) — KMC, Kangaroo Mother Care; SD, Standard Deviation Table 2 shows pain scores and recovery time across all intervention groups. Baseline (120 seconds before heel lance) NIPS scores for all neonates were 0. After heel lancing, the breastfeeding group had the lowest initial (2 seconds post-procedure) pain response (2.17±1.76), followed by KMC (2.29±1.69) and oral dextrose (2.61±1.72), though the differences were not statistically significant (p=0.25). Table 2: Pain scores and recovery time Outcome Breastfeeding (n=80) KMC (n=80) Oral Dextrose (n=80) p-value NIPS at 2 sec, Mean±SD 2.17±1.76 2.29±1.69 2.61±1.72 0.25 NIPS at 10 sec, Mean±SD 0.50±1.27 0.64±1.34 0.84±1.40 0.28 NIPS at 10 sec, 95% CI 0.22–0.78 0.34–0.94 0.53–1.15 — NIPS change (2→10 sec) 1.68±1.41 1.65±1.41 1.77±1.28 0.83 Recovery time (sec), Mean±SD 7.61±12.74 8.40±11.15 11.39±12.99 0.13 Recovery time (sec), Median 3.0 5.5 7.5 — KMC, Kangaroo Mother Care; NIPS, Neonatal Infant Pain Scale; SD, Standard Deviation; CI, Confidence Interval At 10 seconds post-procedure, pain reduction was observed across all groups. Breastfeeding achieved the lowest NIPS scores (0.50±1.27), followed by KMC (0.64±1.34) and oral dextrose (0.84±1.40). At 120 seconds, all neonates’ NIPS scores returned to baseline (NIPS=0). Adjusted Analysis (ANCOVA) After controlling for age and birth weight category, the adjusted mean NIPS scores at 2 seconds were 2.28 for breastfeeding, 2.15 for KMC, and 2.66 for oral dextrose group. Adjusted mean NIPS scores at 10 seconds were 0.62 for breastfeeding, 0.48 for KMC, and 0.87 for oral dextrose group (figure 2). The difference remained statistically non-significant after adjustment (p=0.16 at 2 seconds; p=0.22 at 10 seconds). Age was a significant variable affecting pain scores at both time points (p=0.004 at 2 seconds; p=0.027 at 10 seconds), with older neonates showing higher pain responses across all intervention groups (figure 3). Birth weight category was not significant after controlling for age. After adjustment, the KMC group showed lower pain scores than the breastfeeding group (0.48 vs. 0.62 at 10 seconds), compared to unadjusted scores where the breastfeeding group showed lower scores (0.50 vs. 0.64). Table 3: NIPS scores adjusted to age and birth weight categories Time Point Variable F p-value 2 seconds Intervention group 1.86 0.16 Age (days) 8.23 0.004 Birth weight category 0.48 0.49 10 seconds Intervention group 1.54 0.22 Age (days) 4.93 0.027 Birth weight category 0.05 0.83 Discussion This observational study compared breastfeeding, kangaroo mother care (KMC), and oral dextrose for pain relief in neonates. All three interventions were associated with low pain scores, with no statistically significant differences in mean NIPS scores between groups. Breastfeeding for procedural pain relief Shah et al.’s Cochrane review of 20 studies concluded that breastfeeding or breast milk should be used for procedural pain, with oral glucose/sucrose showing similar effectiveness. 28 Efe and Savaşer found no significant difference in NIPS scores between breastfeeding (1.9±1.9) and sucrose (1.2±1.3), both significantly lower than control (6.4±1.6). 35 Our findings confirm comparable effects, with NIPS scores of 2.17±1.76 for breastfeeding and 2.61±1.72 for oral dextrose at 2 seconds, and 0.50±1.27 and 0.84±1.40 respectively at 10 seconds. Okan et al. demonstrated that both skin-to-skin contact alone and breastfeeding with skin-to-skin contact significantly reduced pain responses compared to no intervention. 36 Pain relief from breastfeeding is mediated via oral stimulation through suckling, gustatory stimulation from milk taste, olfactory stimulation from breast milk odor, tactile warmth from skin contact, and auditory stimulation from maternal heartbeat. 37 Campbell-Yeo et al. noted significantly higher effectiveness of breastfeeding compared to breast milk alone. 38 Kangaroo mother care for procedural pain relief A meta-analysis of six studies conducted by Sharma et al. found significant benefit of KMC over standard care for pain reduction. 39 Chidambaram et al. reported significantly lower PIPP scores during KMC versus without KMC (4.3 and 3.84 vs. 5.76 and 5.24 at 15 and 30 minutes respectively) for heel-prick pain in preterm neonates. 30 A Cochrane review of 25 studies by Johnston et al. (n=2001 infants) found that skin-to-skin care significantly reduced PIPP scores at 30 seconds (MD -3.21), 60 seconds (MD -1.64), and 90 seconds (MD -1.28) compared to no intervention, though by 120 seconds the difference was no longer significant. 40 These findings align with our observation that all NIPS scores returned to baseline (NIPS=0) by 120 seconds post-procedure. Shukla et al. found significantly lower PIPP scores with KMC (7.7±3.9) versus control (11.5±3.4), concluding that KMC with expressed breast milk should be the first choice for pain control in preterm neonates. 41 Our findings support this recommendation, with KMC showing effective pain reduction comparable to breastfeeding, even in predominantly preterm cohort. Kapoor et al. found comparable PIPP scores between KMC (8.42±1.99) and oral dextrose 50% (8.76±1.84) groups, both significantly lower than nesting (13.08±1.70). 42 This study supports our finding of comparable efficacy between KMC and oral dextrose for heel prick analgesia. Oral dextrose for procedural pain relief Mahmud et al. found significantly lower Modified Behavioral Pain Scale (MBPS) with 10% dextrose (4.31) versus sterile water (6.26), concluding that oral dextrose is cheap and effective for neonatal pain management. 43 Shanthi et al. reported significantly lower pain scores during heel prick in 10% dextrose group compared to expressed breast milk group using PIPP-R scale at all time points: at initiation (4.97±1.42 vs. 7.19±2.0), at 30 seconds (2.36±1.44 vs. 5.5±1.5), and at 60 seconds (1.69±1.53 vs. 4.28±1.65). 44 In contrast, Nayak et al. found no significant difference between 10% dextrose, expressed breast milk, and sterile water for ROP screening pain in preterm neonates, with mean PIPP scores as 9.8±3.3, 11.8±2.8, and 10.2±2.9 respectively. 45 Ravishankar et al. found that 25% dextrose had significantly lower PIPP scores (median=10) compared to 10% dextrose and placebo (median=12 each), during nasogastric tube insertion pain. 46 Ranjbar et al. found significantly better PIPP scores with oral dextrose (7.60±1.17) and facilitated tucking (9.56±1.15) compared to control (12.90±1.14) during heel sticks. 47 These findings suggest that oral dextrose effectiveness may vary with dextrose concentration and pain intensity. A systematic review and network meta-analysis of 103 trials by Abiramalatha et al. found non-nutritive sucking plus sucrose most effective (SMD −3.15), followed by breastfeeding, then skin-to-skin contact. 48 This analysis supports the use of mother-based non-pharmacological pain relief approaches, consistent with our findings. An integrative review of 27 studies conducted by Sutton and Lemermeyer found that sucrose, facilitated tucking, pacifier, skin-to-skin care, and human milk lessened pain during heel sticks, suctioning, nasogastric tube insertions, and echocardiograms. 25 However, all nonpharmacological interventions failed to adequately manage pain during ROP screening, consistent with Nayak et al.’s findings. 45 This review supports the use of nonpharmacological measures for routine NICU procedures. In our study, oral dextrose 10% was effective but showed the slowest pain attenuation and lowest proportion of complete pain relief. The absence of maternal contact and suckling may limit the depth and speed of soothing compared to breastfeeding and KMC, but oral dextrose remains a practical option when maternal interventions are not feasible. 38 Secondary outcomes Recovery time Mean recovery time was shortest for breastfeeding (7.61 seconds), followed by KMC (8.40 seconds), and oral dextrose (11.39 seconds), though differences were not statistically significant (P=0.13). Baijnathan and Arya found significantly shorter recovery with immediate KMC (iKMC) versus radiant warmer care (33.3±20.22 vs 92.43±49.68 seconds) during heel prick. 49 The longer recovery times in their study compared to ours likely reflect differences in population (sick LBW neonates vs. mixed cohort) and recovery definition (heart rate return to baseline vs. NIPS return to baseline). Both studies support faster recovery with maternal-based interventions. Effective and complete pain relief Effective pain relief (NIPS≤3 at 10 seconds) was achieved in similar proportions across groups: 95.0% (breastfeeding), 93.8% (KMC), and 93.8% (oral dextrose). However, complete pain relief (NIPS=0 at 10 seconds) differed significantly: 83.8% (breastfeeding), 78.8% (KMC), and 65.0% (oral dextrose) (P=0.016). This difference reflected a higher proportion of partial responders (NIPS 1–2) in the oral dextrose group. While all three interventions prevented severe pain, breastfeeding and KMC more frequently achieved complete analgesia. ANCOVA findings: age and pain response In our study, the baseline characteristics differed significantly across groups. The breastfeeding group comprised predominantly younger neonates (77.5% neonates <7 days) with normal birth weight (86.3%), while the KMC group comprised predominantly older neonates (80% neonates ≥7 days) with low birth weight (92.5%). This reflects standard clinical practice where breastfeeding was initiated from birth in stable neonates with normal birth weight and mature sucking-swallowing-breathing coordination, while KMC was initiated predominantly in low birth weight neonates after initial stabilization. We applied ANCOVA to adjust for age and birth weight. After adjustment, between-group differences in NIPS remained non-significant (P=0.16 at 2 seconds; P=0.22 at 10 seconds), but age was a significant independent predictor at both 2 seconds (P=0.004) and 10 seconds (P=0.027) post-procedure. Older neonates showed higher pain scores across all three intervention groups, regardless of intervention type. Birth weight category was not a significant predictor after adjustment. Adjusted mean NIPS scores at 10 seconds were 0.62 for breastfeeding, 0.48 for KMC, and 0.87 for oral dextrose. After adjustment, KMC showed lower pain scores than breastfeeding (0.48 vs. 0.62), reversing the unadjusted pattern in which breastfeeding had lower scores (0.50 vs. 0.64). This suggests that the lower NIPS scores of breastfeeding in unadjusted analysis may reflect the younger age of neonates in this group rather than a true intervention effect. This pattern is consistent with developmental studies of pain expression. Johnston et al. (1993) found that full-term neonates showed more differentiated facial and cry responses than premature infants. 50 Another study by Johnston et al. (1995) reported that gestational age significantly affects pain response, with younger infants responding less robustly. 51 In our mixed cohort of term and preterm neonates, older neonates may have demonstrated greater behavioral differentiation and thus more observable pain responses on NIPS, which scores facial expression, cry, and motor parameters. NIPS proved feasible for pain assessment in our setting. Assessment was simple, required no equipment, minimal training, and provided rapid bedside scoring, which supports its use in low- and middle-income settings where advanced monitoring may not be available. 29 Strength and limitations We implemented a standardized procedural protocol with consistent lancet gauge, insertion site and depth (spring loaded lancet), and timing of assessments across all participants. NIPS scoring by a single, trained assessor minimized inter-observer variability. The use of validated NIPS instrument and serial measurements at multiple time points allowed us to plot pain response trajectories. This practical design enhances external validity in similar resource-constrained settings. However, several limitations deserve consideration. The non-randomized, purposive allocation introduced baseline differences between groups—the KMC group comprised predominantly preterm and low birth weight neonates, while the breastfeeding group included mostly term, normal-weight infants. These differences may have confounded outcome comparisons. Gestational age was not recorded, precluding analysis of prematurity effects independent of birth weight. This is particularly relevant given that 92.5% of neonates in the KMC group had low birth weight. The absence of a control group precluded analysis of the natural trajectory of pain resolution compared to intervention groups. Pain assessment was limited to the immediate procedural period (up to 120 seconds), leaving long-term neurodevelopmental outcomes unevaluated. Our study design did not allow blinding; this may have introduced observer bias in subjective components such as facial expression. Finally, the single-center tertiary care setting and exclusion of critically ill neonates requiring intensive care may limit generalizability. Future research scope Future studies should prioritize well-designed RCTs with stratified randomization by gestational age and birth weight to enable balanced groups and valid comparisons. Crossover designs could control for individual variation while improving statistical power. Multi-center trials across diverse settings would enhance generalizability, and combined or sequential interventions (e.g., breastfeeding with oral sucrose, KMC with non-nutritive sucking) warrant further investigation. Long-term follow-up is needed to determine whether effective procedural pain management influences neurodevelopmental outcomes, stress reactivity, or pain sensitivity in later childhood. Implementation research should evaluate strategies for integrating non-pharmacological interventions into routine protocols and identify barriers to adoption in resource-limited settings. Declarations Financial support and sponsorship Nil. Conflicts of interest None. References Barker DP, Rutter N. Exposure to invasive procedures in neonatal intensive care unit admissions. Arch Dis Child Fetal Neonatal Ed. 1995 Jan;72(1):F47-8. doi: 10.1136/fn.72.1.f47. PMID: 7743285; PMCID: PMC2528401. Perry M, Tan Z, Chen J, Weidig T, Xu W, Cong XS. Neonatal Pain: Perceptions and Current Practice. Crit Care Nurs Clin North Am. 2018 Dec;30(4):549-561. doi: 10.1016/j.cnc.2018.07.013. PMID: 30447813; PMCID: PMC6570422. Vinall J, Grunau RE. Impact of repeated procedural pain-related stress in infants born very preterm. Pediatr Res. 2014 May;75(5):584-7. doi: 10.1038/pr.2014.16. Epub 2014 Feb 5. PMID: 24500615; PMCID: PMC3992189. Akesson E, Kjaeldgaard A, Samuelsson EB, Seiger A, Sundström E. 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Early pain experience, child and family factors, as precursors of somatization: a prospective study of extremely premature and fullterm children. Pain. 1994 Mar;56(3):353-359. doi: 10.1016/0304-3959(94)90174-0. PMID: 8022628 Anand KJ, Coskun V, Thrivikraman KV, Nemeroff CB, Plotsky PM. Long-term behavioral effects of repetitive pain in neonatal rat pups. Physiol Behav. 1999 Jun;66(4):627-37. doi: 10.1016/s0031-9384(98)00338-2. PMID: 10386907; PMCID: PMC4211637. Hall RW, Anand KJ. Pain management in newborns. Clin Perinatol. 2014 Dec;41(4):895-924. doi: 10.1016/j.clp.2014.08.010. Epub 2014 Oct 7. PMID: 25459780; PMCID: PMC4254489. Lago P, Garetti E, Merazzi D, Pieragostini L, Ancora G, Pirelli A, Bellieni CV; Pain Study Group of the Italian Society of Neonatology. Guidelines for procedural pain in the newborn. Acta Paediatr. 2009 Jun;98(6):932-9. doi: 10.1111/j.1651-2227.2009.01291.x. PMID: 19484828; PMCID: PMC2688676. 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Epub 2005 Oct 20. PMID: 16242828. Sutton R, Lemermeyer G. Nonpharmacological Interventions to Mitigate Procedural Pain in the NICU: An Integrative Review. Adv Neonatal Care. 2024 Aug 1;24(4):364-373. doi: 10.1097/ANC.0000000000001164. Epub 2024 Jun 22. PMID: 38907705. McPherson C, Miller SP, El-Dib M, Massaro AN, Inder TE. The influence of pain, agitation, and their management on the immature brain. Pediatr Res. 2020 Aug;88(2):168-175. doi: 10.1038/s41390-019-0744-6. Epub 2020 Jan 2. PMID: 31896130; PMCID: PMC7223850. Tessier R, Cristo M, Velez S, Giron M, de Calume ZF, Ruiz-Palaez JG, Charpak Y, Charpak N. Kangaroo mother care and the bonding hypothesis. Pediatrics. 1998 Aug;102(2):e17. doi: 10.1542/peds.102.2.e17. PMID: 9685462. Shah PS, Herbozo C, Aliwalas LL, Shah VS. Breastfeeding or breast milk for procedural pain in neonates. Cochrane Database Syst Rev. 2012 Dec 12;12(12):CD004950. doi: 10.1002/14651858.CD004950.pub3. 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PMID: 31762179. Campbell-Yeo M, Fernandes A, Johnston C. Procedural pain management for neonates using nonpharmacological strategies: part 2: mother-driven interventions. Adv Neonatal Care. 2011 Oct;11(5):312-8; quiz pg 319-20. doi: 10.1097/ANC.0b013e318229aa76. PMID: 22123399. Sharma H, Ruikar M. Kangaroo mother care (KMC) for procedural pain in infants: A meta-analysis from the current evidence of randomized control trials and cross-over trials. J Family Med Prim Care. 2022 Apr;11(4):1250-1256. doi: 10.4103/jfmpc.jfmpc_1383_21. Epub 2022 Mar 18. PMID: 35516684; PMCID: PMC9067180. Johnston C, Campbell-Yeo M, Disher T, Benoit B, Fernandes A, Streiner D, Inglis D, Zee R. Skin-to-skin care for procedural pain in neonates. Cochrane Database Syst Rev. 2017 Feb 16;2(2):CD008435. doi: 10.1002/14651858.CD008435.pub3. PMID: 28205208; PMCID: PMC6464258. Shukla VV, Bansal S, Nimbalkar A, Chapla A, Phatak A, Patel D, Nimbalkar S. Pain Control Interventions in Preterm Neonates: A Randomized Controlled Trial. Indian Pediatr. 2018 Apr 15;55(4):292-296. Epub 2018 Feb 9. PMID: 29428919. Kapoor A, Khan MA, Beohar V. Pain Relief in Late Preterm Neonates: A Comparative Study of Kangaroo Mother Care, Oral Dextrose 50%, and Supine Nesting Position. Int J Appl Basic Med Res. 2021 Jul-Sep;11(3):188-191. doi: 10.4103/ijabmr.IJABMR_584_20. Epub 2021 Jul 19. PMID: 34458123; PMCID: PMC8360213. Mahmud S, Shah SA, Khattak SZ. Neonatal Pain And Preventive Strategies: An Experience In A Tertiary Care Unit. J Ayub Med Coll Abbottabad. 2017 Jan-Mar;29(1):42-44. PMID: 28712171. Shanthi Y, Thandapani K, Krishnakumar R, Thirunavukkarasu BK. Efficacy of 10% Dextrose versus Expressed Breast Milk in Relieving Procedural Pain in Neonates – A Randomized Controlled Trial. J Clin Neonatol. 2024 Jul-Sep;13(3):90-94. doi: 10.4103/jcn.jcn_37_24. Nayak R, Nagaraj KN, Gururaj G. Prevention of Pain During Screening for Retinopathy of Prematurity: A Randomized Control Trial Comparing Breast Milk, 10% Dextrose and Sterile Water. Indian J Pediatr. 2020 May;87(5):353-358. doi: 10.1007/s12098-020-03182-6. Epub 2020 Jan 27. PMID: 31989459; PMCID: PMC7223887. Ravishankar A, Thawani R, Dewan P, Das S, Kashyap A, Batra P, Faridi MM. Oral dextrose for analgesia in neonates during nasogastric tube insertion: a randomised controlled trial. J Paediatr Child Health. 2014 Feb;50(2):141-5. doi: 10.1111/jpc.12392. Epub 2013 Oct 18. PMID: 24134074. Ranjbar A, Bernstein C, Shariat M, Ranjbar H. Comparison of facilitated tucking and oral dextrose in reducing the pain of heel stick in preterm infants: a randomized clinical trial. BMC Pediatr. 2020 Apr 14;20(1):162. doi: 10.1186/s12887-020-2020-7. PMID: 32290829; PMCID: PMC7155270. Abiramalatha T, Ramaswamy VV, Anne RP, Amuji N, Thinesh J, Venkateshwarlu V, Rao VP, Shaik NB, Pullattayil AK, Balachander B, Sivanandhan S, Kumar J, Gupta N, Chawla D, Kumar P, Rao S. Comparative Efficacy of Interventions for Analgesia During Heel Prick in Newborn Infants - A Systematic Review and Network Meta-Analysis. Indian Pediatr. 2024 Sep 15;61(9):851-875. doi: 10.1007/s13312-024-3279-9. PMID: 39193923. Baijnathan I, Arya S. Effect of Immediate Kangaroo Mother Care on Pain in Sick Low Birth Weight Neonates During Heel Prick in Mother–Newborn Intensive Care Unit. Indian J Palliat Care. 2024;30(2):142-147. doi: 10.1177/09732179241237864. Johnston CC, Stevens B, Craig KD, Grunau RVE. Developmental changes in pain expression in premature, full-term, two- and four-month-old infants. Pain. 1993 Feb;52(2):201-208. doi: 10.1016/0304-3959(93)90132-9. PMID: 8455968. Johnston CC, Stevens BJ, Yang F, Horton L. Differential response to pain by very premature neonates. Pain. 1995 Jun;61(3):471-479. doi: 10.1016/0304-3959(94)00213-X. PMID: 7478691. Additional Declarations No competing interests reported. Supplementary Files supplfigure1paintrajectory.jpeg Supplementary figure 1 Mean NIPS scores over time (baseline → 2 seconds → 10 seconds → 120 seconds) supplfigure2recoveryboxplot.jpeg Supplementary figure 2 Distribution of recovery time for each intervention group statisticalanalysis.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-9112827","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":609403405,"identity":"6c1317b8-6b3c-4798-bb6a-3ab204b591c6","order_by":0,"name":"Anjali Gupta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABHElEQVRIie2RMWrDMBSGBQZnUelqCLVOUHhGEDrlGJ2fMcSLAx49tMFQ8B2Ch17BIdCQTRBIFh9A0A7xDdSphRZa2YZ0qZy1UH2DEOL/+N9DhFgsfxMkR+hvgNmX317E8ZyCreISkqra4Z2C53t65XVZOGF+ejFwWeaNwvQ+3LCHZk1rN3683TW6Zepf578r3ouIPIRDuC1czmlG56vnGWgl4hNhqJGIWtmH1Z5MOK29+arEVhHhk0FhEqP3Xhm9fVwUEAdlrAYVkDjTLXdaoRAsC0Q2ToZbAq3c6En4tkhSULUIqnGSCgTzLr5MIqk+F1cb51DprxSMlfFaqWzqG9cnFPWxI3AatUuCId0xaiOLnwzLh9IWi8XyH/kGai1taASZfFoAAAAASUVORK5CYII=","orcid":"","institution":"Sawai ManSingh Medical College and Hospital","correspondingAuthor":true,"prefix":"","firstName":"Anjali","middleName":"","lastName":"Gupta","suffix":""},{"id":609403406,"identity":"52afdf45-a8df-4d17-9417-6101aead6cff","order_by":1,"name":"Ramesh Choudhary","email":"","orcid":"","institution":"Sawai ManSingh Medical College and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ramesh","middleName":"","lastName":"Choudhary","suffix":""},{"id":609403407,"identity":"35e428e8-4aaa-4052-b46b-8425329854c4","order_by":2,"name":"Chetan Meena","email":"","orcid":"","institution":"Sawai ManSingh Medical College and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chetan","middleName":"","lastName":"Meena","suffix":""},{"id":609403408,"identity":"780c8cc9-d552-43f7-99f9-ed8a17cfca3e","order_by":3,"name":"Vijendra Garg","email":"","orcid":"","institution":"Sawai ManSingh Medical College and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Vijendra","middleName":"","lastName":"Garg","suffix":""},{"id":609403409,"identity":"f9607bf1-f104-4a8c-a369-f0679a0bdc6c","order_by":4,"name":"Sourabh Agrawal","email":"","orcid":"","institution":"Sawai ManSingh Medical College and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sourabh","middleName":"","lastName":"Agrawal","suffix":""}],"badges":[],"createdAt":"2026-03-13 09:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9112827/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9112827/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105297623,"identity":"e63b9dbd-c9da-4551-bcfc-1dc4240866fe","added_by":"auto","created_at":"2026-03-24 13:21:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80626,"visible":true,"origin":"","legend":"\u003cp\u003eStudy flow\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9112827/v1/2ee86e5f2b5fb00ab42679c2.png"},{"id":105297625,"identity":"918f3dae-8520-4439-979c-0486d49fb389","added_by":"auto","created_at":"2026-03-24 13:21:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57195,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of unadjusted vs. adjusted (for age and birth weight) mean NIPS scores at 2 seconds and 10 seconds\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9112827/v1/fc9a2bd366884cb5b78255ad.png"},{"id":105297646,"identity":"66c0c574-479b-477a-8d9a-a4b82aa94c03","added_by":"auto","created_at":"2026-03-24 13:21:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111236,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between neonatal age (days) and peak pain response (NIPS at 2 seconds); X→age in days; Y→NIPS at 2 seconds (immediate pain response); The regression lines with positive slopes indicate that older neonates show higher pain scores across all intervention groups\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9112827/v1/25e6fff303d22324b29ad465.png"},{"id":105297621,"identity":"4c92c0a9-91ee-4eeb-9fc1-6f965182ff70","added_by":"auto","created_at":"2026-03-24 13:21:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52564,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of neonates achieving pain relief at 10 seconds\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9112827/v1/579acd05e6c9d17c636b2107.png"},{"id":105903947,"identity":"827371e3-b620-4a3a-bded-d5772253fd84","added_by":"auto","created_at":"2026-04-01 09:59:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1060332,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9112827/v1/39972ce3-f9cf-41f1-98ef-513127b9a4b4.pdf"},{"id":105297622,"identity":"a1eb1ce1-e7ed-4702-bf2f-e59221608c29","added_by":"auto","created_at":"2026-03-24 13:21:00","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":289969,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 1 Mean NIPS scores over time (baseline → 2 seconds → 10 seconds → 120 seconds)\u003c/p\u003e","description":"","filename":"supplfigure1paintrajectory.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9112827/v1/e851d2f4054f783f8e4ec54d.jpeg"},{"id":105564191,"identity":"d507aee8-9a83-4367-bf94-5471a0e33a71","added_by":"auto","created_at":"2026-03-27 12:48:57","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":217812,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 2 Distribution of recovery time for each intervention group\u003c/p\u003e","description":"","filename":"supplfigure2recoveryboxplot.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9112827/v1/1e769cf269a2bbef8cd7af84.jpeg"},{"id":105297645,"identity":"36ed7cb1-0c7c-4099-8fa8-ea1f91235911","added_by":"auto","created_at":"2026-03-24 13:21:00","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12375,"visible":true,"origin":"","legend":"","description":"","filename":"statisticalanalysis.docx","url":"https://assets-eu.researchsquare.com/files/rs-9112827/v1/71a445976f0c05fc19f206ea.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Effectiveness of Non-Pharmacological Pain Management Interventions in Neonates: A Cross- Sectional Observational Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeonates admitted to intensive care units undergo numerous painful procedures daily. Studies report an average of 7–17 painful procedures per day, with some neonates experiencing over 300 procedures during their NICU stay.\u003csup\u003e1,2,3\u003c/sup\u003e Common procedures include heel lancing, venipuncture, endotracheal suctioning, and intravenous cannulation. Although clinically necessary, procedural pain has important implications for both immediate well-being and long-term neurodevelopmental outcomes of the infant.\u003c/p\u003e\n\u003cp\u003eThe neurobiological basis of pain perception is established early in fetal life. By the second trimester, nociceptors, sensory neurons, and cortical pain-processing centers are functional. Ionotropic glutamate receptors involved in nociceptive transmission (AMPA and kainate) are expressed early in the fetal life.\u003csup\u003e4\u003c/sup\u003e Therefore, neonates possess the capability to detect and transmit painful stimuli.\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHowever, neonatal pain differs from adult pain. Immature pain pathways increase the vulnerability to painful stimuli. Structural and functional differences in neonatal spinal cord sensory connections can enhance and prolong the effects of both noxious and innocuous sensory inputs. Normally, pain signals use AMPA receptors, but with repeated painful stimulation, NMDA receptors activate and cause central sensitization and hypersensitivity. This immature but hyperreactive NMDA system, combined with underdeveloped inhibitory control, causes the neonate to experience exaggerated pain.\u003csup\u003e5,6\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAt the spinal level, dorsal horn neurons occupy a larger receptive field area compared to adults.\u003csup\u003e7,8\u003c/sup\u003e As a result, a given stimulus activates a broader neuronal population, producing a more diffuse sensory response. In addition, the descending inhibitory pathways are immature at birth, limiting endogenous analgesic attenuation of persistent noxious stimuli.\u003csup\u003e9,10\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eBecause neonates can detect painful stimuli but have limited capacity to modulate them, painful procedures activate their stress response. This manifests as surges in catecholamines and cortisol; increases in heart rate, blood pressure, and respiratory rate; oxygen desaturation; and disrupted glucose metabolism.\u003csup\u003e11,12\u003c/sup\u003e These changes increase the risk of complications such as intraventricular hemorrhage and hypoxia and may contribute to poor weight gain and delayed healing, particularly in preterm and critically ill neonates.\u003c/p\u003e\n\u003cp\u003eSuch sensitization may persist beyond the NICU stay. Neonates exposed to repeated heel lances in the first 24 to 36 hours exhibit more intense responses during subsequent venipuncture.\u003csup\u003e13\u003c/sup\u003e In preterm and extremely low birth weight infants, greater exposure to invasive procedures is associated with less mature pain responses and subtle differences in autonomic regulation and behavioral recovery.\u003csup\u003e14,15\u003c/sup\u003e Infants who underwent painful circumcision showed amplified pain responses to vaccination at 4 and 6 months compared to uncircumcised infants.\u003csup\u003e16\u003c/sup\u003e Children with neonatal pain exposure and poor maternal sensitivity were found to have higher somatization at 4.5 years, suggesting that early pain's long-term effects may be influenced by the quality of parent-child interaction.\u003csup\u003e17\u003c/sup\u003e Animal models further support these findings, demonstrating decreased pain thresholds and altered stress-related behavior after repetitive neonatal pain.\u003csup\u003e18\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ePharmacological pain management in neonates remains limited because immature hepatic and renal function can make drug metabolism unpredictable and increase accumulation risk,\u003csup\u003e19\u003c/sup\u003e and because opioids such as morphine and fentanyl carry risks of respiratory depression, hypotension, and potential neurotoxicity.\u003csup\u003e20,21\u003c/sup\u003e These limitations have driven interest in non-pharmacological alternatives such as breastfeeding, kangaroo mother care (KMC), and oral dextrose. These interventions act through complementary mechanisms: endogenous opioid pathway activation, stress regulation with vital-sign stabilization, and comforting sensory input that competes with pain signaling.\u003csup\u003e22\u003c/sup\u003e Analgesic effects may be mediated via endogenous endorphin release at the spinal level\u003csup\u003e23\u003c/sup\u003e and via neuropeptide systems that potentiate opioid activity.\u003csup\u003e24\u003c/sup\u003e These approaches are physiologically compatible with the developing nervous system, feasible without specialized equipment, and carry minimal risk of cumulative toxicity.\u003csup\u003e25,26\u003c/sup\u003e Breastfeeding and kangaroo mother care may also promote calming through skin-to-skin contact, maternal scent, and bonding, strengthening parent–infant relationships.\u003csup\u003e27,28\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe Neonatal Infant Pain Scale (NIPS) provides a validated, practical tool for bedside pain assessment.\u003csup\u003e29\u003c/sup\u003e It evaluates six parameters—facial expression, crying, breathing pattern, arm movement, leg movement, and state of arousal—yielding scores from 0 to 7, with scores ≥3 indicating significant pain requiring intervention. Its simplicity and rapid scoring time make it particularly suitable for busy clinical environments and low-resource settings.\u003c/p\u003e\n\u003cp\u003eWhile individual non-pharmacological interventions have demonstrated efficacy in reducing procedural pain, direct comparative studies evaluating their relative effectiveness remain limited. Most evidence derives from placebo-controlled trials of single interventions. This gap is particularly pronounced in low- and middle-income settings, where implementation feasibility is a primary consideration.\u003c/p\u003e\n\u003cp\u003eWe compared breastfeeding, kangaroo mother care, and oral dextrose for heel-lance pain relief in neonates. The primary objective of our study was to compare NIPS scores across groups at standardized time points; secondary objectives were recovery time to baseline, and the proportion of neonates achieving effective and complete pain relief.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch3\u003eStudy Design and Setting\u003c/h3\u003e\n\u003cp\u003eThis was a cross-sectional observational study conducted at a tertiary care referral center in the state of Rajasthan from August 2023 to December 2024. The facility is a public care center with high patient turnover and nearly 100% bed occupancy. The center has 378 neonatal beds across 9 neonatal care units, with a patient-to-nurse ratio of 5.5:1 per shift.\u003c/p\u003e\n\u003ch3\u003eStudy Participants\u003c/h3\u003e\n\u003cp\u003eAll neonates admitted to the neonatal care units, requiring heel lancing for blood glucose monitoring were screened for eligibility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInclusion:\u003c/em\u003e Term and preterm neonates aged 0–28 days, delivered under spinal anesthesia if cesarean, and hemodynamically stable, were included in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExclusion:\u003c/em\u003e Neonates with perinatal asphyxia or intracranial bleeding; those who received sedatives, muscle relaxants, analgesics, or antiepileptics within the preceding 24 hours; those with hypoglycemia, defined as blood glucose levels of ≤ 45 mg/dl,\u003csup\u003e34\u003c/sup\u003e diagnosed during heel prick; those with acute conditions requiring critical care, surgery, or continuous sedation; those exposed to maternal opioid use during pregnancy or labor; those with congenital malformations affecting pain perception or expression; and those whose parents refused informed consent were excluded.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample size\u003c/em\u003e was calculated for comparing mean NIPS scores between two groups using the formula: n = (Z₁₋α/₂ + Z₁₋β)² × 2σ² / d². Based on Efe and Savaşer,\u003csup\u003e35\u003c/sup\u003e we assumed a standard deviation (σ) of 1.9 and a minimum clinically meaningful difference (d) of 1.0 point: n = (1.96 + 0.84)² × 2 × 1.9² / 1.0² = 7.84 × 7.22 = 57 per group. The sample size was increased to 80 per group to enhance statistical power for the three-group comparison and to account for potential dropouts.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSampling and group allocation:\u003c/em\u003e We used convenience sampling to enroll eligible neonates during routine clinical rounds. The pain relief intervention was selected by the treating pediatric consultant as part of standard clinical practice. Neonates receiving breastfeeding were included in the breastfeeding group (n=80), those in the KMC nursery were included in the KMC group (n=80), and those for whom maternal interventions were not feasible—mother not present, breastfeeding not allowed due to prematurity, or feeding restricted for medical reasons—received oral dextrose as per unit protocol (n=80).\u003c/p\u003e\n\u003ch3\u003eProcedures\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eBreastfeeding protocol:\u003c/em\u003e Mother positioned comfortably holding her baby in cradle hold; breastfeeding initiated at least 1 minute before the procedure; good latch confirmed before heel lance; breastfeeding continued throughout and after the procedure.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKMC protocol:\u003c/em\u003e Neonate placed prone on mother’s bare chest between breasts, wearing only a diaper and covered with KMC gown and cotton sheet; skin-to-skin contact initiated at least 15 minutes before the procedure and maintained throughout.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOral dextrose protocol:\u003c/em\u003e 2 mL of 10% dextrose was administered using a pacifier made from autoclaved cotton rolled in sterile autoclaved bandage, given at least 1 minute before heel lance; neonate positioned supine in radiant warmer.\u003c/p\u003e\n\u003cp\u003eA standardized painful stimulus was given—heel lancing using a spring-loaded lancet—for clinically indicated blood glucose monitoring. The baby’s foot was held stable, heel cleansed with surgical spirit and allowed to dry. The lateral aspect of the baby’s heel was punctured using an automatic spring-loaded lancet. The prick was instantaneous; blood was then collected onto a test strip for bedside glucometer measurement (2–5 seconds), followed by gentle pressure with sterile gauze for hemostasis. The researcher documented NIPS scores at standardized time points without influencing care decisions.\u003c/p\u003e\n\u003ch3\u003ePain Assessment\u003c/h3\u003e\n\u003cp\u003eNIPS assessment was performed by a single trained pediatric resident doctor who observed the neonate throughout the procedure. The resident’s role was limited to pain assessment; the heel lancing procedure was performed by trained nursing staff responsible for routine neonatal procedures. Neither the resident assessing NIPS nor the nursing staff performing the procedure were blinded to the pain relief intervention, as the nature of the interventions (breastfeeding, KMC, oral dextrose) precluded blinding in this study design.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTraining of assessor:\u003c/em\u003e Prior to data collection, the pediatric resident received hands-on training sessions of approximately 30 minutes per day for six consecutive days, from pediatric consultants, including scoring of neonates during routine procedures. Training continued until the resident achieved consistent NIPS scores, verified by two independent pediatric consultants.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTraining of nursing staff:\u003c/em\u003e Nursing staff responsible for routine neonatal procedures received separate hands-on training in lancet-based heel prick technique.\u003c/p\u003e\n\u003ch3\u003eData Collection\u003c/h3\u003e\n\u003cp\u003eBaseline data collected included demographic characteristics (age, sex, birth weight category), clinical condition, and pre-procedure NIPS assessment.\u003c/p\u003e\n\u003ch3\u003eOutcome Measures\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eThe primary outcome\u003c/em\u003e was NIPS score measured at four time points: baseline (120 seconds before heel prick), immediate response (2 seconds post-procedure), early recovery (10 seconds post-procedure), and late recovery (120 seconds post-procedure).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSecondary outcomes\u003c/em\u003e were recovery time, defined as duration for NIPS to return to baseline, for which the examiner continuously assessed NIPS after the heel prick until the scores returned to 0, and recorded the time of return to baseline score; proportion of neonates achieving effective pain relief (NIPS≤3 at 10 seconds); proportion of neonates achieving complete pain relief (NIPS=0 at 10 seconds).\u003c/p\u003e\n\u003ch3\u003eStatistical Analysis\u003c/h3\u003e\n\u003cp\u003eData were compiled using Microsoft Excel. Categorical variables were expressed as frequencies and percentages; continuous variables as mean ± standard deviation, or median and range where appropriate. Chi-square tests were used for categorical comparisons, one-way ANOVA for continuous variables across groups, and independent t-tests for pairwise comparisons. Because of statistically significant differences in age and birth weight categories across all groups, analysis of covariance (ANCOVA) was performed with intervention group as independent variable, NIPS score as dependent variable, and age (days) and birth weight category (LBW vs. normal) as covariates; adjusted means were calculated using estimated marginal means. All statistical analysis was done using R version 4.5.1 (R Core Team, 2025),\u003csup\u003e31\u003c/sup\u003e with the Companion to Applied Regression (car) package for ANCOVA\u003csup\u003e32\u003c/sup\u003e and Estimated Marginal Means (emmeans) package for adjusted means.\u003csup\u003e33\u003c/sup\u003e The full reproducible code is available in supplementary materials. P-value of \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e\n\u003ch3\u003eEthics\u003c/h3\u003e\n\u003cp\u003eWritten informed consent was obtained from all mothers before enrollment. The study protocol was approved by the Institutional Ethics Committee (Reference no. MC/EC/2023/368; Date: 18/01/2024) and conducted in accordance with the Declaration of Helsinki. Heel prick procedures done in this study were clinically indicated, and no additional heel pricks were done for study purposes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 312 neonates were screened for eligibility during the study period. Of these, 72 were excluded and 240 were enrolled and allocated to three intervention groups. All 240 completed the study with no post-enrollment exclusions or missing data (figure 1).\u003c/p\u003e\n\u003cp\u003eThe study groups differed significantly in age distribution (p\u0026lt;0.001), and birth weight categories (p\u0026lt;0.001). Clinical condition profiles also varied across the intervention groups (Table 1).\u003c/p\u003e\n\u003cp\u003eTable 1: Baseline demographic characteristics\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"611\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBreastfeeding (n=80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKMC (n=80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOral Dextrose (n=80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal (N=240)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAge (days), Mean±SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.69±6.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15.09±7.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8.70±8.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.82±8.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e—\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e62 (77.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e16 (20.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e47 (58.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e125 (52.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e≥7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18 (22.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e64 (80.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e33 (41.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e115 (47.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e46 (57.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e47 (58.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e52 (65.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e145 (60.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34 (42.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e33 (41.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e28 (35.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e95 (39.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBirth weight\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e≥2500 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e69 (86.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6 (7.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e39 (48.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e114 (47.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;2500 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e11 (13.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e74 (92.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e41 (51.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e126 (52.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical conditions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRespiratory distress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e13 (16.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e64 (80.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e52 (65.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e129 (53.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNeonatal jaundice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7 (8.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8 (10.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e16 (20.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e31 (12.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.067\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRefusal to feed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1 (1.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8 (10.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10 (12.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e19 (7.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInfant of diabetic mother\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1 (1.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0 (0.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3 (3.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4 (1.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e—\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eKMC, Kangaroo Mother Care; SD, Standard Deviation\u003c/p\u003e\n\u003cp\u003eTable 2 shows pain scores and recovery time across all intervention groups. Baseline (120 seconds before heel lance) NIPS scores for all neonates were 0. After heel lancing, the breastfeeding group had the lowest initial (2 seconds post-procedure) pain response (2.17±1.76), followed by KMC (2.29±1.69) and oral dextrose (2.61±1.72), though the differences were not statistically significant (p=0.25).\u003c/p\u003e\n\u003cp\u003eTable 2: Pain scores and recovery time\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"610\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOutcome\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBreastfeeding (n=80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKMC (n=80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOral Dextrose (n=80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNIPS at 2 sec, Mean±SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.17±1.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.29±1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.61±1.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNIPS at 10 sec, Mean±SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.50±1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.64±1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.84±1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNIPS at 10 sec, 95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.22–0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.34–0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.53–1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e—\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNIPS change (2→10 sec)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.68±1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.65±1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.77±1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRecovery time (sec), Mean±SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.61±12.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8.40±11.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e11.39±12.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRecovery time (sec), Median\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e—\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eKMC, Kangaroo Mother Care; NIPS, Neonatal Infant Pain Scale; SD, Standard Deviation; CI, Confidence Interval\u003c/p\u003e\n\u003cp\u003eAt 10 seconds post-procedure, pain reduction was observed across all groups. Breastfeeding achieved the lowest NIPS scores (0.50±1.27), followed by KMC (0.64±1.34) and oral dextrose (0.84±1.40). At 120 seconds, all neonates’ NIPS scores returned to baseline (NIPS=0).\u003c/p\u003e\n\u003ch3\u003eAdjusted Analysis (ANCOVA)\u003c/h3\u003e\n\u003cp\u003eAfter controlling for age and birth weight category, the adjusted mean NIPS scores at 2 seconds were 2.28 for breastfeeding, 2.15 for KMC, and 2.66 for oral dextrose group. Adjusted mean NIPS scores at 10 seconds were 0.62 for breastfeeding, 0.48 for KMC, and 0.87 for oral dextrose group (figure 2). The difference remained statistically non-significant after adjustment (p=0.16 at 2 seconds; p=0.22 at 10 seconds). Age was a significant variable affecting pain scores at both time points (p=0.004 at 2 seconds; p=0.027 at 10 seconds), with older neonates showing higher pain responses across all intervention groups (figure 3). Birth weight category was not significant after controlling for age. After adjustment, the KMC group showed lower pain scores than the breastfeeding group (0.48 vs. 0.62 at 10 seconds), compared to unadjusted scores where the breastfeeding group showed lower scores (0.50 vs. 0.64).\u003c/p\u003e\u003cp\u003eTable 3: NIPS scores adjusted to age and birth weight categories\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"528\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime Point\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e2 seconds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eIntervention group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eAge (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e8.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eBirth weight category\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e10 seconds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eIntervention group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eAge (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e4.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003eBirth weight category\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25%;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis observational study compared breastfeeding, kangaroo mother care (KMC), and oral dextrose for pain relief in neonates. All three interventions were associated with low pain scores, with no statistically significant differences in mean NIPS scores between groups.\u003c/p\u003e\n\u003ch3\u003eBreastfeeding for procedural pain relief\u003c/h3\u003e\n\u003cp\u003eShah et al.’s Cochrane review of 20 studies concluded that breastfeeding or breast milk should be used for procedural pain, with oral glucose/sucrose showing similar effectiveness.\u003csup\u003e28\u003c/sup\u003e Efe and Savaşer found no significant difference in NIPS scores between breastfeeding (1.9±1.9) and sucrose (1.2±1.3), both significantly lower than control (6.4±1.6).\u003csup\u003e35\u003c/sup\u003e Our findings confirm comparable effects, with NIPS scores of 2.17±1.76 for breastfeeding and 2.61±1.72 for oral dextrose at 2 seconds, and 0.50±1.27 and 0.84±1.40 respectively at 10 seconds.\u003c/p\u003e\n\u003cp\u003eOkan et al. demonstrated that both skin-to-skin contact alone and breastfeeding with skin-to-skin contact significantly reduced pain responses compared to no intervention.\u003csup\u003e36\u003c/sup\u003e Pain relief from breastfeeding is mediated via oral stimulation through suckling, gustatory stimulation from milk taste, olfactory stimulation from breast milk odor, tactile warmth from skin contact, and auditory stimulation from maternal heartbeat.\u003csup\u003e37\u003c/sup\u003e Campbell-Yeo et al. noted significantly higher effectiveness of breastfeeding compared to breast milk alone.\u003csup\u003e38\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eKangaroo mother care for procedural pain relief\u003c/h3\u003e\n\u003cp\u003eA meta-analysis of six studies conducted by Sharma et al. found significant benefit of KMC over standard care for pain reduction.\u003csup\u003e39\u003c/sup\u003e Chidambaram et al. reported significantly lower PIPP scores during KMC versus without KMC (4.3 and 3.84 vs. 5.76 and 5.24 at 15 and 30 minutes respectively) for heel-prick pain in preterm neonates.\u003csup\u003e30\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eA Cochrane review of 25 studies by Johnston et al. (n=2001 infants) found that skin-to-skin care significantly reduced PIPP scores at 30 seconds (MD -3.21), 60 seconds (MD -1.64), and 90 seconds (MD -1.28) compared to no intervention, though by 120 seconds the difference was no longer significant.\u003csup\u003e40\u003c/sup\u003e These findings align with our observation that all NIPS scores returned to baseline (NIPS=0) by 120 seconds post-procedure.\u003c/p\u003e\n\u003cp\u003eShukla et al. found significantly lower PIPP scores with KMC (7.7±3.9) versus control (11.5±3.4), concluding that KMC with expressed breast milk should be the first choice for pain control in preterm neonates.\u003csup\u003e41\u003c/sup\u003e Our findings support this recommendation, with KMC showing effective pain reduction comparable to breastfeeding, even in predominantly preterm cohort.\u003c/p\u003e\n\u003cp\u003eKapoor et al. found comparable PIPP scores between KMC (8.42±1.99) and oral dextrose 50% (8.76±1.84) groups, both significantly lower than nesting (13.08±1.70).\u003csup\u003e42\u003c/sup\u003e This study supports our finding of comparable efficacy between KMC and oral dextrose for heel prick analgesia.\u003c/p\u003e\n\u003ch3\u003eOral dextrose for procedural pain relief\u003c/h3\u003e\n\u003cp\u003eMahmud et al. found significantly lower Modified Behavioral Pain Scale (MBPS) with 10% dextrose (4.31) versus sterile water (6.26), concluding that oral dextrose is cheap and effective for neonatal pain management.\u003csup\u003e43\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eShanthi et al. reported significantly lower pain scores during heel prick in 10% dextrose group compared to expressed breast milk group using PIPP-R scale at all time points: at initiation (4.97±1.42 vs. 7.19±2.0), at 30 seconds (2.36±1.44 vs. 5.5±1.5), and at 60 seconds (1.69±1.53 vs. 4.28±1.65).\u003csup\u003e44\u003c/sup\u003e In contrast, Nayak et al. found no significant difference between 10% dextrose, expressed breast milk, and sterile water for ROP screening pain in preterm neonates, with mean PIPP scores as 9.8±3.3, 11.8±2.8, and 10.2±2.9 respectively.\u003csup\u003e45\u003c/sup\u003e Ravishankar et al. found that 25% dextrose had significantly lower PIPP scores (median=10) compared to 10% dextrose and placebo (median=12 each), during nasogastric tube insertion pain.\u003csup\u003e46\u003c/sup\u003e Ranjbar et al. found significantly better PIPP scores with oral dextrose (7.60±1.17) and facilitated tucking (9.56±1.15) compared to control (12.90±1.14) during heel sticks.\u003csup\u003e47\u003c/sup\u003e These findings suggest that oral dextrose effectiveness may vary with dextrose concentration and pain intensity.\u003c/p\u003e\n\u003cp\u003eA systematic review and network meta-analysis of 103 trials by Abiramalatha et al. found non-nutritive sucking plus sucrose most effective (SMD −3.15), followed by breastfeeding, then skin-to-skin contact.\u003csup\u003e48\u003c/sup\u003e This analysis supports the use of mother-based non-pharmacological pain relief approaches, consistent with our findings.\u003c/p\u003e\n\u003cp\u003eAn integrative review of 27 studies conducted by Sutton and Lemermeyer found that sucrose, facilitated tucking, pacifier, skin-to-skin care, and human milk lessened pain during heel sticks, suctioning, nasogastric tube insertions, and echocardiograms.\u003csup\u003e25\u003c/sup\u003e However, all nonpharmacological interventions failed to adequately manage pain during ROP screening, consistent with Nayak et al.’s findings.\u003csup\u003e45\u003c/sup\u003e This review supports the use of nonpharmacological measures for routine NICU procedures.\u003c/p\u003e\n\u003cp\u003eIn our study, oral dextrose 10% was effective but showed the slowest pain attenuation and lowest proportion of complete pain relief. The absence of maternal contact and suckling may limit the depth and speed of soothing compared to breastfeeding and KMC, but oral dextrose remains a practical option when maternal interventions are not feasible.\u003csup\u003e38\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eSecondary outcomes\u003c/h3\u003e\n\u003ch4\u003eRecovery time\u003c/h4\u003e\n\u003cp\u003eMean recovery time was shortest for breastfeeding (7.61 seconds), followed by KMC (8.40 seconds), and oral dextrose (11.39 seconds), though differences were not statistically significant (P=0.13). Baijnathan and Arya found significantly shorter recovery with immediate KMC (iKMC) versus radiant warmer care (33.3±20.22 vs 92.43±49.68 seconds) during heel prick.\u003csup\u003e49\u003c/sup\u003e The longer recovery times in their study compared to ours likely reflect differences in population (sick LBW neonates vs. mixed cohort) and recovery definition (heart rate return to baseline vs. NIPS return to baseline). Both studies support faster recovery with maternal-based interventions.\u003c/p\u003e\n\u003ch4\u003eEffective and complete pain relief\u003c/h4\u003e\n\u003cp\u003eEffective pain relief (NIPS≤3 at 10 seconds) was achieved in similar proportions across groups: 95.0% (breastfeeding), 93.8% (KMC), and 93.8% (oral dextrose). However, complete pain relief (NIPS=0 at 10 seconds) differed significantly: 83.8% (breastfeeding), 78.8% (KMC), and 65.0% (oral dextrose) (P=0.016). This difference reflected a higher proportion of partial responders (NIPS 1–2) in the oral dextrose group. While all three interventions prevented severe pain, breastfeeding and KMC more frequently achieved complete analgesia.\u003c/p\u003e\n\u003ch3\u003eANCOVA findings: age and pain response\u003c/h3\u003e\n\u003cp\u003eIn our study, the baseline characteristics differed significantly across groups. The breastfeeding group comprised predominantly younger neonates (77.5% neonates \u0026lt;7 days) with normal birth weight (86.3%), while the KMC group comprised predominantly older neonates (80% neonates ≥7 days) with low birth weight (92.5%). This reflects standard clinical practice where breastfeeding was initiated from birth in stable neonates with normal birth weight and mature sucking-swallowing-breathing coordination, while KMC was initiated predominantly in low birth weight neonates after initial stabilization. We applied ANCOVA to adjust for age and birth weight.\u003c/p\u003e\n\u003cp\u003eAfter adjustment, between-group differences in NIPS remained non-significant (P=0.16 at 2 seconds; P=0.22 at 10 seconds), but age was a significant independent predictor at both 2 seconds (P=0.004) and 10 seconds (P=0.027) post-procedure. Older neonates showed higher pain scores across all three intervention groups, regardless of intervention type. Birth weight category was not a significant predictor after adjustment.\u003c/p\u003e\n\u003cp\u003eAdjusted mean NIPS scores at 10 seconds were 0.62 for breastfeeding, 0.48 for KMC, and 0.87 for oral dextrose. After adjustment, KMC showed lower pain scores than breastfeeding (0.48 vs. 0.62), reversing the unadjusted pattern in which breastfeeding had lower scores (0.50 vs. 0.64). This suggests that the lower NIPS scores of breastfeeding in unadjusted analysis may reflect the younger age of neonates in this group rather than a true intervention effect.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis pattern is consistent with developmental studies of pain expression. Johnston et al. (1993) found that full-term neonates showed more differentiated facial and cry responses than premature infants.\u003csup\u003e50\u003c/sup\u003e Another study by Johnston et al. (1995) reported that gestational age significantly affects pain response, with younger infants responding less robustly.\u003csup\u003e51\u003c/sup\u003e In our mixed cohort of term and preterm neonates, older neonates may have demonstrated greater behavioral differentiation and thus more observable pain responses on NIPS, which scores facial expression, cry, and motor parameters.\u003c/p\u003e\n\u003cp\u003eNIPS proved feasible for pain assessment in our setting. Assessment was simple, required no equipment, minimal training, and provided rapid bedside scoring, which supports its use in low- and middle-income settings where advanced monitoring may not be available.\u003csup\u003e29\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eStrength and limitations\u003c/h3\u003e\n\u003cp\u003eWe implemented a standardized procedural protocol with consistent lancet gauge, insertion site and depth (spring loaded lancet), and timing of assessments across all participants. NIPS scoring by a single, trained assessor minimized inter-observer variability. The use of validated NIPS instrument and serial measurements at multiple time points allowed us to plot pain response trajectories. This practical design enhances external validity in similar resource-constrained settings.\u003c/p\u003e\n\u003cp\u003eHowever, several limitations deserve consideration. The non-randomized, purposive allocation introduced baseline differences between groups—the KMC group comprised predominantly preterm and low birth weight neonates, while the breastfeeding group included mostly term, normal-weight infants. These differences may have confounded outcome comparisons.\u003c/p\u003e\n\u003cp\u003eGestational age was not recorded, precluding analysis of prematurity effects independent of birth weight. This is particularly relevant given that 92.5% of neonates in the KMC group had low birth weight.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe absence of a control group precluded analysis of the natural trajectory of pain resolution compared to intervention groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePain assessment was limited to the immediate procedural period (up to 120 seconds), leaving long-term neurodevelopmental outcomes unevaluated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur study design did not allow blinding; this may have introduced observer bias in subjective components such as facial expression. Finally, the single-center tertiary care setting and exclusion of critically ill neonates requiring intensive care may limit generalizability.\u003c/p\u003e\n\u003ch3\u003eFuture research scope\u003c/h3\u003e\n\u003cp\u003eFuture studies should prioritize well-designed RCTs with stratified randomization by gestational age and birth weight to enable balanced groups and valid comparisons. Crossover designs could control for individual variation while improving statistical power.\u003c/p\u003e\n\u003cp\u003eMulti-center trials across diverse settings would enhance generalizability, and combined or sequential interventions (e.g., breastfeeding with oral sucrose, KMC with non-nutritive sucking) warrant further investigation.\u003c/p\u003e\n\u003cp\u003eLong-term follow-up is needed to determine whether effective procedural pain management influences neurodevelopmental outcomes, stress reactivity, or pain sensitivity in later childhood. Implementation research should evaluate strategies for integrating non-pharmacological interventions into routine protocols and identify barriers to adoption in resource-limited settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eFinancial support and sponsorship\u003c/h3\u003e\n\u003cp\u003eNil.\u003c/p\u003e\n\u003ch3\u003eConflicts of interest\u003c/h3\u003e\n\u003cp\u003eNone.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarker DP, Rutter N. Exposure to invasive procedures in neonatal intensive care unit admissions. Arch Dis Child Fetal Neonatal Ed. 1995 Jan;72(1):F47-8. doi: 10.1136/fn.72.1.f47. PMID: 7743285; PMCID: PMC2528401.\u003c/li\u003e\n\u003cli\u003ePerry M, Tan Z, Chen J, Weidig T, Xu W, Cong XS. Neonatal Pain: Perceptions and Current Practice. Crit Care Nurs Clin North Am. 2018 Dec;30(4):549-561. doi: 10.1016/j.cnc.2018.07.013. 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PMID: 7478691.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"neonatal pain, non-pharmacological pain relief, breastfeeding, kangaroo mother care, skin-to-skin contact, oral dextrose, neonatal infant pain scale, procedural pain","lastPublishedDoi":"10.21203/rs.3.rs-9112827/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9112827/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eBackground: \u003c/em\u003eNeonates in intensive care units undergo numerous painful procedures. Non-pharmacological interventions offer safe approaches to reduce procedural pain, but comparative studies evaluating their relative effectiveness are limited, particularly in low-resource settings.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethods: \u003c/em\u003eWe enrolled 240 neonates requiring heel lancing for blood glucose monitoring. Neonates were allocated to three intervention groups: breastfeeding, kangaroo mother care (KMC), and oral dextrose. Pain was assessed using the Neonatal Infant Pain Scale (NIPS) at baseline and at 2, 10, and 120 seconds post-procedure.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResults: \u003c/em\u003eAll three interventions effectively reduced procedural pain. Mean NIPS scores at 2 seconds were 2.17±1.76 for breastfeeding, 2.29±1.69 for KMC, and 2.61±1.72 for oral dextrose group (p=0.25). Complete pain relief (NIPS=0 at 10 seconds) differed significantly across groups: 83.8% for breastfeeding, 78.8% for KMC, and 65.0% for oral dextrose (p=0.016). However, after adjusting for age and birth weight, KMC showed lower pain scores than breastfeeding, though the difference was not significant.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConclusion: \u003c/em\u003eAll three non-pharmacological methods were effective in reducing procedural pain. Between-group differences in mean pain scores were not statistically significant. These methods offer safe, accessible options for procedural pain management in neonates.\u003c/p\u003e","manuscriptTitle":"Comparative Effectiveness of Non-Pharmacological Pain Management Interventions in Neonates: A Cross- Sectional Observational Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 13:20:54","doi":"10.21203/rs.3.rs-9112827/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":"a193ee07-7871-4fca-b533-7d7fc655aa6c","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T16:55:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 13:20:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9112827","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9112827","identity":"rs-9112827","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}