Early versus Delayed Fortification of Human Milk in Preterm Neonates: A Randomised Controlled Trial

Early versus Delayed Fortification of Human Milk in Preterm Neonates: A Randomised Controlled Trial | 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 Early versus Delayed Fortification of Human Milk in Preterm Neonates: A Randomised Controlled Trial Kaifi Siddiqui, Gunjana Kumar, Surendra Singh Meena, Nirupa Chandorkar, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7596338/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 Preterm very low birth weight infants are at high risk of postnatal growth failure due to limited nutrient reserves and high metabolic demands. Human milk fortification is essential to optimize growth, but the timing of initiation remains controversial, with concerns regarding feeding tolerance and metabolic complications. Objectives To compare the effects of early versus delayed initiation of human milk fortification on feeding tolerance, growth outcomes, and neonatal morbidities in preterm infants. Methods This randomised controlled Trial enrolled 66 preterm neonates ≤34 weeks of gestation, who were grouped between an Early Fortification Group (n = 32), in which fortification was initiated once enteral feeds reached 60 ml/kg/day and baby tolerated for next two feeds, and a Late Fortification Group (n = 34), in which fortification was initiated at enteral feeds of 100 ml/kg/day and tolerated for next two feeds . Baseline maternal and neonatal characteristics were recorded. The primary outcome was the time to achieve full enteral feeds. Secondary outcomes included feeding intolerance, time to regain birth weight, growth velocity, duration of intravenous fluids, hospital stay, gestational age at discharge, incidence of extrauterine growth restriction, and other neonatal morbidities. Results The two groups were comparable at baseline. The mean time to reach full enteral feeds was 10.06 ± 2.14 days in the Early Fortification Group and 11.00 ± 3.62 days in the Late Fortification Group, with no statistically significant difference (p = 0.215). Feeding intolerance occurred in 21.9% of the early group and 32.4% of the late group (p = 0.339). Birth weight was regained earlier in the early group (12.21 ± 6.14 days) compared with the late group (15.23 ± 6.87 days), showing a trend towards significance (p = 0.065). Growth velocity in terms of weight, length, and head circumference was similar in both groups. The mean duration of hospital stay was 36.37 ± 18.08 days in the early group and 38.82 ± 14.73 days in the late group (p = 0.548). The mean gestational age at discharge was significantly higher in the early fortification group (37.09 ± 1.89 weeks vs. 36.13 ± 1.65 weeks, p = 0.031). Extrauterine growth restriction was observed in 24.2% of the overall cohort, with no significant difference between groups (21.9% vs. 26.5%, p = 0.663). No cases of necrotizing enterocolitis ≥ Stage 2b or retinopathy of prematurity requiring treatment were seen. Osteopenia of prematurity occurred in 34.4% of the early group and 35.3% of the late group (p = 0.938). The incidence of sepsis, patent ductus arteriosus, periventricular leukomalacia, and intraventricular hemorrhage was similar in both groups. Conclusion Early initiation of human milk fortification was safe, well tolerated, and not associated with increased feeding intolerance or gastrointestinal morbidity. Although most growth outcomes were comparable, early fortification demonstrated a favorable trend toward earlier regaining of birth weight and was associated with a significantly higher gestational age at discharge. These findings suggest that early fortification may be beneficial in moderately preterm infants without compromising safety. Figures Figure 1 Figure 2 Introduction Preterm infants are often deprived of normal in-utero nutrient accretion and at risk of nutritional emergency which results in interruption of growth leading to high rate of extrauterine growth restriction (EUGR) at discharge, ranging from 23% in infants born at 34 weeks' gestation to 71% in those born at 23 weeks' gestation 1 . It is fundamentally non-physiological to deprive an infant of any gestation of enteral feeding, and hence early and aggressive nutrition is the norm as each day without enteral nutrition increases the likelihood of EUGR 2 . Mothers own milk (MOM) despite being the ideal feed for their offsprings, alone it often fails to meet the high protein requirements of very low birth weight (VLBW) infants, limiting lean mass deposition and growth 3 , hence without fortification, it is insufficient in protein, calcium, and phosphorus—nutrients essential for bone and tissue accretion 4 . On average, unfortified human milk provides 67 kcal and 1.1 g protein per 100 mL, while human milk with human milk fortifier (HMF) provides 80 kcal and 2 g protein per 100 mL. Early HMF addition may help bridge the protein–energy gap, reducing risks of postnatal growth failure and adverse neurodevelopment. However, this benefit needs to be balanced against the risk associated with exposing the immature gut to the high osmolarity of fortified feeds that, in turn, increases the risk of feed intolerance and Necrotising enterocolitis (NEC).European Milk Bank Association (EMBA) Working group and National Neonatology Forum India, 2022 guidelines have recommended the addition of HMF for preterm neonates with birth weight < 1800g to as early as the feed volume of 50–80 ml/kg/day 5 .Tradition and lack of robust evidence have led to variable practices 6 . Commonly, most clinicians prefer adding HMF once the infant is on full enteral feeds, generally defined as 150 ml/kg/day 6 . .There is paucity of literature on early fortification of human milk, especially in the Indian context. Hence, the randomised controlled trial was planned to assess the safety and effects on growth, of early fortification of human milk versus late fortification, in preterm infants. Materials and Methods Subjects and settings This prospective, open label, randomised controlled trial was conducted in the Department of Neonatology at the National Institute of Medical Sciences and Research, Jaipur, India, over 18 months. The trial was registered with the Clinical Trials Registry of India (Reference no. CTRI/2024/04/065921 dated 18/04/2024). Preterm neonates born with birth weight between 1000–1600 grams, hemodynamically stable and feeds being started within 48 hours of life, were screened for eligibility. Babies fulfilling the inclusion criteria were enrolled and randomised within the first 48 hours of life, after taking informed consent from parents. Neonates were excluded if they had any of the following - antenatally diagnosed GI malformation, absence or reversal of end-diastolic flow on antenatal umbilical artery Doppler, presence of major congenital anomalies or chromosomal abnormalities at birth, need of more than one vasopressor support for more than 48 hrs ,leading to failure to start feeds uptill day 5 of life, ,perinatal asphyxia in neonates with APGAR score < 4 at 5 minutes, died or were expected to die within 72 hours, and whose mother not intended or was unable to provide mother’s own milk. Randomisation and intervention Randomisation was performed using computer-generated block randomisation with variable block sizes done by a statistician not a part of the study. Allocation was concealed using sequentially numbered, opaque sealed envelopes opened after enrolment. Randomisation was performed in blocks of 4 on strata defined by birth weight 1000 to 1250 g, and 1251 to 1600 g. Separate block randomization schemes were prepared for each of the strata and performed centrally. The investigators were not aware of the block size. After enrolment, the eligible neonates were randomized into 2 groups by the study investigator. In Early Fortification Group A(n = 32) : Fortification was initiated once the neonate was receiving 60 ml/kg/day of expressed breast milk and had tolerated for next two feeds (Table 1 ). Group B (n = 34) : Fortification was initiated once the neonate was on 100 ml/kg/day of expressed breast milk and had tolerated the same for next two feeds.. Table 1 Feeding policy Day 1 20 Day 2 40 Day 3 60 +/- HMF GROUP 1 Day 4 80 Day 5 100 +/- HMF GROUP 2 Day 6 120 Day 7 140 Day 8 150 Day 9 150 Table 2 Baseline Characteristics of Study Population Characteristic Early Fortification (n = 32) Late Fortification (n = 34) p-value Neonatal variables Gestational age (weeks), mean ± SD 31.86 ± 3.10 30.75 ± 1.98 0.087 Birth weight (g), mean ± SD 1347.1 ± 262.4 1299.7 ± 211.9 0.422 Length (cm), mean ± SD 41.9 ± 4.0 40.7 ± 3.0 0.160 OFC (cm), mean ± SD 29.1 ± 2.8 28.1 ± 1.5 0.060 Male sex, n (%) 17 (53.1) 19 (55.9) 0.822 AGA, n (%) 22 (68.8) 28 (82.4) 0.197 SGA, n (%) 10 (31.2) 6 (17.6) Maternal variables Maternal age (years), mean ± SD 26.4 ± 4.9 25.7 ± 4.5 0.525 Multigravida, n (%) 24 (75.0) 27 (79.4) 0.699 Maternal comorbidities Preeclampsia, n (%) Oligohydramnios, n (%) GDM, n (%) Others, n (%) 4 (12.5%) 3 (9.4%) 2 (6.3%) 4 (12.5%) 2 (5.9%) 2 (5.9%) 1 (2.9%) 8 (23.5%) 0.694 Mode of delivery Vaginal, n (%) Cesarean section, n (%) 15 (46.9%) 17 (53.1%) 16 (47.1%) 18 (52.9%) 0.988 Table 3 Primary Outcome: Time taken to reach full feeds Variable Early(n = 32) (Mean ± SD) Late(n = 34) (Mean ± SD) Mean Difference ± SE p-value Mean age of initiation of feeds (hours) 24.10 ± 2.14 20.78 ± 2.84 3.32 ± 0.7 0.32 Day of Attainment of Full Feeds 10.06 ± 2.14 11.00 ± 3.61 -0.935 ± 0.746 0.215 Table 4 Secondary outcome Variable Early(n = 32) n(%) Late(n = 34) n(%) p-value Feed Intolerance 7 (21.9%) 11 (32.4%) 0.339 PMA to achieve full feeds weeks(Mean ± SD) 33.00 ± 4.27 32.32 ± 1.84 0.402 Birth weight regained at Day of life(gms) (Mean ± SD) 12.21 ± 6.14 15.23 ± 6.87 0.060 EUGR at discharge(days) 7 (21.9%) 9 (26.5%) 0.663 Table 5 Comparison of Anthropometric parameters at discharge variables in Early vs Late Fortification group Variable Early(n = 32) (Mean ± SD) Late(n = 34) (Mean ± SD) Mean Difference ± SE) p-value Rate of Weight gain velocity at discharge (g/kg/day) 12.96 ± 4.24 12.779 ± 5.38 0.189 ± 1.198 0.875 Rate of OFC Gain velocity at discharge (cm/wk) 0.54 ± 0.170 0.55 ± 0.17 -0.0092 ± 0.043 0.832 Rate of Length Gain velocity at discharge (cm/wk) 0.74 ± 0.175 0.70 ± 0.20 0.0349 ± 0.047 0.464 In both groups, the feeds were advanced by 20 ml/kg/day, until a maximum enteral feed of 150ml/kg/day was attained and maintained for 24 hours. As a unit policy, the total parenteral nutrition was initiated in the form of glucose infusion, aminoven and lipids for all babies with birth weight 1600 grams. Once full feeds were reached i.e. 150 ml/kg/day and sustained for 24 hours, feeds were increased to a maximum of 180 ml/kg/day based on the growth and daily weight gain of the baby. All efforts were made to provide MOM( mothers own milk ) to babies. Babies who had received > 70% (8 feeds) of EBM were enrolled in the study. Feeding protocol Feeding was initiated within 2 hours of life, and were given as 2-hourly, bolus feeds. Pre-feed abdominal girth charting was performed only in cases when there was suspicion of abdominal distension. Baby was strictly monitored for feed intolerance (vomiting, abdominal distension), apnea, signs of sepsis and NEC. Any baby whose feeds were discontinued in view of feed intolerance, feeds were re-initiated and advanced as per the clinician’s directive till the baby was enrolled in the clinical trial. Enteral feeds were given via orogastric tubes in preterm infants 32 weeks’ gestation, oral spoon feeds were tried, and if taken well was continued along with breastfeeds. Cue-based individualised feeding was followed as a part of the unit protocol. As the feed volume was advanced i.e. >60% of the required fluid, parental nutrition, including aminoven and lipids, was decreased, and only glucose infusion was continued. For giving TPN, a central line was placed. Fortification was done bedside by the nursing staff, following all aseptic precautions. In both groups, the HMF used was the same throughout the study period (Lactodex powder, 1g sachet). 1g of the powdered sachet was added to 25 ml of human milk as a standard fortification norm to maintain the osmolarity. Enrolled infants were monitored as per the unit protocol for vital parameters, assessed regularly for feed intolerance and anthropometric measurements were also assessed regularly. Supplements were added as per the requirement of neonates. The subjects were followed up till discharge or death. Outcome variables and their measurements Primary Outcome- To compare the time taken to reach full feeds (150ml/kg/day sustained for at least 24 hrs) in both groups. Secondary outcomes- To compare the outcome in terms of incidence of feed intolerance, incidence of NEC (as per Bell’s stage ≥ 2), Incidence of Sepsis, Total duration of intravenous fluid usage, Time of regaining birth weight, Total duration of hospital stay, Weight gain per kg per day at 1 month of age, Extrauterine growth retardation (EUGR) at 36 weeks PMA, Duration of hospital stay, All-cause mortality, Incidence of neonatal morbidities including, patent ductus arteriosus, severe intraventricular haemorrhage (grade III and IV), periventricular leukomalacia, and retinopathy of prematurity Ethics approval and consent to participate Ethical approval was obtained from the Institutional Ethics Committee. Written informed consent was taken from parents prior to enrolment. Sample Size Calculation and Statistical analysis Sample size was determined based on the previous year’s data from our unit, where the mean duration of Parenteral nutrition (PN), a surrogate of attainment of full feeding was attained at 14 ± 5.5 days in very low birth weight neonates. With the current intervention, the need for a mean duration of parenteral nutrition in VLBW neonates was expected to demonstrate a reduction of 20% of PN days (3 days earlier). Thus, assuming the same SD (5.5) with an α error of 5% and a power of 80%, a total of 64 subjects (32 in each arm) were required to be enrolled. Statistical analysis was done using the appropriate software version of SPSS. Results were analyzed as per the ‘Intention to Treat’ analysis. Descriptive stats were used wherever applicable. Continuous outcomes were summarized using mean and standard deviation. Student t-test was used to compare normally distributed continuous data. For all comparisons, p< 0.05 was considered significant. Results A total of 102 preterm neonates were screened for eligibility, of which 36 were excluded, 66 met the inclusion criteria and were randomized into two arms. The Early HMF fortification group (n=32) received fortification once the neonate tolerated 60 ml/kg/day of expressed breast milk for two consecutive feeds, while the Late HMF fortification group (n=34) received fortification once the neonate tolerated 100 ml/kg/day of expressed breast milk for two consecutive feeds(figure 1) The baseline maternal and neonatal characteristics were comparable between groups (Table 2). The primary outcome(Table 3, Figure 2- the time taken to reach full feeds (150ml/kg/day sustained for at least 24 hrs) was a mean of 10.06 ± 2.14 days in the early fortification group and 11.00 ± 3.62 days in the late fortification group, with a mean difference between the groups was -0.94 ± 0.75 days, (p = 0.215). While early fortification was associated with a trend towards earlier achievement of full enteral feeds, the difference compared to late fortification was not statistically significant. Among secondary outcomes (Table 4), the incidence of feed intolerance, postmenstrual age (PMA) at achievement of full feeds, Duration of IVF used (days), the weight at discharge and EUGR at discharge were comparable in both groups. The day of life at which birth weight was regained was slightly earlier in the early group compared to the late group but the mean gestational age at discharge was significantly less in the late fortification group. The anthropometric parameters including rate of weight gain, OFC gain and length gain, at discharge were also comparable in both groups. Among neonatal morbidities (Table 5, incidence of Shock, and hemodynamically significant patent ductus arteriosus, incidence of sepsis, osteopenia of prematurity was comparable across the two groups. Incidence of PVL was more in the late fortification group but not significant. There were no cases of IVH ≥ (grade III), NEC, severe retinopathy of prematurity. The mean duration of hospital stay was more in the late fortification group (p = 0.548). Discussion Optimal nutritional support remains a cornerstone of care in preterm and very low birth weight (VLBW) infants, where growth failure is strongly associated with adverse neurodevelopmental outcomes. 7 Although human milk is universally accepted as the ideal source of nutrition for the newborns, its intrinsic protein and caloric content often fall short of meeting the heightened metabolic demands of premature infants. 8 Human milk fortification is therefore is now the standard practice in neonatal intensive care. 9 However, the optimal timing of fortification continues to have no consensus without many studies Concerns over early initiation largely focus on potential risks of feeding intolerance or necrotizing enterocolitis (NEC), whereas its advocates emphasize enhanced nutrient delivery, faster achievement of growth milestones, and reduced incidence of extrauterine growth restriction (EUGR). Our randomized controlled trial of early versus delayed fortification in preterm infants of birth weight 1000-1600 grams is another step to find out a evidence based suitable timing of fortification of human milk . The primary outcome of our study was the time taken to achieve full enteral feeding. Infants in the Early Fortification Group attained full feeds in 10.06 ± 2.14 days, compared with 11.00 ± 3.62 days in the Late Fortification Group. Although the mean difference of −0.94 ± 0.75 days favored the Early group, the result did not reach statistical significance (p = 0.215). Nonetheless, the trend is clinically meaningful. Our findings align with Gupta et al. (2024) 10 , who reported almost identical times to full feeds in early and late groups (5.56 vs. 5.38 days), and with the trial by Shah et al. 11 , where the median duration to full feeds was 20 days in both groups (p = 0.45). Similarly, Alyahya et al. 12 found no significant impact of timing of fortification on achievement of full feeds. Collectively, these studies suggest that early fortification does not delay progression to full enteral feeding and, in certain cohorts, may be associated with modestly faster advancement. 10,11,12 Feeding intolerance, the most cited safety concern in this context, was not increased by early fortification in our trial. Intolerance occurred in 21.9% of infants in the Early group compared with 32.4% in the Late group (p = 0.339). Although statistically non-significant, this pattern suggests atleast that early fortification is not harmful. Similar observations were reported by Gupta et al. 10 , who found intolerance in 40% of early-fortified infants versus 29% in the late group, though not significant. Alizadeh et al. 13 also reported higher intolerance in early-fortified infants, without significance, while Shah et al. 11 documented similar episode counts between groups (58 vs. 57). The meta-analysis by Hilditch et al. 14 further consolidates these findings, showing little or no difference in feeding intolerance when fortification was initiated even at ≤40 mL/kg/day versus later at 75 mL/kg/day. Importantly, no cases of NEC ≥ stage 2b were observed in our study, echoing the safety profile consistently reported in the literature. 10,11,13,14 An important clinical observation in our study was the time to regain birth weight. Neonates in the Early Fortification Group regained birth weight in 12.21 ± 6.14 days compared to 15.23 ± 6.87 days in the Late group, with the difference approaching statistical significance (p = 0.065). This observation is supported by Gupta et al. 10 , who also reported earlier weight regain in early-fortified infants (10.13 vs. 11.26 days). Mechanistically, earlier fortification ensures greater protein and caloric intake during the critical early weeks of adaptation, supporting more rapid recovery of intrauterine growth trajectories. This is further supported by the work of Shah et al. 11 , who documented significantly higher protein intake in early-fortified neonates during the first three weeks of life, with cumulative four-week intake markedly superior (98.6 vs. 89.6 g/kg, p < 0.001). At discharge, weight gain velocity in our cohort was comparable between groups (12.96 ± 4.24 vs. 12.78 ± 5.38 g/kg/day, p = 0.875). Similarly, length gain (0.74 ± 0.18 vs. 0.70 ± 0.20 cm/week, p = 0.464) and head circumference gain (0.54 ± 0.17 vs. 0.55 ± 0.17 cm/week, p = 0.832) showed no significant differences. These findings are consistent with Alyahya et al. 12 , who reported similar growth velocities across groups, and with Wynter et al. 16 , who observed non-significant trends toward better growth in the early group. Alizadeh et al. 13 found no significant differences in Z scores for weight or length but noted a non-significant trend toward greater head growth in early-fortified infants. Conversely, Gupta et al. 10 reported significantly higher median gains in weight (11.16 vs. 8.99 g/kg/day), length (1.0 vs. 0.88 cm), and head circumference (0.77 vs. 0.62 cm) favoring early fortification. Salas et al. 15 , in a cohort of extremely preterm neonates (median GA 26 weeks), also reported superior outcomes in the Early group, including higher weight (+131 g, p = 0.03), fat-free mass (+103 g, p = 0.03), and length (+0.9 cm, p = 0.04). These findings suggest that the magnitude of benefit from early fortification may vary depending on the maturity of the cohort studied, with extremely preterm infants deriving the greatest advantage. Other clinical outcomes in our study were reassuring. The mean duration of hospital stay did not differ significantly (36.37 vs. 38.82 days, p = 0.548). Interestingly, gestational age at discharge was significantly higher in the Early group (37.09 ± 1.89 vs. 36.13 ± 1.65 weeks, p = 0.031), which may indicate better readiness for discharge among early-fortified infants. EUGR occurred in 21.9% of Early and 26.5% of Late group infants (p = 0.663), a pattern similar to Gupta et al. 10 , who also reported EUGR in approximately one-fifth to one-third of infants in both groups. Neonatal morbidities, including sepsis, PDA, periventricular leukomalacia, and osteopenia of prematurity, showed no significant differences, reinforcing the safety of early fortification. Notably, no infant developed severe intraventricular hemorrhage, NEC ≥ stage 2b, or retinopathy requiring treatment. The strengths of this study include its randomized controlled design, comparable baseline characteristics, and detailed outcome analysis across nutritional and clinical parameters. In summary, our findings add to the growing evidence that early fortification of human milk is both safe and effective. It does not increase the risk of feeding intolerance or major morbidities and may confer clinically relevant benefits such as earlier regaining of birth weight and improved maturity at discharge. While growth velocities at discharge were comparable, studies in more immature cohorts have demonstrated clearer advantages, underscoring the importance of tailoring fortification strategies to gestational age. Larger, multicenter trials with longer-term follow-up are warranted to confirm these findings and to evaluate their impact on neurodevelopment. There were few limitations in our study :The study was underpowered and was limited by a smaller sample size.Although Randomization and allocation concealment were done, blinding was not feasible due to the study design.The study was conducted on all preterms, with birth weight 1000-1600 gms ,without any specific gestation range and so its results cannot be extrapolated to ELBWs and ELGANs.Such studies which involve the use of exclusive human milk for feeding , it should be done at a centre which has its indigenous milk bank or can procure milk from them on demand. Conclusion Early fortification of human milk (@ 60 ml/kg/day in preterm neonates with birth weight 1000-1600 gms was safe and well tolerated. While trends toward improved growth (weight gain and reduced EUGR) were observed but they were not statistically significant, hence larger multicenter studies may be required to confirm these findings. Based on this study, early fortification may be considered a feasible strategy for improving nutritional outcomes in preterm neonates. Declarations Funding: there was no funding for this study Author Contribution K.S. Did the data collection and prepared the manuscriptG.K. Helped in statistics and preparing the manuscriptN.C. Did the help in preparing figures and tablesG.S. Did the help in preparing figures and tablesJ.K.-planned the study , advised in preparing the plan for execution of the study Acknowledgement Special acknowledgment to my wife Dr Sheeba Farooqui, without her support this study could not have been possible. References Clark RH, Thomas P, Peabody J. Extrauterine growth restriction remains a serious problem in prematurely born neonates. Pediatrics. 2003;111(5):986–90. Freitas BA, Priore SE, Lima LM, Franceschini SC. Extrauterine growth restriction: universal problem among premature infants[Restrição de crescimento extrauterino: problema universal entre os]. 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Underwood MA. Human milk for the premature infant. Pediatr Clin North Am. 2013;60(1):189-207. doi:10.1016/j.pcl.2012.09.008. Arslanoglu S, Corpeleijn W, Moro G, Braegger C, Campoy C, Colomb V, et al. Donor human milk for preterm infants: current evidence and research directions. J Pediatr Gastroenterol Nutr. 2013;57(4):535-42. doi:10.1097/MPG.0b013e3182a3af0a. Gupta G, Murugesan A, Thanigainathan S, Adhisivam B. Does early fortification of human Milk decrease time to regain birth weight as compared to late fortification among preterm infants —a randomized controlled trial. Indian Journal of Pediatrics. 2025 Jun;92(6):592-7. Shah SD, Dereddy N, Jones TL, Dhanireddy R, Talati AJ. Early versus delayed human milk fortification in very low birth weight infants - a randomized controlled trial. Journal of Pediatrics. 2016;174:126-31. [DOI: 10.1016/j.jpeds.2016.03.056] [PMID: 27112041] Alyahya S, Alayed N, Alghuraybi S, Alshahrani F, Alsulaimi F, Almutairi B. Human milk fortification and use of infant formulas to support growth in neonatal intensive care unit infants. Nutrients [Internet]. 2023 Oct 19 [cited 2025 Sep 10];15(20):4443. Available from: https://doi.org/10.3390/nu15204443. Alizadeh Taheri P, Sajjadian N, Asgharyan Fargi M, Shariat M. Is early breast milk fortification more effective in preterm infants?: a clinical trial: Early and late fortification. Journal of perinatal medicine. 2017;27;45(8):953-7. Hilditch C, Keir A, Collins CT, Middleton P, Gomersall J. Early versus delayed introduction of human milk fortification in enterally fed preterm infants: A systematic review and meta‐analysis. Journal of Paediatrics and Child Health. 2022;58(1):30-8. Salas AA, Gunawan E, Nguyen K, Reeves A, Argent V, Finck A, Carlo WA. Early human milk fortification in infants born extremely preterm: a randomized trial. Pediatrics. 2023 Sep 1;152(3):e2023061603. Wynter Z, Gorham JA, Thompson AB, Mundy C, Waller JL, Stansfield BK. Immediate fortification of human milk with a bovine milk-derived human milk fortifier in very low birth weight infants: a randomized clinical trial. Journal of Perinatology. 2024 Nov;44(11):1591-6. Additional Declarations No competing interests reported. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7596338","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":533037365,"identity":"ebd17f63-9f5f-4b8a-bdf0-e31dd15c4529","order_by":0,"name":"Kaifi Siddiqui","email":"","orcid":"","institution":"National Institute of Medical Sciences and Research","correspondingAuthor":false,"prefix":"","firstName":"Kaifi","middleName":"","lastName":"Siddiqui","suffix":""},{"id":533037366,"identity":"00a15e8c-7d5b-4c21-8616-dfff2f623b36","order_by":1,"name":"Gunjana Kumar","email":"","orcid":"","institution":"National Institute of Medical Sciences 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1","display":"","copyAsset":false,"role":"figure","size":171487,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7596338/v1/dc03ace4725c5c87e58c9d50.png"},{"id":94410567,"identity":"250c3498-6dc8-46b3-9deb-4297ed34af21","added_by":"auto","created_at":"2025-10-27 14:04:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrimary Outcome: Time taken to reach full feeds\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7596338/v1/b6b7342659d23017e61aabde.png"},{"id":95228305,"identity":"503d15ef-e0ab-4507-8a5a-27c3245f32b9","added_by":"auto","created_at":"2025-11-05 16:33:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":991611,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7596338/v1/d3796a6b-5feb-4583-8108-9a66809b8892.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Early versus Delayed Fortification of Human Milk in Preterm Neonates: A Randomised Controlled Trial","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreterm infants are often deprived of normal in-utero nutrient accretion and at risk of nutritional emergency which results in interruption of growth leading to high rate of extrauterine growth restriction (EUGR) at discharge, ranging from 23% in infants born at 34 weeks' gestation to 71% in those born at 23 weeks' gestation\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt is fundamentally non-physiological to deprive an infant of any gestation of enteral feeding, and hence early and aggressive nutrition is the norm as each day without enteral nutrition increases the likelihood of EUGR\u003csup\u003e2\u003c/sup\u003e. Mothers own milk (MOM) despite being the ideal feed for their offsprings, alone it often fails to meet the high protein requirements of very low birth weight (VLBW) infants, limiting lean mass deposition and growth\u003csup\u003e3\u003c/sup\u003e, hence without fortification, it is insufficient in protein, calcium, and phosphorus\u0026mdash;nutrients essential for bone and tissue accretion\u003csup\u003e4\u003c/sup\u003e. On average, unfortified human milk provides 67 kcal and 1.1 g protein per 100 mL, while human milk with human milk fortifier (HMF) provides 80 kcal and 2 g protein per 100 mL. Early HMF addition may help bridge the protein\u0026ndash;energy gap, reducing risks of postnatal growth failure and adverse neurodevelopment. However, this benefit needs to be balanced against the risk associated with exposing the immature gut to the high osmolarity of fortified feeds that, in turn, increases the risk of feed intolerance and Necrotising enterocolitis (NEC).European Milk Bank Association (EMBA) Working group and National Neonatology Forum India, 2022 guidelines have recommended the addition of HMF for preterm neonates with birth weight\u0026thinsp;\u0026lt;\u0026thinsp;1800g to as early as the feed volume of 50\u0026ndash;80 ml/kg/day\u003csup\u003e5\u003c/sup\u003e.Tradition and lack of robust evidence have led to variable practices\u003csup\u003e6\u003c/sup\u003e. Commonly, most clinicians prefer adding HMF once the infant is on full enteral feeds, generally defined as 150 ml/kg/day\u003csup\u003e6\u003c/sup\u003e. .There is paucity of literature on early fortification of human milk, especially in the Indian context. Hence, the randomised controlled trial was planned to assess the safety and effects on growth, of early fortification of human milk versus late fortification, in preterm infants.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSubjects and settings\u003c/h2\u003e\u003cp\u003eThis prospective, open label, randomised controlled trial was conducted in the Department of Neonatology at the National Institute of Medical Sciences and Research, Jaipur, India, over 18 months. The trial was registered with the Clinical Trials Registry of India (Reference no. CTRI/2024/04/065921 dated 18/04/2024). Preterm neonates born with birth weight between 1000\u0026ndash;1600 grams, hemodynamically stable and feeds being started within 48 hours of life, were screened for eligibility. Babies fulfilling the inclusion criteria were enrolled and randomised within the first 48 hours of life, after taking informed consent from parents. Neonates were excluded if they had any of the following - antenatally diagnosed GI malformation, absence or reversal of end-diastolic flow on antenatal umbilical artery Doppler, presence of major congenital anomalies or chromosomal abnormalities at birth, need of more than one vasopressor support for more than 48 hrs ,leading to failure to start feeds uptill day 5 of life, ,perinatal asphyxia in neonates with APGAR score\u0026thinsp;\u0026lt;\u0026thinsp;4 at 5 minutes, died or were expected to die within 72 hours, and whose mother not intended or was unable to provide mother\u0026rsquo;s own milk.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRandomisation and intervention\u003c/h3\u003e\n\u003cp\u003eRandomisation was performed using computer-generated block randomisation with variable block sizes done by a statistician not a part of the study. Allocation was concealed using sequentially numbered, opaque sealed envelopes opened after enrolment. Randomisation was performed in blocks of 4 on strata defined by birth weight 1000 to 1250 g, and 1251 to 1600 g. Separate block randomization schemes were prepared for each of the strata and performed centrally. The investigators were not aware of the block size. After enrolment, the eligible neonates were randomized into 2 groups by the study investigator. \u003cb\u003eIn Early Fortification Group A(n\u0026thinsp;=\u0026thinsp;32)\u003c/b\u003e: Fortification was initiated once the neonate was receiving 60 ml/kg/day of expressed breast milk and had tolerated for next two feeds (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cb\u003eGroup B (n\u0026thinsp;=\u0026thinsp;34)\u003c/b\u003e: Fortification was initiated once the neonate was on 100 ml/kg/day of expressed breast milk and had tolerated the same for next two feeds..\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFeeding policy\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay 2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay 3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60 +/- HMF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGROUP 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay 4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay 5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100 +/- HMF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGROUP 2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay 6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay 7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay 8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay 9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBaseline Characteristics of Study Population\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEarly Fortification (n\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLate Fortification (n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eNeonatal variables\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGestational age (weeks), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.087\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBirth weight (g), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1347.1\u0026thinsp;\u0026plusmn;\u0026thinsp;262.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1299.7\u0026thinsp;\u0026plusmn;\u0026thinsp;211.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.422\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLength (cm), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e41.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.160\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOFC (cm), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.060\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale sex, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17 (53.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19 (55.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.822\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAGA, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22 (68.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28 (82.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.197\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSGA, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 (31.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 (17.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMaternal variables\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaternal age (years), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.525\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMultigravida, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24 (75.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27 (79.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.699\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaternal comorbidities\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePreeclampsia, n (%)\u003c/p\u003e\u003cp\u003eOligohydramnios, n (%)\u003c/p\u003e\u003cp\u003eGDM, n (%)\u003c/p\u003e\u003cp\u003eOthers, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4 (12.5%)\u003c/p\u003e\u003cp\u003e3 (9.4%)\u003c/p\u003e\u003cp\u003e2 (6.3%)\u003c/p\u003e\u003cp\u003e4 (12.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (5.9%)\u003c/p\u003e\u003cp\u003e2 (5.9%)\u003c/p\u003e\u003cp\u003e1 (2.9%)\u003c/p\u003e\u003cp\u003e8 (23.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.694\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMode of delivery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVaginal, n (%)\u003c/p\u003e\u003cp\u003eCesarean section, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15 (46.9%)\u003c/p\u003e\u003cp\u003e17 (53.1%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16 (47.1%)\u003c/p\u003e\u003cp\u003e18 (52.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.988\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimary Outcome: Time taken to reach full feeds\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEarly(n\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLate(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMean Difference\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMean age of initiation of feeds (hours)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e24.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e20.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDay of Attainment of Full Feeds\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e10.06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-0.935\u0026thinsp;\u0026plusmn;\u0026thinsp;0.746\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.215\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSecondary outcome\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEarly(n\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e\u003cp\u003en(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLate(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\u003cp\u003en(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFeed Intolerance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7 (21.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11 (32.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.339\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePMA to achieve full feeds weeks(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e33.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.402\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBirth weight regained at Day of life(gms) (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.21\u0026thinsp;\u0026plusmn;\u0026thinsp;6.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15.23\u0026thinsp;\u0026plusmn;\u0026thinsp;6.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.060\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEUGR at discharge(days)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7 (21.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9 (26.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.663\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of Anthropometric parameters at discharge variables in Early vs Late Fortification group\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEarly(n\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLate(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMean Difference\u0026thinsp;\u0026plusmn;\u0026thinsp;SE)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRate of Weight gain velocity at\u0026nbsp;discharge (g/kg/day)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.96\u0026thinsp;\u0026plusmn;\u0026thinsp;4.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e12.779\u0026thinsp;\u0026plusmn;\u0026thinsp;5.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.189\u0026thinsp;\u0026plusmn;\u0026thinsp;1.198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.875\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRate of OFC Gain velocity at discharge (cm/wk)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e-0.0092\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.832\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRate of Length Gain velocity at discharge (cm/wk)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.0349\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.464\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn both groups, the feeds were advanced by 20 ml/kg/day, until a maximum enteral feed of 150ml/kg/day was attained and maintained for 24 hours. As a unit policy, the total parenteral nutrition was initiated in the form of glucose infusion, aminoven and lipids for all babies with birth weight 1600 grams.\u003c/p\u003e\u003cp\u003eOnce full feeds were reached i.e. 150 ml/kg/day and sustained for 24 hours, feeds were increased to a maximum of 180 ml/kg/day based on the growth and daily weight gain of the baby. All efforts were made to provide MOM( mothers own milk ) to babies. Babies who had received\u0026thinsp;\u0026gt;\u0026thinsp;70% (8 feeds) of EBM were enrolled in the study.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFeeding protocol\u003c/strong\u003e\u003cp\u003eFeeding was initiated within 2 hours of life, and were given as 2-hourly, bolus feeds. Pre-feed abdominal girth charting was performed only in cases when there was suspicion of abdominal distension. Baby was strictly monitored for feed intolerance (vomiting, abdominal distension), apnea, signs of sepsis and NEC. Any baby whose feeds were discontinued in view of feed intolerance, feeds were re-initiated and advanced as per the clinician\u0026rsquo;s directive till the baby was enrolled in the clinical trial.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eEnteral feeds were given via orogastric tubes in preterm infants\u0026thinsp;\u0026lt;\u0026thinsp;32 weeks of gestation initially followed by spoon feeds once the infant was ready for oral feeds. In preterm infants\u0026thinsp;\u0026gt;\u0026thinsp;32 weeks\u0026rsquo; gestation, oral spoon feeds were tried, and if taken well was continued along with breastfeeds. Cue-based individualised feeding was followed as a part of the unit protocol. As the feed volume was advanced i.e. \u0026gt;60% of the required fluid, parental nutrition, including aminoven and lipids, was decreased, and only glucose infusion was continued. For giving TPN, a central line was placed. Fortification was done bedside by the nursing staff, following all aseptic precautions.\u003c/p\u003e\u003cp\u003eIn both groups, the HMF used was the same throughout the study period (Lactodex powder, 1g sachet). 1g of the powdered sachet was added to 25 ml of human milk as a standard fortification norm to maintain the osmolarity.\u003c/p\u003e\u003cp\u003eEnrolled infants were monitored as per the unit protocol for vital parameters, assessed regularly for feed intolerance and anthropometric measurements were also assessed regularly. Supplements were added as per the requirement of neonates. The subjects were followed up till discharge or death.\u003c/p\u003e\n\u003ch3\u003eOutcome variables and their measurements\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003ePrimary Outcome-\u003c/b\u003e To compare the time taken to reach full feeds (150ml/kg/day sustained for at least 24 hrs) in both groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSecondary outcomes-\u003c/b\u003e To compare the outcome in terms of incidence of feed intolerance, incidence of NEC (as per Bell\u0026rsquo;s stage\u0026thinsp;\u0026ge;\u0026thinsp;2), Incidence of Sepsis, Total duration of intravenous fluid usage, Time of regaining birth weight, Total duration of hospital stay, Weight gain per kg per day at 1 month of age, Extrauterine growth retardation (EUGR) at 36 weeks PMA, Duration of hospital stay, All-cause mortality, Incidence of neonatal morbidities including, patent ductus arteriosus, severe intraventricular haemorrhage (grade III and IV), periventricular leukomalacia, and retinopathy of prematurity\u003c/p\u003e\u003ch3\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eEthical approval was obtained from the Institutional Ethics Committee. Written informed consent was taken from parents prior to enrolment.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eSample Size Calculation and Statistical analysis\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eSample size\u0026nbsp;\u003c/strong\u003ewas determined based on the previous year\u0026rsquo;s data from our unit, where the mean duration of Parenteral nutrition (PN), a surrogate of attainment of full feeding was attained at 14 \u0026plusmn; 5.5 days in very low birth weight neonates. With the current intervention, the need for a mean duration of parenteral nutrition in VLBW neonates was expected to demonstrate a reduction of 20% of PN days (3 days earlier). Thus, assuming the same SD (5.5) with an \u0026alpha; error of 5% and a power of 80%, a total of 64 subjects (32 in each arm) were required to be enrolled.\u003c/p\u003e\n\u003cp\u003eStatistical analysis was done using the appropriate software version of SPSS. Results were analyzed as per the \u0026lsquo;Intention to Treat\u0026rsquo; analysis. Descriptive stats were used wherever applicable. Continuous outcomes were summarized using mean and standard deviation. Student t-test was used to compare normally distributed continuous data. For all comparisons, p\u0026lt; 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 102 preterm neonates were screened for eligibility, of which 36 were excluded, 66 met the inclusion criteria and were randomized into two arms. The Early HMF fortification group (n=32) received fortification once the neonate tolerated 60 ml/kg/day of expressed breast milk for two consecutive feeds, while the Late HMF fortification group (n=34) received fortification once the neonate tolerated 100 ml/kg/day of expressed breast milk for two consecutive feeds(figure 1)\u003c/p\u003e\n\u003cp\u003eThe baseline maternal and neonatal characteristics were comparable between groups (Table 2). The primary outcome(Table 3, Figure 2- \u0026nbsp;the time taken to reach full feeds (150ml/kg/day sustained for at least 24 hrs) was a mean of 10.06 ± 2.14 days in the early fortification group and \u0026nbsp;11.00 ± 3.62 days in the late fortification group, with a mean difference \u0026nbsp;between the groups was -0.94 ± 0.75 days, (p = 0.215).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile early fortification was associated with a trend towards earlier achievement of full enteral feeds, the difference compared to late fortification was not statistically significant.\u003c/p\u003e\n\u003cp\u003eAmong secondary outcomes (Table 4), the incidence of feed intolerance, postmenstrual age (PMA) at achievement of full feeds, Duration of IVF used (days), the weight at discharge and EUGR at discharge were comparable in both groups. The day of life at which birth weight was regained was slightly earlier in the early group compared to the late group but the mean gestational age at discharge was significantly less in the late fortification group.\u003c/p\u003e\n\u003cp\u003eThe anthropometric parameters including rate of weight gain, OFC gain and length gain, at discharge were also comparable in both groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong neonatal morbidities (Table 5, incidence of Shock, and hemodynamically significant patent ductus arteriosus, incidence of sepsis, osteopenia of prematurity was comparable across the two groups. Incidence of PVL was more in the late fortification group but not significant. There were no cases of IVH ≥ (grade III), NEC, severe retinopathy of prematurity.\u003c/p\u003e\n\u003cp\u003eThe mean duration of hospital stay was more in the late fortification group (p = 0.548).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOptimal nutritional support remains a cornerstone of care in preterm and very low birth weight (VLBW) infants, where growth failure is strongly associated with adverse neurodevelopmental outcomes.\u003csup\u003e7\u003c/sup\u003e Although human milk is universally accepted \u0026nbsp;as the ideal source of nutrition for the newborns, its intrinsic protein and caloric content often fall short of meeting the heightened metabolic demands of premature infants.\u003csup\u003e8\u003c/sup\u003e Human milk fortification is therefore is now the standard practice in neonatal intensive care.\u003csup\u003e9\u003c/sup\u003e However, the optimal timing of fortification continues to have no consensus without many studies \u0026nbsp;Concerns over early initiation largely focus on potential risks of feeding intolerance or necrotizing enterocolitis (NEC), whereas its advocates emphasize enhanced nutrient delivery, faster achievement of growth milestones, and reduced incidence of extrauterine growth restriction (EUGR). Our randomized controlled trial of \u0026nbsp;early versus delayed fortification in preterm infants of birth weight 1000-1600 grams is another step to find out a evidence based suitable \u0026nbsp;timing of fortification of human milk . The primary outcome of our study was the time taken to achieve full enteral feeding. Infants in the Early Fortification Group attained full feeds in 10.06 ± 2.14 days, compared with 11.00 ± 3.62 days in the Late Fortification Group. Although the mean difference of −0.94 ± 0.75 days favored the Early group, the result did not reach statistical significance (p = 0.215). Nonetheless, the trend is clinically meaningful. Our findings align with Gupta et al. (2024)\u003csup\u003e10\u003c/sup\u003e, who reported almost identical times to full feeds in early and late groups (5.56 vs. 5.38 days), and with the trial by Shah et al.\u003csup\u003e11\u003c/sup\u003e, where the median duration to full feeds was 20 days in both groups (p = 0.45). Similarly, Alyahya et al.\u003csup\u003e12\u003c/sup\u003e found no significant impact of timing of fortification on achievement of full feeds. Collectively, these studies suggest that early fortification does not delay progression to full enteral feeding and, in certain cohorts, may be associated with modestly faster advancement.\u003csup\u003e10,11,12\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003eFeeding intolerance, the most cited safety concern in this context, was not increased by early fortification in our trial. Intolerance occurred in 21.9% of infants in the Early group compared with 32.4% in the Late group (p = 0.339). Although statistically non-significant, this pattern suggests atleast that early fortification is not harmful. Similar observations were reported by Gupta et al.\u003csup\u003e10\u003c/sup\u003e, who found intolerance in 40% of early-fortified infants versus 29% in the late group, though not significant. Alizadeh et al.\u003csup\u003e13\u003c/sup\u003e also reported higher intolerance in early-fortified infants, without significance, while Shah et al.\u003csup\u003e11\u003c/sup\u003e documented similar episode counts between groups (58 vs. 57). The meta-analysis by Hilditch et al.\u003csup\u003e14\u003c/sup\u003e further consolidates these findings, showing little or no difference in feeding intolerance when fortification was initiated even at ≤40 mL/kg/day versus later at 75 mL/kg/day. Importantly, no cases of NEC ≥ stage 2b were observed in our study, echoing the safety profile consistently reported in the literature.\u003csup\u003e10,11,13,14\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003eAn important clinical observation in our study was the time to regain birth weight. Neonates in the Early Fortification Group regained birth weight in 12.21 ± 6.14 days compared to 15.23 ± 6.87 days in the Late group, with the difference approaching statistical significance (p = 0.065). This observation is supported by Gupta et al.\u003csup\u003e10\u003c/sup\u003e, who also reported earlier weight regain in early-fortified infants (10.13 vs. 11.26 days). Mechanistically, earlier fortification ensures greater protein and caloric intake during the critical early weeks of adaptation, supporting more rapid recovery of intrauterine growth trajectories. This is further supported \u0026nbsp;by the work of Shah et al.\u003csup\u003e11\u003c/sup\u003e, who documented significantly higher protein intake in early-fortified neonates during the first three weeks of life, with cumulative four-week intake markedly superior (98.6 vs. 89.6 g/kg, p \u0026lt; 0.001). At discharge, weight gain velocity in our cohort was comparable between groups (12.96 ± 4.24 vs. 12.78 ± 5.38 g/kg/day, p = 0.875). Similarly, length gain (0.74 ± 0.18 vs. 0.70 ± 0.20 cm/week, p = 0.464) and head circumference gain (0.54 ± 0.17 vs. 0.55 ± 0.17 cm/week, p = 0.832) showed no significant differences. These findings are consistent with Alyahya et al.\u003csup\u003e12\u003c/sup\u003e, who reported similar growth velocities across groups, and with Wynter et al.\u003csup\u003e16\u003c/sup\u003e, who observed non-significant trends toward better growth in the early group. Alizadeh et al.\u003csup\u003e13\u003c/sup\u003e found no significant differences in Z scores for weight or length but noted a non-significant trend toward greater head growth in early-fortified infants. Conversely, Gupta et al.\u003csup\u003e10\u003c/sup\u003e reported significantly higher median gains in weight (11.16 vs. 8.99 g/kg/day), length (1.0 vs. 0.88 cm), and head circumference (0.77 vs. 0.62 cm) favoring early fortification. Salas et al.\u003csup\u003e15\u003c/sup\u003e, in a cohort of extremely preterm neonates (median GA 26 weeks), also reported superior outcomes in the Early group, including higher weight (+131 g, p = 0.03), fat-free mass (+103 g, p = 0.03), and length (+0.9 cm, p = 0.04). These findings suggest that the magnitude of benefit from early fortification may vary depending on the maturity of the cohort studied, with extremely preterm infants deriving the greatest advantage.\u003cbr\u003e\u0026nbsp;\u003cbr\u003eOther clinical outcomes in our study were reassuring. The mean duration of hospital stay did not differ significantly (36.37 vs. 38.82 days, p = 0.548). Interestingly, gestational age at discharge was significantly higher in the Early group (37.09 ± 1.89 vs. 36.13 ± 1.65 weeks, p = 0.031), which may indicate better readiness for discharge among early-fortified infants. EUGR occurred in 21.9% of Early and 26.5% of Late group infants (p = 0.663), a pattern similar to Gupta et al.\u003csup\u003e10\u003c/sup\u003e, who also reported EUGR in approximately one-fifth to one-third of infants in both groups. Neonatal morbidities, including sepsis, PDA, periventricular leukomalacia, and osteopenia of prematurity, showed no significant differences, reinforcing the safety of early fortification. Notably, no infant developed severe intraventricular hemorrhage, NEC ≥ stage 2b, or retinopathy requiring treatment.\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u0026nbsp;The strengths of this study include its randomized controlled design, comparable baseline characteristics, and detailed outcome analysis across nutritional and clinical parameters.\u0026nbsp;\u003cbr\u003e\u0026nbsp;In summary, our findings add to the growing evidence that early fortification of human milk is both safe and effective. It does not increase the risk of feeding intolerance or major morbidities and may confer clinically relevant benefits such as earlier regaining of birth weight and improved maturity at discharge. While growth velocities at discharge were comparable, studies in more immature cohorts have demonstrated clearer advantages, underscoring the importance of tailoring fortification strategies to gestational age. Larger, multicenter trials with longer-term follow-up are warranted to confirm these findings and to evaluate their impact on neurodevelopment.\u003c/p\u003e\n\u003cp\u003eThere were few limitations in our study \u0026nbsp;:The study was underpowered \u0026nbsp;and was limited by a smaller sample size.Although \u0026nbsp;Randomization and allocation concealment were done, blinding was not feasible due to the study design.The study \u0026nbsp;was conducted on all \u0026nbsp;preterms, with birth weight 1000-1600 gms ,without any specific gestation range and so its results cannot be extrapolated to ELBWs and ELGANs.Such studies which involve the use of exclusive human milk for feeding , it should be done at a centre which has its indigenous milk bank or can procure milk from them on demand.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEarly fortification of human milk (@ 60 ml/kg/day in preterm neonates with birth weight 1000-1600 gms was safe and well tolerated. While trends toward improved growth (weight gain and reduced EUGR) were observed but they were not statistically significant, hence larger multicenter studies may be required to confirm these findings. Based on this study, early fortification may be considered a feasible strategy for improving nutritional outcomes in preterm neonates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding: there was no funding for this study\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eK.S. Did the data collection and prepared the manuscriptG.K. Helped in statistics and preparing the manuscriptN.C. Did the help in preparing figures and tablesG.S. Did the help in preparing figures and tablesJ.K.-planned the study , advised in preparing the plan for execution of the study\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eSpecial acknowledgment to my wife Dr Sheeba Farooqui, without her support this study could not have been possible.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eClark RH, Thomas P, Peabody J. Extrauterine growth restriction remains a serious problem in prematurely born neonates. Pediatrics. 2003;111(5):986\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eFreitas BA, Priore SE, Lima LM, Franceschini SC. Extrauterine growth restriction: universal problem among premature infants[Restri\u0026ccedil;\u0026atilde;o de crescimento extrauterino: problema universal entre os]. Revista de Nutricao. 2016;29(1):53-64.\u003c/li\u003e\n\u003cli\u003eMorales Y, Schanler RJ. Human milk and clinical outcomes in VLBW infants: how compelling is the evidence of benefit? Seminars in Perinatology. 2007;31(2):83\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eNutritional needs of the preterm infant. In: Kleinman R, ed. Pediatric nutrition handbook. Elk Grove, IL: American Academy of Pediatrics; 2013. \u003c/li\u003e\n\u003cli\u003eNational Neonatology Forum. Clinical Practice Guidelines on Feeding of Low Birth Weight Neonates. India: NNF;2022. Available from: https://www.nnfi.org/assests/pdf/cpg-guidelines/FeedingofLBW.\u003c/li\u003e\n\u003cli\u003eKlingenberg C, Embleton ND, Jacobs SE, O\u0026apos;Connell LA, Kuschel CA. Enteral feeding practices in very preterm infants: an international survey. Archives of Disease in Childhood-Fetal and Neonatal Edition. 2012;97(1):56-61.\u003c/li\u003e\n\u003cli\u003eAmerican Academy of Pediatrics Committee on Nutrition. Nutritional needs of preterm infants. In: Kleinman RE, Greer FR, editors. Pediatric Nutrition. 7th ed. Elk Grove Village (IL): American Academy of Pediatrics; 2014. p. 83-122.\u003c/li\u003e\n\u003cli\u003eUnderwood MA. Human milk for the premature infant. Pediatr Clin North Am. 2013;60(1):189-207. doi:10.1016/j.pcl.2012.09.008.\u003c/li\u003e\n\u003cli\u003eArslanoglu S, Corpeleijn W, Moro G, Braegger C, Campoy C, Colomb V, et al. Donor human milk for preterm infants: current evidence and research directions. J Pediatr Gastroenterol Nutr. 2013;57(4):535-42. doi:10.1097/MPG.0b013e3182a3af0a.\u003c/li\u003e\n\u003cli\u003eGupta G, Murugesan A, Thanigainathan S, Adhisivam B. Does early fortification of human Milk decrease time to regain birth weight as compared to late fortification among preterm infants \u0026mdash;a randomized controlled trial. Indian Journal of Pediatrics. 2025 Jun;92(6):592-7.\u003c/li\u003e\n\u003cli\u003eShah SD, Dereddy N, Jones TL, Dhanireddy R, Talati AJ. Early versus delayed human milk fortification in very low birth weight infants - a randomized controlled trial. Journal of Pediatrics. 2016;174:126-31. [DOI: 10.1016/j.jpeds.2016.03.056] [PMID: 27112041]\u003c/li\u003e\n\u003cli\u003eAlyahya S, Alayed N, Alghuraybi S, Alshahrani F, Alsulaimi F, Almutairi B. Human milk fortification and use of infant formulas to support growth in neonatal intensive care unit infants. \u003cem\u003eNutrients\u003c/em\u003e [Internet]. 2023 Oct 19 [cited 2025 Sep 10];15(20):4443. Available from: https://doi.org/10.3390/nu15204443. \u003c/li\u003e\n\u003cli\u003eAlizadeh Taheri P, Sajjadian N, Asgharyan Fargi M, Shariat M. Is early breast milk fortification more effective in preterm infants?: a clinical trial: Early and late fortification. Journal of perinatal medicine. 2017;27;45(8):953-7.\u003c/li\u003e\n\u003cli\u003eHilditch C, Keir A, Collins CT, Middleton P, Gomersall J. Early versus delayed introduction of human milk fortification in enterally fed preterm infants: A systematic review and meta‐analysis. Journal of Paediatrics and Child Health. 2022;58(1):30-8.\u003c/li\u003e\n\u003cli\u003eSalas AA, Gunawan E, Nguyen K, Reeves A, Argent V, Finck A, Carlo WA. Early human milk fortification in infants born extremely preterm: a randomized trial. Pediatrics. 2023 Sep 1;152(3):e2023061603.\u003c/li\u003e\n\u003cli\u003eWynter Z, Gorham JA, Thompson AB, Mundy C, Waller JL, Stansfield BK. Immediate fortification of human milk with a bovine milk-derived human milk fortifier in very low birth weight infants: a randomized clinical trial. Journal of Perinatology. 2024 Nov;44(11):1591-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7596338/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7596338/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreterm very low birth weight infants are at high risk of postnatal growth failure due to limited nutrient reserves and high metabolic demands. Human milk fortification is essential to optimize growth, but the timing of initiation remains controversial, with concerns regarding feeding tolerance and metabolic complications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo compare the effects of early versus delayed initiation of human milk fortification on feeding tolerance, growth outcomes, and neonatal morbidities in preterm infants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis randomised controlled Trial enrolled 66 preterm neonates ≤34 weeks of gestation, who were grouped \u0026nbsp;between \u0026nbsp;an Early Fortification Group (n = 32), in which fortification was initiated once enteral feeds reached 60 ml/kg/day and baby \u0026nbsp;tolerated for next two feeds, and a Late Fortification Group (n = 34), in which fortification was initiated at enteral feeds of 100 ml/kg/day and tolerated for next two feeds \u0026nbsp;. Baseline maternal and neonatal characteristics were recorded. The primary outcome was the time to achieve full enteral feeds. Secondary outcomes included feeding intolerance, time to regain birth weight, growth velocity, duration of intravenous fluids, hospital stay, gestational age at discharge, incidence of extrauterine growth restriction, and other neonatal morbidities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe two groups were comparable at baseline. The mean time to reach full enteral feeds was 10.06 ± 2.14 days in the Early Fortification Group and 11.00 ± 3.62 days in the Late Fortification Group, with no statistically significant difference (p = 0.215). Feeding intolerance occurred in 21.9% of the early group and 32.4% of the late group (p = 0.339). Birth weight was regained earlier in the early group (12.21 ± 6.14 days) compared with the late group (15.23 ± 6.87 days), showing a trend towards significance (p = 0.065). Growth velocity in terms of weight, length, and head circumference was similar in both groups. The mean duration of hospital stay was 36.37 ± 18.08 days in the early group and 38.82 ± 14.73 days in the late group (p = 0.548). The mean gestational age at discharge was significantly higher in the early fortification group (37.09 ± 1.89 weeks vs. 36.13 ± 1.65 weeks, p = 0.031). Extrauterine growth restriction was observed in 24.2% of the overall cohort, with no significant difference between groups (21.9% vs. 26.5%, p = 0.663). No cases of necrotizing enterocolitis ≥ Stage 2b or retinopathy of prematurity requiring treatment were seen. Osteopenia of prematurity occurred in 34.4% of the early group and 35.3% of the late group (p = 0.938). The incidence of sepsis, patent ductus arteriosus, periventricular leukomalacia, and intraventricular hemorrhage was similar in both groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEarly initiation of human milk fortification was safe, well tolerated, and not associated with increased feeding intolerance or gastrointestinal morbidity. Although most growth outcomes were comparable, early fortification demonstrated a favorable trend toward earlier regaining of birth weight and was associated with a significantly higher gestational age at discharge. These findings suggest that early fortification may be beneficial in moderately preterm infants without compromising safety.\u003c/p\u003e","manuscriptTitle":"Early versus Delayed Fortification of Human Milk in Preterm Neonates: A Randomised Controlled Trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 13:26:52","doi":"10.21203/rs.3.rs-7596338/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":"02b94927-80fe-4d91-9272-855bec24effc","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T14:53:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-26 13:26:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7596338","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7596338","identity":"rs-7596338","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}