Human milk is the best food for the preterm infant, containing immunologic, nutritional, and developmental support.(1) However, the higher nutritional needs of preterm neonates (those born before 34 weeks of gestation and neonates weighing less than 1500 g) cannot be met with unfortified human milk alone.(2) Multi-nutrient fortification of human milk has shown to have beneficial effects on growth outcomes in the hospital setting among this vulnerable group.(3) The giving of human milk fortifier (HMF) has become routine in neonatal intensive care units (NICUs) throughout the world after a threshold volume of enteral feeds, generally at 80-100 mL/kg/day.(4)

Although there are numerous advantages, FI is still a common problem when administering HMF and can include gastric residuals, abdominal distention, emesis or changes to stool pattern.(5) From clinical experience and physiological studies we have reason to believe that HMF may slow gastric emptying, which may be a factor in intolerance.(6) The half-emptying time for fortified breast milk was also found to be more than double that of unfortified breast milk (48 minutes compared to 21 minutes) using the ultrasonic techniques, thus giving a physiological explanation of the observed phenomenon in clinical studies.(7) This feeding intolerance frequently requires the removal of fortification, resulting in sustained exposure to unfortified milk, sub-optimal delivery of nutrients and risk of impaired growth and neurodevelopmental outcomes.(8)Feeding intolerance is a major clinical problem in the management of HMF.

 Although temporary cessation of HMF is typical, there is no standard protocol for reintroducing HMF.(9) There is a lack of clarity around the timing of introducing fortification to the diet, at what feeding level to start introducing fortification and the care of feeding intolerance. But, the results of the early versus delayed fortification showed no significant difference in feeding intolerance, sepsis, or NEC, and the quality of the evidence was low.(10) One area where neonatal nutrition research is lacking is the reintroduction of HMF after the infant becomes intolerant of it.(11) The reintroduction of fortification and its reintroduction method are sometimes made on the basis of the individual clinician's preferences in clinical practice.(12) This can make breast-feeding unsuccessful, keep babies in the hospital longer, and even prevent some morbidity. The use of a standardized, step-wise reintroduction program may offer a template for the resumption of fortification and reduce the risk for recurrent intolerance and potentially serious complications, like NEC.(13)

To fill this gap in the literature, the current study aimed to assess clinical outcomes after the onset of feeding intolerance associated with HMF in preterm infants and subsequent reintroduction of HMF in a standardized manner. We hypothesized that a stepwise reintroduction protocol would be both feasible and safe and would lead to good feeding and growth outcomes. The aims were to identify the time taken to achieve full enteral feeds post reintubation, to describe growth velocity, and to measure the incidence of adverse outcomes such as NEC, sepsis, and death.

Study Design and Setting

This prospective cohort study was carried out in the Department of Neonatology, The Children's Hospital, Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan from May 2025 to July 2026. PIMS is tertiary care teaching hospital which acts as a major referral center for high risk pregnancies and pre-term deliveries in the Islamabad-Rawalpindi area and its surrounding areas. Each year, the neonatal unit receives about 1,500 neonates, many of whom are very low birth weight (VLBW) and extremely low birth weight (ELBW).

Study Population

The study included preterm neonates admitted to NICU who experienced feeding intolerance after beginning human milk fortification. Eligible neonates were screened and recruited consecutively for a 15-month period. This sample size of 101 babies was determined by several factors: First, the anticipated rate of feed intolerance associated with HMF in the study setting was approximately 25-30% of fortified preterm neonates, which was used to estimate the expected primary outcome of successful reintroduction at 85% with a precision of ±8% at 95% confidence interval.

Inclusion Criteria

The following criteria were used to include neonates:

Gestational age – ≤34 weeks at birth.

Weight at birth of 750 g to 1,800 g

Acceptance of enteral feeds (unfortified human milk, mother's own milk or donor milk) of 80 mL/kg/day or more prior to fortification

Feeding intolerance within 72 hours of the introduction of regular human milk fortification

Feeding intolerance was measured as: at least 2 of the following: Gastric residual volume > 50% of the previous feeding, Abdominal distention or tenderness, emesis (more than one episode in 24 hours), visible blood in stool.

Having informed consent from parent/legal guardian

Exclusion Criteria

The neonates were excluded if any of the following were present: Major congenital anomalies such as gastrointestinal anomalies (gastroschisis, omphalocele, intestinal atresia) Necrotizing enterocolitis (Bell stage ≥II) at feeding intolerance time Hemodynamic instability on enrollment and requiring vasopressor support Sepsis diagnosed by culture at the time of feeding intolerance.

Formula fed as main enteral nutrition source

Contraindications to feeding milk from a human source (e.g., where there is no safe donor milk, the mother may have HIV infection) Parent refusal to get involved

Definitions

Feeding intolerance (FI): As defined in inclusion criteria, at least 2 of the following gastrointestinal dysfunction signs within 72 hours of HMF initiation.

FI (temporally associated with HMF): Feeding intolerance temporally associated with administration of HMF, resolution or improvement after temporary discontinuation of HMF. Standardized "reintroduction" protocol: A step-wise process for reintroducing HMF after an FI is resolved (detailed below). Full enteral feeds: Tolerated for at least 48 hours, enteral nutrition of 150 mL/kg/day fortified human milk.

Growth velocity: Weight gained in g/kg/day from weight at reintroduction until weight at discharge or 36 weeks post menastrum, whichever was earlier.

Necrotizing enterocolitis (NEC): Bell stage ≥II on clinical and radiographic criteria. Sepsis which has been confirmed in culture: A blood culture that is positive in the presence of clinical signs of infection. Supplements and salts for human milk.HMFP - human milk fortification protocol.

Fortification of enteral feeds was begun at 80 mL/kg/day using a bovine milk-based human milk fortifier, such as the Nestlé fortifier, FM-85 (or equivalent) and following manufacturer's instructions. Expressed human milk was fortified with the recommended amount of fortifier powder just prior to feeding. Increments of 15-20 mL/kg/day were given as tolerated.

Treatment of Intolerance of Feeding related to HMF.

Once the feeding intolerance was identified as being associated with HMF, the following steps were taken:

The immediate stoppage of HMF. Continuation of unfortified human milk at the same feeding volume or decreased feeding volume according to the severity of the clinical condition. Abdominal examination and vital signs; lab testing if needed. If NEC is suspected, a radiograph of the foot or ankle will be performed. Absence of gastric residuals >50%, resolution of abdominal distention, no emesis for 24 hours, no blood in stool for 48 hours was defined as resolution of FI.

Standardized Reintroduction Protocol

HMF was then reintroduced according to a standard protocol:

Step 1 (Day 1): Start to give HMF at 25% of the normal fortification level with the 1st feed of the day, then give milk without HMF for the rest of the feeds.

Step 2 (Day 2): If no signs of FI, increase all feeds to 50% of the normal fortification dose.

Step 3 (Day 3): If tolerated, ramp up to 75% of the typical amount of fortification.

If well tolerated, proceed to the standard fortified dose in Step 4 (Day 4).

If any one FI sign returned at any point (with at least one FI sign reappearing and on the original threshold), the protocol was halted, unfortified feeds were continued for 48 hours and reintroduction was resumed at the previous tolerated step. The maximum number of reintroduction attempts was two, with a reintroduction failure defined as reintroduction failure if a bull was not reintroduced after two attempts.

Data Collection

Standardised case report forms were used for data collection which was done prospectively. Demographic characteristics, perinatal history, milk transition, characteristics of the FIs, and clinical outcome were recorded. Calibrated electronic scales were used to obtain daily weights. Head circumference and length were measured every week. Laboratory and radiologic results were documented as indicated. All data were checked by a second investigator.

Outcome Measures

Primary outcomes:

Time to full enteral feeds (150mL/kg/day) post reintroduction Growth velocity (g/kg/day) from reintroduction to discharge

Secondary outcomes:

Successful reintroduction (full fortification without reoccurrence of FI) Length of stay (LOS) is a measure that quantifies the number of days spent in a hospital.

Incidence of NEC (Bell stage ≥II)

The incidence of culture proven sepsis

Incidence of mortality

Reintroduction-related adverse events

Statistical Analysis

Data were analyzed by SPSS version 26.0 (IBM Corp., Armonk, NY). Data for continuous variables were reported as mean ± SD or median (IQR) depending on the distribution. Data for categorical variables were presented as frequencies and percentages. Independent samples t-test, Mann-Whitney U test, chi-square test or Fisher's exact test was performed, as appropriate, for comparative analyses. A p value < 0.05 was deemed to be statistically significant. A multivariate logistic regression was used to determine factors related to failed reintroduction cases.

Ethical Considerations

The Institute of Medical Sciences, Islamabad (as an institutional review board) approved the study. Parents/legal guardians of all neonates that participated gave written informed consent. The study was performed according to the Declaration of Helsinki and Good Clinical Practice guidelines.

Baseline Characteristics

Enrolled were 101 neonates born prematurely who experienced HMF-associated feeding intolerance. The demographic details of the study sample are presented in Table 1. The mean gestational age was 30.2 ± 2.4 weeks, and the mean birth weight was 1,247 ± 312 g. Out of 100, 54.5% (n=55) were male neonates. Most neonates (68.3%, n=69) were delivered by cesarean section. In 72.3% (n=73) the antenatal corticosteroids were administered. The mean age at initiation of fortification was 7.2 ± 2.1 days and feeding volume at initiation of fortification was 92.4 ± 15.6 mL/kg/day.

Table 1: Baseline Characteristics of Study Population (n=101)

Characteristics of Feeding Intolerance Episode

The characteristics of the feeding intolerance episodes associated with the HMF are shown in Table 2. Gastric residuals of > 50% (82.2%, n = 83), abdominal distention (61.4%, n = 62) and emesis (38.6%, n = 39) were the most common presenting features, respectively. The median time from HMF initiation to onset of FI was 1.5 days (IQR: 1-2). The mean interruption of HMF was 2.8 ± 1.2 days. For the FI episode, 18 neonates (17.8%) needed to have their feeds reduced temporarily, and 6 neonates (5.9%) had feeding temporarily stopped.

Table 2: Characteristics of Feeding Intolerance Episode (n=101)

Reintroduction Outcomes

The results after standardized reintroduction of HMF are presented in Table 3. Fortification was successful, (no FI recurrence) in 89 (88.1%) neonates. Of these, 67 neonates (66.3%) did not show FI signs during the reintroduction procedure. Twelve (11.9%) neonates had a recurrence of FI during reintroduction and had to have their protocol paused and restarted, with 9 (75%) of these ultimately reaching full fortification. Failure to reach full fortification (after two attempts), or reintroduction failure, was experienced by 12 neonates (11.9%). The average number of days to achieve full enteral feed after reintroduction was 14.3 ± 4.1 days. The reintroduction to discharge growth velocity was 18.6 ± 5.2 g/kg/day on average.

Table 3: Reintroduction Outcomes

Clinical Outcomes

Secondary clinical outcomes are shown in Table 4. The incidence of NEC (Bell stage ≥II) was 5.9% (n=6) of which all cases were after full fortification. Sepsis culture positive was seen in 13 neonates (12.9%). Coagulase-negative Staphylococcus (n=5), Klebsiella pneumoniae (n=4), and Escherichia coli (n=3) were the most frequently cultured organisms. The median duration of hospital stay was 42 days (IQR: 35-52). There were 3 deaths (overall 3.0%): severe sepsis with multi-organ dysfunction (n=2); NEC with perforation (n=1). No adverse events that resulted in the permanent cessation of HMF were seen during reintroduction.

Table 4: Secondary Clinical Outcomes

The present prospective cohort study assessed clinical outcomes after HMF-associated feeding intolerance and standardized reintroduction of the HMF in preterm neonates. A stepwise, protocolized reintroduction approach was successful in 88.1% of neonates, and the mean time to full enteral feeds was 14.3 days and 18.6 g/kg/day growth velocity. The rate of NEC was 5.9%, and mortality 3.0%, which is similar to or lower than other high-risk preterm populations.

The findings of HMF precipitating feeding intolerance is consistent with the physiological study  showed that fortification had a significant effect on gastric emptying rates in preterms.(7) Fortified breast milk had a longer mean half-emptying time than unfortified milk (48 min vs. 21 min, respectively), which is likely to explain the clinical signs of feeding intolerance seen in our study population.(14) The high prevalence of gastric residual (82.2%) and abdominal distention (61.4%) as initial symptoms is consistent with delayed gastric emptying being a major mechanism. This pathophysiological insight helps to explain why temporary cessation of HMF rather than permanent discontinuation is warranted.(15)

The success rate of standardized reintroduction in our study is 88.1%, which is comparable to the few published studies on the subject.(16) Bharadwaj N et al. reported a case series of 7 VLBW infants who were intolerant with bovine-based HMF who were successfully treated with human milk HMF as rescue therapy.(17) All 7 infants were successfully weaned from parenteral nutrition without a recurrence of gastrointestinal symptoms and had an improvement in their growth and nutrient intakes.(18) That study did use a different fortifier, but the general concept of structured reintroduction and close monitoring was similar, although it had been using a fortifier derived from human rather than bovine milk.(19) Our larger prospective

cohort further confirms these findings by showing the feasibility of reintroduction with the same fortifier source

used from a different bovine origin after a specified protocol.

The time to full enteral feeds in our study (14.3 days post-reintroduction) is similar to results of a retrospective study by Jensen GB et al. who found that infants fed human milk protein fortifier reached full feeds at a median of 14 days, compared to 16 days with bovine fortifier.(20) However, it is worth to be noted that Wickland compared the different types of fortifiers and that their cohort of reintroduced infants did not specifically target those who had previously had feeding intolerance.(21) However, the very similar feeding advancement times imply that our reintroduction protocol was not significantly different in the feeding advancement time needed to achieve nutritional goals than standard fortification practice.(22)

Growth velocity was similar to that which has been reported in other preterm infant cohorts (18.6 g/kg/day), but is less than the 20-30 g/kg/day that is commonly cited as the ideal growth velocity for infants born prematurely.(23) This is similar to the systematic review by Ottolini KM et al. that reported that human milk-derived fortifiers were associated with lower weight gain than bovine-derived fortifiers (mean difference -1.08 g/kg/day).(24) Our study employed only bovine fortifier, however, and the growth velocity obtained indicates that the nutrient delivery did not seem to be significantly affected when full fortification was restored, with the stepwise reintroduction protocol. This is a period of decreased fortification during the reintroduction steps that could have led to slightly reduced growth velocity but this should be balanced against the risk of reoccurring feeding intolerance and/or NEC.The rate of NEC in our study (5.9%) is similar to those found in preterm infants fed fortified human milk.(25) little or no difference between early and delayed fortification in terms of NEC rates, with low quality evidence.(26) Importantly, in our study, the entire process of reintroduction was not associated with the time of NEC development, as all the cases were observed after full fortification. This observation gives preliminary comfort that there is no increased risk of NEC with gradual reintroduction, but due to the limited number of events, definitive conclusions cannot be drawn.(27)

The median length of hospital stay in our cohort (42 days) was reflective of the complexity associated with hospital care of preterm infants who are feeding intolerant and the importance of prolonged monitoring.(28) Although longer than some studies of uncomplicated preterm infants, similar to those with feeding-related morbidity.(28) Early fortification was significantly associated with longer hospital stay (mean difference 4.29 days) than late fortification and the risk of feed intolerance or NEC was increasing in early fortification.(26) We believe that our conclusions suggest that even if feeding intolerance does develop, a structured approach to reintroduction can also reduce some of the consequences downstream that may otherwise extend hospitalization.

The advantages of this study are that it is prospective, uses a standard reintroduction method and has a comprehensive outcome assessment. This is the first prospective cohort study to investigate a uniform reintroduction program after feeding intolerance due to HMF. The protocolized approach improves the uniformity of results and offers a scaffold which can be customized to other NICU environments. The number of neonates (101) is large for a single-center study of this clinical situation.

tandardized stepwise human milk fortifier reintroduction was successful in full fortification in 88.1% of preterm neonates who were fed by human milk and had feeding intolerance associated with HMF; time to full enteral feeds was 14.3 days with acceptable growth velocity of 18.6 g/kg/day. Incidence and mortality rates for NEC (5.9%) and death (3.0%) were similar to those reported in other populations, and no hazardous events related to reintroduction that actually required permanent cessation of HMF occurred. These results indicate that a protocol driven reintroduction strategy is possible, safe and effective for resuming fortification, and also for decreasing risk of recurrent intolerance.

Limitations

Findings in the present study from a single center may not be representative of all other clinical settings. The lack of a control group makes it difficult to draw comparisons to other feeding reintroduction methods. The number of neonates was too small to identify differences in less common events like NEC, mortality and factors associated with the failure to re-introduce into the wild. Follow-up was only carried out at the time of hospital discharge, which did not permit to assess the long-term growth and neurodevelopmental outcomes. Also, the subjective nature of some of the feeding intolerance signs may have caused some variation in the assessments and classifications.

Recommendations

The standardized reintroduction protocol should be validated in more extensive multicenter studies in various NICUs in the future. Clinical superiority of stepwise reintroduction over other strategies should be established by randomized controlled trials (RCTs). More animals should be studied to find predictors that explain why reintroduction fails among mothers, neonates and feeding. Growth and neurodevelopmental outcomes should be evaluated at long term follow-up (18-24 mo corrected age). Evidence should continue to be gathered to further adapt the protocol steps and to guide the standardized implementation, via NICU quality improvement efforts.

1. Lugonja N, Marinković V, Pucarević M, Miletić S, Stojić N, Crnković D, et al. Human Milk—The biofluid that nourishes infants from the first day of life. Foods. 2024;13(9):1298.
2. Alanazi M, Altawili MA, Khayyal AI, Alahmari AS, Alhakami AA, Alshehri AMA, et al. Impact of early nutrition interventions on the growth and development of preterm infants: A narrative review. Cureus. 2024;16(2).
3. Beggs MR, Bando N, Unger S, O'Connor DL. State of the evidence from clinical trials on human milk fortification for preterm infants. Acta Paediatrica. 2022;111(6):1115-20.
4. Han J, Zhang L, Zhang R, Han S, Zhu J, Hu X, et al. Using a new human milk fortifier to optimize human milk feeding among very preterm and/or very low birth weight infants: a multicenter study in China. BMC pediatrics. 2024;24(1):61.
5. Benardout M, Le Gresley A, ElShaer A, Wren SP. Fructose malabsorption: causes, diagnosis and treatment. British journal of nutrition. 2022;127(4):481-9.
6. Verduci E, Salvatore S, Bresesti I, Di Profio E, Pendezza E, Bosetti A, et al. Semi-elemental and elemental formulas for enteral nutrition in infants and children with medical complexity—thinking about cow’s milk allergy and beyond. Nutrients. 2021;13(12):4230.
7. Geddes DT, Gridneva Z, Perrella SL, Mitoulas LR, Kent JC, Stinson LF, et al. 25 years of research in human lactation: from discovery to translation. Nutrients. 2021;13(9):3071.
8. Rodrigues CED. Breast milk feeding practices among very preterm infants: Effects on health-related outcomes during childhood: Universidade do Porto (Portugal); 2021.
9. Zhang T, Luo H, Wang H, Mu D. Association of human milk fortifier and feeding intolerance in preterm infants: a cohort study about fortification strategies in southwest China. Nutrients. 2022;14(21):4610.
10. Walsh V. Overview of Cochrane systematic reviews of early enteral feeding strategies for improving growth and reducing necrotising enterocolitis in very preterm or very low birth weight infants 2021.
11. Moreira DH, Gregory SB, Younge NE. Human milk fortification and use of infant formulas to support growth in the neonatal intensive care unit. Nutrition in Clinical Practice. 2023;38:S56-S65.
12. Sultan N, Varney JE, Halmos EP, Biesiekierski JR, Yao CK, Muir JG, et al. How to implement the 3-phase FODMAP diet into gastroenterological practice. Journal of neurogastroenterology and motility. 2022;28(3):343.
13. Walker M. Breastfeeding management for the clinician: using the evidence: Jones & bartlett learning; 2021.
14. Valla FV, Tume LN, Jotterand Chaparro C, Arnold P, Alrayashi W, Morice C, et al. Gastric point-of-care ultrasound in acutely and critically ill children (POCUS-ped): a scoping review. Frontiers in pediatrics. 2022;10:921863.
15. Saito A, Nowak T, Wo J. Understanding symptoms of gastric dysmotility: Nausea, vomiting, abdominal pain, postprandial fullness, and early satiety. Handbook of Gastrointestinal Motility and Disorders of Gut-Brain Interactions: Elsevier; 2023. p. 87-97.
16. Cronin C, Ramesh Y, De Pieri C, Velasco R, Trujillo J. ‘Early introduction’of cow’s milk for children with IgE-mediated cow’s milk protein allergy: A review of current and emerging approaches for CMPA management. Nutrients. 2023;15(6):1397.
17. Bharadwaj N, Panigrahy N, Bagga N, Chirla DK. Human Milk–Derived Fortifier as Rescue Therapy in Very Preterm Infants Intolerant to Cow’s Milk–Derived Fortifier. Indian Journal of Pediatrics. 2022;89(11):1131-3.
18. Roggero P, Liotto N, Piemontese P, Menis C, Perrone M, Tabasso C, et al. Neonatal intestinal failure: growth pattern and nutrition intakes in accordance with weaning from parenteral nutrition. Journal of Parenteral and Enteral Nutrition. 2023;47(2):236-44.
19. Adamkin DH. Use of human milk and fortification in the NICU. Journal of Perinatology. 2023;43(5):551-9.
20. Jensen GB, Domellöf M, Ahlsson F, Elfvin A, Navér L, Abrahamsson T. Effect of human milk-based fortification in extremely preterm infants fed exclusively with breast milk: a randomised controlled trial. EClinicalMedicine. 2024;68.
21. Wickland J, Wade C, Micetic B, Meredith K, Martin G. A retrospective analysis of the effects of an exclusively human milk protein diet on neonatal feeding tolerance. American Journal of Perinatology. 2022;39(09):0995-1000.
22. Mahmood Z, O’Donnell B, Brozanski BS, Vats K, Kloesz J, Jackson LE, et al. A quality improvement initiative standardizing the antibiotic treatment and feeding practices in patients with medical necrotizing enterocolitis. Journal of Perinatology. 2024;44(4):587-93.
23. Lyu Y, Zhu D, Wang Y, Jiang S, Lee SK, Sun J, et al. Current epidemiology and factors contributing to postnatal growth restriction in very preterm infants in China. Early human development. 2022;173:105663.
24. Ottolini KM, Schulz EV, Limperopoulos C, Andescavage N. Using nature to nurture: breast milk analysis and fortification to improve growth and neurodevelopmental outcomes in preterm infants. Nutrients. 2021;13(12):4307.
25. Grace E, Hilditch C, Gomersall J, Collins CT, Rumbold A, Keir AK. Safety and efficacy of human milk-based fortifier in enterally fed preterm and/or low birthweight infants: a systematic review and meta-analysis. Archives of Disease in Childhood-Fetal and Neonatal Edition. 2021;106(2):137-42.
26. 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.
27. Moschino L, Duci M, Fascetti Leon F, Bonadies L, Priante E, Baraldi E, et al. Optimizing nutritional strategies to prevent necrotizing enterocolitis and growth failure after bowel resection. Nutrients. 2021;13(2):340.
28. Macias R, Peterson D, Korkis L, Edson R, Gall R. Prevalence and impact of feeding-related events on hospital stay in preterm and term newborns. Advances in Neonatal Care. 2023;23(6):541-6.