Submitted:
15 July 2026
Posted:
21 July 2026
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Abstract
Background
Cow's milk-derived fortifiers (CMDFs) are widely used to improve the nutritional adequacy of human milk for very preterm infants. Although generally well tolerated, some infants develop feeding intolerance. Identifying these infants may facilitate more individualized nutritional strategies.
Objective
To identify clinical characteristics associated with adverse feeding events following CMDF use in infants receiving donor human milk (DHM) and to identify infants who may derive greater benefit from an exclusive human milk diet (EHMD).
Methods
This retrospective nationwide cohort study used the Japanese Human Milk Bank Database. Infants receiving DHM followed by CMDF were included. Clinical characteristics were compared between infants with and without adverse feeding events. Multivariable logistic regression analysis identified independent risk factors.
Results
Among 2,587 infants receiving DHM followed by CMDF, 2,476 had complete data. Adverse feeding events occurred in 315 infants (12.7%), most commonly abdominal distension, increased gastric residuals, vomiting, and suspected cow's milk protein allergy. Infants with adverse feeding events had lower gestational age and birth weight and required longer to reach an enteral feeding volume of 100 mL/kg/day. After adjustment, only delayed achievement of 100 mL/kg/day remained independently associated with adverse feeding events. Routine clinical variables showed limited predictive ability (AUC 0.611).
Conclusions
Most preterm infants tolerate CMDF well. However, infants with delayed progression to enteral feeding may derive additional benefit from EHMD. These findings support preferential use of EHMD in selected high-risk infants while complementing its established benefits in reducing major neonatal morbidities.
Keywords:
human milk fortification
; donor human milk
; Cow’s milk-derived fortifier
Introduction
Human milk is the recommended enteral nutrition for very preterm infants because it reduces major neonatal morbidities, including necrotizing enterocolitis (NEC), late-onset sepsis (LOS), bronchopulmonary dysplasia (BPD), and retinopathy of prematurity (ROP). When mothers' own milk (MOM) is unavailable or insufficient, pasteurized donor human milk (DHM) is recommended as the preferred alternative.
However, although human milk provides numerous bioactive components that are not available in formula, it does not fully meet the nutritional requirements of very preterm infants. Even preterm MOM alone is insufficient to provide adequate amounts of protein, calcium, and phosphorus to support optimal growth. Because pasteurization and donor selection further reduce the concentrations of several nutrients, DHM has an even lower nutritional content than preterm MOM. Therefore, human milk fortification is considered an essential component of nutritional management for very preterm infants.
Currently, CMDFs are widely used to increase protein and mineral intake and improve postnatal growth. Although these fortifiers are generally well tolerated, feeding intolerance and gastrointestinal adverse events are occasionally observed following fortification [1,2,3,4,5,6]. Previous studies comparing EHMD with CMDF have primarily focused on NEC and major neonatal morbidities [7,8,9]. However, little is known about which infants are most susceptible to adverse feeding events after bovine-derived fortification. Identifying these high-risk infants is clinically important because universal implementation of EHMD remains challenging owing to limited availability and higher costs. A precision nutrition strategy that selectively provides EHMD to infants at greatest risk may maximize both clinical benefit and resource utilization.
Therefore, the aim of this nationwide study was to identify clinical characteristics associated with adverse feeding events following bovine-derived human milk fortification among infants receiving donor human milk and to determine which infants may preferentially benefit from an exclusive human milk diet.
Methods
Study design and data source
This retrospective cohort study was conducted using the nationwide Japanese Human Milk Bank Database, which prospectively collects clinical information on infants receiving pasteurized DHM supplied by the Nippon Foundation Human Milk Bank. The database includes demographic characteristics, nutritional management, clinical outcomes, and adverse events recorded by NICUs. Data collected between February 2018 and June 2026 were included in the present study. Data completeness and consistency were reviewed before analysis using the same database quality assurance procedures described previously.
Study population
Infants were eligible if they
- received DHM supplied by the Japanese Human Milk Bank
- subsequently received CMDF
- had information regarding adverse events after fortification.
Infants with missing information regarding adverse events or key baseline variables were excluded from comparative analyses.
Unlike our previous study evaluating the timing of formula introduction [10], the present study was designed to investigate clinical factors associated with adverse events following CMDF rather than feeding initiation practices.
Nutritional Management
All infants received DHM according to each NICU's standard feeding protocol. Because DHM alone does not provide sufficient protein and mineral intake for very preterm infants, CMDFs were introduced during enteral feeding.
The exact day of fortifier initiation was unavailable in the database. However, participating NICUs generally followed the recommendations of the Japanese Society of Neonatal Health and Development guidelines [11], in which CBDF is initiated after enteral feeding reaches approximately 100 mL/kg/day, provided that feeding tolerance is satisfactory. Therefore, the day on which enteral feeding reached 100 mL/kg/day was included as a surrogate marker of gastrointestinal maturation and nutritional progression.
Outcomes
The primary outcome was the occurrence of adverse feeding events after initiation of CMDFs.
Reported adverse events included abdominal distension, increased gastric residuals, vomiting, bloody stool, suspected cow's milk protein allergy, necrotizing enterocolitis, and other gastrointestinal symptoms. Infants were classified into an adverse-event group and a non-adverse-event group according to the database records.
Variables
Baseline variables included gestational age, birth weight, sex, and the day reaching an enteral feeding volume of 100 mL/kg/day. The day reaching an enteral feeding volume of 100 mL/kg/day was selected because it reflects the progression of enteral nutrition and is widely used in neonatal nutritional management as the threshold for initiating human milk fortification.
Statistical Analysis
Continuous variables were compared using the Student's t-test or Mann–Whitney U test, as appropriate.
Categorical variables were compared using the χ² test or Fisher's exact test.
Variables considered clinically relevant, including gestational age, birth weight, sex, and the day reaching 100 mL/kg/day, were entered into a multivariable logistic regression model to identify independent factors associated with adverse feeding events.
Receiver operating characteristic (ROC) curve analysis was performed to evaluate the discriminatory ability of routinely available clinical variables.
Results
Study population (Table 1)
A total of 2,587 infants received donor human milk followed by bovine-derived human milk fortification during the study period. Of these, 2,476 infants had complete information regarding adverse feeding events and were included in the final analysis. Adverse feeding events were documented in 315 infants (12.7%), whereas 2,161 infants (87.3%) experienced no documented adverse feeding events following fortification.
Baseline characteristics
Infants who experienced adverse feeding events had significantly lower gestational age and birth weight than those without adverse events. They also required a significantly longer time to achieve an enteral feeding volume of 100 mL/kg/day. No significant differences were observed in sex distribution between the two groups (Table 2).
Values are presented as median (interquartile range) or n (%).
Types of adverse feeding events
Abdominal distension was the most frequently reported adverse event, followed by increased gastric residuals, vomiting, and suspected cow's milk protein allergy. Necrotizing enterocolitis was uncommon, occurring in only six infants (Figure 1).
Adverse feeding events according to maturity
The incidence of adverse feeding events increased with decreasing gestational age and birth weight (Table 3, Figure 2). Infants born before 24 weeks' gestation and those weighing <750 g at birth demonstrated the highest incidence of adverse feeding events.
Multivariable analysis
In multivariable logistic regression analysis, a longer time to achieve an enteral feeding volume of 100 mL/kg/day remained independently associated with adverse feeding events. In contrast, gestational age, birth weight, and sex were not independently associated with adverse feeding events after adjustment for potential confounding variables (Table 4).
Receiver operating characteristic analysis demonstrated that routinely available clinical variables had only modest discriminatory ability for predicting adverse feeding events, with the multivariable model yielding an area under the curve of 0.611 (Figure 3).
Discussion
The present nationwide study demonstrated three principal findings. First, most preterm infants tolerated bovine-derived human milk fortification without clinically significant adverse feeding events. Second, adverse feeding events were more common among infants with lower gestational age and birth weight. Third, after adjustment for potential confounding variables, delayed progression to an enteral feeding volume of 100 mL/kg/day remained independently associated with adverse feeding events, whereas gestational age and birth weight were no longer independent predictors.
The higher incidence of adverse feeding events among the most immature infants is biologically plausible because gastrointestinal immaturity is common in extremely preterm infants. However, after adjustment for potential confounding variables, only delayed progression to full enteral feeding remained independently associated with adverse feeding events. This finding suggests that delayed achievement of an enteral feeding volume of 100 mL/kg/day may better reflect functional gastrointestinal maturation, successful intestinal adaptation, and feeding tolerance than gestational age or birth weight alone.
Our findings do not suggest that CMDFs should be avoided. On the contrary, most infants tolerated CMDFs without clinically significant adverse feeding events. Therefore, our findings should not be interpreted as supporting universal replacement of CMDFs.
EHMD has already been shown to reduce major neonatal morbidities, including NEC, BPD, ROP, and LOS, and accumulating evidence also suggests favorable effects on long-term neurodevelopment. These established benefits remain the primary rationale for the use of EHMD in extremely preterm infants. The present study does not challenge this evidence. Rather, our findings provide an additional clinical perspective by suggesting that, among infants receiving DHM, those at increased risk of feeding intolerance following CMDFs may derive even greater benefit from EHMD because of its potential to improve feeding tolerance while preserving the well-established advantages of an EHMD [12,13,14,15,16]. Our findings are also consistent with the clinical observations reported by Sandhu et al., who described successful transition to an EHMD in very low birth weight infants demonstrating feeding intolerance to CMDF [17]. All infants subsequently achieved full enteral feeding without recurrence of gastrointestinal symptoms while improving nutritional intake and growth. These findings suggest that EHMD may serve as an effective rescue strategy for infants who cannot tolerate CMDFs.
Importantly, routinely available clinical variables demonstrated only modest discriminatory ability (AUC 0.611), indicating that gestational age and birth weight alone are insufficient for identifying infants who would derive the greatest benefit from EHMD. Therefore, decisions regarding preferential use of EHMD should not rely solely on chronological maturity but should also consider the infant's overall clinical condition and feeding tolerance. Further prospective studies are needed to validate these findings and establish evidence-based criteria for identifying infants who are most likely to derive additional benefit from EHMD.
Limitations
This study has several limitations. First, because this was a retrospective analysis of a nationwide human milk bank database, the exact timing of CMDF initiation was unavailable. Although participating NICUs generally followed Japanese and international neonatal nutrition guidelines recommending initiation of fortification after enteral feeding reached approximately 100 mL/kg/day, we were unable to confirm the precise timing of fortification for individual infants. Therefore, the day on which enteral feeding reached 100 mL/kg/day was used as a surrogate marker of nutritional progression and gastrointestinal maturation.
Second, adverse events were identified from database records. Although standardized definitions were recommended, variations in clinical judgment and reporting among institutions may have resulted in some degree of misclassification.
Third, although lower gestational age and birth weight were associated with adverse events, these variables alone were insufficient to accurately identify infants at greatest risk. The decision to preferentially use an EHMD should therefore not rely solely on chronological maturity but should consider the infant's overall clinical condition and feeding tolerance. Further prospective studies are required to refine patient selection.
Conclusion
Most preterm infants tolerated CMDF without clinically significant adverse events. However, infants with delayed gastrointestinal maturation, reflected by slower advancement to full enteral feeding, may derive additional benefit from an EHMD. Rather than replacing CMDFs universally, our findings support a precision human milk nutrition approach in which EHMD is preferentially considered for carefully selected high-risk infants.
Institutional Review Board Statement
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Showa University School of Medicine (Approval No. 21-084-A).
Informed Consent Statement
Written informed consent was waived by the Institutional Review Board because this was a retrospective observational study using anonymized clinical data. Instead, an opt-out approach was adopted, allowing participants or their legal guardians to decline participation.
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Table 1.
Study population.
| Characteristic | Value |
|---|---|
| CMDF exposed infants | 2587 |
| Analyzed infants with AE information | 2476 |
| Adverse events | 315 |
| No adverse events | 2161 |
| Missing AE information | 111 |
| AE rate among analyzed infants (%) | 12.7 |
Table 2.
Baseline characteristics of infants with and without adverse feeding events.
| Variable | No AE n | No AE median (IQR) | AE n | AE median (IQR) | P value |
|---|---|---|---|---|---|
| Gestational age, weeks | 2154 | 28.7 (26.3–30.9) | 315 | 27.9 (25.9–30.0) | <0.001 |
| Birth weight, g | 2161 | 994.0 (753.0–1264.0) | 315 | 872.0 (645.5–1189.0) | <0.001 |
| Day reaching 100 mL/kg/day | 1971 | 9.0 (7.0–12.0) | 285 | 11.0 (8.0–15.0) | <0.001 |
| Male sex | 2160 | 1128 (52.2%) | 315 | 159 (50.5%) | 0.562 |
Table 3.
Incidence of adverse feeding events according to gestational age and birth weight.
| Gestational age | AE n | Total n | AE rate (%) | Birth weight | AE n | Total n | AE rate (%) |
|---|---|---|---|---|---|---|---|
| <24 wk | 30 | 179 | 16.8 | <750 g | 120 | 656 | 18.3 |
| 24–25 wk | 50 | 367 | 13.6 | 750–999 g | 78 | 650 | 12.0 |
| 26–27 wk | 79 | 529 | 14.9 | 1000–1249 g | 54 | 538 | 10.0 |
| 28–29 wk | 75 | 581 | 12.9 | ≥1250 g | 63 | 632 | 10.0 |
| ≥30 wk | 81 | 813 | 10.0 |
Table 4.
Multivariable logistic regression analysis for adverse feeding events following bovine-derived human milk fortification.
Table 4.
Multivariable logistic regression analysis for adverse feeding events following bovine-derived human milk fortification.
| Variable | Adjusted OR | 95% CI | P value |
|---|---|---|---|
| Gestational age (per 1-week increase) | 0.974 | 0.911–1.041 | 0.439 |
| Birth weight (per 100-g increase) | 0.949 | 0.892–1.011 | 0.104 |
| Male sex | 0.934 | 0.726–1.203 | 0.598 |
| Days to reach 100 mL/kg/day (per 1-day increase) | 1.021 | 1.006–1.037 | 0.007 |
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