This systematic review and meta-analysis was conducted in accordance to PRISMA guidelines [23].

Search strategy and search criteria: All authors independently searched the databases including PubMed, Embase, Cochrane Central Register of Controlled Trials, other clinical trial registries, Google Scholar, Scopus, Web of Science and hand searching of conference proceedings from inception to December 2019 for peer-reviewed publications in English language. The electronic search strategy included a combination of keywords along with their representative medical subjects headings (MeSH) terms. Details of search strategy are provided in Web Appendix 1. Reference list of all articles whose full texts were screened, was also checked to find additional articles.

We included randomized or quasi-randomized controlled trials (RCT) evaluating the effects of HM fortified with IMF as intervention, compared to unforti-fied or HMF-fortified HM on growth rate, duration of hospital-stay and other clinically relevant outcomes in VLBW preterm infants. Non-English publications were excluded.

The primary outcome was assessment of velocity of gain in weight, length, and head circumference (HC). Secondary outcomes were duration of hospital stay, incidences of FI and NEC, time to reach full feeds, concen-tration of nutritional biomarkers (calcium, phosphorous, blood urea nitrogen, prealbumin, albumin, alkaline phos-phatase) and cost of intervention.

Data extraction and quality assessment: Two authors independently extracted data using a pre-designed pro-forma. Disagreement, if any, was resolved by discussion with third author. Study details including location and year of study, number of infants and their characteristics, details of feeding including fortification and outcomes relevant to the study were noted. Quality of studies were assessed independently by all authors, for each study, using the risk of bias (ROB) criteria outlined in the Cochrane Handbook for Systematic Reviews of Interventions [24] in the domains of random sequence generation, allocation concealment, blinding of partici-pants and personnel, blinding of outcome assessment, incomplete outcome data, selective outcome reporting, and other bias.

Statistical analysis: Statistical analysis was performed using Review Manager version 5.4 (The Cochrane Colla-boration, 2020). Out-come variables were calculated as risk ratio (RR) with 95% confidence interval (CI) for dichotomous data and mean differences (MD) with 95% CI for continuous data. Standardized mean differences (SMDs) were calculated where outcomes had different measurement instruments. Studies reporting dispersion of outcomes in range was converted to standard deviation using established mathematical models [25]. Results were pooled using either fixed or random effects model based on hetero-geneity which was assessed using the I² statistic. Grading of recommendations assessment, development and evaluation (GRADE) approach [26] was applied to assess the quality of evidence for predefined outcomes.

Screening and inclusion of studies are summarized in Fig.1. Four full-text articles [18-21] and one abstract [22] were selected for this systematic review including a total of 423 VLBW preterm infants.

Fig. 1 PRISMA flow diagram.

The characteristics of included studies are summarized in Table 1. The birth weight of the preterm VLBW infants included in the studies, ranged from 500g to 1499g. Fortification of HM with IMF was the intervention in all five trials. The time to start fortification varied from 100 mL/kg/d [18,19,22] to150 mL/kg/d of enteral feed [21]. Willeitner, et al. [20] introduced fortification as early as at 60 mL/kg/d, at the discretion of the treating team. In three studies [18,20,22] the comparator was HMF, while other two studies [19,21] used unfortified HM. Web Fig. 1 depicts ROB graph summarizing each ROB item as percentage across all studies while Web Fig. 2 summarizes ROB for each included study.

All included studies evaluated weight gain velocity as primary outcome. Four studies [19,20-22], described weight gain velocity in terms of g/kg/day, while Khorana, et al. [18] reported weight gain as g/day. Overall, pooled effects of all five studies on weight gain velocity was statistically not significant (SMD 0.27 g/kg/day; 95% CI: -0.08 to 0.62) (Fig. 2a). Sensitivity analysis was done due to difference in comparators. IMF fortification was found to cause a statistically significant increase in the rate of weight gain (MD 02.03 g/day; 95% CI: 1.15 to 2.92) compared to unfortified HM. Using HMF as comparator, SMD of weight gain velocity was similar (SMD -0.01 g/day; 95% CI: -0.27 to 0.25).

Fig. 2 Forest plot showing meta-analysis of the effect of infant milk fortification on the velocity of weight gain (2a), length gain (2b) and head circumference (HC) gain (2c).

Four studies [18,19,21,22] with 353 participants reported data regarding rate of increase in length and HC. The pooled effect with respect to velocity of gain in length was not statistically significant (MD 0.07 cm/week; 95% CI: -0.02 to 0.16) (Fig. 2b). On sensitivity analysis, when compared to unfortified HM, IMF fortification resulted in significantly higher rate of gain (MD 0.12 cm/week; 95% CI: 0.02 to 0.22), but failed to show difference when compared with HMF (MD -0.03 cm/week; 95% CI: -0.15 to 0.08). Similarly, the pooled effect with respect to velocity of gain in HC was not statistically significant (MD 0.05 cm/week; 95% CI: -0.01 to 0.11) (Fig. 2c). On sensitivity analysis, when compared to unfortified HM, IMF fortification resulted in significantly higher rate of gain (MD 0.08 cm/week; 95% CI: 0.03 to 0.13), but failed to show difference when compared with HMF (MD -0.04 cm/week; 95% CI: -0.14 to 0.06).

FI, reported in two studies [19,21] (n=208), showed no difference in risk between IMF and HMF fortification versus no fortification of HM (RR 2.29; 95% CI: 0.61to 8.59) (Web Fig. 3a). Though HMF fortification showed apparently higher rates of NEC [18,20], the RR was not statistically significant for either suspected NEC (RR 0.37; 95% CI: 0.07 to 1.95) (Web Fig. 3b) or confirmed NEC (RR 0.25; 95% CI: 0.04 to 1.39) (Web Fig. 3c).

Three studies [18,19,21] including 231 participants, showed that the length of hospital stay of neonates with IMF was significantly reduced (MD -4.38 days; 95% CI: -7.39 to -1.37) (Web Fig. 3d). Two studies [18,19] (n=83) found no significant difference with respect to time to achieve full enteral feeding, between those receiving formula fortified HM and those on either HMF fortified or unfortified HM. (Web Fig. 3e). Effect of fortification on nutritional biomarkers were reported by two studies [18,21]. No significant effect on BUN nor albumin levels was observed (Web Fig.3f, 3g).

Though four of the studies favored IMF intervention in terms of cost, this economical aspect was not studied as an outcome in any of them. The data presentation was not uniform and therefore, could not be pooled.

The quality of evidence pooled from included studies was assessed using GRADE approach and summary of findings table was generated on GRADE pro GDT software (Evidence Prime Inc.) (Web Appendix 2).

This systematic review and meta-analysis of five RCTs, including a total of 423 VLBW preterm infants, did not show any significant benefit of IMF fortification of HM over combined HMF fortification/no fortification, on growth velocity, with respect to weight, length and HC. On sensitivity analysis for the same parameters, IMF and HMF fortifications were comparable, whereas IMF fortification was significantly better than unfortified HM, quality of evidence (QOE) being very low. No significant difference was noted in the incidences of FI/NEC and levels of nutritional biomarkers like BUN and albumin (QOE: very low). Pooled data from three trials, showed a significant reduction in duration of hospital stay favoring IMF fortification (QOE, very low). This reduction was probably because the comparator in two of these studies was unfortified HM.

There are several limitations in the included trials. The study by El Sakka, et al. [19] was quasi-randomized with an unclear methodology. Still this study was included as its outcome measures met our inclusion criteria. The gestational age varied among the studies, with one trial [21] excluding late preterm infants. No data were available regarding long term growth and developmental outcome. Formulas and HMFs preparations used were from different manufacturers, though the protein and energy content were similar. Another area of discrepancy was non-uniform timing of initiation of fortification in included trials, which might have affected growth. The most important concern for implementation of IMF fortification in routine practice is increase in osmolarity with risk of FI and NEC. Only one trial [21] measured osmolarity of HM after IMF fortification and found it below 400 mOsm/L, the recommended upper safety limit of American Academy of Pediatrics [27]. Though no difference in the incidences of FI and NEC was noted, none of the studies was adequately powered to detect the difference. None of the trials had individualized the fortification by analysis of HM macronutrients. IMF measurement technique for fortification was described by only one study [22].

A relatively limited number of studies, with high ROB and statistical heterogeneity in this systematic review limit the generalizability of this meta-analysis. Variability in the time of initiation of feed, the maximum feeding volume and continuation of IMF as ‘bridge feeding’ when EBM was unavailable [20] probably limited the impact of the intervention on growth outcomes. Further, subgroup analysis based on gestation or birth weight could not be done because of unavailability of raw data. Not all biomarkers of nutrition could be evaluated due to lack of measured values. Data regarding cost could not be pooled as there was no uniformity in presentation.

To summarize, a very-low quality evidence suggests that IMF fortification of HM is superior to unfortified HM and may be a safe alternative for bovine HMFs for short term growth of VLBW preterm infants, especially in resource-limited settings. Larger well-designed studies with strict monitoring of complications including NEC with a focus on long-term outcomes are needed.

Acknowledgement: Dr. Poonam Singh for assistance in revising the manuscript.

Contributors: MK: conceptualized the review, literature search, data analysis and manuscript writing; JU: literature search, data analysis and manuscript writing; SB: conceptualized the review, literature search, data analysis and manuscript writing.

Funding: None; Competing interest: None stated.

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