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Chen et al. Microbiome Res Rep 2025;4:8 https://dx.doi.org/10.20517/mrr.2024.44 Page 15 of 18

Our review entails multiple strengths, including the categorization of studies involving infants exclusively
human milk-fed and those with mixed feeding regimens. This stratification enabled us to mitigate possible
bias by the potential effects caused by any consumption of PF. Furthermore, since both gestational age and
chronological age may influence microbiota composition, we reported the PMA in Figures 2 and 3 for easier
comparison when analyzing stool samples. However, this approach oversimplifies the potential impact of
individual maturity on gut microbiome composition. Limitations of this review include the inability to
provide a quantitative synthesis due to the considerable heterogeneity in reported outcome measures in the
original studies. This includes variations in the use of different diversity indices, the use of different gut
microbiome analysis techniques, and the preferred reporting styles for taxonomic outcomes. Additionally,
most studies did not account for the mode of delivery which may have a confounding effect on microbiota
composition. Several studies excluded infants with adverse neonatal outcomes such as mortality, NEC, and
sepsis, possibly omitting infants with the most aberrant gut microbiome development, thus potentially
biasing the results of this review. However, this can be seen as an advantage as well, where relatively well-

doing preterm infants form a more homogeneous group with less external influences, so that the influence
of milk type is better recognized.

The absence of randomized controlled trials (RCTs) comparing MOM to DHM is a significant limitation of
reviews on this topic. The reliance on observational studies necessitates cautious interpretation of findings
due to potential confounding variables. Future research should link health outcomes to gut microbiota
changes attributable to feeding with DHM vs. MOM. In addition, the mechanisms through which MOM
exerts its benefits to the infant should be investigated and used to improve existing DHM processing
techniques to increase benefits. For instance, Holder pasteurization results in significant loss of bioactive
compounds like lactoferrin, cytokines, and growth factors, which are vital for a healthy gut microbiome in
infants . Alternative pasteurization techniques such as high-temperature short-time pasteurization, high-
[60]
pressure pasteurization, and Ultraviolet C may better preserve these compounds and thereby affect the
microbiome in infants, although studies on this matter are sparse . A novel concept of inoculation of
[61]
DHM with bits of MOM shows promising in-vitro results in restoring the human milk microbiome [62,63] .
However, in-vivo studies are needed to fully assess the effects of inoculated DHM on clinically important
outcomes.

In conclusion, our review reveals several differences in gut microbiota development of preterm infants fed
predominantly DHM vs. those fed predominantly MOM. The gut microbial composition shows distinct
clustering based on feeding type, with the DHM group potentially displaying lower alpha diversity levels. At
the compositional level, a DHM diet is associated with an increased abundance of the Staphylococcaceae and
Clostridiaceae family and a lower abundance of Bacteroidetes and the Bifidobacterium genus. The
differences persist throughout the follow-up period examined in the included studies. Importantly, these
observed differences have been associated with adverse health outcomes both in the short and long term,
such as an increased incidence of NEC, sepsis, and neurodevelopmental delay beyond infancy.

DECLARATIONS
Acknowledgments
Chen J would like to express her gratitude to the Chinese Scholarship Council for providing the living
expenses during her PhD studies in the Netherlands.

Authors’ contributions
Conceptualization: Chen J, van Wesemael AJ, van den Akker CHP, de Meij TGJ