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Chen et al. Microbiome Res Rep 2025;4:8 https://dx.doi.org/10.20517/mrr.2024.44 Page 13 of 18

Khumbare et al. used calprotectin levels in stool as a marker for gut inflammation and showed it was higher
[34]
in the DHM group (P = 0.02) .
In the study by Morais et al., fecal alkaline phosphatase (ALP) activity was measured as a supposed potential
[37]
biomarker for NEC. They stated no difference in ALP activity between the DHM and MOM groups .

Parra-Llorca et al. observed no significant differences in estimated metabolic profiles of the gut microbiome
[35]
using predictive functional profiling between the DHM and MOM groups .

Clinical outcomes
Three studies in this review reported differences in health outcomes between the DHM and the MOM
cohorts. Morais et al. did not observe differences in growth between the two groups on the 26th day of
[37]
life . However, Ford et al. showed growth velocity and body weight at a PMA of 36 weeks to be lower in
the DHM group (both adjusted P-values < 0.01). In addition, they showed the incidence of the composite
outcome of NEC, spontaneous intestinal perforation, sepsis, severe bronchopulmonary dysplasia, and death
[33]
to be higher in the DHM group (adjusted P-values < 0.02) . On the contrary, Piñeiro-Ramos et al. showed
no statistically significant difference in NEC incidence between the groups .
[32]
DISCUSSION
In this systematic review, we observed differences in the fecal microbiome of preterm infants fed
predominantly with DHM, compared to those predominantly fed with MOM. These differences manifest in
both diversity and composition across various taxonomic levels and are reported across several time points,
from as early as the second day of life to approximately 36 weeks PMA. In the following sections, we will
discuss these findings in more detail.

Our review provides evidence indicating that preterm infants predominantly fed with DHM exhibit reduced
alpha diversity in their gut microbiome compared to those fed predominantly with MOM. However, this
evidence is solely documented within subgroup B, which encompasses studies incorporating formula
feeding alongside MOM or DHM. Therefore, the inferred effect could also be related to the quantity of
formula milk received, rather than the difference between MOM and DHM feeding. These findings align
with existing evidence on the microbiome composition of infants fed PF vs. MOM, reporting higher alpha
diversity in the latter cohorts . The only study that described an increase in alpha diversity in the DHM
[39]
group focused only on Bifidobacterium species, rather than assessing overall diversity .
[31]

The included studies employed different metrics for alpha diversity evaluation, including the Shannon- or
Chao1-indices and OTU richness. Several studies reported multiple measures for alpha diversity with
conflicting outcomes. For example, Morais et al. showed a difference in the Chao1 index, but not in
Shannon index findings, underscoring that the results on alpha diversity are dependent not only on the
[37]
analyzed cohort, but also on the selected metric . This discrepancy limits the comparability of published
data.

Our review yields mostly consistent evidence for differences in beta diversity, with distinct clustering of the
gut microbiome based on feeding type from the tenth day of life onwards. These differences are apparent in
both subgroups A and B. The two studies assessing the effects of multiple demographic factors on the Bray-
Curtis dissimilarity index, showed that feeding type contributed more to the observed difference than other
factors tested, such as gestational age, birth weight, postnatal age, or antibiotic administration [19,36] .
Contradicting results were present in the study by Morais et al., which found no differences in beta diversity