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

location of the study, demographic data, duration of study, proportion of predominant feeding type (MOM
or DHM), number of participants, time points of fecal sample collection, total numbers of samples analyzed,
and microbiota analysis method. The main outcome data that were extracted included alpha and beta
diversity and microbial composition at different taxonomic levels. When reported, clinical outcomes and
other fecal analysis results were extracted as they might provide relevant insights into the mechanistic effects
the microbiome differences could have on health outcomes.

Outcomes measures and data presentation
To acquire a robust assessment of the impact of feeding MOM or DHM on intestinal microbiota, and to
mitigate potential bias caused by the effects of any consumption of PF, two subgroups were delineated based
on the infants’ feeding regimen from birth till full enteral feeding achieved, or up to the postmenstrual age
(PMA) of 36 weeks:

● Subgroup A: studies where infants received 100% human milk (either MOM or DHM)
○ MOM group: received > 50% MOM
○ DHM group: received > 50% DHM

● Subgroup B: studies where infants received a combination of human milk (MOM or DHM) and PF
○ MOM group: received > 50% MOM (with < 50% DHM and/or PF)
○ DHM group: received > 50% DHM (with < 50% MOM and/or PF)

The MOM group serves as the reference group in all text, tables, and figures, unless stated otherwise.

Risk of bias and quality assessment
The risk of bias in longitudinal studies was assessed using the Newcastle-Ottawa Scale (NOS) for
[30]
observational studies . This instrument measures four domains: participant selection, comparability,
exposure, and outcome. It can be modified to better fit the specific subject of interest. The NOS used for this
review was modified by the authors to suit this review and is found in Supplementary File 2. The scoring is
based on the number of stars, with longitudinal studies being eligible for a maximum score of nine stars.

Data synthesis
Due to the expected heterogeneity of study designs and outcomes, we did not aim to provide a meta-
analysis.

RESULTS
Included studies
The search process is shown in Figure 1. A total of 1,028 articles were identified through the database
search. After removing duplications, 717 articles were initially screened by title and abstract, of which 671
articles could be excluded. In total, 46 articles underwent full-text review, of which 38 articles were
excluded. Thus, a total of 8 original studies could be included in our qualitative synthesis [19,31-37] . Backward
snowballing did not yield any additional suitable articles.

Description of the studies
Characteristics of the eight included studies are summarized in Table 1 and depicted in Figure 2. Seven out
of the eight studies were prospective observational cohorts. The remaining study by Kumbhare et al. was a
randomized, controlled, open-label trial comparing bovine milk-based vs. human milk-based fortifier added
to MOM or, if insufficiently available, to DHM . However, the authors also described fecal microbiota
[34]
results for two post-hoc cohorts where infants were stratified whether they had predominantly received