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Chen et al. Microbiome Res Rep 2025;4:8 https://dx.doi.org/10.20517/mrr.2024.44 Page 3 of 18

The aim of this systematic review is to assess and provide a qualitative synthesis of available literature that
reports on the effects of DHM vs. MOM on the gut microbiota composition in preterm infants, with specific
attention to microbial diversity and taxonomic composition.

METHODS
Registration
This systematic review was registered at PROSPERO international prospective register of systematic reviews
under number CRD42022358080. It was conducted according to the guidelines of the preferred reporting
[26]
items for systematic review and meta-analysis protocols (PRISMA) .

Eligibility criteria
We included studies describing the fecal microbiota composition of preterm infants born < 37 weeks of
gestation, with data available for at least two distinct groups of infants. One group received > 50% MOM,
while the other group received > 50% DHM, at least until achieving full enteral feeding. Study cohorts in
which infants had received predominantly formula feeding (defined as > 50% of total enteral volume)
during this period were excluded. The use of human milk fortifiers with protein from bovine origin was not
a criterion for exclusion. Studies using only conventional culturing methods were excluded, since this
technique does not cover the entire gut microbiota composition . Since molecular culture-independent
[27]
[28]
techniques for gut microbiota analysis became available from 1990 onwards , only studies published since
then were eligible for inclusion. Given the ethical considerations and impossibilities of randomizing the
provision of MOM, our literature search focused on observational studies and other relevant research
designs.

Information sources and search strategy
A comprehensive search was conducted in MEDLINE (Ovid), Cochrane Library, and EMBASE (Ovid) by
J.C., A.v.W., and a medical information specialist (N.P.D.). Searches were restricted to articles published in
English and focused on human subjects. The final search was completed on January 15th, 2024. Literature
search strategies were developed using a combination of controlled terms and titles and abstract words
related to the concepts: preterm infants, enteral feeding, human milk, DHM, and gut microbiota. The search
terms were combined using Boolean operators AND and OR. No data limit was applied. Duplicates were
[29]
removed with DedupEndNote (version 1.0.1) . The full search strategy is included in
Supplementary File 1. In addition, the International Clinical Trials Registry Platform Search Portal and
ClinicalTrials.gov were searched for ongoing or recently completed trials and PROSPERO was searched for
ongoing or recently completed systematic reviews. Backward snowballing techniques were used to identify
potentially relevant articles from the references of included articles.

Study selection and data extraction
Two authors (J.C and A.v.W) independently screened the titles and abstracts retrieved from the search
against the predefined inclusion and exclusion criteria. Full reports for all titles that appeared to meet the
inclusion criteria or where there was any uncertainty were obtained. Review author pairs screened the full-
text reports and decided whether these met the inclusion criteria. We recorded the reasons for excluding
trials.

Standardized forms were used to extract data from the original studies by J.C. and A.v.W. Inconsistencies
were resolved by discussion with T.G.M. and C.H.P.A. or study authors were contacted if there were
important uncertainties. We extracted the following data: primary author, year of publication, geographic