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Page 12 of 20 Horwell et al. Microbiome Res Rep 2025;4:1 https://dx.doi.org/10.20517/mrr.2024.32

This raises two interesting points. Firstly, if the associated health differences seen in observational studies
are causally related to the microbiomic differences, the effect occurs during a very short period of time. This
has been referred to as the “critical window”. Animal studies have shown that during this window, the
immune system is exposed to microbial antigens via specific pattern recognition receptors (e.g., Toll-Like
Receptors), and primed to tolerate these specific species that direct the immune system in later life, with
evidence suggesting that the microbiota need to follow a specific sequence of colonisation to ensure an
optimal cascade of immune and metabolic development [199-204] . Furthermore, several experiments using
germ-free mice that were exposed to microbiota before weaning demonstrated less inflammation, compared
to those exposed later in life [70,205] . Innate-like T cells appear to be crucial. These cells develop immunological
effector characteristics before thymic emergence, and recent work has shown that these cells are particularly
responsive to early-life antigen exposure, thereby promoting the maintenance of tissue homeostasis [206,207] .
Early exposure to commensal microbes correlates to the abundance of mucosal-associated invariant T cells
(MAIT) and NK cells, and the absence of these early life antigen exposures cannot be compensated for in

later life, with work showing that these T cell populations are vital for maintaining homeostasis and
[206]
preventing excessive inflammation . As an example, one murine study showed that Bacteroidetes (a genus
associated with breastfeeding) increased colonic CD4 cytotoxic T lymphocytes and increased FoxP3
+
protein expression, polarising the immune system away from Th2 type allergic response, in favour of Th1-
mediated anti-inflammatory IL-10 production . Evidence in humans supports this theory of a critical
[208]
window of opportunity. For instance, cohort studies have shown that children who live in rural
environments have a lower prevalence of IBD and asthma in later life compared to those born in urban
areas; however, this protective effect is diminished if the child moves into a rural location after the critical
window (i.e., is not exposed from birth), and the protective effect is absent in adults [148,209,210] .

Secondly, if this critical window of opportunity during the first 3 to 5 years of life is proved to be true - and
based on the available evidence, it is our opinion that this is a reasonable position to have - the highly
dynamic infant microbiome offers a potential opportunity for health intervention. Building on public health
advice that already exists (e.g., breastfeeding), interventions could be used to modulate the microbiome. We
have discussed a Finnish group that has already shown that using soil from a forest transplanted to urban
day care facilities changes the microbiome of the attendees, but large long-term studies need to be
performed to see if this can influence health outcomes. Other approaches, such as using similar
environmental inoculants in the home or using probiotics, have also been suggested, although the impact of
this has not been assessed. Animal models are being employed in increasing numbers in an attempt to
elucidate what specific microbial populations are important in orchestrating beneficial health outcomes,
which in the future may be able to increase the precision of any microbiomic intervention.

CONCLUSION
There has been a remarkable increase in knowledge over a short period of time, covering the subtleties of
the infant microbiome and its variation depending on specific external stimuli. It is clear that the first five
years of life are a highly complex and dynamic period for the infant microbiome. We have described how
this is influenced by numerous variables in a highly orchestrated and dynamic ecosystem that follows a
similar pattern across different global populations. An individual’s microbiome can be seen to diverge from
birth, with CS being associated with an altered microbiome compared to VD, although the mechanism of
this difference is not fully established. It is apparent that the largest single effector of the microbiome is
breastfeeding, with very clear evidence that once this ceases, the microbiome of the infant quickly starts to
change to that of an adult. Very few mechanistic studies have been performed to study these processes in
detail. As such, observational data have been employed to infer how the environment influences the
microbiome. Several studies have identified key external influences on the infant’s microbiome, but