Review article

The maternal gut microbiome during pregnancy

and offspring allergy and asthma
Yuan Gao, BSc,a,b,c Ralph Nanan, Dr med Habil,d Laurence Macia, PhD,d,e Jian Tan, PhD,d,e Luba Sominsky, PhD,a,b
Thomas P. Quinn, MD, PhD,f Martin O’Hely, PhD,a,g Anne-Louise Ponsonby, MBBS, FAFPHM, PhD,g,h,i,w
Mimi L. K. Tang, MBBS, FRACP, PhD,g,i,j,w Fiona Collier, PhD,a Deborah H. Strickland, PhD,k,l Poshmaal Dhar, PhD,a
Susanne Brix, MSc, PhD,m Simon Phipps, PhD,n,o,p Peter D. Sly, MBBS, MD, DSc,p,q Sarath Ranganathan, MD, PhD,g,i,j
Jakob Stokholm, MD, PhD,r,s Karsten Kristiansen, PhD,t,u,v Lawrence E. K. Gray, MBBS, PhD,a,b* and
Peter Vuillermin, MBBS, BMedSci, FRACP, PhDa,b,w* Geelong, Sydney, Melbourne, Perth, Crawley, Herston, St Lucia,
and Parkville, Australia; Copenhagen, Kongens Lyngby, and Slagelse, Denmark; and Shenzhen, China

Environmental exposures during pregnancy that alter both the and metabolic products of the maternal gut microbiome can
maternal gut microbiome and the infant’s risk of allergic promote the expression of microbial pattern recognition
disease and asthma include a traditional farm environment and receptors, as well as thymic and bone marrow hematopoiesis
consumption of unpasteurized cow’s milk, antibiotic use, relevant to regulatory immunity. At birth, transmission of
dietary fiber, and psychosocial stress. Multiple mechanisms maternally derived bacteria likely leverages this in utero
acting in concert may underpin these associations and prime the programming to accelerate postnatal transition from a TH2- to
infant to acquire immune competence and homeostasis TH1- and TH17-dominant immune phenotype and maturation of
following exposure to the extrauterine environment. Cellular regulatory immune mechanisms, which in turn reduce the
child’s risk of allergic disease and asthma. Although our
understanding of these phenomena is rapidly evolving, the field
From athe Institute for Physical and Mental Health and Clinical Transformation, Deakin
University, and bthe Child Health Research Unit, Barwon Health, Geelong; cthe Fac-
is relatively nascent, and we are yet to translate existing
ulty of Health and Medical Sciences, University of Copenhagen, Copenhagen; dthe knowledge into interventions that substantially reduce disease
Charles Perkins Center and ethe School of Medical Sciences, Faculty of Medicine risk in humans. Here, we review evidence that the maternal gut
and Health, The University of Sydney, Sydney; fthe Applied Artificial Intelligence microbiome impacts the offspring’s risk of allergic disease and
Institute, Deakin University, Geelong; gMurdoch Children’s Research Institute, hthe
asthma, discuss challenges and future directions for the field,
Florey Institute, ithe University of Melbourne, and jRoyal Children’s Hospital, Mel-
bourne; kthe Telethon Institute, Perth; lthe University of Western Australia, Crawley; and propose the hypothesis that maternal carriage of Prevotella
m
the Department of Biotechnology and Biomedicine, Technical University of copri during pregnancy decreases the offspring’s risk of allergic
Denmark, Kongens Lyngby; nQIMR Berghofer Medical Research Institute, Herston; disease via production of succinate, which in turn promotes
o
the School of Biomedical Sciences, Faculty of Medicine, pAustralian Infectious Dis- bone marrow myelopoiesis of dendritic cell precursors in the
eases Research Centre, and qthe Children’s Health and Environment Program, Child
Health Research Centre, The University of Queensland, St Lucia; rCOPSAC, Copen-
fetus. (J Allergy Clin Immunol 2021;148:669-78.)
hagen Prospective Studies on Asthma in Childhood, Herlev and Gentofte Hospital,
University of Copenhagen, Copenhagen; sthe Department of Pediatrics, Slagelse Hos-
Key words: Gut microbiome, fetal immunity, allergy, asthma
pital, Slagelse; tBGI-Shenzhen and uChina National Genebank, Shenzhen; vthe
Laboratory of Genomics and Molecular Biomedicine, Department of Biology, Univer-
sity of Copenhagen, Copenhagen; and wthe Centre For Food Allergy Research, Mur- The modern environment, lifestyle, and diet have altered the
doch Children’s Research Institute, Parkville. composition and function of the human gut microbiome,1-11 which
*Co-senior authors. in turn plays a central role in early-life immune development.12,13
Supported by National Health and Medical Research Council of Australia grants
Although most research regarding the gut microbiome and allergic
1082307; 1147980; 1134812; 1166319.
Disclosure of potential conflict of interest: M. O’Hely, A.-L. Ponsonby, M. L. K. Tang, F. disease has focused on the postnatal period,13-15 the maternal gut
Collier, S. Ranganathan, and P. Vuillermin have a financial interest in the biotech microbiome during pregnancy promotes fetal immune develop-
company Prevatex Pty Ltd, which seeks to develop technology regarding maternal and ment,16,17 and appears to influence the expression of allergic dis-
infant carriage of Prevotella copri (PCT2016905378; PCT/AU2019/050878). M. L. K. ease in the offspring.18,19 A range of mouse and human studies
Tang is an inventor on the patent ‘‘Methods and compositions for determining and for
minimizing the likelihood of development of allergy in infants’’ (pending and licensed
support the putative construct shown in Fig 1: In a traditional envi-
to Prevatex Limited). ronment, adequate exposure to environmental microbes, the
Received for publication June 15, 2021; revised July 19, 2021; accepted for publication absence of antibiotics, adequate dietary fiber, and lower levels of
July 19, 2021. maternal psychological stress and mental illness decrease the risk
Available online July 24, 2021.
of offspring allergic disease and asthma via their impacts on the
Corresponding author: Lawrence E. K. Gray, MBBS, PhD, or Peter Vuillermin, MBBS,
BMedSci, FRACP, PhD, The Institute for Mental and Physical Health and Clinical mother’s gut microbiome and its products. The potential underlying
Translation (IMPACT) Barwon Health, Geelong, VIC 3220, Australia. E-mail: pathways include the alignment between maternal and infant im-
Lawrence.gray@barwonhealth.org.au. Or: peter.vuillermin@deakin.edu.au. munity; transplacental passage of microbial metabolites and com-
The CrossMark symbol notifies online readers when updates have been made to the ponents; and possible seeding of a fetoplacental microbiome.
article such as errata or minor corrections
0091-6749/$36.00
Collectively, these result in increased expression of microbe-
Ó 2021 American Academy of Allergy, Asthma & Immunology associated molecular pattern (MAMP) recognition receptors, pro-
https://doi.org/10.1016/j.jaci.2021.07.011 motion of thymic regulatory T (Treg) cells, and the establishment

669
670 GAO ET AL J ALLERGY CLIN IMMUNOL
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Abbreviations used
Farm children demonstrate prenatal ‘‘priming’’ characterized
DC: Dendritic cell in functional studies of cord blood by higher expression of the
EV: Extracellular vesicle microbial pattern recognition Toll-like receptor (TLR)7 and
SCFA: Short-chain fatty acid TLR8 receptors,37 equipping them to respond to viral-based sig-
TLR: Toll-like receptor nals during early infancy, as well as increased Treg-cell number
Treg: Regulatory T and function, required to control inflammatory responses to path-
ogens and environmental stimuli.38 Numerous studies have
shown that both increased TH1/IFN-g responses,39,40 increased
Treg cells, and enhanced levels of neutrophils37 at birth predict
of dendritic cell (DC) networks in the gut, lung, and other tissues. decreased risk of allergic disease and asthma.41-44 Consistent
Inoculation of the infant gut with the maternal microbiota during with prenatal priming, farm children demonstrate accelerated
and after birth leverages the antenatal programming to accelerate progression from TH2/eosinophil-skewed immunity at birth,
the transition from TH2 to TH1, interferon regulatory transcription which is associated with susceptibility to both infections and
factor 7, and TH17-dominant immunity required for pathogen atopy, to a more balanced and competent immune state character-
clearance, and promotes DC induction of Treg cells in the gut ized by robust antimicrobial responses to pathogens, and
and lung required to calibrate inflammatory responses. enhanced Treg-cell function required for maintenance of immune
There are however fundamental gaps in current knowledge. homeostasis and control of excessive inflammation.37 Further
Few human studies have evaluated direct microbial transfer studies are required to evaluate the contribution of the maternal
between mother and child, nor directly examined the relationship microbiome to the ‘‘farm effect.’’
between the maternal gut microbiome and offspring allergic
disease and asthma.18,19 No such studies have incorporated meta-
genomic measures; nor have human studies demonstrated media- Antibiotics
tion pathways linking environmental factors, via maternal Antibiotics have profound and lasting effects on the gut
microbiome, and infant immunity to clinical outcomes. Rela- microbiome.45 Maternal treatment with antibiotics during preg-
tively little is known about the multitude of factors driving fetal nancy has been linked to increased eczema,46 food allergy,46-48
immune development, few human studies have shown that and asthma49-51 in offspring. Evidence of a dose-response be-
microbiome-related interventions during pregnancy alter tween maternal treatment with antibiotics and offspring asthma
offspring immunity, and overall, findings regarding probiotics in human birth cohort studies52 and experimental models53
supplementation during pregnancy are limited and conflicting. strengthens the case for causality. Antibiotic treatment during
In this context, there is a clear need for improved evidence to pregnancy may influence both the maternal and infant gut micro-
progress and translate the potential of manipulating the maternal biome in mice,54 so it is difficult to delineate the relative impor-
microbiome to prevent offspring allergic disease and asthma. tance of the prenatal and postnatal periods. It is also impossible
to exclude confounding by indication, in particular antibiotic
treatment of maternal respiratory infections, which may relate
EXPOSURES IMPACTING BOTH MATERNAL GUT to maternal asthma, and thus shared maternal/infant genetic
MICROBIOTA AND OFFSPRING ALLERGY risk. From large register-based studies, where it is also possible
Farming environment to evaluate associations with antibiotic exposure to the mother
Studies in Europe, Asia, the United States, and Africa have around the pregnancy period55 and by applying siblings design,56
shown that early-life exposure to a traditional farm environment is evidence of antibiotic use in pregnancy as a causal link to later
associated with reduced risk of allergic disease and asthma.20-26 disease disappears. Instead, these studies suggest that a suscepti-
Although prenatal and postnatal exposures were highly correlated bility to infections may be the factor inherited, because the
in each of these studies, the Prevention of Allergy Risk factors for mother’s use of antibiotics outside the pregnancy period was
Sensitization In children related to Farming and Anthroposophic just as important a risk factor for asthma in the child, and a higher
Lifestyle (PARSIFAL) cohort was large enough to show that prena- risk of disease was also found in siblings unexposed to maternal
tal farm exposure may be more protective than postnatal exposure.20 antibiotics during pregnancy.
Although the ‘‘farm effect’’ appears to relate to exposure to
livestock20,27,28 and a diverse range of environmental mi-
crobes,29-32 several studies have shown that maternal consumption Diet
of unprocessed milk is also relevant.27,33,34 Building on this, a Although the relationship between maternal diet and offspring
recent study in mice showed that ingestion of raw versus pasteur- allergic disease remains controversial, recent work has added to
ized cow’s milk increased butyrate-producing Clostridiales in the evidence that a maternal diet high in vegetables is associated
maternal fecal samples, decreased Proteobacteria, and conferred with a reduced risk of offspring allergic disease in humans.57 Diet
protection against IgE-mediated food allergy in the offspring.35 has an important impact on gut microbiota composition and meta-
The risk of Listeria monocytogenes infection associated with bolic function.8 Ecological studies have consistently shown asso-
ingestion of raw milk is an important consideration, particularly ciations between a preagrarian or hunter-gatherer diet and
in pregnancy; thus, specific understanding of the underlying path- lifestyle, and increased diversity of the gut microbiota, and
ways is required to inform more targeted and safe interventions. increased production of specific microbial metabolites, in partic-
Although traditional farming is becoming uncommon in Western- ular, short-chain fatty acids (SCFAs).1,2,9 In each of these studies,
ized populations, the same protection from allergic disorders has the hunter-gatherer communities had a diet high in plant-derived
been observed in rural living environments, where extended biodi- fibers,58 which have a central role in shaping the composition and
versity36 has been suggested as a mediator. metabolic activity of the gut microbiome.10 A maternal diet high
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FIG 1. Putative pathways linking environmental factors, the maternal gut microbiome during pregnancy,
early-life immune development, and protection against allergic disease and asthma. IRF7, Interferon regu-
latory transcription factor 7; MAMPs, microbe-associated molecular patterns.

in fiber is associated with increased diversity and richness of gut responses to ovalbumin challenge in the offspring.64 Although the
microbiota during pregnancy, with increases in specific taxa underlying mechanisms are not fully characterized, SCFA induc-
including Holdemania, Roseburia, and Lachnospira and Copro- tion of Treg cells may be relevant,65 and human studies investi-
coccus.59-61 Studies in mice have shown that a diet low in fiber gating maternal diet-by-microbiome pathways to enhanced
leads to intergenerational depletion in the diversity of gut micro- infant immune competence are required.
biota, which only partially recovers following reinstitution of a
high-fiber diet.62 This suggests that the low-fiber diet, which is
now endemic in many parts of the world, is contributing to the Psychosocial stress and mental illness
loss of our ‘‘ancestral’’ gut microbes. Anaerobic bacteria in the Maternal psychosocial stress and mental illness during preg-
gut ferment fiber to produce SCFAs, which have potent local nancy are associated with increased risk of allergy and asthma in
and systemic immune effects63 and can cross the placenta. In the offspring.66-68 Stress-induced release of glucocorticoids has
mice, a maternal diet high in fiber attenuates inflammatory airway been suggested to mediate these effects,69 but the impact of
672 GAO ET AL J ALLERGY CLIN IMMUNOL
SEPTEMBER 2021

psychological stress and anxiety on the maternal gut microbiome stimulating DCs and enhancing DC-mediated T-cell induction
may also be relevant.11,70-72 In support of this, animal studies have and cytokine production.89 Further studies are needed to investi-
demonstrated that maternal psychosocial stress before and during gate the impact of these metabolites of the maternal microbiome
pregnancy may alter both the mothers’ and infants’ intestinal mi- on fetal immune development and offspring allergic disease.
crobiota, in part mediating the effects of maternal stress on
immune development of the offspring.73 Related studies are
needed in humans. Bacterial components
In mice, repeated administration of the bacterial lysate OM-85
during pregnancy attenuates aeroallergen-induced asthma-like
POTENTIAL MECHANISMS responses in the offspring.90 This finding is consistent with earlier
Maternal-fetal immune alignment work showing that maternal oral intake of Acinetobacter lwoffii
Preparing the fetal immune system in utero for the extrauterine F78, derived from farm (barn) dust, during pregnancy attenuated
environment is germane for survival, with maternally conferred aeroallergen-induced inflammation in the offspring, and that this
immune protection against environmental pathogens representing process was TLR-dependent.91 The offspring of mothers treated
a key defense mechanism. In this context, maternal transfer of hu- with OM-85 during pregnancy demonstrate increased capacity
moral immunity, specifically of maternal IgG through an active for expansion and functional activation of Treg cells in the airway
mechanism enabled through neonatal Fc receptors, has been mucosa in response to aeroallergen challenge.90 This enhanced
well established.74 Through this process serum IgG levels of the regulatory capacity is underpinned by a dramatic expansion of
term newborn are equal to or even exceed maternal levels. Of bone marrow myeloid progenitors during late pregnancy, which
note, maternal IgG transfer of gut microbial components to the in turn supply the establishment of tissue DC networks that
offspring during gestation also plays a crucial role in fetal innate mediate local immune surveillance and homeostasis.90 Subse-
immune development in mice.17 Postpartum, transfer of maternal quent studies have shown that maternal OM-85 treatment is asso-
immunoglobulins continues through lactation, but these anti- ciated with transcriptional activation of cellular cholesterol
bodies confer mucosal immunity only.75 biosynthesis pathways (mediated via the mevalonate pathway)
In contrast, for cellular immunity it has been a long-held view and expansion of myeloid progenitors (myelopoiesis) and down-
that the fetus holds an immune privileged status, keeping the fetal stream conventional DCs displaying enhanced functional matura-
and maternal immune cells separate, allowing physiologic tion within fetal bone marrow.92 The establishment of DC
development of the semiallogenic fetus without triggering in- networks in late gestation can be considered a form of immune
flammatory responses.76 Immune tolerance toward the fetus is up- training—a process in which innate immune cells exhibit a sus-
held by a series of highly effective mechanisms, with the placenta tained state of enhanced functionality. Recently, it has also been
providing a barrier preventing immune rejection and/or traf- demonstrated that treatment of pregnant mice with OM-85 during
ficking of immune cells across the maternal-fetal interface.77 pregnancy protects offspring against mouse-adapted rhinovirus,
The latter concept has been challenged by studies providing reducing cellular inflammation and clinical severity of lung dis-
compelling evidence that traces of maternal cells can be found ease.93 Studies are needed to determine whether maternal gut mi-
in fetal organs including the thymus78 and vice versa.79 This early crobiota and their products promote fetal immune development
exchange seems to shape the offspring’s immune response and via the same pathways.
can in part explain the better acceptance of maternal versus
paternal organ transplants.80
There is also evidence for alignment of maternal and fetal Bacterial extracellular vesicles
lymphocytes, specifically of Treg cells. This alignment is likely Like other cells, gut bacteria produce extracellular vesicles
linked to soluble factors that cross the placenta from the mother to (EVs), which are blebs of plasma membrane carrying a cargo of
the fetus and effectively influence the size of the fetal Treg-cell intracellular products such as proteins, nucleic acids, and lipids.94
pool81,82 and metabolites of the maternal gut microbiome are There is growing interest in the role of EVs in mediating the im-
likely candidates.83 mune effects of gut bacteria. For example, the activation of TLR2
by Bacteroides fragilis has been shown to be mediated by the
release of EVs.95 In postnatal mouse studies, the administration
Bacterial metabolites of Lactobacillus plantarum EVs prevented the development of
The SCFAs butyrate, propionate, and acetate are by-products of atopic dermatitis,96 whereas the administration of EVs from Bifi-
dietary fiber fermentation by gut bacteria.58 In mouse models, dobacterium longum prevented food allergy.97 Given that EVs are
maternal SCFAs attenuate inflammatory airway response in small structures that can be actively transported from the mother
offspring.64 Although the underlying mechanism is unknown, to the fetus,98 studies are needed to evaluate whether EVs pro-
SCFAs have potent immunomodulatory properties including in- duced by maternal gut microbiota impact fetal immune
duction of Treg cells from naive T cells via dendritic cells development.
(DCs) either through epigenetic changes by inhibiting histone de-
acetylases64,84,85 or through the activation of specific G-protein–
coupled receptor (GPR)43.86 Recently, acetate has been identified In utero bacterial colonization
as an important factor in fetal thymic development, linked to the The presence of bacteria in sites including placenta, amniotic
expression of the autoimmune regulator, which is known to fluid, and meconium was reported in 2011,99,100 and has since
contribute to Treg generation.87 Succinate is another immune- been the subject of intense debate. Subsequent studies have iden-
active microbial metabolite that is increased in animals fed a tified bacteria in placenta,101-105 amniotic fluid,106-111 and meco-
high-fiber and high-fat diet.88 Succinate binds GPR91, nium,103,104,112-114 and 1 study found that maternal consumption
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of probiotics and contact with furry pets during pregnancy may there is currently no evidence that either reduces the baby’s risk
each influence the microbiota present in meconium.115 of allergic disease or asthma.
Ascension and translocation through the choriodecidual
barriers is one potential mechanism of in utero bacterial coloniza-
tion.102,108,116,117 Alternatively, microbes derived from the DIRECT LINKS BETWEEN MATERNAL GUT
maternal intestine and oral cavity may be translocated by hema- MICROBIOTA AND OFFSPRING ALLERGY IN
togenous spread to the placenta during implantation,17,102 due HUMANS
to higher intercellular junctional permeability and/or DC trans- As far as we are aware, only 2 published studies have directly
port.118 Translocation of bacteria to the maternal circulation, investigated the relationship between the maternal gut microbiota
via the epithelial gaps of the intestine and the oral mucosa, is and offspring allergic disease in humans. A small study (n 5 60)
enhanced during pregnancy, and this enables the transfer of low from 2012 used culture-based techniques to show that higher total
numbers of bacteria to possibly seed the placenta.102,118-120 aerobes and Enterococci in maternal fecal samples predicted
Although some studies suggest that the presence of bacterial increased risk of infant wheeze.19 More recently, we used 16S
DNA in pregnancy tissues may relate to contamination of samples gene amplicon sequencing to demonstrate that maternal carriage
or reagents,104,105,121 it remains a possibility that a fetoplacental of Prevotella copri during pregnancy predicts decreased IgE-
microbiome plays a role in fetal immune development. The meco- mediated food allergy in the offspring.18 Importantly, maternal
nium microbiota was found to reflect microbial composition of rather than infant carriage drove this association. Prevotella car-
the amniotic fluid,122 suggesting that the fetal intestine could riage is virtually ubiquitous in preagrarian, hunter-gatherer popu-
become colonized via ingestion of small quantities of amniotic lations,1,2,9 and the genus is among the so-called ancestral
fluid during the antenatal period and thus the meconium micro- microbes present in the human gut at least as far back as the Nean-
biome could be a proxy for the fetoplacental microbiome. Of rele- derthal period,7,138 and which are now disappearing in Western-
vance, a study from the Canadian Healthy Infant Longitudinal ized populations.1-7 P copri is the predominant Prevotella
Development (CHILD) cohort suggests that the metabolic diver- species within the human gut microbiome. Our study showed
sity of meconium collected at birth predicts both the infant’s post- that reduced household size and the recent exposure to antibiotics
natal gut microbiota and the risk of allergic sensitization at 1 were associated with decreased maternal carriage of P copri.18
year.123 Moreover, a recent study found live bacterial strains The link between household size and P copri carriage is consis-
including Staphylococcus and Lactobacillus in fetal tissue, which tent with previous studies,139 and intriguing given the
induced in vitro activation of memory T cells in fetal mesenteric well-known, yet still unexplained, association between larger
lymph nodes, supporting a role for microbial exposure in fetal im- household size and decreased allergic disease.140 P copri is an
mune programming.124 important producer of succinate,88 which can stimulate function
and migration of DCs.89 Previous studies have shown that the
metabolic products of the gut microbiome stimulate bone marrow
hemopoiesis of DC precursors, which then migrate to peripheral
Vertical transmission of gut microbiota at birth tissues including the lung, attenuating the features of allergic air-
In infants delivered vaginally, the maternal gut microbiome is ways disease.141 Considering the mounting evidence regarding
the largest donor of the infant-acquired strains125,126 whereas the the role of DC hemopoiesis in late pregnancy in prevention of
maternal vaginal transfer appears of minor importance.127 Strain- allergic disease,90-92 we hypothesize that maternal carriage of P
level analyses of mother-infant pairs revealed that 50% of the copri, via production of succinate, promotes the establishment
microbial species in the infant gut on the day of delivery were of fetal DC networks, conveying protection against allergic dis-
transmitted from the mother’s microbiome, and this fraction ease and asthma (Fig 2).
was relatively stable over the next 4 months.125 The largest contri-
bution at birth was from the mothers’ gut, accounting for 22%,
followed by the vagina (16%), the oral cavity (7%), and the PROBIOTIC SUPPLEMENTATION DURING
skin (5%).125 Moreover, the maternal gut microbial strains were PREGNANCY
more persistent in the infant’s gut and more stably detected Recent meta-analyses have found no consistent beneficial
than those obtained from other maternal or environmental effects from probiotic supplementation (either prenatal or post-
sources.125,126,128 natal or both) for prevention of eczema142 or asthma.143 These in-
It is therefore unsurprising that mode of birth profoundly consistencies may be due to differences in the study populations,
influences the infant’s gut microbiome.128-133 Although the link the probiotic strains, using a single strain rather than a complex
between mode of birth and allergic disease remains controversial, mixture, and administration protocols. Although several trials
a recent study found that increased risk of asthma following cesar- found that combined maternal and infant probiotic supplementa-
ean delivery was apparent in only those children who retained a tion with various strains of Lactobacillus reduced the risk of al-
‘‘cesarean section signature’’ in the gut microbiome to age 1 lergy,144 others found no benefit145,146; and the only trial of
year.134 Strain-level analyses directly implicate the interruption Lactobacillus in pregnancy alone was negative.147 Potential ben-
of maternal-infant transmission in this effect.125,126,133,135 Inter- efits may be modified by mode of delivery. One trial randomized
ventions proposed to offset the alteration in gut microbiota high-risk mothers to receive either a probiotic mixture (L rhamno-
induced by cesarean section include dietary supplementation sus GG ATCC 53103 5, L rhamnosus LC705 DSM 7061, Bifido-
with a mix of probiotics during pregnancy and to the infants,136 bacterium breve Bb99 DSM 13692, and Propionibacterium
and postnatally, orally delivered fecal microbiota transplantation freudenreichii ssp. shermanii JS DSM 7076) or placebo twice
from their mothers.137 Both techniques appear to ameliorate birth daily starting from 36 weeks of gestation.148 Their infants then
mode–associated differences in infant intestinal microbiota, but received the same probiotic combination mixed with galacto-
674 GAO ET AL J ALLERGY CLIN IMMUNOL
SEPTEMBER 2021

FIG 2. Hypothesized pathway by which maternal carriage of Prevotella copri trains fetal immunity,
conveying protection against subsequent allergic disease and asthma. IRF7, Interferon regulatory transcrip-
tion factor 7.

oligosaccharides once daily from birth until 6 months. Treatment and postnatal life are both multimodal and temporal in nature.
with either probiotic mixture was associated with a reduced risk As such, their analyses and interpretation pose formidable chal-
of allergen sensitization and eczema among children born via ce- lenges, and methodological frameworks built specifically for
sarean section but not among those born vaginally. Another trial modeling time-varying multiomic data are required.152 The
found that maternal ingestion of probiotic milk containing Lacto- compositional nature of microbiome data also has important im-
bacillus acidophilus LA-5, Bifidobacterium lactis Bb12, or L plications.153 This is generally addressed through various forms
rhamnosus in pregnancy was associated with a slightly reduced of preprocessing steps, the choice of which can substantially
relative risk of atopic eczema at 6 months and allergic rhinitis be- impact the results,154 making quantitative metagenomics a prefer-
tween 18 and 36 months compared with no consumption during able strategy, whenever possible.
pregnancy.149 Robust consideration of confounding bias and mediation
Importantly, to date there have been no trials of supplementa- pathways also poses an important, but neglected, challenge for
tion with Gram-negative organisms, which may be relevant given the field. Microbiome data are highly dimensioned, and the
that oral administration of the Gram-negative Acinetobacter lwof- determinants of specific components/metrics are largely un-
fii F78 in mice resulted in decreased allergy in the offspring.91 known; thus, a priori identification of common causes of expo-
This benefit was TLR-dependent, suggesting that LPS in the sures and outcomes (confounders) is largely speculative. One
cell membrane of Gram-negative organisms may be important. approach to this dilemma is to apply the so-called disjunctive
Another consideration is that, in general, probiotic strains that cause criterion,155 which has an elegant proof and removes the
do not colonize the bowel are specifically chosen; that is, they need for previous knowledge of common causes. Others argue
do not live permanently and multiply in the gastrointestinal tract. that a strictly a priori–defined approach to estimating causal ef-
It is possible that active growth and multiplication is required to fects using highly dimensioned data risks missing important sig-
produce the metabolites that influence fetal and neonatal immune nals and that one should therefore conduct multiple analyses
development, with subsequent reduction in offspring allergic aimed to build up layers of causal features and exclude noncausal
disease. explanations such as bias, chance, or reverse causation.156
Whichever approach is taken to preprocessing and analysis of
human multiomics data, it is crucial to formulate clear research
FUTURE DIRECTIONS questions, and to then choose methods that are appropriate. This is
Correcting the absence of deep whole metagenome sequencing a highly complex task requiring clinical, biological, bio-
data from human prebirth cohorts incorporating robust allergy informatic, biostatistical, and epidemiological input. Reporting
and asthma outcomes is a priority. In contrast to 16S rRNA gene from human studies should include effect sizes both with and
amplicon, deep metagenomic sequencing provides sufficient without adjustment along with a clear description of the causal
resolution at the species and strain level to infer the functional framework that underlies the model(s) that has been applied, and
capacity and nutrient interactions of a given bacterium.150 Esti- the sensitivity of the findings to key assumptions. Importantly, the
mation of absolute abundances of microbiota may also provide risk of false discoveries in highly dimensioned data must be
more accurate information than currently used measures of rela- controlled for and replication is crucial.
tive abundance.151 In principle, the integration of metagenomics, Progressing from correlation in human microbiome studies to
downstream measures of microbiome activity including metabo- causation and effective interventions requires mechanistic in-
lomics, transcriptomics, proteomics, and lipidomics with various sights.157 Preclinical studies have generally involved the trans-
immune measures has significant promise. However, multiomic plantation of bacteria from humans with and without disease
data generated using separate assays over the course of prenatal into germ-free mice and comparative analysis of pathological
J ALLERGY CLIN IMMUNOL GAO ET AL 675
VOLUME 148, NUMBER 3

outcomes. Although this approach has merit, it is of concern that 14. Depner M, Taft DH, Kirjavainen PV, Kalanetra KM, Karvonen AM, Peschel S,
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