Like the hands that pull the strings at a marionette puppet show, our resident microbes influence the day-to-day running of virtually all of our biological systems. For instance, host-microbe interactions during the first three years of life are particularly important for the development of the immune system, and perturbances to the gut microbiota, or dysbiosis, during this critical time in early life can have long-lasting detrimental effects on health. Until very recently, this microbe-mediated immune education was thought to be initiated when a newborn baby leaves the relatively sterile environment of the uterus and is seeded with its mother’s microbes, but work from our group and others in the last few years has shown that the maternal microbiome can exert its influence even earlier—on the gestating fetus.
In the mid-2010s, when we started our work on the role of the maternal microbiome during pregnancy, there was no direct evidence that mom’s resident bacteria affected the developing baby. However, it seemed naive to think that the influence of the maternal microbiota would start only at birth. We knew that maternal antibodies crossed the placenta to protect the fetus from infection, and we suspected these antibodies could also direct the maturation of the immune system. We also knew that commensal bacteria, in addition to pathogenic microbes, trigger the development of antibodies. Moreover, we thought that perhaps microbial products or metabolites could also be transferred to initiate exposure to a microbial world even before the baby is colonized with his or her own microbiome at birth. Now, just five years later, we know this to be the case: both bacteria-produced molecules and maternally derived antibodies appear to drive immune development in utero.
Many unanswered questions remain, and we are far from understanding the long-term significance of this phenomenon. However, it seems increasingly possible that our mothers’ microbiomes may, to some extent, shape our health and well-being before we are born.
Developmental origins of health and disease
During delivery, babies are exposed to the maternal microbiota. Children born by the vaginal route are colonized by vaginal and fecal microbes, while those born by Caesarean section are instead predominantly seeded by the maternal skin microbiota and sometimes hospital-acquired microbes. The importance of this vertical microbial transfer is apparent in the higher rates of immune, metabolic, and neurodevelopmental disorders among babies born by C-section.
See “The Maternal Microbiome”
Manipulating the microbiota of pregnant mice can modify the function of the offspring’s immune systems and alter their disease outcomes.
After birth, it takes several years for a child’s microbiota to fully develop and diversify. This is a dynamic process, heavily influenced by external factors including hygiene, antibiotic usage, and diet, including human oligosaccharides from the mother’s milk. In the same time frame, the offspring’s immune system is undergoing intense development and maturation and is highly susceptible to microbial imprinting. Exposing young children to antibiotics during this critical window has been associated with an increased susceptibility to several diseases, likely due to the indirect effects that these drugs have on immune development.
In 2013 and 2014, as researchers began to question the role of the maternal microbiota during pregnancy, several groups launched epidemiological and animal studies to examine whether in utero antibiotic exposure poses a similar risk to a child’s health. Fetuses don’t have their own microbiota, so any microbe-mediated immune education that may happen in the womb falls to the resident microbes of the gestating parent, and antibiotic exposure during pregnancy would disrupt this.
Sure enough, exposing pregnant mice to antibiotics can modify the function of the offspring’s immune systems and alter their disease outcomes. This was first demonstrated in 2015, when Youjia Hu and colleagues from Yale University showed that prenatal antibiotic exposure influenced the development of type 1 diabetes in the offspring. Since then, a number of other studies have similarly demonstrated effects on offspring of manipulating the microbiota of pregnant mice, either with antibiotics or dietary intervention. Researchers have observed such results in a variety of models, with implications not only for diabetes, but for susceptibility to asthma, obesity, and colitis, as well as the progression of autism-like behaviors.
In most circumstances, manipulating the maternal microbiota changes which microbial communities are passed from mother to offspring at birth, and subsequently alters immune development in the neonatal host. It has therefore proven challenging to attribute the immune phenotypes described in most of these studies to the maternal microbiota directly, as opposed to those mediated by the newly seeded microbiota of the neonate. Fortunately, we are beginning to tease these differences apart, and tantalizing hints are emerging that the maternal microbiota educates the immune and nervous systems of gestating offspring remotely.
Maternal microbiota shapes infant antibody repertoire
For decades, we have known that antibodies are passed from parent to fetus during pregnancy. They play a huge role in protecting babies from infection both before and after birth. Antibodies continue to be passed postnatally through breast milk, which is one of the many reasons breastfeeding is more beneficial to a baby’s immune system than formula is, and why immunizing a person against pathogens during pregnancy or lactation can protect her baby from infections. However, we are only now beginning to appreciate the role the maternal microbiota plays in shaping the antibody repertoire. We are also gaining new insights into other roles for antibodies beyond binding to pathogens and protecting from infections.
By adulthood, the intestine is home to the body’s largest collection of immune cells. As it is continuously exposed to enormous amounts of foreign antigens, derived from both the microbiota and our diet, the intestinal immune system must learn to tolerate innocuous food components and symbiotic microbes while retaining the ability to mount a successful defense against harmful pathogens. Moreover, all microbes can quickly become harmful if they enter the bloodstream, even those that are considered symbiotic when living in the gut. Antibodies such as immunoglobulin A (IgA), secreted by B cells at the mucosal surface lining the intestines, are transported across the gut epithelium into the lumen, where they bind to microbes and prevent them from passing through the intestinal epithelial barrier.
The immune system of a newborn infant is inexperienced. The fetus is not thought to be colonized with its own bona fide microbial communities. Traditionally, researchers thought that maternal antibodies transferred across the placenta were specific to infectious microbes that might infect the baby while its own immune system was still developing. We now know that maternally derived antibodies can also bind commensal bacteria—and that this helps to keep these nonpathogenic bacteria from crossing the epithelial barrier as a newborn’s gut is rapidly colonized by a vast array of unfamiliar microbes.
Antibodies transferred in breast milk can interact directly with the microbial inhabitants of the infant gastrointestinal (GI) tract, where they keep populations of commensal species in check and ensure that the microbes stay in the gut lumen where they belong, thereby preventing the inappropriate activation of the local adaptive immune response. This transfer of antibodies might partly explain why newborn babies fed breast milk are less susceptible to developing necrotizing enterocolitis (NEC), a severe and often fatal inflammation of the colon that can occur when babies are born prematurely.
The reason that preterm babies are highly susceptible to NEC is unclear, but it is widely postulated to be a consequence of their immature immune systems overreacting to the abrupt colonization of their GI tracts—something that full-term babies typically handle without issue. In 2019, Kathyayini Gopalakrishna and colleagues at the University of Pittsburgh Medical Center Children’s Hospital demonstrated the importance of bacteria-specific IgA antibodies in preventing an overexpansion of Enterobacteriaceae—a classic hallmark of NEC—in the guts of preterm babies. These and other results imply that immune education in the final weeks before birth is important for babies’ immune systems to tolerate friendly bacteria. Maternally derived antibodies appear to put the brakes on inflammatory pathways to protect the gut from unnecessary damage when first exposed to the microbial world.
As well as functioning in the GI tract, commensal-targeting antibodies from breast milk can be actively transported across the epithelial barrier of a baby’s intestine and into its circulation, ultimately disseminating throughout the body. At least a portion of these anticommensal antibodies can cross-react with pathogens. Wen Zheng and colleagues at Harvard Medical School hypothesize that this transepithelial transfer of cross-reacting anticommensal antibodies, from breast milk to the infant bloodstream, could explain the observed protection of neonatal mice from systemic pathogen infection. While it is unclear whether the transfer of antibodies across the placenta in utero contributed to protection in this model, it seems likely that they acted in concert with antibodies transported via breast milk to protect the neonate.
The Many Effects of Microbes on Offspring
During pregnancy, the body is subject to numerous changes. The composition of the gut microbiome shifts, metabolism changes, and the gut epithelium becomes more permeable. These alterations facilitate interactions between the immune system and gut microbiota, leading to the production of microbe-specific antibodies that are transferred across the placenta to the developing fetus, and later via the milk to the nursing offspring.
The maternal microbiota, and the external factors that shape it, influence which immunomodulatory metabolites are produced and transferred to offspring, where they support immune education and otherwise influence development, helping to protect offspring from allergic asthma, metabolic syndrome, and likely other inflammatory diseases later in life. After birth, maternally derived antibodies help newborns tolerate the bacterial colonization of their own GI tracts, while simultaneously protecting them from enteric and systemic infections.
@Studio Lindalu, Linda Lubbersen
External factors affect the composition of the maternal microbiota (for example, diet, antibiotics, and other drugs).
The antibody-mediated transfer of metabolites increases antimicrobial peptide production, which strengthens the epithelial barrier to prepare the gut for microbial colonization.
The permeability of intestinal epithelium increases during pregnancy, facilitating interactions between the microbiota and a mother’s immune system.
SCFAs transferred from mom travel to the fetal thymus, where they trigger epigenetic changes that nudge T cells toward becoming regulatory as opposed to inflammatory later in life.
The maternal microbiota shapes the repertoire of commensal-targeting antibodies, which cross the placenta and are transferred in breast milk to her offspring.
SCFAs also have various effects on the heart, pancreas, thymus, and other organs.
Gut microbes produce various metabolites, including short-chain fatty acids (SCFAs) and immunomodulatory compounds, some of which are bound by antibodies and transferred to the fetus.
@Studio Lindalu, Linda Lubbersen
Commensal-targeting antibodies promote tolerance during bacterial colonization, largely by keeping gut bacteria inside the intestinal lumen or ushering them back in when they do escape.
If microbes that escape the intestinal lumen are not immediately ushered back in, antibodies from mom tag them for efficient elimination to limit inflammation.
@Studio Lindalu, Linda Lubbersen
As the pups age, the immune education that they received in utero and shortly after birth protects the animals from inappropriate inflammatory reactions that can lead to allergy, metabolic syndrome, or other health consequences.
Microbial metabolites also influence infant immunity
The maternal microbiota is not a one-trick pony; it does more to shape the offspring’s immune system than induce the production of antibodies that are shared with the newborn. By breaking down the food we eat, and molecules secreted by other resident microbes, intestinal microbes produce a wealth of metabolites with wide-ranging immune-modulatory functions. At least some of these are passed from parent to child during gestation and breastfeeding.
Best characterized are the short chain fatty acids (SCFAs), derived from the fermentation of dietary fiber by intestinal microbes. The amounts and types of SCFAs that are produced in the parent’s gut and transferred to her baby depend on the maternal microbiome, which is in turn shaped by her diet. When pregnant people eat a diet rich in fiber, SCFA-producing microbes thrive, and increased amounts of SCFAs are transferred to the developing fetus. These compounds may influence the maturation of the fetal immune system—specifically, the development of regulatory T cells (Tregs), which help quiet runaway inflammation.
Tregs are crucial for protecting our bodies from autoimmune diseases, as well as from allergies and asthma. They also teach our immune systems to tolerate food and friendly bacteria. Although they self-renew with time, Tregs are long-lived and their progeny will likely be present throughout the life of the host. So, if the maternal microbiota influences the development or maturation of these cells, this could have far-reaching implications for the health of the offspring.
In 2017, Akihito Nakajima and colleagues at Juntendo University in Tokyo reported that three-day-old mouse pups had more Tregs in their thymuses and spleens if their mothers had been fed a high-fiber diet, compared with pups of mothers on a low-fiber diet. The pregnant dams that ate more fiber had increased amounts of the SCFAs acetate, propionate, and butyrate in their feces, as well as increased butyrate in their blood, and their pups had increased SCFAs, especially acetate, in their blood at day 11 of life. The authors suggest that SCFAs produced by the maternal microbiota may act remotely to influence T cell education in the developing thymus, accounting for this increase in Tregs. However, mothers fed a high-fiber diet had differences in their own microbiota, meaning that their babies would likely be seeded with different microbes that might also contribute to circulating SCFAs. More work is needed to establish if it was really the maternal SCFAs transferred during gestation, as opposed to the altered composition of the offspring’s microbiomes, that caused the varying numbers of Tregs in the mouse pups.
In addition to those that are produced in the thymus around the time of birth, another group of Tregs can develop from naive T cells in the periphery, and these are equally important for preventing autoimmunity. Alison Thorburn and colleagues at Monash University have shown that the process happens more efficiently when the SCFA acetate is transferred from mother to fetus across the placenta. By increasing transcriptional accessibility of the gene encoding FoxP3, the master regulator of Tregs, maternally derived acetate permanently altered naive T cells in the fetal thymus. This process skewed T cell differentiation toward a regulatory phenotype, as opposed to an inflammatory one, following antigen exposure later in life, thereby protecting mice from developing asthma. Importantly, the authors performed a variety of cohousing and cross-fostering experiments to rule out contributions from milk metabolites and the offspring’s own microbiota in driving this protective phenotype. That is not to say that the transfer of SCFAs in milk doesn’t contribute to protection. But in this study, maternal acetate in the mice’s milk was not sufficient to confer asthma protection, whereas transfer of maternal acetate across the placenta was.
Until very recently, this microbe-mediated immune education was thought to be initiated at birth when a newborn baby leaves the relatively sterile environment of the uterus.
The study by Thorburn and colleagues, published in 2015, was arguably the first to concretely and convincingly demonstrate the long-term effects of the maternal microbiota on disease susceptibility in the offspring. A recent study by Ikuo Kimura of the Tokyo University of Agriculture and Technology and colleagues showed that the placental transfer of propionate, another SCFA that is regulated by the microbiota, could reduce susceptibility of the offspring to obesity and metabolic syndrome in response to a high-fat diet later in life. These phenotypes, which were driven by interactions between maternal SCFAs and their receptors in the developing fetus, could implicate the maternal microbiota in the risk of type 2 diabetes in the offspring.
We predict these two pioneering articles will be the first of many, particularly when one considers the extent of inflammatory diseases that can be controlled and suppressed by Tregs and the wide range of immunomodulatory effects mediated by SCFAs. Moreover, SCFAs are not the only group of microbial metabolites that offspring receive in utero. When working at the University of Bern, our research group demonstrated that a broad range of metabolites are transferred during pregnancy and lactation. We colonized germ-free mice with a friendly species of E. coli that was genetically engineered to be unable to replicate without supplementation with essential amino acids. This enabled us to restrict colonization to a precise period during pregnancy, allowing time for the mothers to return to germ-free status before giving birth to germ-free pups. The metabolites passed from mom to pup changed the immune cell profile in the neonatal intestine and increased the transcription of genes involved in antimicrobial defense. These processes reinforced the integrity of the epithelial barrier, so that when the germ-free offspring were colonized later in life, friendly gut bacteria were prevented from escaping the confines of the intestine to invade the host.
Surprisingly, we found that antibodies were necessary for optimal metabolite transfer, both across the placenta and via breast milk. Specifically, antibodies apparently bound to metabolites in the circulation after pregnant mice were fed E. coli. This sheds new light on more than 100 years of antibody research—in addition to protecting against infection, these molecules appear to serve as chaperones of immune-modulating metabolites. If the mothers lacked antibodies, the effect of the maternal microbiota was largely absent; metabolites required maternal antibodies for their transfer.
We are far from fully understanding the extent to which microbial molecules are passed from mother to child, or to what degree they imprint upon the developing immune system. Our group found hundreds of metabolites in the organs of fetal mice and the milk of their mothers, and our model only involved a single species of microbe. In a natural microbiome with diverse assemblages of microbes, the range of metabolites transferred would likely be even more extensive.
A new view of the maternal microbiome
Most of the research into the effects of the maternal microbiota on offspring has focused on immune education. However, there are some studies emerging that imply that these microbes may have further-reaching consequences. Epidemiological studies have tentatively linked maternal diet and antibiotic exposure to the development of neurodevelopmental disorders in children.
Researchers have recently begun to investigate these observations using animal models. Two separate studies, one published in 2018 by Morgane Thion and colleagues from the Université PSL in Paris and the other in 2020 by Helen Vuong and colleagues from the University of California, Los Angeles, showed that the colonization status of a pregnant mouse can influence gene expression in the brain of her prenatal offspring. The study from Thion’s group linked these changes to differences in microglial phenotype and abundance during critical developmental stages, while Vuong’s team documented the stunted development of the nerves connecting the thalamus to the cerebral cortex in the offspring of germ-free or antibiotic-treated mice. Collectively, these studies provide preliminary evidence that a mother’s microbiota may modulate neurodevelopment in her offspring, possibly even protecting them from neurological disorders later in life.
In the last century, the incidence of neurological, inflammatory, and metabolic disorders has risen dramatically. What healthier way to flatten these curves than to nip them in the bud—or, more accurately, in the womb? The research to date adds weight to the developmental origins of health and disease (DOHaD) hypothesis, which suggests that prenatal and perinatal exposure to environmental factors can influence disease susceptibility later in life, and puts the maternal microbiome on the map as one important environmental factor to consider.
The implications of these studies are extensive. For one, they could provide evidence for guidance about maintaining a healthy microbiota throughout gestation—for example, by eating a fiber-rich diet or avoiding unnecessary antibiotic use. Moreover, they could inspire microbial manipulation strategies tailored to prospective parents with genetic susceptibilities to certain diseases. Such prophylactic therapies could be designed to curtail the establishment of such diseases before they begin by ensuring the healthiest possible immune development during gestation. The more information we glean on how the maternal microbiota shapes neonatal development and future disease susceptibility, the more likely we are to be able to prevent certain disorders altogether.
Kathy D. McCoy is a professor in the Snyder Institute of Chronic Diseases and the Cumming School of Medicine at the University of Calgary. Carolyn A. Thomson is a postdoctoral fellow in McCoy’s lab, which studies microbiome-immune interactions in health and disease.