Maternal Microbial Factors that Affect the Fetus and Subsequent Offspring

Abstract

The maternal microbiome has emerged as an important area of investigation. While birth is a critical timepoint for initial colonization of the newborn, the fetus resides in the womb surrounded by multiple unique colonized niches. The maternal microbiome has recently been shown to be associated with several morbidities in offspring. Understanding the multiple bacterial niches within the pregnant woman and how they interact with the fetus in-utero can lead to novel therapies to improve the health of offspring. In this review, we provide an overview of the available literature on normal bacterial colonization within the individual niches of the pregnant woman and the known associations with outcomes in offspring, including a discussion of the controversy of in-utero colonization.

Introduction

The mother and fetus are intimately intertwined, with the fate of the fetus being entirely dependent on maternal delivery of nutrition, immunity and physical protection. More recently, a unique level of complexity in this synergy has emerged with the unraveling of the contribution of maternal bacterial colonization on fetal and offspring health. Maternal microbial alterations are associated with a range of outcomes in offspring, which include preterm birth¹, altered susceptibility to sepsis², necrotizing enterocolitis³, adverse neurodevelopmental outcomes⁴, atopy, metabolic syndrome⁵ and obesity.⁶ The range of potential mechanisms include alteration of the maternal microbiome by maternal diet, changes in microbial metabolite exposure to the fetus in utero, differential in-utero fetal colonization and altered acquisition of the microbiome at birth.

In this review, we summarize several aspects of the relationship between microbes, the pregnant woman and her fetus that affect the outcome of the newborn. The “holobiont” of pregnancy consists of the triad of the woman, fetus and microbiota.⁷ We will address some components of this special relationship including how it evolves during pregnancy and how maternal diet and medications affect this holobiont with resultant potential for altered outcomes.

Site-specific alterations in maternal bacterial colonization associated with offspring outcomes

While it is clear that there are several microbial niches in the human body, we will focus on those of the vagina, maternal oral cavity, and maternal intestine and uterus (Table 1).

Table 1 -.

Changes in the maternal microbiome during pregnancy and association with offspring outcomes.

Maternal Niche	Changes during pregnancy	Associated with offspring outcomes	Specific notes
Vagina	↓ Diversity during pregnancl70 - Returns to normal at term	Preterm labor	Lactobacillus-poor microbiome associated with preterm birth11
Intestinal track	Mixed data: Changes from 1st to 3rd trimester15,16 or Remains stable during pregnancy11,17	Altered offspring microbiome Necrotizing enterocolitis Adverse neurodevelopmental outcomes Metabolic disease, including obesity	Proteobacteria may be increased in the third trimester15 Decrease in Bacteroidetes and Actinobacteria16 Placental microbiome has signatures from the gut microbiome30
Oral cavity	Increased number of bacteria during 1st and 2nd trimester29	Preterm birth Small for gestational age infants	Candida increased in the 3rd Trimester29
Placenta	Bacteria found in preterm human placental tissue as early as 28 weeks Mixed data: Bacterial DNA found in healthy term human placenta71 Or No placental microbiome48	Preterm birth	Placental microbiome has signatures from the maternal oral and gut microbiomes30 Studies that showed a placental microbiome may be result of contamination47,48

Vaginal Microbes

Predominant microbes in the non-pregnant vagina in most women of European descent consist of four Lactobacillus species, but African American and Hispanic women have a non-Lactobacillus dominant microbiome.⁸ During pregnancy, the lower vaginal tract diversity decreases. Around term, the vaginal microbiota trends back to a pre-pregnant composition.

Studies suggest that an inflammatory response originating from the decidua leads to preterm labor. A widely accepted concept is that microbes residing in the vagina ascend to the choriodecidual membranes, where they translocate into amniotic fluid.^{9, 10} Relman et al¹¹, in a case-control cohort of 40 women, found that a Lactobacillus-poor vaginal microbiome in pregnant women had a stronger association with preterm delivery than Lactobacillus-dominated vaginal microbiomes. A similar study by Hymen et al also found a relationship between the vaginal microbiota and preterm birth, associated with ethnicity of the mothers.¹ Even though this is a commonly held belief, an alternative explanation is that inflammation in decidua is not a cause of preterm labor, but labor is the cause of the inflammation.¹² This remains an area of controversy however, with other studies contradicting the notion that the microbes originating from the vagina are the origin of preterm labor.¹³

A more recent large-scale study reported multi-omic profiles that related preterm delivery to lower levels of Lactobacillus crispatus, higher Sneathea amnii, certain Prevotella species and nine additional taxa. These studies also associated preterm-birth-associated microbial taxa with higher proinflammatory cytokines in vaginal fluid.¹⁴

Maternal Gastrointestinal Microbes

Several clinical studies have examined the dynamism of the maternal gut microbiome during pregnancy, evaluating how bacterial populations change from the first to the third trimester, with mixed conclusions. Ley et al¹⁵ evaluated the fecal bacteria of 91 pregnant women during pregnancy and found that the gut microbiota changed from the first to the third trimester, with an increase in Proteobacteria and Actinobacteria. Another study evaluated 41 pregnant women with gestational diabetes and compared stool samples collected during the 2^nd trimester to samples collected at 38 weeks’ gestation and similarly found a difference in bacterial populations over trimesters, with an increase in diversity, but a reduction in Bacteroidetes and Actinobacteria¹⁶. In contrast, a recent study examined 1479 pregnant women and found that the microbiota did not change significantly through pregnancy, but that changes in the microbiota were instead associated with age, pre-pregnancy BMI and gestational conditions.¹⁷ Similarly, Relman et al¹¹, in a case-control study evaluating 49 pregnant women, also found gut bacterial composition and diversity remained stable during pregnancy. While the studies seem to have conflicting results, there may be modifiable factors that are not captured in these studies which may have had a significant impact on the microbiome including maternal nutrition, exercise and exposure to pets.

Clinical studies evaluating alterations of the maternal microbiome during pregnancy, based on maternal BMI have more consistent findings. Several studies have found that maternal pre-pregnancy BMI is associated with an altered microbiome, in particular demonstrating that the obese and overweight microbiome profile is unique from the microbiome of women with normal or lean BMI during pregnancy^17–20. Among these studies, a decrease in Bifidobacterium was found in obese women^{17, 20} and women who had excessive weight gain during pregnancy^{18, 20}.

One significant potential confounder for the variability in human studies is the challenge of adequately capturing nutritional intake and macronutrient composition during pregnancy. Several studies in non-gravid women and men have clearly demonstrated that diet alters the microbiome²¹ and obese and overweight individuals also have a unique microbiome²². Non-human studies more directly demonstrate that maternal diet during pregnancy alters the offspring microbiome. Gohir et al²³ exposed mice to either high fat diet or regular diet prior to mating and during pregnancy. They found that the pregnant mice in the high fat diet group had very distinct and significant differences in gut bacterial populations, with many increases in taxa belonging to the phylum Firmicutes. The study design allowed the authors to conclude that it was primarily the pre-pregnancy changes in the microbiome as a result of diet that resulted in this difference. Another murine study showed that maternal high fat diet, and not obesity, can change the microbiome of offspring, with an increase in the phylum Firmicutes³. Another elegant mouse study by Kimura et al showed that the maternal gut microbiome modulates short chain fatty acid (SCFA) production and can pass from intestine into the maternal blood stream across the placenta and into the developing fetus.⁵ They further demonstrate that a high fiber diet and maternal antibiotic exposure can alter the gut microbiota and consequently SCFAs and alter the metabolic profile in offspring. There are only a limited number of human studies evaluating the specific role of maternal diet but these generally support a role for diet in affecting both the maternal and offspring microbiome.²⁴

Maternal Oral Cavity Microbes

Studies on the maternal oral microbiome are limited. There is some evidence that poor maternal oral health is associated with increased risk of preterm birth²⁵, pre-eclampsia²⁶ and delivering small-for-gestational age infants²⁷. Up to 1 billion bacteria representing more than 500 distinct microbial species can be found in areas of dental plaque and inflammation and these may play a role in the pathogenesis.²⁸ Fujiwara et al found when comparing 132 healthy pregnant women to 51 healthy nonpregnant women that pregnancy increased the total number of live bacteria in the oral cavity with species variation during the first and second trimester.²⁹ Interestingly, they also found that Candida was more frequently found in the oral mycobiome in the third trimester of pregnancy.

Several factors have been shown to affect the oral microbiome and oral health, including nutrition, smoking, immune status and hygiene.²⁸ The possibility of a pathogenic or invasive oral microbiome penetrating the placenta and fetal tissues as a potential cause of preterm birth has been raised. Gomez-Arango et al looked at the oral, gut and placental microbiome in 37 overweight and obese pregnant women and found that the placental microbiome had signatures from both the maternal gut and oral microbiomes, but it was most similar to the pregnant oral microbiome.³⁰ More studies are needed to further identify the role of the maternal oral microbiome on outcomes in offspring.

Maternal placenta and decidual tissue colonization

Sterile Womb Controversy

The century-old paradigm that the fetus resides in a sterile milieu and that the newborn only attains its microbiota after exposure to the extra uterine environment is being challenged by compelling evidence,^{31, 32,33, 34} that may have major implications for health and disease during both fetal and subsequent life.^{35, 36} Prior to recent evidence, microbes detected in the uterine cavity were thought of as pathogenic. ³⁷ The concept that that the placenta might harbor commensal microbes was largely ignored. With the advent of the Human Microbiome Project, the development of techniques that identified microorganisms without needing to culture them altered this concept. ^38–44

One pioneering study identified bacterial DNA in what was otherwise thought to be healthy placental tissue.³⁹ If these are actually commensals, could they be involved in early development of the embryo and fetus and/ influence the individual’s postnatal health and or even transgenerational health via epigenetic mechanisms? Additional studies found “non-sterile” placentas using both microscopic⁴⁵ and DNA sequencing-based techniques.⁴⁶ Microbes found in placentas were most often found that were not associated with inflammatory responses (no white blood cells or lymphocytes or other immune cells) in their proximity.

Studies that support commensal microbes in the womb have stimulated considerable controversy and continue to be a matter of contention related to several technical experimental factors including use of appropriate positive and negative controls.³⁴,^{47, 48} Although much of the controversy focuses on non-culture based techniques, viable microbes have been cultured from human placenta, amniotic fluid, and umbilical cord blood samples and some of these concerns are being effectively debated.^{38, 49}

Microbes in babies’ first stool (meconium) as evidence for a fetal microbiome

Unlike the newborn feces expelled several days after delivery, meconium is representative of the in-utero rather than the extra uterine environment. Meconium consists largely of intestinal epithelial cells, lanugo (fine, soft hair, especially that which covers the body and limbs of a human fetus or newborn), mucus, amniotic fluid, bile and water.

Several studies in the past decades have demonstrated that meconium of newborns both at term and at preterm harbored bacteria. Most of these studies were done using DNA sequencing techniques (showing at least that microbial DNA was present)^{43, 50, 51} but some studies also used culture based techniques that found not only microbial DNA but live microbes.⁴²

Is the presence of microbes in meconium significant from a clinical standpoint?⁵² If microbes are acquired in the fetal intestine, they could play a role in development of the intestinal mucosal immune system even before the infant is passed from the womb. Although the newborn infant does not have a fully developed immune system, several components of the innate and adaptive immune systems are present at the time of and shortly after birth. Albeit they continue a maturational trajectory, but this suggests that emergence from the womb does not suddenly turn on the development of the immune system.⁵²

During normal pregnancy, the mother maintains immune tolerance to the fetus. Dysbiosis could affect this balance and lead to a response that leads to prematurely eliminating the fetus. The origin of the fetal intestinal microbes that comprise meconium is unclear, but the fetus swallows large quantities of amniotic fluid.⁵³ Under certain circumstances, the amniotic fluid may harbor microbes, and the quantity and types of microbes present correspond to the presence of inflammatory mediators as well as preterm delivery.^{54, 55} When swallowed by the fetus, it is likely that this reaches the highly immunoreactive areas of the gastrointestinal tract,⁵⁶ which responds to bacterial components, such as lipopolysaccharide (LPS), more robustly than does the mature intestine.⁵⁷ Although, spontaneous preterm delivery has been attributed to inflammation with a placental origin, it is possible that the fetal gastrointestinal tract is the source of the inflammatory response syndrome that triggers spontaneous preterm birth. ³⁶ Further studies are needed to test this hypothesis.

If there is a non-sterile womb, where do the microbes come from?

The womb is a relatively isolated structure and not readily accessible to microbes. Studies suggest that bacteria might originate as ascending microbes from the mothers’ vaginal tracts.^{9, 10, 37, 58} Others suggest they originate from the mother’s gastrointestinal tract via simple translocation between the cells that line the intestinal tract.⁵⁹ It is also possible that they are transported to the placenta and fetus via the bloodstream by specialized cell types.⁶⁰

As previously mentioned, the placenta harbors microbes that resemble those found in the mothers’ oral cavity.^{30, 39} The fact that in both of these studies intestinal microbes, including E. coli, was also represented in the placental samples is of interest, and suggests that maternal intestine is also contributing. Whether oral microbes may play a role in preterm labor remains a speculation.

Maternal bacterial products affecting fetal tissues

The maternal microbiome may also alter outcomes in offspring by modifying exposure of the fetus to microbe-derived substrates or hormones. One clinical study evaluating obese and overweight pregnant women found that fasting metabolic hormone levels differed between groups and some hormone levels correlated with alterations in the abundance of specific bacterial groups.¹⁹ Specifically, they found that adipokine levels were strongly correlated with Ruminococcaceae and Lachnospiraceae, while insulin levels positively correlated with the genus Collinsella. This suggests the potential of altered hormonal exposure to the fetus in utero. A recent murine study by Vuong et al found that the maternal microbiome was involved in early embryonic brain development, where depletion of the maternal microbiome altered over 300 genes in the embryonic brain, resulting in decreased axonal volume and persistent disrupted neurobehavioral responses in adult offspring.⁴ They elegantly demonstrated that the maternal microbiome alters the bioavailability of metabolites in the fetal brain. The potential that the maternal microbiome exerts an effect on the fetus via modulation of microbial derived metabolite exposure is intriguing and warrants further investigation.

Other Sources of Maternally Transmitted Microbes in the Newborn

Microbes acquired via vaginal delivery, mothers’ milk, and skin lead to a highly diverse ecosystem in newborn babies shortly after birth. Thus, vertical transmission of microbes from the mother occurs, even when invoking the possibility of a sterile womb.⁶¹ These microbes contain the metabolic machinery to produce a myriad of bioactive metabolites that are crucial for host development and also have potential to alter development in the lifetime of the individual and perhaps even transgenerational by epigenetic means.

Maternal probiotics as a potential modulator of the maternal microbiome

While studies are limited, a few investigators have evaluated the use of probiotics during pregnancy as a way to “improve” the maternal microbiota, with the potential for preferentially benefitting the mother and offspring. Rutten et al evaluated 341 mothers who reported taking various probiotics during pregnancy and compared with 2150 mothers who reported no use of probiotics during pregnancy. They found that during the first year of life, there was no difference in respiratory tract infections, gastrointestinal infections or constitutional eczema. Dotterud et al performed a randomized controlled trial where pregnant and lactating women received either probiotic milk or placebo and evaluated offspring until 2 years old for development of atopic dermatitis.⁶² They found that supplementation with probiotic milk at the end of pregnancy and during lactation reduced the risk of development of atopic dermatitis in offspring. Several studies in rodents have demonstrated benefit with the use of maternal probiotics in offspring including protection from development of hypertension⁶³ and cardiovascular dysfunction⁶⁴, reduction of air pollutant induced airway inflammation⁶⁵, promotion of intestinal⁶⁶ and brain development⁶⁷ and anxiolytic effects^{68, 69}.

Conclusions

The maternal microbiome is unique, with changes in each microbial niche occurring in the pregnant state. Studies to date demonstrate some evidence that the maternal microbiome plays an important role in maternal and infant health. Future research focused on the mechanisms of how the maternal microbiome exerts its impact on the fetus and offspring may lead to therapeutics aimed at modulating the microbiome for benefit to the offspring. Future interventions may include the use of probiotics, diet modification and nutrient supplementation for benefit of both the mother and offspring.