The Developing Microbiome of the Fetus and Neonate: A Multiomic Approach

Introduction

In the past two decades, DNA-based technologies have identified a myriad of difficult-to-culture microbes in the human body, including the gastrointestinal tract, skin, vaginal tract, urethra, and reproductive tract. In addition to the DNA-based technologies, newly developed bioinformatics are being developed that integrate microbial metagenomes, genomics, transcriptomes, proteomes, and metabolomes (or multi-omics) into system-based schema that more clearly illustrate the pathophysiology of diseases. Problems seen during pregnancy and the neonatal period beg for application of these newly developed techniques, which will enable better understanding of their pathophysiology in order to improv prevention, diagnosis, and treatment.

In this chapter, we will summarize some of the most recent studies pertaining to in utero microbial environment and how “omic” perturbations may result in preterm birth. The effects of mode of delivery on the developing microbiota-host-immune system interactions, and the effects of environmental factors such as antibiotic usage and diet will also be reviewed. In addition to the consequences of these early perturbations on health and disease in the individual, we will also speculate on transgenerational effects.

Is the Uterus “Sterile”?

Microbial exposure in utero can be at different levels. One involves direct exposure via live reproducing microbes that reside in niches such as the placenta, amniotic fluid, or fetal gastrointestinal tract. The other is indirect exposure to microbial components that are passed via the bloodstream to the placenta and fetus. A combination of these is also possible.

The widely held concept that the in utero environment under normal conditions is “sterile” continues to be a matter of controversy. It is most commonly thought that the presence of microbes in the uterine cavity implies an infectious process, but there is considerable information that this may not be the case. In fact, a nonsterile environment is seen in numerous nonhuman animals and may be quite beneficial in humans as well. When considered from the perspective of immunologic regulation and tolerance, exposure to certain microbes may play a vital role in the maintenance of pregnancy and has implications for the early developmental processes that lead to the hosts’ subsequent ability to counteract infections, experience normal brain development, or develop autoimmunity.

Despite the presence of various barriers in the maternal-fetal interface, it is difficult to exclude the possibility of placental and/or fetal exposure to microbial components that originate from the maternal vagina, gastrointestinal tract, oral cavity, or skin. However, more recent data support that microbes likely harbor and colonize the placenta, amniotic fluid, and fetal gastrointestinal tract. ^{, , ,} This may have major implications for the production of various metabolites with high physiologic activity. These include metabolites such as serotonin, very important in neurodevelopment, and short-chain fatty acids, which are very important in terms of immune development and epigenetic mechanisms. ^{, , ,} Other studies suggest that the microbiology may be important for the development of the heart.

The pregnant woman harbors microbes in her gastrointestinal tract, mouth, vaginal tract, skin, and other niches. Studies have shown that the vaginal tract harbors different sets of microbes depending on location in the vagina (upper vs. lower) and stage of gestation. ^, Previous studies have suggested that ascending microbes may translocate through the choriodecidual membrane into the amniotic fluid. The extent to which this occurs under healthy conditions is unclear. However, studies evaluating amniotic fluid microbes using both culture- and nonculture-based studies support their presence, with the greatest quantity found in preterm versus term deliveries. Studies of the placental microbiome have also suggested the presence of microbes that vary in their taxonomy depending on the stage of pregnancy.

Even if one invokes the possibility of a sterile womb, microbes or microbial components transferred from maternal sites such as the intestine likely signal and interact with the fetal immune system. Studies have demonstrated that inoculation of germ-free pregnant mice with a single microbe result in major postnatal modifications of the innate immune system, with enhanced responses in mice whose mothers were exposed to the microbes during pregnancy. Thus, the environment in which the mother resides may be very important in terms of her microbial composition, the interaction between her microbes, their components and/or metabolites, and the developing fetus. Maternal diet, and medications such as antibiotics, antacids, stress, and other environmental factors, likely play major roles in these microbial host interactions.

Although the most recent data suggest that the fetal environment is not sterile, challenges to this concept are being made because of the possibility that some studies had inadequate controls, where a “kitome” or environmental contaminant is detected. ^{, ,} In other words, microbes are present in the ambient environment or in the kits that are utilized to evaluate the microbiota in certain environments. Therefore, when low biomass samples are analyzed (as present in placenta and amniotic fluid), it is possible that the microbes detected in these niches may actually be a part of the background (“blank”) environment rather than resident microbes in such an environment. Although it appears from a large number of recent studies that there actually is an in utero microbiome, future studies with better quality control techniques will be necessary. ^,

The Vaginal Microbiome: Implications for Mode Of Delivery

There is a body of literature supporting a lack of microbial diversity, with a predominance of Lactobacillus crispatus , in the healthy vaginal tract in mothers who deliver their babies at term. ^, This is reflected by a study of term and preterm infants’ first gastric aspirate, wherein those born at term exhibited a preponderance of L. crispatus, and those born preterm had a wider variety of microbes with a relative scarcity of L. crispatus. This may have implications in terms of the pathophysiology of some forms of spontaneous preterm birth.

Could microbes acquired by the newborn during vaginal delivery differ from those acquired during cesarean section delivery, and what might be the implications of these differences? Since the beginning of the early 20th century, cesarean deliveries have become more common. In several areas of the world, they are the most common form of delivery. Although there are certainly indications for many of these cesarean sections, the high variability between countries and hospitals suggests that some cesarean sections are not medically indicated. With this in mind, the question has been raised about whether the high incidence of cesarean sections may be a trigger that increases the risk for obesity, autoimmune disease, infectious diseases, and allergic and atopic diseases. Epidemiologic studies support that cesarean section deliveries are associated with a greater risk for these problems. Several studies support that differences in the acquisition of microbes do occur depending on the mode of delivery. ^, Whether this difference is directly secondary to the mode of delivery or whether other mitigating factors are involved—including high antibiotic use in cesarean-delivered babies, the longer duration of hospital stays for these babies, and longer time to enterally feed cesarean-delivered babies—has yet to be determined. ^, Whether major differences actually occur depending on mode of delivery is not supported by at least one study, where no significant differences were seen depending on mode of delivery at 6 weeks after birth ( Fig. 87.1 ). ^, Sources of the neonatal microbiome likely originate from numerous sources. Whether the difference in microbial colonization in cesarean section versus vaginally delivered infants has a role in health and disease is still not known, but it is potentially important from the perspective of therapeutics.

The notion that the differences in acquisition of vaginal microbes during vaginal delivery may play a role in prevention of certain diseases has led to the practice of “seeding” of the vaginal microbes into the baby’s mouth using a swabbing technique, whereby the vaginal microbes of the mother are put onto a gauze pad and then swapped into the cesarean section–delivered newborn’s mouth. A small preliminary study supports that vaginal microbes can be transferred using this technique. Whether this has implications in terms of health and disease remains speculative, and there are ongoing studies addressing this question. However, the safety of this procedure has been questioned, and recommendations against it have been made by obstetrical societies because of the possibility of seeding the newborn with potentially pathogenic microbes.