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The Microbiome and Development: A Mother’s Perspective Kathleen M. Antony, MD1
1 Division of Maternal-Fetal Medicine, Department of Obstetrics and
Gynecology, Baylor College of Medicine 2 Department of Molecular and Human Genetics, Bioinformatics Research Lab, Baylor College of Medicine, Houston, Texas 3 Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas Semin Reprod Med 2014;32:14–22
Abstract
Keywords
► microbiome ► pregnancy ► Human Microbiome Project (HMP) consortium
Jun Ma, PhD1,2
Kjersti M. Aagaard, MD, PhD1,2,3
Address for correspondence Kjersti M. Aagaard, MD, PhD, Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine and Department of Molecular and Cell Biology, Department of Molecular Physiology and Biophysics, National School for Tropical Medicine, Center for Metagenomics and Microbiome Research, Center for Reproductive Medicine, John M. Eisenberg Center for Health Outcomes Research, Bionformatics Research Lab at the HGSC, Translational Biology and Molecular Medicine and Co-Director, Baylor College of Medicine MSTP program, 1 Baylor Plaza, Houston, TX 77030 (e-mail: [email protected]).
Dysbiosis of the microbiome has been associated with type II diabetes mellitus, obesity, inflammatory bowel disorders, and colorectal cancer, and recently, the Human Microbiome Project Consortium has helped to define a healthy microbiome. Now research has begun to investigate how the microbiome is established, and in this article, we will discuss the maternal influences on the establishment of the microbiome. The inoculation of an individual’s microbiome is highly dependent on the maternal microbiome, and changes occur in the maternal microbiome during pregnancy that may help to shape the neonatal microbiome. Further, we consider how mode of delivery may shape the developing microbiome of a neonate, and we end by discussing how the microbiome may impact preterm birth and the possibility of bacterial colonization of the placenta. Although the current literature demonstrates that the transformation of the maternal microbiome during pregnancy effects the establishment of the neonatal microbiome, further research is needed to explore how the microbiome shapes our metabolism and developing immune system.
It is established that humans have a unique and varied relationship with the microbes in our environment. We form symbiotic relationships with these microbes in the form of mutualistic (both organisms benefit), commensal (one organism benefits while the other is left unharmed), or parasitic (one organism benefits while the other is harmed) relationships. Recently, these microbes have been the focus of many studies. In particular, dysbiosis of the microbiome has been associated with type 2 diabetes mellitus, obesity, inflammatory bowel diseases, and colorectal cancer.1–18 Therefore, the Human Microbiome Project (HMP) Consortium investigated what constitutes a healthy microbiome.19–24 Although these studies uncovered the profile of a healthy microbiome, they resulted in questioning how the human microbiome is established and how the human microbiome is influenced. Naturally, our laboratory and others have turned
Issue Theme The Microbiome and Reproduction; Guest Editors, James H. Segars, MD, and Kjersti M. Aagaard, MD, PhD
to our first exposure to the outside world, our mothers. Recent work has demonstrated that women’s microbiomes undergo changes during pregnancy25,26 and that these changes in turn influence the microbiomes of offspring.26,27 The findings of the HMP Consortium and recent investigations of maternal and neonatal microbiomes of various body sites are summarized in ►Table 1. We will discuss in this article these changes and how the maternal microbiome can have long-lasting effects on children’s microbiome and health.
The Microbiome during Pregnancy Over the course of a normal, healthy pregnancy, the body undergoes profound anatomic, physiologic, and biochemical changes which result from the influences of hormonal and
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DOI http://dx.doi.org/ 10.1055/s-0033-1361818. ISSN 1526-8004.
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Amanda L. Prince, PhD1
Seminars in Reproductive Medicine
Amniotic fluid, placenta, maternal endocervix
Amniotic fluid
Amniotic fluid, vagina, and dental plaque
Douvier et al/199957
Han et al/200658
Bearfield et al/200259
Bacterial culture
Bacterial culture and PCR
16S rRNA
Bacterial culture
qPCR
16S rRNA
DGGE and TGGE
Murine study
Cross-sectional/gravidae
Cross-sectional/gravidae
Case study/gravidae
Cross-sectional/infants
Cross-Sectional/infants
Cross-sectional/infants
Case control, longitudinal/infants
Cross-sectional/gravidae and newborns
Longitudinal/gravidae in first and third trimesters
Longitudinal, stool transplant into mice/gravidae in first and third trimesters
Cross-sectional/gravidae
Longitudinal/nongravidae
Study design/population
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F. nucleatum isolated from preterm births colonized the placenta of pregnant mice
Few gravidae tested positive for Streptococcus spp. or F. nucleatum in the vagina, but these bacterium where mostly found in the oral cavity of gravidae testing positive in the amniotic fluid
Amniotic fluid was colonized with oral bacteria of the Bergeyella species
Placenta colonized with the oral bacteria, Capnocytophaga sputigena
Infants are colonized differentially depending on mode of delivery
Infants born via an emergency cesarean had the highest bacterial diversity while infants born via an elective cesarean had to the lowest bacterial diversity
Infants born vaginally have a higher bacterial diversity than infants born via cesarean
Probiotics can be vertically transmitted to infants
Vaginally delivered infants acquired bacterial communities that resembled their own mother’s microbiota. Cesarean delivered infants’ microbiota reflected skin communities, but not necessarily their mothers’
Microbiome composition differed between normal weight and overweight gravidae and differed between gravidae with normal weight gain and excessive weight gain
Stool in the third trimester of pregnancy showed signs of inflammation and energy loss. When transferred into mice, third trimester stool induced greater adiposity and insulin insensitivity than first trimester stool
Characterized healthy gravidae
Characterized healthy population
Findings
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Placenta, liver, spleen, amniotic fluid, fetus
Stool
Penders et al/200638
Han et al/200460
Stool
Stool
Azad et al/201337
Biasucci et al/2008
16S rRNA
Stool
Schultz et al/200427
36
16S rRNA
Maternal: skin, oral mucosa, vagina Neonatal: skin, oral mucosa, nasopharyngeal aspirate, and meconium
Dominguez-Bello et al/201035
16S rRNA
16S rRNA
16S rRNA and qPCR
Stool
Vaginal introitus, posterior fornix, and midvagina
16S rRNA and WGS
Technique(s)
Collado et al/200832
Koren et al/201226
Aagaard et al/2012
Skin, nares, oral, vagina
NIH HMP Consortium/201220
25
Site
Authors/year
Table 1 Microbiome literature reviewed
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physical fluctuations. These alterations affect every organ of the body and occur in concert with changes in the microbiome. To date, only two body sites have been specifically examined for pregnancy-related changes in the microbiome: the vagina and the gut25,26 (►Fig. 1). During pregnancy, increased vascularity and hyperemia develop in the skin of the vulva and the mucosa of the vagina. The vaginal mucosa increases in thickness while the underlying smooth muscle cells hypertrophy and the connective tissue relaxes. There is a concomitant increase in cervical secretions with a decrease in the vaginal pH, which has long been observed to be a result of the increased production of lactic acid by Lactobacillus acidophilus. However, until recently, the dynamic nature of the vaginal microbiome during pregnancy has not otherwise been characterized. Robust knowledge of the microbiota present may help explain differences in vaginal cytokine levels, variations in pH, and differential susceptibility to infections, such as human immunodeficiency virus (HIV). Hence, we recently cataloged the “normal” microbiota signature during pregnancy across gestational age strata using cultivation-independent, molecular-phylogenetic techniques, namely, 16S rRNA gene-based sequencing.25 DNA was isolated from the vagina (introitus, midvagina, and posterior fornix) and the V5V3 region of 16S rRNA genes were sequenced (454FLX titanium platform).25 Sixty-eight samples from 24 healthy gravidae at 18 to 40 weeks gestation were systematically compared with 301
Figure 1 The maternal microbiome. During pregnancy, changes in the gut and vaginal microbiomes have been documented (signified in black). 25,26 However, recent studies suggest that the placental may be inoculated by the oral microbiome (signified in red). In addition, since the gut and vaginal microbiomes undergo changes during pregnancy, we may also discover that changes occur in the microbiomes of the nasal cavity, the oral cavity, the skin, and the placenta. These changes in the maternal microbiome during pregnancy may be critical in influencing the developing neonatal microbiome.
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Cross-sectional/gravidae Histopathology Placenta Stout et al/201365
Histopathology, FISH Steel et al/200564
Fetal membranes
Seminars in Reproductive Medicine
Abbreviations: DGGE, denaturing gradient gel electrophoresis; FISH, fluorescence in situ hybridization; PCR, polymerase chain reaction; TGGE, temperature gradient gel electrophoresis; WGS, whole-genome shotgun.
Intracellular bacteria were found in the basal plate of the placenta regardless of chorioamnionitis diagnosis
Bacteria was detected in the fetal membranes at all stages of pregnancy Cross-sectional/gravidae
Oral bacterium from human is able to colonize the murine placenta 16S rRNA Placenta Fardini et al/2010
Authors/year
61
Technique(s)
Murine study
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Site
Study design/population
Findings
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Table 1 (Continued)
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nonpregnant controls.25 When compared with the nonpregnant vaginal microbiota, the vaginal community is uniquely structured during pregnancy, and the changes could not be attributed to alterations in body mass index (BMI), race/ ethnicity, or other clinical confounders.25 Our group also found that the vaginal microbial community differed by gestational age and proximity to the cervix with the microbial community structure resembling that of the nonpregnant community in the latter weeks of gestation.25 Further longitudinal studies in pregnancy to examine individuals’ microbiome at multiple vaginal sites throughout pregnancy are needed to more robustly ascertain the dynamic nature of the vaginal microbiome. At a genus and species level, pregnancy was associated with an overall decrease in species diversity and in richness marked by the dominance of Lactobacillus, Clostridiales, Bacteriodales, and Actinomycetales.25 The specific species that were discriminated or enriched in pregnancy, in the context of diminished community richness and diversity, were able to be detected using robust analytic methods. More specifically, there was enrichment of Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus johnsonii.25 The enrichment of these species is of probable biologic significance. For example, L. jensenii anaerobically metabolizes glycogen, which is increased with rising estrogen levels, thereby contributing to the acidic vaginal environment. In addition, L. jensenii may have surface-associated proteins that inhibit sexually transmitted infections, including infection by Neisseria gonorrhea.28 Both L. jensenii and L. crispatus are strong hydrogen peroxide producers and have been hypothesized to protect against bacterial vaginosis, which has been posited as a risk factor for preterm birth and HIV infection.29,30 L. johnsonii produces Lactacin F, a bacteriocin, which can kill other Lactobacillus species (spp.) as well as Enterococcus spp. in the gastrointestinal (GI) tract.31 Its predominance in the vagina in pregnancy may play a role in preserving the integrity of the community to reduce the risk of ascending infection or preterm birth. Alternatively, L. johnsonii may also be important for establishing the neonatal upper GI microbiota, which may help infants digest milk. Pregnancy is also characterized by changes in the gut microbiota. Bacterial load is reported to increase over the course of gestation, and the composition also changes with pregnancy progression.26,32 Using limited 16S rRNA and quantitative polymerase chain reaction (qPCR), a study by Collado et al found significant differences in microbiota composition according to BMI states and gestational weight gain.32 Specifically, they found significantly higher numbers of Bacteroides and Staphylococcus aureus in the overweight state with lower numbers of Clostridium histolyticum in the first trimester.32 Normal versus excessive weight gain in pregnancy also exhibited differential microbiota communities with each kilogram of weight gain associated with an increase in Bacteroides counts by 0.006 log units.32 In addition, excessive weight gain was negatively associated with Bifidobacterium genus numbers.32 The presence of Bifidobacterium correlates positively with normalization of inflammatory status and improves glucose tolerance,4 which may play
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an important role in the newborn gut. Using self-collected specimens in the first trimester, third trimester, and postpartum, Koren et al examined changes in the gut microbiome during pregnancy and found significant remodeling over the course of pregnancy.26 They found that phylogenetic diversity within an individual (α-diversity) decreased with advancing gestation, and that this occurred regardless of BMI and diabetes status.26 They also analyzed between-sample variation (β-diversity) analysis and found the β-diversity in the first trimester to be similar to nonpregnant controls, whereas the β-diversity in the third trimester was much higher.26 Further, women with above-average BMI or diabetes did not drive this change.26 Along with this overall increase in diversity between mothers, there was an overall increase in Proteobacteria and a decrease in Faecalibacterium.26 Abundance of Proteobacteria is often associated with inflammatory conditions.33 Faecalibacterium is a butyrate producer with anti-inflammatory effects and is depleted in inflammatory bowel disease.34 In sum, the stool in the third trimester of pregnancy resembles stool from inflammatory disease states. Koren et al then took third trimester stool and transplanted it into germ-free mice to see if it would induce greater adiposity and inflammation than first trimester stool.26 They found that innoculation of germ-free mice with third trimester stool did induce higher levels of inflammation and higher levels of adiposity than recipients of first trimester stool.26 There was also a statistically significant increase in blood glucose levels following an oral glucose tolerance test among third trimester stool recipients.26 Thus, pregnancy is associated with changes in the gut microbiome that resemble disease-associated dysbiosis in nonpregnant individuals; however, these changes may promote energy storage and fetal growth in pregnancy. Further, these microbial changes may be the result of alterations in the immune system at the intestinal mucosal surface, hormonal fluctuations, or factors not yet appreciated. In addition, transformations in the microbiome that occur during pregnancy may be a combination of many factors, but the studies described above demonstrate that the host is able to manipulate the gut microbiota to promote these changes, presumably for a favorable host outcome.
Establishment of the Microbiome The changes described above in the vaginal and enteric microbiomes during pregnancy indicate that these alterations may be important for influencing the establishment of the neonatal microbiome. Previously, Schultz et al determined that the probiotic Lactobacillus rhamnosus strain GG could be vertically transmitted to infants via a vaginal birth (4/4) and a cesarean (1/2) birth.27 Accordingly, this strain was absent in the stool from infants born to mothers not taking this probiotic.27 Although this study included a limited number of gravidae, the results suggested that mode of delivery may influence the vertical transmission of the microbiome from mother to infant. As the delivery of infants via cesarean section continues to rise, investigators have started to study how the microbiome of infants is influenced by the mode of delivery. When examining the microbiome of Seminars in Reproductive Medicine
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Figure 2 Mode of delivery may determine microbial diversity in offspring. Recent studies have shown the mode of delivery influences the diversity of the neonatal microbiome. (A) Infants with the most diverse microbiome are delivered by emergency cesarean while infants born vaginally or via a scheduled cesarean have the least microbial diversity. (B) A representative principle components analysis (PCoA) plot that displays how clustering of infants born via a scheduled cesarean (orange), vaginally (blue), or via an emergency cesarean (purple) may look if studied concomitantly in a next-gen sequencing study.
neonates, Dominguez-Bello et al observed that infants born vaginally had a microbiome resembling the maternal vagina while infants born via cesarean section had a microbiome resembling maternal skin.35 Further, the diversity of bacterial flora is diminished in infants born via cesarean when compared with infants born vaginally.36,37 This loss of diversity is accompanied by an increase in Clostridium difficile and a decrease in Bifidobacterium spp., Lactobacillus spp., and Bacteroides spp., particularly B. fragilis, in neonates born via cesarean delivery when compared with vaginally born infants.37–39 A Canadian group took these studies further and examined how the microbiomes of neonates born vaginally, by elective cesareans, or by emergency cesareans compared. As seen with the studies mentioned previously, this group saw the lowest bacterial diversity in infants born by an elective cesarean delivery; however, infants born by an emergency cesarean delivery had the greatest bacterial diversity37 (►Fig. 2). This may be attributed by the exposure of these neonates to many aspects of their mothers’ microbiome during delivery. The inoculation of an individuals’ microbiota due to mode of delivery may have a long-lasting impact on an individuals’ health. Previously, Schultz et al demonstrated that probiotics taken by mothers could be found in the stool of infants born vaginally for up to 24 months postpartum.27 Thus, the vertical transmission of the microbiome from mother to child can be long lasting. Further, a Finnish study demonstrated that children born via cesarean had a significantly higher incidence of allergy and asthma when compared with children born vaginally.40 Another study in The Netherlands found that infants with a higher prevalence of C. difficile in their stool had an increased risk of asthma, eczema, and food allergens although they did not see any significant associations with mode of delivery.41 Thus, the inoculation of neonatal microbiota may help shape a developing immune system, thereby influencing the occurrence of atopic disease. Seminars in Reproductive Medicine
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Studies have shown differences in the immune system composition of infants delivered vaginally or by cesarean. Interestingly, infants born vaginally have a higher number of neutrophils and a lower number of lymphocytes when compared with infants delivered by cesarean.42 These differences may be due to the rupture of membranes that occur during a vaginal birth and may influence the establishment of the microbiome. However, when examining the associations between the microbiome and the immune system, we begin to have a chicken-and-egg scenario. For example, the influence of the microbiome in the developing immune system has been tested in a mouse model. Here, mice 3 weeks postpartum were given kanamycin to eliminate gram-positive bacteria from the enteric microbiome.43 The authors found that these mice had increased serum levels of immunoglobulin E (IgE) and IgG1 while decreasing the serum levels of IgG2a and the cellularity of the spleen and Peyer Patches.43 In a separate study, this group went on to replenish the microbiome of kanamycin-treated mice with either Lactobacillus acidophilus, Enterococcus faecalis, or Bacteroides vulgatus probiotics.44 Although the cellularity of the spleen and Peyer Patches was restored, the serum antibody levels were rescued to varying degrees by the probiotic bacteria.44 Interestingly, none of the probiotics provided reduced IgG1 serum levels in kanamycintreated mice.44 Thus, the colonization of the microbiome early in life appears to have long-lasting effects on the immune system. If the microbiota can affect the immune system in this manner, it will be interesting to determine in the future how the microbiome influences other physiological processes. In addition to mode of delivery, breastfeeding or formulafeeding has been shown to greatly impact the microbiome. Similar to mode of delivery, changes in C. difficile and Bacteroides have been documented in formula-fed versus breastfed infants.37 Thus, it is not only the initial inoculation of the infant with the maternal flora that influences an offspring’s
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microbiome but also this continual inoculation from contact with our mothers. Although we mentioned the impact of breastfeeding versus formula-feeding on the microbiome briefly here, this subject will be described in detail in another article of this issue.45
The Microbiome and Preterm Birth In the developed world, preterm birth accounts for a significant proportion of infant morbidity and mortality,46 yet, the underlying etiology of preterm birth is not fully understood. Intrauterine infection is often seen in association with preterm birth, and it is thought that an ascending infection from the vagina to the placenta causes the inflammation and preterm premature rupture of membranes (PPROM) that results in a preterm birth.47,48 For instance, the human placenta expresses the Toll-like receptors (TLRs) -2, -4, and -10, and caspase activity was found to increase in the presence of TLR-2 and -10 agonists.49,50 In addition, endocervical and ectocervical cell lines were found to produce the inflammatory cytokines interleukin (IL)-6 and IL-8 in response to lipopolysaccharide (LPS).51 Cytokine production was reduced with the use of dexamethasone, an anti-inflammatory drug.51 In addition, a study by Han et al utilizing DNA amplification techniques to detect 16S rRNA found that almost half of women presenting with symptoms of preterm labor tested positive for bacterium while control subjects did not.52 Further, patients who demonstrated symptoms of preterm birth also had significantly reduced levels of glucose with correspondingly increased levels of IL-6 when compared with control subjects.52 However, women undergoing preterm birth with positive bacterial cultures of the amniotic fluid were found to have significantly increased levels of the immunosuppressive cytokine, IL-10,53 which may be due to the maternal immune response shifting to a Th2 phenotype during pregnancy.54–56 Thus, although the maternal immune system shifts during pregnancy, the placenta retains the ability to respond to pathogens. Although the cause of placental infection has focused on an ascending infection,47,48 this dogma has been recently challenged. A case study involving a patient undergoing preterm labor found that the amniotic fluid was colonized with Capnocytophaga sputigena, a bacterium normally found in the oral cavity.57 In addition, upon investigating the colonization of amniotic fluid with bacterium, Han et al discovered high levels of Bergeyella spp. in the amniotic fluid in a patient undergoing preterm labor; additionally, the placenta showed clinical signs of infection.58 However, this bacterium was not found to be present in the vagina but was detected in the subgingival plaque.58 In an expanded study, Bearfield et al examined the presence of Fusobacterium nucleatum and Streptococcus spp. in the buccal gingival, vagina, and amniotic fluid of pregnant women.59 Although many of the gravidae had bacterial colonization of the amniotic fluid, these two strains of bacteria were primarily found in the oral cavity and not the vagina.59 These studies demonstrated that an ascending infection may not be the main culprit in placental infections.
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Further, recent work in mice suggests that oral bacterium colonizing the placenta hematogenously is the root cause of infections associated with preterm birth.60 For instance, F. nucleatum, which is normally found in the oral cavity, has been cultivated from women who underwent preterm birth.60 Han et al went on to intravenously inoculate pregnant mice with the F. nucleatum isolated and found that placentas of these mice were colonized with F. nucleatum.60 Although pregnant mice inoculated with F. nucleatum did undergo a term birth, this treatment resulted in a high incidence of stillbirth.60 Interestingly, F. nucleatum failed to colonize in other organs of the mouse (i.e., spleen or liver),60 which may be due to the protection of the maternal immune system to prevent rejection of the fetus. Although F. nucleatum was able to infect the murine placenta, the ability of this bacterium to infect the human fetus was recently studied. In this study, the DNA encoding bacterial 16S rRNA was amplified and sequenced from the amniotic fluid of patients.52 In addition to the presence of F. nucleatum, Sneathina sanguinegens, Streptococcus agalactiae, Bacteroides ureolyticus, Shigella spp., Ureaplasma parvum, Leptotrichia amionii, Clostridiales order, Peptostreptococcus spp., Citrobacter koseri, Bergeyella spp., Bacteroides fragilis, and Prevotella bivia were detected.52 Therefore, F. nucleatum is not the only bacterium able to colonize the placenta. In an independent study, Fardini et al pooled saliva samples from multiple donors and inoculated pregnant mice I.V. with these samples.61 Within 24 hours, the placenta was colonized with various bacteria.61 Altogether, these data demonstrate that microbial infections of the placenta that induce preterm birth may originate from the oral cavity. These studies provide evidence to promote further study of the potential for a placental microbiome.
The Placental Microbiome The placenta serves as a metabolic and endocrine organ with diverse functions and roles in health and disease, and maintenance of its integrity is paramount to fetal growth, development, and survival.62 Conventional wisdom is that the placenta is sterile, and therefore, the majority of intrauterine infections originate in the lower genital tract and ascend into this “sterile” placental environment.47,63 However, as described above, bacteria have been detected in the amniotic fluid of gravidae that underwent a preterm birth.52,57–59 In addition, animal studies have demonstrated that I.V. infections of oral bacteria can result in the bacterial colonization of the placenta.60,61 In these scenarios, it was hypothesized that the placenta was a sterile environment with the exception of infections that resulted in preterm birth. Yet, bacteria have been detected deep within human fetal membranes by in situ hybridization. For example, a study by Steel et al discovered that placental tissue, derived from an elective term cesarean section, had organisms present in 70% of membranes, which implies that the presence of bacteria does not induce preterm labor per se.64 Thus, this study demonstrates that there may be a more complicated relationship between bacteria and the host’s inflammatory response.64 Similarly, a recent crossSeminars in Reproductive Medicine
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sectional study of 195 patients demonstrated gram-positive and -negative intracellular occult bacteria harbored in the basal plate of 27% of placenta.65 Importantly, while these bacteria were observed with a high prevalence among preterm deliveries < 28 weeks, there was no statistically significant difference between the presence of bacteria among subjects with or without antepartum sexually transmitted infections, urinary tract infections, Group B streptococcus colonization, or chorioamnionitis.65 As the presence of bacteria are not associated with genitourinary infections or colonizations, there must be an alternate source; indeed, the majority of DNA-based detected taxa in the placenta are not found in the urogenital tract but rather are found in the oral cavity.58,60 For example, F. nucleatum is a gramnegative anaerobe ubiquitous to the oral cavity, and it is associated with periodontal disease. It has also been isolated from amniotic fluid in women with preterm labor and with PPROM.66,67 And as described above, studies have demonstrated that this bacterium can have devastating effects on neonatal survival. An interesting idea to postulate with recent studies involving term gravidae is the placenta harbors its own, unique microbiome. If this hypothesis were true, then understanding how the placental microbiome differs between preterm and term gravidae could be of vital importance in preterm birth treatment (►Fig. 1). However, further characterization of the placental microbiome in infected, noninfected, term, and preterm human pregnancies via 16S rRNA sequencing and whole-genome shotgun (WGS) sequencing is desperately needed. Such characterization can then be compared with human body sites as described by the HMP Consortium to ascertain whether the same principles are relevant in humans.20
Conclusions and Thoughts In this review, we have discussed the changes in the maternal microbiome during pregnancy, how the maternal microbiome may influence the establishment of the neonatal microbiome, and the role of the host and placental microbiome in preterm birth. As new discoveries are made, there is much to learn about the role of the microbiome in health and disease. For instance, many of the studies described throughout this review exclusively use 16S rRNA sequencing, and many previous studies cultured bacterium before amplification and/or sequencing. The major caveat to these types of studies is that only the most prevalent bacterium may be detected. However, the onset of WGS sequencing is allowing for a greater sensitivity in detecting microbes and may help further our knowledge in not only the bacteria living within us but also other microbes, such as viruses or fungi. In light of these new methods of examining the microbiome, longitudinal studies of gravidae are more feasible. Although we know that the vaginal and enteric microbiomes undergo changes during pregnancy,25,26 further understanding the maternal micorbiome during pregnancy may help prevent preterm births. For instance, with the developing microbiome field, we can further examine the role of periodontal disease and oral infection in preterm birth. Although Seminars in Reproductive Medicine
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previous studies have shown that oral bacteria can be detected in the amniotic fluid of some preterm patients,52,57–59 there are a significant number of preterm births where a bacterial infection is not found utilizing 16S rRNA sequencing.52,59 Thus, with the availability of WGS, we may be able to detect bacterial infections in all preterm pregnancies. In addition, nonbacterial microbes, such as viruses and fungi, may be involved in the onset of preterm birth. Hopefully, these studies will help us in treating and in preventing preterm birth worldwide. In addition to further examining preterm birth, studies of our microbiome naturally result in questions pondering the development of our immune system. In separate studies, we see that mode of delivery can be correlated to atopic diseases,40,41 and recent studies have actually shown that the neonatal microbiome can differ based on mode of delivery.35,37 Thus, with immune cell types, such as mucosalassociated invariant T cells, depending on microbes for their maintenance and/or development,68 we begin to wonder how these early changes in the neonatal microbiome may influence these cells. Alternatively, in a chicken-and-egg scenario, the early immune system may influence the development of the microbiome, and there are studies that describe the differing constituents of the immune system based on mode of delivery.42 With the high prevalence of atopic disease in the Western world, understanding the interactions of the microbiome and the immune system could be critical for treatment. Therefore, these inquiries are beginning to take center stage in both immunology and microbiome research. To answer the question proposed here involving preterm births, the microbiome, and the developing immune system, prospective longitudinal studies with serial sampling of mothers and newborns throughout pregnancy, parturition, and postnatally are needed to delineate the cause-and-effect of alterations in body-site microbiota. In addition to further examining the role of the microbiome in preterm birth, associations with immune status and hormonal changes are also needed to further examine how the host promotes or fails to promote a microbiome conducive to meet the host’s needs, both in terms of fostering a continuing pregnancy or meeting the metabolic needs of a growing fetus. However, the studies presented here demonstrate that the maternal microbiome can have dramatic influences on the developing fetus and the establishment of the neonatal microbiome.
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infantile colonization. J Pediatr Gastroenterol Nutr 2004;38(3): 293–297 Spurbeck RR, Arvidson CG. Lactobacillus jensenii surface-associated proteins inhibit Neisseria gonorrhoeae adherence to epithelial cells. Infect Immun 2010;78(7):3103–3111 Hillier SL, Krohn MA, Klebanoff SJ, Eschenbach DA. The relationship of hydrogen peroxide-producing lactobacilli to bacterial vaginosis and genital microflora in pregnant women. Obstet Gynecol 1992;79(3):369–373 Atashili J, Poole C, Ndumbe PM, Adimora AA, Smith JS. Bacterial vaginosis and HIV acquisition: a meta-analysis of published studies. AIDS 2008;22(12):1493–1501 Pridmore RD, Berger B, Desiere F, et al. The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci U S A 2004;101(8):2512–2517 Collado MC, Isolauri E, Laitinen K, Salminen S. Distinct composition of gut microbiota during pregnancy in overweight and normal-weight women. Am J Clin Nutr 2008;88(4):894–899 Mukhopadhya I, Hansen R, El-Omar EM, Hold GL. IBD-what role do Proteobacteria play? Nat Rev Gastroenterol Hepatol 2012;9(4): 219–230 Sokol H, Pigneur B, Watterlot L, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A 2008;105(43):16731–16736 Dominguez-Bello MG, Costello EK, Contreras M, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A 2010;107(26):11971–11975 Biasucci G, Benenati B, Morelli L, Bessi E, Boehm G. Cesarean delivery may affect the early biodiversity of intestinal bacteria. J Nutr 2008;138(9):1796S–1800S Azad MB, Konya T, Maughan H, et al; CHILD Study Investigators. Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months. CMAJ 2013;185(5):385–394 Penders J, Thijs C, Vink C, et al. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 2006; 118(2):511–521 Grönlund MMM, Salminen SS, Mykkänen HH, Kero PP, Lehtonen OPO. Development of intestinal bacterial enzymes in infants— relationship to mode of delivery and type of feeding. APMIS 1999; 107(7):655–660 Kero JJ, Gissler MM, Grönlund M-MM, et al. Mode of delivery and asthma—is there a connection? Pediatr Res 2002;52(1):6–11 van Nimwegen FA, Penders J, Stobberingh EE, et al. Mode and place of delivery, gastrointestinal microbiota, and their influence on asthma and atopy. J Allergy Clin Immunol 2011;128(5):948–955, e1–e3 Grönlund MM, Nuutila J, Pelto L, et al. Mode of delivery directs the phagocyte functions of infants for the first 6 months of life. Clin Exp Immunol 1999;116(3):521–526 Oyama N, Sudo N, Sogawa H, Kubo C. Antibiotic use during infancy promotes a shift in the T(H)1/T(H)2 balance toward T(H)2-dominant immunity in mice. J Allergy Clin Immunol 2001;107(1): 153–159 Sudo N, Yu XN, Aiba Y, et al. An oral introduction of intestinal bacteria prevents the development of a long-term Th2-skewed immunological memory induced by neonatal antibiotic treatment in mice. Clin Exp Allergy 2002;32(7):1112–1116 La Tuga MS, Stuebe A, Seed P. A review of the source and function of microbiota in breast milk. Semin Reprod Med 2014;35(1):68–73 Simmons LE, Rubens CE, Darmstadt GL, Gravett MG. Preventing preterm birth and neonatal mortality: exploring the epidemiology, causes, and interventions. Semin Perinatol 2010;34(6):408–415 Goldenberg RL, Hauth JC, Andrews WW. Intrauterine infection and preterm delivery. N Engl J Med 2000;342(20):1500–1507 Gonçalves LF, Chaiworapongsa T, Romero R. Intrauterine infection and prematurity. Ment Retard Dev Disabil Res Rev 2002;8(1):3–13 Seminars in Reproductive Medicine
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plays a role in mediating bacterial peptidoglycan-induced trophoblast apoptosis. Am J Reprod Immunol 2013;69(5):449–453 Abrahams VM, Bole-Aldo P, Kim YM, et al. Divergent trophoblast responses to bacterial products mediated by TLRs. J Immunol 2004;173(7):4286–4296 Nold C, Anton L, Brown A, Elovitz M. Inflammation promotes a cytokine response and disrupts the cervical epithelial barrier: a possible mechanism of premature cervical remodeling and preterm birth. Am J Obstet Gynecol 2012;206(3):e1–e7 Han YW, Shen T, Chung P, Buhimschi IA, Buhimschi CS. Uncultivated bacteria as etiologic agents of intra-amniotic inflammation leading to preterm birth. J Clin Microbiol 2009;47(1): 38–47 Greig PC, Herbert WN, Robinette BL, Teot LA. Amniotic fluid interleukin-10 concentrations increase through pregnancy and are elevated in patients with preterm labor associated with intrauterine infection. Am J Obstet Gynecol 1995;173(4): 1223–1227 Christiansen OB. Reproductive immunology. Mol Immunol 2013; 55(1):8–15 Zenclussen AC. Adaptive immune responses during pregnancy. Am J Reprod Immunol 2013;69(4):291–303 Martinez FF, Cervi L, Knubel CP, Panzetta-Dutari GM, Motran CC. The role of pregnancy-specific glycoprotein 1a (PSG1a) in regulating the innate and adaptive immune response. Am J Reprod Immunol 2013;69(4):383–394 Douvier S, Neuwirth C, Filipuzzi L, Kisterman JP. Chorioamnionitis with intact membranes caused by Capnocytophaga sputigena. Eur J Obstet Gynecol Reprod Biol 1999;83(1):109–112 Han YW, Ikegami A, Bissada NF, Herbst M, Redline RW, Ashmead GG. Transmission of an uncultivated Bergeyella strain from the oral
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cavity to amniotic fluid in a case of preterm birth. J Clin Microbiol 2006;44(4):1475–1483 Bearfield C, Davenport ES, Sivapathasundaram V, Allaker RP. Possible association between amniotic fluid micro-organism infection and microflora in the mouth. BJOG 2002;109(5):527–533 Han YWY, Redline RWR, Li MM, Yin LL, Hill GBG, McCormick TST. Fusobacterium nucleatum induces premature and term stillbirths in pregnant mice: implication of oral bacteria in preterm birth. Infect Immun 2004;72(4):2272–2279 Fardini Y, Chung P, Dumm R, Joshi N, Han YW. Transmission of diverse oral bacteria to murine placenta: evidence for the oral microbiome as a potential source of intrauterine infection. Infect Immun 2010;78(4):1789–1796 Sood R, Zehnder JL, Druzin ML, Brown PO. Gene expression patterns in human placenta. Proc Natl Acad Sci U S A 2006; 103(14):5478–5483 Romero R, Schaudinn C, Kusanovic JP, et al. Detection of a microbial biofilm in intraamniotic infection. Am J Obstet Gynecol 2008; 198(1):e1–e5 Steel JH, Malatos S, Kennea N, et al. Bacteria and inflammatory cells in fetal membranes do not always cause preterm labor. Pediatr Res 2005;57(3):404–411 Stout MJ, Conlon B, Landeau M, et al. Identification of intracellular bacteria in the basal plate of the human placenta in term and preterm gestations. Am J Obstet Gynecol 2013;208(3):e1–e7 Chaim W, Mazor M. Intraamniotic infection with fusobacteria. Arch Gynecol Obstet 1992;251(1):1–7 Hill GB. Preterm birth: associations with genital and possibly oral microflora. Ann Periodontol 1998;3(1):222–232 Treiner E, Duban L, Bahram S, et al. Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature 2003;422(6928):164–169
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The Microbiome and Development: A Mother’s Perspective Kathleen M. Antony, MD1
1 Division of Maternal-Fetal Medicine, Department of Obstetrics and
Gynecology, Baylor College of Medicine 2 Department of Molecular and Human Genetics, Bioinformatics Research Lab, Baylor College of Medicine, Houston, Texas 3 Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas Semin Reprod Med 2014;32:14–22
Abstract
Keywords
► microbiome ► pregnancy ► Human Microbiome Project (HMP) consortium
Jun Ma, PhD1,2
Kjersti M. Aagaard, MD, PhD1,2,3
Address for correspondence Kjersti M. Aagaard, MD, PhD, Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine and Department of Molecular and Cell Biology, Department of Molecular Physiology and Biophysics, National School for Tropical Medicine, Center for Metagenomics and Microbiome Research, Center for Reproductive Medicine, John M. Eisenberg Center for Health Outcomes Research, Bionformatics Research Lab at the HGSC, Translational Biology and Molecular Medicine and Co-Director, Baylor College of Medicine MSTP program, 1 Baylor Plaza, Houston, TX 77030 (e-mail: [email protected]).
Dysbiosis of the microbiome has been associated with type II diabetes mellitus, obesity, inflammatory bowel disorders, and colorectal cancer, and recently, the Human Microbiome Project Consortium has helped to define a healthy microbiome. Now research has begun to investigate how the microbiome is established, and in this article, we will discuss the maternal influences on the establishment of the microbiome. The inoculation of an individual’s microbiome is highly dependent on the maternal microbiome, and changes occur in the maternal microbiome during pregnancy that may help to shape the neonatal microbiome. Further, we consider how mode of delivery may shape the developing microbiome of a neonate, and we end by discussing how the microbiome may impact preterm birth and the possibility of bacterial colonization of the placenta. Although the current literature demonstrates that the transformation of the maternal microbiome during pregnancy effects the establishment of the neonatal microbiome, further research is needed to explore how the microbiome shapes our metabolism and developing immune system.
It is established that humans have a unique and varied relationship with the microbes in our environment. We form symbiotic relationships with these microbes in the form of mutualistic (both organisms benefit), commensal (one organism benefits while the other is left unharmed), or parasitic (one organism benefits while the other is harmed) relationships. Recently, these microbes have been the focus of many studies. In particular, dysbiosis of the microbiome has been associated with type 2 diabetes mellitus, obesity, inflammatory bowel diseases, and colorectal cancer.1–18 Therefore, the Human Microbiome Project (HMP) Consortium investigated what constitutes a healthy microbiome.19–24 Although these studies uncovered the profile of a healthy microbiome, they resulted in questioning how the human microbiome is established and how the human microbiome is influenced. Naturally, our laboratory and others have turned
Issue Theme The Microbiome and Reproduction; Guest Editors, James H. Segars, MD, and Kjersti M. Aagaard, MD, PhD
to our first exposure to the outside world, our mothers. Recent work has demonstrated that women’s microbiomes undergo changes during pregnancy25,26 and that these changes in turn influence the microbiomes of offspring.26,27 The findings of the HMP Consortium and recent investigations of maternal and neonatal microbiomes of various body sites are summarized in ►Table 1. We will discuss in this article these changes and how the maternal microbiome can have long-lasting effects on children’s microbiome and health.
The Microbiome during Pregnancy Over the course of a normal, healthy pregnancy, the body undergoes profound anatomic, physiologic, and biochemical changes which result from the influences of hormonal and
Copyright © 2014 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0033-1361818. ISSN 1526-8004.
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Amanda L. Prince, PhD1
Seminars in Reproductive Medicine
Amniotic fluid, placenta, maternal endocervix
Amniotic fluid
Amniotic fluid, vagina, and dental plaque
Douvier et al/199957
Han et al/200658
Bearfield et al/200259
Bacterial culture
Bacterial culture and PCR
16S rRNA
Bacterial culture
qPCR
16S rRNA
DGGE and TGGE
Murine study
Cross-sectional/gravidae
Cross-sectional/gravidae
Case study/gravidae
Cross-sectional/infants
Cross-Sectional/infants
Cross-sectional/infants
Case control, longitudinal/infants
Cross-sectional/gravidae and newborns
Longitudinal/gravidae in first and third trimesters
Longitudinal, stool transplant into mice/gravidae in first and third trimesters
Cross-sectional/gravidae
Longitudinal/nongravidae
Study design/population
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F. nucleatum isolated from preterm births colonized the placenta of pregnant mice
Few gravidae tested positive for Streptococcus spp. or F. nucleatum in the vagina, but these bacterium where mostly found in the oral cavity of gravidae testing positive in the amniotic fluid
Amniotic fluid was colonized with oral bacteria of the Bergeyella species
Placenta colonized with the oral bacteria, Capnocytophaga sputigena
Infants are colonized differentially depending on mode of delivery
Infants born via an emergency cesarean had the highest bacterial diversity while infants born via an elective cesarean had to the lowest bacterial diversity
Infants born vaginally have a higher bacterial diversity than infants born via cesarean
Probiotics can be vertically transmitted to infants
Vaginally delivered infants acquired bacterial communities that resembled their own mother’s microbiota. Cesarean delivered infants’ microbiota reflected skin communities, but not necessarily their mothers’
Microbiome composition differed between normal weight and overweight gravidae and differed between gravidae with normal weight gain and excessive weight gain
Stool in the third trimester of pregnancy showed signs of inflammation and energy loss. When transferred into mice, third trimester stool induced greater adiposity and insulin insensitivity than first trimester stool
Characterized healthy gravidae
Characterized healthy population
Findings
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Placenta, liver, spleen, amniotic fluid, fetus
Stool
Penders et al/200638
Han et al/200460
Stool
Stool
Azad et al/201337
Biasucci et al/2008
16S rRNA
Stool
Schultz et al/200427
36
16S rRNA
Maternal: skin, oral mucosa, vagina Neonatal: skin, oral mucosa, nasopharyngeal aspirate, and meconium
Dominguez-Bello et al/201035
16S rRNA
16S rRNA
16S rRNA and qPCR
Stool
Vaginal introitus, posterior fornix, and midvagina
16S rRNA and WGS
Technique(s)
Collado et al/200832
Koren et al/201226
Aagaard et al/2012
Skin, nares, oral, vagina
NIH HMP Consortium/201220
25
Site
Authors/year
Table 1 Microbiome literature reviewed
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physical fluctuations. These alterations affect every organ of the body and occur in concert with changes in the microbiome. To date, only two body sites have been specifically examined for pregnancy-related changes in the microbiome: the vagina and the gut25,26 (►Fig. 1). During pregnancy, increased vascularity and hyperemia develop in the skin of the vulva and the mucosa of the vagina. The vaginal mucosa increases in thickness while the underlying smooth muscle cells hypertrophy and the connective tissue relaxes. There is a concomitant increase in cervical secretions with a decrease in the vaginal pH, which has long been observed to be a result of the increased production of lactic acid by Lactobacillus acidophilus. However, until recently, the dynamic nature of the vaginal microbiome during pregnancy has not otherwise been characterized. Robust knowledge of the microbiota present may help explain differences in vaginal cytokine levels, variations in pH, and differential susceptibility to infections, such as human immunodeficiency virus (HIV). Hence, we recently cataloged the “normal” microbiota signature during pregnancy across gestational age strata using cultivation-independent, molecular-phylogenetic techniques, namely, 16S rRNA gene-based sequencing.25 DNA was isolated from the vagina (introitus, midvagina, and posterior fornix) and the V5V3 region of 16S rRNA genes were sequenced (454FLX titanium platform).25 Sixty-eight samples from 24 healthy gravidae at 18 to 40 weeks gestation were systematically compared with 301
Figure 1 The maternal microbiome. During pregnancy, changes in the gut and vaginal microbiomes have been documented (signified in black). 25,26 However, recent studies suggest that the placental may be inoculated by the oral microbiome (signified in red). In addition, since the gut and vaginal microbiomes undergo changes during pregnancy, we may also discover that changes occur in the microbiomes of the nasal cavity, the oral cavity, the skin, and the placenta. These changes in the maternal microbiome during pregnancy may be critical in influencing the developing neonatal microbiome.
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Cross-sectional/gravidae Histopathology Placenta Stout et al/201365
Histopathology, FISH Steel et al/200564
Fetal membranes
Seminars in Reproductive Medicine
Abbreviations: DGGE, denaturing gradient gel electrophoresis; FISH, fluorescence in situ hybridization; PCR, polymerase chain reaction; TGGE, temperature gradient gel electrophoresis; WGS, whole-genome shotgun.
Intracellular bacteria were found in the basal plate of the placenta regardless of chorioamnionitis diagnosis
Bacteria was detected in the fetal membranes at all stages of pregnancy Cross-sectional/gravidae
Oral bacterium from human is able to colonize the murine placenta 16S rRNA Placenta Fardini et al/2010
Authors/year
61
Technique(s)
Murine study
Prince et al.
Site
Study design/population
Findings
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Table 1 (Continued)
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nonpregnant controls.25 When compared with the nonpregnant vaginal microbiota, the vaginal community is uniquely structured during pregnancy, and the changes could not be attributed to alterations in body mass index (BMI), race/ ethnicity, or other clinical confounders.25 Our group also found that the vaginal microbial community differed by gestational age and proximity to the cervix with the microbial community structure resembling that of the nonpregnant community in the latter weeks of gestation.25 Further longitudinal studies in pregnancy to examine individuals’ microbiome at multiple vaginal sites throughout pregnancy are needed to more robustly ascertain the dynamic nature of the vaginal microbiome. At a genus and species level, pregnancy was associated with an overall decrease in species diversity and in richness marked by the dominance of Lactobacillus, Clostridiales, Bacteriodales, and Actinomycetales.25 The specific species that were discriminated or enriched in pregnancy, in the context of diminished community richness and diversity, were able to be detected using robust analytic methods. More specifically, there was enrichment of Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus johnsonii.25 The enrichment of these species is of probable biologic significance. For example, L. jensenii anaerobically metabolizes glycogen, which is increased with rising estrogen levels, thereby contributing to the acidic vaginal environment. In addition, L. jensenii may have surface-associated proteins that inhibit sexually transmitted infections, including infection by Neisseria gonorrhea.28 Both L. jensenii and L. crispatus are strong hydrogen peroxide producers and have been hypothesized to protect against bacterial vaginosis, which has been posited as a risk factor for preterm birth and HIV infection.29,30 L. johnsonii produces Lactacin F, a bacteriocin, which can kill other Lactobacillus species (spp.) as well as Enterococcus spp. in the gastrointestinal (GI) tract.31 Its predominance in the vagina in pregnancy may play a role in preserving the integrity of the community to reduce the risk of ascending infection or preterm birth. Alternatively, L. johnsonii may also be important for establishing the neonatal upper GI microbiota, which may help infants digest milk. Pregnancy is also characterized by changes in the gut microbiota. Bacterial load is reported to increase over the course of gestation, and the composition also changes with pregnancy progression.26,32 Using limited 16S rRNA and quantitative polymerase chain reaction (qPCR), a study by Collado et al found significant differences in microbiota composition according to BMI states and gestational weight gain.32 Specifically, they found significantly higher numbers of Bacteroides and Staphylococcus aureus in the overweight state with lower numbers of Clostridium histolyticum in the first trimester.32 Normal versus excessive weight gain in pregnancy also exhibited differential microbiota communities with each kilogram of weight gain associated with an increase in Bacteroides counts by 0.006 log units.32 In addition, excessive weight gain was negatively associated with Bifidobacterium genus numbers.32 The presence of Bifidobacterium correlates positively with normalization of inflammatory status and improves glucose tolerance,4 which may play
Prince et al.
an important role in the newborn gut. Using self-collected specimens in the first trimester, third trimester, and postpartum, Koren et al examined changes in the gut microbiome during pregnancy and found significant remodeling over the course of pregnancy.26 They found that phylogenetic diversity within an individual (α-diversity) decreased with advancing gestation, and that this occurred regardless of BMI and diabetes status.26 They also analyzed between-sample variation (β-diversity) analysis and found the β-diversity in the first trimester to be similar to nonpregnant controls, whereas the β-diversity in the third trimester was much higher.26 Further, women with above-average BMI or diabetes did not drive this change.26 Along with this overall increase in diversity between mothers, there was an overall increase in Proteobacteria and a decrease in Faecalibacterium.26 Abundance of Proteobacteria is often associated with inflammatory conditions.33 Faecalibacterium is a butyrate producer with anti-inflammatory effects and is depleted in inflammatory bowel disease.34 In sum, the stool in the third trimester of pregnancy resembles stool from inflammatory disease states. Koren et al then took third trimester stool and transplanted it into germ-free mice to see if it would induce greater adiposity and inflammation than first trimester stool.26 They found that innoculation of germ-free mice with third trimester stool did induce higher levels of inflammation and higher levels of adiposity than recipients of first trimester stool.26 There was also a statistically significant increase in blood glucose levels following an oral glucose tolerance test among third trimester stool recipients.26 Thus, pregnancy is associated with changes in the gut microbiome that resemble disease-associated dysbiosis in nonpregnant individuals; however, these changes may promote energy storage and fetal growth in pregnancy. Further, these microbial changes may be the result of alterations in the immune system at the intestinal mucosal surface, hormonal fluctuations, or factors not yet appreciated. In addition, transformations in the microbiome that occur during pregnancy may be a combination of many factors, but the studies described above demonstrate that the host is able to manipulate the gut microbiota to promote these changes, presumably for a favorable host outcome.
Establishment of the Microbiome The changes described above in the vaginal and enteric microbiomes during pregnancy indicate that these alterations may be important for influencing the establishment of the neonatal microbiome. Previously, Schultz et al determined that the probiotic Lactobacillus rhamnosus strain GG could be vertically transmitted to infants via a vaginal birth (4/4) and a cesarean (1/2) birth.27 Accordingly, this strain was absent in the stool from infants born to mothers not taking this probiotic.27 Although this study included a limited number of gravidae, the results suggested that mode of delivery may influence the vertical transmission of the microbiome from mother to infant. As the delivery of infants via cesarean section continues to rise, investigators have started to study how the microbiome of infants is influenced by the mode of delivery. When examining the microbiome of Seminars in Reproductive Medicine
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Figure 2 Mode of delivery may determine microbial diversity in offspring. Recent studies have shown the mode of delivery influences the diversity of the neonatal microbiome. (A) Infants with the most diverse microbiome are delivered by emergency cesarean while infants born vaginally or via a scheduled cesarean have the least microbial diversity. (B) A representative principle components analysis (PCoA) plot that displays how clustering of infants born via a scheduled cesarean (orange), vaginally (blue), or via an emergency cesarean (purple) may look if studied concomitantly in a next-gen sequencing study.
neonates, Dominguez-Bello et al observed that infants born vaginally had a microbiome resembling the maternal vagina while infants born via cesarean section had a microbiome resembling maternal skin.35 Further, the diversity of bacterial flora is diminished in infants born via cesarean when compared with infants born vaginally.36,37 This loss of diversity is accompanied by an increase in Clostridium difficile and a decrease in Bifidobacterium spp., Lactobacillus spp., and Bacteroides spp., particularly B. fragilis, in neonates born via cesarean delivery when compared with vaginally born infants.37–39 A Canadian group took these studies further and examined how the microbiomes of neonates born vaginally, by elective cesareans, or by emergency cesareans compared. As seen with the studies mentioned previously, this group saw the lowest bacterial diversity in infants born by an elective cesarean delivery; however, infants born by an emergency cesarean delivery had the greatest bacterial diversity37 (►Fig. 2). This may be attributed by the exposure of these neonates to many aspects of their mothers’ microbiome during delivery. The inoculation of an individuals’ microbiota due to mode of delivery may have a long-lasting impact on an individuals’ health. Previously, Schultz et al demonstrated that probiotics taken by mothers could be found in the stool of infants born vaginally for up to 24 months postpartum.27 Thus, the vertical transmission of the microbiome from mother to child can be long lasting. Further, a Finnish study demonstrated that children born via cesarean had a significantly higher incidence of allergy and asthma when compared with children born vaginally.40 Another study in The Netherlands found that infants with a higher prevalence of C. difficile in their stool had an increased risk of asthma, eczema, and food allergens although they did not see any significant associations with mode of delivery.41 Thus, the inoculation of neonatal microbiota may help shape a developing immune system, thereby influencing the occurrence of atopic disease. Seminars in Reproductive Medicine
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Studies have shown differences in the immune system composition of infants delivered vaginally or by cesarean. Interestingly, infants born vaginally have a higher number of neutrophils and a lower number of lymphocytes when compared with infants delivered by cesarean.42 These differences may be due to the rupture of membranes that occur during a vaginal birth and may influence the establishment of the microbiome. However, when examining the associations between the microbiome and the immune system, we begin to have a chicken-and-egg scenario. For example, the influence of the microbiome in the developing immune system has been tested in a mouse model. Here, mice 3 weeks postpartum were given kanamycin to eliminate gram-positive bacteria from the enteric microbiome.43 The authors found that these mice had increased serum levels of immunoglobulin E (IgE) and IgG1 while decreasing the serum levels of IgG2a and the cellularity of the spleen and Peyer Patches.43 In a separate study, this group went on to replenish the microbiome of kanamycin-treated mice with either Lactobacillus acidophilus, Enterococcus faecalis, or Bacteroides vulgatus probiotics.44 Although the cellularity of the spleen and Peyer Patches was restored, the serum antibody levels were rescued to varying degrees by the probiotic bacteria.44 Interestingly, none of the probiotics provided reduced IgG1 serum levels in kanamycintreated mice.44 Thus, the colonization of the microbiome early in life appears to have long-lasting effects on the immune system. If the microbiota can affect the immune system in this manner, it will be interesting to determine in the future how the microbiome influences other physiological processes. In addition to mode of delivery, breastfeeding or formulafeeding has been shown to greatly impact the microbiome. Similar to mode of delivery, changes in C. difficile and Bacteroides have been documented in formula-fed versus breastfed infants.37 Thus, it is not only the initial inoculation of the infant with the maternal flora that influences an offspring’s
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microbiome but also this continual inoculation from contact with our mothers. Although we mentioned the impact of breastfeeding versus formula-feeding on the microbiome briefly here, this subject will be described in detail in another article of this issue.45
The Microbiome and Preterm Birth In the developed world, preterm birth accounts for a significant proportion of infant morbidity and mortality,46 yet, the underlying etiology of preterm birth is not fully understood. Intrauterine infection is often seen in association with preterm birth, and it is thought that an ascending infection from the vagina to the placenta causes the inflammation and preterm premature rupture of membranes (PPROM) that results in a preterm birth.47,48 For instance, the human placenta expresses the Toll-like receptors (TLRs) -2, -4, and -10, and caspase activity was found to increase in the presence of TLR-2 and -10 agonists.49,50 In addition, endocervical and ectocervical cell lines were found to produce the inflammatory cytokines interleukin (IL)-6 and IL-8 in response to lipopolysaccharide (LPS).51 Cytokine production was reduced with the use of dexamethasone, an anti-inflammatory drug.51 In addition, a study by Han et al utilizing DNA amplification techniques to detect 16S rRNA found that almost half of women presenting with symptoms of preterm labor tested positive for bacterium while control subjects did not.52 Further, patients who demonstrated symptoms of preterm birth also had significantly reduced levels of glucose with correspondingly increased levels of IL-6 when compared with control subjects.52 However, women undergoing preterm birth with positive bacterial cultures of the amniotic fluid were found to have significantly increased levels of the immunosuppressive cytokine, IL-10,53 which may be due to the maternal immune response shifting to a Th2 phenotype during pregnancy.54–56 Thus, although the maternal immune system shifts during pregnancy, the placenta retains the ability to respond to pathogens. Although the cause of placental infection has focused on an ascending infection,47,48 this dogma has been recently challenged. A case study involving a patient undergoing preterm labor found that the amniotic fluid was colonized with Capnocytophaga sputigena, a bacterium normally found in the oral cavity.57 In addition, upon investigating the colonization of amniotic fluid with bacterium, Han et al discovered high levels of Bergeyella spp. in the amniotic fluid in a patient undergoing preterm labor; additionally, the placenta showed clinical signs of infection.58 However, this bacterium was not found to be present in the vagina but was detected in the subgingival plaque.58 In an expanded study, Bearfield et al examined the presence of Fusobacterium nucleatum and Streptococcus spp. in the buccal gingival, vagina, and amniotic fluid of pregnant women.59 Although many of the gravidae had bacterial colonization of the amniotic fluid, these two strains of bacteria were primarily found in the oral cavity and not the vagina.59 These studies demonstrated that an ascending infection may not be the main culprit in placental infections.
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Further, recent work in mice suggests that oral bacterium colonizing the placenta hematogenously is the root cause of infections associated with preterm birth.60 For instance, F. nucleatum, which is normally found in the oral cavity, has been cultivated from women who underwent preterm birth.60 Han et al went on to intravenously inoculate pregnant mice with the F. nucleatum isolated and found that placentas of these mice were colonized with F. nucleatum.60 Although pregnant mice inoculated with F. nucleatum did undergo a term birth, this treatment resulted in a high incidence of stillbirth.60 Interestingly, F. nucleatum failed to colonize in other organs of the mouse (i.e., spleen or liver),60 which may be due to the protection of the maternal immune system to prevent rejection of the fetus. Although F. nucleatum was able to infect the murine placenta, the ability of this bacterium to infect the human fetus was recently studied. In this study, the DNA encoding bacterial 16S rRNA was amplified and sequenced from the amniotic fluid of patients.52 In addition to the presence of F. nucleatum, Sneathina sanguinegens, Streptococcus agalactiae, Bacteroides ureolyticus, Shigella spp., Ureaplasma parvum, Leptotrichia amionii, Clostridiales order, Peptostreptococcus spp., Citrobacter koseri, Bergeyella spp., Bacteroides fragilis, and Prevotella bivia were detected.52 Therefore, F. nucleatum is not the only bacterium able to colonize the placenta. In an independent study, Fardini et al pooled saliva samples from multiple donors and inoculated pregnant mice I.V. with these samples.61 Within 24 hours, the placenta was colonized with various bacteria.61 Altogether, these data demonstrate that microbial infections of the placenta that induce preterm birth may originate from the oral cavity. These studies provide evidence to promote further study of the potential for a placental microbiome.
The Placental Microbiome The placenta serves as a metabolic and endocrine organ with diverse functions and roles in health and disease, and maintenance of its integrity is paramount to fetal growth, development, and survival.62 Conventional wisdom is that the placenta is sterile, and therefore, the majority of intrauterine infections originate in the lower genital tract and ascend into this “sterile” placental environment.47,63 However, as described above, bacteria have been detected in the amniotic fluid of gravidae that underwent a preterm birth.52,57–59 In addition, animal studies have demonstrated that I.V. infections of oral bacteria can result in the bacterial colonization of the placenta.60,61 In these scenarios, it was hypothesized that the placenta was a sterile environment with the exception of infections that resulted in preterm birth. Yet, bacteria have been detected deep within human fetal membranes by in situ hybridization. For example, a study by Steel et al discovered that placental tissue, derived from an elective term cesarean section, had organisms present in 70% of membranes, which implies that the presence of bacteria does not induce preterm labor per se.64 Thus, this study demonstrates that there may be a more complicated relationship between bacteria and the host’s inflammatory response.64 Similarly, a recent crossSeminars in Reproductive Medicine
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sectional study of 195 patients demonstrated gram-positive and -negative intracellular occult bacteria harbored in the basal plate of 27% of placenta.65 Importantly, while these bacteria were observed with a high prevalence among preterm deliveries < 28 weeks, there was no statistically significant difference between the presence of bacteria among subjects with or without antepartum sexually transmitted infections, urinary tract infections, Group B streptococcus colonization, or chorioamnionitis.65 As the presence of bacteria are not associated with genitourinary infections or colonizations, there must be an alternate source; indeed, the majority of DNA-based detected taxa in the placenta are not found in the urogenital tract but rather are found in the oral cavity.58,60 For example, F. nucleatum is a gramnegative anaerobe ubiquitous to the oral cavity, and it is associated with periodontal disease. It has also been isolated from amniotic fluid in women with preterm labor and with PPROM.66,67 And as described above, studies have demonstrated that this bacterium can have devastating effects on neonatal survival. An interesting idea to postulate with recent studies involving term gravidae is the placenta harbors its own, unique microbiome. If this hypothesis were true, then understanding how the placental microbiome differs between preterm and term gravidae could be of vital importance in preterm birth treatment (►Fig. 1). However, further characterization of the placental microbiome in infected, noninfected, term, and preterm human pregnancies via 16S rRNA sequencing and whole-genome shotgun (WGS) sequencing is desperately needed. Such characterization can then be compared with human body sites as described by the HMP Consortium to ascertain whether the same principles are relevant in humans.20
Conclusions and Thoughts In this review, we have discussed the changes in the maternal microbiome during pregnancy, how the maternal microbiome may influence the establishment of the neonatal microbiome, and the role of the host and placental microbiome in preterm birth. As new discoveries are made, there is much to learn about the role of the microbiome in health and disease. For instance, many of the studies described throughout this review exclusively use 16S rRNA sequencing, and many previous studies cultured bacterium before amplification and/or sequencing. The major caveat to these types of studies is that only the most prevalent bacterium may be detected. However, the onset of WGS sequencing is allowing for a greater sensitivity in detecting microbes and may help further our knowledge in not only the bacteria living within us but also other microbes, such as viruses or fungi. In light of these new methods of examining the microbiome, longitudinal studies of gravidae are more feasible. Although we know that the vaginal and enteric microbiomes undergo changes during pregnancy,25,26 further understanding the maternal micorbiome during pregnancy may help prevent preterm births. For instance, with the developing microbiome field, we can further examine the role of periodontal disease and oral infection in preterm birth. Although Seminars in Reproductive Medicine
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previous studies have shown that oral bacteria can be detected in the amniotic fluid of some preterm patients,52,57–59 there are a significant number of preterm births where a bacterial infection is not found utilizing 16S rRNA sequencing.52,59 Thus, with the availability of WGS, we may be able to detect bacterial infections in all preterm pregnancies. In addition, nonbacterial microbes, such as viruses and fungi, may be involved in the onset of preterm birth. Hopefully, these studies will help us in treating and in preventing preterm birth worldwide. In addition to further examining preterm birth, studies of our microbiome naturally result in questions pondering the development of our immune system. In separate studies, we see that mode of delivery can be correlated to atopic diseases,40,41 and recent studies have actually shown that the neonatal microbiome can differ based on mode of delivery.35,37 Thus, with immune cell types, such as mucosalassociated invariant T cells, depending on microbes for their maintenance and/or development,68 we begin to wonder how these early changes in the neonatal microbiome may influence these cells. Alternatively, in a chicken-and-egg scenario, the early immune system may influence the development of the microbiome, and there are studies that describe the differing constituents of the immune system based on mode of delivery.42 With the high prevalence of atopic disease in the Western world, understanding the interactions of the microbiome and the immune system could be critical for treatment. Therefore, these inquiries are beginning to take center stage in both immunology and microbiome research. To answer the question proposed here involving preterm births, the microbiome, and the developing immune system, prospective longitudinal studies with serial sampling of mothers and newborns throughout pregnancy, parturition, and postnatally are needed to delineate the cause-and-effect of alterations in body-site microbiota. In addition to further examining the role of the microbiome in preterm birth, associations with immune status and hormonal changes are also needed to further examine how the host promotes or fails to promote a microbiome conducive to meet the host’s needs, both in terms of fostering a continuing pregnancy or meeting the metabolic needs of a growing fetus. However, the studies presented here demonstrate that the maternal microbiome can have dramatic influences on the developing fetus and the establishment of the neonatal microbiome.
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