Women and Their Microbes: The Unexpected ...

TIMI 1490 No. of Pages 17

Review

Women and Their Microbes: The Unexpected Friendship Jessica A. Younes,1,* Elke Lievens,2,3 Ruben Hummelen,4 Rebecca van der Westen,5 Gregor Reid,6,7 and Mariya I. Petrova2,3,* Communities of microbiota have been associated with numerous health outcomes, and while much emphasis has been placed on the gastrointestinal niche, there is growing interest in the microbiome specific for female reproductive health and the health of their offspring. The vaginal microbiome plays an essential role not only in health and dysbiosis, but also potentially in successful fertilization and healthy pregnancies. In addition, microbial communities have been isolated from formerly forbidden sterile niches such as the placenta, breast, uterus, and Fallopian tubes, strongly suggesting an additional microbial role in women’s health. A combination of maternally linked prenatal, birth, and postnatal factors, together with environmental and medical interventions, influence early and later life through the microbiome. Here, we review the role of microbes in female health focusing on the vaginal tract and discuss how male and female reproductive microbiomes are intertwined with conception and how mother–child microbial transfer is a key determinant in infant health, and thus the next generation.

Trends Understanding and identifying more relevant biomarkers will be a step forward in diagnosing and ultimately treating dysbiosis in women’s health. There is a possible role for microbes during fertilization, implantation, and gestation. Overlap of vaginal and penile microbiomes in shared sexual partners could have strong implications for reproductive and urogenital health. Forbidden female niches such as the breast, bladder, placenta, and amniotic fluid, are not sterile as previously thought. Microbial seeding, metabolic and immunological priming of the infant is thought to already happen in utero.

Humans As Holobionts Given the crucial role of the microbiome in human physiology, humans have been described as holobionts (see Glossary) or communities composed of the host and its symbiotic microbes, rather than individuals [1]. Interestingly, this combination of the host genome and microbiome increases genetic variation and phenotypic plasticity, enabling the holobiont to increase its overall fitness [2] (Box 1). Consequently, bacteria might arguably play an essential role in producing reproductively fit and healthy offspring in addition to influencing the overall health of an individual. Of the many studies carried out on the microbial populations of humans, most notably using 16S rRNA gene high-throughput sequencing, very few have focused exclusively on women’s health. Indeed, vaginal health of women is often stigmatized and underresearched, despite it being recognized as critical for public health, overall well-being, and human reproduction [3]. In order to emphasize the unique aspects of the female microbiome – and to examine what is known, and identify what gaps exist – two scientific conferences termed ‘Women & Their Microbes’ were held in Amsterdam in 2015 and 2016. In this review, we discuss how microbes impact the lives of women and their offspring.

The Impact of Microbes on the Vaginal Niche Genetic Basis for the Key Role of Lactobacilli The human vagina is an underappreciated organ that is not merely a passageway for vaginal discharge, menses, sperm, and neonates, but can profoundly affect the health of generations. The vaginal microbiome (VMB) is dominated mainly by Lactobacillus spp., and depletion of these organisms is associated with several important adverse conditions, including but not limited to preterm birth, pelvic inflammatory disease, increased risk and transmission of sexually

Trends in Microbiology, Month Year, Vol. xx, No. yy

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Winclove Probiotics, 11 Hulstweg, 1032 LB Amsterdam, The Netherlands 2 KU Leuven, Centre of Microbial and Plant Genetics, Kasteelpark Arenberg 20, 3001 Leuven, Belgium 3 University of Antwerp, Department of Bioscience Engineering, Antwerp, Belgium 4 McMaster University, Department of Family Medicine, 100 Main Street West Hamilton, ON L8P 1H6, Canada 5 University Medical Center Groningen, Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands 6 Human Microbiology and Probiotics, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario, N6A 4V2, Canada 7 Departments of Microbiology & Immunology, and Surgery, The University of Western Ontario,

http://dx.doi.org/10.1016/j.tim.2017.07.008 © 2017 Elsevier Ltd. All rights reserved.

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Box 1. Host Genetic Shaping of the Human Microbiome Multiple external factors, such as diet, antibiotic exposure, and social contact, are known to influence the composition of the human microbiome. In addition, evidence is emerging that host genetic variation is associated with microbiome composition in the gut [110,111]. In general, host genes associating with specific gut microbial taxa are related to immune regulation, diet preference, and metabolism [112]. Related to immunity, the NOD2 risk allele that confers a high risk of developing inflammatory bowel disease (IBD) appeared to be associated with an altered intestinal microbiome characterized by an increased relative abundance of species of the family Enterobacteriaceae [113]. Further, microbiome composition correlated with host genetic variation in immunity-related pathways, such as leptin, melatonin, JAK/ Stat, chemokine, and CXCR4 signaling. Moreover, these associations were especially enriched in host genes that have been associated with microbiome-related diseases, such as IBD and obesity [111]. Related to diet preference, an interesting correlation was found between host genetic variation in the LCT gene, encoding the lactase enzyme that cleaves lactose, and the abundance of Bifidobacterium in the gut. Given that genetic variants of LCT are linked to lactase persistence, and that lactase persisters harbor fewer bifidobacteria compared to lactase nonpersisters, these variants may dictate an individual’s milk product consumption which, in turn, may regulate the abundance of Bifidobacterium [111,112]. The authors also show that host genomic regions associated with the microbiome have high levels of genetic differentiation among human populations. This is suggestive of host genomic adaptation to population-specific microbiomes likely controlled by environmental conditions [111]. Related to metabolism, Christensenellaceae, a family belonging to the phylum Firmicutes, was identified as the most heritable taxon in a study using identical twins, and this family was enriched in lean individuals. Moreover, addition of Christensenella minuta, a cultured member of the Christensenellaceae, to an obese-associated microbiome and subsequent transplantation to germ-free mice resulted in reduced weight gain compared to no addition of C. minuta. These findings provide evidence that host genetics can influence gut microbiome composition and, in turn, can shape the individual’s phenotype [114]. Future research on how to reshape the gut microbiome to reduce disease risk within the context of an individual’s genotype might provide new treatment approaches for obesity-related diseases [115].

transmitted infections, infertility, and multiple stigmatizing symptoms that affect quality of life [4]. Intriguingly, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii appear to dominate the vagina of most healthy women [5,6]. Yet there are no known genomic explanations why other Lactobacillus species with access to the urogenital tract do not colonize and dominate as well, such as Lactobacillus helveticus or Lactobacillus acidophilus. Of note, a fifth non-Lactobacillus-dominated microbial community has also been reported in healthy women and is characterized by strictly anaerobic bacteria, such as Atopobium, Dialister, Gardnerella, Megasphaera, Prevotella, Peptoniphilus [5]. However, as there are dynamic shifts across the menstrual cycle and also across the female lifecycle (Box 2), the role of this microbial community in maintaining vaginal health is still under discussion. The genetic basis for the key role of lactobacilli in the maintenance of vaginal health holds several clues to support the above long-held notion. It has been suggested that certain keystone species have adapted over time to allow for optimal survival in the vaginal niche [7,8]. For instance, various stress proteins, sortase-dependant proteins, utilization pathways of mannose/glycogen, toxin–antitoxin systems, and transcription factors are more often detected in vaginal lactobacilli as compared to lactobacilli from the gastrointestinal tract [8]. As an illustration of this adaptation, the genome of L. iners contains several genes putatively involved in optimal survival in the fluctuating vaginal environment, such as an iron–sulfur cluster, alkaline shock proteins, various heat-shock proteins, cold-shock proteins, universal stress proteins, and several s-factors [7]. However, thus far, these unique protein families have not yet been detected by comparative functional genomics in the vaginal species L. crispatus, L. gasseri, L. iners, and L. jensenii [8]. The genomes of vaginal Lactobacillus species are significantly smaller and with lower GC content than those of other closely related lactobacilli residing in the gastrointestinal tract and in dairy products [8]. This observation suggests that these particular Lactobacillus species show some degree of adaptation to a host-dependent lifestyle. Certain gene clusters are overexpressed or underexpressed in the genome of vaginal Lactobacillus species as compared to nonvaginal strains, also suggesting a preference for a functional symbiotic existence [8]. For instance, genes involved in intracellular trafficking, secretion, vesicular transport, translation, ribosomal structure and biogenesis, cell cycle control, cell division, and chromosome repair are overexpressed in vaginal lactobacilli. On the other hand,

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London, Ontario, N6A 5C1, Canada

*Correspondence: [email protected] (J.A. Younes) and [email protected], [email protected] (M.I. Petrova).


Box 2. Changes in the Vaginal Microbiome during a Woman’s Life

Glossary

The composition of the vaginal microbiota changes drastically over a woman’s lifecycle. During perinatal development, residual maternal estrogen induces thickening of the vaginal epithelium and the deposition of glycogen in the epithelial cells. Through exfoliation of epithelial cells, glycogen is released, thereby favoring glucose-fermenting microorganisms [116,117]. Postnatally, the maternal estrogen is metabolized, resulting in a thinning of the mucosa, reduction in glycogen- and glucose-fermenting microorganisms, and ecological selection for a wide range of aerobes and facultative anaerobes. The vaginal microbiota during childhood is mostly dominated by Gram-negative anaerobe bacteria, including Bacteroides, [371_TD$IF]Fusobacterium, Veillonella, some Gram-positive anaerobic bacteria, including Actinomyces, [372_TD$IF]Bifidobacterium, Peptococcus, Peptostreptococcus, and Propionibacterium, as well as some aerobic bacteria such as Staphylococcus aureus, Staphylococccus epidermidis, Streptococcus viridans, and Enterococcus faecalis [118,119]. Typical for the vaginal microbiota of prepuberal girls is the low abundance of lactobacilli, Gardnerella vaginalis, and Prevotella bivia [119]. With the beginning of puberty, under estrogenic control, the vaginal epithelium once again thickens, selecting for glucose-fermenting microorganisms. The microbiome of adolescent girls is similar to the vaginal microbiota of adult women and is dominated by Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii [120]. During menopause, characterized with a decline in estrogen, the microbiome is dominated by L. crispatus, L. iners, G. vaginalis, and Prevotella and a lower abundance of Candida, Mobiluncus, Staphylococcus, Bifidobacterium, and Gemella [121]. Therefore, the vaginal microbiome is remarkably similar to the premenopausal state, although increased microbial diversity is also observed [122]. Longitudinal analysis showed that the postmenopausal vaginal microbiome is remarkably stable, with no significant change in diversity, whereas the premenopausal microbiome changes markedly over time, switching from one to another community type during the menstrual period. For example, the vaginal communities dominated by L. crispatus often transform to community states dominated by L. iners, or to community state IV-A characterized by a modest proportion of either L. crispatus, L.iners, or other Lactobacillus species and a low numbers of strict anaerobes. Community states dominated by L. iners tend to shift more often to community type IV-B (dominated by Atopobium, Provotella, Gardnerella, or Mobiluncus) but in rare cases to IV-A. In comparison, the community state dominated by L. gasseri rarely transitions to other types and tends to stay stable over time.

genes involved in transport and metabolism of nucleotides, lipids, amino acids, coenzymes, and secondary metabolites are underexpressed in those strains [8]. A better understanding of genetic and molecular pathways and processes of vaginal Lactobacillus strains could hold the key not only to uncovering the pathogenesis of vaginal dysbiosis, but also to help to refine diagnostics and guide therapeutic interventions for microbial dysbiosis. However, these findings still do not explain why most VMBs tend to be dominated by only a few of the aforementioned vaginal species and not others. Dominant vaginal Lactobacillus species exert important health-promoting effects to maintain reproductive fitness of the host and to maintain their dominance in the vaginal niche. This is accomplished by various direct and indirect antipathogenic mechanisms such as the production of biochemically active compounds that directly kill or inhibit pathogens. Biophysical mechanisms are also employed, such as the formation of microcolonies that adhere to the epithelial cells and form a physical barrier against pathogen adhesion, and the stimulation of host defense mechanisms against pathogens (Figure 1). For example, the production of lactic acid is accepted as a hallmark beneficial activity of the VMB. Lactic acid has been shown to inactivate or kill a wide range of vaginal pathogens, including Chlamydia trachomatis [9], uropathogenic Escherichia coli [10], Neisseria gonorrhoeae [11], and potentially viral pathogens such as herpes simplex virus type 2 (HSV-2) [12] and HIV-1 [13]. Hydrogen peroxide has also been shown in vitro to damage planktonic vaginal pathogens, and, when combined with lactobacilli-isolated biosurfactants [14], can also increase pathogen sensitivity to certain antibiotics [15]. While the exact nature of the role of vaginal hydrogen peroxide has yet to be confirmed in vivo, these data do suggest a strong protective role of lactobacilli in the vagina. Another postulated mechanism, by which lactobacilli prevent colonization by pathogens, is through adhesion to host cells (Figure 1). Several vaginal Lactobacillus isolates have been shown to block the adhesion of pathogens to vaginal epithelial cells in vitro, such as E. coli [16], Gardnerella vaginalis [17], Klebsiella pneumonia [18], Pseudomonas aeruginosa, Staphylococcus aureus, group B streptococci [18], and Trichomonas vaginalis [19]. Yet again, the health benefits of vaginal lactobacilli have been poorly substantiated by molecular studies. Therefore

Bacterial vaginosis (BV): one of the most common vaginal disorders affecting fertile, premenopausal, and pregnant women. BV is a complex, polymicrobial disorder characterized by the disruption of the vaginal econiche, resulting in a reduction in lactobacilli and an overgrowth of strict or facultative anaerobic bacteria. Colostrum: a form of breast milk produced in late pregnancy just prior to giving birth. It contains a number of immune cells and many antibodies, such as IgA, IgG, and IgM. Dysbiosis: refers to an imbalance in the composition of the vaginal microbiota – such as during bacterial vaginosis and aerobic vaginitis. High-throughput sequencing: also known as next-generation sequencing, it describes a number of different modern sequencing technologies, including, but not limited to, Illumina sequencing, Roche 454 sequencing, Ion torrent, and SOLiD sequencing. Holobiont: the host organism together with all its microbial symbionts, including transient and stable members. Lectins: carbohydrate-binding proteins, which are highly specific for the sugar moieties they recognize. Lectins play an important role in biological recognition phenomena, facilitating protein–sugar interactions and protein–protein interactions, thereby mediating cell–cell, bacteria– cell, and bacteria–bacteria contacts. Meconium: the first stool of the infant, composed of materials ingested in the uterus, such as intestinal epithelial cells, lanugo, mucus, amniotic fluid, bile, and water. Necrotizing enterocolitis (NEC): one of the most commonly seen disorders in premature infants; it is characterized by necrosis of the bowel. If not treated, NEC can lead to morbidity in premature infants. Pattern-recognition receptors (PRRs): proteins expressed by immune cells. They are involved in the recognition of microorganismassociated molecular patterns (MAMPs), allowing for quick responses when invading microorganisms are detected. Probiotics: live microorganisms which, when administered in


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adequate amounts, confer a health benefit on the host. Secretory leukocyte peptidase inhibitor (SLPI): a highly cationic single-chain enzyme found in bronchial, cervical, and nasal mucosa. Its enzymatic activity contributes to the immune response by protecting epithelial surfaces from endogenous proteolytic enzymes.

Figure 1. Mode of Action of Lactobacillus Species. Lactobacilli are able to inhibit pathogens in a direct manner by coaggregation or by producing active components such as bacteriocins, lactic acid, and hydrogen peroxide (H2O2). In addition, they promote the integrity of the epithelium by stimulating mucus secretion and modulating the immune response, which are indirect ways of inhibiting viral and bacterial pathogens.

more studies focusing on these outcomes and mechanisms are required to understand their exact role. Eubiosis, Dysbiosis, and Biofilms Much of the early information about the vaginal microbiome from asymptomatic women was derived from culture studies, clue cells, and Gram stains. Recent genetic-based techniques have provided a better understanding of which species are present in eubiosis, a healthy balance of vaginal microbiome, and this knowledge is evolving rapidly. However, the ‘healthy’ or eubiotic VMB is a moving target. Although the physiological/microbiome conditions in the vagina fluctuate daily [20], most molecular/genetic studies provide only a cross-sectional snapshot of the VMB under distinct circumstances. The presence of certain vaginal species (Atopobium vaginae, Candida albicans ssp., G. vaginalis, Lactobacillus spp., Leptotrichia spp., Megasphaera spp., etc.) in both eubiosis and dysbiosis, in combination with the natural fluctuations, makes it difficult to compare microbiome composition data across the many

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emerging studies. Interestingly, some of these same species identified by genetic methods can be found residing in dysbiotic vaginal biofilms [21,22]. It is then tempting to consider these common species as critical for the transition between health and disease. However, this concept has mostly been studied in vitro, and needs to be confirmed more extensively, especially because genetic and molecular data suggest the existence of multiple lineages [23,24]. Patterns concerning the structural organization of the vaginal microbiome are slowly starting to emerge: asymptomatic women have very thin and sometimes loose vaginal biofilms, whereas women with bacterial vaginosis (BV) tend to have thick adherent biofilms (Box 3) [22]. Certain species, such as G. vaginalis and A. vaginae, have been proposed as primary and secondary BV biofilm colonizers, respectively [25,26]; the former provides a physical scaffold for the latter to establish a mature biofilm structure. Detection of G. vaginalis in extravaginal microbial reservoirs pre-existing a BV infection has been linked to a significantly higher infection risk [27]. This also supports the hypothesis of G. vaginalis being an essential ‘founder’ species necessary to establish an infectious biofilm (Box 3). This hypothesis is not unreasonable as some species are known in vitro to enhance the growth and biofilm formation of other vaginal species, including G. vaginalis [28,29]. It is also interesting that organisms in BV biofilms have been shown in vitro to be better able to resist the natural defence mechanisms of the vagina [21,30] compared to their planktonic counterparts. What is perhaps more important to understand, however, in addition to unraveling the nature of vaginal biofilms, is the contribution of biofilms to the development of vaginal dysbiosis (Figure 2). Three distinct states of VMB dysbiosis have been proposed by Professor Janneke van de Wijgerti[37_TD$IF]. The first state is characterized by high recurrence and by a pathogenic biofilm, possibly instigated by G. vaginalis. The second proposed state of dysbiosis is characterized by a high planktonic bacterial load in which G. vaginalis is commonly isolated, but its colonization and biofilm presence is not necessarily required. This type of VMB dysbiosis is easier to treat with antibiotics, and has a low recurrence rate. The third polymicrobial biofilm state is dense, contains high diversity along with an abundance of potentially pathogenic microorganisms and is highly inflammatory. It is also hypothesized that, because of the unknown nature of the

Box 3. Bacterial Biofilms Microorganisms have developed several strategies to protect themselves from environmental challenges and external toxic factors, one of which is the formation of biofilm communities. Bacteria in biofilms have the ability to sense their own cell density, and to communicate and behave as a population through quorum sensing. This specific regulation is dependent on the nutritional environment, as well as on the maturation stage of the bacterial biofilms. It has been hypothesized – based on extensive work in other microbiome niches, such as the oral cavity – that the many unique features of the microbiome structure in biofilms contribute to treatment failure and up to 70% of long-term recurrence rates within vaginal infections [123]. One such inherent characteristic of biofilms is that their mode of growth is different from that of planktonic bacteria; this enables the biofilm microbes to survive threats to their survival and reproductive abilities [124]. The protective biofilm matrix limits the penetration and diffusion of antimicrobials to the outermost biofilm layers, limiting any killing effect and ensuring that the inner layers are completely protected or only exposed to sublethal levels of antimicrobials [124–126]. Furthermore, mature biofilms can release planktonic and persister cells during their life cycle [124,127] and are less vulnerable to elimination via the immune system [125], contributing to recurrent and recalcitrant infections. Nevertheless, certain compounds and molecules from probiotics have been shown to be beneficial in attacking pathogenic biofilms. Lectin-like molecules isolated from probiotic strains, such as the Lactobacillus rhamnosus strains GG and GR-1, or from the natural vaginal isolate Lactobacillus plantarum CMPG5300, showed the capacity to destroy pathogenic biofilms without killing the pathogens [128,129]. Biosurfactants, which are a heterogeneous group of amphiphilic compounds produced by various microorganisms, have also been shown to play a role in preventing pathogenic biofilm formation and disruption. For example, biosurfactants isolated from Lactobacillus fermentum B54 and Lactobacillus paracasei A20 inhibit urogenital bacterial biofilms [130] and various bacterial and fungal biofilms [131], respectively. Therefore, it is tempting to speculate that the use of these purified molecules alone, or as part of living probiotic Lactobacillus strains, might be used in the future as complementary therapy to antibiotics to treat vaginal infections.


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Figure 2. Improving Women’s Health in Relation to Their Microbes. This figure demonstrates the interdependency of the research priorities necessary to improve women’s health.

individual species, this third state is quite difficult to treat. Because the first and the third types of VMB are difficult to treat, and are characterised by high recurrence, the importance of recognizing exactly how a vaginal biofilm influences infections cannot be underestimated. Current diagnostic methods are not yet sensitive enough to identify biofilm-related dysbiosis, and they rely mainly on planktonic microbes and/or shed biofilms adhered to clue cells. Therefore, future research priorities should clearly include structural and theoretical studies to understand eubiotic, and dysbiotic, biofilms and pathogenesis, thereby allowing better differential diagnosis and more targeted treatment options (Figure 2). The Vaginal Environment beyond the Microbiome The vaginal environment is a complex interaction of host cells, symbionts and pathobionts, endosymbionts, and mucosal, endocrine, and immunological factors. Interactions between the microbes and epithelium shape mucosal innate immunity and play a key role in reproductive health [31] (Figure 1). There appears to be an emerging relationship between vaginal cytokine biomarker profiles and the microbial composition of the genital tract. For example, the presence of L. crispatus and L. jensenii is negatively associated with cellular inflammatory markers, and is positively associated with low levels of proinflammatory cytokines such as IL-1a and IL-8 [32]. In the case of L. iners, higher levels of secretory leukocyte peptidase inhibitor (SLPI) [33], an antimicrobial peptide normally depleted in women with dysbiosis such as BV [34,35], can be observed. Doerflinger et al. also discovered that L. crispatus ATCC 38820 does not significantly upregulate pattern-recognition receptor (PRR) signaling pathways in human primary vaginal epithelial cells, whereas L. iners ATCC 5195 does [36]. These data suggest that the microbiome composition plays a unique function in the maintenance of a healthy vagina. Dysbiosis in the VMB affects the mucosal barrier through cervicovaginal inflammation [37], including mucus and epithelial cytoskeleton alterations, changes in protein and peptide balances, and increased concentrations of proinflammatory cytokines. For instance, in women

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with BV, the proinflammatory cytokine IL-1b is elevated, and the SLPI antimicrobial peptide is decreased [38]. In addition, G. vaginalis and A. vaginae, but not Prevotella bivia, stimulated a proinflammatory response by upregulating the production of cytokines (IL-1b, IL-6, IL-8, TNFa) and chemokines (MIP-1b, RANTES), some antimicrobial peptides (i.e., defensins such as hBD2) and membrane-associated mucins when tested on vaginal or cervical epithelial cell monolayers and tissue models [36,39]. Similar results were observed in women with BV [38], suggesting a distinct pathogenicity of certain BV species. As a result of these observations, specific cytokine patterns have emerged as potential genital tract biomarkers to indicate vaginal health or inflammation. For example, IL-8 and the IL-1RA-to-IL-1 ratio have been suggested as vaginal immune balance biomarkers [40]. Although biomarkers can elucidate further relationships, the genital tract is unique, and various biomarkers can differ based on location, time point, stimulus, and sample type. Indeed, multiple factors can influence cytokine levels such as physiologic variation (e.g., menstrual cycle, sexual intercourse, pregnancy, contraceptive method) as well as technical variation (e.g., sampling method and accompanied sample dilution, sample processing, assay linearity) [40,41].

The Role of Microbes in Reproduction The Contribution of Female and Male Microbiomes during Conception Despite the assumption that reproduction takes place in an aseptic environment, the apparent importance of microbes from conception to delivery is slowly emerging. There have been recent discoveries of reproductive microbiomes associated with male and female tissues that seem to be involved in human reproduction [3] (Figure 3, Key Figure). Coupled with the emerging role of the human microbiomes in reproduction, are possibilities for interventions to improve fertility, conception, healthy pregnancy, and microbial seeding of the infant, and to prevent preterm birth. Although essential for successful conception, the male seminal fluid microbiome has been neglected. Recent sequencing analysis has revealed more diverse communities with lower bacterial concentrations as compared to the vaginal microbiome [42]. Using molecular detection techniques, several studies identified Corynebacterium [43–46], Gardnerella [46,47], Lactobacillus [42,43,45–47], Prevotella [43,44,46,47], Pseudomonas [47], Streptococcus [42,44,45], and Veillonella [42–44] among the most abundant species in the seminal microbiome. Although community types differ among studies, specific bacterial species in the seminal microbiome are highly associated with semen health and fertility. For example, the presence of Anaerococcus, Prevotella, and Pseudomonas species has been associated with low-quality semen, while Lactobacillus predominance is associated with healthy semen [42,43,47]. Additionally, the semen of prostatitis patients, a pathology associated with reduced semen quality, harbored a significantly higher species diversity and a lower Lactobacillus count, especially for L. iners. These findings indicate that possibly a similar function as in the VMB might be attributed to Lactobacillus species in the male seminal microbiome [46]. Interestingly, application of probiotic Lactobacillus brevis CD2, Lactobacillus plantarum FV9, and Lactobacillus salivarius FV2 preserved sperm motility and viability in vitro by preventing membrane damage induced by reactive oxygen species [48,49]. This suggests that microbial protection of spermatozoa function also might occur through vaginal lactobacilli in the vagina after intercourse [48], though this has yet to be definitively proven. During heterosexual intercourse, the male seminal microbiome is in association with the VMB, thereby directly influencing the host partner’s health. For example, a significant association between inflammation in the male genital tract and G. vaginalis predominance in the VMB has been detected [42]. Furthermore, the male penile skin and urethral microbiome is significantly


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Key Figure

Microbiome and Related Factors at Different Stages of Human Reproduction

Figure 3. Differentiation is made between maternal (M) and infant (I) microbiomes during childhood. The light blue color represents the factors influencing the microbiome at each stage of human reproduction. The purple boxes describe the species detected during different stages.

more similar to the VMB of their BV-positive female partner as compared to the VMB of other BV-positive women, suggesting sexual exchange of BV-associated bacteria [43,50]. Interestingly, similar trends of microbial influence can be seen in artificial reproduction techniques (ARTs) outcomes. Some studies have investigated the influence of the VMB on in vitro fertilization (IVF) success rates. The VMB on the day of embryo transfer has been shown to affect pregnancy outcome, following IVF, and appeared most successful when the VMB is composed solely of lactobacilli [51]. On the other hand, BV is more prevalent in infertile versus fertile women [52,53], and is associated with increased risk of preclinical pregnancy loss [53] and decreased pregnancy rate in IVF patients [54]. Although other species could be involved, G. vaginalis and A. vaginae were significantly associated with lower pregnancy rates [54]. Moreover, BV treatment of infertile women significantly increased pregnancy rates, indicating a possible role for BV in female infertility [52]. Further exploratory investigations within ART have identified microbes in the ovaries, Fallopian tubes, and the endometrium. Distinct bacterial

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communities appear to colonize the left and right Fallopian tube and ovary as well as different sites within the upper genital tract, such as the proximal Fallopian tube, fimbriae, and ovarian surface [55]. Regarding community composition, Lactobacillus species could not be identified in all samples, nor were they the most dominant isolates [55]. The presence of Propionibacterium and Streptococcus species in ovarian follicular fluid was associated with lower embryo transfer rates and pregnancy rates in both fertile and infertile women compared to women with Lactobacillus species [55]. Perhaps most strikingly of all: the endometrium also appears to harbor its own unique microbiota, with Bacteroidetes (Flavobacterium spp.) and Firmicutes (Lactobacillus spp.) as dominant phyla in healthy women [56,57]. Endometrial microbiota composition correlated with implantation rates during IVF and live birth rates [58]. Analysis of the uterine microbiome of women with recurrent reproductive failure, and without visible uterine anomaly, appeared to be dominated by Bacteroides species [57]. Furthermore, nonLactobacillus-dominated endometrial microbiome was negatively associated with pregnancy outcome, and this was more evident with increased Gardnerella and Streptococcus genera [58]. Given the responsiveness of the host’s immune system to the microbiome, an intriguing question arises whether the uterine microbiome can influence the mother’s immune-mediated quality control to either accept or reject conceptus [59,60]. In the absence of infection and stress, the female Th1/Th17 immune response is suppressed to increase the likelihood of pregnancy onset [59]. This concept is based on the mother’s immunological capacity to sense reproductive fitness of the semen, the integrity of placental trophoblast, as well as reproductive tract infection and stress. While current knowledge on the collective reproductive microbiome remains scarce, future studies are needed to focus on the [374_TD$IF]origin, community dynamics and identification of biomarker species associated with positive and negative reproductive outcomes. Infant Health Starts with Microbial Priming in the Uterus It is especially interesting that not only our genetic makeup but also our microbial makeup might be determined in utero. There is evidence that, during gestation, the fetus experiences intrauterine contact with maternal microbes [61,62]. This low biomass microbial interaction appears to be able to significantly affect the immune and metabolic programming of the fetus [62]. Following fertilization, the placenta not only functions as a source of nutrient exchange between mother and fetus, but also has been reported to harbor a low-abundance, though metabolically rich, microbiome [61] (Figure 3). It is still unclear whether these temporary reproductive structures harbor their own distinct microbiome communities or whether they are, instead, a cumulative reflection of the surrounding microbiome niches. Further studies should focus on determining the exact role and nature of the microbial communities identified thus far in these niches. While E. coli has been found to be the single most prevalent species in the placenta, this microbial community is also composed of species of the Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria phyla and the unique presence of Tenericutes. Variation in the placental microbiome is associated with preterm birth; increased Burkholderia taxa have been found to be enriched in preterm placentas and Paenibacillus taxa in term placentas [61]. Of note, when compared to other microbial niches, the composition of the placenta is most similar to the oral microbiota, including species such as Prevotella tannerae and Neisseria [61]. This is suggestive of bacterial translocation from the oral cavity to the placenta possibly through the increased permeability of the gingival vascular bed in gingivitis [63], which may explain why women with periodontal disease have an increased risk of pregnancy complications [64]. It is worth mentioning that the mother’s gastrointestinal and oral microbiota changes during pregnancy. For example, increased abundance of species of the phyla Actinobacteria and Proteobacteria, as well as a reduction in individual richness, was observed between the first and third trimester in the gut microbiota [65]. In addition, the total


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viable microbial counts in the oral cavity in all stages of pregnancy were higher than those of the nonpregnant women, especially in early pregnancy [66], and levels of the pathogenic bacteria Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in the subgingival plaque were significantly higher compared to the nonpregnant women. Similar to the placenta, the microbial population in amniotic fluid also shows low abundance, low richness, and low diversity. Species of the phylum Proteobacteria were the most prevalent in amniotic fluid, with a high abundance of species belonging to the family Enterobacteriaceae, such as Enterobacter and Escherichia/Shigella [67]. Species of the genera Lactobacillus, Propionibacterium, Staphylococcus, and Streptococcus were also detected in the amniotic fluid and in the placenta [67]. In addition, microbial colonization in the umbilical cord of healthy neonates born by elective cesarean section was also detected, with isolates belonging to the genera Enterococcus, Streptococcus, Staphylococcus, or Propionibacterium [68]. Of note, confirmation that the infant gut microbiota colonization starts in utero is also exemplified by detecting microbial species in the meconium, which share some common features with the amniotic fluid and placenta microbiota [67]. Meconium microbiota shows lower species diversity and is dominated by Streptococcus, unclassified species of the Enterobacteriaceae (mostly Enterobacter and Escherichia), Lactobacillus, and Propionibacterium [67]. Bifidobacteria are also found in the meconium of the infant at days 3, 7, 30, and 90, suggesting that the infant gut is immediately colonized [69], and these strains have been directly linked to the mother’s intestine before delivery [69,70]. Microbial colonization of the uterus, placenta, and amniotic fluid may thus allow the fetus to become tolerant to bacteria after birth through priming [60], and to affect the fetal innate immune gene expression and establish a healthy newborn microbial profile [62]. However, future studies clearly need to focus on in utero colonization, since this subject is under discussion. Interestingly, probiotics may be implemented during perinatal and neonatal development to reduce the infant’s risk of certain noncommunicable diseases, as recently summarized by [3]. It seems rational to consider key fetal developmental milestones in the womb, for example at week 3 (central nervous system development and onset of heartbeat) and during weeks 14–16 (brain development, neonate begins to suck, swallow, and starts breathing movements). Maternal dietary supplementation with iron/folic acid [71] and a-linolenic acid [72] improves brain-related fetal outcomes such as working memory, inhibitory control, and fine motor functioning. Since certain bacterial species can produce conjugated isomers of a-linolenic acid [73], folate [74], and can even manufacture neurochemicals such as serotonin, dopamine, and GABA, there are excellent probiotic candidates to support fetal development [75]. Positive effects of maternal probiotic supplementation on fetal Toll-like receptor gene expression in the fetal gut have been documented [62]. Probiotic use during gestation for some maternal indications has been shown to benefit both mother and infant. These indications include reduction of preterm birth, reduction of gestational diabetes mellitus, prevention of group B Streptococcus colonization, reduction of postpartum depression, alleviation of gastrointestinal-related complaints and antibiotic-associated side effects, heavy metal and pesticide detoxification, and reduction in vaginal and urinary tract infections. For the neonate, reduction in colic, elimination of necrotizing enterocolitis (NEC), atopy, and allergy support, and a reduction in antibiotic resistance – in addition to the development of a healthy immune system and proper maternal microbial priming – would be reasons to consider peri- and neonatal probiotic supplementation. Even days following birth, probiotic treatment has been demonstrated to reduce the mortality rate in NEC, the perilous neonatal gastrointestinal emergency [76]. Probiotic administration during pregnancy has been shown to reduce maternal central adiposity, by improving plasma glucose concentrations and reducing excessive weight gain among children [77–79]. Probiotics given to pregnant mothers

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have also the ability to influence immune targets such as neonatal risk reduction of atopic eczema in term infants [80,81]. There is growing experimental and clinical evidence to support probiotic intervention for metabolic targets [82,83]. However, in a double-blind, placebocontrolled randomized trial, probiotics were unable to reduce maternal fasting glucose [84]. Whether other probiotic strains could be effective remains to be seen. The ability of probiotic strains to process enteral antigens, modulate gut mucin production, innate and adaptive immune regulation, and epithelial development [85,86] further suggests that we must consider the timing and dosage of probiotics use during pregnancy. Ensuring Maternal Microbial Inheritance at Birth As mentioned earlier, the mother acts as the bacterial reservoir for the microbial seeding of the fetus [87]. However, other factors, such as lifestyle, diet, and medical interventions, also shape the infant microbial landscape until it reaches a more stable state around 2–3 years of age [87,88]. Perturbations of the early-life gut microbiota development caused by antibiotic treatment can have lifelong metabolic consequences [88,89], but current knowledge about this is mostly limited to animal studies. Limited duration of low-dose penicillin treatment given to infant mice immediately after birth transiently disrupts the intestinal microbiome composition and body composition [89]. Early life presence of Lactobacillus, Allobaculum, Rikenellaceae, and Candidatus arthromitus in the mouse gut microbiota has been proposed as protective taxa against obesity and impaired ileal immunity. Administration of low-dose penicillin to mother mice shortly before birth and through weaning has led to enhanced metabolic phenotypes compared to antibiotic exposure in early infancy [89]. Thus, possible restoration of lost taxa following antibiotic treatment might provide a strategy against microbe-induced obesity, although metabolic recovery in humans could differ from that in mice since the human microbiome is exposed to higher environmental variation, such as diet and lifestyle [89]. Although a possible causal role of altered neonatal gut microbiota has not yet been determined, prenatal antibiotic exposure in humans during the second or third trimester of pregnancy also increases the risk of childhood obesity [88]. Another frequent medical intervention altering the acquisition of the initial neonatal microbiota is delivery by cesarean section (C-section) [90]. Numerous studies have shown an association between C-section delivery and an increased risk of type 1 diabetes [91,92] and obesity [88,93] among other diseases. Given the fact that the obesity phenotype is transferable by intestinal microbiota transplantation from humans to mice [94], the mother-to-newborn microbial transfer has been suggested to play a role in intergenerational association of obesity in addition to maternal genetics [95]. Since the maternal gut microbiota is changing during pregnancy, and these changes are dependent on weight status, it is possible that maternal body weight codetermines the microbiota composition that will be inherited by their offspring [96]. Although multiple studies have found an association between obese mothers and altered intestinal microbiota in their infants, the influence of the delivery mode was not considered [97,98]. Recent evidence suggests that the delivery mode shapes the initial acquisition of microbiota [99], underlining the importance of this factor in studying mother–infant microbiome inheritance. Not surprisingly, infants delivered vaginally harbor bacterial communities resembling the VMB of the mother, whereas the microbiota of infants delivered by C-section is similar to the skin bacteria of their mother [90] (Figure 3). A recent study demonstrated a different gut microbiota of neonates born by C-section compared to vaginal delivery, independent of maternal weight status. Only during vaginal delivery was an association found between excess maternal prepregnancy weight and an altered neonatal gut microbiome composition [95]. [375_TD$IF]The microbiota of obese mothers was characterized by enriched Bacteroides and depleted Pseudomonas, Enterococcus, and Acinetobacter, and translated to altered metabolic functioning that has been associated with obesity in previous studies [95]. Additionally, using strain-level analysis, Makino et al. have shown that mother-to-infant transmission was found only among vaginally


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born infants, and not among C-section-born infants [69]. Further research is warranted since these study associations only apply to the microbial status directly after birth. Nevertheless, even among women entering pregnancy with excess weight, it appears that vaginal delivery is associated with a lower risk of childhood obesity compared to C-section delivery. The high prevalence of elective C-section births, especially in countries such as Brazil, is a major public health concern. These recently discovered health implications nullify the scheduling advantages and health economic benefits of C-sections, and they warrant strong consideration for policy change to prioritize natural childbirth when possible. When surgical intervention is necessary or inevitable, restoration of the neonate’s microbiome should be considered. To test this principle, a pilot study showed that a sterile gauze, first inoculated in the mother’s vagina, then rubbed over the skin and mouth of C-section-born babies, could partially restore the neonate’s microbiome to one similar to a vaginally born baby by 1 month of age [100]. However, this method does not account for the fecal and skin microbes that also pass to the infant at the time of vaginal birth, and other concerns have been raised about passing potential maternal pathogens to the infant [101]. Nevertheless, such studies have brought to the fore many public health issues associated with maternal and infant health, as well as healthcare costs and the promise of microbiome management [102].

Feeding the Newly Formed Infant Microbiome During the first 2 years of life, the infant acquires microbes from their diet, from the environment, and from people surrounding them. This period is characterized by dramatic dietary changes, new environmental exposures, and rapid development of the immune system, which all have a strong influence on the infant’s microbiome. The type of food (breast milk or formula, and later in life, solid food) is perhaps the most important factor shaping the gut microbiome [99]. Human breast milk plays a critical role in newborn development. Notwithstanding the high interindividual variability, there are around 50 genera (12 predominant, 16 subdominant) and 200 species [103] identified in breast milk, with roughly 103–104 cfu/ml in healthy women. The most commonly isolated genera include Staphylococcus, Streptococcus, Lactococcus, Leuconostoc, Weissella, Enterococcus, Propionibacterium, Lactobacillus, and Bifidobacterium [103]. Urbaniak et al. reported that the most abundant taxa in human milk are Staphylococcus (31%), Enterobacteriaceae (10%), Pseudomonas (17%), Streptococcus (5%), and Lactobacillus (3%) irrespective of gestation, mode of delivery (C-section vs. vaginal), or gender of the newborn [104]. However, antibiotics [105], chemotherapeutic agents, lactational stage [106], post-pregnancy BMI, and weight gain [106] can all influence the composition of human breast milk, and subsequently the infant microbiome. For instance, chemotherapy caused depletion of species of the genera Bifidobacterium, Eubacterium, Staphylococcus, and Cloacibacterium which are associated with health, in favor of Acinetobacter, Xanthomonadaceae, and Stenotrophomonas [107]. Milk microbiome changes during lactation itself have also been observed. Weissella, Leuconostoc, Staphylococcus, Streptococcus, and Lactococcus are dominant in colostrum, while Veillonella, Leptotrichia, and Prevotella are dominant in milk samples collected 1–6 months postpartum. All of these latter species are well recognized oral-cavityrelated species, which could be due to frequent interaction with the infant’s oral microbiota [106]. In addition, a higher abundance of Staphylococcus, namely S. aureus, was reported during the first 6 months of lactation in obese mothers, in addition to a higher abundance of Lactobacillus in the first month, compared to samples from mothers with normal weight [106]. The gut microbiota of formula-fed infants has been reported to be more diverse, with higher proportions of Bacteroides, Clostridium, and Enterobacteriaceae compared with breastfed infants, however with fewer bacterial cells [108]. Martin et al. also observed more diverse gut microbiota of formula-fed infants with higher total bacteria counts when analysing 108 healthy neonates in the first half year of life [99]. The gut microbiota of breastfed infants contains higher

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Box 4. Role of Breast Microbiota in Cancer and Mastitis

Outstanding Questions

The bacterial population present in human breast milk, and in breast tissue, appears to play a role not only in the development of a healthy infant gut microbiota, but also in women’s health. Studies have shown that mammary tissue contains a microbiota regardless of the location within the breast, the presence/absence of breast malignancy, country of origin, age, or history of pregnancy. Proteobacteria has been reported to be the most abundant phylum in breast tissue, unlike other body sites (vagina, oral cavity, skin, and gastrointestinal tract) where members of this phylum are not often isolated. This suggests that breast tissue may have somewhat of a unique microbiota, which might be the result of host microbial adaptation to the fatty acid environment in this tissue. However, the microbiota seems to differ in healthy women compared to those with breast cancer, suggesting an emerging role for bacterial dysbiosis in breast health [132,133]. For example, Xuan et al. have found strains of the genera Sphingomonas and Methylbacterium enriched, or a higher abundance of Escherichia coli, in tissue samples of women with breast cancer compared to healthy controls [132,134]. Analysis of breast tissue from mastitis (inflammation of the breast with or without infection) patients, suggests that opportunistic pathogens, such as the mutually cooperative species Staphylococcus and Corynebacterium, are kept in balance by normal microbiota [135]. These bacteria likely reach the tissue via the skin/nipple and blood, and they are one source of those present in milk. In addition, it appears that dendritic cells transport bacteria to the breast via gut translocation [136], but this remains to be confirmed in future studies. Overall, the breast microbiota needs to be considered as an important factor in maintaining a favorable health–disease balance in the breast.

What are the tipping points that trigger a vaginal pathobiont to become pathogenic? And what does this look like in recurrent infections?

proportions of Bifidobacterium, Lactobacillus, and Bacteroides – which are able to degrade the complex oligosaccharides present in human milk into small sugars (e.g., lactose) so as to utilize them as an energy source (Figure 3). The remaining small sugars can be metabolized by glycolytic microbes present in the gastrointestinal tract of the infant, such as Streptococcus, Staphylococcus, and Enterococcus. With the introduction of solid foods, during weaning, infants are exposed to more complex carbohydrates and other nutrients that drive the development of the gut microbiota. For example, Martine et al. observed that the introduction of solid food was associated with a higher prevalence of an Atopobium cluster and some butyrate-producing bacteria like the Clostridium coccoides group. Of note, the authors did not observe a reduction in bifidobacterial species after the introduction of solid foods, and the prevalence of B. longum subsp. longum was even higher [99]. Post-weaning changes in the microbiota are more pronounced, the microbiota being enriched with species that are prevalent in adults, including Bacteroidetes and Firmicutes from the Clostridia class: Clostridium, Ruminococcus, Faecalibacterium, Roseburia, and Anaerostipes [109]. Clearly, more studies are needed to determine what factors influence the human milk microbiome and how these changes impact neonatal and maternal health (Box 4) (Figure 2).

Are there better ways to detect BV and associated biofilm infections? How can we develop new ways of treating bacterial biofilms based on our knowledge of the female microbiome?

Which comes first in vaginal dysbiosis: overgrowth of vaginal pathogens or the loss of vaginal lactobacilli? How is the stability and resilience of the VMB maintained? Are keystone species or keystone functions more important for a resilient and stable microbiome?

How do microbes actually translocate to the breast tissue? And is there microbial migration between microbiome niches? How to differentiate true dysbiosis from natural fluctuations in the VMB or other microbiome niches? How does this impact diagnostics? What role do the female microbiomes have during reproduction and pregnancy?

Concluding Remarks The wide breadth of research themes covered in this article clearly demonstrates the extent of global relevance and interest in female-related microbes. The resulting health implications – not only for the health and wellbeing of women, but also for their children – strengthen the notion that microbes are among the most important things that we inherit from our mothers. While the knowledge concerning the reproductive microbiomes and their impact on human health is increasing, it is still unclear what can be done to improve women’s health in order to improve the health of children and their subsequent adult health (see Outstanding Questions). In an era where chronic disease is responsible for the majority of healthcare expenditure in the developed world, it would surely be logical to invest in interventions that significantly impact these public health issues by enhancing the quality of life and reducing the economic burden. The origins of many diseases are hugely influenced by the health of the mother, and as friendly and unfriendly microbes clearly play a major role, it seems astounding that research funding from governments and nongovernmental granting agencies is alarmingly sparse and still far from being a priority. It is unacceptable that no breakthroughs have occurred in managing bladder and vaginal infections in over 40 years! Furthermore, the sole and unrelenting use of untargeted antimicrobial agents or unnecessary medical interventions for treating and managing these microbialrelated conditions, suggests that the wellbeing of women is not important. We counter that, without reproductive health, there would be no humanity to make such decisions, and thus, we must start prioritizing the female microbiomes.


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Acknowledgments The organizational committee of ‘Women & Their Microbes’ thanks all the speakers and attendees whose participation and knowledge have made each conference thus far such a success. ‘Women & Their Microbes’ has been financially supported by BioClin B.V., Winclove Probiotics B.V., and Bifodan A/S. J.A. Younes founded the conference in 2015 (www.womenandtheirmicrobes.com) and currently works for Winclove Probiotics. E. Lievens holds a PhD grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen). M. Petrova holds a postdoctoral grant from the Fund for Scientific Research (FWO Vlaanderen).

Resources i

https://www.eiseverywhere.com/file_uploads/9fe8513bac41824a2442d084d2b5e457_JannekevandeWijgert.pdf

References 1.

Gilbert, S.F. et al. (2012) A symbiotic view of life: we have never been individuals. Q. Rev. Biol. 87, 325–341

2.

Bordenstein, S.R. and Theis, K.R. (2015) Host biology in light of the microbiome: ten principles of holobionts and hologenomes. PLoS Biol. 13, e1002226

21. Simhan, H.N. et al. (2005) The vaginal inflammatory milieu and the risk of early premature preterm rupture of membranes. Am. J. Obstet. Gynecol. 192, 213–218 22. Swidsinski, A. et al. (2008) An adherent Gardnerella vaginalis biofilm persists on the vaginal epithelium after standard therapy with oral metronidazole. Am. J. Obstet. Gynecol. 198, 97–106

3.

Reid, G. (2016) Cervicovaginal microbiomes – threats and possibilities. Trends Endocrinol. Metab. 27, 446–454

4.

Petrova, M.I. et al. (2015) Lactobacillus species as biomarkers and agents that can promote various aspects of vaginal health. Front. Physiol. 6, 81

5.

Ravel, J. et al. (2011) Vaginal microbiome of reproductive-age women. Proc. Natl. Acad. Sci. U. S. A. 1, 4680–4687

24. Schellenberg, J.J. et al. (2016) Gardnerella vaginalis subgroups defined by cpn60 sequencing and sialidase activity in isolates from Canada, Belgium and Kenya. PLoS One 11, e0146510

6.

van de Wijgert, J.H. et al. (2014) The vaginal microbiota: what have we learned after a decade of molecular characterization? PLoS One 9, e105998

25. Hardy, L. et al. (2016) A fruitful alliance: the synergy between Atopobium vaginae and Gardnerella vaginalis in bacterial vaginosis-associated biofilm. Sex. Transm. Infect. 92, 487–491

7.

Macklaim, J.M. et al. (2011) At the crossroads of vaginal health and disease, the genome sequence of Lactobacillus iners AB-1. Proc. Natl. Acad. Sci. U. S. A. 108 (Suppl. 1), 4688–4695

8.

Mendes-Soares, H. et al. (2014) Comparative functional genomics of Lactobacillus spp. reveals possible mechanisms for specialization of vaginal lactobacilli to their environment. J. Bacteriol. 196, 1458–1470

26. van der Veer, C. (2016) The cervicovaginal microbiota in women notified for Chlamydia trachomatis infection: a case-control study at the sexually transmitted infection outpatient clinic in Amsterdam, The Netherlands. Clin. Infect. Dis. 64, 24–31

9.

Gong, Z. et al. (2014) Lactobacilli inactivate Chlamydia trachomatis through lactic acid but not H2O2. PLoS One 9, e107758

10. Juarez Tomas, M.S. et al. (2003) Growth and lactic acid production by vaginal Lactobacillus acidophilus CRL 1259, and inhibition of uropathogenic Escherichia coli. J. Med. Microbiol. 52, 1117–1124

23. Harwich, M.D., Jr et al. (2010) Drawing the line between commensal and pathogenic Gardnerella vaginalis through genome analysis and virulence studies. BMC Genomics 11, 375

27. Marrazzo, J.M. et al. (2012) Extravaginal reservoirs of vaginal bacteria as risk factors for incident bacterial vaginosis. J. Infect. Dis. 205, 1580–1588 28. Castro, J. et al. (2016) Escherichia coli and Enterococcus faecalis are able to incorporate and enhance a pre-formed Gardnerella vaginalis biofilm. Pathog. Dis. 74, ftw007 29. Machado, A. and Cerca, N. (2015) Influence of biofilm formation by Gardnerella vaginalis and other anaerobes on bacterial vaginosis. J. Infect. Dis. 212, 1856–1861

11. Graver, M.A. and Wade, J.J. (2011) The role of acidification in the inhibition of Neisseria gonorrhoeae by vaginal lactobacilli during anaerobic growth. Ann. Clin. Microbiol. Antimicrob. 10, 8

30. Patterson, J.L. et al. (2007) Effect of biofilm phenotype on resistance of Gardnerella vaginalis to hydrogen peroxide and lactic acid. Am. J. Obstet. Gynecol. 197, 170–177

12. Conti, C. et al. (2009) Inhibition of herpes simplex virus type 2 by vaginal lactobacilli. J. Physiol. Pharmacol. 60 (Suppl. 6), 19–26

31. Fichorova, R.N. et al. (2013) The villain team-up or how Trichomonas vaginalis and bacterial vaginosis alter innate immunity in concert. Sex. Transm. Infect. 89, 460–466

13. Shukair, S.A. et al. (2013) Human cervicovaginal mucus contains an activity that hinders HIV-1 movement. Mucosal Immunol. 6, 427–434 14. Sgibnev, A. and Kremleva, E. (2016) Influence of hydrogen peroxide, lactic acid, and surfactants from vaginal lactobacilli on the antibiotic sensitivity of opportunistic bacteria. Probiotics Antimicrob. Proteins 9, 131–141 15. Srinivasan, U. et al. (2009) Vaginal and oral microbes, host genotype and preterm birth. Med. Hypotheses 73, 963–975 16. Osset, J. et al. (2001) Assessment of the capacity of Lactobacillus to inhibit the growth of uropathogens and block their adhesion to vaginal epithelial cells. J. Infect. Dis. 183, 485–491 17. Mastromarino, P. et al. (2002) Characterization and selection of vaginal Lactobacillus strains for the preparation of vaginal tablets. J. Appl. Microbiol. 93, 884–893 18. Zarate, G. and Nader-Macias, M.E. (2006) Influence of probiotic vaginal lactobacilli on in vitro adhesion of urogenital pathogens to vaginal epithelial cells. Lett. Appl. Microbiol. 43, 174–180 19. Phukan, N. et al. (2013) The adherence of Trichomonas vaginalis to host ectocervical cells is influenced by lactobacilli. Sex. Transm. Infect. 89, 455–459 20. Ma, B. et al. (2012) Vaginal microbiome: rethinking health and disease. Annu. Rev. Microbiol. 66, 371–389

14


32. Kyongo, J.K. et al. (2012) Searching for lower female genital tract soluble and cellular biomarkers: defining levels and predictors in a cohort of healthy Caucasian women. PLoS One 7, e43951 33. Nikolaitchouk, N. et al. (2008) The lower genital tract microbiota in relation to cytokine-, SLPI- and endotoxin levels: application of checkerboard DNA–DNA hybridization (CDH). APMIS 116, 263– 277 34. Balkus, J. et al. (2010) Effects of pregnancy and bacterial vaginosis on proinflammatory cytokine and secretory leukocyte protease inhibitor concentrations in vaginal secretions. J. Pregnancy 2010, 385981 35. Dezzutti, C.S. et al. (2011) Performance of swabs, lavage, and diluents to quantify biomarkers of female genital tract soluble mucosal mediators. PLoS One 6, e23136 36. Doerflinger, S.Y. et al. (2014) Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner. J. Infect. Dis. 209, 1989–1999 37. Borgdorff, H. et al. (2016) Cervicovaginal microbiome dysbiosis is associated with proteome changes related to alterations of the cervicovaginal mucosal barrier. Mucosal Immunol. 9, 621–633


38. Mitchell, C. and Marrazzo, J. (2014) Bacterial vaginosis and the cervicovaginal immune response. Am. J. Reprod. Immunol. 71, 555–563

62. Rautava, S. et al. (2012) Probiotics modulate host–microbe interaction in the placenta and fetal gut: a randomized, double-blind, placebo-controlled trial. Neonatology 102, 178–184

39. Rose, W.A. et al. (2012) Commensal bacteria modulate innate immune responses of vaginal epithelial cell multilayer cultures. PLoS One 7, e32728

63. Zaura, E. et al. (2014) Acquiring and maintaining a normal oral microbiome: current perspective. Front. Cell. Infect. Microbiol. 4, 85

40. Fichorova, R.N. et al. (2011) Baseline variation and associations between subject characteristics and five cytokine biomarkers of vaginal safety among healthy non-pregnant women in microbicide trials. Cytokine 55, 134–140

64. Prince, A. (2012) The bitter taste of infection. J. Clin. Invest. 122, 3847–3849

41. Fichorova, R.N. et al. (2015) A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation. Toxicol. Appl. Pharmacol. 285, 198–206

66. Fujiwara, N. et al. (2017) Significant increase of oral bacteria in the early pregnancy period in Japanese women. J. Invest. Clin. Dent. 8, 10

42. Mandar, R. et al. (2015) Complementary seminovaginal microbiome in couples. Res. Microbiol. 166, 440–447 43. Hou, D. et al. (2013) Microbiota of the seminal fluid from healthy and infertile men. Fertil. Steril. 100, 1261–1269 44. Jarvi, K. et al. (1996) Polymerase chain reaction-based detection of bacteria in semen. Fertil. Steril. 66, 463–467 45. Kiessling, A.A. et al. (2008) Detection and identification of bacterial DNA in semen. Fertil. Steril. 90, 1744–1756 46. Mandar, R. et al. (2017) Seminal microbiome in men with and without prostatitis. Int. J. Urol. 24, 211–216 47. Weng, S.L. et al. (2014) Bacterial communities in semen from men of infertile couples: metagenomic sequencing reveals relationships of seminal microbiota to semen quality. PLoS One 9, e110152 48. Barbonetti, A. et al. (2011) Effect of vaginal probiotic lactobacilli on in vitro-induced sperm lipid peroxidation and its impact on sperm motility and viability. Fertil. Steril. 95, 2485–2488 49. Barbonetti, A. et al. (2013) Soluble products of Escherichia coli induce mitochondrial dysfunction-related sperm membrane lipid peroxidation which is prevented by lactobacilli. PLoS One 8, e83136 50. Zozaya, M. et al. (2016) Bacterial communities in penile skin, male urethra, and vaginas of heterosexual couples with and without bacterial vaginosis. Microbiome 4, 16 51. Hyman, R.W. et al. (2012) The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer. J. Assist. Reprod. Genet. 29, 105–115 52. Salah, R.M. et al. (2013) Bacterial vaginosis and infertility: cause or association? Eur. J. Obstet. Gynecol. Reprod. Biol. 167, 59–63 53. van Oostrum, N. et al. (2013) Risks associated with bacterial vaginosis in infertility patients: a systematic review and metaanalysis. Hum. Reprod. 28, 1809–1815 54. Haahr, T. et al. (2016) Abnormal vaginal microbiota may be associated with poor reproductive outcomes: a prospective study in IVF patients. Hum. Reprod. 31, 795–803 55. Pelzer, E.S. et al. (2013) Microorganisms within human follicular fluid: effects on IVF. PLoS One 8, e59062 56. Franasiak, J.M. et al. (2016) Endometrial microbiome at the time of embryo transfer: next-generation sequencing of the 16S ribosomal subunit. J. Assist. Reprod. Genet. 33, 129–136 57. Verstraelen, H. et al. (2016) Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene. PeerJ 4, e1602 58. Moreno, I. et al. (2016) Evidence that the endometrial microbiota has an effect on implantation success or failure. Am. J. Obstet. Gynecol. 215, 684–703 59. Robertson, S.A. (2010) Immune regulation of conception and embryo implantation – all about quality control? J. Reprod. Immunol. 85, 51–57 60. Sisti, G. et al. (2016) Maternal immunity and pregnancy outcome: focus on preconception and autophagy. Genes Immun. 17, 1–7 61. Aagaard, K. et al. (2014) The placenta harbors a unique microbiome. Sci. Transl. Med. 6, 237ra65

65. Koren, O. et al. (2012) Host remodeling of the gut microbiome and metabolic changes during pregnancy. Cell 150, 470–480

67. Collado, M.C. et al. (2016) Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid. Sci. Rep. 6, 23129 68. Jimenez, E. et al. (2005) Isolation of commensal bacteria from umbilical cord blood of healthy neonates born by cesarean section. Curr. Microbiol. 51, 270–274 69. Makino, H. et al. (2011) Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant, determined by multilocus sequencing typing and amplified fragment length polymorphism. Appl. Environ. Microbiol. 77, 6788–6793 70. Makino, H. et al. (2013) Mother-to-infant transmission of intestinal bifidobacterial strains has an impact on the early development of vaginally delivered infant’s microbiota. PLoS One 8, e78331 71. Christian, P. et al. (2010) Prenatal micronutrient supplementation and intellectual and motor function in early school-aged children in Nepal. JAMA 304, 2716–2723 72. Chang, C.Y. et al. (2009) Essential fatty acids and human brain. Acta Neurol. Taiwan. 18, 231–241 73. Ogawa, J. et al. (2005) Production of conjugated fatty acids by lactic acid bacteria. J. Biosci. Bioeng. 100, 355–364 74. Rossi, M. et al. (2011) Folate production by probiotic bacteria. Nutrients 3, 118–134 75. Ganguli, K. et al. (2015) Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut. Pediatr. Res. 77, 528–535 76. Janvier, A. et al. (2014) Cohort study of probiotics in a North American neonatal intensive care unit. J. Pediatr. 164, 980–985 77. Ilmonen, J. et al. (2011) Impact of dietary counselling and probiotic intervention on maternal anthropometric measurements during and after pregnancy: a randomized placebo-controlled trial. Clin. Nutr. 30, 156–164 78. Laitinen, K. et al. (2009) Probiotics and dietary counselling contribute to glucose regulation during and after pregnancy: a randomised controlled trial. Br. J. Nutr. 101, 1679–1687 79. Luoto, R. et al. (2012) Impact of maternal probiotic-supplemented dietary counseling during pregnancy on colostrum adiponectin concentration: a prospective, randomized, placebocontrolled study. Early Hum. Dev. 88, 339–344 80. Huurre, A. et al. (2008) Impact of maternal atopy and probiotic supplementation during pregnancy on infant sensitization: a double-blind placebo-controlled study. Clin. Exp. Allergy 38, 1342–1348 81. Kukkonen, K. et al. (2007) Probiotics and prebiotic galactooligosaccharides in the prevention of allergic diseases: a randomized, double-blind, placebo-controlled trial. J. Allergy Clin. Immunol. 119, 192–198 82. Tremaroli, V. et al. (2015) Roux-en-Y gastric bypass and vertical banded gastroplasty induce long-term changes on the human gut microbiome contributing to fat mass regulation. Cell Metab. 22, 228–238 83. Vrieze, A. et al. (2012) Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome. Gastroenterology 143, 913–916 84. Lindsay, K.L. et al. (2014) Probiotics in obese pregnancy do not reduce maternal fasting glucose: a double-blind, placebo-controlled, randomized trial (Probiotics in Pregnancy Study). Am. J. Clin. Nutr. 99, 1432–1439


15


85. Neish, A.S. (2009) Microbes in gastrointestinal health and disease. Gastroenterology 136, 65–80 86. Rijkers, G.T. et al. (2010) Guidance for substantiating the evidence for beneficial effects of probiotics: current status and recommendations for future research. J. Nutr. 140, 671S–676S 87. Mueller, N.T. et al. (2015) The infant microbiome development: mom matters. Trends Mol. Med. 21, 109–117 88. Mueller, N.T. et al. (2015) Prenatal exposure to antibiotics, cesarean section and risk of childhood obesity. Int. J. Obes. (Lond) 39, 665–670 89. Cox, L.M. et al. (2014) Altering the intestinal microbiota during a critical developmental window has lasting metabolic consequences. Cell 158, 705–721 90. Dominguez-Bello, M.G. et al. (2010) Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc. Natl. Acad. Sci. U. S. A. 107, 11971–11975

108. Bezirtzoglou, E. et al. (2011) Microbiota profile in feces of breastand formula-fed newborns by using fluorescence in situ hybridization (FISH). Anaerobe 17, 478–482 109. Backhed, F. et al. (2015) Dynamics and stabilization of the human gut microbiome during the first year of life. Cell Host Microbe 17, 690–703 110. Bevins, C.L. and Salzman, N.H. (2011) The potter’s wheel: the host’s role in sculpting its microbiota. Cell. Mol. Life Sci. 68, 3675–3685 111. Blekhman, R. et al. (2015) Host genetic variation impacts microbiome composition across human body sites. Genome Biol. 16, 191 112. Goodrich, J.K. et al. (2016) Cross-species comparisons of host genetic associations with the microbiome. Science 352, 532–535 113. Knights, D. et al. (2014) Complex host genetics influence the microbiome in inflammatory bowel disease. Genome Med. 6, 107

91. Bonifacio, E. et al. (2011) Cesarean section and interferoninduced helicase gene polymorphisms combine to increase childhood type 1 diabetes risk. Diabetes 60, 3300–3306

114. Goodrich, J.K. et al. (2014) Human genetics shape the gut microbiome. Cell 159, 789–799

92. Cardwell, C.R. et al. (2008) Caesarean section is associated with an increased risk of childhood-onset type 1 diabetes mellitus: a meta-analysis of observational studies. Diabetologia 51, 726–735

116. Paavonen, J. (1983) Physiology and ecology of the vagina. Scand. J. Infect. Dis. Suppl. 40, 31–35

93. Li, H.T. et al. (2013) The impact of cesarean section on offspring overweight and obesity: a systematic review and meta-analysis. Int. J. Obes. (Lond) 37, 893–899 94. Ridaura, V.K. et al. (2013) Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 341, 1241214 95. Mueller, N.T. et al. (2016) Birth mode-dependent association between pre-pregnancy maternal weight status and the neonatal intestinal microbiome. Sci. Rep. 6, 23133 96. Collado, M.C. et al. (2008) Distinct composition of gut microbiota during pregnancy in overweight and normal-weight women. Am. J. Clin. Nutr. 88, 894–899 97. Collado, M.C. et al. (2010) Effect of mother’s weight on infant’s microbiota acquisition, composition, and activity during early infancy: a prospective follow-up study initiated in early pregnancy. Am. J. Clin. Nutr. 92, 1023–1030 98. Galley, J.D. et al. (2014) Maternal obesity is associated with alterations in the gut microbiome in toddlers. PLoS One 9, e113026 99. Martin, R. et al. (2016) Early-life events, including mode of delivery and type of feeding, siblings and gender, shape the developing gut Microbiota. PLoS One 11, e0158498 100. Dominguez-Bello, M.G. et al. (2016) Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer. Nat. Med. 22, 250–253 101. Cunnington, A.J. et al. (2016) ‘Vaginal seeding’ of infants born by caesarean section. BMJ 352, i227

115. Ley, R.E. (2015) The gene–microbe link. Nature 518, S7

117. Gregoire, A.T. et al. (1971) The glycogen content of human vaginal epithelial tissue. Fertil. Steril. 22, 64–68 118. Dei, M. et al. (2010) Vulvovaginitis in childhood. Best Pract. Res. Clin. Obstet. Gynaecol. 24, 129–137 119. Randelovic, G. et al. (2012) Microbiological aspects of vulvovaginitis in prepubertal girls. Eur. J . Pediatr. 171, 1203–1208 120. Yamamoto, T. et al. (2009) Bacterial populations in the vaginas of healthy adolescent women. J. Pediatr. Adolesc. Gynecol. 22, 11–18 121. Gupta, S. et al. (2006) Vaginal microflora in postmenopausal women on hormone replacement therapy. Indian J. Pathol. Microbiol. 49, 457–461 122. Hummelen, R. et al. (2011) Vaginal microbiome and epithelial gene array in post-menopausal women with moderate to severe dryness. PLoS One 6, e26602 123. Bradshaw, C.S. et al. (2006) High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. J. Infect. Dis. 193, 1478–1486 124. Donlan, R.M. and Costerton, J.W. (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15, 167–193 125. Cerca, N. et al. (2005) Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry. J. Antimicrob. Chemother. 56, 331–336

102. Clemente, J.C. and Dominguez-Bello, M.G. (2016) Safety of vaginal microbial transfer in infants delivered by caesarean, and expected health outcomes. BMJ 352, i1707

126. Swidsinski, A. et al. (2015) Polymicrobial Gardnerella biofilm resists repeated intravaginal antiseptic treatment in a subset of women with bacterial vaginosis: a preliminary report. Arch. Gynecol. Obstet. 291, 605–609

103. Jost, T. et al. (2015) Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment and gut health. Nutr. Rev. 73, 426–437

127. Hardy, L. et al. (2017) Bacterial biofilms in the vagina. Res. Microbiol. http://dx.doi.org/10.1016/j.resmic.2017.02.001 February 21, 2017

104. Urbaniak, C. et al. (2016) Human milk microbiota profiles in relation to birthing method, gestation and infant gender. Microbiome 4, 1

128. Petrova, M.I. et al. (2016) Lectin-like molecules of Lactobacillus rhamnosus GG inhibit pathogenic Escherichia coli and Salmonella biofilm formation. PLoS One 11, e0161337

105. Soto, A. et al. (2014) Lactobacilli and bifidobacteria in human breast milk: influence of antibiotherapy and other host and clinical factors. J. Pediatr. Gastroenterol. Nutr. 59, 78–88

129. Petrova, M.I. et al. (2016) The lectin-like protein 1 in Lactobacillus rhamnosus GR-1 mediates tissue-specific adherence to vaginal epithelium and inhibits urogenital pathogens. Sci. Rep. 6, 37437

106. Cabrera-Rubio, R. et al. (2012) The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery. Am. J. Clin. Nutr. 96, 544–551

130. Velraeds, M.M. et al. (2000) Inhibition of uropathogenic biofilm growth on silicone rubber in human urine by lactobacilli – a teleologic approach. World J. Urol. 18, 422–426

107. Urbaniak, C. et al. (2014) Effect of chemotherapy on the microbiota and metabolome of human milk, a case report. Microbiome 2, 24

131. Gudina, E.J. et al. (2010) Antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20. Lett. Appl. Microbiol. 50, 419–424

16



132. Urbaniak, C. et al. (2014) Microbiota of human breast tissue. Appl. Environ. Microbiol. 80, 3007–3014 133. Urbaniak, C. et al. (2016) The microbiota of breast tissue and its association with breast cancer. Appl. Environ. Microbiol. 82, 5039–5048 134. Xuan, C. et al. (2014) Microbial dysbiosis is associated with human breast cancer. PLoS One 9, e83744

135. Ma, Z.S. et al. (2015) Network analysis suggests a potentially ‘evil’ alliance of opportunistic pathogens inhibited by a cooperative network in human milk bacterial communities. Sci. Rep. 5, 8275 136. Rescigno, M. et al. (2001) Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol. 2, 361–367


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