**5. Microbiome of the reproductive tract**

As more is learned about the human microbiome, it is becoming evident that it meaningfully affects the physiologic function of virtually every organ where bacteria are present.

The human body is colonized with an order of magnitude more bacteria than human cells in the body [81]. The majority of published medical literature focuses on the subset of the microbiome involved in pathogenesis, and only a subset focuses on the physiologic role that the microbiome plays.

The importance of this was recognized in 2001 at the time of the human genome published [82], when scientists called for a "second human genome project" that would investigate the normal microbiome colonies at various sites to understand the synergistic interactions between the microbiome and its host [83]. Several initiatives commenced worldwide, and in the United States the Human Microbiome Project led by the National Institutes of Health was launched in 2007, using high throughput sequencing technologies to characterize the human micro‐ biome in 250 normal healthy volunteers at multiple body sites [81].

The female reproductive tract has long been known to have an active microbiome (Figure 4). Although the greatest focus has been on the vaginal milieu, data have been accumulating for decades demonstrating that the remainder of the female reproductive axis is not sterile. In fact, with more than 20 studies completed, virtually all of them have found that there is a small but active microbiome in the uterine cavity.

Importantly, many of these studies obtained their samples at the time of surgery with the use of transfundal collection techniques where there was no potential for contamination from transiting the vagina or endocervical canal. The majority of these studies were done with the use of traditional culture techniques to identify any bacteria that were present.

More recently, metagenomic techniques are confirming earlier findings and providing a more comprehensive definition of the endometrial microbiome.

Reproduced with the permission from Franasiak et al. [100]. Copyright© 2015.

**Figure 4.** The human microbiome affects all facets of reproduction from gametogenesis, to fertilization and embryo mi‐ gration, to implantation with implications in early pregnancy failure, to involvement in late pregnancy loss, and poor obstetric outcomes during gestation and parturition in terms of intrauterine infection and preterm birth, among other things. A more complete characterization of this complex symbiosis is imperative as we understand its implications in human health and disease.

Interestingly, the microbiome extends above the endometrial cavity. Some studies have demonstrated bacteria in the fallopian tubes of women without obvious tubal pathology. Additional studies have demonstrated that the intra-follicular milieu may have an active microbiome in some patients.

Finally, there are now studies showing that the microbiome of the male reproductive axis is more complex than previously appreciated. The addition of metagenomic tools allowed descriptions of much broader and more complex microbiome, even in men without evidence of acute or chronic inflammation of their reproductive tract.

As the microbiome of the female and male reproductive axis has become more clearly defined, studies evaluating the clinical impact on ART treatment have followed. Given the influence which the microbiome has in virtually every organ systems, it is not surprising that subtle changes in the microbiome are associated with meaningful changes in gamete quality and ultimate clinical outcomes. In some cases, changes in the microbiome may provide insight into previously unexplained treatment failure [82].

#### **5.1. Diagnosis**

Interestingly, the microbiome extends above the endometrial cavity. Some studies have demonstrated bacteria in the fallopian tubes of women without obvious tubal pathology. Additional studies have demonstrated that the intra-follicular milieu may have an active

**Figure 4.** The human microbiome affects all facets of reproduction from gametogenesis, to fertilization and embryo mi‐ gration, to implantation with implications in early pregnancy failure, to involvement in late pregnancy loss, and poor obstetric outcomes during gestation and parturition in terms of intrauterine infection and preterm birth, among other things. A more complete characterization of this complex symbiosis is imperative as we understand its implications in

Finally, there are now studies showing that the microbiome of the male reproductive axis is more complex than previously appreciated. The addition of metagenomic tools allowed descriptions of much broader and more complex microbiome, even in men without evidence

As the microbiome of the female and male reproductive axis has become more clearly defined, studies evaluating the clinical impact on ART treatment have followed. Given the influence which the microbiome has in virtually every organ systems, it is not surprising that subtle changes in the microbiome are associated with meaningful changes in gamete quality and ultimate clinical outcomes. In some cases, changes in the microbiome may provide insight into

microbiome in some patients.

human health and disease.

118 Genital Infections and Infertility

of acute or chronic inflammation of their reproductive tract.

Reproduced with the permission from Franasiak et al. [100]. Copyright© 2015.

previously unexplained treatment failure [82].

It is important to briefly recognize that microbiome data are procured in one of two ways: culture-based or sequencing-based technology.

The various techniques available in metagenomics (fingerprinting, DNA microarrays, targeted sequencing, and whole genome sequencing) supply both strengths and weaknesses depending upon the primary purpose of the analysis.

Much of the early work describing the human microbiome comes from culture-based approaches using the 16S rRNA analysis of highly conserved genes as a way to character‐ ize the diversity of the microbiome in a given environment [84]. However, data from the vaginal microbiome suggest that many organisms can not be identified with the use of culture-based techniques, which results in underestimating the diversity of the ecosystem as well as failing to identify potentially important organisms when describing their relation‐ ship to health and disease [85]. Thus, culture-based data, though still informative, must be interpreted within the limits of the technology.

Data presented more recently have relied on 16S rRNA gene sequencing, specifically the hypervariable regions within the gene, which serves as a molecular fingerprint down to the genus and species level [86]. Although to date, data that describe the microbiome of the reproductive tract have not widely used this technique, metagenomics is becoming an increasingly widespread approach to describing the microbiome [87]. Using this method, also termed community genomics, analysis of microorganisms occurs by means of direct extraction and cloning of DNA from a grouping of organisms. It allows analysis that extends beyond phylogenetic descriptions and attempts to study the physiology and ecology of the microbiome [82].

The study of the microbiome and its relationship to the efficiency of conception and early pregnancy maintenance is just beginning. Although there have been efforts to distinguish between normal or favorable microbiomes and those that impair or limit clinical outcomes, early investigations are also identifying alterations in several physiologic processes. These alterations provide insight into reproductive failure in some patients. They may also provide the foundational information to guide the development of new therapeutic interventions that could improve outcomes and previously recalcitrant clinical circumstances.

The association between clinically evident infection, inflammation, and altered reproductive function is well established. Much of this inflammation involves secretion of a number of proinflammatory cytokines and growth factors secreted by immune cells, which are activated in response to the presence of apparent pathogens. In the case of small shifts in the microbiome, the resulting subtle changes in the local milieu are typically not clinically evident but may remain clinically meaningful; however, the exact molecular mechanisms are not well charac‐ terized.

Accumulations of a particular interleukin or some other cytokine are described, but detailed mechanisms are still lacking. It is possible that the influence of some components of the microbiome is not via direct interaction with the local organ system.

The microbiome of the vagina is typically dominated by Lactobacilli [88]. In fact, a normal milieu is defined by the presence of specific subspecies of Lactobacilli that are capable of acting as probiotics and inhibiting the overgrowth of other bacterial species. For example, Lactobacilli species capable of producing high levels of H2O2 are generally considered to be most favorable. This demonstrates an important concept that some components of the microbiome's principal function may be to alter or limit some other component of the microbiome. A direct interaction with the actual tissue may occur but is not essential.

It is becoming increasingly evident that the aggregate microbiome is not a simple accumulation of free-floating bacteria on the surface of a human tissue. In many cases, complex threedimensional lattices are formed, which may have one layer or may have an inner and an outer layer.

A protective outer coating composed of polysaccharide, nucleic acid, and protein may develop. At times, these biofilms may inhibit immune detection and reduce the effectiveness of antimicrobial treatment [89]. These three-dimensional structures spread across the surfaces of the tissues where they are located and are termed biofilms.

Biofilms are the subject of intensive investigation and may have important physiologic and pathophysiologic roles. Biofilms are routinely present in the vagina but commonly extend into the endometrial cavity [90] and even up into the fallopian tubes (Figure 5). Although no definitive conclusions regarding the role of biofilms of the reproductive axis have been established, it is important to understand that the relationship between the microbiome and the mullerian system may be more complex than the simple presence or absence of various species or bacteria or even their relative concentration. The interactions that lead to different biofilms and their subsequent impact on reproduction will provide important topics for future investigation.

The influence of the microbiome, most prevalent in the mullerian system, may extend to the remainder of the reproductive axis and may even affect gametogenesis. Indeed, ovarian follicles may have an active microbiome. Some investigators have found that some bacteria may adversely influence follicular development and may even inhibit gonadotropin respon‐ siveness. Similarly, the male reproductive axis may be adversely affected, with subtle changes in the microbiome being associated with altered semen parameters [82].

Most studies characterizing the influence of the microbiome on ART and clinical outcomes are largely association studies. Detailed mechanistic studies that could lead to new therapeutic approaches are possible but remain to be done.

#### **5.2. Vaginal microbiome**

In contrast to the Human Microbiome Project, which investigated normal healthy volunteers, several investigators have looked at the link between the vaginal microbiome and infertility in patients undergoing various forms of ART [82].

One such study prospectively analyzed 152 patients undergoing IVF. Of the 152 patients, 133 (87.5%) tested positive for one or more microorganisms and 19 (12.5%) tested completely

The microbiome of the vagina is typically dominated by Lactobacilli [88]. In fact, a normal milieu is defined by the presence of specific subspecies of Lactobacilli that are capable of acting as probiotics and inhibiting the overgrowth of other bacterial species. For example, Lactobacilli species capable of producing high levels of H2O2 are generally considered to be most favorable. This demonstrates an important concept that some components of the microbiome's principal function may be to alter or limit some other component of the microbiome. A direct interaction

It is becoming increasingly evident that the aggregate microbiome is not a simple accumulation of free-floating bacteria on the surface of a human tissue. In many cases, complex threedimensional lattices are formed, which may have one layer or may have an inner and an outer

A protective outer coating composed of polysaccharide, nucleic acid, and protein may develop. At times, these biofilms may inhibit immune detection and reduce the effectiveness of antimicrobial treatment [89]. These three-dimensional structures spread across the surfaces of

Biofilms are the subject of intensive investigation and may have important physiologic and pathophysiologic roles. Biofilms are routinely present in the vagina but commonly extend into the endometrial cavity [90] and even up into the fallopian tubes (Figure 5). Although no definitive conclusions regarding the role of biofilms of the reproductive axis have been established, it is important to understand that the relationship between the microbiome and the mullerian system may be more complex than the simple presence or absence of various species or bacteria or even their relative concentration. The interactions that lead to different biofilms and their subsequent impact on reproduction will provide important topics for future

The influence of the microbiome, most prevalent in the mullerian system, may extend to the remainder of the reproductive axis and may even affect gametogenesis. Indeed, ovarian follicles may have an active microbiome. Some investigators have found that some bacteria may adversely influence follicular development and may even inhibit gonadotropin respon‐ siveness. Similarly, the male reproductive axis may be adversely affected, with subtle changes

Most studies characterizing the influence of the microbiome on ART and clinical outcomes are largely association studies. Detailed mechanistic studies that could lead to new therapeutic

In contrast to the Human Microbiome Project, which investigated normal healthy volunteers, several investigators have looked at the link between the vaginal microbiome and infertility

One such study prospectively analyzed 152 patients undergoing IVF. Of the 152 patients, 133 (87.5%) tested positive for one or more microorganisms and 19 (12.5%) tested completely

in the microbiome being associated with altered semen parameters [82].

approaches are possible but remain to be done.

in patients undergoing various forms of ART [82].

**5.2. Vaginal microbiome**

with the actual tissue may occur but is not essential.

the tissues where they are located and are termed biofilms.

layer.

120 Genital Infections and Infertility

investigation.

Reproduced with the permission from Swidsinski et al.Reproductive tract microbiome in ART. Fertil Steril. Copy‐ right© 2015.

**Figure 5.** Polymicrobial biofilm dominated by Gardnerella attached to the endometrium. The left panel shows follicu‐ lar and the right panel shows luteal endometrium.

negative for bacterial contamination. The most common microorganisms identified were *Lactobacillus* species, *Staphylococcus* species, and Enterobacteriaceae, including *Escherichia coli*, Klebsiella, and Proteus. Outcomes data showed that implantation rates were 12.4% in those with one or more bacteria present versus 14% in those completely negative (P<0.001).

Additionally, patients testing positive for Enterobacteriaceae and *Staphylococcus* had lower pregnancy rates than the negative culture group. Although this study provided some insight into the microbiome during IVF treatment, it highlighted the limitations associated with culture-based technology for evaluation of the microbiome. The fact that 12.5% of patients were completely negative for bacterial contamination suggests that the culture-based techni‐ que significantly underrepresents both the presence and the diversity of the microbiome at the time of ET.

A subsequent study with the use of 16S sequencing technology took a more robust look at the vaginal microbiome in the infertile patient undergoing IVF [90]. The investigators approached the study design with the hypothesis that, given that the vaginal microbiome has changes during the normal menstrual cycle with varied estrogen levels in the physiologic range [91], controlled ovarian hyperstimulation required to achieve success in IVF would also affect the vaginal microbiome [82].

#### **5.3. Uterine microbiome**

Just as alterations in the microbial environment affect vaginal health, these fluctuations may also predispose to upper genital tract infection, such as PID.

Direct culture or sequencing of samples from upper genital tract structures has been performed less frequently than the evaluation of vaginal samples, but available data confirm that BVassociated bacteria can be isolated from the upper genital tract [92].

In a study of 45 women with laparoscopically confirmed acute salpingitis (cases) and 44 women seeking bilateral tubal ligation (controls), 16S rDNA PCR detected bacteria in the fallopian tubes of 24% of cases and none of the controls [93]. Several of the specimens contained bacteria associated with BV, such as *Atopobium vaginae*, as well as *Leptotrichia* species and *N. gonorrhoea*.

The identification of causal microorganisms in upper genital infection is important for understanding disease pathogenesis and provides insight into why some cases are resistant to conventional treatment. The presence of certain organisms in the vagina is physiologic, and bacteria may also exist in the upper cervix and uterus during states of health.

In a study, by performing quantitative PCR on endometrial and upper cervical swabs from 58 women undergoing hysterectomy for benign conditions, at least one bacterial species was found in the upper genital tract of 95% of the subjects. The most frequently detected species were *Lactobacillus iners* (45%), *Prevotella* species (33%), and *Lactobacillus crispatus* (33%) [94].

An important consideration is that the upper cervix and uterus were grouped together as the ''upper genital tract'';however, these sites may contain different bacterial species or propor‐ tions of bacteria. For instance, there was a statistically significant difference in the proportion of upper genital tract bacteria based on race. African American and Hispanic women were more likely to harbor an upper genital tract microbiome dominated by a non-*Lactobacilli* species (83% and 75%, respectively) compared with Caucasian women (54%). These results mirror those of vaginal microbiome in that non-*Lactobacilli* species were more common in African American and Hispanic women, but the clinical implications of these findings are unclear.

Furthermore, there was no evidence of significant inflammation in the endometrial samples that contained bacteria typically found in the vaginal tract. Possible explanations for the lack of inflammation include vaginal contamination of the uterine samples or that molecular methods detected RNA of non-living organisms that did not affect clinical status.

Alternatively, it remains a possibility that certain bacteria in the upper cervix and uterus may serve important roles in maintaining homeostasis and may not necessarily represent pathology [92].

#### **5.4. Ovarian follicle microbiome**

Human follicular fluids have been extensively cultured and found to have an active micro‐ biome in many patients.

Although some specimens were collected from follicular aspirate attained at the time of transvaginal oocyte retrieval, others were collected laparoscopically [95]. It is not clearly established whether the bacteria that were cultured represent true colonization or merely contamination of the ovarian follicular fluid at the time of puncture for transvaginal oocyte aspiration [96].

Studies simultaneously evaluating the vagina, endocervix, endometrium, fallopian tube, follicular fluid, and peritoneal cavity are lacking.

Current studies looking at the microbiome of the follicle have used culture techniques. Early culture studies have suggested that an active follicular microbiome does affect ART outcomes. Interestingly, the impact of the microbiome is influenced by the clinical diagnosis of the female partner.

Diminished fertilization and development rates as well as reduced transfer and implantation rates have been noted in women with endometriosis, but not in women with ovulatory dysfunction or male-factor infertility [97, 98]. This may suggest that a more complex mecha‐ nism with an altered immune response present in women with endometriosis may produce a different reaction to the presence of an active microbiome and then also influence the devel‐ oping oocyte.

It may also be important to note that an active microbiome is not always a negative find‐ ing. Pelzer et al. [99] noted that outcomes improved when Lactobacilli were present. This is in sharp contrast to the presence of other species, such as *Propionibacterium* and *Actinomy‐ ces*, among others, where impaired clinical outcomes were documented. They also ob‐ served differences in the microbiome between the left and right ovaries, which were attributed to differences in hematogenous spread. The clinical relevance of this finding remains to be fully characterized [82].

At the present time, data are still accumulating regarding the significance of the follicular microbiome and the need for screening. Additional studies, particularly those using metage‐ nomic approaches, are needed.

#### **5.5. ART and microbiome**

**5.3. Uterine microbiome**

122 Genital Infections and Infertility

pathology [92].

**5.4. Ovarian follicle microbiome**

biome in many patients.

Just as alterations in the microbial environment affect vaginal health, these fluctuations may

Direct culture or sequencing of samples from upper genital tract structures has been performed less frequently than the evaluation of vaginal samples, but available data confirm that BV-

In a study of 45 women with laparoscopically confirmed acute salpingitis (cases) and 44 women seeking bilateral tubal ligation (controls), 16S rDNA PCR detected bacteria in the fallopian tubes of 24% of cases and none of the controls [93]. Several of the specimens contained bacteria associated with BV, such as *Atopobium vaginae*, as well as *Leptotrichia* species and *N. gonorrhoea*.

The identification of causal microorganisms in upper genital infection is important for understanding disease pathogenesis and provides insight into why some cases are resistant to conventional treatment. The presence of certain organisms in the vagina is physiologic, and

In a study, by performing quantitative PCR on endometrial and upper cervical swabs from 58 women undergoing hysterectomy for benign conditions, at least one bacterial species was found in the upper genital tract of 95% of the subjects. The most frequently detected species were *Lactobacillus iners* (45%), *Prevotella* species (33%), and *Lactobacillus crispatus* (33%) [94].

An important consideration is that the upper cervix and uterus were grouped together as the ''upper genital tract'';however, these sites may contain different bacterial species or propor‐ tions of bacteria. For instance, there was a statistically significant difference in the proportion of upper genital tract bacteria based on race. African American and Hispanic women were more likely to harbor an upper genital tract microbiome dominated by a non-*Lactobacilli* species (83% and 75%, respectively) compared with Caucasian women (54%). These results mirror those of vaginal microbiome in that non-*Lactobacilli* species were more common in African American and Hispanic women, but the clinical implications of these findings are unclear.

Furthermore, there was no evidence of significant inflammation in the endometrial samples that contained bacteria typically found in the vaginal tract. Possible explanations for the lack of inflammation include vaginal contamination of the uterine samples or that molecular

Alternatively, it remains a possibility that certain bacteria in the upper cervix and uterus may serve important roles in maintaining homeostasis and may not necessarily represent

Human follicular fluids have been extensively cultured and found to have an active micro‐

methods detected RNA of non-living organisms that did not affect clinical status.

bacteria may also exist in the upper cervix and uterus during states of health.

also predispose to upper genital tract infection, such as PID.

associated bacteria can be isolated from the upper genital tract [92].

Data have been gathered on the microbiome at every stage and level of human reproduction from the ovary, follicle and oocyte to testes and semen/spermatozoa and to the fallopian tube, uterus, cervix, and vagina. Both the male and female reproductive tracts exhibit complexity and diversity only realized within the last decade.

Furthermore, it is not enough to simply qualitatively or even quantitatively explore the reproductive tract microbiome using metagenomics. Understanding that these bacteria are not simply free-floating on the surface of tissue, but form their own three-dimensional biofilms with inner and outer layers, adds an additional complexity and could be of great importance if they were further explored. The fact these biofilms exist from the vagina to the fallopian tubes, allows complex and dynamic interactions between the gametes and embryo, as well as the maternal tissue interface.

To date, the assisted reproductive technology literature describing attempts to alter the microbiome in the reproductive tract in order to impact outcomes has been operating on a rudimentary understanding of this complex environment at best. However, this approach, although perhaps to blunt a tool at present, may indeed be an important key to altering both the microbiome and subsequently the immune system as we further explore enhancement of reproductive competence in assisted reproductive technology.

#### **5.6. Conclusion**

Knowledge regarding the interactions between the microbiome and the human reproductive axis is growing rapidly. A deeper understanding of normal physiology, identification of different dysbioses, and characterizing the microbiome's impact on reproductive outcomes promise meaningful enhancements in clinical care. While much has been learned since the early contributions of Semmelweis, the most insightful and powerful findings may lie just ahead [100].
