Human Reproduction Update, Vol.28, No.1, pp.

92–131, 2022
Advance Access Publication on October 27, 2021 doi:10.1093/humupd/dmab035

The role of gut and genital microbiota

and the estrobolome in endometriosis,
infertility and chronic pelvic pain
Mary E. Salliss1,2, Leslie V. Farland 3,4
, Nichole D. Mahnert1,5, and
Melissa M. Herbst-Kralovetz 1,6,*

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1
Department of Obstetrics and Gynecology, University of Arizona—College of Medicine, Phoenix, AZ, USA 2Department of Biology and
Biochemistry, Bath University, Bath, UK 3Department of Epidemiology and Biostatistics, Mel and Enid Zuckerman College of Public Health,
University of Arizona, Tucson, AZ, USA 4Department of Obstetrics and Gynecology, University of Arizona—College of Medicine Tucson,
Tucson, AZ, USA 5Department of Obstetrics and Gynecology, Banner—University Medical Center Phoenix, Phoenix, AZ, USA
6
Department of Basic Medical Sciences, University of Arizona—College of Medicine, Phoenix, AZ, USA

*Correspondence address. Department of Basic Medical Sciences, University of Arizona—College of Medicine, Phoenix, AZ, USA. E-mail:
mherbst1@email.arizona.edu https://orcid.org/0000-0002-8540-5917
Submitted on January 28, 2021; resubmitted on August 25, 2021; editorial decision on September 9, 2021

TABLE OF CONTENTS
................................................................................................................................
• Introduction
Burden of disease—epidemiological significance of endometriosis, infertility, chronic pelvic pain and microbiome
Microbiome
• Methods
• The putative role of gut and vaginal microbiomes in the progression of endometriosis, infertility and CPP
Gut microbiome, estrogen and inflammation
Genital microbiota ascension and inflammation
Estrogen dependence of endometriosis, infertility and CPP
The gut-vagina, gut-brain and hypothalamic-pituitary-gonadal/adrenal axes that may relate to endometriosis, in-
fertility and CPP
• Studies linking microbiome to endometriosis, infertility and CPP
Interpretation of current research
• Methodical limitations of current microbiome studies
Case definition of endometriosis, infertility and CPP
Sampling of participants
Collection of samples
Causation versus association for microbiome studies
• Future—guiding clinical practice and microbiome studies
Leveraging the microbiome for discriminating patients with endometriosis, endometriosis and infertility, or endo-
metriosis and CPP
Screening versus diagnosis
Potential etiologic link between the gut and cervicovaginal microbiomes and endometriosis, infertility and CPP
Future research

BACKGROUND: Endometriosis is a chronic, burdensome condition that is historically understudied. Consequently, there is a lack of
understanding of the etiology of the disease and its associated symptoms, including infertility and chronic pelvic pain (CPP). Endometriosis

C The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved.
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Microbiota in endometriosis, infertility and pelvic pain 93

development is influenced by estrogen metabolism and inflammation, which are modulated by several factors including the microbiome and
the estrobolome (the collection of genes encoding estrogen-metabolizing enzymes in the gut microbiome). Therefore, there is increasing in-
terest in understanding the role of microbiota in endometriosis etiology.
OBJECTIVE AND RATIONALE: To date, there is no cure for endometriosis and treatment options often are ineffective. This manu-
script will review the potential relationship between the microbiome and endometriosis, infertility and CPP and highlight the available data
on the microbiome in relation to endometriosis and its related symptoms. The overarching goal of this manuscript is to inform future
microbiome research that will lead to a deeper understanding of the etiology of the disease and possible diagnostic modalities and treat-
ments. The potential impact of the microbiome on estrogen regulation modulated by the estrobolome, as well as inflammation and other
endometriosis-promoting mechanisms within the genital tract, will be reviewed. The methodological limitations of microbiome-related stud-
ies will be critically assessed to provide improved guidelines for future microbiome and clinical studies.
SEARCH METHODS: PubMed databases were searched using the following keywords: endometriosis AND microbiome, infertility AND
microbiome, pelvic pain AND microbiome, IVF (in-vitro fertilization) AND microbiome, endometriosis AND infertility. Clinical and preclini-

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cal animal trials that were eligible for review, and related to microbiome and endometriosis, infertility or CPP were included. All available
manuscripts were published in 2002–2021.
OUTCOMES: In total, 28 clinical and 6 animal studies were included in the review. In both human and animal studies, bacteria were
enriched in endometriosis groups, although there was no clear consensus on specific microbiota compositions that were associated with
endometriosis, and no studies included infertility or CPP with endometriosis. However, bacterial vaginosis-associated bacteria and
Lactobacillus depletion in the cervicovaginal microbiome were associated with endometriosis and infertility in the majority (23/28) of
studies. Interpretation of endometrial studies is limited owing to a variety of methodological factors, discussed in this review. In addition,
metadata outlining antibiotic usage, age, race/ethnicity, menopausal status and timing of sample collection in relation to diagnosis of
endometriosis was not consistently reported. Animal studies (6/6) support a bidirectional relationship between the gut microbiota and
endometriosis onset and progression.
WIDER IMPLICATIONS: There is evidence that a dysbiotic gut or genital microbiota is associated with multiple gynecologic conditions,
with mounting data supporting an association between the microbiome and endometriosis and infertility. These microbiomes likely play a
role in the gut-brain axis, which further supports a putative association with the spectrum of symptoms associated with endometriosis, in-
cluding infertility and CPP. Collectively, this review highlights the demand for more rigorous and transparent methodology and controls,
consistency across the field, and inclusion of key demographic and clinical characteristics of disease and comparison participants. Rigorous
study designs will allow for a better understanding of the potential role of the microbiome in endometriosis etiology and the relationship to
other disorders of the female reproductive tract.

Key words: estrogen / estrobolome / inflammation / gut microbiota-brain-axis / Lactobacillus / bacterial vaginosis / uterine microbiota /
vaginal microbiota / metabolome / dysbiosis

..
Introduction ..
..
..
Burden of disease—epidemiological ..
..
significance of endometriosis, infertility, ..
..
chronic pelvic pain and microbiome ..
..
Endometriosis, infertility and chronic pelvic pain (CPP) are conditions ..
that primarily affect women of reproductive age and although they ..
..
may coexist, they may also occur independently. Approximately 10%
of women suffer from endometriosis (Parasar et al., 2017), with associ-
ated symptoms that may include dysmenorrhea, pelvic pain, dyspareu-
nia and infertility (Bulletti et al., 2010; Harada, 2013; Sachedina and
Todd, 2020). Much remains to be understood about the pathophysiol-
ogy, natural progression and prognosis of endometriosis (Simoens
et al., 2012). The gold standard diagnosis of endometriosis requires di-
agnostic laparoscopy with excisional biopsy and pathology confirmation
(Zanelotti and Decherney, 2017). The endometriosis fertility index is
also an emerging tool to determine the estimated chance of a sponta-
neous pregnancy following surgery for endometriosis and can be ap-
plied clinically to determine if ART should be considered (Vesali et al.,
2020). This tool incorporates patient characteristics, endometriosis
stage based on the revised American Fertility Score and surgical
findings (Vesali et al., 2020). The mainstay of treatment for
endometriosis is hormonal suppression (Chen et al., 2019), which is
associated with pregnancy prevention as well as unwanted side effects,
and may not be completely successful (Chen et al., 2019; Nnoaham
et al., 2011). Furthermore, recurrence of endometriosis is common
and coexisting conditions, including infertility and CPP, are also difficult
to treat (Giudice, 2010; Warzecha et al., 2020).
Female infertility and subfertility are complex conditions with a signif-
.. icant economic and psychosocial impact (Hasanpoor-Azghdy et al.,
..
.. 2014; ESHRE Capri Workshop Group, 2015; Njagi et al., 2020).
.. There are many different gynecologic diseases that influence infertility
..
.. including endometriosis (Bulletti et al., 2010), ovulatory dysfunction
.. (Urman and Yakin, 2006), tubal factor (Briceag et al., 2015), endocrine
..
.. disruption (Kalro, 2003) and age-related infertility (Zegers-Hochschild
.. et al., 2017). Among women with infertility, up to 25–50% are esti-
..
.. mated to suffer from endometriosis (Bulletti et al., 2010). The etiology
..
.. that explains why infertility is highly prevalent among women with
.. endometriosis is ambiguous and the mechanisms driving infertility are
..
.. also not fully elucidated, as the majority of women with endometriosis
.. are able to conceive (Prescott et al., 2016). Factors that contribute to
..
.. the relation between endometriosis and infertility include anatomic
.. abnormalities related to adhesive disease (Goldberg et al., 2019; Fukui
94
..
et al., 2020) and possibly inflammation (Macer and Taylor, 2012).
Local inflammation may impact endometrial, tubal, ovarian and perito-
neal reproductive function (Weiss et al., 2009; Macer and Taylor,
2012; de Rivero Vaccari, 2020).
CPP is described as noncyclic pelvic pain below the umbilicus lasting
for more than 3–6 months, that is felt most or some of the time
(Dydyk and Gupta, 2021). CPP has an immense impact on day-to-day
functioning, mood, relationships and productivity (Souza et al., 2011;
Cagnacci et al., 2019). No single etiology exists, and pelvic pain is influ-
enced by the urogenital, gastrointestinal (GI), myofascial and nervous
systems (Latthe et al., 2006; Choung et al., 2010; Pastore and
Katzman, 2012; Brawn et al., 2014; Ayorinde et al., 2015). Multiple
conditions or pain syndromes may coexist with CPP including endome-
triosis (Triolo et al., 2013), as well as interstitial cystitis (IC; Persu
et al., 2010), irritable bowel syndrome (IBS; Williams et al., 2005) and
myofascial pelvic pain syndrome. Among women with CPP, up to 60%
are diagnosed with endometriosis (Laufer et al., 1997; Mowers et al.,
2016). Furthermore, pain can become persistent from any etiology and
is exacerbated by central pain sensitization (Kaya et al., 2013).
Inflammation is a common factor associated with endometriosis, in-
fertility and CPP (Giudice, 2010; Li et al., 2017; Mu et al., 2018;
Symons et al., 2018; Dydyk and Gupta, 2021; Kolanska et al., 2020).
Research suggests that women with endometriosis have higher levels
of localized and systemic inflammation (Symons et al., 2018).
Inflammation contributes to infertility owing to both scarring and ana-
tomic distortion of pelvic anatomy, and to its effects on ovulation, fer-
tilization and implantation (Giudice, 2010; Briceag et al., 2015). In
addition, inflammation plays a key role in acute pain and is a risk factor
for the development of chronic pain (Ness et al., 2002; McKinnon
et al., 2015). A deeper understanding of potential impacts of inflamma-
tion, the estrobolome, hormonal dysregulation, the microbiome and
contributing etiologies on CPP symptoms and presentation, would pro-
vide better management strategies and treatment for endometriosis
(Howard, 2003; Simoens et al., 2012; Kwa et al., 2016; Dydyk and
Gupta, 2021; Fig. 1).
The role of the microbiota in possibly driving local and systemic in-
flammation and the relationship to the pathophysiology of multiple gy-
necologic conditions remains under investigation (Baker et al., 2018;
Łaniewski et al., 2020). The pathogenesis of endometriosis and comor-
bidities is likely related to the interplay of multiple environmental fac-
tors (Bellelis et al., 2011), genetic factors (Hansen and Eyster, 2010;
Ballester et al., 2012), inflammation, immune dysregulation, hormonal
imbalance (Macer and Taylor, 2012; Benner et al., 2018; Symons et al.,
2018; Laganà et al., 2019; Kolanska et al., 2020; Wang et al., 2020)
and possibly the microbiome. Chronic activation of the immune re-
sponse can result in pelvic inflammation and severe endometriosis can
result in pelvic adhesive disease (Laschke and Menger, 2016). This is
one hypothetical explanation for the symptoms of endometriosis that
can also lead to infertility and CPP, although the contribution of inflam-
mation to all three conditions is unclear (Halme et al., 1984; Li et al.,
2017; Mu et al., 2018; Dera-Szymanowska et al., 2020; Dydyk and
Gupta, 2021). These factors (inflammation, immune dysregulation and
altered hormone levels) may also result from changes in the micro-
biome, as outlined in subsequent sections of this review.
Endometriosis has considerable symptom heterogeneity and associ-
ated disease severity (Zanelotti and Decherney, 2017; Koninckx et al.,
2019a). This can be partially attributed to the lack of consensus
Salliss et al.
.. surrounding disease etiology and the highly invasive and expensive diag-
.. nostic methods that may result in a delayed diagnosis (Husby et al.,
..
.. 2003; Nnoaham et al., 2011; Moen, 2017; As-Sanie et al., 2019). This
... heterogeneity in endometriosis and delay between symptom onset and
.. diagnosis impacts the design and interpretation of studies related to
..
.. the microbiome, as described below.
.. Among women with endometriosis, it is estimated that 65% of
..
.. women experience pain symptoms (Nnoaham et al., 2011). However,
.. pain severity may be discordant with disease burden or disease staging
..
.. (Vercellini et al., 2006). Currently, we have limited ability to determine
.. if endometriosis symptoms are well controlled or whether disease se-
..
.. verity is worsening, since symptom presentation is diverse among indi-
.. viduals, and there is no robust method to assess treatment efficacy
..
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.. (Giudice, 2010). Therefore, new biomarker-based diagnostic methods
..
.. that leverage the microbiome or other microbiota-mediated biomarkers
.. (metabolome, estrobolome and hormones associated with the condi-
..
.. tions) could provide a less invasive, more cost-effective and more accu-
.. rate diagnosis, as well as a means of monitoring of symptoms.
..
.. The selection of treatment for endometriosis is highly dependent on
.. symptom presentation and fertility desires (Barbieri, 1998; Bedaiwy
..
.. et al., 2017; Falcone and Flyckt, 2018). Current hormone suppression
.. treatments that include GnRH analog and antagonist treatment likely
..
.. alter the microbiome (Khan et al., 2014), as GnRH treatments aim to
.. reduce circulating estrogen levels, and estrogen is known to promote
..
.. Lactobacillus dominance in the cervicovaginal microbiome (Miller et al.,
.. 2016; Amabebe and Anumba, 2018; Chen et al., 2019; Gheorghisan-
..
.. Galateanu and Gheorghiu, 2019). Following surgical resection of endo-
..
.. metriosis, the risk for repeat surgery to treat endometriosis-related
.. pelvic pain ranges from 20% to 40% and may be associated with the
..
.. initial extent of disease burden (Wheeler and Malinak, 1983; Parazzini
.. et al., 2005; Shakiba et al., 2008; Taylor and Williams, 2010). Further
..
.. etiological understanding of the disease and a possible role of the
.. microbiome would provide the groundwork to identify more effective
..
.. and less invasive methods that are crucial to adequately treat endome-
.. triosis (Kim and Gluck, 2019; Liu et al., 2020; Sokol et al., 2020; Tan
..
.. et al., 2020).
.. The causes of infertility are complex and multifactorial; however, en-
..
.. dometriosis is a common cause of female infertility, and endometriosis
.. severity may be related to the chance of success of ART (Hamdan
..
.. et al., 2015). Similarly, as a result of the heterogeneous causes of CPP,
..
.. the pathophysiology is also complex, multifaceted and is mostly not
.. well understood. Changes in the microbiome associated with endome-
..
.. triosis may provide useful information to determine which women are
.. at risk of developing infertility or CPP and, further, which women will
..
.. respond to treatments. Unfortunately, there is a lack of understanding
.. of endometriosis and associated conditions, and the relation to the
..
.. microbiome, which hinders the impact that microbiome-based treat-
.. ments can have on gynecologic diseases. It is therefore of paramount
..
.. importance that these conditions are investigated to identify new,
.. highly effective treatments that may be unlocked by a better under-
..
.. standing of the microbiome.
..
..
.. Microbiome
..
.. Gut microbiome
..
.. The microbiome refers to the collection of genomes of microorgan-
.. isms in a particular environment or mucosal site, such as the GI,
Microbiota in endometriosis, infertility and pelvic pain 95

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Figure 1. Role of estrobolome and human microbiome in estrogen-driven diseases. The estrobolome and human microbiome can po-
tentially contribute to hormonal dysregulation that can contribute to estrogen-driven diseases. Estrogen is the key contributing factor to the pathogen-
esis of estrogen-driven disorders, including endometriosis, and comorbidities infertility and pelvic pain. Therefore, when estrogen metabolism is
dysregulated, estrogen-driven diseases are negatively affected, and this can contribute to disease pathogenesis. The estrobolome can alter the circulat-
ing level of estrogen, through deconjugation from its conjugate (glucuronic acid). Therefore, the estrobolome (and microbiome) has the potential to
affect estrogen-mediated disorders. In addition, the microbiome can alter the metabolism of an individual and the systemic/local level of inflammation.
Inflammation is not only a hallmark of cancer but also is associated with a multitude of other conditions that includes endometriosis, infertility and pel-
vic pain. Other associated factors and symptoms that relate endometriosis with either infertility, CPP or host phenotype are included in the appropri-
ate overlapping circle. Arrows in the center of the diagram indicate which factors influence or can contribute to endometriosis, infertility and CPP and
exacerbate the complex interconnected nature of these disorders. In summary, the microbiome is a key component that may contribute to inflamma-
tory and estrogen-driven disorders through dysbiosis in the microbiome and the estrobolome, which is directly affected by dysbiosis in the micro-
biome. In this review, we focused on endometriosis, infertility and CPP that are all associated with estrogen and inflammation. CPP, chronic pelvic
pain.

..
cervicovaginal and pulmonary mucosa (Ravel et al., 2011; Baker et al., ..
2018; Barko et al., 2018; Costa et al., 2018). The term microbiota ..
..
refers to ecological communities of microorganisms predominantly of ..
bacteria, fungi (e.g. yeast), viruses, protozoa and archaea (Ma et al., ..
..
2012; Marchesi and Ravel, 2015), for the purpose of this review, at a ..
mucosal site. The gut microbiome plays a key role in physiological pro-
..
..
cesses, including nutrient absorption, maintaining the integrity of the GI ..
..
lining, regulation of immune and endocrine systems, and protection ..
against pathogenic insults (Baker et al., 2017; Belkaid and Harrison, ..
..
2017). An ‘optimal microbiome’ can maintain the well-being and ho-
meostasis of the individual and this microbiota composition confers
health benefits at mucosal sites (Belkaid and Hand, 2014; Garrett,
2015; Blander et al., 2017).
In addition, the gut microbiota can influence host health through me-
diating changes in the metabolome. The metabolome is composed of
all the metabolites present in a particular environment. A diverse col-
lection of bacteria in the gut provides a varied repertoire of enzymes
and metabolic pathways that contribute to health and homeostasis,
and this state is referred to as eubiosis. Dysbiosis, on the other hand,
refers to a disruption or change in the microbiota composition that
may be associated with disease, and in the gut is reflected by a reduc-
tion in microbiota diversity (Baker et al., 2017). A eubiotic gut
microbiome contributes to homeostasis by fortifying the epithelial bar-
rier through mucus production, tight junction formation, pathogen ex-
clusion, minimizing inflammation and regulating the immune system
(Chase et al., 2015; Shreiner et al., 2015; Laschke and Menger, 2016;
Baker et al., 2017; Łaniewski et al., 2020).
Butyrate, a key short-chain fatty acid in the gut metabolome, is a
source of energy for enterocytes and stimulates proliferation and re-
generation of the epithelium, to further maintain the barrier (Dalile
et al., 2019). Dysbiosis increases inflammation in the intestinal epithe-
.. lium, increases permeability, and ultimately, disruption of barrier func-
.. tion (Shreiner et al., 2015). Dysbiosis-mediated intestinal inflammation
..
.. can be caused by bacterial enterotoxins, such as lipopolysaccharide
.. (LPS), that signals through host toll-like receptor 4 (Khan et al., 2009,
..
.. 2013, 2018). Intestinal inflammation and epithelial barrier dysfunction
.. both contribute to intestinal disorders, such as IBS, where gut dysbiosis
..
.. and intestinal inflammation are common (Shreiner et al., 2015).
..
.. Dysbiosis in the gut can ultimately increase the permeability and dis-
.. rupt the protective function of the intestinal epithelium (Lobionda
..
.. et al., 2019), which can lead to bacterial infiltration as well as local in-
.. flammation, that can contribute to intestinal disorders like IBS
..
.. (Konturek et al., 2011; Menees and Chey, 2018). Overall, eubiosis in
.. the gut is central to maintaining host health, as colonization by health-
96
..
associated bacteria and a diverse variety of organisms contributes to
the regulation of the immune response, maintenance of intestinal bar-
rier function and host metabolism (Shreiner et al., 2015; Menees and
Chey, 2018). Many women with endometriosis suffer from GI disor-
ders, including IBS. As such, it has been hypothesized that the gut
microbiome may play a key role in orchestrating endometriosis and re-
lated systems/disorders.
Estrogen-metabolizing bacteria
The gut microbiota has the ability to regulate circulating estrogen levels
via the estrobolome, which is defined as the collection of genes encod-
ing estrogen-metabolizing enzymes, specifically in the gut microbiome
(Baker et al., 2017; Castaneda et al., 2020; Eslami-S et al., 2020;
Laborda-Illanes et al., 2020). The gut metabolome refers to the metab-
olites or small molecules that come from a biological sample or site.
These metabolites include those from the host organism, environ-
ment/xenobiotics and microbiota-mediated metabolites. The metabo-
lome is the end product of the interaction between our genome and
the environment and reflects health and disease phenotypes that can
be used to reveal the underlying causes of complex diseases (Laschke
and Menger, 2016; Wishart, 2016).
An immense array of metabolic reactions occurs in the intestinal
lumen, one being the deconjugation of estrogen from its conjugate glu-
curonic acid (Baker et al., 2017; Ervin et al., 2019). This reaction
requires the bacterial enzyme b-glucuronidase, found in specific gut
bacteria such as, Escherichia coli, Bacteroides fragilis and Streptococcus
agalactiae (Pollet et al., 2017), which have the ability to deconjugate
glucuronide (Baker et al., 2017; Ervin et al., 2019). The liver conjugates
estrogen, including 17b-estradiol (E2) the predominant estrogen in
humans (Palmisano et al., 2017), with glucuronic acid (glucuronide) and
secretes the glucuronide into bile salts, where the glucuronides are
later released into the intestinal tract for excretion of the unused con-
jugated toxins and hormones (Adlercreutz, 1974). Glucuronide cannot
be reabsorbed into the circulatory system. However, estrogen that has
been deconjugated from glucuronic acid, by the bacterial enzymatic ac-
tion of b-glucuronidase, can be absorbed into the circulatory system as
active estrogen (Laschke and Menger, 2016; Baker et al., 2017; Ervin
et al., 2019).
Changes in the gut microbial composition, and thereby b-glucuroni-
dase activity, could perturb or dysregulate circulating estrogen levels
and drive estrogen-mediated conditions by contributing to hyper- or
hypo-estrogenic states (Fig. 2; Kitawaki et al., 2002; Laschke and
Menger, 2016; Baker et al., 2017). Notably, microbiota could be driv-
ing endometriosis (and possibly CPP and infertility) by promoting in-
flammation and hormonal dysregulation (through the estrobolome),
altering cellular proliferation/apoptosis, metabolism, oxidative stress
and increasing angiogenesis/vascularization (Hakansson and Molin,
2011; Belkaid and Hand, 2014; Baker et al., 2017; Morotti et al., 2017;
Symons et al., 2018; Wang et al., 2020). The estrobolome is an under-
studied area, particularly in the context of endometriosis, infertility and
CPP. Therefore, estrobolome research is likely to uncover novel
insights that could be utilized in future interventions and treatments for
endometriosis and other estrogen-mediated conditions.
Cervicovaginal microbiome
In the cervicovaginal microbiome, Lactobacillus dominance is associated
with optimal gynecologic and reproductive health. Lactobacilli create a
Salliss et al.
.. hostile environment for invading pathogens and dysbiotic bacteria
.. (Doerflinger et al., 2014; Martin and Marrazzo, 2016; Anahtar et al.,
..
.. 2018; Baker et al., 2018; Kroon et al., 2018). Lactic acid, produced by
... Lactobacillus, lowers the vaginal pH to less than or equal to pH 4.5,
.. and this low pH microenvironment is optimal for vaginal health (Ravel
..
.. et al., 2011). To survive and thrive, Lactobacillus spp. require glycogen
.. byproducts (Ma et al., 2012; Muzny et al., 2020), which are provided
..
.. by an estrogen-dominant vaginal epithelium and host amylases (Spear
.. et al., 2015; Muhleisen and Herbst-Kralovetz, 2016; Nunn et al.,
..
.. 2020). In addition, Lactobacillus spp. contribute to homeostasis by oc-
.. cupying this niche (pathogen exclusion) and by production of anti-
..
.. inflammatory cytokines and antimicrobial peptides from epithelial cells,
.. which fortifies the epithelial cell barrier (Yarbrough et al., 2015; Muzny
..
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.. et al., 2020). Lactobacillus and the cervicovaginal epithelium form a
.. symbiotic relationship that can maintain the health of the epithelium
..
.. and a woman’s overall gynecologic health (Ma et al., 2012; Belkaid and
..
.. Hand, 2014; Doerflinger et al., 2014; Shreiner et al., 2015; Baker et al.,
.. 2018; Lobionda et al., 2019; Muzny et al., 2020).
..
.. Bacterial vaginosis (BV) is the most common vaginal disorder in pre-
.. menopausal women and is associated with multiple obstetric and gyne-
..
.. cologic sequelae, such as the increased risk of acquisition of sexually
.. transmitted infections (STIs), infertility, pelvic inflammatory disorder, en-
..
.. dometritis, postprocedural gynecologic infections, chorioamnionitis and
.. preterm birth (Garrett, 2015; Laschke and Menger, 2016; Muzny et al.,
..
.. 2020). BV is characterized by Lactobacillus spp. depletion coupled with
.. overgrowth of anaerobic bacteria that increases the microbiota diversity
..
.. and may include the formation of a resistant polymicrobial biofilm on
.. the cervicovaginal epithelium (Marrazzo, 2006; Martin and Marrazzo,
..
.. 2016; Muzny et al., 2019, 2020). Clinical indicators of BV include vaginal
.. pH above 4.5, amine odor, clue cells (epithelial cells covered with bac-
..
.. teria) and vaginal discharge (Mohammadzadeh et al., 2014).
..
.. BV-associated organisms have been shown to increase levels of
.. proinflammatory cytokines from the cervicovaginal epithelium
..
.. (Schwebke and Weiss, 2002; Doerflinger et al., 2014; Mitchell and
.. Marrazzo, 2014; Martin and Marrazzo, 2016; Gardner et al., 2020;
..
.. Salliss et al., 2021). Through direct interaction with epithelial cells,
.. BV-associated bacteria may also disrupt epithelial barrier function
..
.. (Muzny et al., 2020). Ultimately, in severe BV, the barrier formed by
.. cervicovaginal epithelial cells and protective mucus is degraded, which
..
.. may contribute to or increase the risk of developing other more se-
.. vere conditions (Belkaid and Hand, 2014; Blander et al., 2017; Baker
..
.. et al., 2018; Muzny et al., 2020).
..
.. Upper genital tract microbiome
..
.. Previously, the endometrium was thought to be sterile; however, this
..
.. remains in debate today with two conflicting hypotheses: sterile womb
.. hypothesis and the in-utero colonization hypothesis emerging from the
..
.. advancement in 16S RNA detection methods (Mitchell et al., 2015;
.. Chen et al., 2017; Moreno and Franasiak, 2017; Tao et al., 2017;
..
.. Benner et al., 2018; Leoni et al., 2019; Winters et al., 2019; Silverstein
.. and Mysorekar, 2021). The ‘sterile womb hypothesis’ has been used
..
.. to describe the lack of evidence for a resident uterine microbiome
.. (Perez-Munoz et al., 2017). This debate is beyond the scope of this re-
..
.. view, however, studies to date that have investigated the presence of
.. the uterine microbiome, mostly lack important contamination controls
..
.. and have low sample size study designs, limiting the ability to identify
.. true bacterial signals (Verstraelen et al., 2016; Chen et al., 2017;
Microbiota in endometriosis, infertility and pelvic pain 97

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Figure 2. Gut dysbiosis negatively impacts the estrobolome. Gut dysbiosis negatively impacts the estrobolome and the female reproductive
tract through altering circulating estrogen levels, increasing systemic inflammation and immune cell trafficking. (A) A healthy gut microbiome is com-
posed of a diverse collection of bacteria, whereas Lactobacillus spp. dominance in the cervicovaginal microbiome is characteristic of cervicovaginal
98 Salliss et al.

..
Moreno and Franasiak, 2017; de Goffau et al., 2019; Winters et al., ..
2019; Olomu et al., 2020). The lack of contamination controls, evi- ..
..
dence of bacterial viability and transcervical sample collection limits the ..
interpretation of findings from these microbiome studies (Baker et al., ..
..
2018; Winters et al., 2019). The presence of contamination does not ..
suggest that all bacteria signals are to the result of contamination, how-
ever, without the inclusion of rigorous controls and identifying sources
of contamination, one cannot determine the genuine signals. Herein,
we review reports to date and methodologies related to the endome-
trial/uterine microbiome for studying endometriosis, infertility and
CPP, with the intention of having a positive impact on the number/
types of controls used for extraction, sampling and downstream analy-
sis to promote rigor, transparency and reproducibility among studies.
2004; Perez et al., 2007; Fardini et al., 2010). In addition, evidence has
suggested that cervicovaginal bacteria can ascend from the lower fe-
male reproductive tract to the uterus, when barriers such as cervical
mucus and the cervical plug are breached, thus suggesting the uterus is
not completely sterile (Baker et al., 2018). It is important to note,
.. however, that the upper female reproductive tract and endometrium
..
.. harbor low biomass microbial communities (Baker et al., 2018).
.. The broad impact of the presence of bacteria in the endometrium
..
.. has sparked an intense area of study, which includes the potential influ-
.. ence of the endometrial microbiome on fertility and reproductive out-
..
.. comes, where studies demonstrated relationship between endometrial
..
.. colonization and fecundity (Moreno et al., 2016; Tao et al., 2017;
.. Benner et al., 2018; Leoni et al., 2019; Winters et al., 2019). Despite

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..
However, there is evidence to suggest that bacteria can ascend into ..
the uterus as potential ‘tourists’ and ‘invaders’, as reviewed in Baker ..
..
et al. (2018). First, through the action of the uterine peristaltic pump, ..
bacteria from the lower female reproductive tract may be transported ..
..
into the uterus and beyond (Baker et al., 2018). Sperm are rapidly ..
transported from the cervical canal to the uterus and fallopian tube by ..
..
cervico-fundal uterine peristaltic contractions (Kunz et al., 1997; ..
Zervomanolakis et al., 2007). These contractions change in frequency
..
..
as the proliferative phase progresses and this has been visualized, using ..
labeled macrospheres, to occur within minutes (Kunz et al., 1997).
..
..
Uterine hyperperistalsis, with nearly double the frequency of contrac- ..
..
tions, has been observed in women with infertility and endometriosis
(Leyendecker et al., 1996; Kissler et al., 2006). This hyperperistalsis in
women with endometriosis may not only contribute to the transfer of
detached endometrial cells to the fallopian tubes and peritoneal cavity
but also bacteria from the lower genital tract. Women may experience
uterine dysperistalsis midcycle resulting in undirected and convulsive
contractions, which may contribute to infertility by interfering with
sperm transport, but again may result in high likelihood of transport of
bacteria to the upper genital tract.
Second, hematogenous spread of bacteria could potentially allow
bacteria to transfer from one mucosal site to another (Han et al.,
recent discoveries providing evidence for a nonsterile endometrium,
the presence and characterization of a resident endometrial micro-
biome are still elusive (Benner et al., 2018). Endometrial sterility
remains a controversial topic, primarily owing to studies lacking con-
tamination controls, or by utilizing transvaginal (oocyte retrieval, follicu-
lar fluid collection) or transcervical sample collection methods (IVF
catheter tips, endometrial biopsy), which can introduce contamination
of bacteria from other genital sites and result in uncertainty for data in-
terpretation (Winters et al., 2019). Therefore, future investigations
that determine and characterize bacterial colonization of the endome-
trium should include robust contamination controls and additional vali-
.. dation samples in their study designs, as well as accounting for the
.. limitations of DNA sequencing methods, discussed below.
..
..
..
..
.. Methods
..
..
.. A comprehensive keyword search of the scientific literature available
.. on PubMed was used to identify studies relating the microbiome to en-
..
.. dometriosis, infertility and CPP. Key words used were as follows: ‘en-
.. dometriosis AND microbiome’, ‘infertility AND microbiome’, ‘IVF
..
. AND microbiome’, ‘infertility AND endometriosis’ and ‘pelvic pain

Figure 2. Continued
health. (A1) A diverse, health-associated gut microbiome sustains homeostatic levels of b-glucuronidase activity that maintains homeostatic
circulating estrogen. Also, health-associated microbiota composition aids in minimizing inflammation and maintenance of barrier function. (A2)
Changing circulating estrogen levels result in menstrual cycle regulation and thus could affect fertility. (A3) Estrogen increases the availability of
glycogen in cervicovaginal mucosa. Health-associated Lactobacillus utilizes glycogen byproducts as an energy source in the process of fermenta-
tion, thus because estrogen promotes Lactobacillus dominance, which generates an acidic cervicovaginal environment, estrogen promotes
optimal cervicovaginal health. This maintains low pH and low inflammation in the cervicovaginal microenvironment that is essential for cervico-
vaginal health. (B) Gut dysbiosis (shift from a diverse to less diverse microbiome) contributes to local and systemic inflammation by upregula-
tion of proinflammatory cytokines, including TNF and recruitment of immune cells. (B1) Increased b-glucuronidase activity in the gut (caused
by dysbiosis), increases circulating estrogen. In addition, increased pro-inflammatory cytokines and immune cell trafficking (via mucosal-associ-
ated lymphoid tissue) can cause dysbiosis at the cervicovaginal mucosa. TNF, tumor necrosis factor. (B2) Increased estrogen could overstimu-
late endometrial tissue and lead to tubal factor infertility, menstrual cycle irregularities, or contribute to uterine inflammation and consequently
CPP. In the context of endometriosis, increased estrogen levels could increase stimulation of lesions and increase symptom severity. (B3)
Systemic inflammation characterized by increased immune cell trafficking and upregulation of proinflammatory cytokines (caused by gut dysbio-
sis), can increase the inflammation at the cervicovaginal mucosa. This can lead to perturbation from Lactobacillus dominance toward disease-as-
sociated polymicrobial biofilms. This can lead to an increase in pH, to pH >4.5, that is associated with cervicovaginal disease states. Equally,
elevated estrogen could increase glycogen availability for Lactobacillus and lead to bacterial overgrowth. Estrogen can also stimulate growth and
proliferation of cervicovaginal cells. Therefore, it is possible that an excess of estrogen could contribute to cancers of the female reproductive
system. CPP, chronic pelvic pain.
Microbiota in endometriosis, infertility and pelvic pain
..
AND microbiome’. All applicable clinical and preclinical animal model
studies that were retrieved by these PubMed keywords searches and
published between 2002 and 2021 were included. Searches were con-
ducted between March 2020 and March 2021. All literature included
was written in English. Studies were not excluded based on date of
publishing, as all eligible studies were published in this timeframe.
The putative role of gut and
vaginal microbiomes in the
progression of endometriosis,
infertility and CPP
Gut microbiome, estrogen and
inflammation
It is postulated that eubiosis in the gut microbiome can regulate
homeostatic circulating estrogen, and a shift to dysbiosis can perturb
this equilibrium and contribute to estrogen-dependent disease states
(Fig. 2). Evidence for an influence of the estrobolome has been
obtained in cases of colon and breast cancer (estrogen-mediated can-
cers), where higher b-glucuronidase activity in the gut was associated
with these pathologic conditions (Kim and Jin, 2001; Kwa et al., 2016;
Parida and Sharma, 2019; Komorowski and Pezo, 2020; Fig. 2B).
Endometriosis, infertility and CPP are all estrogen-associated disorders,
so it is possible that variations in the estrobolome could also promote
the incidence of these conditions (Kim and Jin, 2001; Ervin et al.,
2019). In addition, estrogen may influence fecundity directly by modify-
ing the thickness and quality of the endometrium, factors that could af-
fect implantation of the embryo (Fig. 2B;Liu et al., 2015; Zhang et al.,
2018). Estrogen and phytoestrogen bind estrogen receptors, which are
subsequently internalized and function as transcription factors promot-
ing the transcription of genes involved in cellular growth (Laschke and
Menger, 2016; Baker et al., 2017). Dysbiosis of the gut microbiome
can influence the amount of circulating estrogen in the body, through
dysregulation of the estrobolome, and could lead to increased symp-
tom severity for endometriosis, infertility and CPP (Fig. 2B;Liu et al.,
2015; Baker et al., 2017).
The gut microbiome and the host coexist naturally, and gut bacteria
help to maintain epithelial and host health (Hakansson and Molin,
2011; Belkaid and Hand, 2014). Mucus thickness, transepithelial barrier
function and immune response are all modulated and maintained by
the gut microbiota and are characteristic of a healthy epithelium
(Hakansson and Molin, 2011; Belkaid and Hand, 2014; Blander et al.,
2017). Hence, dysbiosis increases the risk for several disease states by
altering the microenvironment (Kim and Jin, 2001; Hakansson and
Molin, 2011; Schwabe and Jobin, 2013; Garrett, 2015; Komorowski
and Pezo, 2020). Dysbiotic bacteria can digest the intestinal protective
mucus layer and interact directly with enterocytes, typically leading to
an increase in local and systemic inflammation (Blander et al., 2017;
Fig. 2B). In addition, dysbiosis can lead to immune dysfunction and dys-
regulate intestinal barrier function that can lead to the transmigration
of the gut microbiota and bacterial ligands (Hakansson and Molin,
2011; Belkaid and Hand, 2014). Gut microbiota influence mucosal T-
cell composition and function (TH1, TH17, TReg, etc.) and therefore
dysbiosis impacts this balance of mucosal immune cells, triggering
99
.. inflammation and disease (Hooper et al., 2012). Elevated levels of
.. immune mediators, and intestinal tissue damage caused by dysbiosis,
..
.. are symptoms of various disease states, including IBS and GI cancer
... (Hakansson and Molin, 2011; Konturek et al., 2011; Pellissier et al.,
.. 2014; Chenoll et al., 2019). IBS and GI cancers are often caused by ep-
..
.. ithelial inflammation and epithelial barrier disruption, to the result of
.. gut dysbiosis (Garrett, 2015; Lobionda et al., 2019). IBS can be a com-
..
.. mon comorbid condition in women with endometriosis and CPP,
.. which suggests a possible link between the conditions (Williams et al.,
..
.. 2005; Matheis et al., 2007; Seaman et al., 2008). Higher levels of
.. systemic inflammation are associated with endometriosis, and likely
..
.. infertility and CPP (Laschke and Menger, 2016; Mu et al., 2018).
.. Therefore, there is the possibility that the gut microbiota can contrib-
..
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.. ute to these disorders by contributing to or dysregulating an inflamma-
..
.. tory feedback loop.
..
..
.. Genital microbiota ascension and
..
.. inflammation
..
.. The female reproductive tract is interconnected, and cervicovaginal
.. bacteria are able to ascend to the uterus (Baker et al., 2018; Suff et al.,
..
.. 2018), however, cervicovaginal bacteria ascension to the uterus is at
.. least partly inhibited by physical, chemical and microbiological barriers
..
.. in the cervix. The ascension of cervicovaginal microbiota to the upper
.. female reproductive tract may cause damaging inflammation at this
..
.. site, thereby contributing to endometriosis, infertility, CPP and symp-
.. toms associated with these conditions (Baker et al., 2018). Ascension
..
.. of BV-associated bacteria to the endometrium could lead to the eleva-
.. tion of proinflammatory mediators as has been shown using 3D in-vitro
..
.. models of the human endometrium (Łaniewski et al., 2017; Ilhan et al.,
.. 2020). Endometriosis is associated with higher levels of inflammation;
..
.. thus, the genital microbiome could contribute to endometriosis etiol-
..
.. ogy and symptoms through contributing to elevated proinflammatory
.. mediators.
..
.. In addition to altering levels of proinflammatory mediators and
.. promoting chronic inflammation, bacteria may alter the hallmarks of
..
.. cancer, including proliferative capacity of the epithelial cells, apoptosis,
.. oxidative stress, epithelial barrier function, genomic instability and
..
.. dysregulation of metabolism, as documented in two recent reviews
.. (Ilhan et al., 2019; Muzny et al., 2020). Many of these hallmarks of can-
..
.. cer align with aspects of endometriosis including chronic inflammation,
.. cellular proliferation/alteration in apoptosis, oxidative stress, neo-an-
..
.. giogenesis and estrogen-dependence (Witz, 1999; Lebovic et al., 2001;
.. Rocha et al., 2013; Scutiero et al., 2017; Klemmt and Starzinski-Powitz,
..
.. 2018; Symons et al., 2018; As-Sanie et al., 2019; Wang et al., 2020).
..
.. Therefore, the microbiome may impact both cancer and endometriosis
.. by dysregulation of similar mechanisms. A ‘bacterial contamination’ hy-
..
.. pothesis has been proposed and suggests that bacterial LPS could be
.. the initial trigger for the growth and regulation of endometriosis (Khan
..
.. et al., 2009, 2013, 2015, 2018; Azuma et al., 2017; Keyama et al.,
.. 2019; Leonardi et al., 2020). As such, more studies are required to
..
.. better understand the specific bacterial-mediated mechanisms that
.. could be linked to etiology and severity of endometriosis.
..
.. There is also evidence suggesting that colonization of the endome-
.. trium by dysbiotic bacteria or a lack of Lactobacillus dominance in the
..
.. lower genital tract could negatively impact fertility (Moreno et al.,
.. 2016; Hashimoto and Kyono, 2019; Kitaya et al., 2019; Kyono et al.,
100
..
2019; Tsonis et al., 2020). Prior research has suggested that ..
Lactobacillus dominance in the cervicovaginal microenvironment is opti-
mal for embryo implantation, therefore, dysbiosis in the lower female
reproductive tract could impact fertility (Africa et al., 2014; Moreno
et al., 2016; Campisciano et al., 2017). Thus, ascension of dysbiotic
bacteria from the cervix to endometrium may negatively influence the
fecundity of an individual.
Estrogen dependence of endometriosis,
infertility and CPP
Endometriosis pain symptoms are likely associated with the presence ...
of systemic and local inflammation, local neurologic dysfunction owing ..
to direct innervation at the lesion, central sensitization of the periph- ..
eral nervous system (Anaf et al., 2000; Gheorghisan-Galateanu and ..
Gheorghiu, 2019), and the location and/or depth of lesions ..
(Fauconnier et al., 2002). Estrogen plays a key role in the pathophysiol- ..
ogy of endometriosis (Barbieri, 1998; Kitawaki et al., 2002; Baker ..
et al., 2017; Ladanyi et al., 2019), and may additionally impact fertility ..
particularly when estrogen levels are abnormal. The menstrual cycle is ..
regulated by the cyclic production of estrogen and progesterone, ..
which is important for the receptivity of the endometrium (Morotti ...
et al., 2017). Therefore, dysregulation of estrogen production or free ...
estrogen deconjugated by the gut microbiota could lead to dysregula- ...
tion of the menstrual cycle, and impact the receptivity of the endome- ...
trium for a fertilized embryo (Lobo and Carmina, 2000). Unidentified ..
mechanisms of estrogen regulation, such as the estrobolome, should ..
be examined in more detail to develop novel and effective treatments ..
for the management of endometriosis-mediated pain disorders.
The contribution of estrogen to CPP is more difficult to define ..
(Hassan et al., 2014), since CPP is typically a condition that manifests ..
from other disorders, such as dysmenorrhea and endometriosis ..
(Howard, 2003), with contributions related to central pain sensitization ..
(Kaya et al., 2013). However, estrogen can dysregulate nervous system ..
signaling pathways that are involved in pain reception (Paredes et al., ..
2019; Taylor et al., 2019). Furthermore, treatment with GnRH agonist ...
(GnRHa) results in improved pain management among women with ...
CPP, with and without endometriosis (Barbieri, 1998; Bedaiwy et al., ...
2017; Sachedina and Todd, 2020). Control of circulating estrogen at ..
homeostatic levels is important to maintain gynecologic health and suc- ..
cessful reproductive outcomes, and deviation from homeostatic regula- ..
tion of estrogen metabolism can contribute to estrogen-driven disease ..
states (Kim and Jin, 2001; Kitawaki et al., 2002; Kwa et al., 2016; Baker ..
et al., 2017; Ervin et al., 2019; Parida and Sharma, 2019; Komorowski ..
and Pezo, 2020).
The gut-vagina, gut-brain and
hypothalamic-pituitary-gonadal/adrenal axes ...
that may relate to endometriosis, infertility ...
and CPP
Some bacterial metabolites produced by the gut microbiota can inter- ...
act with host receptors that can then alter the host’s brain activity and ...
impact neural transmission, pain generation, inflammation or hormone ...
release (Lal et al., 2001; Bercik et al., 2012; Carabotti et al., 2015; ...
Chichlowski and Rudolph, 2015; Moloney et al., 2016; Baker et al., ..
2017; Bonaz et al., 2017; Breit et al., 2018; Menees and Chey, 2018; .
Salliss et al.
Guo et al., 2019; Amabebe and Anumba, 2020). These metabolites
.. are called neuroactive compounds and include neurotransmitters such
..
.. as serotonin (Baj et al., 2019). The hypothalamus and pituitary gland
... modulate and orchestrate the production of a variety of hormones
.. (Baj et al., 2019; Jones et al., 2020), including GnRH. The hypothala-
..
.. mus secretes the hormone GnRH and is targeted to the pituitary
.. gland, where GnRH causes secretion of LH and FSH into the systemic
..
.. circulation (Hiller-Sturmhöfel and Bartke, 1998; Malyala et al., 2005;
.. Skorupskaite et al., 2014; Watanabe et al., 2014; Marques et al.,
..
.. 2000). LH and FSH target the ovaries to stimulate follicular develop-
.. ment and estrogen secretion (Hiller-Sturmhöfel and Bartke, 1998). The
hypothalamus-pituitary-ovarian axis (HPO) is used to describe the
.
. regulation of circulating estrogen (Mikhael et al., 2019). Several studies
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have revealed the impact of the gut microbiome and neuroactive
.
. metabolites on neurological signaling and brain health (Shreiner et al.,
2015; Rogers et al., 2016; Schmidt et al., 2018; Zhao et al., 2018; Baj
.
et al., 2019; Dalile et al., 2019) but, to our knowledge, little research
.
has extended that work to endometriosis, infertility and CPP (Laschke
.
and Menger, 2016; Perrotta et al., 2020; Qi et al., 2021; Svensson
.
et al., 2021). All three conditions have a neurologic component and
.
. the involvement of the gut microbiome with endometriosis, infertility
and CPP has been postulated, but not studied in sufficient depth
(Brawn et al., 2014; Carabotti et al., 2015; Chichlowski and Rudolph,
2015; Baj et al., 2019; Qi et al., 2021; Svensson et al., 2021).
Neuroactive metabolites can affect the brain and signaling processes
.
directly or indirectly and involve the interaction of the gut microbiome
.
with nerves in the brain. This bidirectional communication is referred
.
to as the gut-brain-axis (GBA; Fig. 3). First, the microbiota can affect
..
.. the host brain signaling directly, by the absorption of microbial gut
metabolites (neuroactive compounds) into the circulatory system, such
.
as serotonin (Hanley and Van de Kar, 2003; Paredes et al., 2019; Jones
.
et al., 2020), gamma-aminobutyric acid (Watanabe et al., 2014;
.
Camille Melón and Maguire, 2016; Kanasaki et al., 2017), glutamate
.
(Baj et al., 2019) and short-chain fatty acids (SCFAs; Dalile et al., 2019;
.
Amabebe and Anumba, 2020), where these metabolites translocate to
.
the brain and directly bind neural receptors, which can activate cere-
bral and hypothalamic neurons, including GnRH neurons (Fig. 3(1–4))
(Banks, 2008; Hawkins, 2009; Marques et al., 2000). GnRH neurons
. have been observed to innervate outside the blood brain barrier and
. indicate that neuroactive compounds may not need to cross the blood
.
brain barrier to interact with GnRH neurons (Ganong, 2000; Banks,
.
2008; Hawkins, 2009; Benarroch, 2011; Ciofi, 2011; Prevot, 2011;
.
Plant, 2015; Bentivoglio et al., 2018; Baj et al., 2019).
.
The vagus nerve (VN) plays a role in the regulation of the immune
..
.. response, where the afferent (leading to the brain) and efferent (leading
.. to the gut) VNs participate in an inflammatory reflex (Pavlov and
..
.. Tracey, 2012; Pellissier et al., 2014; Bonaz et al., 2017; Breit et al.,
.
2018; Collins, 2020). In this inflammatory reflex, activation of the effer-
ent VN results in lower levels of local proinflammatory cytokines (in the
.. gut) and protection of barrier function in the epithelium, by engaging
.. the sympathetic nervous system (Pavlov and Tracey, 2012; Bonaz et al.,
.
2017, 2018; Bonaz, 2020). However, gut disorders such as IBS maintain
high levels of proinflammatory cytokines in the intestine (Pavlov and
Tracey, 2012; Moloney et al., 2016; Bonaz et al., 2017; Breit et al.,
2018; Collins, 2020) and are associated with gut dysbiosis (Bonaz et al.,
. 2017; Breit et al., 2018; Fukui et al., 2018). Therefore, possibly through
. dysbiosis of the gut microbiome, the VN inflammatory reflex is
Microbiota in endometriosis, infertility and pelvic pain 101

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Figure 3. Changes in neuroactive compound/protein production caused by gut dysbiosis can cause increases in GnRH. (1) A dysbi-
otic gut microbiome contains less varied bacterial, and a possible altered neuroactive metabolites/peptides, profile, (2) that can be absorbed into the
circulatory system. (3) Interactions with hypothalamic neurons that secrete GnRH and CRH (corticotropin-releasing hormone). Gamma-amino bu-
tyric acid (GABA), glutamic acid and serotonin (gut metabolites) can activate the secretion GnRH from GnRH neurons. (4) GnRH signals the pituitary
gland to secrete LH and FSH into the circulatory system. LH secretion has two modes: pulsatile secretion and in the form of a surge. It is possible that
gut dysbiosis can dysregulate GnRH production and therefore LH production from the pituitary. (5) The gut microbiome can increase cortisol, a stress
102 Salliss et al.

dysregulated (Pavlov and Tracey, 2012; Pellissier et al., 2014; Casado-

.. further investigate the contribution of the gut and cervicovaginal micro-
..
Bedmar and Keita, 2020) and has the potential to contribute to other .. biota to endometriosis, infertility and CPP.
..
inflammatory disorders, such as intestinal cancers, endometriosis, infer- ..
tility and CPP. ..
..
In addition to the GBA, the HPO, moderates secretion of LH and .. Studies linking microbiome
FSH in response to feedback mechanisms, and environmental and ..
.. to endometriosis, infertility
internal stimuli, including neuroactive compounds, as mentioned earlier, ..
and has been observed to influence central and visceral pain generation .. and CPP
..
(Kaya et al., 2013; Brawn et al., 2014; Carabotti et al., 2015; ..
Marques et al., 2000; Qi et al., 2021). Pain sensitization is a key hall-
.. Interpretation of current research
..
mark of CPP and endometriosis-associated CPP (Triolo et al., 2013; .. Recent reports have detailed a relationship between the gut and cervi-
..
Brawn et al., 2014; Arzani et al., 2020; Lin et al., 2020). Alternatively, .. covaginal microbiomes with endometriosis, infertility and CPP, but
changes in circulating LH/FSH could potentially contribute to hormonal .. studies were not consistent in their methods of analysis and participant

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..
dysregulation (Qi et al., 2021) and this has been observed for patients
with polycystic ovary syndrome (PCOS), another reproductive condi-
tion associated with complex hormonal alterations (Badawy and
Elnashar, 2011; Zhang et al., 2019).
The influence of the GBA, HPO and gut-vagina axes has not been
fully investigated in relation to endometriosis, infertility and CPP, where
the influence of gut-brain axis in pain generation has dominated our
understanding (Brawn et al., 2014; Carabotti et al., 2015; Ravel and
Brotman, 2016; Guo et al., 2019; Mikhael et al., 2019; Lin et al., 2020;
Qi et al., 2021; Svensson et al., 2021). We hypothesize that because
of the direct and indirect ability of the gut microbiota to stimulate
GnRH neurons in the hypothalamus, LH and FSH production can be
dysregulated (thus estrogen secretion and follicular development)
when the gut is in dysbiosis (Hanley and Van de Kar, 2003;
Chichlowski and Rudolph, 2015; Mittal et al., 2017; Guo et al., 2019;
Jones et al., 2020; Silva et al., 2020; Qi et al., 2021). This dysbiosis
could therefore impact estrogen-associated disorders, such as endo-
metriosis, infertility and CPP (Hanley and Van de Kar, 2003; Herde
et al., 2011; Prevot, 2011; Watanabe et al., 2014; Plant, 2015; Mittal
et al., 2017; Marques et al., 2000; Jones et al., 2020; Baj et al., 2019;
Qi et al., 2021; Svensson et al., 2021). In addition, the potential of gut
dysbiosis to impact dysbiosis in the cervicovaginal microbiome through
the gut-vagina axis, and its contribution to endometriosis, infertility and
CPP could reveal complex interactions of the microbiota with these
disease states. Studies have demonstrated a relationship between obe-
sity and the vaginal microbiome composition. Obese women, exhibit
low Lactobacillus dominance and a higher abundance of anaerobic and
BV-associated bacteria (Dialister species, Anaerococcus vaginalis and
Prevotella timonensis), and higher levels of local immune mediators (IL-1
and IL-8) (Saadatian-Elahi et al., 2016; Si et al., 2017). The GBA, HPO
and the gut-vagina axes would be novel and interesting topics to
.. information presented, which made comparisons difficult and conclu-
.. sions hard to discriminate (Tables I and II). This review aims to
..
.. evaluate the current literature on these topics, summarize existing
.. knowledge, critically evaluate study designs and presentation, and iden-
..
.. tify opportunities for future research that could reveal novel interven-
.. tions and diagnostics for endometriosis, infertility and CPP.
..
..
.. Endometriosis and the microbiome
..
.. Research to date has reported various associations of specific bacteria
.. sampled from the vagina, cervix, endometrium, follicular fluid and gut
..
.. with endometriosis and infertility. Additional research could reveal a
.. potential role for the microbiota in the pathogenesis of endometriosis,
..
.. infertility and CPP. No studies in humans have allowed for causal rela-
.. tions to be drawn between the cervicovaginal/gut microbiome and the
..
.. development of endometriosis, infertility and CPP.
.. Two similar studies investigated the vaginal microbiota composition
..
.. of women with endometriosis compared to control women undergo-
.. ing laparoscopic surgery for other gynecologic diagnoses and found no
..
.. difference in the vaginal microbiome of the lower reproductive tract
.. between endometriosis cases and controls (Hernandes et al., 2020;
..
.. Wei et al., 2020). However, one study found a higher abundance of
..
.. Anaerococcus in the vaginal microbiome of women with endometriosis
.. (n ¼ 35) compared to women without (n ¼ 24; Perrotta et al., 2020).
..
.. Four studies assessed the microbial composition of cervical samples
.. from patients with endometriosis (Akiyama et al., 2019; Ata et al.,
..
.. 2019; Chen et al., 2020; Wei et al., 2020; Table I). One study com-
.. pared healthy controls (n ¼ 20) with women diagnosed (surgically or
..
.. with MRI) with endometriosis (n ¼ 36) and found no differences in the
.. cervical microbiome, and found the microbiota was predominantly
..
.. composed of Lactobacillus spp. (Chen et al., 2020). A study by
.. Akiyama et al. (2019) found that, compared to women with fibroids or
..
. other benign ovarian tumors (without endometriosis), women with

Figure 3. Continued
hormone that is secreted from the adrenal glands. Cortisol can contribute to chronic inflammation and pain sensitivity that can in turn both
contribute to chronic pain. Studies have shown an increased secretion of cortisol as a direct result of bacterial metabolites. Increased cortisol
levels in the body can lower the pain tolerance of an individual that is a key feature of CPP. Also, cortisol can cause increases in systemic in-
flammation and dysbiosis of the gut microbiome that can, in turn, potentially negatively impact estrogen and cortisol production. (6) LH stimu-
lates estrogen production from the ovaries; therefore, dysregulation of LH production could affect downstream estrogen production. Estrogen
is a key hormone where dysregulation could contribute to cervicovaginal dysbiosis or have negative impacts on estrogen-driven disorders such
as endometriosis, and comorbidities, infertility and CPP. Solid lines indicate processes that have been studied, and dotted lines indicate hypo-
thetical processes that require further investigation. CPP, chronic pelvic pain.
Table I Human clinical trials that investigated the association between the microbiome and endometriosis or chronic pelvic pain.

Study Clinical study participant Disease definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall study
demographics definition collected controls to kit controls collection identify bacterial identified as being conclusions
detect potential signal associated with
contamination endometriosis group
...................................................................................................................................................................................................................................................................................................
Interstitial cystitis definition:
Abernethy et al., Participants: Diagnosis was based on uri- Women without genito- Transurethral urine NR NR N/A RNA samples am- Transurethral sample: Patients with interstitial
Obstet Gynaecol, N ¼ 20 Controls nary frequency and suprapu- urinary or pelvic com- sample plified by PCR  Less likely to contain cystitis:
2017 N ¼ 20 patients diagnosed bic or bladder pain for a plaints, and are age- Sequenced with L. acidophilus in dis-  Had a less diverse
with interstitial cystitis minimum of 6 months matched within 3 years Illumina MiSeq ease group com- urinary microbiome
Age Range: 21–65 years old pared to control  less likely to possess
Race/Ethnicity/Country of group. Lactobacillus
Origin: NR  Lactobacillus acidophi-  higher levels of proin-
Menopausal status: NR lus was associated flammatory cyto-
Exclusion criteria: Subject had with less severe kines, specifically
recurrent or current urinary scores on the inter- IL-4.
tract infection, urethral stitial cystitis symp-
strictures, iatrogenic cysti- toms index.
tis, bladder augmentation,
Microbiota in endometriosis, infertility and pelvic pain

autoimmune disorder, neu-

romuscular disease, non-
skin cancer, high-grade cer-
vical dysplasia, current
pregnancy or current antibi-
otic use
Endometriosis Definition:
Akiyama et al., Am J Participants: Confirmation by laparoscopic Women who had fib- Cervical Mucus NR NR Before confirmation by Next generation se- Cervical: Microbiota distribution
Reprod Immunol, N ¼ 30 with endometriosis visualization roids or benign ovarian Laparoscopic surgery quencing (NGS) of  Enterobacteriaceae was similar in cervical
2019 N ¼ 39 without tumor other than V5-V6 region of the Streptococcus mucus of women with
endometriosis endometriosis 16S rRNA gene  Pseudomonas and without endometri-
Age Range: 20–44 years  Corynebacterium osis regardless of men-
Race/Ethnicity/Country of strual cycle phase
Origin: NR
Menopausal status:
Premenopausal
Exclusion criteria: Subjects has
gynecological malignancies,
pelvic inflammatory disease,
BV, or endocrine disorders
including thyroid diseases
and diabetes mellitus.
Ata et al., Sci Rep, Participants: Histologic confirmation Asymptomatic women 5 ml stool sample NR Controls for DNA Samples taken before if V3 and V4 16S Vaginal/Cervical:  Similar vaginal, cervi-
2019 N ¼ 14 stage 3/4 who presented for rou- Vaginal and endocervical extraction and undergoing surgery for rRNA gene regions  Absence of cal and intestinal
endometriosis tine well-woman visit or swabs sequencing endometriosis amplified with Atopobium microbiota composi-
N ¼ 14 healthy controls preconceptional metagenomic se-  Gardnerella tion between stage
Age Range: 18–45 years counseling quencing library  Streptococcus 3/4 endometriosis
Race/Ethnicity/Country of preparation, then  Escherichia and control groups
Origin: NR sequenced  Shigella  Differences at genus
Menopausal status:  Ureoplasma level stated in the
Premenopausal Gut: previous column
Exclusion criteria: Participants  Shigella
were pregnant ever, of  Escherichia
postmenopausal status,
taken probiotics or antibi-
otics 8 weeks prior, have an
abnormal pap smear in the
last 3 years, have inflamma-
tory bowel disease,

Continued
103

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Table I Continued
Study Clinical study participant
demographics
...................................................................................................................................................................................................................................................................................................
functional bowel disease,
history of GI cancer or sur-
gery, acute or severe GI
symptoms that require
medical treatment, or GI
infection.
Chen et al., Nat Participants:
Commun, 2017 N ¼ 95
Age Range: ‘Of reproductive
age’
Race/Ethnicity/Country of
Origin: Asian, Chinese
Menopausal status:
Premenopausal
Exclusion criteria: Subjects
presented with vaginal in-
flammation, severe pelvic
adhesion, any acute inflam-
mation, cancer, endocrine
or autoimmune disorders,
no recent use of antibiotics.
Chen et al., Ann Participants: Laparoscopic visualization or
Transl Med, 2020 N ¼ 20 with endometriosis transvaginal ultrasound and
N ¼ 36 controls MRI
Age Range: 18–45 years
Race/Ethnicity/Country of
Origin: Chinese
Menopausal status:
Premenopausal
Exclusion criteria: Patients
have BV, cervical inflamma-
tion, pelvic inflammatory
disease, any acute inflam-
mation, cancer and autoim-
mune disorders, pregnancy,
patients with an IUD, anti-
biotic use or hormone re-
placement therapy within
30 days, sexual activity for
48 h, douching, use of vagi-
nal medications within 5
days, and patients in men-
strual phase of cycle at the
time of sample collection
Hernandes et al., Participants:
Diagnostics (Basel), N ¼ 11 controls
2020 N ¼ 10 with endometriosis
Age Range: 18–50
Race/Ethnicity/Country of
Origin: Brazilian
Menopausal status:
Premenopausal
Exclusion criteria: Women
were previously diagnosed
with autoimmune,
104
Disease definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall study
definition collected controls to kit controls collection identify bacterial identified as being conclusions
detect potential signal associated with
contamination endometriosis group
NR NR Cervical Mucus, swabs Sterile PBS, sterile NR Lower reproductive Region V4-V5 16S No species identified Existence of distinct bac-
from: lower third of va- physiological saline tract: Samples were rRNA gene ampli- that are possibly associ- terial communities
gina, posterior fornix, en- and dry swabs were taken on the day of con sequencing ated with endometriosis throughout female re-
dometrium, left and right taken from the pre- the clinical visit productive tract indicat-
fallopian tubes and peri- operative skin of Upper reproductive ing the upper female
toneal fluid from Pouch controls, as well as tract: Samples were reproductive tract is not
of Douglas dry swabs of sur- collected during sur- sterile.
geons gloves gery (average
2.81 days after clinical
visit)
Women with infertility Cervical swabs and pos- NR NR Swabs collected before Region V3–V4 Cervical swabs:  Lactobacillus abun-
(without endometriosis terior fornix swabs surgical diagnosis rRNA gene Illumina  Predominantly dance did not vary
confirmed by laparo- sequencing Lactobacillus between groups
scopic visualization), leio-  Gardnerella  Endometriosis-
myoma, ovarian  Streptococcus adenomyosis group
borderline tumor and  Atopobium had higher preva-
teratoma.  Pseudomonas lence of Atopobium in
Posterior fornix swabs: cervical swab
 Predominantly samples
Lactobacillus
 Gardnerella
 Streptococcus
 Atopobium
 Pseudomonas
Endometriosis identified with Control group included Vaginal fluid, eutopic en- NR Negative controls Samples taken during V3–V4 regions am- Vaginal fluid, eutopic en-  No significant differ-
laparoscopic visualization, as women undergoing lapa- dometrium (obtained used throughout Laparoscopy plified by PCR dometrium and endo- ences found in diver-
well as histologic confirmation roscopic surgery for be- with curette) and endo- DNA extraction, Sequenced with metrial lesion: sity between control
nign gynecologic diseases metrial lesion tissue sam- amplification, se- Illumina MiSeq  Similar profiles be- and endometriosis
or elective tubal ligation ples (obtained during quencing and tween all sites groups
surgery) analysis Most abundant  Deep lesions pre-
organisms: sented with a differ-
 Lactobacillus ent composition of
 Gardnerella bacteria (lower
 Streptococcus Lactobacillus, and
 Prevotella higher abundance of
Salliss et al.
Continued
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Table I Continued
Study Clinical study participant Disease definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall study
demographics definition collected controls to kit controls collection identify bacterial identified as being conclusions
detect potential signal associated with
contamination endometriosis group
...................................................................................................................................................................................................................................................................................................
inflammatory and/or neo- Deep endometrial Alishewanella,
plastic disease, and who lesions: Enterococcus and
had used antibiotics/anti-  Less Lactobacillus Pseudomonas)
mycotics in 30 days prior to Higher abundance of:
sample collection  Alishewanella
 Enterococcus
 Pseudomonas
Khan et al., Hum Participants: Confirmed with Laparoscopic Control group had no Vaginal Smears NR DMSO used as a Samples taken during BV score assessed Endometrial:  pH >4.5 for women
Reprod, 2014 N ¼ 73 with endometriosis visualization evidence or history of Endometrial Smears negative control Laparoscopic with gram-staining,  Gardnerella with endometriosis
N ¼ 55 without endometriosis, but oper- Vaginal pH during extraction visualization bacteria identified  a-Streptococcus  GnRHa treatment
endometriosis ated on for dermoid, se- with culturing  Enterococci was significantly and
Age Range: 21–51 years rous, mucinous cysts, methods  Escherichia coli independently associ-
Race/Ethnicity/Country of adenoma or uterine ated with intrauterine
Origin: NR myoma. microbial coloniza-
Menopausal status: tion and occurrence
Microbiota in endometriosis, infertility and pelvic pain

Premenopausal
Exclusion criteria: NR
Pelzer et al., Hum Participants:
Reprod, 2011 N ¼ 18 Fertile women with in-
fertile male partners
(control)
N ¼ 16 Women with
endometriosis
N ¼ 14 Women with PCOS
N ¼ 9 Couples with history of
genital tract infection
N ¼ 14 idiopathic infertility
Age Range: 31–42 years
Race/Ethnicity/Country of
Origin: NR
Menopausal status:
Premenopausal
Exclusion criteria: None, in-
cluded if they fit the groups,
and if they were undergoing
fully stimulated assisted re-
productive technology.
Perrotta et al., Participants:
Reprod Sci, 2020 N ¼ 24 controls
N ¼ 35 with endometriosis
Age Range: 21–49 years
Race/Ethnicity/Country of
Origin: 61% white, 17%
black, 22% mixed race
Menopausal status:
Premenopausal
Exclusion criteria: Participants
were receiving hormone
treatments (GnRH analogs,
progestogens, combined
oral contraceptives) or anti-
biotics 3 months prior to
sample collection, systemic
infection, history of
Women undergoing IVF with
a diagnosis of endometriosis,
PCOS, history of genital tract
infections, or idiopathic
infertility
Confirmed presence of endo-
metriomas with imaging
(transvaginal ultrasound or
MRI scans), or previous lapa-
roscopic visualization.
of endometritis in
women with
endometriosis
Fertile women whose in- Follicular fluid NR NR NR Culturing methods Follicular Fluid:  Follicular fluid not
fertility was attributed to Vaginal Secretion and cytokine  Lactobacillus iners sterile
the male partner analysis  Actinomyces spp.,  Colonizing microor-
 Corynebacterium ganisms in follicular
 auromucosum, fluid were associated
 Fusobacterium spp., with women with
Peptoniphilus endometriosis
 asaccharolticus,
 Peptostreptococcus
spp.,
Propionibacterium
spp.,
 Prevotella,
 Staphylococcus spp.
 Candida parapsilosis
Control group included Vaginal swabs NR NR Samples collected Illumina two-step Vaginal swabs:  Findings suggested
women who do not have 2 months prior to laparo- sequencing of the  Increased that the vaginal
endometriosis and any scopic surgery V4 region of the Anaerococcus in en- microbiome may
inflammatory condition, 16S rRNA gene dometriosis groups. predict the stage of
but underwent laparo-  Anaerococcus was disease when endo-
scopic surgery for other highly predictive for metriosis is present
gynecologic indications rASRM stages 1–2 and  Accurately predicted
3–4 rASRM stages 1–2
compared to rASRM
stages 3–4 (with ma-
chine learning
analysis)
105

Continued

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Table I Continued
Study Clinical study participant
demographics
...................................................................................................................................................................................................................................................................................................
autoimmune disorders, ac-
tive vaginosis, history of
sexually transmitted dis-
eases and contraction of
HIV, pregnancy or malig-
nant neoplasia
Wei et al., Ann Clin Participants:
Microbiol Antimicrob, N ¼ 14 controls
2020 N ¼ 36 with endometriosis
Age Range: 23–44 years
Race/Ethnicity/Country of
Origin: Chinese
Menopausal status:
Premenopausal
Exclusion criteria: Patients had
recorded use of hormonal
drugs or antibiotics within
6 months and vagina medi-
cations within 3 months.
NR, not reported; N/A, not applicable; PGM, personal genome machine; rASRM, revised American Society for Reproductive Medicine; GnRHa, GnRH agonist; PCOS, polycystic ovary syndrome; BV, bacterial vaginosis; GI, gastrointestinal;
IUD, intrauterine device.
Disease definition Comparison group
definition
Participants undergoing lapa- Women undergoing lap-
roscopic visualization of pelvic aroscopic surgery due to
endometriosis, and stage gynecologic benign
identified with endometriosis tumors (ovarian tera-
staging method. toma, serous cystade-
noma and uterine
fibroids), and no symp-
toms of endometriosis
present (confirmed with
surgery), regular men-
strual cycle, no: acute in-
flammation, malignant
tumors and autoimmune
diseases.
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106
Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall study
collected controls to kit controls collection identify bacterial identified as being conclusions
detect potential signal associated with
contamination endometriosis group
Swabs used to collect Negative samples NR Samples taken during V4–V5 16S rRNA Lower reproductive There were only signifi-
lower reproductive tract collected with the Laparoscopic surgery gene regions ampli- tract: cant differences in micro-
samples (lower third of same instruments fied and sequenced Dominated by biome composition in
the vagina, posterior vag- used in sampling with Ion Torrent Lactobacillus in both the upper female repro-
inal fornix, cervical mu- patients, but specific PGM sequencing control and endome- ductive tract, comparing
cus). Upper reproductive components sam- triosis groups. control and endometri-
tract samples collected pled are not stated Cervical mucus: osis study participants
during surgery (endome-  Decrease
trial lavage and perito- Lactobacillus
neal lavage) abundance
 Increase in
Veillonellaceae in
women with
endometriosis
Endometrial lavage:
Higher abundance of:
 Prevotella,
 Veillonella,
 Atopobium,
 Veillonellaceae
Peritoneal lavage: Higher
abundance of:
 Prevotella
 Veillonella
 Atopobium
 Veillonellaceae
 Comamonas
Salliss et al.
Table II Human clinical trials that investigated the association between the microbiome and infertility or IVF.
Study Clinical study participant
demographics
...................................................................................................................................................................................................................................................................................................
Amato et al., Open Participants:
Forum Infect Dis, N ¼ 23 Couples with idiopathic
2020 infertility
Age Range: 32–40 years old
Race/Ethnicity/Country of Origin:
Caucasian
Menopausal status: Premenopausal
Exclusion criteria: Excluded if
patients had PCOS, endometri-
osis, tubal pathology and/or
oligoasthenoteratozoospermia
Babuet al., J Clin Participants:
Diagn Res, 2017 N ¼ 84 Healthy controls
N ¼ 116 Women with infertility
problems
Age Range: 18–45
Race/Ethnicity/Country of Origin:
Asian, Indian
Menopausal status: Premenopausal
Exclusion criteria: Excluded if pa-
tient has a chronic autoimmune
or inflammatory condition, any
oral or topical antimicrobial
within the prior month, women
using any intrauterine contracep-
tive device/hormonal contra-
ceptive, vaginal discharge,
itching, burning and dysuria
Bernabeu et al., J Participants:
Assist Reprod Genet, N ¼ 31 Undergoing ART
2019 Age Range:
NR
Race/Ethnicity/Country of Origin:
NR
Menopausal status: NR
Exclusion criteria: Excluded if
patients have uterine malforma-
tions, untreated hydrosalpinx,
known factors for implantation
failure, defined as implantation
failure following transfer of at
least three good-quality embryos
over at least two cycles were
excluded
Campisciano et al., J Participants:
Cell Physiol, 2017 N ¼ 13 Diagnosed infertility,
N ¼ 14 Idiopathic infertility,
N ¼ 30 Healthy controls,
N ¼ 39 BV control
Age Range: 32–40 years old
Race/Ethnicity/Country of Origin:
Caucasian
Menopausal status: Premenopausal
Infertility definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall conclusions
definition collected controls to kit controls collection identify bacterial identified as being
detect potential signal associated with
contamination infertility
Primary idiopathic infertility Women underwent hys- Vaginal swabs and NR NR N/A Illumina MiSeq se- Vaginal swabs:  Idiopathic infertile
terosonosalpingogram to Seminal Fluid quencing of V3–V4 Lactobacillus crispatus was women showed a dif-
assess tubal patency regions of the 16S the only factor associ- ferent average com-
rRNA gene ated with successful position of vaginal
implantation microbiome com-
pared to healthy
controls.
 Lactobacillus crispatus
was the only bacte-
rium correlated with
successful
implantation
Women were already under- Women with regular Vaginal swabs NR Negative controls N/A Gram-staining of Vaginal swabs:  Women who suffer
going gynecological/infertility menstrual cycles and no used in DNA ex- sample was scored  Healthy controls from infertility
Microbiota in endometriosis, infertility and pelvic pain
treatment gynecological disorders traction and se- with Nugent scoring composed of mostly showed higher fre-
quencing steps to assess consis- Lactobacillus quency of asymp-
tency with BV, and  Micrococcus tomatic vaginosis and
culturing methods  Enterococcus higher abundance of
 Staphylococcus spp. BV-associated bacte-
 Infertility group com- ria, when compared
posed mainly of to healthy controls
Candida spp. and  This study proposed
Enterococcus that women under-
 Escherichia coli going infertility treat-
 Depletion of ment, should also
Lactobacillus species. have their vaginal
flora screened.
Women with a diagnosis of in- NR Vaginal samples NR NR NR Illumina MiSeq se- Vaginal:  Lactobacillus domi-
fertility, indication of ART and quencing of V3–V4 There were 2 main nant profiles were as-
at least 3 months since last an- regions of the 16S clusters: sociated with
tibiotic treatment. rRNA gene  Lactobacillus domi- successful pregnancy
nance, Gardnerella,  Stability of the vaginal
Clostridium, microbiome was as-
Staphylococcus and sociated with suc-
Dialister cessful pregnancy.
 All other Genera
Idiopathic infertility; diagnosed Healthy women (n ¼ 30) Cervical-Vaginal fluid NR NR N/A Ion Torrent se- Cervical-Vaginal fluid:  b-diversity varied be-
infertility defined as (women without BV and women quencing of the V3 Diseased groups had tween all four groups
attending ART clinics for ART diagnosed with BV 16S rRNA region higher a-diversity and  A microbiome com-
treatment) (n ¼ 39) this correlated with position that resem-
less abundance of bles women with BV
Lactobacillus in these was also found in
groups, and more women with idio-
pathic infertility
107
Continued
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Table II Continued
108

Study Clinical study participant Infertility definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall conclusions
demographics definition collected controls to kit controls collection identify bacterial identified as being
detect potential signal associated with
contamination infertility
...................................................................................................................................................................................................................................................................................................
Exclusion criteria: Excluded if pa- growth of anaerobic  Results suggest as-
tient was pregnant, not of repro- bacteria: specifically sessment and identifi-
ductive age, currently using  Gardnerella vaginalis cation of specific
tobacco or alcohol, currently us-  vaginae microorganisms,
ing contraceptive methods, has  Ureaplasma parvum could improve diag-
had hospitalization or use of sys- nosis and treatments
temic medication for chronic dis- of infertility
eases, or use of antibiotics/
probiotics (oral or topic) within
6 months prior to sample
collection
Carosso et al., J Participants: All patients undergoing IVF NR Vaginal swab and em- NR Ultrapure DNase/ Samples collected Next generation se- Vaginal swabs: increase Controlled ovarian stim-
Assist Reprod Genet, N ¼ 15 women undergoing IVF and had no history of infertility bryo transfer catheter tip RNase free water 6 months prior to quencing (NGS) of in: ulation and progesterone
2020 treatment or failed pregnancy used as blank con- IVF treatment the V3, V4 and V6  Prevotella supplementation during
Age Range: <43 years trol used to exclude region of the 16S  Escherichia coli IVF significantly changes
Race/Ethnicity/Country of Origin: contamination rRNA gene  Shigella spp. the composition of the
Caucasian Catheter tip: vaginal and catheter tip
Menopausal status: Premenopausal  Proportion of microbiota, and could af-
Exclusion criteria: Excluded if Lactobacillus reduced. fect endometrial recep-
patients have used antibiotics,  Increase in Prevotella tivity and placentation
have a recent history of cervico- and Atopobium
vaginal infection, recurrent mis-
carriage, no previous repeated
implantation failure, absence of
tubal or uterine pathology, and
GI disease
Fu et al., mBio, 2020 Participants: Women with unexplained re- Women undergoing em- Vaginal swabs NR NR Swabs collected on Illumina MiSeq se- Vaginal swabs: Vaginal Lactobacillus
N ¼ 27 women with repeated im- peated implantation failure bryo transfer for the first the day of embryo quencing of V3–V4  RIF group had higher dominance was positive
plantation failure (RIF) time (by frozen embryo transfer before the regions of the 16S a-diversity compared correlated with preg-
N ¼ 40 controls transfer) procedure rRNA gene to controls nancy rate.
Age Range: 28–37 years Higher abundance of:
Race/Ethnicity/Country of Origin:  Streptococcus
Asian, Chinese  Atopobium
Menopausal status: Premenopausal  Prevotella
Exclusion criteria: Excluded if  Bifidobacterium
patients present with male factor
infertility, ovulation and men-
strual disorders, genital tract or-
ganic lesions, women with BV,
antibiotic use within 2 weeks of
study and systemic diseases
Haahr et al., Hum Participants: Women undergoing IVF NR Vaginal Swabs (Posterior NR NR NR qPCR with Vaginal: qPCR can be used to
Reprod, 2016 N ¼ 130 Infertile Patients treatment Fornix) Bacterial-specific  Gardnerellavaginalis characterize the vaginal
Age Range: Vaginal pH primers of charac-  Atopobium vaginae microbiome of IVF
NR teristic BV-associ- patients and may be
Race/Ethnicity/Country of Origin: ated bacteria, with promising tool for diag-
90% Caucasian ROC curve analysis nosing abnormal vaginal
Menopausal status: Premenopausal microbiomes and for
Exclusion criteria: Excluded if vagi- prediction of clinical
nal sample has been collected pregnancy in IVF
more than 2 months before em- treatment
bryo transfer of prescription of
antibiotics within a month before
inclusion

Continued
Salliss et al.

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Table II Continued
Study Clinical study participant
demographics
...................................................................................................................................................................................................................................................................................................
Haahr et al., J Infect Participants:
Dis, 2019 N ¼ 120 patients undergoing IVF
Age Range: 20–42 years old
Race/Ethnicity/Country of Origin:
NR
Menopausal status: NR
Exclusion criteria: None
Hashimoto et al., J Participants:
Infertility definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall conclusions
definition collected controls to kit controls collection identify bacterial identified as being
detect potential signal associated with
contamination infertility
Women were already attend- NR Vaginal swabs NR NR N/A Illumina MiSeq se- Vaginal samples: qPCR is less expensive,
ing a fertility clinic for IVF ther- quencing of the V4  3 major community simpler and a better pre-
apy; undergoing IVF treatment region of the 16S state types were dictor for reproductive
rRNA gene, and identified: outcomes in patients un-
qPCR with  Dominated by L. dergoing IVF therapy
Bacterial-specific crispatus,
primers of charac-  Dominated by L.
teristic BV-associ- iners,
ated bacteria, with  A diverse community
ROC curve analysis type.
 These did not signifi-
cantly correlate with
reproductive
outcome.
Women undergoing IVF NR Endometrial Samples— NR Ultrapure DNase/ NR Illumina MiSeq se- Endometrial IUI catheter: The endometrial bacte-
Microbiota in endometriosis, infertility and pelvic pain

Assist Reprod Genet, N ¼ 99 IVF Patients treatment with IUI catheter RNase free water quencing of the V1–  Atopobium rial profiles of pregnant
2019 Age Range: Less than 40 years used as blank con- V2, V3–V5 regions  Gardnerella cases with dysbiotic en-
Race/Ethnicity:/Country of Origin trol used to exclude of the 16S rRNA  Streptococcus dometrium were compa-
NR contamination gene rable with those of
Menopausal status: Premenopausal nonpregnant cases
Exclusion criteria: Excluded if par-
ticipants have severe male fac-
tor, hydrosalpinx, uterine
malformation, submucosal my-
oma or antibiotic usage within a
month prior to embryo transfer;
excluded if had symptoms of
vaginitis or endometriosis
Kitaya et al., Participants: Patients undergoing infertility Infertile women with a Endometrial biopsy taken NR Ultrapure DNase/ N/A NGS sequencing of Endometrial fluid:  Endometrial fluid
Mediators Inflamm, N ¼ 28 RIF group examinations (hysterosalpin- history of RIF, and with a curette, endome- RNase free water the V4 region of the Burkholderia spp. microbiota had in-
2019 N ¼ 18 Control (Undergoing first gogram, hysteroscopy, thyroid women undergoing their trial fluid—with MedGyn used as blank con- 16S rRNA gene creased bacterial
IVF attempt) functions, thrombophilic and first IVF attempt. pipette IV and Vaginal trol used to exclude load than the vaginal
Age Range: immunological functions). The secretions contamination. A swabs microbiome in
Mean age in years was 38.7 RIF group was described as mix of both controls and
Race/Ethnicity:/Country of Origin: serial negative pregnancy tests Pseudomonas, RIF group
NR after attempted implantation Escherichia,  Only the endometrial
Menopausal status: NR of five or more good-quality Salmonella, Listeria, fluid microbiome
Exclusion criteria: Not excluded if embryos/blastocysts Bacillus, showed significant
patients had pre-existing condi- Lactobacillus, variation in micro-
tions, such as endometriosis or Enterococcus, and biota composition
PCOS, but these were yeasts when the RIF group
recorded, patient cohorts in- Saccharomyces and and is compared to
cluded patients undergoing IVF Cryptococcus was the control group
treatment used an amplifica-
tion control.
Komiya et al., J Clin Participants: Women undergoing IVF Women that were not Fecal samples NR NR NR Illumina MiSeq se- 1st Stage fecal samples: Predictive factors for
Biochem Nutr, 2020 1st Stage: treatment undergoing ART quencing of V3–V4  Higher abundance of: pregnancy by a decrease
N ¼ 18 infertile women regions of the 16S  Barnesiellaceae in the abundance of
N ¼ 18 fertile controls rRNA gene  Phascolarctobacterium. Paraprevotella and Blautia,
2nd Stage: Lower abundance of: with an increase in the
N ¼ 12/18 infertile patients under- Stenotrophomonas, abundance of
going ART received partially hy-  Streptococcus Bifidobacterium
drolyzed guar gum as a fiber  Roseburia 2nd Stage
supplement fecal samples:

Continued
109

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Table II Continued
110

Study Clinical study participant Infertility definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall conclusions
demographics definition collected controls to kit controls collection identify bacterial identified as being
detect potential signal associated with
contamination infertility
...................................................................................................................................................................................................................................................................................................
Age Range: 32–40 years  Increased abundance
Race/Ethnicity/Country of Origin: of:
Asian, Japanese  Bifidobacterium asso-
Menopausal status: Premenopausal ciated with fiber
Exclusion criteria: NR supplementation
 Decreased abun-
dance of:
 Blautia
 Paraprevotella associ-
ated with fiber
supplementation
Kong et al., Front Participants: Women undergoing IVF NR Vaginal secretion (using NR NR Vaginal secretion Illumina sequencing Vaginal secretion:  Age and endome-
Med, 2020 N ¼ 475 infertility patients treatment sterile suction tube) collected before IVF of vaginal secretion  Lower abundance of trium thickness signif-
Age Range: 27–39 years samples Lactobacillus icantly affected
Race/Ethnicity/Country of Origin:  higher abundance of pregnancy success of
NR Gardnerella and IVF patients
Menopausal status: Premenopausal Prevotella  High levels of LH, es-
Exclusion criteria: Excluded if  Identified from un- trogen and proges-
patients have an interruption or successful IVF terone observed in
discontinuation of treatment for treatment the pregnant out-
any reason, abnormal ovarian come group of IVF
function, abnormal cervical cy- patients.
tology, abnormal semen exami-
nation of partner, or other
diseases combined with infec-
tious diseases, malignant tumors,
severe diabetes, severe liver and
kidney disease were excluded,
cervical treatment and vaginal ir-
rigation within 7 days, sexual ac-
tivity within 2 days and patients
that used antibiotics within
15 days.
Kyono et al., Reprod Participants: Women undergoing IVF Women not undergoing Endometrial samples— NR Ultrapure DNase/ NR Illumina MiSeq se- Endometrial: Not clear evidence of a
Med Biol, 2018 N ¼ 79 IVF patients treatment IVF treatment, but with IUI catheter RNase free water quencing of the V1–  Atopobium Lactobacillus dominant
N ¼ 23 Controls agreed to participate in used as blank con- V2, V3–V5 regions  Bifidobacterium microbiome, with re-
Age Range: Below 45 years old the study trol used to exclude of the 16S rRNA  Gardnerella spect to pregnancy
Race/Ethnicity/Country of Origin: contamination gene  Megasphaera outcome.
Asian, Japanese  Sneathia
Menopausal status: Premenopausal  Prevotella
Exclusion criteria: Excluded if the  Staphylococcus
participants had complaints that  Streptococcus
suggested vaginitis or
endometriosis
Liu et al., Clin Chem, Participants: Women with unexplained re- NR Endometrial fluid col- NR NR NR Illumina MiSeq se- Endometrial tissue: Data suggest the differ-
2018 N ¼ 25 with unexplained recurrent current miscarriage (explained lected with a double lu- quencing of the V4  Hungatella ence of microbiota colo-
miscarriage as the loss of at least three men ET catheter, and region of the 16S  Oxyphotobacteria nization between
Age Range: 20–40 years old consecutive pregnancies be- endometrial biopsy rRNA gene  Verrucomicrobiaceae endometrial fluid and tis-
Race/Ethnicity/Country of Origin: fore 24 weeks of gestation) specimen  Brevundimonas sue (and sampling from
Asian, Japanese  Candidatus both), provide a more
Menopausal status: Premenopausal  Aquiluna complete insight of mi-
Exclusion criteria: Excluded if there  Flavobacterium crobial colonization
was presence of hydrosalpinx,  Tumeriella
uterine pathology (e.g. fibroids),  Flavobacterium
endometrial polyp, or
Salliss et al.

Continued

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Table II Continued
Study Clinical study participant Infertility definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall conclusions
demographics definition collected controls to kit controls collection identify bacterial identified as being
detect potential signal associated with
contamination infertility
...................................................................................................................................................................................................................................................................................................
intrauterine adhesions and medi-
cal conditions that affect the im-
Endometrial
mune system (e.g. systemic
lupus erythematosus)., smokers, fluid:
abnormal menstrual cycles, ab-  Finegoldia
normal uterus, use of estrogen  Anaerococcus
or progestogen hormonal ther-  Clostridoides
apy or intrauterine contraceptive
device within 2 months
Morena et al., Am J Participants: Participants were infertile and NR 1st Study—Endometrial NR NR N/A Pyrosequencing of Non-Lactobacillus domi-  Different bacterial
Obstet Gynecol, 1st Study— were undergoing IVF fluid—using a catheter the V3–V5 regions nant endometrial micro- communities were
2016 N ¼ 13 fertile women—endome- treatment. and syringe, and of the 16S rRNA biome can significantly found between uter-
trial and vaginal microbiota Vaginal aspirates gene decrease implantation ine and vaginal
2nd Study— 2nd Study—Endometrial success. cavities
N ¼ 22 fertile women—hormonal fluid  Uterine cavity de-
Microbiota in endometriosis, infertility and pelvic pain

regulation 3rd Study—Endometrial fined as Lactobacillus

3rd Study— fluid dominated
N ¼ 35 infertile patients undergo-  Non-Lactobacillus
ing IVF dominated micro-
Age Range: 18–40 years old biome, in a receptive
Race/Ethnicity/Country of Origin: endometrial micro-
NR biome, significantly
Menopausal status: Premenopausal reduced implantation
Exclusion criteria: success
1st Study—Excluded if patients had
irregular menstrual cycles, BMI
outside of 19–29 kg/m2 and
patients who used antibiotics/
probiotics 1 month prior to the
study
2nd Study—
Excluded if patients
had irregular menstrual
cycles and patients
who used antibiotics/
probiotics 1 month
prior to the study
3rd Study—
Excluded patients if
BMI is not 19–29 kg/
m2, at least 1 good-
quality embryo was
successfully implanted
with ART and patients
who used antibiotics/
probiotics 1 month
prior to the study
Riganelli et al., Front Participants: Included participants with dif- NR Vaginal fluid (with cyto- NR NR Samples collected Illumina metage- Vaginal fluid:  Results highlight a pu-
Cell Infect Microbiol, N ¼ 34 infertile women ferent infertility etiologies brush) and endometrial at Day 21 of men- nomics sequencing tative relationship be-
2020 Age Range: 22–43 years (tubal occlusion (7/34), biopsy (collected with strual cycle and of the V3–V5 tween vaginal/
Lactobacillus-domi-

Continued
111

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Table II Continued
112

Study Clinical study participant Infertility definition Comparison group Sample type Environmental DNA extraction Timing of sample Method to Key bacteria Overall conclusions
demographics definition collected controls to kit controls collection identify bacterial identified as being
detect potential signal associated with
contamination infertility
...................................................................................................................................................................................................................................................................................................
Race/Ethnicity/Country of Origin: endometriosis (3/34), ovula- IUI catheter inside an- before starting the regions of the 16S nance associated with endometrial micro-
Caucasian tory disorder (9/34) and idio- other catheter) IVF treatment rRNA gene pregnant groups biota and reproduc-
Menopausal status: Premenopausal pathic infertility for at least Enrichment of: tive success
Exclusion criteria: Excluded if 1 year (13/34)).  G. vaginalis  Limited, given that 4
patients had male factor infertil- All participants had undergone  Escherichia fergusonii of 34 participants
ity, risk of pelvic inflammatory ART of second level with im-  Streptococcus achieved pregnancy
disease, cervical carcinoma, on- plantation failure dentisani
going pregnancy, anomalous Endometrial biopsy:
uterine bleeding, and subject of  Higher abundance of:
intrauterine procedures in  Kocuria dechangensis
6 months prior to study.  L. gasseri
Wee et al., J Obstet Participants: Patient reported current infer- NR Vaginal, Cervical and NR NR N/A Illumina metage- Vaginal samples:  Dominant commu-
Gynaecol, 2018 N ¼ 31 tility treatments for more than Endometrial biopsy sam- nomics sequencing Ureaplasma abundance nity members were
Age Range: 35–48 one year, or has previously re- ples taken with a curette of the V3–V5 Cervical samples: generally consistent
Race/Ethnicity/Country of Origin: quired ART for successful regions of the 16S Gardnerella abundance across cervical, vagi-
NR pregnancy rRNA gene Endometrial biopsy: nal and endometrial
Menopausal status: NR Similar composition to microbiomes
Exclusion criteria: Not excluded vaginal and cervical  Although there were
based on any criteria, but patient samples, but bacteria generally lower
demographics were noted (e.g. were generally lower amounts of bacteria
endometriosis patients, contra- in endometrial in the endometrium.
ceptive use, etc., the cohort samples  Tenascin-C was cor-
consisted of women who were related with miscar-
already attending for gynecologi- riage history
cal or fertility care.
Zhao et al., Participants: Infertility definition: women Women who were not Posterior fornix swab NR NR NR Illumina HiSeq 2500 Posterior fornix swab:  No changes were
mSystems, 2020 N ¼ 30 women with secondary with secondary infertility pregnant, no clinically sig- (one taken during the sequencing of the Increased abundance of: observed in the
infertility nificant treatments within first 3 days of follicular V1–V2 region of the  Atopobium microbiome of infer-
N ¼ 92 fertile controls 4 weeks at the start of development and one af- 16S rRNA gene  Aerococcus tile women in rela-
Age Range: 23–42 years the study ter 6 days of GnRH ago-  Bifidobacterium tion to GnRH
Race/Ethnicity/Country of Origin: nist treatment followed Decreased abundance treatment
NR by r-hCG administration of:  Differences were ob-
Menopausal status: Premenopausal 34–36 h before sample  Lactobacillus served between en-
Exclusion criteria: Excluded if collection) dometriosis and
patients had signs or symptoms control groups (re-
of cervical, uterine or tubal infec- gardless of
tion, HIV negative, no family ge- treatment)
netic disease, no clinically
obvious inflammation, no sexual
activity within 2 weeks of the
study

NR, not reported; N/A, not applicable; qPCR, quantitative PCR; ROC, receiver operating characteristics; RIF, repeated implantation failure; ET, embryo transfer; BV, bacterial vaginosis; GI, gastrointestinal; PCOS, polycystic ovary syndrome.
Salliss et al.

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Microbiota in endometriosis, infertility and pelvic pain
..
endometriosis had an enrichment of Enterobacteriaceae, Streptococcus, ..
Pseudomonas, Corynebacterium in cervical samples. In a similar study ..
..
(same control and endometriosis group demographics), Veillonellaceae ..
was found to be enriched with a reduction in Lactobacillus in cervical ...
samples from women with endometriosis (Wei et al., 2020). Another ..
..
study compared women with histologically confirmed endometriosis to ..
women who presented for a well-woman visit and similarly found cer- ..
..
vical enrichment of Streptococcus, as well as enrichment of Gardnerella, ..
Escherichia, Shigella and Ureaplasma and the absence of Atopobium in ..
..
the endometriosis group, compared to the control group (Ata et al., ..
2019; Table I). All of the organisms stated here are associated with ..
..
non-Lactobacillus dominance in the cervicovaginal microbiome, where ..
Gardnerella and Atopobium are common genera that colonize the cervi-
..
..
covaginal epithelium of women with BV (Fredricks et al., 2005). ..
Therefore, it is unexpected that one study would find an absence of
..
..
Atopobium in women with endometriosis, especially since Atopobium is ..
one of the most potent inducers of inflammation in the cervicovaginal
..
..
tract (Doerflinger et al., 2014). Also, considering dysbiosis is associated ..
with cervicovaginal disease states, and potentially endometriosis, infer-
..
..
tility and CPP, it is unusual that Atopobium was found to be decreased ..
..
with the endometriosis group (Ata et al., 2019). Additionally, ..
Streptococcus, Shigella, Escherichia and Ureaplasma are not BV- ..
..
associated organisms, but are considered to be opportunistic patho-
gens or ‘pathobionts’ in the cervicovaginal microbiome, they can con-
tribute to pelvic inflammatory disease, can be isolated from the gut
microbiome, and are associated with changes in estrogen (related to
menopause; Shreiner et al., 2015; Muhleisen and Herbst-Kralovetz,
2016; van de Wijgert, 2017; van de Wijgert et al., 2020). Also,
Streptococcus (Moreno et al., 2018), Shigella (Koninckx et al., 2019b),
Escherichia (Koninckx et al., 2019b) and Ureaplasma (Moreno et al.,
2018) have been associated with chronic endometritis, which is also
correlated with endometriosis (Takebayashi et al., 2014). Therefore,
these bacteria may contribute to endometriosis by mediating inflamma-
tion through chronic endometritis.
Research comparing women with laparoscopically confirmed endo-
metriosis (n ¼ 73) to women undergoing other surgery for dermoid,
serous, or mucinous ovarian cysts (n ¼ 55) was the only study to mea-
sure vaginal pH, and found that women with endometriosis had a sta-
tistically significant increase in cervicovaginal pH to >4.5 (Khan et al.,
2014) compared to women without endometriosis (Table I). A cervi-
covaginal pH of >4.5 is a key characteristic and clinical indicator of BV
(Gorodeski et al., 2005). Therefore, the higher prevalence of BV-
associated organisms and elevated cervicovaginal pH observed in
women with endometriosis would suggest that dysbiotic cervicovaginal
microbiota could contribute/result in the symptoms and/or onset of
endometriosis, possibly reflecting ascension of these organism to the
upper female reproductive tract (Baker et al., 2018).
The presence of the endometrial microbiome has been an ongoing
debate, especially considering it is not clear whether the results reflect
contamination from cervicovaginal bacteria (when samples are col-
lected transervically; Baker et al., 2018) or detection of background
DNA signals related to the molecular characterization of endometrial
samples (Winters et al., 2019). Generally, the studies reviewed here
have found more difference between the endometrial microbiota com-
positions of endometriosis and control groups, compared to the vagina
and cervix. One study by Khan et al. (2014), described above, addi-
tionally compared the endometrial microbiota compositions sampled
113
with endometrial swabs, and found higher abundances of Gardnerella,
Streptococcus, Enterococcus and E. coli in the endometrium of women
with endometriosis (Table I). These organisms are associated with
menopause and depletion of estrogen (Muhleisen and Herbst-
Kralovetz, 2016), which could indicate altered levels of circulating es-
trogen in patients, that contribute to endometriosis pathogenesis.
However, Enterococcus and E. coli are gut colonizers and would suggest
that samples may have been contaminated with gut bacteria, which the
authors acknowledged (Khan et al., 2014). Another study found higher
abundance of Prevotella, Veillonellaceae and Atopobium in the endome-
trium of women with endometriosis, when compared to women with-
out endometriosis. Prevotella, Veillonellaceae and Atopobium are all also
isolated from women with BV (Wei et al., 2020). The higher preva-
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lence of BV-associated bacteria has, to date, been observed in both
the cervicovaginal and endometrial microbiota of women with endo-
metriosis (Bailey and Coe, 2002; Khan et al., 2014; Akiyama et al.,
2019; Ata et al., 2019; Chadchan et al., 2019; Table I), and would sug-
gest that specific BV-associated organisms are consistently associated
with endometriosis.
In addition, Khan et al. (2014) also subjected participants both with
endometriosis (n ¼ 20 out of total 73) and without endometriosis
(n ¼ 19 out of total 55) to GnRHa therapy for 4–6 months prior to
.. collection of vaginal pH, endometrial smear and biopsy samples (col-
.. lected with swabs). As expected, vaginal pH was elevated in all
..
.. patients that used GnRHa therapy compared to women that did not,
.. regardless of endometriosis diagnosis. Women without endometriosis
..
.. using GnRHa treatment therapy had enrichment in the endometrium
.. of Gardnerella and E. coli, while women with endometriosis had enrich-
..
.. ment for Gardnerella, Enterococci and E. coli, and a higher occurrence of
.. endometritis compared to women without endometriosis and sub-
..
.. jected to GnRHa treatment (Table I). GnRHa treatments reduce circu-
.. lating estrogen to target pain symptoms associated with endometriosis
..
.. (Bedaiwy et al., 2017). Estrogen impacts glycogen and is key in pro-
.. moting Lactobacillus growth in the cervicovaginal tract (Muhleisen and
..
.. Herbst-Kralovetz, 2016). Therefore, reducing circulating estrogen with
.. GnRHa treatment will likely negatively impact the growth of health-
..
.. associated Lactobacillus, and could possibly lead to menopausal-like
.. dysbiosis, and contribute to vaginal and endometrial inflammation. The
..
.. study documented a higher occurrence of immunohistochemical endo-
.. metritis (endometrial inflammation) in patients with endometriosis who
..
.. used GnRHa treatment. Therefore, the elevated vaginal pH and enrich-
..
.. ment of bacteria associated with menopause (Muhleisen and Herbst-
.. Kralovetz, 2016) is not unexpected, and supports the fact that GnRHa
..
.. treatment could induce menopausal-like dysbiosis in the cervicovaginal
.. tract and may promote cervicovaginal inflammation. This finding also
..
.. highlights the importance of collecting detailed information on current
.. or recent hormonal treatment exposures for study participants in en-
..
.. dometriosis studies.
.. The microbiota of follicular fluid has also been assessed in relation
..
.. to endometriosis (Pelzer et al., 2011). One study compared five
.. groups of participants (Table I), and included patients with endometri-
..
.. osis (n ¼ 16), fertile women with infertile male partners (n ¼ 18),
.. women with PCOS (n ¼ 14), couples with a history of genital tract in-
..
.. fection (n ¼ 9) and women with idiopathic infertility (n ¼ 14; Pelzer
.. et al., 2011). Sample contamination was assessed by comparing follicu-
..
.. lar fluid samples with bacteria found in vaginal fluid samples, where
.. bacteria present in both locations was considered contamination, and
114
..
bacteria present only in follicular fluid samples were considered a true ..
bacterial signal of follicular fluid. However, further environmental sam- ..
..
pling, such as blank instrument and practitioner samples were not ..
taken. Follicular fluid samples from all five groups contained bacteria, ...
and covered a large variety of species including Lactobacillus iners, ..
..
Peptoniphilus, Fusobacterium and Prevotella (Pelzer et al., 2011). The ..
study found that the mere presence of bacteria in follicular fluid was ..
..
significantly associated with decreased fertility of the individual, as well ..
as with endometriosis (Pelzer et al., 2011). All bacteria analyzed from ..
..
their samples were either BV-associated or were non-Lactobacillus gen- ..
era. However, follicular fluid was collected at the same time as transva- ..
..
ginal oocyte retrieval with a sterile needle, and hence has the potential ..
for cross-contamination with vaginal bacteria. Given that the identified
..
..
bacteria were all BV-associated organisms isolated from the vaginal ..
microbiome (Ceccarani et al., 2019; Muzny et al., 2020), it is possible
..
..
that these samples were contaminated with vaginal bacteria, and fur- ..
ther supports the need for robust contamination controls and method-
..
..
ologies to differentiate between true and false bacterial signals. Despite ..
this, the presence of these BV-associated bacteria in the samples could
..
..
still influence or be a result of endometriosis, and further supports a ..
..
relationship of the microbiome with endometriosis and infertility. ..
Ovarian cells express TLRs that recognize bacterial endotoxins, such ..
..
as LPS, which could lead to the production of pro-inflammatory media- ..
tors and growth factors, promoting progression of endometriosis ..
..
(Khan et al., 2016).
The relationship between the gut microbiome and endometriosis is
under studied, however, women who suffer from endometriosis are
often lean (Farland et al., 2017), which may suggest a metabolic contri-
bution from the gut microbiome in the pathogenesis of endometriosis.
One study assessed the gut microbiota composition of women with
and without endometriosis by collecting rectal swabs at a depth of
3 cm and found no difference in the gut microbiome of endometriosis
groups and healthy controls (Perrotta et al., 2020). In contrast, another
study compared the gut microbiome of healthy controls (n ¼ 14) and
patients with advanced-stage (stages 3 or 4) endometriosis (n ¼ 14),
and found that women with endometriosis had higher levels of
Escherichia and Shigella (Ata et al., 2019; Table I). Recently, a reported
association between anogenital distance and endometriosis may also
explain the frequency or likelihood of the transfer of these organisms
from the rectum to the vagina (Mendiola et al., 2016). Another study
that utilized mice as the model found the same higher abundances of
Escherichia and Shigella in the endometriosis groups (Hantschel et al.,
2019; Table II).
Escherichia-Shigella dominance in the gut microbiota was predicted to
be more proinflammatory and is associated with patients with acute
gastroenteritis, which is also accompanied by high levels of inflamma-
tion (Casta~no-Rodrı́guez et al., 2018). Since endometriosis is an inflam-
matory disorder, it is possible that Escherichia-Shigella dominance in the
gut could be associated with endometriosis and systemic inflammation,
as has been identified by these preclinical and clinical studies (Ata
et al., 2019; Hantschel et al., 2019). Another animal study that utilized
the Rhesus macaque model of endometriosis found a reduction in in-
testinal Lactobacillus spp., and an increase in gram-negative anaerobes
among macaques with endometriosis compared to macaques without
endometriosis (Bailey and Coe, 2002; Table III). The authors suggested
that there was a nonspecific overgrowth of gut bacteria that shifted be-
cause of changes in gut motility related to endometriosis (Bailey and
Salliss et al.
Coe, 2002). Gram-negative anaerobes are actually very common in
the gut owing to the low oxygen environment and are not always
pathogenic (Kim and Jin, 2001). However, a shift in the composition of
the gut microbiome can impact the estrobolome or metabolism, and
this may have downstream effects on estrogen-driven diseases such as
endometriosis, and endometriosis comorbidities, infertility and pelvic
pain.
The potential contribution of the Firmicutes/Bacteroidetes ratio to
host health is generally a controversial topic (Magne et al., 2020), al-
though it has been implicated in obesity (Magne et al., 2020). Two ani-
mal studies demonstrated opposing associations between
endometriosis and the Firmicutes/Bacteroidetes ratio (Magne et al.,
2020). The first study that compared healthy control mice (n ¼ 20) to
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mice with induced endometriosis (n ¼ 16) found that a higher
Firmicutes/Bacteroidetes ratio and higher abundance of Bifidobacterium
were associated with endometriosis groups (Yuan et al., 2018). The
second study investigated the effect of metronidazole treatment on en-
dometriosis-induced mouse models and found that a lower Firmicutes/
Bacteroidetes ratio was associated with endometriosis groups (n ¼ 4)
compared to healthy controls (n ¼ 4; Chadchan et al., 2019). All three
bacterial classifications (Firmicutes, Bacteroidetes and Bifidobacterium)
have genes related to glucuronidase activity, thus could be indicative of
an altered estrobolome and dysregulated estrogen metabolism in mice
with endometriosis compared to controls. Also, metronidazole has
.. anti-inflammatory properties, which may have facilitated the observed
.. reduction in endometriosis disease after metronidazole treatment
..
.. (Bjarnason et al., 1992; Pradhan et al., 2016; Chadchan et al., 2019).
.. Additional studies are needed to better understand the estrobolome
..
.. and its role in endometriosis.
.. In addition to measurements from the reproductive tract, one study
..
.. investigated the microbial composition of deep endometrial lesions
.. (n ¼ 10; collected during laparoscopic surgery) compared to control
..
.. endometrial biopsy samples (n ¼ 11; collected transcervically with a cu-
.. rette) (Hernandes et al., 2020). As part of the study protocol vaginal,
..
.. swabs were also collected. No differences were identified (vaginal or
.. endometrium samples) between controls and endometriosis groups,
..
.. except in the deep endometrial lesions of endometriosis patients,
.. where they found increased abundance of Alishewanella, Enterococcus
..
.. and Pseudomonas, and reduction in Lactobacillus (Hernandes et al.,
.. 2020). The data from endometrial lesions indicate that there may be
..
.. bacteria directly affecting lesion inflammation and growth, similar to the
..
.. tumor-promoting features of oncobacteria (Laborda-Illanes et al.,
.. 2020), and this should be investigated further. Comparisons of the
..
.. microbiota composition of peritoneal fluid collected during laparo-
.. scopic surgery from women with endometriosis (n ¼ 36) and controls
..
.. without endometriosis (n ¼ 14) revealed an enrichment of Prevotella,
.. Veillonellaceae, Atopobium and Comamonas in the endometriosis group
..
.. relative to controls (Wei et al., 2020). Again, the majority of these
.. bacteria are BV-associated microorganisms (Biagi et al., 2009; Muzny
..
.. et al., 2020) and therefore could implicate ascension of dysbiotic bac-
.. teria to the upper genital tract in the pathogenesis of endometriosis.
..
.. Studies so far have identified the enrichment of BV-associated bacte-
.. ria, pathobionts and gut bacteria in the genital microbiome of patients
..
.. with endometriosis compared to healthy controls. While alterations in
.. the Firmicutes/Bacteroidetes ratio and higher abundance of
..
.. Bifidobacterium were associated with study participants suffering from
.. endometriosis compared to healthy controls, results from the gut
Table III Animal studies that investigated the association between the gut microbiota and endometriosis.
Study
...................................................................................................................................................................................................................................................................................................
Bailey et al., Hum
Reprod, 2002
Cao et al., Evid
Based Complement
Alternat Med, 2020
Chadchan et al.,
Hum Reprod, 2019
Hantschel et al.,
PLoS One, 2019
Animal model Sample type Endometriosis model Method to identify bac- Key bacteria identified in Overall conclusions
collected terial signal endometriosis animal
model
Groups: Fecal samples and The study used macaques Culturing methods (with dif- Fecal samples:  Endometriosis is corre-
N ¼ 18 intestinal tissue that exhibited naturally oc- ferential and selective agars)  Significant reduction in lated with an adjusted
Age: 10–31 years old curring endometriosis Lactobacilli gut microbiome in
Animal species/genetic back-  Higher abundance of gram- macaques
ground: Macaca mulatta negative anaerobes and fac-  Macaques with endome-
Hormones used: None ultative anaerobes triosis were more likely
Exclusion criteria: Control ani- to have intestinal
mals had no symptoms of en- inflammation
dometriosis for 2 years prior
to the study.
Participants: Fecal pellets Autologously transplanted/ Illumina MiSeq of the V3–V4 Fecal pellets: The therapeutic mecha-
Microbiota in endometriosis, infertility and pelvic pain
N ¼ 24 Endometriosis group sutured 5 mm endometrial regions of the 16S rRNA  a-diversity decreased nisms of both letrozole and
N ¼ 8 control tissue segment to left peri- gene  Firmicutes/Bacteroidetes ratio Shaofu Zhuyu decoction
Age: 6- to 8-week old toneal wall and allowed to increased were related to the restora-
Animal species/genetic back- establish for 4 weeks, be-  Ruminococcaceae abundance tion of the gut microbiota
ground: Sprague Dawley rats fore treatment with letro- decreased
Hormones used: Estradiol valer- zole or Shaofu Zhuyu
ate 24 h before endometriosis decoction (Chinese herbal
inducing surgery treatment)
Exclusion criteria: No exclusion
criteria, but estrus stage moni-
tored daily with vaginal
smears (tissue transplanted
during estrus stage)
Groups: Fecal pellets, perito- Endometrial tissue from es- Illumina MiSeq of the V1–V9 Fecal pellets:  Oral antibiotic treat-
N ¼ 5 without endometriosis neal fluid, endo- trus stage mice was autolo- regions of the 16S rRNA  Bacteroidetes were in higher ment reduces endome-
N ¼ 5 with endometriosis þ metriotic lesions gously transplanted to the gene abundance triosis progression in
vehicle peritoneal wall  Firmicutes were in a lower mice, by reducing spe-
N ¼ 4 with endometriosis and abundance cific bacteria
subjected to broad spectrum Peritoneal fluid:  Oral gavage with feces
antibiotics (vancomycin, neo-  Inflammatory response as from mice with endo-
mycin, metronidazole and measured by macrophage metriosis restored
ampicillin) marker iba1, IL-1b, TNF-a, endometriotic lesion
Age: 10 weeks old IL-6 and TGF-b1 were re- growth and inflamma-
Animal species/genetic back- duced by treatment with tion in metronidazole-
ground: C57BL/6 mice broad spectrum antibiotics treated mice
Hormones used: None  Endometriotic lesions:
Exclusion criteria: No exclusion Treatment with metronida-
criteria zole only exhibited smaller
ectopic lesions relative to
control mice
Groups: Fecal pellets Transplanted uterine tissue Illumina MiSeq of the V4–V5 Fecal pellets: Fecal bacterial composition
N ¼ 8 Controls from 4 C57BL/6-TgN regions of the 16S rRNA Induction of endometriosis in was unchanged by the
N ¼ 8 Induced endometriosis (ACTB-EGFP)10sb/J donor gene mice did not result in
115
Continued
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Table III Continued
Study Animal model
...................................................................................................................................................................................................................................................................................................
Age: 12–16 weeks old
Animal species/genetic back-
ground: C57BL6 wild-type
mice and transgenic C57BL/
6-TgN (ACTB-EGFP)10sb/J
donor mice
Hormones used: None
Exclusion criteria: Mice were
excluded if they were not in
the estrus stage.
Ni et al., Am J Participants:
Reprod Immunol, N ¼ 16 Endometriosis group
2020 N ¼ 20 control
Age: 6 weeks old
Animal species/genetic back-
ground: C57BL/6J mice
Hormones used: Estrogen solu-
tion on Days 1, 4 and 7 (from
initiation of endometriosis
model—endometriosis frag-
ments were transplanted on
Day 8)
Exclusion criteria: No exclusion
criteria, but estrus stage moni-
tored daily with vaginal
smears (tissue transplanted
during estrus stage)
Yuan et al., Hum Groups:
Reprod, 2018 N ¼ 16 Endometriosis group
N ¼ 20 control
Age: NR
Animal species/genetic back-
ground: C57BL6 mice
Hormones used: Estradiol ben-
zoate, 1 week before sacrific-
ing the mice for uterine horn
collection
Exclusion criteria: Mice were
not excluded, but endometrial
tissue was not injected until
the mouse reached estrus
stage, which was monitored
by vaginal smears collected
every morning for 2 weeks
NR, not reported; TNF, tumor necrosis factor; TGF, transforming growth factor; ACTB-EGFP, beta-actin/enhanced green fluorescent protein.
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Sample type Endometriosis model
collected
mice, into the peritoneal
cavity of 8 C57BL/6 wild-
type mice. All cells but hair
and red blood cells express
GFP, for visualization of
implanted tissue
Feces from cecum Mice injected with estrogen
solution on Days 1, 4 and 7
and endometrial fragments
were transplanted on Day
8. Donor mice were sacri-
ficed, then 1 mm  1 mm 
1 mm endometrial frag-
ments were implanted intra-
peritoneally with syringe
aspiration. Estrogen solution
was injected again the day
after endometriosis induc-
tion, Days 11 and 14. After
3 weeks of normal growth
mice were euthanized to re-
trieve feces from the cecum
segment.
Fecal pellets Induced by intraperitoneal
injections of endometrial tis-
sue, collected from sacri-
ficed donor mice
Method to identify bac-
terial signal
Illumina MiSeq of the V3–V4
regions of the 16S rRNA
gene
Illumina TruSeq of the V4
region of the 16S rRNA
gene
116
Key bacteria identified in Overall conclusions
endometriosis animal
model
pronounced dysbiosis or induction of endometriosis
changes in bacterial (in early lesion formation)
composition
Feces from cecum: The abnormal fecal metabo-
 Decreased gut microbiota lites (secondary bile acid
diversity biosynthesis and alpha-lino-
 Decreased Firmicutes/ lenic acid pathways), which
Bacteroidetes ratio are influenced by dysbiosis,
Increased abundance of: characteristics of endome-
 Allobaculum triosis mice and could pro-
 Akkermansia vide important metrics to
 Parasutterella distinguish the disease
 Rikenella
Decreased abundance of:
 Lactobacillus
 Bacteroides
 Lachnospiraceae
Fecal pellets: Mice with the persistence of
 Endometriosis and control lesions for 42 days develop
groups shared similar rich- distinct composition of gut
ness/diversity microbiota.
 Elevated Firmicutes/
Bacteroidetes ratio
 Bifidobacterium increased
Salliss et al.
Microbiota in endometriosis, infertility and pelvic pain
..
microbiome were still somewhat inconclusive. Overall, this evidence ..
suggests that colonization of the genital tract by BV-associated bacteria ..
..
and pathobionts has been associated with endometriosis. However, ..
two human studies found changes in the gut microbiome in endometri- ...
osis; therefore, the requirement for additional studies is evident. ..
..
Furthermore, no differences have been identified either in estrogen- ..
metabolizing bacteria in the gut, or in b-glucuronidase activity, when ..
..
comparing women with and without endometriosis, despite estrogen ..
being key to endometriosis pathophysiology. ..
..
..
Infertility and the microbiome ..
We found 19 studies (Table II) that have investigated the relation of
..
..
microbiota composition of cervicovaginal samples and endometrial ..
..
microbiota samples to fertility and embryo implantation success of ..
patients undergoing ART. Patterns emerged in this body of research ..
..
allowing for a clearer consensus to be drawn from these studies. One ..
study investigated the cervicovaginal microbiome and infertility ..
..
(Campisciano et al., 2017): this was achieved through collecting cervi- ..
covaginal fluid and comparing healthy controls (n ¼ 30) to three dis- ..
..
eased groups that included patients with diagnosed infertility (n ¼ 13), ..
idiopathic infertility (n ¼ 14) or BV (n ¼ 39), and found that a higher ..
..
a-diversity and Lactobacillus depletion are associated with all three dis- ..
eased groups and reflect an association of dysbiosis in the cervicovagi- ..
..
nal fluid microbiome with infertility. Gardnerella was also connected to
patients who suffer with infertility and is a ubiquitous and key BV-
associated bacterium (Wee et al., 2018). These data suggest that cervi-
covaginal dysbiosis could contribute to infertility (Africa et al., 2014;
Davis, 2016; Haahr et al., 2016; Babu et al., 2017; Amato et al., 2020;
López-Moreno and Aguilera, 2020; Tomaiuolo et al., 2020).
Collectively, these studies (Table II) found that the genital microbiome
of women suffering with infertility had higher abundances of BV-
associated bacteria, as well as an association of successful pregnancy
with Lactobacillus dominance.
Studies have found a range of bacteria colonizing the vaginal and en-
dometrial mucosa, that are key microbiota in the development of BV/
vaginal dysbiosis, and were associated with infertility (Pelzer et al.,
2011; Haahr et al., 2016; Moreno et al., 2016; Campisciano et al.,
2017; Kroon et al., 2018; Wee et al., 2018; Bernabeu et al., 2019;
Haahr et al., 2019; Kyono et al., 2019; Amato et al., 2020; Table II).
For example, nine studies that investigated the vaginal microbiota com-
position of women undergoing IVF treatment found the following BV-
associated bacteria to be associated with unsuccessful pregnancy:
Atopobium, Gardnerella, Streptococcus, Megasphaera, Sneathia, Prevotella,
Finegoldia, as well as bacteria that are found in the anus (Anaerococcus,
Bifidobacterium, E. coli and Shigella; Haahr et al., 2016; Hashimoto and
Kyono, 2019; Kyono et al., 2019; Carosso et al., 2020; Fu et al., 2020;
Kong et al., 2020; Riganelli et al., 2020; Table I). Bifidobacterium, E. coli
and Shigella are typically found in the anus, which may indicate that
transfer of gut bacteria to the vagina could be higher in women with
infertility and possibly influenced by a shorter anogenital distance, men-
tioned earlier as a potential risk factor for endometriosis (Shreiner
et al., 2015). Bacteria found in vaginal samples were also common in
endometrial samples collected transcervically via IVF or an IUI catheter
tip obtained from women undergoing IVF treatment. The specific com-
position was non-Lactobacillus dominant (Kyono et al., 2018) and con-
tained BV-associated bacteria, which suggests that vaginal bacteria may
cross-contaminate endometrial samples collected transcervically with a
117
catheter, and may reflect a combination of both vaginal and endome-
trial microbiota sample compositions.
Differences in the cervicovaginal microbiota associated with infertility
have also been investigated in two studies. The first study compared
the reproductive tract microbiome (collected with swabs and use of
sterile speculum) of fertile control women (n ¼ 16) with infertile
women (n ¼ 15), and reported a higher abundance of Gardnerella in
the infertile group (Wee et al., 2018). Another, larger study (n ¼ 30
fertile controls and n ¼ 92 women with secondary infertility) found
that the infertile group had a reduction in Lactobacillus, but increases in
Atopobium, Aerococcus and Bifidobacterium (Zhao et al., 2020). Both
studies provide evidence that dysbiosis in the cervical microbiome neg-
atively impacts the fertility of women, as indicated by the reduction in
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Lactobacillus and increases in BV-associated bacteria, namely
Gardnerella and Atopobium (Biagi et al., 2009; Verstraelen et al., 2016;
Muzny et al., 2020). In addition, the presence of the gut bacterium
Bifidobacterium (O’Callaghan and van Sinderen, 2016) suggests that the
gut-vagina axis, anogenital distance and translocation of these bacteria
to the genital tract may play a role in contributing to infertility.
Unique bacterial species and signatures have been identified in the
endometrial fluid, collected transcervically, of women with infertility
compared to women without infertility, or had unexplained miscar-
riage; specifically, Burkholderia spp.(Kitaya et al., 2019), Hungatella,
.. Oxyphotobacteria, Verrucomicrobiaceae, Brevundimonas, Candidatus,
..
.. Aquiluna, Flavobacterium, Finegoldia, Anaerococcus, Clostridoides (Liu et al.,
.. 2018) and Kocuria dechangensis (Riganelli et al., 2020) were enriched in
..
.. women with infertility. The majority of these species are not typically
.. isolated from the cervicovaginal microenvironment and could also indi-
..
.. cate a contamination signal from other mucosal sites, the collection en-
.. vironment or contamination from DNA extraction kits (e.g. the
..
.. ‘kitome’; Chen et al., 2017; Anahtar et al., 2018; Kroon et al., 2018;
.. Ceccarani et al., 2019; Olomu et al., 2020). However, these studies
..
.. did not include contamination controls that would have allowed for
.. identification of potential contamination signals (Moreno et al., 2016;
..
.. Verstraelen et al., 2016; Kitaya et al., 2019; Liu et al., 2019). One study
.. that compared the endometrial aspirates (collected with a catheter
..
.. and syringe inserted 6 cm into the uterine cavity) of patients with suc-
.. cessful and unsuccessful pregnancy by ART found that a lack of
..
.. Lactobacillus spp. significantly decreased implantation success (Moreno
.. et al., 2016). The same conclusion was supported further by a similar
..
.. study that collected endometrial microbiota samples from the tip of a
..
.. catheter, that had been inserted into the uterus (Carosso et al., 2020).
.. In a separate study by Amato et al. (2020), researchers compared the
..
.. vaginal microbiota of women with or without infertility and found that
.. Lactobacillus dominance was the only microbial factor that led to im-
..
.. plantation success, which was also the conclusion reached from three
.. additional studies (that collected vaginal secretion, endometrial aspi-
..
.. rates and biopsy) that compared the microbiome to pregnancy success
.. of infertile cohorts undergoing ART (Moreno et al., 2016; Kong et al.,
..
.. 2020; Riganelli et al., 2020). These studies compared the vaginal micro-
.. biome (sampled with swabs) to pregnancy outcome of IVF patients
..
.. (n ¼ 475; Kong et al., 2020) and (n ¼ 34) controls (Riganelli et al.,
.. 2020). Collectively, Lactobacillus dominance in the genital microbiome
..
.. appears to play a role in promoting successful pregnancy in women un-
.. dergoing ART, such as IVF.
..
.. One study by Komiya et al. (2020) assessed changes in the gut
.. microbiome (collected stool) of women with (n ¼ 18) and without
118 Salliss et al.

..
infertility (n ¼ 18), which highlights the paucity of studies investigating .. demonstrate clear causal links between the microbiome and the path-
the association of the gut microbiome with infertility. The group diag- .. ogenesis of endometriosis, infertility or CPP.
..
nosed with infertility showed a higher abundance of Barnesiellaceae and ..
Phascolarctobacterium, and a reduction in Stenotrophomonas, ...
Streptococcus and Roseburia (Komiya et al., 2020). Several groups of ..
.. Methodical limitations of
bacteria are part of the gut microbiome (Barnesiellaceae, ..
Phascolarctobacterium and Roseburia) and have previously been associ- .. current microbiome studies
..
ated with an active lifestyle (Bressa et al., 2017; Wu et al., 2017), indi- ..
cating a potential contribution of metabolism in the pathophysiology of .. Case definition of endometriosis, infertility
.. and CPP
infertility. ..
Infertility could potentially be orchestrated through elevated levels of
.. Human studies
..
inflammation and other changes in the local microenvironment that .. The majority of human studies reviewed defined endometriosis
cause remodeling through, for example, mucus degradation by BV-
..
.. through surgical confirmation (visualization and/or histologic evalua-

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associated bacteria. These physiological changes elicited by the genital .. tion), however, several studies (Table I) did not clearly articulate the
microbiota have been postulated to play a role in other pathologies in-
..
.. methods used for diagnosing endometriosis (Pelzer et al., 2011; Chen
cluding preterm birth and gynecological cancers (Muzny et al., 2019; .. et al., 2017). Differences in endometriosis diagnostic criteria may rep-
..
Łaniewski et al., 2020). Also, bacterial endotoxins, such as LPS, are .. resent differences in severity of symptoms, lesion location, hormonal
able to induce an innate immune response through interaction with .. therapy utilization and thereby differences in the microbiome (Molina
..
TLR4, where LPS presence in menstrual fluid was associated with a de- .. et al., 2020). It is vital for studies to state the diagnostic method and
creased pregnancy rate (Kamiyama et al., 2004). With regard to infer- .. criteria used for diagnosing endometriosis as well as the exclusion cri-
..
tility, these microbiota-mediated mechanisms could impact embryo .. teria for other conditions, to ensure that any bacterial associations/
implantation, which is also supported by the numerous studies demon- .. causations are exclusively related to endometriosis and not to other
..
strating that Lactobacillus dominance in the genital microbiome is con- .. underlying diseases or therapies for endometriosis.
sistently associated with successful pregnancy (Moreno et al., 2016; .. Similarly, clinical studies that investigated the association of the
..
Amato et al., 2020). On the other hand, BV-associated bacteria could .. microbiome with infertility often did not record any criteria for diagno-
alter the local microenvironments, including the metabolic environ-
.. sis of infertility, however, most included women undergoing fertility
..
ment, to those that may be less favorable for implantation and fertility. .. treatment. Infertility is usually defined as 12 months of unprotected in-
..
CPP and the microbiome .. tercourse without conception and may be caused by a variety of un-
.. derlying gynecologic conditions, including endometriosis (Matzuk and
To date, no studies have examined the microbiome of women with ..
.. Lamb, 2008) or may be attributed to the male partner. When re-
CPP exclusive of overlapping conditions of endometriosis, IC or IBS. .. search studies of infertility combine participants who have different un-
One study that investigated the urinary microbiome and IC ..
.. derlying conditions causing their infertility, the study results may be
(Abernethy et al., 2017) found that the urinary microbiome of patients .. inaccurate or more reflective of the dominant infertility diagnosis within
with IC was less likely to contain Lactobacillus acidophilus. Lactobacillus ..
.. the sample. Indeed, this may lead to misclassification or obstruction of
acidophilus was also associated with less severe symptom scores on .. true patterns of association among certain rarer conditions that cause
the IC index (Abernethy et al., 2017). An association between gut dys- ..
.. infertility. Some microbiome studies used samples from women under-
biosis and IBS exists (Matheis et al., 2007; Menees and Chey, 2018) .. going ART/IVF as a proxy for infertility (Wee et al., 2018).
and would suggest that CPP can be influenced by the gut microbiome ..
.. Importantly, fertility treatments can influence the host microbiome,
or specific bacteria within the microbiome. Two other studies have .. and therefore, should be described and considered when interpreting
been able to link the microbiome with pain generation, by identifying a
..
.. results from these studies.
dysmenorrhea-associated elevation of prostaglandin levels in menstrual ..
fluid, and LPS-mediated increases in several proinflammatory mediators
.. Animal studies—methods to produce a model of endometriosis
..
as well as the pain mediators cyclo-oxygenase 2 and prostaglandin syn- .. In our review, we found five animal studies (Table III) that model en-
thetase 2 (Khan et al., 2012; Azuma et al., 2017). In addition, local in-
..
.. dometriosis using inbred mice (wild-type C57BL/6). Studies differed in
flammatory pain mediators and microbial ligands involved in pain .. their methods of creating the endometriosis model to study the gut
..
generation and perception can cause uterine dysperistalsis that can im- .. microbiome (Yuan et al., 2018; Chadchan et al., 2019; Hantschel et al.,
pact pelvic pain. However, further research into the role of the genito- .. 2019; Cao et al., 2020; Ni et al., 2020). All five studies obtained endo-
..
urinary and gut microbiomes on the development and progression of .. metrial tissue from mice in the estrus stage that was monitored with
CPP and associated conditions is required before the microbiome can .. either vaginal lavage and analyzed with phase-contrast microscopy, or
..
be associated with the pathogenesis of CPP. .. with vaginal smears. The first study used four transgenic (green fluores-
Overall, studies to date have attempted to elucidate a contribution .. cent protein) donor mice (C57BL/6-TgN ACTB-EGFP10sb/J) to ob-
..
of the cervicovaginal/endometrial/gut microbiomes to the pathogene- .. tain 2 mm endometrial fragments for i.p. injection into wild-type mice
sis of endometriosis, infertility and CPP. Some of the findings outlined .. (mice were left for 21 days before sample collection), and they found
..
above identify associations with bacteria that have been linked to con- .. enrichment of Escherichia and Shigella in the gut microbiome of mice
tamination in other reports (de Goffau et al., 2019). Therefore, more .. with induced endometriosis compared to mice without endometriosis
..
robust studies that employ rigorous controls, phenotypic characteriza- .. (Hantschel et al., 2019). The second study used a similar model, ex-
tion, longitudinal sampling and rich patient metadata are required to
.. cept they used 3 mm endometrial fragments that were sutured onto
Microbiota in endometriosis, infertility and pelvic pain
..
the peritoneal wall, and mice were left for 24 h after the
endometriosis-inducing surgery before administering antibiotics through
drinking water for 21 days prior to sample collection (Chadchan et al.,
2019). This study found higher abundance of Bacteroidetes and lower
abundance of Firmicutes in the gut microbiome of mice with endometri-
osis compared to mice without endometriosis (Chadchan et al., 2019).
The third study used a similar model to the two previous studies ex-
cept that they used endometrial debris of 1 mm  1 mm  1 mm,
injected estrogen solution on Days 1, 4, 7 (on Day 8 endometrial tis-
sue was transplanted), 11 and 14, and allowed the model to develop
for 3 weeks before euthanazing to collect fecal pellets (Ni et al., 2020).
This study found that the gut microbiome of the endometriosis mouse
model had a lower a-diversity, lower Firmicutes/Bacteroidetes ratio,
higher abundance of Allobaculum, Akkermansia, Parasutterella, Rikenella
and lower abundance of Lactobacillus, Bacteroides and Lachnospiraceae
(Table III).
The fourth study used a different model that involved autologous
transplantation of 1 mm endometrial fragments injected i.p., and they
found that mice with endometriosis had developed distinct gut micro-
biota compositions that comprised mainly a higher Firmicutes/
Bacteroidetes ratio and higher abundance of Bifidobacterium, 42 days af-
ter the induction of endometriosis (Yuan et al., 2018). In addition, a
study by Cao et al. (2020), also used autologous transplantation of en-
dometrial tissue, where 5 mm  5 mm endometrial fragments were su-
tured to the peritoneal wall and endometriosis was established after
4 weeks. At this time, fecal pellets were collected and results showed
that the endometriosis-induced mice had a higher Firmicutes/
Bacteroidetes ratio in their gut microbiome as well as lower abundance
of Ruminococcaceae. Therefore, all five studies provide evidence for an
association of the gut microbiome with endometriosis (Table III).
One study that investigated gut bacteria and glucuronidases found
that a particular Bacteroidetes species, namely B. fragilis, as well as the
genera Firmicutes, Actinobacteria and Proteobacteria, encoded a b-glucu-
ronidase enzyme (Pollet et al., 2017). The above murine studies also
found a higher prevalence of Bacteroidetes in the gut microbiome of
mice with induced endometriosis, although the specific species were
not identified (Chadchan et al., 2019). This could indicate a potential
role for the Bacteroidetes or even the Firmicutes/Bacteroidetes ratio, in
the pathophysiology of endometriosis, infertility and CPP, which has
not yet been investigated.
In addition, the human studies reviewed here have isolated several
bacteria associated with endometriosis that are part of these b-glucu-
ronidase taxa such as Escherichia, Shigella and Streptococcus (Pollet
et al., 2017; Koedooder et al., 2018; Kyono et al., 2018; Ata et al.,
2019). Although these were enriched in the genital tract, and not the
gut microbiome, further analysis could reveal the function of b-glucu-
ronidase activity in the genital tract or possibly estrogen absorption
across the genital epithelium that impacts the microenvironment.
Potentially, b-glucuronidase enzyme activity could be regulated or influ-
enced by surrounding bacteria, and by extension the microbiota com-
position could influence b-glucuronidase activity and expression within
the microbiome, through intra- and intermicrobial interactions and
signaling.
CPP—need for future research
As a multifactorial condition, CPP is difficult to study. Specific to the
microbiome, it is challenging to discern between changes that lead to
119
.. disease incidence versus changes that are a result of the disease. One
.. study that investigated the urinary microbiome and IC which can be as-
..
.. sociated with CPP found that the urinary microbiome differed between
... symptomatic patients and healthy controls (Abernethy et al., 2017). In
.. addition, patients with IC possessed elevated levels of cytokines, which
..
.. may contribute to inflammation and pain, CPP and other pain syn-
.. dromes, including endometriosis (Persu et al., 2010; Uppal et al., 2011;
..
.. Triolo et al., 2013; Ayorinde et al., 2015; Junttila, 2018). Future studies
.. that investigate the relationship of the microbiome with CPP should
..
.. clearly identify the co-occurring conditions with CPP (Fig. 1).
..
..
.. Sampling of participants
..
.. Many factors are known to influence the microbiome including demo-
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..
.. graphic and lifestyle factors such as age, race/ethnicity, diet, BMI, med-
.. ical history, pregnancy, childbirth, sexual and hygiene practices, genetic
..
.. variation, cultural habits, menstrual cycle, hormones, physical activity
.. and the environment (Neggers et al., 2007; Hickey et al., 2012; Gupta
..
.. et al., 2017; Aleman and Valenzano, 2019; Li et al., 2019; Xu et al.,
.. 2019; Molina et al., 2020). Therefore, it is imperative for studies of the
..
.. microbiome to consider a number of factors when sampling partici-
.. pants and record this patient metadata to aid in reporting and inter-
..
.. pretation of the findings. Participants in the existing research studies
..
.. ranged in age from 18 to 65 years (Table I). While the majority of
.. studies recruited premenopausal participants, some studies included
..
.. postmenopausal women. Menopausal status is important to consider
.. in microbiome studies, as the reduction of estrogen that occurs during
..
.. menopause can influence the cervicovaginal microbiome composition
.. (Muhleisen and Herbst-Kralovetz, 2016; Baker et al., 2017). From the
..
.. standpoint of microbiome research, all studies should categorize partic-
.. ipants by menopausal status.
..
.. Overall, information on participant race and ethnicity was poorly
.. recorded in the existing literature; however, some information could
..
.. be inferred based on country of study origin (Table I). Moreover, most
.. research was conducted among homogeneous populations, which may
..
.. be partially driven by known barriers to accessing reproductive health
..
.. care (Bougie et al., 2019). This information should be consistently
.. documented in microbiome studies, and future research should strive
..
.. to recruit large, racially/ethnically diverse cohorts for sample
.. collection.
..
.. Studies should also routinely collect detailed clinical information and
.. past medical history of procedures that breech the cervical barrier, as
..
.. well as current/recent medication use. For example, information on
.. history of STIs, dilation and curettage, and intrauterine devices or
..
.. other gynecologic procedures is important to document in study popu-
.. lations as these conditions/procedures may introduce bacteria into the
..
.. uterus. In our review of the literature, we found 12/31 studies that ex-
..
.. cluded participants who used antibiotics within the past 30 days. The
.. use of antibiotics, especially broad spectrum antibiotics, can impact the
..
.. microbiome and therefore can confound and impact the results if not
.. considered, or reported correctly. Most studies we found did not in-
..
.. clude information on current or prior medication use in relation to
.. timing of sample collection. Hormonal treatments, particularly hor-
..
.. mone suppressive treatments for some gynecologic conditions, may in-
.. fluence the microbiome (Achilles et al., 2018; Fosch et al., 2018);
..
.. therefore, information about treatment regimens for both the diseased
.. population and the comparison group is critical for studies in this field.
120
..
From an epidemiological perspective, it is also important to ..
consider the generalizability of the study sample. There are known ..
..
barriers to receiving an endometriosis diagnosis (Nnoaham et al., ..
2011), which may lead to racial/ethnic and economic disparities in ...
who is being studied if sampling occurs among a convenient clinical ..
..
sample of women undergoing surgery for endometriosis (Bougie ..
et al., 2019; Farland and Horne, 2019). There are also known dis-
..
..
parities in accessing fertility care and treatment by race/ethnicity, ..
socioeconomic status and health behaviors (Farland et al., 2016).
..
..
Therefore, a convenient study sample recruited from a fertility clinic ..
..
or from women undergoing gynecologic surgery may not be ..
representative of the general population, which may lead to issues ..
..
of generalizability and selection bias. ..
In addition to clearly defining an appropriate overall study popula- ..
..
tion, it is also important to clearly define a useful comparison group. ..
This is especially important in studies of gynecological and reproductive ..
..
conditions where comparison groups are often drawn from convenient ..
samples of patients that may be presenting clinically for other benign ..
..
indications. Among the studies that investigated the association of the ..
microbiome with endometriosis, there was no consistently defined
..
..
comparison group across studies. For example, some studies of endo- ..
..
metriosis utilized comparison groups that included women with fib-
roids, benign tumors or had surgery for ovarian cysts, while excluding
women that presented with vaginal inflammation, cancer, endocrine
disorders or autoimmune disorders. In some studies of infertility,
patients were excluded if they had vaginitis, endometriosis, uterine
malformation, fibroids, endometrial polyps, intrauterine adhesions, sys-
temic lupus erythematosus, PCOS, tubal pathology or used tobacco or
alcohol (Table I;Khan et al., 2014). Heterogeneity in the comparison
group across studies may lead to biased conclusions (Shafrir et al.,
2018). An ideal comparison group for microbiome studies should be
free of the disease of interest and free of any other conditions that are
known to influence the microbiome.
Collection of samples
Timing of sample collection
To date, nearly all human studies included in this review have been
cross-sectional. Four studies (80%) of endometriosis collected vagi-
nal and cervical samples before or during laparoscopic diagnostic
surgery (Khan et al., 2014; Chen et al., 2017; Akiyama et al., 2019;
Ata et al., 2019; Table I). Among microbiome and infertility studies,
none had recorded when the samples were collected relative to in-
fertility diagnosis. In addition, most studies failed to identify specific
timing of the menstrual cycle at sample collection. No study was
able to prospectively compare microbiome measurements taken be-
fore disease incidence. Given the cross-sectional nature of these
studies, whether these differences in the microbiome are a cause of
the disorder or a consequence of the disorder is yet to be fully
elucidated.
Variation in description of sample collection and the inclusion of
controls (contamination controls, cultivation samples, etc.)
Vaginal samples were collected with swabs across all studies, and typi-
cally employed a sterile speculum followed by a vaginal swab (Kyono
et al., 2018; Kitaya et al., 2019). However, some vaginal samples in-
cluded vaginal aspirates and were collected with a sterile catheter and
Salliss et al.
‘direct vision’ (Moreno et al., 2016). Generally, cervical samples col-
lected vaginally used a sterile speculum in combination with swabbing,
but care was taken to not touch vaginal walls when collecting samples.
However, cervical sample collection with swabs is unlikely to effectively
prevent vaginal contamination, as it is almost impossible to collect cer-
vical samples without vaginal contamination, given the anatomic posi-
tion. Also, very few of the reviewed studies included adequate
environmental and DNA extraction controls that would allow for
more accurate and diligent interpretation of microbiome study results.
Endometrial samples are also susceptible to cervicovaginal contami-
nation, when collected transcervically. Endometrial samples in the pre-
sented studies were collected using a variety of different techniques
and protocols that took specific measures to limit or avoid cervicovagi-
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nal contamination. For example, in one study endometrial biopsy sam-
ples were collected with a double sheathed catheter, where the outer
catheter sheath protects the inner sample collecting sheath from cervi-
covaginal bacterial contamination, but the sheathed catheter must still
pass through this area and is therefore subject to contamination (Liu
et al., 2018, 2019). In another study, endometrial fluid samples were
collected using a catheter and a syringe, where the cervix was cleansed
prior to catheter insertion, and syringe pressure was dropped upon re-
moval, to prevent cervicovaginal contamination (Moreno et al., 2016).
..
.. Finally, another method to collect an endometrial sample involved
.. careful aspiration with an IUI catheter using sterile technique, and care
..
.. was taken not to touch the cervicovaginal walls, however, this is still a
.. source of potential contamination (Kyono et al., 2018). Precautions
..
.. such as vaginal disinfection and careful collection should be adopted in
.. future studies, with the inclusion of negative controls that can be sub-
..
.. tracted from the test samples (Baker et al., 2018) or used to generate
.. reasonable cutoffs for sequencing depth for the bioinformatics pipeline
..
.. and during microbiome analysis, which is particularly important for low
.. abundance sites (Eisenhofer et al., 2019; Haahr et al., 2020). In addi-
..
.. tion, collecting both cervicovaginal and endometrial samples that could
.. be used to compare bacteria present in the lower reproductive tract
..
.. with endometrial bacteria and assess cross-contamination with endo-
.. metrial samples would be valuable. Overall, consistent protocols need
..
.. to be generated to limit cervicovaginal contamination and offer more
.. consistent collection methods.
..
.. In addition to contamination at the stage of sample collection, analy-
.. sis of samples can introduce bacterial DNA and produce false results
..
.. (de Goffau et al., 2019). Bacterial DNA is present in DNA extraction
..
.. kits and reagents used in the extraction process, reagents used in the
.. amplification of DNA for sequencing, the environment (operating
..
.. room, pathology lab, laboratory, etc.) and equipment used in sequenc-
.. ing DNA samples (the ‘kitome’; de Goffau et al., 2019; Olomu et al.,
..
.. 2020). Therefore, contamination can be amplified and analyzed as a
.. signal as these analyses are highly sensitive to the presence of any bac-
..
.. terial DNA that may be false (de Goffau et al., 2019). Very few studies
.. (8/28) allowed for possible contamination by including positive, and/
..
.. negative controls. Notably, only one study included some kind of envi-
.. ronmental sampling (Chen et al., 2017; Tables I and II), however, these
..
.. controls are essential for differentiating between true and false bacte-
.. rial signals, as well as validation of bacterial signals. For example, ‘nega-
..
.. tive controls’ that are able to provide information on bacterial signals
.. that are a result of the kitome/contamination include the sequencing
..
.. of a blank swab, blank extraction (sequencing of an extraction with no
.. sample, to assess bacterial signals present in the reagents and
Microbiota in endometriosis, infertility and pelvic pain 121

..
equipment used to extract sample DNA), and blank library (exclusion ..
of extraction protocol and use of DNA-free water the obtain the re- ..
..
sultant blank library; Kim et al., 2017; Poussin et al., 2018; Hornung ..
et al., 2019). ‘Positive controls’ can include the use of a synthetic com- ...
munity, although the composition of the studied microbiome is needed ..
..
to be able to match the positive control to the study (Kim et al., 2017;
Hornung et al., 2019). Therefore, the inclusion of controls becomes
even more important when there are low counts of open reading
frames, as contamination and false positives make up a greater propor-
tion of the measured bacterial DNA signal (Kim et al., 2017). Overall,
future microbiome-related studies should take sample contamination
into account as part of their study design.
Finally, to further provide clear evidence and support of bacteria
dysbiotic change in the gut microbiome (Chadchan et al., 2019).
Treatment with oral metronidazole then reduced endometriosis patho-
genesis by reducing specific bacteria that may have been contributing
to disease progression (Chadchan et al., 2019). Following metronida-
zole treatment, the investigators administered an oral fecal gavage
.. from mice with endometriosis to the metronidazole-treated mice,
.. which restored endometriotic lesion growth and inflammation
..
.. (Chadchan et al., 2019). While human studies are currently unable to
..
.. demonstrate that the microbiome may play a role in causing endome-
.. triosis, animal studies suggest that the gut microbiota may lead to
..
.. endometriosis and disease progression. However, the detailed
.. bacterial-mediated mechanisms have not been defined. Yet, bacterial-
..
.. associated signaling pathways, including LPS and TLR4, the metabo-

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found in microbiome studies at low abundance sites (Mitchell et al., ..
.. lome, estrobolome and immune mediators can be speculated to play a
2015), confirmation via microscopy or cultivation in vitro would be ideal .. role in inducing a host environment that can promote endometriosis,
for future research (Winters et al., 2019). Microscopy (FISH, special- ..
.. and the potential bidirectional interplay between endometriosis, infer-
ized staining or scanning electron-based approaches) can be utilized to .. tility and CPP, and the microbiome (Table III).
confirm that sequencing signals are the result of bacteria, and not de- ..
..
rived from other entities (Winters et al., 2019). In addition, some bac- ..
teria may have infiltrated a particular mucosal site and failed to ..
.. Future—guiding clinical practice
colonize, and this is referred to as the viability bias of sequencing ..
..
approaches (Lagier et al., 2018; Winters et al., 2019). Therefore, com- .. and microbiome studies
bining multiple methods can be useful for cross-validating findings and ..
yielding an accurate reflection of the microbiota composition for a
.. Leveraging the microbiome for
..
given sample (Winters et al., 2019), which is particularly important for .. discriminating patients with endometriosis,
low abundance sites.
..
.. endometriosis and infertility, or
..
.. endometriosis and CPP
Causation versus association for ..
microbiome studies .. Previous studies that have investigated the association between the
.. microbiome, endometriosis and infertility have found significant differ-
Longitudinal studies that assess incident endometriosis, infertility and/
..
.. ences between women with and without the disease; however, many
or CPP are required in order to characterize the contribution of the ..
.. studies possess important methodological limitations that limit their in-
microbiome to disease etiology. The field is currently unable to draw .. ternal and external validity. Future research is needed to further eluci-
conclusions about whether changes in the microbiome cause these ..
.. date specific microbiota compositions that may be associated with
conditions, given the limitations of cross-sectional study designs. .. endometriosis, infertility or CPP. In addition, microbiota-mediated
However, cross-sectional research with rigorous methodology and vali- ..
.. metabolites, which could also relate to local inflammation or tissue
dation of results could reveal novel and effective diagnostics and treat- ..
.. damage, could be utilized in the development of a more effective diag-
ments resulting in improved management of the symptoms .. nosis modality and treatment options for all three conditions.
characteristic of endometriosis, infertility and CPP, and improve the ..
quality of life for many women who suffer from these conditions. .. Metabolome-based studies should investigate the influence of dysbiosis
.. on the estrobolome, variations in SCFAs, damage-associated metabo-
The animal studies examined in this review provided evidence to ..
suggest that gut dysbiosis may have led to endometriosis. The animal
.. lites such as matrix metalloproteinases, angiogenesis factors, inflamma-
.. tory metabolites and bacterial endotoxins. Potentially, specific
studies discussed above induced endometriosis by transplantation of ..
endometrial tissue to the uterine wall. First, two studies found that in-
.. microbiota and microbiota-mediated metabolite compositions could
..
duction of endometriosis caused a decrease in gut microbiome diver- .. help to stratify patients with heterogeneous symptoms, overcoming
sity that is indicative of dysbiosis (Cao et al., 2020; Ni et al., 2020).
.. this obstacle for patients who are clinically complex and difficult to
..
Also, various bacteria were enriched in the endometriosis groups in- .. identify and diagnose.
.. In addition to the microbiota, the estrobolome could be a useful
cluding Bifidobacterium, Allobaculum, Akkermansia, Parasutterella and ..
Rikenella (Yuan et al., 2018; Ni et al., 2020). To our knowledge, human .. tool to identify patients at high risk of developing endometriosis, to de-
.. velop novel diagnostic methods for endometriosis and in discriminating
clinical studies have not identified these bacteria; however, specific bac- ..
teria may play a role in disease onset and progression of endometri- .. disease severity. This information can further be used to risk stratify
..
osis, infertility and CPP. One study even stated that induction of .. patients with a specific constellation of symptoms to determine an ap-
endometriosis led to a distinct gut microbiota composition, consisting .. propriate treatment strategy, which is inclusive of both patient symp-
..
of elevated Bifidobacterium and Bacteroidetes/Firmicutes ratio (Yuan .. toms and stage of disease over time. A stronger understanding of the
et al., 2018). Another animal study found that the endometriosis group .. relationship of disease etiology and changes in the microbiota, estrobo-
..
had a lower Bacteroidetes/Firmicutes ratio (Ni et al., 2020). Even more .. lome, hormonal dysregulation, angiogenesis and inflammation will pro-
convincingly, one study found that induction of endometriosis caused a
.. vide key biological insights that may guide the development of more
122 Salliss et al.

..
precise and effective treatment strategies for endometriosis, infertility ..
and CPP. ..
..
..
Screening versus diagnosis ..
..
Similar to using the microbiome and estrobolome in the discrimination
..
..
of heterogeneous symptoms, the microbiome and estrobolome could
be used to noninvasively diagnose or predict which women will experi-
ence endometriosis, infertility or CPP. With regard to endometriosis,
this could significantly reduce the diagnostic delay (average of 7 years)
in a minimally invasive fashion and thereby the morbidity associated
with endometriosis. If additional studies reveal consistent microbiome
compositions or estrobolome molecular discriminants to be associated
allow for stronger associations between bacteria cataloguing, identifica-
tion of key bacteria species or multiple species that may be associated
with endometriosis with and without CPP, and infertility. We recognize
that each of these conditions is multifactorial and driven by a combina-
tion of genetic, environmental and microbial factors that culminate in
.. chronic inflammation, changes in estrogen, epithelial proliferation and
.. remodeling and angiogenesis that manifest in pathology. Notably, many
..
.. of these downstream factors may be microbiota-mediated changes in
.. the local and distal microenvironments, similar to oncogenesis
..
.. (Łaniewski et al., 2020). The identification of microbial signatures asso-
.. ciated with these conditions could allow for the development of nonin-
..
.. vasive diagnostics that would decrease the diagnostic delay for
..

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with endometriosis, CPP and infertility, these could be used in combi- .. endometriosis and resulting comorbidities. In addition, novel interven-
nation with symptom profiles to better diagnose the presence of the .. tions and therapeutics that target and modulate the microbiome (pre-
..
disease in a noninvasive way. Furthermore, this ‘omics’ strategy may .. biotics, bacterial therapeutics or metabolites) could be developed to
be used to assess or predict the severity of disease. These molecular .. enhance quality of life for those women who suffer from endometri-
..
signatures would enable more effective and efficient application of .. osis, CPP and infertility.
treatments for patients who already suffer from endometriosis, infertil-
..
..
ity or CPP, or are at high risk of developing these disorders. ..
Therefore, the microbiota-mediated byproducts and/or estrobolome
..
.. Data availability
could be used as a new diagnostic method that is less invasive (Baker ..
et al., 2017).
.. The data underlying this article are available in the article.
..
..
..
Potential etiologic link between the gut .. Acknowledgments
and cervicovaginal microbiomes and ..
..
endometriosis, infertility and CPP .. The authors would like to thank Dr Pawel Laniewski for critical review
.. of the manuscript. In addition, we thank Dr Michael Foley and the
The gut microbiome has a profound influence on host health and can ..
.. Department of Obstetrics and Gynecology at the University of College
negatively impact host homeostasis through dysbiosis, an altered estro- .. of Medicine-Phoenix for supporting the Bath University Placement
bolome, elevation of local and systemic inflammation, the HPO axis
..
.. Program.
and the GBA. Despite the understudied nature of the estrobolome, ..
HPO and GBA in relation to endometriosis, infertility and CPP, current
..
..
evidence suggests that there could be a role for GBA in the pathogen- ..
.. Authors’ roles
esis of endometriosis, infertility and/or CPP. ..
The cervicovaginal microbiome also has profound effects on wom- .. M.M.H.-K., L.V.F,, and N.D.M. conceived and discussed the original
..
en’s health that can occur through dysbiosis and divergence from .. hypothesis. M.E.S. discussed the original hypothesis, wrote the first
Lactobacillus dominance. The relation of the cervicovaginal microbiome ..
.. draft of the manuscript, drafted and edited the figures and tables,
to endometriosis, infertility and CPP has not been studied extensively, .. compiled, reviewed and edited the bibliography, and revised the
yet cross-sectional studies have found differences in the cervicovaginal ..
.. manuscript. M.M.H-K., L.V.F., and N.D.M. provided guidance and
and endometrial microbiomes of patients with both endometriosis and .. feedback on writing, figures and tables, and read and revised the
infertility. Therefore, robust clinical studies will ultimately lead to new ..
.. manuscript. M.M.H.-K. supervised M.E.S. and the writing process.
diagnostic tools, preventative interventions and more effective treat- .. All authors read, revised and approved the final version of the
ments by determining the association or causation of the metabolome, ..
.. manuscript.
estrobolome and cervicovaginal and/or endometrial microbiomes with ..
endometriosis, infertility and CPP.
..
..
..
.. Funding
Future research ..
..
Future research should begin with samples that are carefully curated .. The authors would like to acknowledge the Women Investing in
and collected, and are linked to phenotypically well-characterized .. Science and Health (WISH) at Banner Health Foundation for grant sup-
..
cohorts of women with and without endometriosis, CPP and infertility, .. port (to M.M.H.-K.) related to this manuscript.
as outlined above. Herein, we have outlined best practices for the col- ..
..
lection of samples for microbiome analyses and the rich patient meta- ..
data that are required, along with the appropriate and rigorous ..
.. Conflict of interest
controls to better understand the role of the microbiome in endome- ..
triosis. Additional studies are needed that address these concerns to
.. The authors declare no competing interests.
Microbiota in endometriosis, infertility and pelvic pain 123
..
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