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Systematic Review

Endometrial Microbiome and Its Correlation to Female


Infertility: A Systematic Review and Meta-Analysis
Panagiota Foteinidou *, Maria Exindari, Dimitrios Chatzidimitriou and Georgia Gioula

Microbiology Department, School of Medicine, Faculty of Health Science, Aristotle University of Thessaloniki,
54636 Thessaloniki, Greece; mexidari@auth.gr (M.E.); dihi@auth.gr (D.C.); ggioula@auth.gr (G.G.)
* Correspondence: pfoteinid@auth.gr

Abstract: The endometrial cavity was considered sterile until the second half of the 20th century.
Through modern technological advances and the sequencing of the bacterial 16S rRNA gene, it was
proven that the area possesses its own unique microbiome, which can be categorised into two types,
Lactobacillus-dominant (LD, with a Lactobacillus spp. abundance percentage greater than 90%) and non-
Lactobacillus-dominant (non-LD, with a Lactobacillus spp. abundance percentage smaller than 90%),
with other species like Bifidobacterium, Gardnerella, Prevotella, and Streptococcus also being prominent.
The aim of this study was to investigate the possible correlation of the endometrial microbiome
to female infertility, through the identification and appraisal of studies published in the databases
PubMed, Web of Science, and Scopus. Moreover, 12 studies met the research criteria, including the
analysis of endometrial fluid or tissue samples from infertile women through PCR, culturomics-based,
or NGS methods. According to most of these studies, a eubiotic LD-type microbiome seems to be
best for maximising endometrial receptivity and pregnancy chances, whereas a dysbiotic non-LD-
type microbiome, with increased α-diversity and a higher number of pathogens, has a harmful
effect. There were few studies that presented contradictory results without, however, a satisfactory
explanation. Thus, more time and a greater number of studies are required to clarify contradictions
and achieve more certain results.

Keywords: microbiome; endometrium; infertility; systematic review; Lactobacillus


Citation: Foteinidou, P.; Exindari, M.;
Chatzidimitriou, D.; Gioula, G.
Endometrial Microbiome and Its
Correlation to Female Infertility: A
1. Introduction
Systematic Review and Meta-Analysis.
1.1. The Microbiome
Acta Microbiol. Hell. 2024, 69, 14–28.
https://doi.org/10.3390/ Humans have co-evolved with trillions of microorganisms that colonise the human
amh69010004 body and create a complex, adaptive and dynamic ecosystem, which is fully attuned to the
constantly changing physiology of the host [1]. The human microbiome is defined as the
Academic Editor: Thomas Walsh
trillions of commensal microbial cells hosted by every human—primarily bacteria, but also
Received: 30 January 2024 archaea, viruses, bacteriophages and fungi—and their genomes [2,3]. There has been some
Revised: 17 February 2024 confusion in the definition of the human microbiome, due to the very subtle difference in
Accepted: 20 February 2024 the terms “microbiota” (the microbial taxa associated with humans) and “microbiome” (the
Published: 26 February 2024 catalogue of these microbes and their genes), though in most cases, the two terms are used
interchangeably [2]. A microbiome can be found in every niche of the human body that
has been examined [3], even in organs that were traditionally considered sterile, such as
the lungs [4] and the stomach [5], and the largest microbiome in terms of the number of
Copyright: © 2024 by the authors.
microorganisms it contains is found in the intestinal tract and, more specifically, mainly in
Licensee MDPI, Basel, Switzerland.
the large intestine [2]. In contrast to the host’s genome, which remains relatively stable, the
This article is an open access article
microbiome and its genome are, as previously mentioned, dynamic, and undergo changes
distributed under the terms and
conditions of the Creative Commons
during a human’s development due to environmental factors, such as their diet, use of
Attribution (CC BY) license (https://
antibiotics, way of birth, past infections, etc., but also as a response to disease [6]. The recent
creativecommons.org/licenses/by/ advances in DNA sampling and sequencing techniques have given answers to a number of
4.0/). questions regarding the composition of the microbiome of every niche of the human body,

Acta Microbiol. Hell. 2024, 69, 14–28. https://doi.org/10.3390/amh69010004 https://www.mdpi.com/journal/amh


Acta Microbiol. Hell. 2024, 69 15

the formation of microbial communities of various degrees of complexity, their correlation


to the microbiomes of other species [7], and, most importantly, their correlation to different
diseases [6]. Dysbiosis in a microbial community can be described as a disturbance in
the balance of the community’s ecology which causes or intensifies a health problem.
Thus, dysbiosis in the human microbiome has been associated with a plethora of diseases
and conditions, such as irritable bowel syndrome, diabetes, allergies, asthma, and even
cancer [1]. More specifically, in the case of reproduction and fertility-related conditions, like
endometriosis, studies have shown that the gastrointestinal, neuroendocrine and immune
system interact and play an important part. The gut microbiome has been shown to
influence the progression of endometriosis, with the endometriotic microbiome consisting
of the genera Prevotella, Bautia, and Bifidobacterium [8]. A dysbiotic gut microbiome has also
been shown to affect the pathological process of polycystic ovary syndrome (PCOS), with
lower α- and β-diversities and an increased abundance of Bacteroides, Parabacteroides, and
Clostridium [9]. The characterisation of the human microbiome and the factors that affect
the composition and evolution of the microorganisms that it is composed of was the main
goal of the Human Microbiome Project (HMP) as a logical extension of the Human Genome
Project (HGP) [10]. The HMP, which started in 2007 and was set up by the National Health
Institutes of the U.S.A., has produced a total of 2.3 Tb of metagenomic data about the
16S ribosomal RNA (16S rRNA). The use of the 16S rRNA gene in the identification and
taxonomy of bacteria began in the 1980s by C. Woese, as it was proven that it makes for an
excellent molecular chronograph. It is defined by a great degree of conservation, which
stems from the importance of the gene as a crucial element of cell function, and very few
other genes are as conserved as this [11]. It is clear that big projects like the HMP and its
European equivalent, MetaHIT, already offer a deeper understanding of the biology and
the clinical importance of the human microbiome and its genome [12].

1.2. The Endometrial Microbiome


The data that originally described the healthy microbiome of the female reproduc-
tive system were derived from studies performed exclusively on the vagina and how its
microbiome changes throughout the reproductive years of a woman’s life and during the
menstrual cycle [13]. As a result, the endometrial cavity was considered to be a sterile field
until the second half of the 20th century [14], and the prevailing opinion at the time was that
it was protected from chemical and mechanical trauma and the invasion of microorganisms
by the cervical mucus plug [15]. However, recent studies on the area, and on the upper
reproductive tract in general, prove that the endometrium possesses its own unique micro-
biome, which has greater species diversity than that of the lower reproductive tract [16].
The results of studies on women of reproductive age who underwent hysterectomy led to
the categorisation of the endometrial microbiome into two main types, according to their
microbial composition: Lactobacillus-dominant (LD, with a Lactobacillus spp. abundance
percentage greater than 90%) and non-Lactobacillus-dominant (non-LD, with a Lactobacillus
spp. abundance percentage smaller than 90%). Other species prominent in the samples
include Bifidobacterium, Gardnerella, Prevotella, and Streptococcus [14]. Further analysis and
organisation of the aforementioned species in communities showed a negative correlation
of Lactobacillus with Gardnerella, Bifidobacterium, and Atopobium and a positive correlation
with the commensal genera Clostridium and Streptomyces [14]. The endometrial microbiome,
as shown by the analyses of samples from women before the start of in vitro fertilisation
(IVF), plays an important role in the reproductive process, as women with an LD-type
microbiome had higher chances of a successful implantation, full-term pregnancy and
live birth, while women with an abundance of other genera and a decreased presence of
Lactobacillus in their microbiome were significantly less likely to achieve pregnancy and
more likely to suffer a miscarriage [17]. Moreover, it has been proven that the presence of a
non-LD-type microbiome is also associated with pathological conditions and diseases of
the endometrium, such as endometriosis [18] and chronic endometritis [19]. Nonetheless,
Acta Microbiol. Hell. 2024, 69 16

an agreement on the microbial composition of the healthy microbiome, or the existence of a


core microbiome, has not yet been reached [18].

1.3. Analysis of the Endometrial Microbiome


All data published until now concerning the analysis of the endometrial microbiome,
and mainly the bacteria found in it, have been extracted using one of two method types:
culture-based or sequencing-based methods. Culture-based methods, which are the ones
traditionally used, include cultivation of the samples in appropriate conditions and identi-
fication of the bacteria found, either by the substances they produce or via characterisation
of conserved genes in their 16S rRNA. However, analyses of the vaginal microbiome have
shown that several microorganisms are impossible to cultivate, and unsuccessful cultiva-
tion, in many cases, fails to fully reveal the diversity of the microorganisms present in the
sample [20]. For these reasons, methods based on sequencing the 16S rRNA gene are increas-
ingly being used lately. This gene’s sequence is approximately 1550 base pairs (bp) long,
consisting of conserved and variable regions, and the many intraspecific polymorphisms
present are sufficient for identifying and classifying strains all the way to the sub-species
level [11]. The most common sequencing methods are next-generation sequencing (NGS)
or high-throughput sequencing methods, which allow the sequencing of large DNA or
RNA molecules, up to 30,000–50,000 bp long, in real time. NGS methods are based on using
an engineered DNA polymerase attached to the DNA to be sequenced at the bottom of a
well, which incorporates nucleotides, labelled with different phosphor-linked fluorophores
for differential detection, to the growing chain. When a nucleotide is incorporated into
the growing chain, light is emitted, which enables the identification of the nucleotide due
to its colour, and then the fluorophore is released, allowing for the incorporation of the
next nucleotide. The whole process occurs simultaneously in up to one million wells on
a single microchip, producing sequences of a total length of 10,000–15,000 bp (equivalent
to a data volume of up to 7.6 Gb) [21]. In the case of the 16S rRNA gene in microbiome
analyses, universal primers are used, which are complementary to the conserved region
at the beginning of the gene and also to either the region at approximately 540 bp or the
end of the gene, whereas the variable regions in between [11], mainly the V4 hypervari-
able region [22,23], are used for bacterial differentiation. In recent years, there has also
been an increase in the use of fourth-generation sequencing methods, which include the
identification of the individual nucleotides of a single-strand DNA molecule as it passes
through a small-diameter nanopore, according to the different electrical signal each one
produces. This way, the production of even larger data volumes is possible, utilising less
time and space [21,24]. However, in the case of the endometrium, which is characterised by
a microbiome of relatively low biomass and a high risk of DNA from contamination being
present, an appropriate protocol, which combines both high reliability and low cost, has
not yet been designed [24].

2. Methods and Materials


2.1. Information Sources and Search Strategy
This systematic review was conducted according to the PRISMA guidelines (preferred
reporting items for systematic reviews and meta-analyses. The literature search about
the endometrial microbiome and infertility was performed using the databases PubMed,
Scopus, and Web of Science, with the last search date being October 2023. The search
strategy was accordingly modified for each database, taking into consideration alternate
spellings, synonyms for the keywords used, and changes in terminology over the years,
and MeSH (medical subject headings) terms were also used where possible, in order to
increase the specificity of the search. Furthermore, the references of the relevant studies
were hand-searched to ensure a more thorough coverage of the topic.
The search strategy formed for the PubMed database was as follows: (“Microbiota”
[MeSH terms] OR microbiot* [All fields] OR microbiom* [All fields]) AND (“Endometrium”
[MeSH terms] OR endometrial [All fields] OR endometrium [All fields]) AND (“Infertility”
Acta Microbiol. Hell. 2024, 69 17

[MeSH terms] OR infertil* [All fields] OR fertil* [All fields] OR steril* [All fields]). The
number of retrieved results was 177.
For the Scopus database, the search strategy was formed as follows: (endometrium OR
endometrial) (Title, Abstract, Keywords) AND (microbiot* OR microbiom*) (Title, Abstract,
Keywords) AND (infertil* OR fertil* OR steril*) (Title, Abstract, Keywords). The number of
retrieved results was 261.
Finally, for the Web of Science database, the search strategy was formed as follows:
(endometrium OR endometrial) (Topic) AND (microbiot* OR microbiom*) (Topic) AND
(infertil* OR fertil* OR steril*) (Topic). The number of retrieved results was 230.

2.2. Results Screening and Eligibility Criteria


First, the retrieved results were imported into the reference management software
EndNote Online Classic (Clarivate Analytics (former Thomson Reuters), Philadelphia,
U.S.A.), where duplicate results were identified and deleted. Then, the relevant results
were screened by their titles and abstracts, and the full texts of all eligible studies were
retrieved, as well as those of articles where eligibility could not be decided based solely
on their title and abstract. The final stage of the results management was picking the
studies that met all the eligibility criteria after studying their full texts when necessary. The
eligibility criteria set for this review were as follows:
• Only articles of studies;
• Publication date: 2020 or later;
• Language: English;
• Subjects: humans;
• Analysis of the endometrial microbiome either exclusively or in combination with the
microbiome of other parts of the reproductive tract;
• Correlation to infertility and/or the outcome of IVF treatment.
Exclusionary criteria, corresponding to the aforementioned eligibility criteria, were:
• Reviews, systematic reviews, books or chapters of books and other types of text;
• A publication date of 2019 or earlier;
• Language of the article other than English;
• Animal species as subjects of the studies;
• Analysis and focus on the microbiome of other organs and systems of the human body
besides that of the female reproductive tract;
• Correlation of the microbiome to pathological conditions or diseases of the female
reproductive system (e.g., endometriosis, endometritis, etc.).

2.3. Data Extraction


The data extraction from each study was conducted using the Microsoft Excel program.
The relevant data extracted were:
• Author(s);
• Publication date;
• Country where the study was conducted;
• Aim of the study;
• Basic demographic data;
• Sample types;
• Analysis method;
• Basic data and results from the analyses;
• Correlation to infertility and/or IVF treatment outcome.

3. Results
3.1. Search Results
The search strategy described previously retrieved a total of 668 results. After combin-
ing the results from all databases and deleting duplicates, there were 338 unique results
3. Results
3.1. Search Results
Acta Microbiol. Hell. 2024, 69 18
The search strategy described previously retrieved a total of 668 results. After com-
bining the results from all databases and deleting duplicates, there were 338 unique re-
sults
left,left,
whichwhich
werewere
thenthen screened
screened for for their
their relativity
relativity to the
to the search
search criteria.
criteria. Then,
Then, thethe
irrel-
irrelevant results were also removed, and the full texts of the 258
evant results were also removed, and the full texts of the 258 remaining articlesremaining articles were
were
retrieved,
retrieved, which
whichwere
werethen
then further assessed for
further assessed foreligibility
eligibilityaccording
according to the
to the criteria
criteria de-
described
scribed in Section 2.2. Finally, 12 studies’ articles were chosen to be included
in Section 2.2. Finally, 12 studies’ articles were chosen to be included in this review, which in this re-
view, which contained all the necessary data about the endometrial microbiome
contained all the necessary data about the endometrial microbiome analysis on infertile analysis
onwomen
infertileandwomen and its potential
its potential correlation
correlation to pregnancy
to pregnancy achievement
achievement or theoroutcome
the outcome
of IVF
of treatment,
IVF treatment, and all of the relevant data were extracted for further studying.
and all of the relevant data were extracted for further studying. The full selection The full
selection and retrieval process is shown
and retrieval process is shown in Figure 1. in Figure 1.

Figure 1. PRISMA
Figure flow
1. PRISMA diagram.
flow diagram.

3.2.3.2. Publication
Publication Characteristics
Characteristics
From
From 20202020 until
until October
October 2023,
2023, 12 12 studies
studies havehave been
been published
published about
about endometrial
endometrial mi-mi-
crobiomeanalysis
crobiome analysisonon infertile women
womenand anditsits
correlation
correlationto pregnancy
to pregnancyachievement and/or
achievement
the outcome
and/or of IVF
the outcome of treatment and and
IVF treatment werewere
therefore included
therefore includedin this review.
in this FiveFive
review. of them
of
(41.67%) were published in 2023, four (33.33%) in 2022, two (16.67%)
them (41.67%) were published in 2023, four (33.33%) in 2022, two (16.67%) in 2021, and in 2021, and only one
(98.33%) in 2020. With regard to the country where each study was
only one (98.33%) in 2020. With regard to the country where each study was conducted, conducted, three studies
eachstudies
three (25%) were conducted
each (25%) wereinconducted
Japan andin Italy,
Japan twoand
(16.67%)
Italy, intwo Spain, and one
(16.67%) each (8.33%)
in Spain, and
oneineach
Turkey, Russia,
(8.33%) China and
in Turkey, the U.S.A.
Russia, The the
China and main goal The
U.S.A. of these
mainstudies
goal ofwas thestudies
these analysis
wasand thestudy of the
analysis andendometrial
study of the microbiome,
endometrialeither exclusively
microbiome, or along
either with the
exclusively vaginal
or along
and/or cervical microbiome, in women with infertility, and mainly in cases with repeated
implantation failure (RIF). In some studies, a comparison to the microbiome of healthy
(fertile) women was performed, with the ultimate goal always being the investigation of
its microbial composition and its potential correlation to pregnancy achievement and the
Acta Microbiol. Hell. 2024, 69 19

outcome of IVF treatment or embryo implantation in cases where these were performed.
The basic publication characteristics of each study are shown in Table 1.

Table 1. Basic publication characteristics of the studies included.

Reference
Author(s) Year Country Aim
Number
Shunsaku Fujii, Takaaki Evaluation of the correlation of age and
2023 Japan [25]
Oguchi microbiome to endometrial receptivity.
Identification of specific microbial
Takuhiko Ichiyama communities in the vaginal and endometrial
2021 Japan [23]
et al. microbiomes as potential biomarkers for
implantation failure.
Correlation of the disruption of the vaginal
Ozlem Sezer et al. 2022 Turkey and endometrial microbiome to unexplained [26]
infertility.
Comparison of the endometrial microbiome
Francisca Maria Lozano
2023 Spain between patients with and without RIF before [27]
et al.
undergoing IVF.
Comparison of the qualitative and
quantitative species abundance of bacteria,
Mark Jain et al. 2023 Russia [28]
viruses and fungi in vaginal, cervical and
endometrial fluid samples of infertile women.
Investigation of the effect of the balance
between Lactobacillus and other pathogens in
Maho Miyagi et al. 2022 Japan [29]
the vaginal and endometrial microbiome on
IVF outcomes of infertile patients.
Comparison of the vaginal and endometrial
microbiomes of women undergoing IVF, and
Marco Reschini et al. 2022 Italy [30]
correlation to the possibility of a successful
pregnancy.
Investigation of the possible effect of the
Immaculada Moreno
2022 U.S.A. composition of the endometrial microbiome [17]
et al.
on the reproductive result.
Use of culturomics-based methods for
Federica Cariati et al. 2023 Italy endometrial microbiome analysis, and [31]
correlation to pregnancy rates.
Description and comparison of the vaginal
and endometrial microbiomes between
Maria del Carmen
2021 Spain women with and without a successful [32]
Diaz-Martinez et al.
pregnancy after IVF, as well as between
women with and without RIF.
Investigation of structural differences
between the vaginal and endometrial
Lucia Riganelli et al. 2020 Italy [33]
microbiome to define potential biomarkers
related to implantation failure.
Recording of the endometrial microbiome
Yixuan Zou et al. 2023 China profiles of women with RIF, and investigation [34]
into the use of antibiotics on these patients.

3.3. Population Characteristics


The studies in this review included a total of 1229 infertile patients, and three of
them also included a total of 65 control patients (not facing fertility problems). All studies
excluded patients with secondary infertility, which could be attributed to pathological
causes such as endometriosis, polyps and other masses and lesions in the endometrial
Acta Microbiol. Hell. 2024, 69 20
Acta Microbiol. Hell. 2024, 69, FOR PEER REVIEW 7

cavity, underlying infections, etc. The age range of the patients was between 18 and
50 years, and no studies presented any further demographic data. In every study, the
12 consecutive months of natural efforts, or as infertile with RIF, meaning infertile with a
patients were characterised either as infertile, meaning unable to achieve clinical pregnancy
history of at least three failed IVF attempts and good-quality embryo transfers. Moreover,
after 12 consecutive months of natural efforts, or as infertile with RIF, meaning infertile with
the studies can also be split into those where IVF or embryo transfer was performed as
a history of at least three failed IVF attempts and good-quality embryo transfers. Moreover,
part of them and those that only included microbiome analysis. In total, five studies were
the studies can also be split into those where IVF or embryo transfer was performed as
conducted on infertile patients with a simultaneous IVF attempt, including 549 patients
part of them and those that only included microbiome analysis. In total, five studies were
and 26 controls,
conducted whilepatients
on infertile four studies
with awere conducted
simultaneous onattempt,
IVF patientsincluding
with RIF549with a simulta-
patients and
neous
26 IVF attempt,
controls, while fourincluding
studies387 patients
were and 18
conducted oncontrols.
patientsOne
withstudy was aconducted
RIF with simultaneouswith
a simultaneous
IVF IVF attempt
attempt, including on patients
387 patients andwith and without
18 controls. OneRIF. With
study wasregard to the studies
conducted with a
not including an IVF attempt, one of them was conducted on a total of 100 infertile
simultaneous IVF attempt on patients with and without RIF. With regard to the studies patients
not
and one on
including anaIVF
total of 145 patients
attempt, withwas
one of them RIF.conducted
The basic on
population
a total ofcharacteristics of each
100 infertile patients
study
and areonshown
one a totalinofTable 2.
145 patients with RIF. The basic population characteristics of each
study are shown in Table 2.
Table 2. Basic population data of the studies.
Table
Reference Number Patients 2. BasicControls
Total population dataAge
of the studies.
(Years) Patients’ Characterisation IVF Attempt
[25] 185 No 25–47 With RIF Yes
Reference Number Patients Total Controls Age (Years) Patients’ Characterisation IVF Attempt
[23] 145 21 N/A With RIF No
[25] 185 No 25–47 With RIF Yes
[26] 26 26 20–45 Infertile Yes
[23] 145 21 N/A With RIF No
[27]
[26] 2726 1826 <45
20–45 With RIF
Infertile Yes
Yes
[28]
[27] 10027 No18 N/A<45 Infertile
With RIF YesNo
[28]
[29] 100
35 NoNo N/A
N/A Infertile
Infertile NoYes
[29]
[30] 5335 NoNo N/A
N/A Infertile
Infertile Yes
Yes
[30] 53 No N/A Infertile Yes
[17]
[17] 342
342 NoNo <50
<50 Infertile
Infertile Yes
Yes
[31]
[31] 9393 NoNo 29–47
29–47 Infertile
Infertile Yes
Yes
[32]
[32] 4848 NoNo 18–50
18–50 With
With and withoutRIF
and without RIF Yes
Yes
[33]
[33] 3434 NoNo 22–43
22–43 With RIF
With RIF Yes
Yes
[34] 141 No <40 With RIF Yes
[34] 141 No <40 With RIF Yes

3.4.
3.4. Sample
Sample Types
TypesandandAnalysis
AnalysisMethods
Methods
The
The main
main type
type ofof sample
sample used
used for
for microbiome
microbiomeanalysis
analysiswaswas endometrial
endometrial fluid,
fluid, which
which
was used in eight studies (66.67%). In three studies (25%), an endometrial
was used in eight studies (66.67%). In three studies (25%), an endometrial tissue sample tissue sample
was
wastaken
takenafter
aftera abiopsy,
biopsy,and in in
and one study
one (8.33%),
study samples
(8.33%), from
samples bothboth
from endometrial fluidfluid
endometrial and
tissue were used. There were some studies that also used vaginal and/or cervical
and tissue were used. There were some studies that also used vaginal and/or cervical sam- samples
for
plesanalysis and and
for analysis comparison,
comparison,butbut
for for
thethe
purposes
purposes ofof
this
thisreview,
review,only
onlythe
theendometrial
endometrial
samples
samples were
were taken
taken into
into consideration.
consideration. A A graph
graph with
with the
the percentage
percentage distribution
distribution ofof the
the
sample types used in the included studies is shown in
sample types used in the included studies is shown in Figure 2. Figure 2.

Sample types

Endometrial fluid Endometrial tissue


Endometrial fluid and tissue

Figure2.
Figure 2. Sample
Sample types
types used
used in
inthe
thestudies.
studies.
Acta Microbiol. Hell. 2024, 69, FOR PEER REVIEW

Acta Microbiol. Hell. 2024, 69 21

Regarding the methods used for DNA analysis after its extraction, the majori
Regarding
studies, and the methods
more used for nine
specifically DNA of analysis
themafter its extraction,
(75%), used NGS the methods
majority offor
the sequen
studies, and more specifically nine of them (75%), used NGS methods for sequencing the
16S rRNA gene. Real-time PCR, wide-spectrum PCR, and MALDI-TOF (matrix-
16S rRNA gene. Real-time PCR, wide-spectrum PCR, and MALDI-TOF (matrix-assisted
laserdesorption/ionisation—time
laser desorption/ionisation—time ofmass
of flight) flight) mass spectrometry
spectrometry afterused
after culture were culture
in were
onestudy
one study each.
each. A graph
A graph with
with the the percentage
percentage distribution
distribution of the DNA of the DNA
analysis methodsanalysis m
used in the included studies is shown in Figure 3.
used in the included studies is shown in Figure 3.

DNA analysis methods

NGS
Real-time PCR
Wide-spectrum PCR
Culture and MALDI-TOF mass spectomectry

Figure
Figure DNA
3. 3. analysis
DNA methods
analysis used inused
methods the studies.
in the studies.
3.5. Data and Results from the DNA Analysis
3.5. According
Data and Results from
to the data thethe
from DNA Analysis
analysis of the microbial DNA extracted from the
samples,According
a non-LD-typeto the data from
microbiome the analysis
was found of thewith
in most women microbial
infertility DNA extracted f
or RIF, with
low percentages of Lactobacillus and a higher abundance of
samples, a non-LD-type microbiome was found in most women with infertilitypathogenic bacterial genera,
primarily Prevotella, Gardnerella, Atopobium, Pseudomonas, Streptococcus and Staphylococcus.
with low percentages of Lactobacillus and a higher abundance of pathogenic bacte
In general, the main trend in patients with infertility problems seems to be the presence of
aera, primarily
dysbiotic Prevotella,
microbiome, Gardnerella,
with looser Atopobium,
and disorganised Pseudomonas,
connections Streptococcus
among microbial species and S
coccus.
in In general,
its communities, whilethe main
it also seemstrend in β-diversity,
that the patients with infertility
meaning problems
the composition of theseems t
microbial communities of the endometrium, plays a more important
presence of a dysbiotic microbiome, with looser and disorganised connections am role in endometrial
receptivity and pregnancy rates rather than the α-diversity, meaning the number of species
crobial species in its communities, while it also seems that the β-diversity, mea
in the communities, as the α-diversity did not significantly differ between infertile patients
composition
and the controls.of the microbial
Regarding communities
the outcome of the
of IVF attempts, in endometrium,
the studies where plays a more im
they were
role in endometrial
performed, in most cases, receptivity and pregnancy
there were successful ratesinrather
pregnancies womenthan with antheLD-type
α-diversity, m
microbiome,
the numberwith a higher in
of species Lactobacillus abundance as
the communities, andthelowα-diversity
percentages did of pathogens.
not significant
However,
between infertile patients and the controls. Regarding the outcome of IVFofattemp
this is not absolute because, in some cases, it was proven that the abundance
Lactobacillus was not as important for pregnancy achievement as it did not differ greatly
studies patients
between where who theygotwere performed,
pregnant and those in who
most didcases, there
not, but were
rather, the successful
presence of pregna
women with
pathogens was ofan LD-type
greater microbiome,
importance, which seems withtoadecrease
higher the
Lactobacillus
chances of aabundance
successful and
centages
IVF attemptofand
pathogens.
pregnancy.However,
Moreover,this it isisworth
not absolute
noting thatbecause, in some
in one study, the cases,
high it was
abundance of species other than Lactobacillus seemed beneficial,
that the abundance of Lactobacillus was not as important for pregnancy achievem as there were higher
pregnancy rates in women with this microbiome type, while another study presented a
did not differ greatly between patients who got pregnant and those who did not
higher number of pregnancies in women with a complete absence of Lactobacillus and a
ther, the
higher presence
IVF failure rate of pathogens
in women with anwas of greater
LD-type importance,
microbiome. The datawhichfrom theseems
DNA to decr
chancesand
analysis ofthe
a successful
basic results IVF attempt
of each study areand pregnancy.
shown in Table 3.Moreover, it is worth noting
one study, the high abundance of species other than Lactobacillus seemed bene
there were higher pregnancy rates in women with this microbiome type, while
study presented a higher number of pregnancies in women with a complete ab
Lactobacillus and a higher IVF failure rate in women with an LD-type microbio
data from the DNA analysis and the basic results of each study are shown in Tab

Table 3. Analysis data and studies’ results.

Reference
Analysis Data Results
Acta Microbiol. Hell. 2024, 69 22

Table 3. Analysis data and studies’ results.

Reference Analysis Data Results


Number
Patients’ categorisation into five distinct microbial
profiles: 40 patients with profile 1 (normal, with
Lactobacillus percentage >90% and absence of The patients with profile 1 had a more receptive
pathogens), 8 patients with profile 2 (abnormal, with endometrium, whereas those with profile 5 had a less
[25] Lactobacillus percentage <90% and absence of receptive endometrium. In general, a non-receptive
pathogens), 32 patients with profile 3 (abnormal, with endometrium was correlated to low Lactobacillus levels
Lactobacillus percentage <90% and presence of and the presence of a dysbiotic microbiome.
pathogens), 49 patients with profile 4 (mildly dysbiotic),
and 56 patients with profile 5 (very low biomass).
The endometrial α-diversity was higher than the vaginal
one, and dysbiosis in the vagina was always noticed
A total of 131 microbial species were detected in the
along with dysbiosis in the microbiome (the 14 genera
endometrial microbiome. In the RIF group, 14 genera
indicative of dysbiosis are the same in both the vagina
[23] (Atopobium, Gardnerella, etc.) were in higher abundance
and the endometrium, so they are likely transferred
than in the control group, whereas the Lactobacillus
upwards), while an abundance of Lactobacillus does not
abundance did not significantly differ between the two.
necessarily correlate to an unsuccessful implantation
and pregnancy.
The percentages of a disorganised microbiome in the
Patients with unexplained infertility had more dysbiotic
reproductive tract differed significantly between fertile
[26] microbiomes, with lower Lactobacillus and higher
and infertile women, with the key factor being the
pathogen percentages than the control group.
reduction in Lactobacillus abundance.
Control group: higher abundance of Lactobacillus
(specifically L. iners) and lower abundance of Prevotella.
Negative correlation of Lactobacillus to pathogens, and
RIF group: high abundance of Lactobacillus, but also high
[27] an abundance of species related to implantation failure
percentages of Prevotella, Gardnerella and Ralstonia. In
in the RIF group.
general, there were no differences in the α-diversity but
a great difference in β-diversity between the two groups.
The total bacterial loads were lower in the endometrial
samples (6.3 × 103 genome copies), with 16% of the
The endometrium of infertile patients has a distinct
samples not containing any bacterial DNA. The
microbial profile compared to the vagina and the cervix,
endometrial samples had a very low α-diversity
[28] and the immunological and biochemical interactions
compared to the vaginal and cervical ones, and the
among members of the microbial communities possibly
abundance of Gardnerella vaginalis, Prevotella bivia, and
Porphyromonas spp. influenced the abundance play an important role in regulating its receptivity.
of Lactobacillus.
Categorisation of patients into four microbial profiles:
22 patients with profile 1 (high Lactobacillus, low
pathogens), 3 patients with profile 2 (high Lactobacillus
and high pathogens), 2 patients with profile 3 (low Higher chances of pregnancy in high Lactobacillus and
[29] Lactobacillus and low pathogens) and 6 patients with low pathogen levels, and lower chances in high
profile 4 (low Lactobacillus and high pathogens). pathogen and low Lactobacillus levels.
Furthermore, 77.3% of patients with profile 1 had a
successful pregnancy, while 83.3% of patients without a
successful pregnancy had profile 4 microbiome.
In total, 8% of patients were found with an LD-type
microbiome, with high percentages of Pelomonas, Relatively low frequency of LD patients, with a higher
[30] Probionabacterium, Pseudomonas, Streptococcus and species diversity rather than Lactobacillus abundance
Escherichia as well. 30% of patients achieved pregnancy, which is beneficial for pregnancy achievement.
with 53% of them having an LD-type microbiome.
The dominant genus in both sample types was
Lactobacillus, with other genera such as Atopobium,
Bifidobacterium, and Gardnerella, etc., also being quite Patients with live births had denser and more tightly
common. Differences were noted in the bacterial connected microbial communities, and some
networks between the two sample types: fluid samples connections were only present in these cases, which
[17] had two connected communities, while tissue samples shows their importance in the IVF outcome. On the
had four, the fluid samples networks were more tightly other hand, these connections were absent in patients
connected than the tissue sample ones, and finally, with failed attempts, who had looser and more
Lactobacillus was both positively and negatively disorganised microbial networks.
correlated to neighbouring genera in fluid samples but
only negatively correlated in tissue samples.
Acta Microbiol. Hell. 2024, 69 23

Table 3. Cont.

Reference Analysis Data Results


Number
In total, 74% of patients had a positive culture of at least
one bacterial species, whereas 26% presented no growth.
The most common phyla were Firmicutes (87.76% of
patients), Proteobacteria (27.94% of patients),
An LD-type microbiome correlated to higher pregnancy
Actinobacteria (10.29% of patients) and Ascomycota (8.82% rates, whereas pathogens, and specifically the genus
[31] of patients). The most dominant genus was Lactobacillus Staphylococcus, were more common in patients with
(37% of pregnant patients versus 5% of non-pregnant failed IVF attempts.
ones), while the phylum Actinobacteria was only present
in non-pregnant patients. There was also a correlation of
the families Staphylococcaceae and Enterobacteriaceae to
failed IVF attempts.
There were no differences in α- and β-diversities
between pregnant and non-pregnant women, and
pregnant women had a higher abundance of
In total, 21 women achieved pregnancy, of which 38.9%
Lactobacillus, Gardnerella, Burklhodena, and Anaerobacillus,
while non-pregnant ones had a higher abundance of had a history of RIF compared to 70% of those who did
Streptococcus, Ralstonia, Prevotella, and Delfia. Regarding not achieve pregnancy. There was a higher diversity in
[32] the history of RIF, there was a higher α-diversity in the endometrial microbiome compared to the vaginal
women without RIF, with differences in β-diversity also one, and a non-LD-type microbiome correlated to lower
being present. The dominant genus in women with RIF rates of successful implantation, pregnancy and live
was Prevotella and the species L. iners and L. jensenii, births.
while in women without RIF, the dominant genus was
Ralstonia and the species L. helveticus and Sneathia amnii.
The endometrial microbiome presents ahigh species
heterogeneity, specifically with the presence of species,
like Kocuna dechagenensis, not found before in the The presence of Lactobacillus in the endometrium is
reproductive tract. The microbiome of pregnant women possibly caused by upwards migration from the vagina,
[33] presented a total absence of Lactobacillus and a high and it creates an unfavourable environment for embryo
abundance of Lachnospiraceae and Enterobacteriaceae, implantation and a successful IVF attempt.
while that of non-pregnant women presented
Lactobacillus dominance and a higher α-diversity.
In total, 69 patients achieved pregnancy, of which 63 had
In total, 20 patients were found with an LD-type pathogens in their microbiome. A non-LD-type
microbiome and 121 with a non-LD-type one. 11.3% of microbiome and pathogens were noted in most patients
patients did not have any pathogenic bacteria in their with RIF. Apart from the presence of pathogens and the
[34] microbiome, and 88.7% of them did. The most common decrease in Lactobacillus, the co-occurrence of most of
species were Streptococcus (72.3% of patients), these pathogens seems to play an important role in the
Staphylococcus (51.8% of patients) and Neisseria disturbance of the microbial ecosystem of the
(47.5% of patients) endometrium and the decrease in successful
implantation and pregnancy chances.

4. Discussion
4.1. Population Characteristics and Demographics
With regards to the age of the patients recruited for the studies, it is only mentioned
in half of the studies. The patients cover almost the entire range of the reproductive
ages (18–50 years old), with the majority of them, however, being 25 to 40 years old. As
the studies investigate the correlation of the microbiome of the endometrium and, in some
cases, of other organs of the reproductive system to female infertility, we can safely deduce
that the rest of the patients whose age is not included in the studies, are also of reproductive
age. Other demographic data are also omitted, apart from the countries where the studies
were conducted, which also constitute the patients’ countries of origin. Regarding these,
half of the studies were conducted in Asian countries and the rest in European countries
(and more specifically, in countries of the European south), apart from one which was
conducted in the U.S.A. It is obvious that the number of countries is quite limited, which
could potentially lead to misleading results. For this reason, it is suggested that subsequent
studies on the subject include patients from a larger number of countries, especially African,
American and Oceanian countries, which have not so far been represented. This way, the
results can be more representative, and there can be further investigation on whether
Acta Microbiol. Hell. 2024, 69 24

the country of origin and the lifestyle and standard of living there plays a role in the
composition of the microbiome and infertility. Finally, the population sample sizes were
quite small, with the average being 103 patients per study and the median being 73 patients
per study, which is mainly due to the complexity of the process and the time required to
choose and recruit the appropriate patients and perform all the analyses. Nonetheless,
studies with a significantly larger sample size, to the extent that this is feasible, would
lead to safer and more complete results and would help us decipher if any unusual or
unexpected results are really statistically important or just the result of chance.

4.2. Sample Types


The main sample type used in the studies was fluid from the endometrial cavity,
which, in most cases, was taken using a double-lumen catheter [23,26,28,30–32]. With
this device, the internal catheter easily passes through the external one without making
contact with the vaginal or cervical epithelium, and the fluid is drawn through the syringe
on the other end [30]. In only two cases, the sample was taken using a cell-collecting
brush, specifically a Tao Brush (Cook Medical, Madrid, Spain) in one [27] and a Yuino
Brush (Asuka Pharmaceuticals, Tokyo, Japan) in the other [29]. In the studies which used
tissue from a biopsy as the preferred sample type, this was taken using a Pipelle-type
device (Laboratoire CDD, Paris, France). This device is a flexible polypropylene tube with
an external diameter of 3.1 mm and an internal diameter of 2.6 mm. By removing an
internal piston, negative pressure is created, and the tissue is aspirated in the cannula [35].
All the aforementioned modern techniques are significantly less invasive than traditional
ones, causing minimal pain and discomfort to the patients and eliminating the need for
anaesthesia. Moreover, through the simultaneous ultrasound guidance performed by
experienced technicians, the sample is taken quickly from the exact anatomical spot [30].
Any unusual or unexpected results did not occur only in studies using one or the
other sample type, and thus it cannot be safely said that either sample type is more or less
reliable than the other. The most important point during the sample collection process
is avoiding contamination from the lower reproductive tract organs. For this reason, the
vagina and cervix should be washed out with plenty of saline solution prior to the sample
collection and then carefully dried using a sterile gauze or cotton pieces [29,34]. Also,
extra precaution should be taken during the insertion and removal of the catheter or brush
into and out of the endometrial cavity to avoid contact with the vaginal walls [36]. Lastly,
proper storage of the samples until further analysis is also crucial, and it should be carried
out using sterile containers and appropriate buffer solutions. The samples should then be
stored either in the refrigerator (4 ◦ C) or the freezer (−80 ◦ C), according to how much time
there will be between the collection and analysis stages.

4.3. Sample Analysis Methods


The three types of methods used in the studies are, as previously mentioned, culturomics-
based methods, which include the mass culture of microorganisms and identification using
MALDI-TOF mass spectrometry, PCR-based methods and NGS methods. First, MALDI-
TOF mass spectrometry allows for the identification of microorganisms by species-specific
peptide and protein mass profiles. This method can identify microorganisms up to the
species level, with a high level of accuracy and reliability even in samples with a very
low microorganism biomass. However, it is a method with a high workload as it requires
cultivation and incubation of the microorganisms from the samples, which subsequently
increases the total time and cost of the process. Furthermore, the methods that make use of
the various different forms of PCR achieve detection, identification and quantification of the
microbial species in the samples, in a quick, highly accurate and effective way. Their main
disadvantage, however, is the fact that they require knowledge of the sequences of interest of
the microorganisms under investigation in order for the appropriate primer sequences to be
designed. Lastly, NGS methods, along with the evolution of bioinformatics, allow for DNA
Acta Microbiol. Hell. 2024, 69 25

sequencing straight from the sample, even if only a small amount is available, produce a high
data volume in real time and only require a little amount of space.
From these, it becomes clear that modern molecular methods are the most advanta-
geous, defined by their high degree of sensitivity, specificity and reliability. However, the
endometrial cavity is a niche of the human body whose microbiome is only recently starting
to get more thoroughly explored. Thus, a universal protocol for sample analysis has not
yet been created, and therefore it is up to each researcher to choose the desired methods,
according to the available funds and materials and the specific needs and expected results
of each study.

4.4. Analysis Results and Correlation of the Microbiome to Infertility


In accordance with most of the earlier studies conducted on the topic, the majority
of the studies included in this review present a eubiotic endometrial microbiome, with a
percentage of species of the genus Lactobacillus greater than 90% and very few or no other
species of pathogenic bacteria present, as the most beneficial for increasing the chances of a
successful IVF attempt and pregnancy. The genus Lactobacillus was positively correlated to
the commensal bacteria and negatively correlated to pathogenic ones, and the microbial
networks of patients who achieved a successful pregnancy were found to be denser and
with more and denser microbial relations compared to those of patients with RIF. According
to two of the studies, the mechanisms through which the eubiotic microbiome of the vagina
and the endometrium alike affects endometrial receptivity and chances of a successful
embryo implantation are most possibly immunological, as the immune tolerance of some
cells participating in the immune response (e.g., T-regulatory lymph cells) potentially
affects implantation. When bacteria invade the endometrium and stimulate the pattern
recognition receptors (PRRs) of the epithelial cells, these cells secrete cytokines that affect
the local lymph cell population. Bacteria of the genus Lactobacillus prevent pathogens
from entering by acting on the PRRs of the mucosal cells to regulate the immune response
necessary for embryo implantation [17,29]. Furthermore, bacteria of the genus Lactobacillus
secrete lactic acid, thus creating an acidic environment that inhibits pathogen growth [31].
On the other hand, a dysbiotic microbiome, defined by a decrease in the percentage
of Lactobacillus and other commensal bacteria and an increase in pathogen levels, has
negative effects in IVF outcome. Almost all studies presented similar results regarding
the composition of the dysbiotic microbiome, with the most common genera appearing as
biomarkers of RIF being Gardnerella, Prevotella, Megasphaera, Atopobium, Streptococcus and
Staphylococcus, with most of these coexisting in the microbiome. As the different bacteria
interact and the dense community formed by the Lactobacillus species and other commensal
bacteria is necessary for the stability of the local “ecosystem” in the endometrium, the
presence of pathogens, which also interact with each other, plays an important role in the
disorganisation of this “ecosystem” in patients with RIF. However, it is not clear whether
the presence of a non-LD-type microbiome facilitates the entrance of pathogens in the
endometrium or if the entrance of pathogens is what causes the decrease in the Lactobacillus
percentage [34]. In general, it seems that the increase in α- diversity in a dysbiotic, non-
LD-type microbiome has harmful effects on endometrial receptivity and the chances of a
successful pregnancy, as the lack of a dominant species potentially facilitates colonisation by
many bacterial species, especially pathogens, and creates an adverse environment related
to infertility [32].
Nevertheless, there were studies that presented contradictory results. One of these
showed that the microbiome of patients with lower levels of Lactobacillus was not as
highly correlated to endometrial receptivity compared to patients with a complete lack of
microbiomes, which may indicate that it is the quantity rather than the percentage ratio
of Lactobacillus that affects endometrial receptivity [25]. Another study also agrees with
these results, in which the microbiome of all patients with a successful pregnancy was
found completely lacking the Lactobacillus species, with high levels of Lactobacillus found in
non-pregnant women [33]. In a third study, the Lactobacillus abundance did not significantly
Acta Microbiol. Hell. 2024, 69 26

differ between control patients and ones with RIF, with the successful pregnancy rates also
being similar between patients with a Lactobacillus percentage greater and smaller than
90% [23]. Finally, one study showed a higher α- diversity in the microbiome of pregnant
patients than non-pregnant ones, which led the researchers to assume that more diverse
microbiomes are more beneficial for endometrial receptivity than a non-LD-type one [30].
Therefore, it seems that in some cases, the absence of Lactobacillus and the presence of more
diverse microbiomes does not necessarily lead to dysbiosis, while higher Lactobacillus levels
could even prove to be detrimental to pregnancy chances. However, these studies are only
a small percentage of the total, and as none of them provide a satisfactory explanation, a
larger number of studies are required in order to prove if these results are indeed statistically
important or just the result of chance.

5. Conclusions
This review investigated the composition of the endometrial microbiome and its
potential correlation to female infertility and the outcome of IVF treatment based on the
most recent studies on the topic. The strength of this study is that it examines a very
interesting topic that only recently started getting more attention from researchers, and
since female infertility is a problem many people are struggling with, we feel that this
study would be valuable to researchers and practitioners working in the field of human
reproduction. According to the majority of the studies included in this systematic review,
as well as most of the earlier ones, a eubiotic LD-type microbiome seems to be best for
maximising endometrial receptivity and the chances of a successful pregnancy, whereas a
dysbiotic non-LD-type microbiome, with increased α- diversity and a higher number of
pathogens present, has a harmful effect. On the other hand, there were few studies that
presented contradictory results, without, however, a satisfactory explanation. Thus, also
taking into consideration the fact that studies on the endometrial microbiome are still in
the early stages, there is only a small number of them, from few countries and with small
population sizes, and it is clear that more time and a larger number of studies are needed
in order to decipher contradictions and produce more certain results.

Author Contributions: Conceptualization, P.F. and G.G.; methodology, G.G.; software, P.F.; validation,
P.F., M.E., D.C. and G.G.; formal analysis, P.F.; investigation, P.F. and G.G.; resources, P.F. and G.G.;
data curation, P.F.; writing- original draft preparation, P.F.; writing-review and editing, P.F., M.E.,
D.C. and G.G.; visualisation, P.F., M.E., D.C. and G.G.; supervision, M.E., D.C. and G.G.; project
administration, M.E., D.C. and G.G. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

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