Idiopathic infertility is an extremely exciting and equally frustrating subject for practitioners. In recent years, the research space has focused mainly on the study of oocytes/embryos and endometrium.
The term non-receptive (refractory) endometrium has been used more and more often for patients with repeated good quality embryos implantation failures, in which no organic uterine pathology has been identified(1,2). During endometrial explorations by hysteroscopy, forms of clinically unapparent chronic endometritis have often been identified. Moreover, hysteroscopy experts observed a certain endometrial pattern (stromal oedema, micropolyps, polypoid endometrium, and diffuse hyperaemia) which they also included in the category of chronic endometritis despite negative endometrial cultures. In these patients, a major improvement in post-antibiotic pregnancy rates was observed(3).
In the last decade, the study of the human microbiota has developed a lot. A great gain of this research was the identification of saprophytic cultures in areas long considered sterile, such as the placenta, uterus or fallopian tubes. However, the small volume of these commensal germs was a major impediment to their identification in standard cultures.
The new techniques – next-generation sequencing – coupled with insertion sequencing allowed the exhaustive examination of the genes of bacterial origin proteins, by using mobile DNA elements (transposons)(4). This new approach allowed access to a territory that was virtually uninterrupted by standard technical resources and, thus, opened a new door to understand in intimacy the functionality of the endometrium(4).
The present study aims to review the possible impact of the microbiota in the fertility equation and to examine the data we currently have.
The role of the endometrium in reproduction is extremely important. At the moment of maximum receptivity of the endometrium, the implantation process implicates the involvement of numerous actors and the development of extremely sophisticated phenomena, culminating in embryo-maternal cross-talk(1). The factors responsible for endometrial preparation are very diverse. Attempts have been made over time to detach several functional levels directly involved in the preparation of the endometrium: the immune system, the interleukin system, adhesion molecules and barrier structure compartments system, the local metabolic system and free-radical oxidation system. In this paradigm, any uterine pathology with an impact on reproduction is explained by the alteration of some of these factors. The most intensively studied were organic diseases such as fibroids, polyps or adenomyosis, and their implications on the systems described before(5). More recently, practitioners have identified a new type of pathology defined generically as the refractory endometrium. Behind this condition, there are the traumas after curettage/myomectomy/embolization or after radiotherapy, infections, but also a segment that does not seem to fall into any obvious category – idiopathic alteration. Especially for the latter segment, the vast majority of researchers agreed either on the existence of a primary genetic alteration in some of the systems that control endometrial development, or on subtle aggressors unapparent to standard tests, such as chronic infections or, more recently, uterine dysbiosis(6).
Unfortunately, despite some special efforts, which included testing many parameters, such as leukaemia inhibitory factor, dv/ß3 integrin (ß3), cyclin E, products from homeobox/wnt genes, B cell lymphoma 6, and advancing a significant number of functional tests (Receptive Dx, E tegrity, Endometrial Function Test)(7,8), there could not be identified recurrent changes present in all patients with this diagnosis (or at least in a sufficiently large group), a reason for which it was concluded that under this title (idiopathic refractory endometrium) there are found a lot of subentities.
In this context, the exploration of the uterine flora and the identification of some pathologies at this level can constitute such a subsegment.
The human microbiome is the totality of microorganisms, as well as the related genetic material located on and in the human body. Although the term microbiome was advanced long ago, it was not until the early 2000s that the framework of the Lederberg definition was established(9).
The special attention that this subject enjoys at present has its origin, first of all, in the rethinking of the relationship humans – saprophytic flora, through the neo-Darwinian concept proposed by the biologist Lynn Margulis (“Symbiosis as a Source of Evolutionary Innovation”). In this paradigm, humans appear as a holobiont – a host organism that coexists with other species – bionites. The combined genome is called a hologenoma(10).
It is currently considered that the number of germs belonging to the microbiome exceeds 100 trillion, including bacteria, archaea, protists and fungi. This structure can be associated with a vital superorgan to maintain the health of the host organism. The functions of this body have proven to be extremely diverse. In a schematic manner, a series of major levels can be distinguished: maintaining energy homeostasis, preventing colonization with pathogenic opportunistic germs, developing a degree of tolerance to non-self substances and the formation of intestinal architecture.
The association of commensal bacteria located in the digestive tract has proven to be extremely important in the management of components of nutrients that the host organism was unable to metabolize(11).
In terms of immunity, the microbiota is attributed a major role in inducing and developing its many branches. The constant microbiota – host organism dialogue involves an elastic feedback between the microbiota and the various congenital or acquired components of immunity. The immune system controls the shape and type of microbiota (favouring certain types of colonies), while the microbiota induces fine calibrations of the immune system. The imbalances that appear in this equation open the door to the respective infections, in the opposite direction to minor diseases or allergies(12).
The development of molecular techniques for exploring the microbiota – next-generation sequencing (NGS) technologies, after 2007, combined with the development of homicidal concepts have greatly broadened the horizon of understanding the microbial flora. Thus, microbial structures were identified in niches considered traditionally sterile and, consequently, the functions assigned to the microbiota were reanalyzed, obtaining data that offer additional dimensions. Starting from these elements, the definition of the microbiota has been recently subjected to a constant process of refining. In a joint effort, a group of experts redefined in 2020 the microbiome “as a characteristic microbial community occupying a reasonable well-defined habitat which has distinct physiochemical properties”. “The microbiome not only refers to the microorganisms involved, but also encompass their theatre of activity, which results in the formation of specific ecological niches. The microbiome, which forms a dynamic and interactive micro-ecosystem prone to change in time and scale, is integrated in macro-ecosystems, including eukaryotic hosts, and here crucial for their functioning and health. The microbiota consists of the assembly of microorganisms belonging to different kingdoms (prokaryotes [bacteria, Archaea], eukaryotes [e.g., protozoa, fungi, and algae]), while «their theatre of activity» includes microbial structures, metabolites, mobile genetic elements (e.g., transposons, phages, and viruses), and relic DNA embedded in the environmental conditions of the habitat”(13).
Uterine microbiome and its pathology
The genital tract is probably one of the segments that has benefited mostly from the findings regarding the microbiome.
The arguments in favour of colonizing the upper genital area are diverse. First of all, we have a solid documentation in support of the existence of an ascending current induced by uterine peristalsis in the follicular phase(14). Secondly, the cervical mucus functions only as a barrier to germs in the vagina. At the time of ovulation, the consistency of the mucus becomes very low, which further reduces the efficiency of this protection mechanism. In addition, the semen has its own microbiota that will be engaged in the ascension process, simultaneously with the sperm(15).
The disposition of the bacterial flora on the genital tract differs, however, during the four major stations (vagina, endometrium, fallopian tubes, Douglas sac), both in terms of type and volume.
Thus, at the level of the vagina, the order of size of the flora is 10¹¹, in which the lactobacilli represent >99% of the flora, while at the level of the uterus, the bacterial flora decreases to 107, and is much more diverse. At the level of the fallopian tubes/Douglas sac, the flora is very low and Lactobacillus represents <1%. Including at the level of the intrafollicular environment, commensal germs such as Lactobacillus, Actinomyces, Bifidobacterium, Propionibacterium, Staphylococcus and Streptococcus are described(16).
From the practitioner’s perspective, the uterine segment of the microbiome is the area of greatest interest. Unfortunately, the data reported by the authors who studied this subject differ greatly, especially in terms of germ weight, even though there is agreement for the presence of the main actors, Firmicutes (lactobacilli, Streptococcus, Staphylococcus), Bacteroidetes (Enterobacter – E. coli), Proteobacteria and Actinobacteria(17).
The main shortcomings are represented, on the one hand, by the physiological variations of the flora related to ethnicity, race, eating habits, but also by changes induced by age (the microbiota is probably the organ with the greatest capacity for change over time) or periods of the cycle. The technical difficulties of harvesting, respectively the accessibility, cannot be neglected either, being extremely important in the context of the massive examinations in the general population necessary for a more realistic mapping.
The volume of information on the action of the uterine microbiota on the endometrium is still too small to document which segments are affected in the endometrial preparation process. The peculiarities of the uterine area – low germ volume (compared to other situses), the proximity of a region with extremely rich flora (vagina) and relatively difficult to access (requiring minimally invasive manoeuvres) are important obstacles in the consistent analysis of uterine flora.
However, relatively recent studies in immunology confirm the potential of genital epithelial cells to express a wide range of pattern recognition receptors (PRRs) capable of responding differently and individually to non-self-agents(18). In this sense, Toll-like receptors (TLRs) and NOD-like receptors, essential in both the immediate cellular immune response and in tissue adaptive reactions, are described. The activation of TLR receptors via the microbacterial metabolites will control the size of the bacterial cohort, while the activation of NOD receptors will induce changes in the endometrial cell from its metabolism to its communication systems to modulate gene expression via the underlying transcriptomic modulations, representing grounds for epigenetic inductions.
In the practitioner’s registry, the alterations of the uterine microbiota can create the premises for the development of chronic endometritis and uterine dysbiosis, respectively.
Chronic endometritis is a clinical entity that is difficult to diagnose in the context of a frustrating condition. The examinations imposed by infertility (hysteroscopic balance + cultures + endometrial biopsies) allowed the diagnosis of a much larger number of cases(19).
At present, the most reliable means of diagnosis is considered to be the identification of plasma cells in the endometrium using immunohistochemistry for plasma marker CD138 (also known as syndecan-1, a transmembrane-type heparan sulphate proteoglycan)(20). The bacterial agents involved in the development of corneal endometritis are sensitive to different acute infections – Chlamydia trachomatis, Neisseria gonorrhoea and Mycoplasma. Most often, chronic endometritis has common bacteria, such as Streptococcus species, Escherichia coli, Enterococcus faecalis and Staphylococcus species), Mycoplasma/Ureaplasma species (Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma urealyticum), Pseusonia aerosa, Proteus species, Gardnerella vaginalis, Corynebacterium, and to a much lesser extent Chlamydia trachomatis(3). The actual mechanisms by which the implantation process is altered revolve around the structural alterations of the endometrial test or at least of its functional expression (production of interleukins/adhesins/mucins), respectively of the immune changes. Unlike other territories in the endometrial area, natural killer (NK) cells have a much more consistent role in modulating the receptivity of endometrial cells to progesterone(2).
Starting from the massive involvement of bacteria that make up the microbiota in the process of nutrition by metabolizing many products that are unaffordable to human enzymes, it is easy to speculate that a possible uterine dysbiosis could affect the nutrition of the endometrium.
Clinical data that associate the alteration
of the vaginal and uterine microbiome
The first studies that explored the relationship between uterine microbial flora and infertility used cultures to document infection. The systematic identification of germs at the tip of the catheter used for embryo transfer has been associated with much lower pregnancy rates(21).
The development of molecular techniques for identifying bacterial microflora has greatly broadened the horizon of examination, thus identifying pathogenic germs that could not be identified by classical tests due to low biomass.
Kyano examines the uterine microbiota in 92 patients on the ART protocol according to the dominant (>90%) versus non-dominant (<90%) type of Lactobacillus. The pregnancy rate was higher in the dominant Lactobacillus group (58.9% versus 47.2%), but without reaching the threshold of statistical relevance(22). Morena, in turn, reports a higher implantation rate in the dominant Lactobacillus group versus non-dominant Lactobacillus lot (60.7% versus 23.1%; p<0.001)(19).
Hashimoto reports a higher pregnancy rate and a lower miscarriage rate in patients who had eubiotic flora in the endometrium compared to patients with dysbiotic flora, but without the results being statistically relevant(23).
Brecewell evaluates, in a systematic review, 26 studies focused on the role of the vaginal and uterine microbiota on ART. The results of studies based on vaginal cultures did not identify any negative effects of vaginal dysbiosis. In contrast, studies in which the vaginal microbiota was examined by metagenomics sequencing techniques documented an adverse effect of vaginal flora imbalances on ART outcomes. However, the authors of the review draw attention to the very large discrepancies between the studies and urge caution in interpreting the results(24).
Cicinelli aims to improve implantation rates by administering antibiotics to the group of patients with repeated implantation failures diagnosed with chronic endometritis. Pregnancy rate was 76.3% versus 20% (p<0.0001), while the live birth rate was 65.8% versus 6.6% (p<0.0001). It should also be noted that in the group without chronic endometritis (probably, another cause of repeated failure) the respective live birth rates were 9.5% and 4.5%(25).
It is worth mentioning that, unfortunately, the value of all these studies has been greatly diminished (according to their authors) both by technical problems and by the small number of cases and, consequently, the results must be analyzed with caution.
In sterile patients with resistant endometrium, there is a subgroup in which the alteration of the microbiota may be the central element. In this regard, chronic endometritis is described, histologically confirmed by the presence of plasma cells in the endometrium presenting an altered but nonspecific flora, respectively endometrial dysfunction characterized only by dysbiosis of the uterine microbiota.
Regarding the diagnosis of chronic endometritis, although relatively laborious, the diagnosis is standardized. Unfortunately, the diagnosis of uterine dysbiosis is still in the evaluation stage. At present, there are two molecular tests based on sequencing but which are still being tested: Endometrial Microbiome Metagenomics Analysis – Ingenomix, respectively Endometrial Microbiome Test – Varinos. Both tests are limited to establishing the dominant or non-dominant character of the Lactobacillus flora, without making distinctions related to the rest of the flora.
The treatment of chronic endometritis focuses on the use of antibiotics as opposed to the treatment of dysbiosis in which their use will aggravate the existing imbalance.
In the context of disputes over the germs involved, several formulas are discussed. Starting from the frequent presence of Mycoplasma, most authors propose an initial monotherapy type based on ofloxacin. In case of persistent infection (documented by a new biopsy after one month of treatment), a broad-spectrum combination, such as ofloxacin + metronidazole or cephalosporin + doxycycline + metronidazole and amoxicillin + clavulanic acid, is used(26). The route of administration was oral for most authors, but there were also voices for local administration, such as intrauterine instillation. The reported results were generally optimistic, ranging from very good results (92% curability after monotherapy and 99% after combination therapy) to modest results (28% curability after the first cure, 23% after the second cure, 25% after the third cure, respectively 25% resistant after all three cures)(27).
The adjuvant administration of dexamethasone was tested, but the results were unconvincing.
The use of probiotics has proven, at least so far, to be useful only in vaginal dysbiosis. The administration of lactobacilli is problematic, considering that the share it should have in the uterine flora is not very clear.
Equally, the idea of transferring uterine microflora from a healthy patient (with documented fertility) is viewed with great scepticism in the context of the often very large discrepancies that exist even between healthy women.
We currently have compelling arguments to give the uterine microbiota a significant role in the functionality of the endometrium. Under these conditions, its systematic research in patients with repeated implantation failure becomes mandatory.
The hysteroscopic examination with endometrial biopsies (to confirm chronic endometritis), doubled by the evaluation of the microbiota by molecular techniques, seems to be the most consistent direction for a complete diagnosis.
There is an urgent need for the development of a profile of the physiological uterine microbiota adapted to the age, ethnicity, parity or period of the uterine cycle. It is also necessary to develop reliable tests that are widely applicable for monitoring the uterine flora in different circumstances. In addition, the intervention with probiotics or prebiotics must be refined according to the particularities of the endometrium.
Even though in the short term these desiderata seem to be exaggeratedly optimistic, it is indispensable that the subject play an important role in the agenda of both researchers and practitioners.