The most likely source of microorganisms colonizing the endometrium is bacteria ascending from the vagina ( Figure 1 ). This hypothesis is supported by the study of Swidsinski and colleagues (21), who showed the presence of biofilms of Gardnerella vaginalis on the endometrium and fallopian tubes in patients suffering from bacterial vaginitis. Subsequent years of analysis have shown that the cervical mucus plug, previously considered an impermeable barrier separating the vagina from the endometrium for bacteria, is not effective against all microorganisms. A study by Hansen and colleagues (22) found Ureaplasma parvum bacteria to be present within it. The researchers suggested that this barrier significantly restricts the passage of microorganisms from the vagina to the lower pole of the uterus, but does not block it completely, so the endometrial sterility theory has once again been challenged. Colonization of the endometrium can also be supported by physiological uterine contractions. The peristaltic pump promotes the translocation of sperm from the cervical canal, which may be accompanied by the movement of other particles, including bacteria, especially during the follicular and luteal phases of the cycle, when contractions intensify (23, 24). Dysfunction of uterine peristaltic activity may particularly favor colonization through increased transport caused by increased frequency of contractions (25). Some studies suggest that in addition to bacteria representing the vaginal microflora, the endometrium may also be colonized by blood-spreading microorganisms from the respiratory tract, gastrointestinal tract, or oral cavity (26). It has been shown that bacteria commonly colonizing the upper respiratory tract, such as Haemophilus influenzae or Streptococcus pneumoniae, can also colonize the female reproductive system, with the consequence that pregnancy can be terminated prematurely (27). Results of another study showed a similarity between the placental microbiome and oral microbiome of nonpregnant women (28). Among obese women with common disorders of the intestinal microbiome, a disproportion was more often observed among the population of microorganisms colonizing the vagina, which could result in preterm labor (29).

Similar to the vaginal microbiome, it is commonly believed that the endometrium of healthy women is a niche dominated by bacteria of the genus Lactobacillus. This thesis is supported by numerous studies that have shown the predominance of lactobacilli over other microorganisms residing in the uterine cavity. Results obtained by the team of Mitchell et al. (3) revealed that the species most frequently isolated from sterile endometrial swabs in a group of 58 women undergoing hysterectomy were Lactobacillus iners, Prevotella spp. and Lactobacillus crispatus. The study, led by Franasiak (4), found a predominance of microorganisms from the genera Flavobacterium and Lactobacillus in the material collected from 33 patients. Miles (5) also identified lactic acid bacilli as the microorganisms most frequently isolated from the endometrium of patients undergoing hysterectomy. Moreno et al. (6), based on a study of endometrial fluid collected from 52 women revealed that the endometrial cavity was most abundantly colonized by bacteria of the genus Lactobacillus, followed by Gardnerella, Bifidobacterium, Streptococcus and Prevotella. Based on the results of their own experience, supported by available literature sources, the researchers proposed a method for classifying the microorganisms residing in the uterine cavity, distinguishing a microflora dominated by Lactobacillus (LD), which is one in which bacteria of this genus make up more than 90% of the population, and NLD, where the dominance of lactic acid bacilli over other species is less. Tao et al. (8, 16) observed predominance of lactic acid bacilli oscillating between 70-90% relative to other taxa. The dominance of bacteria of the genus Lactobacillus within the endometrial microbiota was also confirmed by Wee and colleagues (8), who showed that the uterine cavity is characterized by a relatively lower microbial abundance compared to the vaginal microflora. Research on the endometrial microflora was also undertaken by Bednarska-Czerwińska (9), who identified 22 microbial species within it with a definite dominance of the genus Lactobacillus over other taxa, as well as a team of Lüll and colleagues (10), who also demonstrated a predominance of lactobacilli in endometrial fluid and tissue fragments collected from 25 patients.

Despite the prominent results, it is still hard to determine the definite molecular pattern of microorganisms residing in healthy endometrium. Some studies have found no lactobacilli in the reproductive tract of healthy, asymptomatic women, which may suggest that the dominance of lactobacilli over other microorganisms residing in the endometrium is not always associated with a condition indicative of abnormalities. Verstraelen and colleagues (11), in a study conducted on a group of 19 healthy women, showed a dominance of Bifidobacteria, while lactic acid bacilli prevailed in only four cases. Pseudomonas spp., Acinetobacter spp., Vagococcus spp. and Sphingobium spp. were the dominant species among the microorganisms isolated from the endometrium by the team of Chen and colleagues (12). Similar results were obtained by Winters (13), who identified Acinetobacter, Pseudomonas, Cloacibacterium, and Comamonadaceae. In shotgun sequencing conducted by the team of Li and colleagues (14), Pseudomonas sp., Propionilbacterium sp., Streptococcus sp., and Moraxella sp. were described as dominant taxa. The study by Sola-Leyva (15) showed a predominance of Klebsiella pneumoniae, Clostridium botulinum and Pasterurella multocida, over other species isolated from the endometrium.

The reasons for the differences in the results obtained can be traced to physiological stimuli at the individual level, as well as technical ones, resulting from the methodology of the study. There are many factors, such as medical history, endocrine disorders, genetic conditions, age, ancestry or behavioral variables, that can directly and indirectly affect the composition of a patient’s microbiota. Studies have shown that the microbiome of women suffering from various types of reproductive disorders, such as endometriosis, endometrial cancer, polyps, or infertility, had a different composition than that observed in patients without similar abnormalities (30). It has also been shown that the abundance and biodiversity of microorganisms inhabiting the female reproductive system are subject to changes throughout a woman’s life and are significantly reduced during the menopausal period (31). Some studies also suggest the role of ethnicity as a potential factor affecting the composition of the microbiota of the female URT. Fettweis et al. (32) showed that the microbiota of women of European descent was significantly less species-diverse and poorer than that of African-American women. Zhou et al. (33) reported that the types of vaginal communities found in Japanese women were similar to those found in both African-American and European women. However, with few studies and significant differences in results, it is difficult to say whether ethnicity, rather than geographical area and the resulting behavioral and cultural differences may have a significant impact on the composition of the female reproductive tract microbiome. Changes in the composition of the female reproductive tract microbiome may also be caused by certain physiological conditions, such as pregnancy (34). Methodological and technical factors can also contribute to the discrepancy in results. One of the biggest obstacles in attempting to determine the composition of the endometrial microbiota is the limited ability to obtain high-quality material for testing. Most experiments are conducted on swabs taken from patients through the cervical canal, which carries a significant risk of contaminating samples with vaginal bacteria (35). Although studies conducted on tissues from patients undergoing hysterectomy have a lower risk of contamination, the composition of the microbiota of such patients, may differ from the physiological one, as such women have abnormalities, pathological conditions and diseases that may affect it (3). Another major limitation of most studies is the difficulty in obtaining study material, resulting in small sample sizes and reduced statistical value of the results (36). While the development of 16S rRNA variable regions (V) sequencing techniques has allowed for much more accurate studies of the human microbiome, this method also has its limitations. Available research protocols vary in terms of the amplification primers, sequencing primers or sequencing techniques used, which can contribute to discrepancies in the results obtained (37). In addition, the detection of bacterial genetic material is not always indicative of the presence of a viable microorganism in a given niche (38). An additional factor potentially explaining inconsistencies in reported prevalence of Lactobacillus dominance in the endometrial microbiota is the concept of functional redundancy among bacterial taxa. Even when Lactobacillus sp. is not taxonomically dominant, taxa such as Gardnerella and Bifidobacterium may perform analogous functions such as lactic acid production, modulation of pH, or competitive exclusion of pathogens, thereby preserving endometrial homeostasis. Inclusion of this functional perspective could help reconcile discordant findings across studies (16). Due to inconsistencies in the results obtained and the number of variables that must be considered when interpreting them, we cannot yet unequivocally define the composition of a healthy endometrial microbiota. However, based on the available data, we can make some generalizations. The microorganisms statistically most common in the endometrial cavity are bacteria belonging to types: Bacteroidetes, Proteobacteria, Actinobacteria and Firmicutes, among which the genus Lactobacillus, whose dominance within the endometrium has been confirmed by numerous research groups, deserves special attention. Lactobacilli are also a major component of the bacteria found in the vagina, but although the composition of the two habitats has some similarities, they are not identical. The endometrium has its unique microbiota (39).

It may seem impossible for the microorganisms to survive in this highly variable environment due to the menstrual cycle, but research clearly indicates that the endometrium has its own microbiota. Maintaining a balance between commensal bacteria and pathogens in this particular niche is essential for its healthy activity. Based on the studies of gut microbiota, in 2010, Hooper and Macpherson (40) described three types of immune barriers needed to maintain a balance between the gut microbiota and the host. The first barrier is the anatomical limitation of the exposure of commensal microorganisms to the host immune system. The next barrier is the immune mediators that limit direct contact of microorganisms with the epithelium, and the last one is the ability to rapidly detect and inactivate pathogenic microorganisms once any of the barriers are broken. Each of the above-mentioned conditions is initially fulfilled by the endometrial environment. The microbial homeostasis of the endometrium is guarded by the immune system, which takes care to maintain a certain limit of invasion of commensal microorganisms and elimination of pathogens (41).

One potential role of the endometrial microbiota is to modulate immune responses within the niche through several mechanisms, such as competition, receptor stimulation and modulation of epithelial barrier function. Both endometrial stroma cells and epithelial cells are equipped with several receptors that allow them to recognize foreign molecular patterns of pathogenic microorganisms. Recognition of foreign antigens leads to the activation of the inflammatory response, resulting in the secretion of a wide range of specific chemokines that are sensed by appropriate subsets of immune cells. They also stimulate the synthesis of PRR receptors. Moreover, commensal microorganisms restrict the pathogen invasion by depleting the environment of nutrients (42). The uterine cavity microbiome may also contribute to supporting the genomic stability of the epithelium by modulating transcription factors (30). Microbes can also strengthen the physical barrier of the endometrium by stabilizing the mucosal layer, promoting repair processes or secreting antimicrobial substances (43). The endometrial microbiome can also influence the phenotype of immune cells in the uterine cavity, which, in addition to their defensive role, play a key role in embryo implantation by providing local immune tolerance to both fetal and paternal antigens, as well as trophoblast invasion (44).

The immune system plays a crucial role in maintaining homeostasis within the female URT, protecting it from pathogens and preventing pathological changes ( Figure 2 ). Although immune cells are distributed differently across various regions of the reproductive system ( Table 1 ), the most abundant are T lymphocytes, dendritic cells (DCs), natural killer (NK) cells, neutrophils, and mast cells which collectively orchestrate tissue remodeling, immune surveillance, and regulation of local inflammation, particularly during the secretory phase of the menstrual cycle. Dominance of these particular immune cells within the endometrium suggests that the immune landscape of this niche is predominantly shaped by cellular immunity, with innate immune cells constituting the majority of leukocyte populations. In contrast, humoral immunity plays a supplementary role, with low numbers of B lymphocytes and plasma cells producing limited amounts of secretory IgA and IgG in the endometrial milieu. This cellular predominance ensures rapid and localized immune responses while maintaining immune tolerance required for successful implantation (45).

While the number of immune cells in individual parts of the URT is relatively constant, the number of cells in the uterus is sensitive to changes in sex hormone levels caused by the ovarian cycle. Cyclical hormonal changes during the menstrual cycle may affect immune responses, which is crucial for promoting endometrial receptivity, necessary for embryo implantation (46). The normal ovulatory menstrual cycle depends on the proper functioning of a mature hypothalamic-pituitary-ovarian axis and the precise regulation of hormonal feedback mechanisms ( Figure 3 ). The cycle is traditionally divided into two main phases: the follicular phase and the luteal phase, followed by menstrual bleeding. These phases are characterized by fluctuations in progesterone (P4), oestradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels. Each cycle culminates in the development of mature follicles and the release of an oocyte, with menstruation occurring in the absence of fertilization. During the follicular phase, oestrogen levels gradually increase until a surge in LH secretion from the pituitary triggers ovulation. If fertilization does not occur, oestrogen levels rapidly decline within 24 hours. LH also prompts granulosa cells to shift from converting androgens to producing progesterone, leading to elevated progesterone levels throughout the luteal phase. In the absence of implantation, progesterone levels drop after 14 days, resulting in menstrual bleeding. This consistent pattern of a two-week estrogen-dominant environment, followed by a two-week progesterone-dominant phase during the female reproductive years, provides an opportunity to study the physiological role of these hormones in cellular homeostasis in an in vitro context (73, 74).

Experimental studies have shown that the immune cell composition in the endometrium undergoes dynamic changes during the menstrual cycle, particularly during menstruation. Immune cells, including T-helper cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), natural killer (NK) cells, and macrophages, fluctuate in response to hormonal changes. There is a marked infiltration of macrophages during menstruation, which play a key role in tissue remodeling and the clearance of apoptotic cells. Additionally, dendritic cells are involved in initiating immune responses and show dynamic changes in number and activation state throughout the menstrual cycle. These fluctuations of immune cells are tightly regulated by hormonal signals, primarily estrogen and progesterone, and contribute to maintaining immune homeostasis in the endometrium, which is essential for reproductive health. Both DCs and macrophages reach their peak number during the late secretory phase (75). Other cells playing an important role during menstruation are endometrial neutrophils. Through the elastase release resulting in the activation of extracellular metalloproteinase, neutrophils promote the destruction of endometrial tissue. This allows intimal remodeling and intimal vascular repair (62). The T cell level is subject to fluctuations during the menstrual cycle, representing 50% of the leukocyte population during the proliferative phase and decreases to <10% by the late secretory phase (76). NK cells, crucial for immunosurveillance, also exhibit increased activity during menstruation, contributing to regulating endometrial shedding and tissue repair. The T-regulatory cell levels (Tregs), which are involved in immune tolerance, are generally reduced during menstruation, reflecting the heightened inflammatory response necessary for endometrial breakdown. When it comes to endometrial mast cells, their abundance in the endometrium is relatively low, and the number of cells does not undergo significant changes during the menstrual cycle (63).

The survival of commensal microbiota within the endometrium is determined by the presence of Toll-like receptors (TLRs), which enable the differentiation of pathogenic microorganisms from the physiological microbiota, thus strengthening the endometrial defense barrier. These receptors can be found on the surface of columnar cells lining the upper FRT and on immune cells such as macrophages, dendritic cells, neutrophils or NK cells (77–79).

TLRs represent a group of trans-membrane receptors belonging to the family of pattern recognition receptors (PRRs). In their structure, one can differentiate an extracellular domain, a very short trans-membrane domain and a cytoplasmic domain. The extracellular domain is composed of leucine-rich repeats and is responsible for the binding of a well-defined ligand by a specific receptor. It plays a key role in recognizing pathogen-associated molecular patterns (PAMPs), such as bacterial or viral nucleic acids, LPS, lipoproteins, or peptidoglycan (80). The cytoplasmic domain, also known as the Toll/IL-1 receptor (TIR) domain due to its homologous nature to the cytoplasmic domain of the human IL-1 type 1 receptor, has cysteine units at its ends, which are responsible for initiating signaling cascades in the cell, ultimately leading to the activation of transcriptional nuclear factor κB (NF-κB), crucial for gene expression of a wide range of immune response-related molecules such as cytokines, chemokines, costimulatory molecules or adhesins (56, 81). Thus, the role of TLRs is not limited to a receptor function during the detection of molecular patterns of microorganisms but is also expressed through their key role in the activation of the inflammatory response through the secretion of pro-inflammatory cytokines. It is worth noting that cytokine secretion resulting from TLR receptor activation can also indirectly induce the differentiation of Th1-type lymphocytes, as well as activate monocytes and NK cells, which will involve the secretion of further inflammatory mediators such as interleukin-2 (IL-2), IL-12, IL-15, IL-18, tumor necrosis factor α (TNF-α), interferons α (IFN-α) and β (IFN-β). TLRs activated on the surface of B lymphocytes, in turn, lead to direct activation of these cells, resulting in their secretion of antibodies, which are precise tools to combat pathogenic microorganisms. TLR receptors thus play their role by combining specific and nonspecific immune responses (82–84). Among mammals, we identify 11 different TLR receptors, the presence of which within the endometrium for most of them has been confirmed by studies. Their expression in the uterine cavity is subject to changes depending on the woman’s physiological state or the phase of the menstrual cycle (85–87). In order to recognize a specific ligand pair or a specific microbial ligand TLRs can form homodimers, meaning they have two identical receptor chains or heterodimers consisting of 2 different receptor chains, which are formed by TLR-2 and TLR-1 or TLR-6. TLR1, TLR2, TLR4, TLR5, and TLR6 mainly recognize bacterial products present on the surface of microbial cells, whereas TLR3, TLR7, TLR8 and TLR9 tend to recognize nucleic acids (88, 89). Without a doubt, TLRs are a key element in the induction of inflammation through the detection and recognition of foreign molecular patterns, resulting in the activation of transcription factors responsible for the secretion of inflammatory mediators. However, their role is not limited to pathogen detection and control. TLR receptors also condition host immune tolerance to commensal microbiota inhabiting particular FRT niches. These microorganisms are recognized by TLRs, but the immune response is not directed against them. Thus, one can say that TLRs are also a kind of a specific identifier, enabling the survival of selected microorganisms within the endometrium (90, 91).

In a situation where the microbial load is too high or rich in representatives of certain taxa, excessive tissue destruction and immune stimulation can occur, leading to an imbalance of homeostasis between bacteria, which is dysbiosis. It is not known whether dysbiosis is a cause or an effect of the disease. However, there is no doubt that disorders within the composition of the endometrial microbiome are associated with the occurrence of abnormalities in the physiology of the female reproductive system (18).

Disruption of the endometrial microbiota homeostasis may result in pathological conditions such as endometritis, defined as chronic inflammation caused by bacterial pathogens. Due to its asymptomatic course, it is an infrequently diagnosed condition that can be closely associated with recurrent implantation failure (RIF) or pregnancy loss. It is estimated that endometritis can affect up to 45% of infertile patients (92). Another disorder underlying dysbiosis in the reproductive system is endometriosis, characterized by the presence of endometrial tissue outside the uterine cavity, resulting in pelvic pain or infertility. It is believed that this disease affects up to 10% of women of reproductive age. The causes of the incidence of endometriosis are not yet fully understood, but it is thought that it may have a bacterial origin (93). This theory derives from a study showing increased incidence of the isolation of microorganisms from the genus Actinomyces, Corynebacterium, Enterococcus, Fusobacterium, Gardnerella, Prevotella, Propionibacterium, Staphylococcus, Streptococcus or E. coli from menstrual blood and endometrial samples of endometriosis patients, compared to a control group, in which mainly lactic acid bacteria were observed. Among the abnormalities underlying dysbiotic disorders in the uterine cavity, endometrial neoplasia is also receiving increasing attention. An enrichment of certain taxa has been observed among patients suffering from hyperplasia and cancer, suggesting an inflammatory role for certain bacteria in carcinogenesis (94).

Endometrial cancer is currently the most commonly diagnosed malignancy affecting women in developed countries, and an increasingly problematic gynecological cancer. It is estimated that there were nearly 400,000 new cases worldwide in 2018, and according to the International Agency for Research on Cancer, the incidence rate of this cancer, by 2040, will increase by up to more than 50% worldwide (95).

Histologically, one can differentiate between 2 types of EC: estrogen-dependent (I type), which accounts for 10-20% of all cases, and non-estrogen-dependent (II type), which accounts for 80-90% (7). Among the risk factors for endometrial cancer, the most cited are high Body Mass Index (BMI), disorders related to hormonal imbalance, diabetes, or hypertension. Excessively high blood pressure can inhibit apoptosis, which can promote the growth of cancer cells. Studies also indicate a lower incidence rate of endometrial cancer among patients who have children. Hormonal changes during pregnancy usually result in increased production of progesterone, which is attributed to a protective effect against endometrial cells, which may account for the lower incidence of cancer among patients who have children. The most cited risk factor, however, is obesity, which is attributed to an inflammatory role that may contribute to the induction of neoplastic processes, as well as hyperinsulinemia resulting from excessive body weight, which is a common predecessor to diabetes. Persistently high blood insulin concentrations have mutagenic effects and can cause an increase in the body’s levels of bioavailable estrogens by decreasing the concentration of sex hormone-binding globulin (SHBG) (96). The effect of obesity is also very often caused by disturbances in the intestinal microbiota, which plays a significant role in the modulation of the reproductive endocrine system through interactions with insulin, androgens, or estrogens - hormones crucial for the carcinogenesis of the female reproductive tract, which can also affect the composition of the microorganisms inhabiting the uterine cavity.

In 1863, the prominent German pathologist Rudolf Virchow observed the presence of lymphocytes in tumor tissues, which allowed him to hypothesize that chronic inflammation links to the onset of cancer. More than 150 years later, we dispose of evidence that enables us to confirm that his hypothesis was correct (97). It is estimated that up to 13-20% of cancer cases diagnosed each year are associated with infections, the main effect of which is inflammation (98, 99). Usually, during an acute inflammatory response caused by the pathogens, complete elimination of microorganisms occurs, which contributes to the restoration of homeostasis. However, in some infections, elimination of the pathogen does not happen, and the secreted pro-inflammatory factors can promote a chronic inflammatory process that, if sustained over a long period, may pose a risk for cancer development. Microorganisms, including bacteria and viruses, have long been suspected of a potential role in cancer induction and development, but identifying the specific species that could be responsible for such processes and the underlying mechanisms has so far been a challenge. To date, the International Agency for Research on Cancer has classified only one bacterial species, Helicobacter pylori, as a human carcinogen (100). Infection with this microorganism can result in inflammation of the gastric mucosa, which in a chronic state can lead to peptic ulcer disease and, consequently, also cancer (101). A similar relationship has been demonstrated for Fusobacterium sp., particularly the species of Fusobacterium mortiferum, Fusobacterium nucleatum or Fusobacterium necrophorum and colon cancer. The F. nucleatum bacterium is responsible for activating the nuclear factor NF-κB, a key factor regulating the course of the inflammatory response, which in the dysbiosis stage can cause the development of chronic inflammation (102). Another study showed that in the case of this cancer, the genus Porphyromonas, too, can induce the process of carcinogenesis (103). Admittedly, numerous literature data highlight the key role of inflammation in the processes of the FRT, such as the menstrual cycle, ovulation, and implantation, emphasizing that the endometrium is a dynamic environment whose physiological function depends on cyclic processes in which inflammation is alternately induced to then become self-silenced. Unfortunately, when inflammation becomes a chronic condition, it can endanger a woman’s health (104).

The link between chronic inflammation has so far also been demonstrated for endometrial cancer. The role of inflammation in EC was first pointed out by Modugno et al. (105) who showed a link between the inflammatory environment in endometrial tissue, the production of inflammatory mediators, and the development and progression of endometrial cancer. In a case-control study performed by Doddus et al. (106) in 2011, it was shown that one of the key inflammatory mediators, TNF, was responsible for proliferation and involved in angiogenesis of the tumor cells studied. In another study, Dossus et al. (107) examined the relationship between the presence of various biomarkers such as hormones, growth factors and cytokines in the pre-diagnostic blood of patients and the occurrence of endometrial cancer. The study demonstrated an association between the presence of TNF-α, TNF receptors 1 and 2, IL-6, and C-reactive protein (CRP) and an increased risk for endometrial cancer in postmenopausal women. Another study by Dossus et al. (108) showed a strong association between elevated cytokine levels and increased risk of endometrial cancer, thus indicating that endometrial cancer may have an inflammatory basis. A similar relationship was demonstrated in a study by Trabert et al. (109), in which the levels of 64 inflammatory biomarkers in the blood were evaluated and showed that EC risk was highest among women with the highest levels of inflammatory markers including adipokines, inflammatory cytokines, angiogenic factors, acute phase proteins and vascular endothelial growth factor-A (VEGF-A). The study by Liu et al. (110) collected and sequenced endometrial biopsy specimens from 130 infertile women with confirmed chronic endometritis and showed that malignancy was not only associated with chronic inflammation but also with an increased abundance of bacterial taxa such as Dialister, Bifidobacterium, Prevotella, Gardnerella and Anaerococcus, shedding new light on the role of microorganisms in the induction of inflammation during EC.

Certain bacteria in the endometrial environment may play a key role in the initial stages of inflammation by inducing an excessive inflammatory response ( Figure 4 ) (111). Molecular patterns of pathogenic bacteria stimulate the activation of metabolic pathways by TLR receptors, resulting in the production of pro-inflammatory cytokines. Under physiological conditions, activation of the inflammatory response by receptors is a favorable situation, as it allows our immune system to fight off the intruder. However, in chronic dysbiosis, TLRs may co-create a tumor-promoting environment by initiating and enhancing the inflammatory response or promoting cellular angiogenesis (112). Experimental studies have shown that Toll-like receptors, the key components of the innate immune system, play a significant role in the early stages of tumorigenesis in EC. TLRs, particularly TLR-4, are involved in the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), which initiate the inflammatory response. This activation leads to the upregulation of pro-inflammatory cytokines, such as TNF-α and IL-6, contributing to development of the microenvironment that promotes tumor cell adhesion and invasion (113). Specifically, TLR-4 activation has been implicated in enhancing the adhesion of endometrial cancer cells to the extracellular matrix and epithelial cells, facilitating their early attachment and colonization (114). Additionally, TLR signaling has been shown to influence the recruitment of immune cells to the site of infection or tumor development, further promoting the inflammatory milieu that supports cancer progression (115). These findings suggest that TLRs are not only involved in immune responses but may also act as key mediators in the initiation and progression of endometrial cancer.

Activated TLRs may also play a key role in angiogenesis by stimulating the secretion of VEGF, which promotes tumor vascularization. The presence of blood vessels enables significant tumor growth because of the nutrients and oxygen supplied. However, the vessels formed under the influence of VEGF are characterized by both functional and structural differences, compared to those found in healthy tissues. These differences result in increased hypoxia of the tumor-affected tissue. Hypoxia, in turn, stimulates further VEGF synthesis and tumor growth (116). The relationship between increased synthesis of VEGFs along with selected TLRs and the degree of tumor progression was confirmed in a study conducted on a sample of 123 neoplastic endometrial samples, which managed to show a significant correlation between the expression of selected TLRs along with VEGRs and the degree of clinical progression and tumor pathology (117).

Recognition of bacterial molecular patterns by TLR receptors may also result in overactivation of NF-κB. NF-κB is a protein complex involved in the synthesis of many pro-inflammatory cytokines, playing a key role in regulating the inflammatory response. However, the ability of the NF-κB factor to inhibit apoptosis, induce proliferation, and enhance angiogenesis suggests its important role in tumor progression (118). Indeed, a study using immunohistochemical methods conducted on a group of 59 patients diagnosed with endometrial cancer, benign endometrial pathology, and with endometrial hyperplasia showed that the group with EC had significantly higher levels of NF-κB factor expression, compared to the other groups (119). Another study on a group of patients with endometrial cancer showed that the group of cancer patients had significantly higher serum levels of NF-κB, compared to the group of healthy patients (120).

Cytokines are a diverse group of soluble proteins released mainly by cells of the immune system in response to infection, inflammation, or tissue damage. To date, the secretion of the cytokines IL-6, IL-17, as well as transcription of genes coding inflammatory factors such as IL-8 (121) has been observed in tumor-lesioned endometrial tissues in which the presence of microorganisms has been confirmed. IL-6 is one of the main factors involved in the inflammatory response against pathogens. It plays an important role in the immune response, induction of inflammation, hematopoiesis, and progression of many cancers, including gynecological cancers. A study showed that the presence of IL-8 and IL-6 in the ovarian cancer setting was significantly correlated with a poorer prognosis of patients (122). This relationship was also confirmed in endometrial cancer. A study by Che et al. (123) showed that the presence of IL-6 in the tumor cell environment correlates with the invasion and migration abilities of endometrial cancer cell lines, suggesting a role for cytokines in tumor progression. Another study showed that IL-6 indirectly stimulates the proliferation, migration, and invasion of endometrial cancer cells and downregulates the expression of protooncogenes in cancer cells (124). IL-8 also plays an important role in tumor angiogenesis in the course of breast, ovarian, cervical, and also endometrial cancers. Overexpression of IL-8 causes infiltration of large numbers of leukocytes to the tumor by chemotaxis. Attracted immune cells release growth factors in the tumor microenvironment, thus promoting tumor growth and proliferation (125). A study by Ciortea et al. (126) found significantly elevated levels of IL-8 in a group of 44 patients with endometrial cancer compared to a group without gynecologic pathology.

Activation of TLR receptors, including TLR-2, also contributes to the induction of oxygen free radicals (ROS) (127). ROS are a group of byproducts of normal cellular metabolism such as superoxide anion (O^2-), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH^-). Maintained in a physiological state, ROS act as signaling molecules that regulate the course of numerous reactions, including growth factor signaling, proliferation or adaptation to hypoxia (128). However, excessive levels of ROS in the cellular environment can have pathological effects. Indeed, ROS play a key role in post-transcriptional gene regulation, modulating cellular development, differentiation, proliferation, and apoptosis, being some of the key inflammatory mediators in cancer progression (129). Indeed, a study of 305 EC patients showed that their ROS levels were significantly higher than those of healthy controls (130). In addition, the results of a recent Lopez-Mejia study suggest that oxidative stress is increased in uterine serous carcinoma (USC), a more aggressive form of EC, and promotes metastasis (131). Chronic inflammatory response within the endometrium can also lead to overexpression of a wide range of inflammatory factors and mediators, including the enzyme cyclooxygenase-2 (COX-2). Under physiological conditions, this enzyme is a key component in the prostaglandins (PGs) synthesis, including prostaglandin E2 (PGE2), formed by the COX-2-induced metabolism of arachidonic acid. Synthesis of PGE2 leads to increased metabolic and inflammatory processes, as well as inhibition of apoptosis, which may result in the initiation of angiogenesis, leading to tumor formation (132). Also, the overgrowth of certain strains within the uterine cavity, including G. vaginalis, the presence of which within the tumor-transformed endometrium is confirmed by several studies (121, 133–140), can lead to overexpression of cyclooxygenase-2. In addition to its ability to initiate COX-2 synthesis, the bacterium is also responsible for vaginolysin (VLY) synthesis, a bacterial toxin that can serve as an inflammation-inducing agent and exhibit promiscuous activity against epithelial cells (141).

Dysbiotic disorders within the endometrial microbiota and persistent chronic inflammation can also lead to the destabilization of the host genome. Chronic inflammation resulting from a disruption of microbial homeostasis within the endometrium provides an environment that promotes damage to the host’s genetic material. Metabolic changes resulting from mutations or damage to host DNA can result in cell cycle disruption, inhibition of apoptosis or stimulation of proliferation of malignant cells (142, 143). Among other things, the changes can be associated with overexpression of COX1 and COX2 enzymes, nitric oxide, and ROS. High concentrations of these factors in the tumor microenvironment promote damage to the genetic material of host cells, which can result in the formation of mutations (144). Microorganisms can also cause damage to host genetic material by secreting products of secondary metabolism, including toxins, or altering the pH of the environment (2). According to the model proposed by Knudson, carcinogenesis requires at least two allelic mutations in a single gene. These mutations include genes involved in cell cycle control or genomic DNA quality. Such single mutations could, of course, lead to the development of cancer, but more mutations are usually required. Chronic inflammation provides a highly mutagenic environment that promotes changes at the epigenetic level. The resulting mutations can lead to abnormalities in cellular metabolism, such as proliferation or apoptosis, processes crucial to the course of carcinogenesis (144, 145).

Differences in the metabolism of cancer cells compared to unaltered tissue were demonstrated in 2017 by Altadill and colleagues (146), who compared the metabolomics of EC tissue samples from 39 patients and tissues from a sample of 17 healthy women. They observed abnormalities in lipids, kynurein, endocannabinoids and the RNA editing pathway in cancer patients. Further studies, in turn, showed that adenosine deaminase acting on RNA2 (ADAR2) is overexpressed in cancer cells, possibly contributing to carcinogenesis. Unfortunately, this study does not indicate whether these changes may be the effect of the presence of pathogenic microorganisms within the tumor-lesioned tissue. Another study by Chen et al. (137) provided ample evidence for the role of microorganisms in modulating the metabolic pathways of host cells. In the first part of the study, the team showed elevated levels of representatives of the genera Firmicutes, Proteobacteria, Tenericutes, Actinobacteria, and Bacteroidetes among EC patients, and, even more significantly, the microorganisms participated in the metabolic conversion of N-acetyl-β-glucosaminyl to 6-sulpho-sialyl Lewis x epitope and promoted the 6-sulpho-biosynthesis of sialyl Lewis x epitope, a molecule responsible for promoting metastasis by adhering tumor cells to endothelial cells. The study was also the first to confirm the presence of these molecules in the endometrium. In addition, the study proved that the microorganisms whose increased presence was noted in tumor-lesioned EC tissue had an inhibitory effect on N-Glycan synthesis and were associated with activation of the Apelin signaling pathway, whose association with increased risk of EC was confirmed in a study by Yang et al. (147). The results presented not only indicate the role of microbes in modulating the metabolism of host endometrial cells but also provide interesting data on the potential role of dysbiotic disorders in metastasis. The evidence presented here suggests that the potential role of microorganisms in the induction of tumorigenic processes should not be downplayed.

The correlation between endometrial dysbiosis and endometrial cancer is indicated by the results of studies from the last decade ( Figure 5 ). The link between the occurrence of EC and the presence of specific bacterial strains within the altered tissues was first demonstrated by Walther-António (133). The experiment was conducted on a group of 17 women who had undergone a hysterectomy for an established cancer. The results of next-generation sequencing of the 16S rDNA V3-V5 region revealed that the presence of Atopobium vaginae and Porphyromonas sp. bacteria in combination with high vaginal pH was statistically associated with endometrial cancer.

In another study of 148 women undergoing hysterectomy for benign lesions, endometrial hyperplasia or endometrial cancer, Walsh et al. (134) also identified Porphyromonas somerae as the most predictive microbiological marker of endometrial cancer. Moreover, Caselli et al. (135) showed that P. somerae and A. vaginae induced the release of pro-inflammatory cytokines by human endometrial cells after 24 hours of co-culture. As indicated by the results of the above studies, enrichment in certain taxa was observed among both hyperplasia and cancer patients, suggesting an inflammatory role of specific microorganisms in carcinogenesis. Nowadays, a growing number of studies point to the presence of particular strains as potential biomarkers of endometrial cancer. In the stage of dysbiosis, microorganisms, especially certain pathogenic taxa, can stimulate immunopathological processes and promote destabilization of the host genome. In a recent study, Li et al. (136) confirmed that increasing Prevotella abundance in endometrial tissue, specifically when correlated with elevated serum levels of D-dimers (DD) and fibrin degradation products (FDP), may be an important factor associated with carcinogenesis. A study by Lu et al. (121) found a correlation between increased Micrococcus abundance and IL-6 and IL-17 micro-RNA (miRNA) levels in endometrial cancer patients, suggesting a pro-inflammatory role for these microorganisms in carcinogenesis. Another study indicated significant enrichment of representatives of the genera Firmicutes, Proteobacteria, Tenericutes, Actinobacteria, and Bacteroidetes in the tumor-lesioned endometrium of EC patients (137). In contrast, in a study conducted on a group of 28 postmenopausal women undergoing hysterectomy, Wang et al. (138) showed that although Lactobacillus and Gardnerella were the predominant taxa in both tumor tissue samples and adjacent non-tumor tissue, only the group of sections taken from cancer patients was enriched in Prevotella, Atopobium, Anaerococcus, Dialister, Porphyromonas and Peptoniphilus. An interesting result was revealed by Chao et al. (139), whose study showed an increased relative abundance of plastic-degrading bacteria, Bacillus pseudofirmus and Stenotrophomonas rhizophila, in endometrial washings from patients with EC or endometrial hyperplasia. In a recent study, Kuźmycz et al. (140) not only found that the group of EC patients exhibited higher microbial diversity compared to the control group but also indicated higher abundance of pathogenic taxa such as Streptococcus, Anaerococcus, Prevotella, Gardnerella, Peptoniphilus, and Porphyromonas in malignant samples. In this study, the authors emphasized the role of Anaerococcus, which has emerged as a taxon that differentiates microbiomes isolated from women with EC and those with benign lesions, possibly representing a pathogenic cofactor of EC and at the same time a potential biomarker of carcinogenesis process. The above results suggest a potential role of selected pathogens in the induction of inflammatory-mediated carcinogenesis processes in EC. However, due to the heterogeneity of the study procedures and the results obtained, we cannot yet conclusively determine whether and how microorganisms induce pathological processes within the uterine cavity ( Table 2 ). However, there is no doubt that dysbiotic disorders within the endometrium should not be underestimated.

Over the past 20 years, our knowledge of the human microbiome and its role in physiology has increased significantly. Research into the composition and function of microorganisms residing in various niches of the human body proved that the microbiota plays an extremely important role in our physiology. Particular importance in the proper functioning, but also in the pathogenesis of many diseases, has been attributed in recent years to gut microbiota (148, 149).

Balanced gut microbiota is composed of a diverse group of microorganisms belonging to 4 main phyla: Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria, from which more than 90% of species consist of Bacteroidetes and Firmicutes phyla (150). A balance between commensal microbes inhabiting the gut is essential for their proper function in maintaining the epithelial barrier, improving digestion and regulating adaptive and innate immune responses (151, 152). The epithelial barrier is described as a monolayer of cells joined through protein complexes which separates the commensal bacteria in the gut from the underlying tissues. Additional protection against excessive microbial invasion is provided by mucus covering the epithelial layer. Its production and degradation are susceptible to changes in the composition of gut microbiota (153). Both inflammation and changes in the composition of the local microbiota can contribute to disturbances in mucus secretion and composition, leading to increased susceptibility to infection. Interaction between gut microbiota and intestinal epithelium is enabled by PRRs. Their presence leads to continuous immune signaling, the regulation of which is essential for maintaining intestinal homeostasis (154).

The intestinal microbiota is also responsible for the production of short-chain fatty acids (SCFAs) - metabolites produced in the process of fermentation of dietary fibers, proteins, and other substrates. By lowering the intestinal pH, SCFAs promote the growth of beneficial bacteria, such as Lactobacilli and Bifidobacteria, while inhibiting the colonization of pathogenic microorganisms like Clostridium and Escherichia coli (155). SCFAs also support the integrity of the intestinal barrier by stimulating the regeneration of epithelial cells and the production of mucus and antimicrobial peptides, which help prevent the translocation of toxins and pathogens into the bloodstream. Furthermore, SCFAs modulate the immune response by promoting the maturation and expansion of Tregs in the colon, which suppress local inflammatory reactions to the microbiota. They also play a role in the proliferation of innate lymphoid cells (ILC3), which release IL-22, a cytokine crucial for the production of antimicrobial molecules by epithelial cells (156). Through these mechanisms, SCFAs contribute to protecting against inflammation, metabolic disorders, and infections, thereby playing a central role in gut health and immune regulation (157, 158). However, the impact of the gut microbiome has a significant impact not only on the gastrointestinal system but also on other physiological systems in the body (159). It has been shown to influence the central nervous system (CNS), where gut-derived metabolites and immune signaling pathways modulate brain function and behavior, a phenomenon known as the gut-brain axis (160, 161). Microbiota also plays a crucial role in regulating the immune system, affecting innate and adaptive immune responses (161). Moreover, gut microbes influence metabolic processes, and the growing evidence suggests their prominent role in affecting cardiovascular health by modulating blood pressure and cholesterol levels (162). Thus, the microbiota represents a complex and integral component that interacts with multiple organ systems, shaping overall health. Unfortunately, the systemic role of the microbiota in shaping the physiological homeostasis of so many systems also means that any disturbance within the gut microbial balance can cause disorders within systems seemingly unrelated to the digestive system (163). Studies indicate that imbalances in the gut microbiota can directly or indirectly lead to numerous pathologies, including depression, hypertension, atherosclerosis, rheumatoid arthritis and even cancer (164, 165). By affecting lipid metabolism and insulin sensitivity, it can also contribute to the development of conditions like diabetes and obesity – one of the main risk factors in the immunopathogenesis of EC (166). Obesity has been identified as a significant risk factor for the development of endometrial cancer through several biological mechanisms. These mechanisms include chronic hyperinsulinemia, elevated insulin-like growth factor (IGF) levels, and increased estrogen production (96). Hyperinsulinemia reduces the levels of IGF-binding proteins (IGFB1 and IGFB2), leading to higher free IGF-1 bioavailability, which in turn promotes mitogenesis and inhibits apoptosis in endometrial cells. Additionally, increased estradiol levels not only stimulate endometrial cell proliferation and prevent cell death but can also enhance local IGF-1 production in the endometrial tissue (167). Moreover, hyperinsulinemia can exacerbate tumorigenesis in estrogen-sensitive tissues by decreasing the concentration of sex hormone-binding globulin, which increases the bioavailability of estrogens (168). Estrogen therapy, which is commonly used to manage menopausal symptoms, has been associated with an increased risk of endometrial cancer due to its stimulatory effects on the endometrial lining (169). Recent research also suggests that the gut microbiota may play an indirect role in endometrial cancer development, possibly by influencing metabolic and immune pathways that interact with hormonal regulation and tumorigenesis (170).

The gut microbiome, particularly a subset of microbial genes known as the estrobolome, plays a pivotal role in regulating estrogen metabolism. The estrobolome consists of microbial genes, products of which are capable of metabolizing estrogens, primarily through the enzymatic action of β-glucuronidase. Estrogens, predominantly produced by the ovaries and adipose tissue, circulate in the body, mostly in a conjugated form. In this state, they are bound to glucuronic acid or sulfate, which renders them inactive and unable to bind to estrogen receptors. However, the action of microbial enzymes, specifically β-glucuronidase, deconjugates these estrogens, releasing the free, bioactive form capable of binding to estrogen receptors located throughout various tissues, including the endometrium (157). This enzymatic activity is crucial because only the unbound, free estrogens are biologically active and capable of exerting their physiological effects on cells.

In the gut, the microbiome influences the bioavailability of estrogen by modulating the process of conjugation and deconjugation. The estrobolome is, therefore, a key player in determining the balance of active estrogen in the body, with direct implications for hormonal regulation (161, 171). When the gut microbiome is in balance, the production of β-glucuronidase is tightly controlled, leading to a healthy level of active estrogen. However, dysbiosis can lead to an overproduction of β-glucuronidase, which increases the conversion of conjugated estrogens into their free, active forms. This shift can result in elevated levels of estrogen in circulation, which may stimulate excessive cell proliferation and inhibit apoptosis processes (144). In the context of endometrial cancer, this microbial-driven estrogenic activity is significant.

Increased estrogen levels, whether due to endogenous sources like adipose tissue in obesity or hormonal therapies, can lead to endometrial hyperplasia, a precursor to endometrial carcinoma (172). The role of the estrobolome in controlling the availability of bioactive estrogen underscores the intricate connection between the gut microbiome and the risk of developing hormone-related cancers. Disruption in the estrobolome’s activity can create an environment in which estrogen levels are abnormally high, promoting the initiation and progression of endometrial cancer (161, 162). Furthermore, the estrobolome may also influence the broader gut-vaginal microbiome axis, with changes in microbial composition in the gut potentially altering the vaginal microbiota and influencing the local estrogen metabolism in the reproductive tract (173). This interconnected microbial environment can synergistically affect the production of pro-inflammatory cytokines such as IL-6 and TNF-α, which in turn upregulate enzymes involved in estrogen synthesis, creating a feedback loop that sustains elevated estrogen levels. Such changes in estrogen homeostasis are a key factor in the development and progression of endometrial cancer (114). Indeed, study by Beratis et al. indicated that the increase in β-glucuronidase titer, as well as its enzymatic activity, corresponded to the process of clinical deterioration in patients with gynecologic cancer or pelvic inflammatory disease compared to the healthy control group (174). In another study, using Mendel randomization analysis, specific taxa of gut bacteria were shown to be significantly associated with the risk of developing EC. Strains belonging to the Erysipelotrichaceae and Bifidobacteriaceae families, among others, known to produce enzymes such as β-glucuronidase, correlated with increased EC risk, which may be related to their involvement in estrogen reactivation. These findings suggest that the gut microbiota - through enzymatic activity affecting hormonal metabolism - may play an important role in the pathogenesis of endometrial cancer (175). Another study showed that endometrial dysbiosis, characterized by reduced Lactobacillus abundance and increased β-glucuronidase activity, is associated with enhanced estrogen receptor ERβ expression and increased levels of pro-inflammatory cytokines. Since β-glucuronidase is involved in estrogen reactivation, its excessive activity may promote inflammation and endocrine signaling disruption in the endometrium. These findings suggest that disruption of the microbiota and estrobolome function may interact in the pathogenesis of estrogen-dependent diseases, including potentially EC (176). Therefore, understanding the role of the estrobolome and its influence on estrogen metabolism provides important insights into the pathogenesis of cancer and highlights potential therapeutic avenues that target the microbiome to modulate estrogen levels and reduce cancer risk.