The role of dysbiosis in shaping host immunity in endometrial cancer development.

For almost a century, due to the work of Henry Tissier from 1900, the female upper reproductive tract (URT) was considered sterile ( 1 ). The presence of bacteria in this area has been linked to pathologies and the development of disease states. In terms of sterility, the FRT was divided into three sectors, the non-sterile vagina, the cervical mucus plug, which provides a barrier to microorganisms, and the sterile endometrium and fallopian tubes ( 2 ). That was until the second half of the 20th century, when the “sterile endometrium” theory was increasingly challenged by case reports of patients in whom the presence of microorganisms in the uterine cavity was confirmed using culture methods ( 3 – 15 ). Unfortunately, the majority of these studies were performed on the material obtained from swabs, which posed a significant risk of contamination. Moreover, studies based on culture methods did not reflect the biodiversity of microorganisms inhabiting the uterine cavity, as only 1% of bacteria residing in the human body can grow on synthetic media ( 16 ). The development of modern sequencing techniques, including those based on metagenomic analyses, allowed the identification of tested material to be carried out with greater efficiency, less expense and higher precision than in the case of cultivation methods ( 17 ). It is becoming clear that each part of the URT harbors its unique microbiome. Microbial balance within the female URT is a key element of the symbiotic relationship between microorganisms and the host. Microbial homeostasis is guarded by the immune system, which maintains a certain limit of invasion of commensal microorganisms and eliminates pathogens. Interactions between immune cells, cytokines and hormones are important factors enabling the maintenance of mucosal homeostasis. However, when the microbial load is too high or rich in representatives of certain taxa, excessive tissue destruction and immune stimulation may occur, leading to an imbalance of homeostatic relationships between bacteria. It is not known whether dysbiosis is a cause or effect of the disease, but disorders within the composition of the endometrial microbiome are associated with the occurrence of abnormalities in the physiology of the FRT ( 18 ). Dysbiotic alterations within the female URT microbiome represent a bacterial factor that is increasingly considered in the context of a potential risk factor for cancerous lesions. Disruption of microbial homeostasis can cause chronic inflammation, leading to a variety of immunopathologies and tumor-inducing diseases. Microbes can also promote the destabilization of the host genome by releasing toxins that can damage its genetic material. The presence of certain bacterial species can contribute to altering the expression of genes encoding proteins involved in the inflammatory response, proliferation, apoptosis, and modification of the secretion of inflammatory factors. Consequently, these events can ultimately disrupt physiological processes and promote the development of disease states, including cancer ( 19 , 20 ).

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. Findings indicating the potential role of pathogenic bacteria in the development of endometrial cancer over the past 10 years. Created in BioRender. Wierzbińska, W. (2025) https://BioRender.com/fqwiu4m . 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. The comparison of the methodologies and key findings in studies of EC microbiota. 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.

Considering current research, the growing body of evidence suggests that microbial dysbiosis, both in the endometrial environment and in the gut, plays a critical role in the immunopathology of endometrial cancer. An excessive load of pathogenic microorganisms can trigger a cascade of inflammatory responses due to the activation of Toll-like receptors and the subsequent production of proinflammatory cytokines like IL-6 and IL-8. This persistent inflammation contributes to an environment conducive to tumor progression, characterized by angiogenesis, oxidative stress, and DNA damage, which ultimately supports carcinogenesis. The upregulation of TLRs, NF-κB, and cyclooxygenase enzymes further highlights the importance of chronic inflammation in EC pathogenesis. Moreover, the gut microbiome’s impact on systemic inflammation and estrogen metabolism, particularly through the estrobolome, is a key link to EC development. Dysbiosis in the gut can disrupt the balance of microbial enzymes, such as β-glucuronidase, leading to the increased bioavailability of active estrogens. This hormonal imbalance, in combination with obesity-related factors, can drive endometrial hyperplasia and promote the initiation of cancer. The interaction between gut-derived metabolites, immune signaling, and estrogen regulation underscores the interconnectedness of the microbiota in shaping both immune responses and hormonal homeostasis, key factors in EC risk. All these arguments strongly support the importance of dysbiosis in the immunopathogenesis of EC. However, the available literature data on the role of EC microbiota in tumor initiation and progression features a wide variety of research protocols used and consequently, results obtained. Standardization of the research protocols would allow studies to be conducted with greater replicability and accuracy, which is crucial for understanding the role of microorganisms in the pathogenesis and progression of EC. More research is needed not only to identify the molecular basis of microbial activity in this niche but also to understand the mechanisms that regulate it. Restoring microbial balance in both the reproductive tract and the gut may provide a promising approach to modulate the inflammatory and hormonal environments that drive EC. Targeting microbial dysbiosis through diet, probiotics, or other microbiome-directed therapies could offer new avenues for the prevention and treatment of this hormonally driven malignancy. Further research into the specific microbial species involved, their mechanisms of action, and their influence on host metabolism and immune responses will be essential to developing such interventions. New possibilities and strategies for EC prevention and therapy support, as well as new diagnostic techniques, may also result from this knowledge.

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 ). The origin of endometrial microbiota. The diagram illustrates the potential sources of microorganisms colonizing the endometrial environment, highlighting the role of the anatomical, mechanical and haematogenic factors. 1 – Dysfunction of uterine peristaltic activity can promote colonization through enhanced transport caused by increased frequency of contractions. 2 – Microorganisms entering from the vagina. 3 – The cervical mucus suppository limits but does not completely block the passage of microorganisms from the vagina 4 – The peristaltic pump supports the transport of sperm cells from the cervix, which may be accompanied by a movement of other factors, including bacteria. 5 – Endometrium can also be colonized by microorganisms spread by blood that originate from the oral cavity (A) , respiratory tract (B) or gastrointestinal tract (C) . Created in BioRender. Wierzbińska, W (2025). https://BioRender.com/xmrvrud . 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 ). Endometrial immune cells and their role in sustaining immunological homeostasis. The interaction between these cells plays a crucial role in maintaining a balanced immune environment, which is essential for both fertility and successful pregnancy. • Neutrophils secrete IL-1-9, which contributes to early inflammation and defense against pathogens while also influencing the recruitment of other immune cells to the site of infection or tissue injury. • Mast Cells: secrete trypsin and chymotrypsin, enzymes that participate in tissue remodeling and inflammatory responses. Their role is particularly important in regulating vascular permeability and mediating allergic or immune responses within the endometrium. • B cells within the endometrial immune microenvironment secrete IL-6, IL-10, IL-17, and GM-CSF. These cytokines play diverse roles, including modulating local immune responses, regulating inflammation, and supporting the survival and function of other immune cells, such as macrophages and T cells. • uNK cells are central to the endometrial immune response, secreting a variety of factors, including VEGF, PEGF, angiopoietin, TNF-α, IFN-γ, and GM-CSF. These cytokines and growth factors are crucial for angiogenesis, tissue remodeling, and activating other immune cells like macrophages and CD8+ T cells. • CD8+ T Cells secrete IFN-γ, TNF-α, perforins, granzymes, IL-4 and IL-12, participating in immune surveillance and destroying infected or altered cells. Their activation is modulated by uNK cells and other immune cells, contributing to the balance between defense and immune tolerance. • Macrophages of the endometrium secrete IL-1, IL-10, IL-15, prostaglandins, MCP-1 and IL-1β, which influence local inflammation, tissue repair, and the recruitment of other immune cells. Macrophages also act as key regulators of the immune response, being able to activate endothelial cells to produce HBD-2, an antimicrobial peptide that further contributes to local immune defense. • DCs expressing MHC-II play a crucial role in antigen presentation and the initiation of adaptive immune responses. Their interaction with T cells helps to shape the endometrial immune landscape, promoting tolerance or active immune defense depending on the tissue requirements. • CD4+ T cells grouped into four subtypes are pivotal in regulating immune responses: o Treg cells are induced when TGF-β levels in the environment are high; they secrete IL-10, IL-35 and TGF-β, ensuring immune tolerance and preventing excessive inflammation. ○ TH17 are being activated when TGF-β levels are low; TH17 cells secrete IL-17, playing a role in pro-inflammatory responses and the recruitment of neutrophils and other immune cells. ○ Th1 secrete IFN-γ, IL-2, TGF-α, and TGF-β. These cells are involved in promoting immune defense and activating macrophages to clear pathogens or damaged cells. ○ Th2 secrete IL-13, IL-10, and IL-4, promoting tissue repair and helping to modulate the immune response to avoid excessive inflammation, contributing to the maintenance of homeostasis. Created in BioRender. Wierzbińska, W. (2025) https://BioRender.com/4xl6gb7 . Immune cells crucial to function of endometrium. The most abundant immune cells residing in the endometrium are the uterine natural killer cells (uNK cells). The number of cells in the endometrium undergoes changes related to the menstrual cycle, increasing during the secretory phase. Also, the quantity of uNK cells rises in early pregnancy, representing up to 70% of all hematopoietic cells and gradually decreases in the middle and late gestation ( 46 , 47 ). uNK cells are distinguished by lower cytotoxicity, fewer receptors responsible for activation and an increased number of inhibitory receptors compared to peripheral blood NK (pNK) cells, which may be related to the role of these cells in successful implantation ( 48 ). Moreover, the array of cytokines secreted by uNK cells differs from the mediators secreted by pNK. In addition to interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), or granulocyte-macrophage colony-stimulating factor (GM-CSF), uNK are also capable of secreting proangiogenic factors like vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and angiopoietin 2 ( 49 , 50 ). In addition, uNK cells can modulate not only local inflammatory responses by producing inflammatory mediators but also induce and activate macrophages and cytotoxic T cells ( 51 ). Unfortunately, there is a lack of studies that clearly indicate what effect menopause has on the number and phenotype of uNK cells, however, the number of pNK cells decreases with age, which may suggest a similar relationship for cells in the uterine cavity ( 52 ). Given the diverse results of previous studies, the effect of age on NK cell cytotoxicity also remains unclear ( 53 – 55 ). Macrophages and dendritic cells (DCs) represent the major antigen-presenting cells (APCs) in the uterine environment, accounting for about 10% of all endometrial immune cells. APCs are one of the key elements activating the inflammatory response by the bacterial molecular pattern and presentation of their antigens to T lymphocytes ( 56 ). Both macrophages and uDCs can be activated by detecting pathogens in the endometrium and starting the process of phagocytosis, internalization, and degradation of the antigen components. They present bacterial peptides to T-cells via major histocompatibility complex (MHC) receptors, which activate the T-cells to initiate a cell-mediated and/or humoral immune response via MHC class II molecules ( 57 ). DCs are the most effective antigen-presenting cells involved in the adaptive immune response activation and mucosal surface immune response regulation. Like macrophages, they are capable of phagocytosis, and thanks to lysosomal enzymes in phagosomes and lysozymes, they can destroy ingested pathogens ( 58 ). Macrophages are the most abundant group of endometrial APCs. Their physiological role in the endometrium is associated with tissue clearance and remodeling ( 59 ). Moreover, together with uNK cells, macrophages are the major cytokine producers in the endometrium. They secrete IL-10, IL-15, IL-1, prostaglandin (PG), and monocyte chemoattractant protein-1 (MCP-1), which induce immunosuppression, differentiation and recruitment of other immune cells ( 60 ). Macrophages also have outstanding phagocytic abilities, enabling them to recognize foreign antigens. Bacterial lipopolysaccharide (LPS) stimulates IL-1b secretion by macrophages, which results in the secretion of human beta-defensin-2 (HBD-2) by cells of the endometrial epithelium to inhibit pathogen invasion ( 61 ). Macrophages also play an important role in the early stages of pregnancy. After fertilization, their number increases rapidly and decreases around the second trimester. In addition to providing the fetus with protection from infection, macrophages are involved in spiral artery remodeling, trophoblast invasion, and implantation ( 50 ). Neutrophils’ presence in the endometrium increases innate immune defense thanks to the secretory capacity of IL 1-9, phagocytosis, production of oxidative compounds and release of antimicrobial peptides ( 62 ). The main role of endometrial mast cells is related to biological defense mechanisms by releasing proteases such as trypsin and chymotrypsin to activate inflammation and the immune response ( 63 ). In a healthy pre-menopausal endometrium, B cells are relatively rare, accounting for only 1% of immune cells. They participate in immune regulation by secreting a wide range of cytokines, including IL-6, IL-10, IL-17, and GM-CSF, and they can differentiate into plasma cells when stimulated by antigens ( 64 ). The main representatives of the adaptive immune response and the third most abundant endometrial immune cells are T cells. Uterine T cells consist of CD8+ (cytotoxic) cells, which make up 66% of this population, and CD4+ (helper) cells, which make up 33% of it. CD4+ T cells population is divided into Th1 cells (5% - 30%), Th2 cells (5%), Tregs (5%) and Th17 cells (2%) ( 65 ). The main role of cytotoxic T cells is to participate in the apoptosis of the target cell. CD8+ T cells can directly initiate apoptosis by secreting cytotoxic perforin proteins and granzymes or by integrating the FasL-CD95 ligand (FasR), resulting in the initiation of apoptosis through the activation of caspase proteases in the intruder cell. In addition to participating in indirect apoptosis, CD8+ T cells can kill target cells by releasing cytokines such as TNF-α, IFN-γ, IL-4, and IL-12, which activate other immune cells in addition to affecting the target cell ( 66 ). During menstruation, endometrial CD8+ T cells can maintain cytolytic activity during the proliferative phase, but activity wanes during the secretory phase ( 67 ). In contrast, during early pregnancy, to maintain immune tolerance to fetal antigens and prevent infection, cells under the influence of hormones reduce the expression level of cytotoxic molecules ( 68 ). CD4+ T cells represent a less abundant, however important, group of cells involved in the production of immune mediators and interaction with other immune cells upon activation by APCs. Th1 cells are responsible for the secretion of IFN‐γ, TNF‐β, IL‐2 and macrophage activation. Moreover, they also produce TNF-a, which is involved in promoting inflammation. Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13, which are mostly involved in humoral immunity and extracellular pathogen infection ( 69 ). Th1 and Th2 cells mutually inhibit each other. Th2s are responsible for secretion of IL-10, a cytokine which, by influencing APCs, inhibits the development of Th1. In turn, Th1 cells produce IFN‐γ, which prevents Th2 cells from being activated ( 70 ). T17 cells are responsible for the secretion of the pro-inflammatory cytokine IL-17. Like Treg cells, they arise from naive CD4+ cells under the influence of TGF-β. Depending on the concentration of cytokine in the microenvironment, naive CD4+ cells differentiate into T17 cells when cytokine concentration is low or Treg cells when cytokine concentration is high ( 71 ). The main role of Treg cells is to reduce inflammation, crucial for the phenomenon of immune tolerance. Immunosuppression occurs when the TCR complex on the surface of Treg cells recognizes the peptide MHC class II (MHCII) on the surface of APC cells, resulting in Treg activation and an increase in their suppressive function. Treg cells express their function by secreting a wide range of immunosuppressive cytokines, including IL-10, IL-35 and TGF-β. In addition, Treg cells have the ability to uptake pro-inflammatory IL-2 from the environment due to high expression of the IL-2 surface receptor ( 72 ). 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 ). Hormonal fluctuations during the menstrual cycle. The graph illustrates the dynamic changes in the level of key reproductive hormones across a typical ovulatory menstrual cycle. Created in BioRender. Kuźmycz, O. and Wierzbińska, W. (2025) https://BioRender.com/es82lve . 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 ).