Eutopic Endometrium Immune Changes Involved in Development and Progression of Endometriosis: A Review

Abstract

Numerous abnormalities of the endometriosis eutopic endometrium contribute to the initiation and development of ectopic lesions. It is also believed that among the complex causes of the disease, systemic immunological disorders play a significant role. Therefore, this literature review aims to summarize the current knowledge on immunological alterations in the endometriosis eutopic endometrium and the impact of these changes on the progression of this disease. The reviewed studies mostly indicated a pro-inflammatory immunological profile within this tissue. This was evidenced by a predominance of M1 macrophages, which have a pro-inflammatory character and elevated levels of pro-inflammatory cytokines such as interleukin-1 (IL-1) or IL-6. Additionally, an increased number of cytotoxic T lymphocytes and a positive correlation between B lymphocyte levels and the presence of endometriosis have been observed. Some changes in T cells and natural killer (NK) cells receptors, which possibly determine endometriosis development, have been described. Several studies have also revealed that patients with endometriosis exhibit reduced presence of dendritic cells in the eutopic endometrium of affected individuals, which may impair uterine cavity clearance during menstruation and contribute to ectopic lesion formation. In summary, current data indicate a pivotal role of the endometrial immune environment in disease progression, but further research is needed to drive development of immunological treatment in endometriosis management.

Introduction

Endometriosis is a common chronic gynecological condition with endometrium-like tissue outside the uterine cavity. Based on the location of the lesions, several types of this disease, including superficial peritoneal endometriosis, ovarian endometriomas, and deep infiltrating endometriosis (DIE), can be distinguished [1–3]. The most common endometriosis symptoms are painful menstruation, severe dyspareunia, and chronic lower-abdominal pain, but endometriosis is also strongly linked to infertility, with a risk twice as high as that in healthy women [4,5].

Despite such a high prevalence of the disease and its bothersome symptoms, the precise pathogenesis of endometriosis has still not been fully elucidated, and several theories have attempted to explain its occurrence. Among them, great importance has been attached to the involvement of immune dysregulation in disease pathogenesis [6]. It involves the ectopic endometrial cells infiltration, which takes place because of retrograde menstruation, which is not the exclusive cause of endometriosis development, and immune alterations play an important role in the development of pathological lesions. Briefly, ectopic endometriotic cells, through the induction of inflammation, change the nature of the cellular infiltration, which further leads to the disease development [7].

The observed systemic immune-based abnormalities in endometriosis have been widely described as an important force participating in disease pathogenesis. An increase in the number of peritoneal neutrophils and macrophages, a decrease in the cytotoxic function of NK cells, and abnormalities in the number of T and B lymphocytes are characteristic of endometriosis patients [8]. Importantly, the immune profile is not homogenous for all tissues and fluids, as some immune cells display different tissue-specific properties [9]. Therefore, it can be hypothesized that the endometriosis eutopic endometrium is also a source of some immune alterations that are unique to this environment, determining the initiation and progression of the disease.

Considering the heterogeneity of endometriosis, further exploration of the immune imbalance of the uterine cavity endometrium may shed new light on the disease development and progression. Knowledge about the nature of immune changes provides insight into the background of the disease and may help in the development of immunotherapy [10]. Thus, our aim was to discuss the impact of the immune environment of the normal endometrium on the formation and evolution of ectopic endometriotic implants.

Abnormal Functioning of Eutopic Endometrium and the Progression of Endometriosis

Despite the seemingly similar cell structure and functioning of the eutopic endometrium in women with and without endometriosis, several biochemical, hormonal, and genetic changes between these groups can be observed [11,12]. Although researchers do not completely agree, some have suggested the affected endometrium has an altered profile of enzymes regulating hormonal balance [13,14]. Several studies found higher levels of aromatase, a crucial enzyme regulating estrogen biosynthesis, in the eutopic endometrium of patients with endometriosis compared to healthy controls [13,15]. Anormal aromatase expression leads to the local production of estrogens, stimulating the growth and maintenance of endometriotic lesions [16]. Estrogen production potentiates inflammation, affecting increased prostaglandin synthesis through COX-2 stimulation and increasing estrogen production, which allows endometrial cells to survive in ectopic sites. Therefore, the inflammatory environment of the endometrium of the uterine cavity may be both a cause and a consequence of increased local estrogen production [17].

The imbalance in the microbiota of the eutopic endometrium and the alterations within the microbiota-gut-reproductive tract axis are other relevant characteristics of endometriosis. Decreased Lactobacillus spp. dominance and increased abundance of pathogenic microorganisms were observed in the normal endometrium of women with endometriosis. When such a microbial shift occurs, the production of pro-inflammatory mediators, including TNF-alpha, increases. As a result, an excessive inflammatory response can damage the eutopic endometrium [18]. Moreover, analogous changes observed in the gut microbiota composition cause an increase in β-glucuronidase secretion, which leads to an increase in estrogen concentrations in the eutopic endometrium and further stimulates endometrial lesion growth [18–20]. The literature also suggests that endometriosis endometria share common characteristics with the endometria of women infected with Chlamydia trachomatis. A similar upregulation profile of effector memory CD8+ T cells in the endometrium of women with Chlamydia-induced endometritis and early-stage endometriosis, as well as suppression of genes responsible for DNA repair and cell cycle regulation, was observed [21].

The chronic inflammation present in endometriosis is also associated with oxidative imbalance [22]. According to Ngô et al, stromal endometrial cells from patients with endometriosis were characterized by enhanced production of superoxide anions when compared to stromal endometrial cells from healthy controls. Moreover, catalase activity, one of the antioxidant enzymes, tended to show an opposite trend. Thus, these changes suggested an aggravated pro-inflammatory milieu in affected women [23]. The main changes in the eutopic endometrium in endometriosis are illustrated in Figure 1.

Figure 1.

Summary of the main changes characteristic of the eutopic endometrium in endometriosis.

Immune Cells in the Eutopic Endometrium

Macrophages

Macrophages are immune cells that participate in the innate and humoral immune response. Their primary functions are phagocytosis and the presentation of antigens to T lymphocytes. They are also involved in tissue remodeling and regeneration. The environment directly affects the phenotype and function of these cells [24,25].

Macrophages can be divided into the M1 group (classically activated macrophages) and the M2 group (alternatively activated macrophages). The former subpopulation has bactericidal and pro-inflammatory effects, while the latter presents anti-inflammatory effects and stimulates angiogenesis and tissue regeneration [26,27].

In the endometrium of healthy women, the number of macrophages varies depending on the menstrual cycle phase, and increases significantly during the perimenstrual period. In contrast, the level of macrophages remains constant in the normal endometrium of diseased patients, which impedes its exfoliation and contributes to the formation of new lesions through the free migration of exfoliated cells into the peritoneal cavity [28].

Although there are no changes in macrophage expression dependent on the cycle phase, several changes in the distribution and functioning of macrophages among patients with endometriosis occur. Firstly, several researchers reported that in the endometriosis eutopic endometrium, there was an imbalance of macrophage subtypes and the predominance of pro-inflammatory M1 and the depletion of M2 was observed [29–31]. Analogously, in a study by Zhu et al, a higher inflammation score for M1 was revealed in the endometrium obtained from individuals with endometriosis [32]. These results indicated that due to the predominance of this phenotype of macrophages, the endometrial cells, via the activation of NF-κB, STAT1, STAT3, or IRF4 pathways, had a greater potential to activate Th1 cells and secrete pro-inflammatory cytokines such as TNF-α, IL1-β, IL-6, IL-12, and IL-23 [27].

According to Poli-Neto et al, the number of macrophages in the eutopic endometrium varies depending on the endometriosis stage. While in the I and II stages of the disease, the endometrium was dominated by M1 macrophages, in stages III and IV, it was dominated by M2 macrophages. In addition to inhibiting the immune response and stimulating angiogenesis, M2 can suppress NK cell action. Such activity promotes immune imbalance, resulting in the formation of endometrial lesions. However, in that study, there was no validation in clinical samples [33].

However, the division of macrophages in endometriosis into anti-inflammatory and pro-inflammatory subsets is not entirely clear. Vallvé-Juanico et al and Ma et al suggested that in endometriosis patients, M2 can develop M1 profile characteristics [29,34]. Vallvé-Juanico et al found that M2 macrophages in eutopic endometrium tended to have phenotype characteristics for M1 [29]. Interestingly, Ma et al found that M2 in endometriosis showed expression of CD40 and CD64, which provided the pro-inflammatory character of these cells, suggesting that M2 in endometrial lesions presents features characterizing pro-inflammatory macrophage subsets via the expression of these molecules [34]. Taken together, these data support the study of the pro-inflammatory character of immune cells and indicate the possibility of a phenotypic switch between macrophage phenotypes; however, they need confirmation in larger study groups.

Xie et al, in their experimental study, focused on other receptors on the surface of macrophages. They found that human acute monocytic leukemia (THP-1) cell-derived macrophages incubated with endometriosis eutopic endometrium and with normal endometrium differed in the subsequent expression of molecules participating in phagocytosis. It was noticed that macrophages treated with eutopic endometrium tended to express higher levels of SIRP-alpha and lower levels of CD36. SIRP-alpha negatively affects signaling molecules, which regulate phagocyte function. It binds CD47, which initiates processes that suppress phagocytosis, so upregulation of this signaling leads to ineffective immune surveillance. In contrast, CD36 mediates phagocytosis; thus, blocking its expression impairs the phagocytic capacity of macrophages. Therefore, an increase in SIRP-alpha expression combined with a decrease in CD36 expression results in ineffective phagocytosis, leading to impaired clearance of ectopic endometrial debris [35].

Jiang et al also focused on the relationship between macrophages and these molecules in endometriosis [36]. They evaluated the effect of macrophage incubation with exosomes derived from the uterine aspirate fluid [36], containing, among others, endometrial cells [37]. Similarly, it was observed that exosomes derived from the endometria of endometriosis patients decreased the expression of CD36, but there was no effect on SIRP-alpha. Surprisingly, such incubation with exosomes caused lower expression of CD80, which is a marker of M1 macrophages. The last result may be confusing, taking into consideration the studies discussed above, but the reason for it may be the limitation of the study group to women with III and IV stages of endometriosis. Moreover, the researchers suggested that downregulated levels of M1 may also be related to endometriosis progression through enhanced escape from immune surveillance [36].

Another issue raised in the literature was the effect of substances secreted by macrophages on the development of endometriosis. Huo et al concluded that macrophages can influence the development of endometriosis by inducing the secretion of certain substances. They found that human endometrial stromal cells (HESC) exposed to medium containing macrophages exhibited increased nicotinamide Nmethyltransferase (NNMT), which promotes epithelial–mesenchymal transition (EMT) and migration through induction of TGF-β1 expression, and also showed that the eutopic endometrium contains more NNMT than the endometrium of healthy women, suggesting the special involvement of this mechanism in endometriosis development [38]. When analyzing the role of macrophages in the development and progression of endometriosis, the impact of inflammatory mediators, cytokines, and chemokines produced by these cells should not be overlooked.

Xie et al proved that interleukin-6 (IL-6), a pro-inflammatory cytokine produced by macrophages, is found in higher concentrations in the uterine cavity endometrium of women with endometriosis than in healthy individuals, and its action induced vascular formation in the early stages of endometriosis [35].

Shao et al, in their in vitro model of endometriosis eutopic endometrium, found that macrophages downregulated interleukin-24 (IL-24) expression. Such action enhanced endometriosis development by increasing the proliferation and invasiveness of endometrial cells. It has also been shown that low levels of IL-24 induced by macrophages increased the expression of COX-2 [39]. Considering that COX-2 is responsible for the expression of PGE2, which can inhibit the phagocytosis capacity of macrophages, such action may contribute to the formation of new endometrial implants [40]. A key role in this signaling axis is played by the NF-κB and JAK/STAT transcriptional pathways, which act synergistically [41,42]. Interestingly, PGE2 initiates a feedback loop through the PI3K/AKT pathway, leading to reactivation of STAT3 and NF-κB [41,42]. Runx2 and Tfap2c are the main genes dependent on JAK/STAT pathways whose expression initiates an effect on macrophage function [43].

Regarding the activity of cytokines, it is also crucial to highlight the role of factors that regulate macrophage activation in the pathophysiology of endometriosis. According to a report by McLaren et al, patients with endometriosis have reduced endometrial levels of interleukin-13 (IL-13), which is a macrophage-inhibitory factor. Its lower levels can result in insufficient macrophage suppression and increased macrophage activity. Levels of another macrophage-inhibitory cytokine, IL-10, have also been investigated, but no differences in concentrations in healthy women and those with endometriosis have been shown [44]. The main macrophage changes in endometriosis are shown in Figure 2.

Figure 2.

Main macrophage changes characteristic of eutopic endometrium in endometriosis.

T Cytotoxic (Tc) Cells

CD8(+) cytotoxic T (Tc) cells play a pivotal role in the destruction of pathological cells and various pathogens [45]. A growing body of evidence suggests that CD8+ T cells are susceptible to hormonal regulation, and while estrogens enhance the inflammatory profile of this cell population, progesterone has the opposite effect [46]. Given the significant impact of these hormones on the development of endometriosis, the role of Tc cells in this disease seems particularly crucial [47].

Several studies found that normal endometrium in patients with endometriosis was characterized by higher levels of CD8+ T cells compared to endometria obtained from healthy individuals [31,48–52]. These findings may correspond with increased inflammation in patients with endometriosis, which enhances disease development. Intensified inflammation induced by CD8+ T cells is assumed to be mainly mediated by activation of the JAK/STAT1 pathway. The targets for this pathway are genes such as Mx1, Mx2, and Ifit3, which modulate the occurrence of inflammatory reactions [43,53]. The involvement of this pathway in the initiation of inflammation provides an opportunity for research on the use of JAK inhibitors in the treatment of endometriosis [54]. However, given the cytotoxic properties of Tc cells, it seems that eutopic endometrial cells, which can form ectopic endometriosis foci, are not recognized by these types of immune cells. Such an increase in the number of CD8+ T cells probably can also induce changes in the infiltration of other immune cells, as the tendency toward more CD8+ T cells causes a local increase in the number of macrophages [55].

Intriguing results were shown by Schmitz et al, who found that CD8+ T cells obtained from the menstrual effluent of endometriosis patients expressed less perforin than in the menstrual effluent from healthy women [56]. Hormonal regulation of perforin may underlie these results, as perforins have been shown to be suppressed under the influence of estrogens [46]. Because perforin expression initiates apoptosis by Tc cells, these findings suggest that in women with endometriosis, lymphocyte cytotoxicity may be impaired in this mechanism [57,58]. Moreover, Tc cells with perforin deficiency display weakened cytotoxicity toward macrophages; therefore, this change in Tc cells may affect these immune cells [59,60].

T Helper (Th) Cells

CD4+ T helper (Th) cells can be subdivided into several subsets, including Th1, Th2, and Th17, based on their ability to express different cytokines. Thus, different subtypes of these cells also play divergent roles in the immune system [61]. Importantly for endometriosis, Th cell subpopulations are susceptible to estrogen and progesterone regulation, and both these hormones promote a shift toward Th2 dominance [62].

When analyzing the role of Th cells in endometriosis, most research teams have focused on the Th17 cell subset (Table 1). These cells fight infections and are critical in initiating autoimmune diseases [63]. Nevertheless, the obtained results regarding Th17 cells in endometriosis are inconsistent. Miller et al found that menstrual effluent from patients with endometriosis has lower levels of Th17 cells [64]. In contrast, Zhan et al, in their most recent study, noticed that the level of Th17 cells was elevated in the endometriosis endometrium compared to healthy individuals [50]. These contrasting results may be due to the methodology used, as Miller et al used a flow cytometry technique, while Zhan et al studied gene sets [50,64].

Table 1.

Summary of the changes in Th17 cells expression in the eutopic endometrium in endometriosis.

Study	Studied material	Method of evaluation	Main results
Zhan et al 2025 [50]	Endometrial samples	Th17 gene sets evaluation	– A higher level of Th17 cells in patients with endometriosis compared to the healthy group
Miller et al 2022 [64]	Menstrual effluent	flow cytometry	– Lower level of Th17 cells in the menstrual effluent obtained from women with endometriosis compared to healthy controls
Le et al 2021 [70]	Endometrial samples	RT-PCR IHC	– Higher RORγt transcript levels in eutopic endometrium from patients with endometriosis compared to endometrial tissue from control patients – Lower levels of eutopic endometrial localization of RORγt positive cells in patients with endometriosis compared to controls (in the absence of HT)
Sisnett et al 2024 [74]	Endometrial samples	RT-qPCR	– Enhanced expression of IL23R (receptor for IL-23) in the endometrium of women with endometriosis compared to healthy controls – Enhanced differentiation of primary CD4+ T cells into Th17 cells after their incubation with IL-23
Chen et al 2012 [75]	Endometrial samples	RT-qPCR IHC Western blot analysis	– Higher GATA-3 expression and decreased expression of T-bet in eutopic endometrium of endometriosis patient

IHC – immunohistochemistry; IL – interleukin; HT – hormonal therapy.

Nevertheless, it seems that the hypothesis of elevated Th17 levels is more plausible, as the main mechanism of action of Th17 cells is based on their secretion of cytokines, including IL-6 and TNF-alpha, which, via the STAT3/NF-KB pathway, enhance cell proliferation [65]. Proliferation dependent on these transcription factors is further regulated by Bcl2, Ier3, Fos, Jun, and Fosl2 [66]. Moreover, a shift toward Th17 cell predominance can strengthen the growth of the Treg population through the action of a TNF-α-TNFR2 signaling pathway [67]. The literature suggests that the action of this pathway is mediated by the K63-dependent ubiquitination and activation of the ubiquitin ligase cAIP1/2 [68].

In addition, this pathway regulates the endometrial receptivity and affects decidualization; thus, it can be hypothesized that this immune cell subset contributes to fertilization. In summary, Th17 cell-based therapy can be effectively targeted for endometriosis-related infertility [69].

A comprehensive analysis of RORγt, a Th17 cell transcription factor, was presented by Le and co-authors in the eutopic endometrium of endometriosis. In the first step, an evaluation of the RORγt transcript expression in the endometrium was performed. They observed the predominance of RORγt over Foxp3 in eutopic endometrium of women with endometriosis, thereby suggesting the pro-inflammatory nature of the tissue, which may exacerbate disease progression. Further, enhanced endometrial expression of RORγt was found in women with endometriosis compared to healthy women, and in untreated women with endometriosis compared to those supplemented with hormonal therapy. According to the authors, single-agent therapy with progesterone was used. It can be concluded that hormonal treatment reduced the expression of this transcription factor, which led to a profile characteristic of a healthy endometrium [70]. It can be hypothesized that progesterone suppresses the IL-6/p-STAT3 pathway, leading to reduced endometrial cell proliferation [71]. Under normal conditions, the expression of the following genes regulates proliferation occurring via this pathway: Bcl-xL, Mcl-1, Bcl-2, Fas, cyclin D1, survivin, c-Myc, VEGF, MMP-2, and MMP-9. It therefore appears that in this situation they are subject to hormonal regulation [72,73].

The observations made by Sisnett et al are also important in the context of Th17 cells. They noticed enhanced expression of IL23R in the endometriosis endometrium compared to healthy women. IL23R serves as a receptor for IL-23. They found that when primary CD4+ T cells were treated with IL-23, their differentiation into Th17 cells was enhanced. Thus, it can be hypothesized that in affected women, the endometrium created better conditions for increased inflammation [74].

Although Th1 and Th2 cells are important elements of the Th family, to the best of our knowledge, only 1 study has focused on the imbalance between these 2 cell subsets in endometriosis. qPCR analysis revealed that GATA-3 mRNA levels were higher in the endometria of women with endometriosis compared to those of healthy individuals. Additionally, an analysis performed by immunohistochemistry confirmed the tendency for GATA-3 expression and found decreased expression of T-bet in diseased eutopic endometrium. Thus, all these results point to the shift toward the Th2 cells in endometriosis, for which GATA-3 is a regulatory transcription factor [75]. Molecules secreted by Th2 cells, such as IL-4 and IL-10, play essential roles in immune regulation. These cytokines, via the STAT3 and STAT6 pathways, enhance polarization toward M2 cells [76]. Macrophage maturation is induced by the ubiquitination or phosphorylation of STAT6 [77].

Additionally, IL-4, through the STAT6 pathway, interacts locally to stimulate endothelial cell proliferation and migration, thereby promoting angiogenesis. These processes occur via activation of transcripts such as IGF-1, VEGF-A, and FGF-2 [78]. Moreover, reports suggest that STAT6-related genes, including zinc finger E-box-binding homeobox (Zeb)1 and Zeb2, play a role in epithelial–mesenchymal transition (EMT) [79], a crucial process in endometriosis development [80].

T Regulatory (Treg) Cells

T regulatory (Treg) cells, which are characterized by the expression of Foxp3, participate in the inhibition of inflammatory responses [81,82]. In normal endometrium, this subset of immune cells plays a pivotal role in maintaining pregnancy and induction of labor [83].

Two studies conducted on animal models have interestingly illustrated changes in eutopic Treg populations in response to endometriosis induction. Braundmeier et al noticed that endometriosis initiation in non-human primates resulted in a decline in the Treg endometrial population. Moreover, they observed that surgical excision of pathological lesions did not affect this trend, suggesting that surgical endometriosis treatment does not affect the Treg population in the endometrium [84]. Slightly different conclusions were formulated by Le et al, who noticed that Treg cells, together with Th17 cells, increased due to the initiation of endometriosis. Nevertheless, as the increase of Th17 cells was greater than the enhancement of Treg cells appearance, the eutopic endometrium seemed to have a more pro-inflammatory nature [85].

Results regarding the number of Tregs in normal endometrium in patients with endometriosis are rather divergent. In addition, different cell populations were evaluated, including total Tregs and activated Tregs. Most studies found that eutopic endometrium in endometriosis patients exhibited lower Treg levels when compared with healthy women [70,86,87]. Importantly, some research teams showed that such a relationship existed only for activated Tregs, thereby assigning special importance to this population of cells [88,89]. Lower levels of Tregs may be associated with inability to inhibit the excessive inflammation that occurs in endometriosis. These reports are also supported by a recent study describing the therapeutic nature of Treg cells transfer in endometriosis, showing that in mice, administration of Treg cells inhibited the development of endometriotic lesions, and that Treg-based therapy lowered the production of Th-dependent pro-inflammatory cytokines [90].

In contrast, several research teams found that endometriosis eutopic endometrium contained higher levels of Tregs in comparison to the endometrium of healthy individuals [34,75,91]. Moreover, the increased production of cytokines secreted by Tregs in the endometrium of patients with endometriosis, as well as increased expression of the molecule CD200, which is responsible for the promotion of Treg cells, suggests the increased activity of these cells in this group of patients [92,93]. It can be hypothesized that a higher number of Tregs in the endometrium of patients with endometriosis is an attempt to attenuate inflammation and inhibit endometriosis progression; however, further research is needed. This relationship may also be supported by the fact that estrogen deficiency has an inhibitory effect on the population of Tregs, so the reverse hormonal profile that is characteristic of endometriosis may increase this population of immune cells [94].

Hou et al found an interesting interaction between Treg cells and macrophages, probably further influencing disease progression. They observed that in patients with endometriosis, the eutopic endometrium was abundant in fibrinogen-like protein 2 (FGL2) and CD32B, a receptor for this molecule. Tregs under the influence of pro-inflammatory cytokines such as IL-6 and IFN-γ were the main producers of FGL2. It has been reported that macrophages treated with FGL2 tended to display more pro-repairing features, which can alleviate endometriosis progression [95]. Therefore, it is seen that the cells of the immune system work together, and it is difficult to analyze them separately.

B Cells

B cells, as a part of the immune system, are responsible for the production of antibodies and act as antigen-presenting cells (APC) [96]. Although B cells are a part of the normal endometrium, where they perform the functions discussed above, their proportion does not exceed 5% of all included immune cells [97]. Schmitz et al reported that despite the small population of these cells in the endometrium, in endometriosis patients, the percentage of cells in menstrual secretions was higher than the percentage of B cells derived from peripheral blood [56]. This suggests that in patients with endometriosis, B cells have a greater effect locally in the uterine cavity than peripherally.

Shih et al noticed a higher concentration of B cells in patients with chronic endometritis. As this state predisposes to the appearance of endometriosis, high levels of these immune cells may contribute not only to the development but also to the initiation of endometriosis [98].

Antsiferova et al and Peng et al presented results in agreement with the above-mentioned hypothesis and found that the presence of B cells in eutopic endometrium is positively correlated with endometriosis [99,100].

Interesting observations on the link between the progression of endometriosis and B lymphocytes in the endometrium were revealed by Xiang et al. Eighteen patients with stage III or IV endometriosis were included in the study. Twelve of the women were treated with surgery and 6 women were untreated. Then, in all patients, the eutopic endometrium was collected and B memory cell levels were determined. It was concluded that surgical treatment of endometriosis caused a decrease in B-cell memory in the endometrium [101]. These results support the hypothesis indicating interconnectedness between immune environments in the endometrium and ectopic lesions.

In addition to the number of B cells in the endometrium, according to Chung et al, lymphocyte B maturation abnormalities can occur in the endometrium of women with endometriosis. They found that the endometrium of endometriosis patients had higher expression of lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF) in comparison to normal endometrium [102]. This factor indirectly inhibits BCL6, which suppresses B-cell maturation [103,104]. Therefore, it can be concluded that in endometriosis patients, B-cell maturation is enhanced. Moreover, the increased amount of LITAF in women with endometriosis predisposes to a decrease in plasma cell differentiation [103]. Given that ectopic endometrial lesions have abundant infiltration of plasma cells, their reduced differentiation in the eutopic endometrium may be a defense mechanism against the disease [105].

In contrast, BCL6 has a protective effect against apoptosis. Thus, an increased amount of LITAF, which inhibits BCL6, can provoke cell death and provide another defense against the spread of endometriotic cells in endometriosis [106].

Natural Killer (NK) Cells

Natural killer (NK) cells participate in cytotoxic reactions through the direct initiation of the destruction of pathological cells, as well as the secretion of a wide range of pro-inflammatory cytokines [107,108]. Under normal conditions, the endometrium contains uterine NK (uNK) cells, which can be divided into 3 subgroups. uNK cells exhibit greater cytokine secretion capacity than cytotoxic properties, and their amount varies depending on the phase of the menstrual cycle [109,110].

Data on differences in the number of NK cells in the endometrium of diseased and healthy women are inconsistent. Some authors revealed similar endometrial NK cell concentrations in the endometrium of patients with endometriosis and healthy women, suggesting there is no significant participation in disease progression [34,111]. There are also reports suggesting that menstrual debris and endometrium from women with endometriosis contained fewer uNK cells, which may be responsible for lower cytotoxic properties of the tissue [98,112,113]. However, some authors observed that enhanced CD45+ NK cells and CD56+ NK cells expression in the endometrium corresponded with greater endometriosis risk [31,100,114]. Thiruchelvam et al reported that such a phenomenon may be a result of enhanced stem cell factor (SCF) production in women with endometriosis [115].

Some research teams have also investigated the role of receptors expressed on the NK cells’ surface in the development of endometriosis. Alimoradi Fard et al evaluated the presence of NK cells exhibiting the expression of a molecule providing their cytotoxic properties, named NKp46 [114]. This receptor mainly acts through the STAT1 and STAT3 signaling pathway [116]. It has been found that there was a lower expression of NKp46+ NK cells in the samples of eutopic endometrium from patients with advanced endometriosis compared to healthy individuals. Decreased levels of NKp46+ NK cells in patients with severe endometriosis probably correspond with their impaired cytotoxic ability, which may result in enhanced dissemination of endometrial cells [114]. Xu et al evaluated the ligands for NKG2D, which conditioned the cytotoxic properties of NK cells [117]. Activation of the MAPK pathway is responsible for such cytotoxic activity [118]. It was found that the expression of ULBP-2, a ligand for NKG2D, was lower in the eutopic endometrium of women with endometriosis compared to the endometrium of healthy individuals. It can result in increased tissue resistance to the cytotoxic effect of NK cells [117]. Both studies suggest that despite the presence of NK cells in the endometrium, changes in the receptors of these cells result in a reduced capacity of the tissue for cytotoxicity [114,117].

Another study, which evaluated the expression of NK cells with another cytotoxic ligand - NKp30+, reached the opposite conclusions, finding that diseased endometrium had greater infiltration of NKp30+ NK cells [119]. Cells with such receptors have a cytotoxic effect through activation of the NF-κB pathway [120]. Nevertheless, the increase in NKp30+ NK cells was explained by the potential expression of this molecule by other immune cells [119].

Although most studies have focused on the number of NK cells in the endometrium, Jørgensen et al have shown that NK cell function in patients with endometriosis may be impaired due to lower expression of the cytokines IP-10, MCP-1, IL-15, and IL-18 secreted by these cells [121].

In addition, it was revealed that NK cells obtained from healthy donors and incubated with endometrial cells obtained from patients with endometriosis expressed similar levels of IFN-γ secretion and had similar levels of degranulation when compared with NK cells incubated with ectopic endometrial cells. These reports suggest that the above-mentioned properties of NK cells were identical in the microenvironment of the eutopic and the ectopic endometrium, which could indicate a common profile of NK cells present in these 2 tissues and confirms the involvement of eutopic endometrium in disease progression [122].

Dendritic Cells

Dendritic cells (DCs) originate in the bone marrow and participate in innate and acquired immune responses. They are antigen-presenting cells (APCs) and serve as an essential link in recognizing and presenting foreign antigens to lymphocytes T [123,124]. They also promote CD4+ lymphocyte polarization toward a Th1 profile [125]. In addition, DCs are involved in the proliferation and activation of NK cells. This is a bidirectional interaction, as NK cells regulate the response of DCs by eliminating them or stimulating their maturation [126]. These immune cells also influence macrophage differentiation through the production of IL-10 and interleukin-12 (IL-12), which promote macrophage differentiation into M2 and M1 profiles, respectively [127]. Therefore, DC imbalance in the endometrium causes a series of changes in different immune cell populations.

Physiologically, during the menstrual cycle, DCs, along with other immune cells, are recruited into the uterine cavity, where they peak during the menstrual phase [128,129].

DCs are classified into various subsets, but the most relevant division is between immature DCs (iDCs) and mature DCs (mDCs). The role of iDC is mainly to trap potential antigens in peripheral tissues, while mDC presents these antigens to T lymphocytes. Immature forms transform into mature ones under the influence of inflammatory factors and antigens, and physiologically, both of them grow in the endometrium during the menstrual phase [130,131].

There is a hypothesis that DC imbalance in the eutopic endometrium results in the formation of endometrial lesions [128,129], and these immune cells enable the capture and subsequent presentation of antigens in the endometrium. A reduction in their levels and their insufficiency facilitates the survival of exfoliated endometrial fragments in the uterus, which consequently promotes the implantation of endometrial implants. In summary, an insufficient increase in the level of DCs during menstruation inhibits the activation of immune processes aimed at effectively cleansing the uterine cavity, which promotes lesion formation and disease progression [129,132].

The study by Schulke et al included 33 patients with endometriosis and 28 healthy women who served as controls. Using immunohistochemistry, they evaluated the expression of CD1+ and CD83+ on DCs in endometrial fragments collected from study participants. While the expression of CD1 was characteristic of iDCs, CD83 occurred on the surface of mDCs. The authors noted differences in iDC and mDC levels in the eutopic endometrium of women with endometriosis compared to the endometrium of healthy women. The study results showed there were fewer mDCs in women with endometriosis compared to the control group. Moreover, an increase of iDCs was found in the endometrium’s basal layer in the cycle’s proliferative phase in patients with endometriosis compared to controls [133].

Maridas et al agreed with the study described above, as they found that late-stage mature endometrial DCs (LAMP-DCs) tended to have lower concentrations in endometrial specimens from endometriosis patients compared to controls [132]. As mature dendritic cells are responsible for effective clearance of the uterine cavity from menstrual debris, such an immune profile in patients with endometriosis may contribute to the existence of persistent cell fragments and thus the progression of endometriosis [134].

In contrast, a meta-analysis by Poli-Neto et al came to the interesting conclusion that the populations of DCs in the eutopic endometrium in endometriosis differ depending on the disease stage. They noted that, according to the data presented above, DC populations were reduced in women with endometriosis compared with healthy women, but only in stage III–IV disease. However, in stage I–II disease, the level of DCs seemed to be higher than in healthy women [33]. This issue was also raised by Hey-Cunningham et al in a prospective cross-sectional cohort study. In endometrial samples obtained from 55 patients with endometriosis and 30 healthy women, they noted that the density of DCs was significantly higher in stage I–II disease than in stage III–IV. The greatest differences in cell levels were noted in the population of CD141+ mDCs, which have an increased ability to capture dead cells. These differences in DC levels depending on the stage of the disease lead to the hypothesis that in mild and moderate stages, there is an increase in their ability to remove exfoliated endometrial fragments. However, in prolonged disease, the immune response is significantly reduced [135].

Immune Cell Interaction Network

When analyzing the role of immune cells in endometriosis, it is crucial to consider not only the impact of individual cell populations but also the numerous interactions that occur between these cell groups, which can modulate their activity. One group of such interactions is those between macrophages and other immune cells. It has been demonstrated that macrophages can suppress the cytotoxic properties of CD8+ T cells [136,137]. Moreover, Yuan et al found that in a hypoxic environment, which also occurs in the vicinity of endometriotic lesions [138], macrophages stimulated the depletion of CD8+ T cells and reduced their proliferation [139]. However, Xiaocui et al found that macrophages stimulate Treg lymphocytes [140]. It was also observed that macrophages exhibit cytotoxic inhibition toward NK cells [141]. This action of macrophages on NK cells likely occurs through the mediation of factors such as IL-6, IL-7, IL-15, and prostaglandins [142]. M2 cells also inhibit the maturation of dendritic cells [137]. Analyzing the above interactions shows that the action of macrophages on other immune system cells involves reducing inflammation.

Another critical group of interactions is those exerted by T cells on other cellular components of the immune system. The action of Treg cells is essential, as they program dendritic cells to reduce the expression of CD80 and CD86 on their surface, thereby inhibiting their activity [128]. Conversely, CD4+ cells can stimulate the maturation of dendritic cells [143]. Treg lymphocytes can also influence macrophages. This action leads to a reduction in the population of pro-inflammatory macrophages and a shift towards those with pro-repair properties [144].

Taking into consideration the significant role of NK cells in endometriosis, interactions between these types of cells are also important. These cells mediate the stimulation of other components of the immune system. On the one hand, NK cells stimulate dendritic cells to mature via the action of IFN-γ. On the other hand, they use the same cytokine to stimulate the differentiation of CD4+ T cells [145].

Cytokines

Cytokines are crucial mediators in inflammatory processes and play a major role in modulating the immune response [146]. They are categorized into pro-inflammatory and anti-inflammatory [147]. In inflammatory diseases, cytokines can induce cellular responses that affect the immune cells’ activity, proliferation, and survival [148]. Through cell proliferation and differentiation, pro-inflammatory and anti-inflammatory cytokines are involved in the development and maintenance of endometriosis [149]. IL-1, IL-6, IL-8, IL-17, IL-23, and TNF-α are classified as pro-inflammatory cytokines [150,151]. Thus, these cytokines were most widely analyzed in endometriosis patients.

When IL-1β, IL-6, IL-8, and TNF-α levels were compared in the endometria of patients with endometriosis and in the endometria of healthy women, interleukin concentrations were shown to be increased in the former group [152–155]. Llarena et al also concluded that higher IL-1β and IL-6 concentrations in patients with endometriosis, compared to healthy women, were present in the endometrium only when patients in the III and IV stages of the disease were involved [156]. All these results indicate a more pro-inflammatory microenvironment of diseased endometrium, which is particularly exacerbated in more advanced stages of the disease.

IL-1RI and IL-1RII, the receptors for IL-1, and the receptor antagonist IL-Ra, are other molecules involved in modulating IL-1 expression [157]. Kharfi et al observed that the expression of IL-1RII was reduced in the eutopic endometrium of patients with endometriosis. As this receptor is involved in the inhibition of IL-1, this cytokine could not be inhibited; therefore, the endometrium of patients with endometriosis could have a more inflammatory profile and increased disease development [158].

Research has also focused on the association between pro-inflammatory cytokines and COX-2 expression. While normal endometrial cells incubated with IL-1β or TNF-α secreted less COX-2, incubation of endometrial cells with IL-1β or TNF-α resulted in increased COX-2 expression [159]. In addition, the exact action for TNF-α was confirmed in another study [160]. Thus, in eutopic endometrial cells, these 2 cytokines, through COX-2 action, can enhance proliferation and attenuate apoptosis of endometrial cells [161].

Sharpe-Timms et al studied the effects of incubating endometrial cells with IL-1β, IL-6, or TNF-α on endometrial haptoglobin (eHp) levels. They proved that eHp increased significantly after treatment with all these cytokines in endometrial cells collected from women with endometriosis [162]. When eHp was introduced into the pelvic cavity, it impaired the function of peritoneal macrophages and enhanced the production of IL-6. Therefore, this is one of the possible mechanisms by which these pro-inflammatory cytokines enhance the progression of endometriosis [163].

Furthermore, the involvement of IL-6 in promoting the NOTCH1 signaling pathway mediated by E-proteins may explain the development of endometriosis lesions [164]. Li et al demonstrated that normal endometrial stromal cells incubated with estrogen showed greater invasiveness through NOTCH1 signaling. These observations are significant given the increased estrogenic environment in endometriosis [165]. Pino et al also explained the possible role of TNF-α in endometriosis progression in their experimental research, finding that eutopic endometrial cells were more responsive to TNF-α-induced MMP-9 expression than normal endometrial cells [166]. MMP-9 was reported to participate in the migration of normal endometrial cells, suggesting that this molecule contributes to disease progression [167].

Some interesting results on the potential effect of cytokines on the treatment of endometriosis were also presented. Bilotas et al found that while incubation of eutopic endometrium with leuprolide acetate (LA) increased apoptotic cells, IL-1β nullified this beneficial treatment effect [168]. IL-8 expression can also be reduced by gonadotropin-releasing hormone agonists (GnRHa) through inhibition of TNF-α-induced NF-κB signaling. Sakamoto et al showed that such intervention suppressed inflammation and progression of endometriosis [169].

Grandi et al conducted an experiment using progestins. Initially, they incubated endometrial cells with TNF-a to induce inflammation. Then, they incubated this mixture with progestins, such as medroxyprogesterone acetate (MPA), norethisterone acetate (NETA), or dienogest (DNG), which resulted in a decrease in IL-6 and IL-8 levels and potentially could decrease disease progression [170]. Lin et al demonstrated that endometrial cells treated with TNF-α inhibitor tended to display lower survival and invasiveness, thus suggesting the possible utility of anti-TNF-α agents in endometriosis suppression [171].

Expression of interleukin-22 (IL-22) and its receptors (IL-22R1 and IL-10R2) was found to be increased in the eutopic endometrium of women with endometriosis compared to healthy individuals. Moreover, recombinant human IL-22 (rhIL-22) incubated with eutopic endometrial cells promotes IL-8 secretion [172]. Although the pro- or anti-inflammatory status of this interleukin is not established, the above results indicate a possible shift toward pro-inflammatory properties, exacerbating disease progression [173].

Some authors have also focused on IL-17. Ahn et al showed an increase in angiogenic, pro-inflammatory, and chemotactic cytokines such as vascular endothelial growth factor (VEGF), IL-8, IL-6, IL-1β, G-CSF, CXCL12, CXCL1, and CX3CL1 in cell lines treated with IL-17A, and laparoscopic removal of endometrial lesions decreased systemic IL-17A levels, but it remains undetermined whether such an effect would also be exerted on eutopic endometrium [174]. In addition, Wu et al reported significant enhancement of the IL-17 signaling pathway in the eutopic endometrium of patients with endometriosis, indicating inflammatory features of the tissue [31].

Future Directions

The changes in the endometrium of patients with endometriosis described above indicate a shift in balance toward pro-inflammatory processes. Researchers have so far mainly focused on analyzing groups of specific immune cells, and this topic seems to have been covered quite extensively in the current literature. Our review of the literature suggests it is important to focus on the expression of receptors and other smaller molecules on the surface of particular immune cell types in the future. Such studies would provide an opportunity for a more in-depth analysis of the processes underlying disease progression. Specific receptors such as NKp46 and IL-1RII or molecules like FGL2 should be of particular interest.

When analyzing the endometrial changes of patients with endometriosis, the collection of eutopic endometrial fragments is technically simple. Therefore, the analysis of immunologic abnormalities within the eutopic endometrium may be more clinically useful than the analysis of ectopic endometrial samples or peritoneal fluid, as eutopic endometrial samples can be collected during a simple biopsy, without the need for surgery.

A close look at new reports of immune changes in the eutopic endometrium may provide a start in developing immunotherapies focused specifically on inhibiting disease progression. So far, anti-TNF therapy has been proven effective in reducing endometriosis lesions in animal models [175]. Similar effects were observed with anti-VEGF therapy [176]. The activation of NK cells, which aims to restore the cytotoxicity of this type of cell, also plays a promising role in endometriosis-targeted immunotherapy [177]. Although no large clinical trials have been conducted to evaluate the efficacy of immunotherapy in endometriosis, it seems that investigating the modulation of the above-mentioned molecules may be valuable.

In the future, in addition to further observation of the changes that occur in the eutopic endometrium, it is important to determine the impact of the immunological changes that occur in the endometrium on fertility because the endometrium provides an environment for the developing embryo. The specific effect of IL-6 on endometrial receptivity or Tregs on implantation should be determined [69,178].

Conclusions

The initiation and progression of endometriosis depend on many biological factors and it is difficult to clearly identify factors that aggravate the disease. The endometriosis eutopic endometrium shows many differences at the immunological level that may play a role in the development of the disease. The predominant role in the process of disease progression is the shift towards a pro-inflammatory endometrium profile, which includes the increased presence of cells and cytokines, leading to such conditions. An in-depth understanding of immunological abnormalities of the endometrium could significantly improve the global management of patients with endometriosis through the diagnostic potential of certain immune cell receptors and novel therapeutic options.