Endometriosis: An Immunologist’s Perspective

1. Introduction

Endometriosis is a prevalent chronic inflammatory condition affecting 10 to 15% of women of reproductive age, which incurs substantial healthcare costs [1,2]. This disorder is characterized by the growth of endometrial tissue outside the uterus, resulting in inflammation and fibrosis. The displaced tissue experiences cyclical changes akin to those of normal endometrial tissue. Multiple risk factors contribute to the onset of endometriosis, including familial history of the disease, nulliparity (the condition of never having given birth), early onset of menstruation (menarche), and exposure to various environmental influences. The condition predominantly affects women aged 25 to 45 [3] and is associated with elevated rates of obstetric complications [4,5] as well as a diminished quality of life [6].

Diagnosis of endometriosis is often delayed, with a typical gap of 7 to 12 years from the onset of symptoms to a surgical diagnosis [7,8]. The condition is systemic, affecting 50–80% of women with pelvic pain, and is a common cause of unexplained infertility. It disrupts the function of the fallopian tubes and interferes with embryo transport, with 25–50% of women undergoing fertility treatments being treated for this condition. Additionally, endometriosis impacts liver and adipose tissue metabolism, which leads to systemic inflammation and altered brain gene expression and contributes to pain sensitization and mood disorders.

Chronic inflammation, immune cell phenotype, and function changes are associated with endometriosis [3,5,9]. There are disturbances in neutrophils, monocytes/macrophages, dendritic cells, NK cells, B cells, and T cells [10]. While benign, endometriosis exhibits cancer-like behaviors, including hyperplasia and invasive growth [10,11]. The eutopic endometrium in affected women exhibits molecular abnormalities, which activate oncogenic pathways and increase the production of estrogen, cytokines, prostaglandins, and metalloproteinases, thereby supporting endometrial implant survival [10,11]. Autoimmunity is also observed in patients with endometriosis [12].

In this review, we discuss the immune system’s involvement in endometriosis, the critical role of cytokines, the mechanisms of inflammation and pain, and a brief overview of anti-inflammatory, cytokine, and anti-cytokine treatments.

2. Endometriosis

The hypothesis of endometriosis encompasses several mechanisms, including retrograde menstruation, metaplasia, and genetic susceptibility [13,14,15]. The Sampson theory explains that during menstruation, endometrial cells can survive and invade pelvic structures through tubal reflux, leading to ectopic lesions. However, it doesn't fully explain the mismatch between the high incidence of blood reflux (90%) and the lower incidence of endometriosis (10%) [16]. No single theory comprehensively accounts for the various clinical presentations and lesions of endometriosis, including those outside the abdominal cavity or in men [17]. Research has also highlighted proangiogenic factors, such as VEGF, IL-1β, and TNF-α, which play a crucial role in the vascularization of endometriosis [18]. Furthermore, patients with carbohydrate antigen 125 levels (≥ 35 U/mL) have a higher risk of pelvic adhesions and more extensive lesions [19].

Endometrial tissue growing outside the uterus causes symptoms like chronic pelvic pain, menstrual pain, painful sex, and infertility in 50% of patients [2,10]. The development of endometriosis may involve factors such as retrograde menstruation, immune response issues, and inflammation triggered by adipokines like leptin [20]. Diet and gut microbiota also play a role in influencing symptoms [21]. Endometrial implants depend on estrogen for growth, and there is often an imbalance between estrogens and progestogens, along with progesterone resistance [22]. Inflammation plays an essential role in the pathophysiology of endometriosis. Inflammation is responsible for pain, tissue remodeling, lesion formation, fibrosis, and infertility (decreased ovarian reserve, reduced oocyte quality, impaired endometrial receptivity) and can promote malignant transformation [5,23,24]

There are three types of endometriosis: superficial peritoneal disease (15–50% of patients), ovarian endometrioma (2–10%), and deep infiltrating endometriosis (20%) [25]. Deep endometriosis features nodules deeper than 5 mm and is the most aggressive form, linked to more significant pain and infertility [14,26]. Symptoms may relate to lesion appearance, and treatment response varies by lesion type, with undifferentiated lesions usually being deep infiltrating [27].

Endometriosis and adenomyosis, while benign, exhibit malignant traits like rapid growth and invasiveness. Transitioning from adenomyosis to a premalignant tumor involves genetic and epigenetic changes [28,29]. Integrin β3 (ITGB3) is upregulated in ectopic endometrial stromal cells from endometriosis patients, promoting cell proliferation and invasion [30,31,32]. ITCH, a ubiquitin E3 ligase involved in endometriosis, is downregulated in this condition and, when overexpressed, enhances the ubiquitination of ITGB3, affecting the proliferation and invasion capabilities of ectopic cells [30,31,32]. The opposing expressions of ITCH and ITGB3 suggest that dysregulation of the ubiquitin process may play a crucial role in endometriosis pathogenesis [32]. Additionally, HOTAIR lncRNA influences the invasion and migration of endometrial stromal cells via the miR-519b-3p/PRRG4 pathway [11].

Endometriosis increases the risk of epithelial ovarian carcinoma, including clear cell, endometrioid, and low-grade serous types [33,34]. Carcinogenesis is linked to an imbalance of reactive oxygen species, antioxidants, and systemic inflammation. Endometriotic cysts have high free iron levels, leading to oxidative stress [35]. Atypical endometriosis, with characteristics between benign and malignant, has been seen in ovarian cancer patients, resembling atypical hyperplasia or intraepithelial neoplasia in the endometrium [36]. A clear connection between the disease and genetic predisposition is observed.

Table 1 summarizes the clinical phenotypes associated with endometriosis, highlighting its relationship with pain, infertility, and potential comorbid medical conditions. Diagnosing this condition can be complex due to its similarities with other clinical entities.

2.1. Genetic and Epigenetic Changes in Endometriosis

Endometriosis has genetic and environmental causes with polygenic inheritance. Relatives of affected individuals are seven times more likely to develop the disease. A twin study indicates that about 52% of disease variance is genetic, and six genetic markers linked to endometriosis have been identified.: CISD2, EFRB, GREB1, IMMT, SULT1E1, and UBE2D3 [37]. Additional analyses have pinpointed associations with loci on chromosomes 7p15.2, 2p25.1 (GREB1), and 12q22 near VEZT [37]. Recent GWAS and integrative-omics analyses highlight the role of immunopathogenesis and key signaling pathways (Wnt, NOTCH, TGFβ) in regulating endometrial cell behaviors in endometriosis [39].

An integrated analysis of DNA profiles was used to analyze candidate genes for ovarian endometriosis. Lei and coworkers [40] were able to show that the most relevant genes for ovarian endometriosis are TMEM184A, GREM2, SFN, KIR3DX1, HPGD, ESR1, BST2, PIK3CG, and RNASE1. Some of these gene candidates are also associated with cancer.

Chou and coworkers [41], studying the genetics of Killer inhibitory receptor (KIR) in Chinese patients with endometriosis, reported an increase in the number of patients with centromeric A/A haplotypes and a decrease in KIR2DL2, an inhibitory gene of the B haplotype. On the other hand, Marin et al. [42] reported a significant association of KIR2DL2 with the risk of deep endometriosis in Euro-descendants [42]. KIR2DL2 is associated with impaired NK cytotoxic activity and clearance of ectopic endometrial cells [41,42]. A Japanese study found a lower frequency of activating KIR3DS1 and a higher frequency of the inhibitory KIR3DL1+/HLA-Bw4+ combination [43]. It can be concluded that extensive studies are required to define the relationship between KIR and endometriosis.

The NOD-like receptors (NLR) pathway fundamentally regulates interleukins, proinflammatory cytokines, and NF-κB activity. Single-nucleotide polymorphisms (SNPs) of the NOD1 and PYDC2 genes were associated with endometriosis, whereas SNPs of the NOS2 and PYDC1 genes were not [44]. Other studies have identified additional cytokine gene polymorphisms associated with the disease, including IL1A rs2856836 and rs2856836 [45] and rs11575812 and rs2069772, and rs2069762 [46], IL-10 (rs1800872) and IFN-γ a13 allele [47], IL-12B rs3212227 [48,49], IL-16 rs11556218 and rs4778889 [50], and rs4778889 [51], IL-17A rs2275913 [52], IL-18 SNP rs1946518 [53], association with severity (TNF, rs1800629), IL-1beta (IL1B, rs1143634), and IL1-Ra (rs2234663) [54], and macrophage migration inhibitory factor (MIF) rs755622 [55]. IL-8 SNP polymorphism rs4073 has been related to pelvic pain in endometriosis [56]. These reports show a clear association with disease and disease severity. However, most studies focused on only one target cytokine, and integrative studies are required.

Women with endometriosis have a two- to threefold increased risk of developing epithelial ovarian cancer, in particular, clear cell and endometrioid types. Notable mutations in ARID1A, PIK3CA, and PTEN may drive the progression from benign endometriosis to cancer [57,58]. Additionally, these cancers may display mutations in K-RAS and β-catenin/Wnt, along with microsatellite instability, indicating shared genetic susceptibility [59,60]. Exome sequencing revealed that 79% of deep endometriosis patients had somatic mutations; nevertheless, these mutations alone are insufficient for malignant transformation [57].

The epigenetic changes associated with endometriosis include DNA methylation and phosphorylation, modifications to histones and non-coding RNA, and chromatin remodeling and organization [61]. Specific epigenetic abnormalities have been described in endometriosis that alter the expression of key transcription factors. For example, hypomethylation of the GATA-binding factor-6, accompanied by overexpression, transforms an endometrial stromal cell into an endometriotic phenotype [61]. Steroidogenic factor-1 overexpression causes excessive estrogen production, which drives inflammation via pathologically high levels of estrogen receptor-β [62].

Some miRNAs serve as biomarkers for endometriosis and could be targets for therapy [63,64]. Some of these miRNAs are shared between endometriosis and atherosclerosis, both diseases are linked [65]. The most relevant miRNA-detected changes in plasma and serum are: 1) increased: miRNA 122, 199a, 125 b-5p, 150-5p, 342-3p, and 451a, 2) decreased miRNA Let-7b, Let-7d, Let-7f, 17-5p, 20c, 20a-5p, and 3613-5p [62,63,64,65]. Studies on non-coding and circular RNA and endometriosis are ongoing [67].

Impaired endometrial decidualization reduces fertility in endometriosis. Transcriptomic profiling shows differences in pathways, including defective BMP/SMAD4 signaling, oxidative stress response, and retinoic acid signaling [66]. Constitutive NF-κB activation in endometriotic lesions promotes inflammation, invasion, and angiogenesis while inhibiting apoptosis [68,69]. Active lesions (red) eventually become fibrotic tissue (white), leading to adhesions [70].

High levels of BCL-6 (a transcription factor) in women with endometriosis are associated with decreased activation of progesterone receptors, resulting in progesterone resistance in the endometrium [71]. The BCL6 gene is significantly upregulated in ectopic tissues compared to controls [72]. mRNA levels of estrogen-related receptors β and γ (ERRβ and ERRγ) were substantially lower in ectopic tissues from patients with severe endometriosis than in the eutopic endometrium of controls [73].

The activation of mutated K-RAS in donor endometrial epithelium and stroma promotes lesion growth in a murine model of endometriosis but is insufficient for cancer transformation [74]. Essential factors for the progression from endometriosis to endometriosis-associated ovarian cancer include somatic mutations in ARID1A, K-RAS, PTEN, and microsatellite instability [75,76]. Overall, there is a link between genetic predisposition and polymorphism for endometriosis, along with other factors under investigation: signal transduction modulation, miRNA, long coding, and circular RNA.

2.2. Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-bound particles that transport regulatory molecules like proteins, miRNAs, and lipids. They consist of small EVs (sEVs), such as exosomes, and large EVs (lEVs), also known as macrovesicles, which are released from various cellular compartments [77]. Gram-positive and Gram-negative bacteria can generate apoptotic bodies and extracellular vesicles (BEVs) [78]. BEV can be formed from the microbiota in the endometrial fluid and can induce the secretion of TNF, IL-6, and IL-17, which are involved in endometriosis [78].

Vesicles of different sizes are found in follicular fluid and affect follicle size, oocyte function, promote granulosa cell proliferation, and cell survival under stress [77,78]. Small and large EVs differ in number, morphology, specific membrane markers, and miRNAs [77,78]. Large EVs influence steroidogenesis by affecting enzyme mRNA levels, stimulating estradiol secretion via the PI3K/AKT pathway. Newly identified mitochondria-derived EVs that contain mitochondrial proteins have a potential role in fertilization [78].

Platelets are key players in endometriosis. They are found in lesions, contribute to fibrosis in damaged tissue [70,79,80], and are associated with extracellular vesicles [81]. Nevertheless, the impact of platelets on endometriosis still requires more research.

2.3. Microbiota and Endometriosis

Microbiota plays a role in establishing and progressing endometriosis [82,83]. The gut microbiota may influence estrogen production and local immune inflammation, promoting endometrial cell proliferation [82,83]. Patients with endometriosis have β-glucuronidase-producing bacteria in their gut microbiome, leading to increased estrogen metabolites [83,84,85]. The endometrial environment and peritoneal cavity microbiota have been linked to endometriosis [86,87,88,89,90,91]. Increased levels of Gardnerella, Streptococcus, Escherichia, Shigella, and Ureaplasma were noted in the cervical microbiota of endometriosis patients [87]. Distinct microbial communities were found in feces and peritoneal fluid, with increased pathogens in peritoneal fluid and reduced protective microbes in feces [88,89]. Endometriosis patients exhibited lower alpha and beta diversity in gut microbiota compared to controls, with significant differences in the abundance of several bacterial classes [90]. The Firmicutes/Bacteroidetes ratio, indicative of dysbiosis, was also higher in endometriosis patients, alongside notable differences in various taxa [90]. The relationship between endometritis and endometriosis has been documented [91,92,93]. Clinical trials targeting dysbiosis and endometrial lesions could benefit cases of recurrent implantation failure and pregnancy loss. [93].

Modulating gut microbiota could potentially slow endometriosis progression. Sobstyl et al. [94] noted that interactions among microbiota and dysbiosis may activate immune cells, producing proinflammatory cytokines that disrupt stem cell homeostasis and affect estrogen levels. Certain gut bacteria, like Bacteroides and Lactobacillus, secrete enzymes that elevate free estrogen levels [95]. An increase in Escherichia coli has been observed in the feces of endometriosis patients, but the interactions between gut, vaginal, and endometrial microbiota remain unclear [93,96].

Patients with chronic pain and endometriosis had lower alpha diversity than controls, showing increased levels of vaginal Streptococcus anginosus and rectal Ruminococcus [95,96]. Guo et al [97] speculated that different gram-negative bacteria, such as Escherichia coli, residing in the vagina could be involved in the pathogenesis of endometriosis in humans. In addition, gut microbiota promotes the progression of endometriosis by influencing peritoneal immune cell populations. Then, the onset and development of endometriosis may be related to the abnormal immune response caused by gut dysbiosis [97].

3. Immune Response in Endometriosis

3.1. Pattern-Recognition Receptors (PRR), Pathogen-Associated Molecular Patterns (PAMP), Damage-Associated Molecular Patterns (DAMP), and Endometriosis

PRRs can be classified into five families: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), retinoic acid-inducible gene I-like receptors (RLRs), and AIM2-like receptors (ALRs) [99,100]. Their activation leads to proinflammatory cytokine, interferon production, phagocytosis, and cell death [99,100]. PAMPs include lipopolysaccharides, flagellin, viral RNA, and fungal cell walls [99,100]. DAMPs are various molecules, such as proteins (e.g., amyloid beta, HSP70), metabolites (e.g., ATP, uric acid), ions (Ca+2, K+), and nucleic acids (self RNA, DNA) [101].

Endometriosis may develop in two distinct phases. The initial wave occurs with an infection and TLR activation. The second wave is characterized by sterile inflammation resulting from oxidative stress and receptor activation by DAMPs [100,101,102].

Increased numbers of highly positive TLR2 B cells and myeloid dendritic cells correlate with the severity of endometriosis in patients [101,102,103]. The concentrations of TLR2 and TLR9 in the peritoneal fluid of individuals with endometriosis were found to be significantly higher when compared to control subjects [104]. Conversely, in a mouse model of endometriosis, Ureoplasma urealyticum infection fosters the progression of endometriosis by enhancing the expression of inflammatory mediators, adhesion molecules, and MMP-2 in peritoneal mesothelial cells via TLR2 signaling [104]. Moreover, ectopic endometriotic lesions significantly increased TLR3 [105,106] and TLR3 and TLR4 [106,107] mRNA expression compared to corresponding eutopic tissues.

Inflammasomes are multi-protein complexes containing nucleotide-binding oligomerization domain-like receptors (NLRs), crucial in activating inflammatory caspases [108]. The nucleotide-binding domain and leucine-rich repeat pyrin domain-containing protein-3 (NLRP3 or NALP3) inflammasome is among the most extensively studied, as it binds to procaspase-1 and facilitates activation [108,109]. Active caspase-1 cleaves pro–IL–1β and pro–IL–18 to generate IL-1β and IL-18, respectively [108,109]. Inflammasome activation by sex hormones, particularly the NLRP3 inflammasome, has been implicated in the pathogenesis of endometriosis [110,111]. In ectopic endometrial tissues, there is an observed upregulation of genes encoding caspase-1, IL-18, and NLRP3 [110]. Granulosa cells in the ovaries of women with endometriosis exhibit elevated expression of the NLRP3 inflammasome at both protein and mRNA levels, accompanied by increased levels of IL-1β and IL-18 in the follicular fluid of these patients, and it is implicated in infertility [112,113]. NLRP3 gene and protein expression levels are markedly elevated in ovarian endometriosis, and treatment utilizing an NLRP3 inhibitor has been shown to reduce the size of ovarian endometriosis lesions in a murine model [114,115].

Interactions between macrophages and endometrial stromal cells via NLRP3 signaling have been shown to enhance the migration of stromal cells and the progression of endometriosis [116]. Notably, NLRP3-deficient mice exhibited smaller endometrial lesions than wild-type mice; however, this difference was reversed upon administering wild-type macrophages [116]. Ectopic endometrial tissues demonstrated significantly higher mRNA levels of IL-18 and IL-6, and IL-1β levels were elevated in endometriotic tissues relative to both eutopic endometrium and controls [117,118]. NLRP3-mediated pyroptosis is linked to fibrosis via TGF-β1, and inhibiting it may reduce fibrosis in endometriosis. [113,119]. Additionally, TRIM24 has been identified as a potential facilitator of endometriosis progression through the NLRP3/caspase-1/IL-1β-mediated pyroptosis pathway [120,121]. Elevated levels of estrogen receptor β in endometriotic lesions have been correlated with increased production of IL-1β, thereby enhancing cell adhesion and proliferation [121]. While progesterone is known to inhibit NLRP3 activation in normal stromal cells via autophagy, this inhibitory effect appears to be diminished in endometriotic cells [122].

NLR family CARD domain-containing 5 (NLRC5) is a negative regulator in endometriosis development by inhibiting inflammation [123,124]. Over-expression of NLRC5 leads to increased autophagy in ectopic endometrial stromal cells, while its inhibition reduces autophagy [124]. NLRC5 expression is notably higher in the ectopic and eutopic endometria of endometriosis patients than in those with leiomyoma, with the highest levels found in the ectopic endometrium and suppresses IL-6 and TNF-α levels [125]. Thus, NLRC5 overexpression inhibits endometriosis's estrogen receptor β-mediated development and inflammatory response [123,124,125].

C-type lectin receptors (CLRs) are essential in the innate immune system, recognizing carbohydrates [126]. In endometriosis patients, peritoneal fluid showed increased mRNAs of CLR MR2 and DAP12 and decreased galectin compared to controls [126,127,128]. Mannose receptor C, type 2 (MRC2), was lower in ectopic endometrial stromal cells than in normal ones [128], while peritoneal dendritic cells in endometriosis expressed high levels of mannose receptor [127]. The receptor for advanced glycation end products (RAGE) is linked to endometriosis and infertility [129]. The soluble receptor (FF sRAGE) potentially influences in vitro fertilization success [129]. The roles of RAGE and CLRs in this context are still being studied.

3.2. Innate Immune Response in Endometriosis

Table 2 and Table 3 provide a general overview of endometriosis's innate and adaptive immune involvement. The aim is to give the reader a summary of the most critical issues in endometriosis.

The innate immune cell response comprises several protein elements, with the complement pathway and defensins being the most relevant. The complement system, a component of innate immunity, contains over 50 proteins that aid in eliminating pathogens, removing immune complexes and apoptotic debris, and participating in processes such as inflammation, adaptive immunity, coagulation, metabolism, tissue regeneration, and host-microbiota symbiosis [130,131]. Table 2 shows several pathway components that have been related to endometriosis. On the other hand, defensins produced by Paneth cells, neutrophils, and epithelial cells have not been involved in endometriosis (Table 2).

Macrophages are crucial in endometriosis physiopathology. Chronic macrophage stimulation and high iron levels in the peritoneal cavity elevate reactive oxygen species in women with endometriosis [132,133,134]. Estrogen prompts peritoneal macrophages to secrete cytokines and prostaglandins through estrogen receptor-β, which decreases MMP-9 activity and inhibits phagocytosis [24]. Upon cell activation, NF-κB p65 phosphorylation induces the transcription of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8), proangiogenic factors VEGF, growth factors like FGF-2, and adhesion molecules [134,135,136], and COX-1 and 2. COX-2 is responsible for the increased concentrations of PGE2 in the peritoneal fluid [137]. The co-culture of macrophages with endometrial stromal cells enhances the proliferation and invasiveness of these endometrial stromal cells [138].

Pro-inflammatory peritoneal fluid in women with endometriosis elevates FasL expression in regurgitated endometrial cells, enhancing Fas-mediated cell death of activated immune cells and aiding immune evasion by endometrial cells [139,140]. Macrophages initiate a regenerative program vital for lesion growth. In patients with endometriosis, peritoneal macrophages exhibit higher iron storage than controls [133,141] and have difficulty managing elevated hemoglobin levels in the peritoneal fluid [141,142]. In women with endometriosis, peritoneal macrophages show heightened proinflammatory markers of the M1 phenotype, while M2 macrophages often shift toward M1 [143,144]. The presence of two subpopulations of macrophages in the lesion was reported using single-cell analysis [145] as well as the role of M2a in fibrogenesis [146]. In the advanced stages of the disease, there is an increase in M2 macrophages and a decrease in the M1 type; the opposite occurs in the initial stages (I-II) [134,147]. Macrophages activated by IL4 can induce epithelial-to-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation through the production of TGF-β1 [134,141].

Uterine NK (uNK) cells express CD56 but no other classical NK cells or T-cell markers. The number of uNK cells changes during the menstrual cycle, pregnancy, and various endometrial pathologies [148]. There is an increase of uNK in the mid-secretory phase [151]. CD56+ cells remain high during early pregnancy and comprise 70% of the lymphocytes at the interface between maternal decidua and the invading trophoblast [148]. Approximately 10% of uNK are CD56+ CD16+, while 90% of the population is CD56+ CD16− in phenotype [148]. In the peripheral blood, 90% of NK cells are CD56+ CD16+ (pNK); CD56+ CD16+ and CD56+ CD16− uNK cells exhibit functional differences. CD16+ cells are cytolytic, whereas CD16− uNK cells secrete cytokines [148]. Activated uterine natural killer (uNK) cells regulate trophoblast invasion into the decidua [148]. The elevation of CD56+ cells is higher in infertile women and pregnancy loss and appears to be directly correlated with pelvic endometriosis [149].

There is limited information on the roles of neutrophils and eosinophils in the endometrium [150] since they are not commonly observed in the endometrium or vagina except for infectious diseases. Nonetheless, mast cells are therapeutic targets for treating endometriosis, inflammation, infertility, and pain. A recent review [151] describes the different experimental treatments involving mast cells in animal models.

Myeloid-derived suppressor cells (MDSCs) are a diverse group of immature myeloid cells, including dendritic cells, granulocytes, and monocyte/macrophage precursors, known for their immunosuppressive properties. They play a significant role in the progression of immunological disorders, such as chronic inflammation and cancer. MDSCs can be categorized into two primary types: polymorphonuclear (PMN) MDSCs, also referred to as granulocyte (G) MDSCs, and monocytic (M) MDSCs. The significant outcome of MDSC expansion is immunosuppression, which may lead to angiogenesis and the secretion of cytokines or growth factors, potentially exacerbating the progression of conditions such as endometriosis [152,153,154,155,156].

Although the proportion of PMN-MDSCs in both peripheral blood and peritoneal fluid was significantly higher in endometriosis patients, the proportion of M-MDSCs did not differ between controls and endometriosis patients [155,156]. On the contrary, an abnormal expansion of M-MDSCs in peripheral blood and peritoneal fluid of patients with endometriosis [156]. Additionally, MDSCs are more abundant in ectopic endometrium than in normal endometrium [157]. M-MDSCs, alongside inflammatory cytokines and exosome miRNA, appear to be involved in the progression of endometriosis [160]. Cysteine-Cysteine Chemokine Receptor 5 (CCR5) and its ligand, CCL5, could drive the progression of endometriosis by increasing the accumulation of MDSC [158]. On the other hand, MDSCs drive the process of endometriosis by enhancing angiogenesis [159].

Another critical issue in endometriosis is the role of immature dendritic cells in the lesions. Those cells are inefficient in antigen presentation and are inducers of tolerogenic responses [160]. The lack of mature dendritic cells in endometriosis is also related to the increase of Tregs, and as stated before, the effectiveness of the Tregs depends on the milieu. Li and coworkers [161] have postulated using dendritic cells for therapeutic use in endometriosis. The effectiveness of this proposal can likely be assessed soon.

Table 3 highlights the role of T and B lymphocytes in the adaptive response in endometriosis. Changes in CD8 populations are essential, while CD4 cell modifications can be categorized into early Th1 and late Th2 responses. Inflammatory lesions promote the production of anti-inflammatory cytokines to balance the inflammatory environment, revealing a complex local cytokine storm beyond just immune cell mediators.

In a recent review by Knez et al. [199], it becomes clear that different Tregs subpopulations- resting Tregs (rTregs; Foxp3loCD45RA+T cells), suppressive aTregs (Foxp3hiCD45RA−), and non-suppressive Tregs (non-Tregs; Foxp3loCD45RA- T cells) should be considered when analyzing Tregs in endometriosis [199]. Tregs expressing CTLA-4 induce tolerogenic responses, while TGFβ and IL-17 are crucial for endometriosis. Tregs interact with T follicular cells, B cells, dendritic cells, and macrophages [199]. The suppressive response involves the induction of M2 macrophages and the production of IL-1, thereby decreasing the inflammatory milieu.

The role of B cells in endometriosis is less clear than that of T cells [200]. The generation of anti-endometrial autoantibodies [201] and the production of IL-17 and, in certain conditions, IL-35, illustrate the complex nature of cell interaction [10,200]. More research is required to understand the role of these cells in endometriosis and their probable link with autoimmune diseases.

4. Cytokines and Endometriosis

Cytokines play a critical role in generating endometriosis since the inflammatory milieu in endometriosis leads to poor quality of oocytes and infertility [6]. However, most of the focus of the published studies has referred to cytokines produced by immune cells, which does not represent the whole picture of events in endometriosis. The local tissue production of cytokines must be considered, as well as the role of adipokines, which may also have a dual role, regional and peripheral [21]. A clear example is the role of leptin in regulating the amount of stored energy by binding to specific neurons in the brain.

Alarmins are key inducers of cytokine release by activating DAMP receptors. HMGB1, a byproduct of cell death, enhances pro-inflammatory cytokine secretion, particularly under hypoxic conditions, making HIF-1α modulation crucial in endometriosis [221,222,223]. HMGB1 is also affected by mediators like prostaglandins [224], while the role of leukotrienes depends on LPS induction in the endometrium [225]. Early animal studies showed reduced endometrial inflammation with leukotriene receptor antagonists [226,227], but the results were inconclusive for human clinical trials.

Lipoxin A4 suppresses inflammation and activates autophagy, which helps reduce the proliferative effects of endometriosis [228,229]. Resolvins, as noted in research by Dimitrieva et al. [229] and Gu et al. [230], also contribute to the management of endometriosis [231]. Additionally, resolvins may offer a promising approach to alleviating the pain related to endometriosis [232].

Despite the enormous efforts in analyzing different types of biomarkers in endometriosis reviewed by Collie and coworkers [233], there is no clear consensus on most metabolites. The authors only reported 3-hydroxybutyrate, lactate, phosphatidic acids, succinate, pyruvate, tetradecenoylcarnitine, hypoxanthine, and xanthine as the most consistent biomarkers. Since these intermediate metabolites can be affected by different pathways, more research is required to determine the metabolic pathways involved. Hypoxia should be carefully analyzed as proposed by Wilson [234].

Ferroptosis represents a significant cellular event in endometriosis [235]. Iron within the endometrioma influences the generation of radical species in conjunction with immune cells that provoke pro-inflammatory and cytokine responses. As highlighted in tumor immunology [236], exploring ferroptosis, radical production, cellular senescence, cell death, and immune exhaustion in endometriosis is paramount. Further research is necessary to identify appropriate pharmacological targets.

Table 4 represents all the critical cytokines involved in endometriosis. It describes a group of proinflammatory and anti-inflammatory cytokines and cytokines involved in angiogenesis, cell growth, chemokines, and inhibitory factors. Other mediators may be part of the physiopathology but have not been studied yet.

Table 5 represents the list of adipokines that have been studied in endometriosis. However, it is essential to note that obesity is not prevalent in patients with endometriosis. Patients with endometriosis have low BMI, and obesity increases its severity [307,308,309,310]. Several hypotheses have been made about the possible role of adipokines in endometriosis [311]. Even though there are disagreements about the relationship between obesity and BMI and endometriosis, most researchers support the proposal of a dual effect of adipokines in the tissue and the central nervous system. More research is required on this topic.

It is important to note that the precise mechanisms underlying the diverse cytokines involved in endometriosis remain unclear. A comprehensive understanding of the chronological progression of this condition is critical for developing novel treatment strategies aimed at reducing both the growth of lesions and associated pain.

5. Mechanisms of Pain in Endometriosis

Endometriosis-associated pain stems from various mechanisms, including nociception, inflammation, and altered pain processing in the nervous system. It is frequently linked to psychological distress and fatigue. Additionally, angiogenesis leads to the growth of nerve fibers that contribute to this pain [323,324]. The size of the lesions appears to be directly correlated with pain intensity, and pain is more severe in patients whose lesions are located on the intestinal wall [325]. However, there is a weak correlation between the graded severity of morphological characteristics and the intensity and character of pain symptoms [326].

Increased levels of the neurotransmitters glutamate and glutamine were found in the anterior insula of endometriosis patients, which enhances connectivity to the prefrontal cortex (where pain-related memories are stored) [327,328]. Other areas of the brain are also affected. According to Eippert et al [329], the periaqueductal gray, which is involved in pain-modulatory pathways, is enlarged in individuals with pain, and measurable changes are observed in the thalamus, insula, and putamen [324,330].

In endometrial lesions, macrophages and nerve fibers interact to promote pain [331,332]. Ectopic endometrial lesions secrete nerve fibers that produce CSF-1 and CCL2, which attract macrophages to the periphery of nerves and regulate their polarization toward the M2 phenotype [333]. On the other hand, macrophages, incubated with CSF-1 and estrogen, produce brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), which stimulate neurite growth from ganglia explants [334].

Endometriosis-related pain is classified as neuropathic or neuroinflammatory [335]. Ectopic endometriotic lesions promote inflammation and disrupt the transmission of inflammatory mediators, altering how nerve fibers process and transmit information [335,336]. Disorders that are characterized by sensory dysfunction, such as overactive bladder syndrome and irritable bowel syndrome, are commonly co-diagnosed with endometriosis [336,337]. These comorbidities suggest a more complex pathophysiology for pain in this condition that cannot be explained by endometrial lesions alone [335,336,337]. Chronic remodeling of the nervous system may occur in shared sensory neural pathways to induce a state of protracted peripheral and central sensitization and chronic pain in patients with endometriosis [337]. Microgliosis, astrogliosis, and enhanced substance P neurokinin-1 receptor immunoreactivity have been observed within the spine in mice with endometriosis, suggesting the development of neuroinflammation and the sensitization of spinal circuitry in this condition [337]. Prostaglandin E2, TNFα, NGF, RANTES, IL-8, and IL-1β are elevated within the peritoneal fluid of endometriosis patients [338]. These mediators can all activate sensory nerve endings directly [11,338,339].

Neuroangiogenesis in ectopic endometriotic lesions explains the transmission of the pain. An increased density of miniature, unmyelinated nerve fibers (sensory afferents, sympathetic, and parasympathetic efferents) has been found in endometrial lesions [337,339]. The local production of VEGF and NGF by macrophages supports neuroangiogenesis [11,339]. On the other hand, the activation of sensory afferent nerves initiates the recruitment of mast cells and the subsequent release of pro-inflammatory cytokines (TNF-α, NGF, PGE2, IL-1β), which contributes to a chronic state of neurogenic inflammation [339]. Neurotrophic factors produced by macrophages, such as Netrin-1, insulin-like growth factor-1, and ten-eleven translocation 3 (TET3), play a role in the pain associated with endometriosis [340,341].

Recently, the role of IL-33 in macrophage-neuron-induced pain has been studied [342]. IL-33 enhanced the release of TNF-α and IL-1β, facilitating macrophage recruitment and neurogenesis in ectopic lesions [342]. IL-33 increased the expression of the transient receptor potential vanilloid 1 (TRPV1), which is responsible for the phenomenon [342]. In women with endometriosis and severe chronic pelvic pain, serum IL-16 levels were higher compared to women with mild pain [52].

Tregs may influence endometriosis pain by modulating macrophages to create a local tolerogenic response, which reduces proinflammatory cytokines, decreases cell migration, and mitigates estrogen's effects on endometriomas [200]. It has recently been found that the meningeal Treg (mTreg) inhibits nociception in female mice [343]. mTreg cells produced enkephalin, which acted on delta opioid receptors in MrgprD+ sensory neurons to reduce pain [343]. However, enkephalin was unnecessary for Treg cell-mediated immunosuppression, and the process depends on sex hormones [343]. One can envision that the understanding of pain in patients with endometriosis is just beginning.

6. Endometriosis and Autoimmunity

There is an association between endometriosis and autoimmune diseases. Women with endometriosis may have a higher risk of conditions such as systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, celiac disease, multiple sclerosis, and inflammatory bowel disease compared to those without endometriosis [12,344,345,346]. Endometriosis shares similarities with autoimmune diseases, featuring elevated cytokines, B cell activation, T and B cell function abnormalities, autoantibody formation, and decreased apoptosis [12]. Women with endometriosis have alterations in B-cell activity and an increased incidence of autoantibodies [12]. These autoantibodies can be directed against various phospholipids, histones, and polynucleotides [12], as well as autoantibodies against the ovary, endometrium, nucleus, smooth muscle, cardiolipins, sperm, laminin, and lupus anticoagulant [12]. Antinuclear antibodies (ANA) in pelvic endometriosis appear to be an immunological secondary effect and do not represent an aggravating factor in patients with pelvic endometriosis [347]. A correlation between the diameter of endometriomas and anti-thyroid peroxidase antibodies has been reported [348]. Patients with endometriosis exhibit elevated transferrin and alpha-2-HS glycoprotein levels in their serum and peritoneal fluid, which may contribute to observed autoimmunity to these proteins [349]. However, no recent reports of these autoantibodies (transferrin and alpha-2-HS glycoprotein) and their impact on the disease exist.

Dotan and coworkers [350] have addressed the issue of SARS-CoV-2 and molecular mimicry in endometriosis. Several other triggers of this process may be observed with viral or bacterial infection and local or gut microbiota. This topic opens an interesting point to address from the pharmacological point of view, preventing autoimmunity.

IgG and complement deposits have been found in the eutopic endometrium in women with endometriosis, corresponding to a decrease in the total serum complement levels [164]. This may have been caused by the ectopic endometrium acting as a foreign trigger that induced an autoimmune response, resulting in infertility [164]. It is not yet clear whether the formation of autoantibodies in endometriosis is a natural response to chronic local tissue destruction or a pathological reaction leading to more generalized autoimmune dysfunction [164].

There exists a singular report regarding the formation of autoantibodies to GM-CSF in individuals diagnosed with deep endometriosis [302]. Nonetheless, it is highly plausible that autoantibodies against additional cytokines are present in patients with endometriosis, considering the unique characteristics of this population. These autoantibodies could significantly impact the equilibrium of cytokines within the microenvironment and may contribute to the diminished immune responsiveness that is often observed in endometriosis. Further investigation into this subject is essential, as it may facilitate the classification of these patients while also providing new therapeutic targets and guidelines for treatment.

Figure 1 represents a general summary of the cells and events in the endometrial lesion. Estrogen, progesterone resistance, and increased adipokine levels also play essential roles in the lesion; however, they were not included due to a lack of space.

7. Immunological Therapies in Endometriosis

Figure 1 provides an overview of the cells and processes involved in endometriosis to understand the complexity of the endometrial lesion. Multiple factors generate autoimmunity based on cell death; however, the possible induction of malignancy, mainly ovarian carcinoma, is also represented.

Endometriosis is typically managed through various therapeutic options, including progestogens, combined oral contraceptives, gonadotropin-releasing hormone antagonists and agonists, androgens, aromatase inhibitors, selective progesterone receptor modulators, selective estrogen receptor modulators, nonsteroidal anti-inflammatory drugs, and/or surgical excision of endometriotic lesions [299,351]. However, many of these treatment modalities are associated with adverse effects, particularly concerning the degree of hypoestrogenism, and there is a notable risk of recurrence following the cessation of therapy. Considering the significant inflammatory component of endometriosis, recent research has increasingly concentrated on utilizing anti-inflammatory and immunomodulatory therapies. Table 6 summarizes therapies for endometriosis with immunomodulatory and anti-inflammatory effects, ranging from common drug treatments to natural products. On the other hand, Table 7 is focused on cytokine inhibition or addition.

A recent review, Zhang et al [352], examined potential therapies targeting immune-associated factors in endometriosis. The objective is to improve the function of NK cells and macrophages. NK cells can be modulated by blocking inhibitory receptors, using cytokines such as IL-2 and IL-12, or through immune checkpoint therapy (anti-PD-1 or anti-PDL-1) [352]. For the moment, this type of therapy has not been tested in patients with endometriosis [352]. Regarding macrophages, potential therapeutic targets include the suppression of the M2 phenotype or activating the M1 phenotype. These two approaches are lacking in the treatment of endometriosis. Another possible therapy option may be using C3 inhibitors or the blockade of C5a and C3a [352]. Anti-IL-33 antibody treatment of the endometriosis mouse model slightly but not significantly reduced peritoneal inflammation and reduced peritoneal cell concentration compared with isotype control [261].

Combining immunophilin suppressors with steroid hormones, such as progesterone, may be a promising approach to treating chronic inflammation associated with endometriosis. Tacrolimus, cyclosporine, progesterone, and analogs can effectively suppress FKBP51, a common target of these agents [353].

Three different drugs that can block ribosome biogenesis, including inhibitors against mTOR/PI3K (GSK2126458) and RNA polymerase I (CX5461 and BMH21), were used in a mouse model with human endometriosis features. The lesion numbers were reduced in treated mice compared to those treated with the vehicle [354].

Other treatment options include drugs with antiangiogenic effects, such as those targeting VEGF (anti-VEGF antibody) or inhibiting tyrosine kinase (Sorafenib, Sunitinib, Pazopanib) [18]. All the studies with these agents are in animal models. Another drug with antiangiogenic effects through VEGF receptor-2 is Cabergoline (a dopamine agonist). In a clinical trial, this drug reduced endometrioma size more effectively than an LHRH agonist [355]. In another small trial, cabergoline decreased pain in patients with endometriosis [356].

Novel therapeutics have been proposed for the management of endometriosis. Iron chelators have demonstrated promising outcomes in animal models [357]. Other strategies aim to ameliorate hypoxic conditions. Sitagliptin has been shown to mitigate hypoxia-induced injury by inhibiting the overproduction of COX-2, PGE2, TNF-α, and IL-6, yielding successful results in animal studies [358,359]. The anti-hypoxic agent myo-inositol trispyrophosphate (IPP) enhances oxygen release from hemoglobin and has effectively inhibited the proliferation of endometrial cells in hypoxic conditions in preclinical models [360]. Research has examined the anti-cancer polypeptide vaccine RESAN, which has been shown to reduce lesion size in mouse models. [361]. No reports in humans on the efficacy of this vaccine due to the absence of clinical trials. The use of extracellular vesicles for the treatment of endometriosis presents a promising avenue, particularly for the delivery of miRNA [362]; nevertheless, this research domain necessitates further exploration and the establishment of consensus among the scientific community.

An interesting proposal has been published that involves modulating kisspeptin neurons, impacting the hypothalamic-pituitary axis and controlling LH and FSH, and consequently, endometrial growth [363]. Since the modulation of pain may involve sex hormones and the production of endorphins and sensory neurons [364], it would be interesting to analyze the induction of endorphins as modulators of endometriosis growth in preclinical models. The topic is engaging, and many patients will benefit from therapy. Future research in this area is anticipated to focus on these patients.

8. Conclusions

Endometriosis is an inflammatory disorder characterized by elements of autoimmunity and a reduced state of immune surveillance. This condition is defined by the abnormal proliferation of functional endometrial glands and stroma located outside the uterine cavity, often resulting in significant pain and infertility. The pathogenesis of endometriosis is multifaceted, involving immunological, hormonal, and genetic factors. Cytokines, adipokines, and growth factors serve as integral components in this process. Furthermore, ectopic endometrium may display functional properties that differ from those of eutopic endometrium. A notable association has been established between endometriosis and ovarian cancer. Autoimmunity is frequently observed in patients diagnosed with endometriosis, and the generation of autoantibodies may be influenced by events occurring within the lesions. Increased iron accumulation, elevated formation of oxygen radicals, and infections (resulting from dysbiotic events within the microbiota) can enhance antigen secretion. Future investigations into molecular mimicry may elucidate the mechanisms underlying the generation of autoimmunity. While anti-inflammatory therapy presents a promising strategy for managing this condition, further clinical studies involving human subjects are necessary to validate its efficacy.