Patients with EMS exhibited the abnormal secretion of Luteinizing Hormone (LH), potentially leading to ovarian dysfunction [39]. Alterations in folliculogenesis among individuals with EMS may contribute to issues such as ovulatory dysfunction, diminished oocyte quality, reduced fertilization rates, the production of lower-grade embryos, and decreased implantation success [6,40]. In an observational in vitro fertilization (IVF) study involving natural cycles, it was observed that patients with minimal-to-mild EMS had significantly longer follicular phases and lower fertilization rates when compared to women with tubal factor infertility and unexplained infertility [39]. Women with EMS were also found to exhibit a slower rate of follicular growth [41] and smaller dominant follicles compared to women with unexplained infertility [42]. Additionally, EMS patients often experience abnormal follicle development, ovulation, and luteal function [43].

EMS is linked to inflammatory changes in the follicular fluid (FF) environment. A case-controlled study comparing patients with EMS to those with other causes of infertility has noted an increased percentage of B lymphocytes, Natural Killer (NK) cells, and monocyte–macrophages in the FF. This suggests potential alterations in the immunological function within the FF of individuals with EMS [44]. Elevated concentrations of interleukins IL-6, IL-1b, IL-10, and Tumor Necrosis Factor-alpha (TNF-alpha), along with decreased Vascular Endothelial Growth Factor (VEGF), have been documented in the FF of EMS patients [45,46,47]. Immunological changes in the follicular fluid (FF) and serum of women with EMS may contribute, at least in part, to the pathological changes associated with infertility in these patients [8]. For instance, VEGF, known for enhancing follicular health and vascularization, is found in reduced levels in women with EMS [46], potentially associated with diminished embryo quality and implantation rates [6].

Notably, significantly elevated concentrations of TNF-alpha in granulosa cell cultures from women with EMS have been reported [48], which may also be linked to infertility [49]. A study demonstrated that the use of etanercept, an immunoglobulin fusion protein that hinders TNF-alpha activity, reduced the severity of EMS and the size of endometriotic foci in an animal study [50]. Unlike the effect on EMS, the inhibition of TNF-α did not appear to affect infertility [51]. However, the use of etanercept before in vitro fertilization (IVF) enhanced the success rate in cases of advanced EMS [51]. Cytokine changes may influence alterations in the granulosa cell cycle, as previously observed in EMS patients [7]. For example, elevated levels of IL-10 were shown to prevent the natural downregulation of p27, leading to a G0 phase arrest [52]. Several other cytokines, elevated in the FF of EMS patients, such as IL-6, IL-1b, IL-8, or IL-1a, likely contribute to various cell cycle abnormalities, further contributing to subfertility in these patients [7].

Additionally, increased levels of IL-6 in the preovulatory follicles of EMS patients have been found to result in decreased aromatase activity via the MAPK signal pathway. This reduced aromatase activity leads to the decreased intrafollicular conversion of androstenedione to estrone and the subsequently diminished conversion of androstenedione to testosterone, which is aromatized to estradiol (E2) [53,54]. This decrease in follicular E2 levels may result in fertility issues, including reduced fertilizing capacity [53]. Changes in progesterone levels have also been observed in the FF of EMS patients, suggesting that altered steroidogenesis likely plays a significant role in EMS-associated infertility. However, a direct relationship between infertility and modified progesterone levels has yet to be firmly established [6]. Some researchers have proposed impaired LH production as the primary pathophysiology underlying impaired ovulation [39]. It has been suggested that Gonadotropin-Surge Attenuating Factor (GnSAF), a small polypeptide primarily produced by small follicles in the FF, contributes to decreased LH levels in EMS patients. GnSAF decreases the ability of E2 to sensitize the pituitary to the gonadotropin-releasing hormone, thereby reducing the pituitary’s potential to produce LH. Since estrogen levels are lower in the FF of EMS patients, the antagonistic actions of GnSAF against LH production are likely to result in suboptimal LH levels and impaired ovulation [55].

Various studies have explored changes in the composition of follicular fluid (FF) in women with EMS, including cytokines [56,57], oxidative stress markers [58,59,60,61,62,63], metals [64], growth factors, prostaglandins [65], and macrophage activation patterns [66]. The evidence of oxidative stress (OS) in the follicular microenvironment of these women [58,59,60,61,62,63] suggests that both their peritoneal fluid (PF) and FF may contain substances detrimental to oocyte competence acquisition. In this regard, studies evaluating the effect of FF from infertile women with EMS on the in vitro maturation of bovine oocytes have shown spindle and chromosomal damage [67], which could be prevented by adding antioxidants to the maturation medium, implying a pro-oxidant microenvironment in the ovarian follicles of these women [68]. These alterations may result from OS-induced damage to oocyte cell structures. Evidence also indicates higher levels of 8-Hydroxy-2-Deoxy-Guanosine (8OHdG) in the FF of infertile women with EMS, suggesting oxidative DNA damage in cumulus-oocyte complexes and a possible mechanism contributing to impaired oocyte quality in these patients [59].

Altered conditions within the peritoneal fluid (PF) environment of individuals with EMS typically lead to the growth, proliferation, and inflammation of ectopic endometrial tissue [69]. Changes in both humoral and cell-mediated immunity have been observed in the peritoneal environment of EMS patients [70].

A pronounced inflammatory response, accompanied by heightened reactive species and cytokines, can create an unfavorable pelvic environment, which is reflected in the composition of PF in these women [71,72,73,74]. Studies have indicated that changes in the PF composition of women with EMS, encompassing alterations in cellular and humoral mediators [75,76,77,78], including pro-inflammatory cytokines such as TNF-alpha, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-13, IL-17, IL-33, Monocyte Chemoattractant Protein (MCP)-1, Macrophage Migration Inhibitory Factor (MIF), and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) [79,80,81,82,83,84,85], chemokines [86], angiogenic factors [84,87,88], and increased activated macrophages, T-lymphocytes, and NK cells [69], may lead to chronic inflammation, lesion proliferation, and local hormonal imbalances, which, in turn, can result in poor oocyte quality, impaired sperm motility, embryo toxicity, and reduced endometrial receptivity [89].

Increased levels of E2 in the PF of EMS patients have been shown to stimulate the cyclo-oxygenase-2 (COX-2) enzyme, which subsequently upregulates Prostaglandin E2 (PGE2) production. Prostaglandin E2 is the most potent stimulator of aromatase expression in endometriotic tissue [52], leading to elevated E2 production, further promoting its own proliferation and growth [7]. E2 and PGE2 then upregulate COX-2, initiating a positive feedback loop, resulting in a sustained endometriotic state [9]. Pregnancy-associated plasma protein (PAPP-A), produced by the endometrium, ovary, and placenta, exhibits protease activity towards insulin-like growth-factor-binding protein-4 (IGFBP-4), which normally suppresses follicular E2 production. The protease activity of PAPP-A reduces Insulin-like Growth-Factor-Binding Protein-4 (IGFBP-4) levels, leading to increased levels of free Insulin-like Growth Factors (IGF), which synergize with Follicle-Stimulating Hormone (FSH). IGF, in conjunction with LH, increases androstenedione and testosterone production, which are then aromatized under the influence of FSH into Estrone (E1) and E2, respectively [54,90]. Typically, this conversion would occur in the follicular aromatase. However, due to decreased aromatase activity in the follicles of EMS patients, the conversion takes place in the endometriotic tissue where there is an abnormally increased expression of aromatase, further accentuating the endometriotic state. This is supported by a study reporting increased PAPP-A levels in the peritoneal microenvironment of EMS patients, with the degree of elevation correlating with disease severity [91].

The production of abundant amounts of Reactive Oxygen Species (ROS) by elevated numbers of macrophages and polymorphonuclear leukocytes in the PF of EMS patients has been documented [92]. Two studies have also found increased ROS in EMS PF, although the levels were not significantly different from those in disease-free controls [93,94]. Based on these studies, it is believed that the development of oxidative stress (OS) in the local peritoneal environment may be one of the contributing factors in the chain of events leading to EMS-associated infertility [95]. It has also been proposed that redox levels may modulate the severity, dynamics, and progression of the disease. The increase in both the number and activity of macrophages in EMS is accompanied by the release of additional cytokines and other immune mediators, such as nitric oxide (NO) [96]. Nitric oxide is a free radical and a regulator of apoptosis [97]. Low levels of NO play an essential role in ovarian function and implantation. However, higher levels of NO and nitric oxide synthase (NOS) are observed in the endometrium of women with EMS [98].

In the PF of women with EMS-associated infertility, the total antioxidant capacity is reduced, and individual antioxidant enzymes, such as superoxide dismutase, are significantly lower [99]. Moreover, lipid peroxide levels are highest among patients with EMS, suggesting a potential role for ROS in the development of EMS [71]. An imbalance between antioxidants and oxidants has been reported in many studies investigating PF from women with EMS. It has also been theorized that ROS may contribute to the formation of adhesions associated with EMS. Although adhesions due to EMS are known to reduce fertility, the precise mechanism remains incompletely understood [100]. Additionally, alterations in folliculogenesis, likely caused by OS, may impair oocyte quality and have been proposed as a cause of subfertility associated with EMS. Levels of the OS marker, 8-hydroxy 1-deoxyguanosine, were higher in patients with EMS compared to those with tubal, male-factor, or idiopathic infertility [101].

OS has also been shown to induce genomic and mitochondrial DNA damage [68], directly resulting in reduced fertility [102]. It was observed that spermatozoa exhibited heightened DNA fragmentation when exposed to peritoneal fluid (PF) from patients with EMS. Moreover, the degree of fragmentation increased in correlation with the stage of EMS and the duration of infertility [103]. Similarly, oocytes displayed increased DNA damage when subjected to PF from EMS patients, with the extent of damage being influenced by the duration of exposure to the PF [104]. As anticipated, embryos cultivated in the PF of EMS patients also exhibited DNA fragmentation [105]. This elevated DNA damage in sperm, oocytes, and resulting embryos is believed to contribute to the higher rates of miscarriages, as well as fertilization and implantation failures, observed in individuals with EMS [103].

The presence of activated macrophages, cytokines, elevated TNF-alpha levels, heightened growth factors, and oxidative stress (OS) within the peritoneal fluid (PF) of infertile women affected by EMS can potentially exert toxic effects on sperm function [105,106,107,108]. These altered factors may lead to reduced sperm motility [107,109], induce sperm DNA fragmentation [108], promote abnormal sperm acrosome reactions [110], disrupt sperm membrane permeability or integrity [111], impair the interaction between sperm and the uterine tube epithelium [112], and hinder sperm–oocyte fusion [106]. These mechanisms represent additional potential contributors to infertility in individuals with EMS.

Uterine receptivity refers to the capacity of the endometrium to facilitate the normal implantation of an embryo [113]. When uterine receptivity is compromised, it can lead to various reproductive issues, ranging from the inability of embryos to implant (resulting in infertility) to incomplete or insufficient implantation (which may result in miscarriages). Histologically, uterine receptivity is established during the mid-luteal phase, commonly referred to as the “window of implantation” (WOI). This critical period is characterized by the occurrence of decidualization, a process that takes place in each menstrual cycle independently of the presence of an embryo [114,115]. However, conditions like EMS can disrupt this delicate process. EMS can interfere with uterine receptivity through various mechanisms, including the dysregulation of important signaling pathways and molecules in endometrial stromal cells, alterations in the expression of genes within the endometrium, changes in cell physiology, and the development of vascular abnormalities, among other factors [114].

In a well-functioning endometrium, the signaling of progesterone (P4) and estrogen is intricately synchronized, following a pattern dictated by the phase of the menstrual cycle. This coordination plays a vital role to maintain a regular menstrual cycle, facilitating successful embryo implantation, and fostering the progression of pregnancy [116]. During the proliferative phase, estrogen stimulates the proliferation of epithelial cells, whereas P4 counters the effects of estrogen, marking the onset of the secretory phase. This phase change prompts stromal cells to undergo decidualization. The disruption in the balance of these hormones—characterized by P4 resistance and an overwhelming influence of estrogen dominance—leads to EMS-associated infertility [116,117].

P4 serves as a critical regulatory hormone, preparing the uterus for implantation and sustaining pregnancy. Its effects hinge on the progesterone receptor (PR), with two subtypes (PR-A and PR-B) acting as ligand-activated transcription factors. Their absence, as seen in PR-specific knockout, leads to pregnancy failure due to unchecked estrogen-driven epithelial cell growth and compromised stromal decidualization [118]. Female mice lacking both PRA and PRB experience reproductive issues, underscoring the importance of epithelial PR expression for successful embryo implantation, the containment of estrogen-driven epithelial proliferation, and stromal decidualization. When the endometrium responds inadequately to P4, it is termed P4 resistance, notably seen in EMS as an inability to activate PR [119]. This receptor expression links to various transcriptional pathways. Chromatin-remodeling protein ARID1A, integral to steroid hormone signaling, potentially regulates PR as a target gene. Reduced ARID1A levels are associated with infertile EMS patients’ endometrium [120].

The P4-triggered Ihh-COUP-TFII signaling axis orchestrates essential epithelial–stromal interactions for embryo implantation and stromal decidualization. Indian Hedgehog (Ihh) ligands engage patched receptors (Ptch1 and Ptch2), heightening COUP-TFII expression in stromal cells. COUP-TFII, an orphan nuclear receptor, influences proper embryo implantation and decidualization. Ihh’s orchestration of uterine receptivity and decidualization, via pathways like Ihh-Ptch1-COUP-TFII-Hand2-Bmp2 and Wnt4 signaling, is hindered in EMS [121]. EMS-associated molecular signaling disruptions, such as KRAS activation and elevated SIRT1/BCL6, may impede Ihh signaling in the endometrium. This signaling axis significantly mediates PR, thus impaired Ihh signaling contributes to EMS-associated progesterone resistance [122].

Wnt signaling, particularly the Wnt/β–catenin pathway, significantly contributes to blastocyst activation, uterine development, and decidualization. During mouse uterine decidualization, Wnt signaling dynamically expresses diverse Wnt ligands, Frizzled Receptors (Fzd), inhibitors (Dkk1, Sfrps), and transcriptional activators (β-catenin, etc.). Bone morphogenetic protein 2 (Bmp2), the downstream of PR, acts as a key paracrine factor, transmitting embryonic adhesion signals from the epithelium to stroma, initiating decidualization. Bmp2’s role in decidualization through FK506-binding proteins (Fkbps) and Wnt ligands is evident in conditional knockout mouse models. Uterine β-catenin knockout impairs endometrial decidualization [123]. PR interacts with nuclear transcription factors to regulate decidual processes. FOXO1 collaborates with PR to promote the expression of decidua-related genes (IGFBP1, PRL). Reduced FOXO1 expression in EMS patients’ secretory endometrium is attributed to hyperactive PI3K/AKT pathways [124,125]. Mutations in PR lead to sterility in mice, characterized by diminished or absent ovulation, uterine hyperplasia, the absence of endometrial decidualization, and the restricted development of mammary glands [116].

Estrogen plays a pivotal role in regulating the transition of the endometrium into a receptive state. In a mice model of delayed implantation treated with P4, researchers observed that the receptive window’s duration was extended with lower estrogen levels, but rapidly shortened with higher concentrations [126]. Thus, the estrogen levels dictate the receptive window’s duration in the uterus. EMS is an estrogen-dependent gynecological disorder characterized by excessive estrogen signaling, leading to an elevated production of 17-β estradiol (E2). This affects both the eutopic endometrium and ectopic sites. Dysregulated 17β-HSD1 expression, rather than aromatase anomalies, has been linked to hyperestrogenism [127].

Maintaining an optimal balance between pro- and anti-inflammatory factors at the maternal–fetal interface is crucial for a successful pregnancy. Excessive inflammation is unfavorable for embryo implantation. Both peripheral blood (PB) and peritoneal fluid (PF) in EMS patients contain an array of substances secreted by endometriotic implants and immune cells, such as growth factors, steroid hormones, and inflammatory agents, creating an inflammatory microenvironment [128,129]. The profile of pro- and anti-inflammatory cytokines evolves dynamically in EMS patients. In EMS, the cytokine profile in PF shifts from unfavorable (e.g., IL-1β, IFN-γ, and TNF-alpha) to favorable (e.g., IL-4, IL-10, and TGFβ) for pregnancy as the condition progresses [130,131]. Elevated levels of TNF-alpha, IL-1, IL-6, and IL-17 in PF are generally observed in EMS patients [83]. Research by Lessey et al. revealed that PF negatively affects the endometrium (specifically LIF and integrin αvβ3) in EMS-afflicted women [132], suggesting that inflammatory cytokines in PF directly impact the endometrium. The study also found higher levels of pro-inflammatory cytokines (IL-1α, IL-1β, and IL-6) in the endometrial fluid of EMS patients [133].

The immune cells implicated in the initiation and progression of endometrial lesions encompass macrophages, neutrophils, NK cells, dendritic cells, and regulatory T cells (Treg). These immune cells release chemokines and cytokines, crucial for communication that influences endometrial receptivity for embryo implantation. Emerging evidence suggests the altered immune status in both endometrium and peritoneal environment in EMS patients, contributing to infertility and pregnancy failure [134,135]. Uterine NK (uNK) cells are crucial for successful embryo implantation and pregnancy. They contribute in embryo bio-sensing and impact endometrial fate decisions during implantation [136]. In cases of EMS, elevated CD16+ uNK cells might lead to an increased infertility risk due to an adverse inflammatory environment affecting implantation and decidualization [137].

Methylation changes in the Human Homeobox A10 (HOXA10) genes are significant as their dysregulation can contribute to EMS. HOXA10 expression is regulated by estrogen and progesterone [130]. These genes are pivotal for normal endometrial changes during the menstrual cycle, governing growth, differentiation, and embryo implantation [137,138]. In EMS patients, reduced HOXA10 expression during the secretory phase leads to decreased uterine receptivity and related infertility [136,138].