Unveiling the fibrotic puzzle of endometriosis: An overlooked concern calling for prompt action

Fibrotic endometriosis overview: knowledge gaps and challenges

Endometriosis is characterized by the persistent occurrence of fibrosis and myofibroblasts within endometriotic lesions, which play a critical role in the disease’s development, making fibrosis a molecular hallmark of endometriosis.²³ Significant scarring is commonly linked to endometriosis.²³ Although the initial onset of endometriosis is associated with the existence of endometrial stromal and glands in abnormal locations, often the endometrial components are soon replaced by fibrotic and smooth muscle components.²⁴ Rectovaginal nodules frequently display glandular epithelium embedded deeply within fibromuscular tissue, devoid of any surrounding stroma.²⁵ Additionally, in 40% of ovarian endometriomas, there is no detection of endometrial epithelium, and the interior of the cyst is covered solely by fibrotic tissue.²⁶ Despite being a crucial pathological feature of the disease, pelvic adhesions generally lack any endometrial components.¹⁴ These adhesions contribute to the pathology of some common symptoms of endometriosis, including chronic pelvic pain, deep dyspareunia, and infertility, which may be influenced by pelvic adhesions.¹⁴ In fibrosis of organs like lungs, liver, and kidney, involvement of TGF-β signaling pathway is well documented.²⁷ TGF-β is an influential growth factor and a chemical that attracts monocytes and is capable of triggering fibrosis and angiogenesis in abnormal growths and promoting the advancement of endometriosis.²⁸ In comparison to normal women, the peritoneal fluid of stage III and IV endometriosis patients exhibits greater levels of TGF-β.²⁹ The process by which endometriosis progresses to a malignant condition remains unknown. However, continuous inflammation, immunological dysregulation, and fibrosis, most likely caused by iron-induced oxidative stress, may lead to genetic changes, which may lead to a malignant feature.^30,31 Fibrosis is believed to be linked to pain, which is the disease’s most common symptom and the principal cause of the patient’s poor quality of life.³² If the underlying mechanisms are uncovered, they may explain why the disease’s morphological characteristics do not match the extent and nature of fibrosis-induced pain sensations.³³ Fibrotic tissue is defined by excessive development of extracellular matrix (ECM) components inside and around inflamed or damaged tissue, and it is a typical and significant phase of tissue repair in all organs. Fibrosis involves activated platelets, macrophages, and myofibroblasts, which result in increased collagen deposition.³⁴ Furthermore, fibrosis occurs with the transition from epithelial to mesenchymal cells in variety of malignancies, which is associated with poor prognosis.³⁵ Fibrosis and smooth muscle metaplasia are two of the main characteristics of endometriosis in women; fibrosis is found surrounding the endometriotic tissue, and the degree of fibrosis is connected with the degree of smooth muscle metaplasia.³⁶ Based on these data, we postulate that fibrotic-based EMTs’ involvement in chronic inflammatory responses may be a factor in the invasive nature of endometriotic lesions. Also, angiogenesis which stimulates endothelial function, vascular permeability, and the emergence of experimental endometriosis, is commonly associated with this heightened invasive and metastatic potential.³⁷ Endometriotic lesions are thought to be “wounds” that undergo repeated tissue injury and repair (ReTIAR) through the processes of smooth muscle metaplasia (SMM), fibroblast-to-myofibroblast trans-differentiation (FMT), and epithelial-mesenchymal transition (EMT). This process ultimately leads to fibrosis and is a common feature of all endometriotic diseases.^31,38 Epithelium-mesenchymal transition (EMT) is characterized by polarised, stationary epithelial cells change into highly motile mesenchymal cells.³⁹ This makes it possible for solitary cells to pass through the basement membrane, grow invasively, and metastasize by both intra- and extravasation. Sampson’s implantation theory states that each of these occurrences is necessary for the development of an endometriotic lesion.⁸

Regretfully, due to differences in opinion over the etiology of the disease, the EMT route has received less attention in the context of endometriosis than it does in cancer research. In recent times, most research on EMT in endometriosis focuses on tissues; very few examine the specific transcription factors involved in EMT signaling that are present in endometriotic cells.^40,41 EMT-related processes in endometriosis have been reported to be much higher in ectopic endometrial lesions than in eutopic endometrium.⁴² As a result, endometriosis etiology may involve EMT. We speculate that EMT and fibrosis processes may be involved in the evolution of endometriosis, given its chronic nature and the possibility of it leading to fibrotic adenomyosis. Additionally, in their mouse model of endometriosis, Modi et al., discovered significant inflammation but no histological fibrosis and no epithelial-mesenchymal transition concluding EMT and fibrosis are not typical in endometriosis.⁴³ Consequently, studies on the molecular pathways based on EMT or possible targets for therapeutic intervention for EMT and fibrosis in endometriosis were stopped due to the unavailability of a animal model of endometriosis that mimicked the human condition. Endometriosis research is mostly based on non-human primate or rodent models due to the apparent limitations and ethical concerns of human experimentation. The available mice models have aided in the investigation into several aspects of the disorder, such as early disease phases,⁴⁴ steroid hormone involvement,⁴⁵ host inflammatory mechanisms,^46,47 oxidative stress,^48,49 neuro-angiogenesis,⁵⁰ and infertility⁵¹ were also studied in mice. However, there is a paucity of information on the development of pre-clinical models that define clinically effective endpoints such as fibrosis or EMT-induced metastasis. Furthermore, 50-70% of drugs that advance to phase II and III of clinical trials are unable to show efficacy.⁵² This suggests that there are not enough disease models to investigate crucial biological processes. To conclude, we want to highlight that an ideal animal model developed specifically to study endometriosis should include the same cellular and pathophysiological pathways and clinical behaviours that are observed in endometriotic patients, such as fibrosis leading to scar formation and EMT linked to invasion and metastasis.

Endometriotic models: Importance of addressing gaps in pre-clinical animal models

According to Greaves et al., endometriosis is currently being studied using two basic approaches: human-based in vitro samples and experimental in vivo animal models. The first type involves experimental in vitro research using tissue biopsies and fluids obtained from resected lesions or aspiration biopsies, such as endometrial and peritoneal explants, endometriotic cell lineages, primary endometrial stromal cells, endometrial stem cells, and immune cells.⁵³ In vivo animal models are essential for assessing drug candidates and for preclinical trial testing. Our knowledge of the early phases of disease development, including the effects of peritoneal microenvironment, inflammatory responses, and steroid responsiveness has improved because to these models.⁵⁴ For several reasons, it is challenging to create in vitro or in vivo models that mimic or represent the salient characteristics observed in endometriotic patients, such as EMT or fibrosis. One, the condition is multifactorial, heterogeneous complexity, as none is certain of the condition’s onset or duration. Second, there are many disease characteristics associated with endometriosis, including peritoneal, deep infiltrative lesions, and ovarian endometrioma. Lastly, endometriosis cannot be modelled based on a particular pathophysiological mechanism.⁵⁵ Furthermore, endometriosis has been connected to genetic,⁵⁶ immunological,⁵⁷ environmental,^58,59 and hormonal changes such as progesterone resistance⁶⁰ and estrogen dependency⁶¹ further posing challenge in creating a suitable animal model (Figure 1).

Figure 1. Schematic representing the local triggers responsible for the development of endometriosis (Created with Biorender.com).

One of the most significant obstacles in endometriosis research is the lack of reliable animal models. Ideally, a disease model should mirror human disease while also allowing researchers to investigate the effects of intrinsic (e.g., genes) and extrinsic (e.g., environment) factors on disease progression. Many previous studies linked fibrosis secondary to the development of endometriosis and there has not been much research on the study of fibrosis. Based on research from animal models of the condition, it became clear that a percentage of women receiving hormone therapy in human trials were not responding to these drugs⁶² requiring surgical lesion removal to alleviate symptoms. Women may have endometriotic lesions that have progressed to a fibrotic state by the time they seek medical attention, rendering treatment ineffective. Significant evidence supporting the process of fibrosis comes from in vitro experiments conducted on humans and from in vivo higher vertebrates such as baboons.

Primate model of endometriosis

Despite a recent surge in endometriosis research, the underlying pathobiology of the disease remains poorly known, implying that animal models of the disorder are crucial for future studies in this field. Non-human primates and higher vertebrates are regarded to be potential candidates for disease research due to their anatomical resemblance to human reproductive organs.⁶³ Controlled experimental investigations on humans are limited because assessing disease prevalence and development necessitates numerous laparoscopies, which are challenging for a variety of reasons. Though endometriosis occurs spontaneously in humans, human investigations have been limited for ethical and practical reasons which is one of the primary reasons being the difficulty of studying the disease. As a result, understanding the mechanisms that cause this disease requires the use of an appropriate animal model. Endometriosis has long been investigated in both primate and non-primate animals. The spontaneous endometriosis of the baboon^64–66 limitation is that baboons have vast and effective mechanisms for clearing and regenerating their peritoneum⁶⁶ the rhesus monkey^67,68 where limitation is the significance of peritoneal cysts in endometriosis pain and discomfort was not investigated. The cynomolgus monkey^69,70 has been described, with the limitations that deep lesions were difficult to diagnose and time course changes in the condition were not investigated. Though non-human primates are an excellent model for studying endometriosis, they are expensive to maintain and are extremely sensitive to captivity. Furthermore, spontaneous endometriosis occurs at a low frequency, limiting the use of primates in research.⁷¹ However, because research facilities for primates are restricted, non-primate experimental animal species, such as mice or rats, are regarded to provide an ideal first-line technique for investigating the etiology of this mysterious disease.

Rodent models of fibrosis

Every menstrual cycle, endometriosis is characterized by the development of new lesions and the advancement of pre-existing lesions. Therefore, additional research is required to comprehend the endometriosis lesion’s natural course and their gradual development.⁷² There is evidence of gradual lesion clearing, but only a small number of studies using mouse models of endometriosis have studied disease induction and regression.^72,73 It is unethical to perform many laparoscopies on endometriosis patients to monitor disease progression. So, longitudinal studies of lesion formation and progression can considerably increase the translational efficiency of pre-clinical model of endometriosis.⁷²

Mice are the most popular experimental animal models due to their ease of gene manipulation, availability, and handling, tissue similarity in vivo, small size and large litter, which make them cost-effective, and their relatively short gestation, which allows for transgenerational examination.⁷⁴ Based on the vast majority of already available research publications, two types of mice models have been successfully used to implant endometriotic lesions. The first approach involves suturing, whereby human endometriotic implants are surgically auto-transplanted into the peritoneum of immunocompromised mice.^75–77 The second approach involves intraperitoneal or subcutaneous implantation of autologous uterine segments into the peritoneum of recipient mice from a syngeneic donor.^50,54,78 Although there are numerous reports describing the spontaneous attachment, growth, and proliferation of endometriotic lesions, these lesions do not accurately reflect human endometriosis because they do not exhibit characteristics like chronic, persistent fibrosis for internal scarring, or invasiveness based on EMT. Moreover, the animal models provide data on the inflammatory processes generated by implanted lesions rather than those caused by endometriosis. Rats can only produce superficial lesions, which are the most fundamental and possibly least clinically significant types of lesions. The inability of any study to recreate fibrotic endometriotic lesions may account for the failure of rat models to yield data relevant to the pathophysiology and treatment of human endometriosis. This situation demonstrates that the preclinical animal studies that have been established are not transferable.⁷⁹ Many studies using rodents as a model for endometriosis have looked at the gene expression patterns of ectopic tissue deposits in rats in an attempt to correlate them with human endometriotic lesions. Chronic inflammation, angiogenesis, and extracellular matrix remodeling have all been found to be common pathways.^80–82 While some aspects of the disease are replicated in the rodent model, all the modifications involve suturing uterine fragments (endometrium plus myometrium) to different sites, which does not accurately represent the formation of lesions from those shed endometrial tissue or the dissemination of menstrual tissue into the peritoneum. It is noteworthy that, particularly in terms of understanding its pathophysiology and treatment options, the current rodent models have not been successful in yielding findings that apply to human endometriosis. Therefore, fibrosis a mostly disregarded component of human endometriosis be taken into consideration.^83,84 However, efforts to translate the results into humans were unsuccessful in offering effective endometriosis treatments. Therefore, developing novel animal models that mirror the continuous fibrotic process seen in endometriotic patients is essential in improving our understanding of the disease. An increasing amount of research has recently brought attention to the role that fibrosis plays in clinical-grade endometriosis. On the other hand, little is known about fibrosis treatment strategies. Therefore, it is critical to develop a fibrotic mouse model of endometriosis, elucidate the regulatory processes behind fibrosis in endometriosis, and identify more precise specific biomarkers for the disease. These markers can also be utilized to find effective therapy targets and identify endometriosis in its early phases (Figure 2).

Figure 2. Schematic illustration of endometriotic lesion milieu and variables expressed contributing to progression of peritoneal endometriosis (Created with Biorender.com).

To mimic the fibrotic scarring observed in endometriosis, many endometriotic fibrotic animal models have been developed (Table 1). Furthermore, new in vivo models that use stromal cells generated from menstrual blood have been created to study endometriosis; these models show enhanced endometriotic cell migration and proliferation.⁶⁰ Many cues, including estrogen stimulation, may trigger the epithelial-mesenchymal transition.⁸⁵ Furthermore, estrogen-induced EMT of Ishikawa cells promotes adenomyosis.⁸⁶ However, it remains still unknown how estrogen causes EMT in endometriosis at the molecular level. To prevent fluctuations in the mice’s estradiol levels during the estrous cycle, the majority of established mouse models use ovariectomized mice.^87–90 As a result, the steady availability of estradiol in the circulation may help to promote lesion establishment and growth. But this makes it impossible to research how estrogen-induced EMT in endometriosis affects fertility like in women with normal circulating estrogen. Therefore, studies of endometriosis produced in intact mice call for more research on the connection between ectopic tissue and fertility.

Human experiment details

In endometriotic lesions, it is known that TGF-β family members, Notch receptor, and bioactive sphingolipid sphingosine 1-phosphate (S1P) cause tissue fibrosis and change signaling pathways.⁹¹ It has been found that NF-κB is activated in endometriotic lesions and peritoneal macrophages, which are essential for the inflammation associated with endometriosis. It has been demonstrated that inhibiting NF-κB lowers the development and progression of endometriosis in women as well as its associated symptoms.⁹² Estrogen can promote the formation and dissemination of endometriosis ectopic lesions by upregulating the expression of the transcription factor Slug in ectopic endothelial cells and inducing the epithelial-mesenchymal transition.⁸⁶ Fibrogenesis in endometriosis may be facilitated by aberrant Wnt/β-catenin pathway activation and reversed by blocking the Wnt/β-catenin pathway.⁹³ TGF-β1 may stimulate the expression of N-cadherin, OCT4, and Snail in ectopic stromal cells, implying that TGF-β1 facilitates cell invasion.⁹⁴ The AKT and ERK signaling pathways may work synergistically to promote the formation of deep endometriotic lesions by increasing endometriotic stromal cell proliferation in a fibrotic milieu in vitro.⁹⁵

By inducing EMT and FMT in endometriotic lesions, platelet-derived TGF-β1 stimulates smooth muscle metaplasia (SMM) and fibrosis.⁹⁶ Evidence suggests that EMT induces fibrogenesis in addition to increasing cellular invasiveness. For instance, TGF-β1/Smad3 signaling pathway, which is driven by platelets, is known to induce EMT and FMT in endometriotic lesions, which eventually results in SMM and fibrosis.⁹⁶ Targeting TGF-β1 may be an effective strategy to prevent fibrosis and adhesion formation since endometriotic cells release TGF-β1, which induces ECM disorganisation and fibrosis in the tissues of ovarian endometriotic patients.⁹⁷ Oxidative stress has been linked to the ADAM17/Notch signaling pathway and perhaps fibrosis, according to a study done on endometriosis patients.⁹⁸ Furthermore, it is known that endometriotic cells of endometriomas express Smad2, Smad3, and Smad4 (as well as their phosphorylated forms), which causes fibrosis and adhesion to ovarian tissues, suggesting a role for TGF- β1/Smad signaling.⁹⁷ FOXP1 uses Wnt signaling to increase fibrosis in endometriosis.⁹⁹ Through their effects on tissue repair, senescence, EMT, FMT, and proliferation of fibroblasts/myofibroblasts, mutations in TP53, PTEN, ARID1A, PIK3CA, KRAS, and PPP2R1A appear to hasten the development and fibrogenesis of endometriosis.¹⁰⁰ A significant increase was observed in the mRNA levels of α-SMA, vimentin, N-cadherin, fibronectin, PAI-1 (Serpine1), Snail, Slug, and LOX.¹⁰¹ Growth factors such TGF-β1, PDGF, EGF, and CTGF are released by activated platelets in lesions, facilitating fibrogenesis in endometriotic patients with deep endometriosis and ovarian endometrioma.¹⁰² NR4A1 is a novel pro-endometriotic transcription factor that accelerates the development of endometriosis.¹⁰³ HOXC8 stimulates TGF-β signaling, which affects adhesion, cell proliferation, migration, and ovarian endometrioma.¹⁰⁴ FAK intracellular non-receptor tyrosine kinase mediates a series of processes in the development of endometriosis, including cell adhesion, inflammatory response, and fibrosis signaling in patients with endometriomas.¹⁰⁵ In ectopic ESCs derived from retrograde menstruation, PGE2/thrombin is known to induce modifications such FMT and EMT, which are linked to fibrotic changes in the lesions.⁴¹ Through EMT and FMT processes, proinflammatory substances such PGE2 and thrombin in retrograde menstrual fluid have been jointly implicated in generating endometriosis fibrosis in endometriotic patients. This suggests potential targets for treatment to mitigate fibrosis.⁴¹ Apart from the fibrotic and EMT markers, numerous processes, such as pyroptosis, NLRP3 inflammasome, and deregulation of the long noncoding RNA MALAT1 are identified to cause fibrosis in endometriotic patients.⁸⁷ It has been discovered that reducing the number of lesions by targeting inflammatory molecules like IL-8 also known to reduce fibrosis and adhesions, highlighting the potential for disease-modifying therapy.¹⁰⁶ While existing research has shed light on the genes involved, there is still a potential to uncover the intricate downstream signaling networks that govern this complex disease. As a result, sophisticated additional approaches, such as knockout models that incorporate high throughput RNA sequencing and omics methodologies should be emphasised to further validate the role and mechanism of fibrotic markers in the development of fibrosis, providing solid proof for the discovery of drugs that hinder, terminate, and reverse fibrosis progression and benefit endometriotic patients.

The interplay of EMT and MMPs in endometriosis

Endometriosis is a common benign gynaecological disease with a high propensity for migration and invasion. The cell-to-cell or cell-ECM connections allow the cells to migrate, invade, and proliferate in new locations. MMPs are linked to adhesion, invasion, and the severity of endometriosis. This indicates that MMPs have a role in extracellular matrix remodeling, which is necessary for the development of ectopic endometriosis lesions.¹⁰⁷ They are also significantly higher in the endometrial and peritoneal fluid of endometriosis patients.^108,109 Matrix metalloproteinases (MMPs) are a family of enzymes that are mostly found in the endometrium’s functional layer. They are secreted by the resident immune cells and stromal fibroblasts, which facilitate the remodeling of the extracellular matrix including collagen, elastins, and other glycoproteins and endometrial disintegration during menstruation. Tissue inhibitors of matrix metalloproteinases (TIMP) are endogenous antagonists that reduce MMP overexpression, and ovarian steroid hormones are known to control MMP activity.¹¹⁰ For early clinical studies of EMT, the nude mouse is a suitable model, particularly for the identification of MMP-2 and TIMP-2, proteins that seem to play a significant role in the pathophysiology of EMT. It has been found that estrogen specifically increases MMP-2 expression to encourage ectopic implantation of the endometrium. Progestin, on the other hand, can suppress TIMP-2 expression, increasing the MMP-2/TIMP-2 ratio and increasing the invasiveness of ectopic endometrium to facilitate implantation.¹¹¹ In ovarian endometriosis, MMP7 facilitated the epithelial-mesenchymal transition; EGF increased MMP7 expression by activating the ERK1-AP1 pathway.^112,113 MMP14 affects the development and function of invadopodia, which in turn modulates mesenchymal cells’ capacity for invasion and migration.¹¹⁴ MMP-2 and MMP-9, two important enzymes involved in the destruction of diverse types of ECM, have been linked to the development of endometriosis by regulating endometrial cell invasion.¹¹⁵ MMP-2 and MMP-9 have been shown to operate as both biomarkers of EMT and triggered factors that contribute to the progression of EMT.¹¹⁶ As a result, we hypothesize that MMPs may be crucial in controlling the endometriosis-related EMT process. However, further research is required to fully understand the connection between MMPs and the epithelial-mesenchymal transition in endometriosis, as there are not enough comprehensive studies on the subject. Despite this, it is apparent that MMPs play a crucial role in collagen production, which is necessary for endometriosis fibrosis to develop gradually.¹⁰⁷ These findings suggest that there may be a precise equilibrium between collagen synthesis and breakdown, which should be investigated further.