The role of fibrosis in endometriosis: a systematic review

Endometriosis is the most prevalent benign gynecologic disease, affecting approximately 190 million people worldwide ( Zondervan et al. , 2020 ). Persons with endometriosis can experience a variety of symptoms, including dysmenorrhea, chronic pelvic pain and subfertility, resulting in a severe decrease in quality of life ( van Aken et al. , 2017 ). Because of the high prevalence of endometriosis and its debilitating effects, endometriosis causes a huge burden, both on an individual as well as at a societal level ( Simoens et al. , 2012 ). Nevertheless, many aspects of this disease remain to be investigated. Endometriosis is characterized by the presence of endometrial-like tissue implants outside the uterine cavity and can be present on the peritoneum, the pelvic organs, in scar tissue after caesarian section and in the thoracic cavity. It is subdivided into three types: peritoneal endometriosis (PER), ovarian endometriotic cysts or endometrioma (OMA), and deep endometriosis (DE). On a histological level, lesions consist of endometrial stromal and epithelial glandular cells and fibrotic deposits ( Camboni and Marbaix, 2021 ). The presence of stromal and epithelial glandular cells in a surgical derived biopsy from visually suspected areas is currently the main criterium of histopathologic diagnosis of endometriosis. However, several leading research groups have recently proposed to highlight fibrosis in the histopathologic definition as well ( Guo, 2018 ; Vigano et al. , 2018 ). Thereby, fibrosis and myofibroblasts, the main extracellular matrix (ECM)-producing cells, are hypothesized to be accountable for endometriosis-related symptoms ( Odagiri et al. , 2009 ; Yan et al. , 2019b ; Garcia Garcia et al. , 2023 ). On the other hand, we know that fibrosis is progressive over time and corresponds with PER lesions changing from red to black to white, which may support the opposite hypothesis that fibrosis is a self-limiting end stage of disease, stopping lesion growth and cyclical bleeding ( Matsuzaki et al. , 1999 ; Zhang et al. , 2016b ). This contrast brings up the question of whether fibrosis is a beneficial or an unfavorable effect. Under normal circumstances, myofibroblasts fulfill an important role in wound healing ( Almadani et al. , 2021 ). By their contraction and production of ECM, myofibroblasts have the ability to congregate wound edges. After tissue homeostasis is reached, myofibroblasts normally go into apoptosis. However, in fibrotic diseases, myofibroblasts persist in an activated state and continue to produce matrix proteins, leading to excess fibrosis ( Adler et al. , 2020 ). This can result in pain and, in more severe cases, even lead to a progressive loss of organ function ( Hutchenreuther and Leask, 2016 ). Because these potent characteristics of myofibroblasts and the fibrotic process have been proposed to influence endometriosis progress and its symptoms, it is important to study the myofibroblasts in lesions to gain more knowledge about the exact role of fibrotic processes in the disease. Fibrosis is defined as the excess deposition of ECM components, mostly collagen, and usually arises during wound healing and inflammation processes ( Kuehlmann et al. , 2020 ). Among the cells forming the stromal compartment of endometriosis, myofibroblasts have a pivotal role as they are responsible for this excessive production of ECM leading to fibrosis ( Adler et al. , 2020 ). The myofibroblasts mainly derive from fibroblasts by a process called fibroblast-to-myofibroblast transdifferentiation (FMT) ( Zhu et al. , 2023 ). Other sources of myofibroblasts are epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT) ( Zhang et al. , 2016a ; Yan et al. , 2020a , b ). After myofibroblastic differentiation, cells can differentiate further into smooth muscle cells, in a process referred to as smooth-muscle-metaplasia (SMM) ( Barcena de Arellano et al. , 2011 ; Ding et al. , 2020b ). Transforming growth factor-β (TGF-β) is a key stimulating factor in myofibroblastic differentiation ( Biernacka et al. , 2011 ). TGF-β signaling is known to be a driver of pathologic fibrosis in several fibrotic diseases like systemic sclerosis, idiopathic pulmonary fibrosis, and liver cirrhosis. The activation of TGF-β signaling can result in the activation of several subsequent pro-fibrotic pathways, among which are Smad, Wingless-related integration (Wnt)/β-catenin, Focal Adhesion Kinase (FAK), and Rho/Rho-associated protein kinase (Rho/ROCK) signaling ( Ji et al. , 2014 ; Meng et al. , 2016 ; Zhao et al. , 2017 ; Distler et al. , 2019 ). As pulmonary fibrosis is currently treated with therapeutics interacting in these pathways with some positive effects, parallels between endometriosis and lung fibrosis show potential for investigation ( Amati et al. , 2023 ). In the initial phase of fibrosis, TGF-β and platelet-derived growth factor (PDGF), among other factors, are released due to a continuous or repetitive process of tissue damage and healing, eventually leading to a new fibrotic steady state ( Adler et al. , 2020 ). In endometriosis, TGF-β has a pivotal role as a pro-fibrotic signaling factor ( Hull et al. , 2012 ; Xiao et al. , 2020 ). It acts as a repetitive signal of tissue injury, potentially triggered by cyclical bleeding as a consequence of the hormonal responsiveness of the endometriotic cells ( Laux-Biehlmann et al. , 2015 ). In this process, platelet activation, macrophage infiltration and neuropeptide secretion may be triggered to further stimulate fibrosis ( Zhang et al. , 2016a ; Duan et al. , 2018 ; Liu et al. , 2019 ; 2020 ). Recently, two reviews focusing on fibrotic pathways in endometriosis have been published ( Vigano et al. , 2020 ; Garcia Garcia et al. , 2023 ). Garcia Garcia et al. , highlighted important differences in fibrotic processes in ovarian as compared to deep endometriosis. EndoMT contributes more to fibrotic development in ovarian endometriosis, whereas sensory nerves and smooth muscle cells are predominantly involved in deep endometriosis. Vigano et al. , provided insight in the cellular processes that are involved in fibrogenesis. Platelets, macrophages, ectopic endometrial cells and sensory nerves produce a variety of cytokines and neuropeptides involved in fibrotic signaling ( Vigano et al. , 2020 ). Closely related to fibrosis is the inflammatory environment of endometriosis. The important role of inflammation in endometriosis is illustrated by a disturbed immune cell composition and an abundance of cytokines in the peritoneal fluid and eutopic endometrium of endometriosis patients ( Vallve-Juanico et al. , 2019 ; Abramiuk et al. , 2022 ). Endometriosis can thus be considered both as a fibrotic and as an inflammatory disease ( Zhang et al. , 2019a ; Vigano et al. , 2020 ). However, these two aspects cannot be seen separately based on their inter-connected modifying effects. A yet unanswered question regarding the inflammatory environment is whether endometriotic implants trigger inflammation in their environment, or whether an inflammatory state in the peritoneal cavity and endometrium supports endometriosis development in susceptible individuals ( Izumi et al. , 2018 ). To date, the published reviews have focused on specific aspects of fibrosis in endometriosis, or on fibrosis in specific endometriosis subtypes. They lack a general overview of fibrosis in endometriosis in in vitro , animal and clinical studies. In this systematic review, a comprehensive overview of the current knowledge about fibrosis in endometriosis is presented by congregating various types of research. The aim of this review is to provide a broad basis for researchers exploring fibrosis as a therapeutic target for endometriosis, as resolving fibrosis is a promising strategy for non-hormonal and non-invasive therapeutic options for endometriosis.

This systematic review was reported according to the PRISMA guidelines for systematic reviews ( Page et al. , 2021 ). The protocol was registered in the PROSPERO database (registration number: CRD42022295727) in December 2021. A systematic literature search was performed in September 2023 in the following databases: Pubmed, Embase, and Web of Science. Keywords as well as title or abstract terms for ‘endometriosis’ and ‘ectopic endometrium’, ‘fibrosis’, ‘myofibroblasts’, ‘collagen’, ‘α-smooth muscle actin’ and their synonyms and related terms were combined. The full search strategy is presented in Supplementary File S1 . No restrictions based on publication date or language were applied in the initial search. Duplicates were excluded using EndNote 20. A cited-reference search was performed to identify potential additional relevant articles. Original studies in English reporting about fibrosis in endometriosis were included in this review. In vitro , animal and human studies were eligible if they reported about the development, presence and/or treatment of fibrosis. Fibrosis as an outcome was defined as the histologic analysis of fibrosis-specific staining or by molecular markers for fibrosis, myofibroblasts or their corresponding genes. These are mainly α-smooth muscle actin (α-SMA, gene symbol: ACTA2 ) and collagen type I ( COL1A1 , COL1A2 ), while including others. Reviews and case reports were excluded, as well as studies exclusively analyzing adenomyosis or eutopic endometrium and studies using immortalized cell lines only. The selection of studies was performed independently by two authors (GV and MG) using Rayyan. The first round of selection was based on screening of title and abstract. Studies selected by at least one author were included for full text screening. In case of inconsistency between the authors after full text screening, eligibility was discussed between them. If inconsistency persisted, a third author (AN) was consulted. During the selection process the reasons for exclusion were reported. Data extraction was performed by one author (GV) and systematically checked by a second author (MG). Quality assessment was performed according to validated risk of bias tools: The MINORS tool was used for the observational studies ( Slim et al. , 2003 ); a modified version of the ROBINS tool was used for the experimental studies with human-derived material ( Sterne et al. , 2016 ; Post et al. , 2020 ); and the SYRCLE tool was used for animal studies ( Hooijmans et al. , 2014 ). Quality assessment was performed by one author (GV) and systematically checked by a second author (MG). In case of inconsistency, the risk of bias was discussed between the authors.

Our search yielded 3441 unique articles. After title and abstract screening 342 articles were included for full text assessment, and 142 articles were ultimately included in our review. The study selection procedure is presented in Fig. 1 . There were 11 articles excluded based on their study type being a review or case report; 45 were excluded based on a their publication type (e.g. conference abstracts); 119 studies were excluded based on not reporting fibrosis, meaning that fibrosis was not assessed or not specifically defined; 28 studies were excluded based on not studying ectopic endometriotic tissue; 8 studies in foreign languages were excluded; and six manually detected duplicates were also removed. From the included studies, 44 were human observational studies and 28 were experimental studies using human-derived tissues. The human studies were subdivided per endometriosis subtype. Peritoneal endometriosis was examined in 5 studies, in 33 ovarian endometriosis in 33 and deep endometriosis in 14. In the remaining 20 human studies, more than one endometriosis subtype was examined. In addition to the studies concerning human subjects or tissue, 75 animal studies were included in this review. Some studies were included in more than one group because both human tissue and animal-based experiments were performed. In every following section, studies assessing outcomes at the tissue level are discussed first, and subsequently we zoomed in on cellular and ultimately molecular levels. We started by discussing human observational studies as these most often assessed outcomes at the tissue level. PRISMA flow diagram: schematic representation of the study selection process. An overview of the 44 human observational studies we included is presented in Table 1 . These studies report findings about histological appearance, cellular composition, pro-fibrotic pathways, and clinical parameters. Observational studies. Studies assessing multiple endometriosis subtypes are only shown in the first section of the table and not in the subsequent following categories to avoid double information. Studies including both observational and intervention approaches are shown in both tables. Information depicted in each table is specific regarding that particular part of the study. The conclusion column shows a conclusion as stated by the authors, so this is including results from both parts of the study. Sample size of number of biopsies is shown, in some cases multiple biopsies from a single patient were included separately. PER, peritoneal endometriosis; OMA, ovarian endometrioma; DE, deep endometriosis; HE, hematoxylin/eosin staining; IHC, immunohistochemistry; IF, immunofluorescence; WB, western blot; RT-qPCR, real-time qualitative polymerase chain reaction; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; COL, collagen; CTGF or CCN2, connective tissue growth factor; SM-MHC, smooth muscle-myosin heavy chain; EMT, epithelial-to-mesenchymal transition; FMT, fibroblast-to-myofibroblast transdifferentiation; SMM, smooth muscle metaplasia; SMC, smooth muscle cell; ER, estrogen receptor; PR, progesterone receptor; ASRM score, American Society of Reproductive Medicine score. Fibrosis affected tissue and its main cell type myofibroblasts were observed in almost all endometriotic lesions. Myofibroblast associated with endometriosis were found to differentiate from epithelial and endothelial cells toward mesenchymal and ultimately smooth muscle-like cells. This was based on observations of an increased expression of mesenchymal and smooth muscle cell markers like vimentin, desmin, and smooth-muscle myosin heavy chain in all studies assessing these markers as main outcome measures, as schematically presented in Fig. 2 ( Anaf et al. , 2000b ; Itoga et al. , 2003 ; Yan et al. , 2020a , b ). In general, this stage of differentiation was observed most extensively toward the periphery of lesions ( Barcena de Arellano et al. , 2011 ; Ibrahim et al. , 2019 ). This was suggested to be associated with continuation of differentiation and outgrowth over time, since more thorough FMT and more extensive fibrosis is found in lesions in adults compared to adolescents ( Guo et al. , 2015b ; Ding et al. , 2020b ). Myofibroblasts throughout the central stromal compartment showed less smooth muscle-like characteristics and rather resembled eutopic endometrial stromal cells, suggesting a different stage of myofibroblastic transdifferentiation ( Konrad et al. , 2018 ). It was suggested that myofibroblast activation, marked by transgelin expression, is at least in some cases triggered by endometrial infection by Fusobacterium nucleatum , which thereby gives myofibroblasts the ability to initiate endometriosis after retrograde menstruation ( Muraoka et al. , 2023 ). The macroscopic aspect and color of peritoneal lesions correlated with the collagen content, which was higher in black compared to red lesions, but was not associated with the amount of SMM ( Matsuzaki et al. , 1999 ; Barcena de Arellano et al. , 2011 ). Furthermore, when comparing different endometriosis subtypes, the most extensive fibrosis and myofibroblast transdifferentiation was observed in deep lesions ( Liu et al. , 2018 ). In ovarian endometriosis, fibrosis did form a well-organized lining between cysts wall and ovarian tissue, whereas in other subtypes fibrosis was more scattered ( Khare et al. , 1996 ). Additionally, more fibrosis was found in the cyst wall of endometrioma in patients treated with hormonal therapy compared to untreated patients ( Tsujioka et al. , 2009 ). Schematic representation of cellular transitions contributing to myofibroblasts in endometriotic lesions . Fibroblast-to-myofibroblast transdifferentiation (FMT), epithelial-to-mesenchymal transition (EMT), and, to a lesser extent, endothelial-to-mesenchymal transition (EndoMT) are sources of myofibroblasts in endometriosis, marked by expression of α smooth muscle actin (α-SMA). In FMT, expression of vimentin increases, and fibroblasts thin and elongate. In EMT, the expression of E-cadherin decreases, and expression of vimentin increases. In EndoMT, expression of CD-31 decreases, and expression of fibroblast-specific protein (FSP1) increases. Smooth-muscle-metaplasia (SMM) can lead to smooth muscle-like cells in endometriosis, expressing desmin and smooth muscle myosin heavy chain (SM-MHC). The origin and detailed characterization of cell types present in endometriotic lesions has been studied based on single-cell RNA profiling. Different myofibroblast and macrophage subpopulations were identified across the different subtypes of endometriosis. An abundance of myofibroblasts, marked by Periostin ( POSTN ), Collagen 6A1 ( COL6A1 ), and Fibronectin ( FN1 ), was found in all subtypes. Myofibroblasts in both PER and DE showed similarities based on their additional specific expression of Secreted frizzled-related protein 1 ( SFRP1 ) and Peptidase domain containing associated with muscle regeneration 1 ( PAMR1 ) or Alpha-2-macroglobulin ( A2M ) and Collagen 4A1 ( COL4A1 ), whilst the main myofibroblasts in OMA showed additional Transmembrane protein 19 ( TMEM19 ) and Tenascin C ( TNC ) expression ( Shin et al. , 2023 ). Zhu et al. , showed the abundance of myofibroblasts and macrophages in OMA. They reported a dominant subpopulation of myofibroblasts characterized by genes involved in TGF-β responsiveness, Wnt signaling, and ECM formation. These myofibroblasts mainly derived from FMT and their proliferative potential was very low. The process of transdifferentiation was accompanied by a decreased expression of hormone receptors for estrogen and progesterone and markers for prostaglandin signaling ( Mechsner et al. , 2005 ; Huang et al. , 2021 ). Collagen types I and IV were found to be the most common type of collagen in endometriosis ( Stovall et al. , 1992 ; Nezhat and Kalir, 2002 ). Besides this, the expression of neural cell adhesion molecule (NCAM) and neuropeptides like substance P (SP) and calcitonin gene-related peptide (CGRP) was positively correlated with increased fibrosis and myofibroblast transdifferentiation, as well as with dysmenorrhea severity, indicating a colocalization between sensory nerves and fibrosis ( Anaf et al. , 2000a ; Bonte et al. , 2002 ; Odagiri et al. , 2009 ; Yan et al. , 2019b ; Zhang et al. , 2019a ). These studies hypothesized that due to this colocalization and the correlation between fibrosis and dysmenorrhea severity, fibrosis plays an important role in pain. Compared to healthy endometrium, α7 nicotinic acetylcholine receptor expression was reduced in endometriosis, and correlated negatively with the extent of fibrosis and dysmenorrhea severity ( Hao et al. , 2022 ). These findings indicated a role for the cholinergic anti-inflammatory pathway in endometriosis, supported by animal experiments of this group, which are discussed below ( Hao et al. , 2021 ). Based on the observational studies markers of Smad and FAK signaling pathways were upregulated ( Shi et al. , 2017 ; Nagai et al. , 2020 ). Upregulation of these signaling pathways was associated with upregulation of TGF-β ( van Kaam et al. , 2008 ). Therefore, these pathways were hypothesized to be important pathways in the etiology of fibrosis in endometriosis. Furthermore, the characteristics of fibrosis show the potential to be used for diagnostic purposes in the future. The potential biomarkers osteopontin, high mobility group box 1 (HMGB1), and hyaluronic acid showed a positive correlation with the extent of fibrosis, in contrast to the association between CA-125 and fibrosis, about which literature reported both a positive and a negative correlation ( Vicino et al. , 2009 ; Cao et al. , 2019 ; Selcuk et al. , 2021 ). The stiffness of fibrotic deposits can be detected with elastosonography, which yields a higher diagnostic accuracy than regular ultrasound examination. However, this is only studied for deep endometriosis ( Xie et al. , 2013 ; Ding et al. , 2020a ). From the perspective of fertility, the extent of fibrosis in ovarian cysts is shown to be independently correlated with a decreased follicular density in the adjacent ovary ( Kitajima et al. , 2011 ). On the other hand, Nie et al. , did not find a correlation between lesional fibrosis present within the ovarian endometriosis cyst or α-SMA expression in endometrioma and serum levels of anti-mullerian hormone (AMH). They did report a correlation between ovarian fibrosis present in the ovarian cortex, probably triggered by the endometrioma, and serum AMH, however, the interplay between lesional and ovarian fibrosis herein is still unclear ( Nie et al. , 2022 ). Experimental studies with human-derived material were performed in 28 studies. An overview is presented in Table 2 . Experimental studies with human-derived material. Eutopic endometrium: eutopic endometrium from endometriosis patients as control. Healthy endometrium: eutopic endometrium from non-endometriosis patients as control, in some studies these patients do have other (gynaecologic) diseases, for example, uterine fibroids or mild cervical dysplasia. PER, peritoneal endometriosis; OMA, ovarian endometrioma; DE, deep endometriosis; HE, hematoxylin/eosin staining; IHC, immunohistochemistry; IF, immunofluorescence; WB, western blot; RT-qPCR, real-time qualitative polymerase chain reaction; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; COL, collagen; CTGF or CCN2, connective tissue growth factor; FN, fibronectin; SM-MHC, smooth muscle-myosin heavy chain; EMT, epithelial-to-mesenchymal transition; FMT, fibroblast-to-myofibroblast transdifferentiation; SMM, smooth muscle metaplasia; SMC, smooth muscle cell; ER, estrogen receptor; PR, progesterone receptor; ASRM score, American Society of Reproductive Medicine score. Most experimental studies with human-derived material were focused on identification of cellular mechanisms and signaling pathways affecting fibrosis. At the tissue level, a high stiffness of the cellular environment was identified as a promoter of fibrogenesis in endometriosis ( Matsuzaki et al. , 2016 ). At the cellular level, an important causative factor is the presence of thrombocytes, as these cells are found to be important in promoting fibrogenesis through secretion of TGF-β ( Zhang et al. , 2016a ). The platelet-inhibiting herbal compound tetramethylpyrazine showed potential to stop pro-fibrotic EMT and FMT and thereby inhibit fibrogenesis ( Huang et al. , 2022b ). A pro-fibrotic effect was also observed from nerves and neural cells, through release of neuropeptides substance P and CGRP ( Yan et al. , 2019b ). The accumulation of iron in OMA can trigger ferroptosis by effects of reactive oxygen species and subsequently cause fibrogenesis ( Zhang et al. , 2022 ). Furthermore, Rho/ROCK, Wnt/β-catenin and Smad are found to significantly affect fibrogenesis. Markers of Rho/ROCK signaling were elevated in endometriosis lesions and inhibition with ROCK antagonists, heparin or fasudil led to a decrease of the fibrotic marker protein expression in vitro ( Yuge et al. , 2007 ; Tsuno et al. , 2009 ; Nasu et al. , 2010 ; Tsuno et al. , 2011 ). The pivotal role of Wnt/β-catenin pathway signaling was shown by the inhibitory effect of either β-catenin inhibitors, or forkhead box protein 1 on expression of pro-fibrotic genes ( Matsuzaki and Darcha, 2013 ; Shao and Wei, 2018 ; Hirakawa et al. , 2019 ). Several interleukins (IL), among which are IL-1, IL-3, IL-6, and IL-10, have also been shown to affect fibrogenesis. IL-1 can have both a pro- or anti-fibrotic effect, depending on the grade of inflammation ( Matsuzaki et al. , 2020 ). IL-3, IL-6 and the normally anti-inflammatory IL-10 drive fibrosis via the dysregulated activation of STAT3 ( Matsuzaki et al. , 2022 , 2023 ). Besides these pathways, several transcriptional factors also have been identified to play a role in fibrogenesis in endometriosis. Transcription factor NR4A1 regulates BCL-2 expression, which resulted in an anti-apoptotic and pro-fibrotic effect ( Zeng et al. , 2018 ; Mohankumar et al. , 2020 ). Flavonoids quercetin and kaempferol are NR4A1 antagonist and showed anti-fibrotic effects in endometriosis ( Zhang et al. , 2023b ). The downregulation of microRNA-214 promoted fibrosis in endometriosis, probably via connective tissue growth factor (CTGF) expression ( Wu et al. , 2018 ; Zhang et al. , 2021 ). We included 75 articles reporting about animal studies. An overview of these studies is presented in Table 3 . Most of these studies aimed to test potential anti-fibrotic therapeutics for endometriosis. Nearly all studies were performed in rodents with induced endometriosis. To establish an endometriosis-like model, various methods were applied. Autologous uterine tissue, donor animal uterine tissue or human endometrial tissue was either surgically transplanted into the recipient’s inner abdominal wall or minced and injected either intraperitoneally or subcutaneously. All these methods led to similar ectopic cystic implants consisting of endometrial-like epithelial and stromal cells and fibrosis. The fibrosis was progressive over time and correlated with markers for EMT and FMT, similar to human endometriotic lesions. Animal studies. N  = 32 (3 * 8, 8 donors); mice. Intraperitoneal injection of donor uterine tissue KLF11 and TGF-βR signaling are important mechanisms in fibrogenesis in endometriosis. KLF11 knockout and a triggered immune response to implants triggers extensive fibrosis N  = 45 (19, 15, 11); mice. Intraperitoneal injection of human endometrium N  = 87 (4 * 10, donors); mice. Intraperitoneal injection of donor uterine tissue N  = 14 (2 * 7); mice. Autologous uterine tissue transplantation N  = 168 (E1 90: 3 *20 + donors), E2 48: 4* 8 +donors E3 30: 2 * 10 +donors); mice. Intraperitoneal injection of donor uterine tissue PF-04418948 an EP2 inhibitor (EP2I) and ONOAE3- 208 an EP4 inhibitor (EP4I); metformin 200 mg/kg/day Substance P for DE lesions model N  = 30 (3 *6 , 12 donors); mice. Intraperitoneal injection of donor uterine tissue N  = 27 (8, 19); mice. Intraperitoneal injection of human endometrium N  = 30 (2 * 8, 2 * 7); mice. Intraperitoneal injection of human endometrium N  = 40 (4 * 10); mice. Autologous uterine tissue transplantation N  = 60 (3 * 10, donors); mice. Donor uterine tissue transplantation N  = 54 (4 * 6, 5 * 6); mice. Subcutaneous injection of human endometrium N  = 20 (2 * 10); rat. Autologous uterine tissue transplantation N  = 124 (E1: 3 * 7, donors; E2: 3 * 8; E3: 4 * 8, donors); mice. E1, E3: Intraperitoneal injection of donor uterine tissue E2: Subcutaneous injection of human endometrium E1: chemical sympathetic denervation by 6-hydroxydopamine (OHDA), sensory denervation by resinaferatoxin (RTX). E2: surgical denervation. E3: substance P, aprepitant N  = 72 (6 * 8, donors); mice. Intraperitoneal injection of donor uterine tissue N  = 75 (E1: 16, 14, donors; E2: 2 * 10, donors); mice. Donor uterine tissue transplantation E1: NRF2−/− knockout donor mice or wild-type donor control. E2: dimethyl-fumarate (DMF) N  = 80 (4 * 10, 4 * 10); mice. Subcutaneous injection of human endometrium N  = 40 (4 * 10); mice. Subcutaneous injection of human endometrium N  = 25 (5 * 5, donors); mice. Donor uterine tissue transplantation N  = 20; mice. Autologous uterine tissue transplantation N  = 16 (2 * 5, 2 * 3); mice. Intraperitoneal injection of donor uterine tissue; Intraperitoneal injected human endometriotic cell line N  = 149 (60, 36, 27, 26); mice. Intraperitoneal injection of donor uterine tissue Fusobacterium nucleatum, Lactobacillus iners or Escherichia coli infection in endometrium of donor mice. Antibiotic treatment of donor or recipient mice by metronidazole (MZ) and chloramphenicol (CP) N  = 25 (13, 12, donors); mice. Intraperitoneal injection of donor uterine tissue N  = 12 (2 * 6, donors); rat. Autologous uterine tissue transplantation N  = 8; Cynomolgus monkey. Spontaneous endometriosis N  = 24 (4 spontaneous, 20 models); Cynomolgus monkey. Autologous endometrial tissue transplantation and intraperitoneal injection N  = 10 (2 * 5); rat. Autologous uterine tissue transplantation N  = 60 (6 * 10); mice. Autologous uterine tissue transplantation N  = 30 (3 * 10, donors); mice. Donor uterine tissue transplantation N  = 40 (4 * 10); mice. Intraperitoneal injection of autologous uterine tissue N  = 17 (2 * 7, 3); rats. Autologous uterine tissue transplantation N  = 18 (3 * 3, donors); rats. Intraperitoneal injection of donor uterine tissue N  = 30 (13, 9, model failed in the rest); rats. Autologous uterine tissue transplantation N  = 20 (12, 8); mice. Autologous uterine tissue transplantation N  = 8; mice. Intraperitoneal and subcutaneous injection of human endometrial tissue N  = 30 (5, 5, 10, donors); mice. Intraperitoneal injection of donor uterine tissue N  = 12 (3 * 4); mice. Intraperitoneal injection of human endometrium N  = 60 +donors (6 * 10); mice. Intraperitoneal injection of donor uterine tissue N  = 120 (2 * 24, 4*8, donors); mice. Intraperitoneal injection of donor uterine tissue N  = 48 (4 * 8, 16 donors), mice. Intraperitoneal injection of donor uterine tissue N  = 95 (4 * 10, 2 * 10, 35 donors); mice. Intraperitoneal injection of donor uterine tissue N  = 60 (2 * 10, 2 * 10, 20 donors); mice. Intraperitoneal injection of donor uterine tissue N  = 16 (6, 10); mice. Donor uterine tissue transplantation N  = 80 (2 * 10, 30, 2 * 15); mice. Autologous uterine tissue transplantation Intraperitoneal injection of human endometrium N  = 22; baboons. Autologous menstrual endometrium intraperitoneal inoculation N  = 50 (3 * 8, 2 * 7, donors); mice. Intraperitoneal injection of donor uterine tissue N  = 90 (2 * 30, donors); mice. Intraperitoneal injection of donor uterine tissue N  = 42 (4 * 7, 14 donors); mice. Intraperitoneal injection of donor uterine tissue N   = 68 (3 * 5, 6, 6 donors; 3 * 9, 14 donors); mice. Donor uterine tissue transplantation N  = 78 (2 * 8, 6 * 10); mice. Donor (E1) or autologous (E2) uterine tissue transplantation E1: Klf11−/− donor lesions implanted to WT mice and vice versa. E2: WT or Klf11−/− mice. Garcinol (histone acetyl transferase inhibitor), suberoyl anilide hydroxamic acide (SAHA, histone deacetylase inhibitor) E1: Lesion size, extent of fibrosis and COL1A1 expression increased in WT mice with Klf11−/− donor lesions compared to vice versa model. E2: In Klf11−/− model treatment with garcinol decreased extent of fibrosis and expression of collagen. In WT model SAHA treatment increased collagen expression and extent of fibrosis N  = 24 (2 * 8, 8 donors); mice. Intraperitoneal injection of donor uterine tissue N  = 124 (7 * 6, 18 donors; 2 * 8, 8 donors; 5 * 5, 15 donors); mice. Intraperitoneal injection of donor uterine tissue PER, peritoneal endometriosis; OMA, ovarian endometrioma; DE, deep endometriosis; WT, wild-type; HE, hematoxylin/eosin staining; IHC, immunohistochemistry; IF, immunofluorescence; WB, western blot; RT-qPCR, real-time qualitative polymerase chain reaction; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; EMT, epithelial-to-mesenchymal transition; FMT, fibroblast-to-myofibroblast transdifferentiation; SMM, smooth muscle metaplasia. In some studies, the specific aim was to evaluate the development of endometriosis in animal models, rather than to test potential therapeutic agents. FMT, an important process in the development of fibrosis, was shown to be a host reaction to ectopic tissue rather than a reaction of the donor tissue itself ( van Kaam et al. , 2008 ). The progressive nature of EMT, FMT, and fibrosis was shown in a baboon endometriosis model, leading to expansion of fibrosis from minor fibrosis at three months to a highly fibrotic lesion at twelve months after endometriosis induction, which strongly arguments for the progressive nature of the disease in human disease as well ( Zhang et al. , 2016b ). In addition to their human observational studies, Muraoka et al. showed that an endometrial infection of donor tissue with F. nucleatum increased lesion size and myofibroblast activation in endometriosis in recipients. This effect was diminished after adequate antibiotic treatment ( Muraoka et al. , 2023 ). These findings support the hypothesis that bacterial infection can affect myofibroblastic transdifferentiation and thereby contribute to endometriosis development. Many animal studies were designed to intervene in the role of different immune cells in endometriotic lesions to achieve an anti-fibrotic effect. Macrophages are known to have a pivotal role in human endometriosis and they are abundantly present in endometriotic implants in animal models as well ( Hull et al. , 2012 ; Nishimoto-Kakiuchi et al. , 2016 ; Luo et al. , 2020 ). Particularly, M2-activated macrophages stimulate fibrogenesis, as shown by increased myofibroblast differentiation, fibrosis and pain behaviour after suppletion of ex vivo differentiated macrophages ( Duan et al. , 2018 ). Inhibition of mast cell activity, among others with fisetin treatment, also decreased fibrotic development of lesions ( Di Paola et al. , 2016 ; Guo et al. , 2021 ; Arangia et al. , 2023 ). Besides this, antibody-based inactivation of B lymphocytes or type 2 innate lymphoid cells led to anti-fibrotic effects in endometriosis models ( Riccio et al. , 2019 ; Miller et al. , 2021 ; Dogan et al. , 2023 ). The immune cell infiltration cascades in endometriosis are partially set in motion because the lesions show a form of tissue injury that normally triggers wound healing mechanisms aiming at resolving the lesion. However, in endometriosis, this wound-healing mechanism fails and triggers repeated platelet aggregation, an important signal for other immune cells to infiltrate the lesions. In this light, many anti-platelet interventions, among which are aggregation inhibitors scutellarin and ozagrel, are shown to reduce macrophage infiltration, lesion growth, and lesional fibrosis in vivo ( Guo et al. , 2015 , 2016 ; Zhang et al. , 2017a ; 2017b ; Ding et al. , 2019 ; Xiao et al. , 2020 ; Huang et al. , 2022b ). Similar to studies in human tissues, sensory nerves and neuropeptides SP and CGRP were identified as pro-fibrotic stimuli ( Liu et al. , 2019 ; Yan et al. , 2019a ). Besides sensory nerves, the autonomous nervous system also influences endometriosis. Sympathetic overstimulation promotes fibrogenesis and activation of nicotinic acetylcholine receptors reducing the development of endometriosis ( Hao et al. , 2021 , 2022 ). To date, the molecular mechanisms of the interaction between endometriosis and nerves are not well understood yet. In nearly all included studies, lesion size and the extent of fibrosis are closely correlated. Strikingly, some studies wherein angiogenesis was inhibited reported beneficial effects with lesion size shrinking but an increase in the extent of fibrosis ( Liu et al. , 2015 ; Buigues et al. , 2018 ). Next to cell type-specific interventions, interventions on cellular and molecular pathways were studied. Endometriotic cells may escape apoptosis via overexpression of the anti-apoptotic BCL-2 family proteins and the lack of apoptosis among senescent cells, as illustrated by successful anti-fibrotic interventions targeting these mechanisms ( Nahari and Razi, 2018 ; Luo et al. , 2020 ; Siracusa et al. , 2021 ). The Wnt/β-catenin and Smad signaling pathways were successfully used as anti-fibrotic targets, and may constitute a potential therapeutic approach ( Matsuzaki and Darcha, 2013 , 2014 ; Hirakawa et al. , 2019 ; Zhang et al. , 2019b ; Shi et al. , 2021 ). Mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling is another pathway where interventions caused a reduction in fibrosis ( Peng et al. , 2022 ; Xia et al. , 2023 ). Transcription regulating mechanisms were also studied in vivo in mice and indicated that transcription factors Klf10 and Klf11 modulated the fibrotic response in endometriosis ( Daftary et al. , 2013 ; Delaney et al. , 2016 ; Zheng et al. , 2016 ; Khan et al. , 2018 ; Grande et al. , 2023 ). MicroRNA miR27b is shown to promote fibrosis, whereas miR214 showed anti-fibrotic effects by interfering in the transcription of NLRP3 ( Kim et al. , 2017 ; Wu et al. , 2018 ). The reduced level of miR214 in endometriosis can increase NLRP3 transcription and thereby trigger an increase of IL-1β release and fibrosis ( Xu et al. , 2023 ). Histone deacetylase 8 was overexpressed in endometriosis and inhibition showed an anti-fibrotic effect ( Zheng et al. , 2023a ; 2023b ). The results of the risk of bias assessment are shown in Tables 4 , 5 , and 6 . The risk of bias of the observational studies was assessed with the MINORS tool. Most of them scored as moderate, although few studies were scored according as having a high risk of bias. The experimental studies with human-derived material were assessed using the ROBINS-I tool and most of them were judged to have a moderate to low risk of bias. The animal studies were assessed using the SYRCLE tool. Many animal studies scored a high risk of bias, mainly due to a lack of reporting details about animal facilities. Bias assessment of observational studies according to MINORS tool. Signaling question numbers correspond with MINORS tool. Score 0: not reported; score 1: reported but inadequate; score 2: reported and adequate. A perfect total score is 16 for non-comparative studies and 24 for comparative studies. Bias assessment of intervention studies according to ROBINS-I tool. Signaling question numbers 1.1-7.3 correspond with ROBINS-I tool, signaling question numbers 1-6 correspond with additional questions for in vitro studies. Scores: Y: yes; PY: probably yes; NR: not reported; PN: probably no; N: no; NA: not applicable. Bias assessment of animal studies according to SYRCLE tool. Signaling question numbers correspond with SYRCLE tool. Scores: Y: yes; PY: probably yes; NR: not reported; PN: probably no; N: no; NA: not applicable.

In conclusion, this review gives a comprehensive overview of the current evidence about fibrosis with regard to endometriosis. This may help in focusing future research on fibrosis in endometriosis.

The current available knowledge about the role of fibrosis in endometriosis is presented in this systematic review. This includes histologic characterization, molecular processes, clinical parameters, and therapeutic strategies. The main findings of this systematic review are as follows. First, the development of fibrosis in endometriosis is accompanied by the dynamic cellular processes of EMT, FMT, and SMM, resulting in myofibroblasts that enable contraction of the extracellular matrix. Various stages of transdifferentiation can be present within a single lesion. Second, platelet aggregation, probably induced by tissue injury triggers pro-fibrotic signaling and immune cell infiltration. TGF-β is a common activator of pro-fibrotic pathways. Potential therapeutic pathways are often based on preventing platelet activation, or inhibition of Smad and Rho/ROCK. Third, fibrosis, nerves, and neuropeptides are histo-anatomically related, show mutual stimulating effects, and correlate with dysmenorrhea and pain behavior, which suggests the relevance of fibrosis in pain. Finally, numerous therapeutics targeting fibrosis have been tested in vitro and in animal models, but none of them have been tested in the light of endometriosis-related fibrosis in human subjects to date. Nevertheless, the successful regression of endometriosis-related fibrosis in animal studies shows the potential for development of successful therapeutics in humans. These findings highlight the importance of fibrosis in endometriosis. They also give insight in the etiology of the fibrotic processes. Following this, it is important to note both the similarities and the differences between endometriosis and other fibrotic diseases, including systemic sclerosis and idiopathic pulmonary fibrosis. Among the spectrum of fibrotic diseases, EMT and FMT are common hallmarks. Cellular injury may cause a new fibrotic steady state wherein myofibroblasts are abundantly present due to both cellular differentiation and recruitment ( Adler et al. , 2020 ). In endometriosis, the cyclical bleeding of the endometriotic implants could act as a stimulus similar to the continuous injury in other fibrotic diseases. This repetitive signaling can stimulate cellular differentiations EMT and FMT via TGF-β release ( Di Gregorio et al. , 2020 ; Wang and Friedman, 2023 ). Next to activated platelets, M2 differentiated macrophages are an important source of this TGF-β release ( Capobianco and Rovere-Querini, 2013 ; Vigano et al. , 2020 ). Smad and Rho/ROCK pathways are activated via TGF-β in idiopathic pulmonary fibrosis or systemic sclerosis ( Knipe et al. , 2015 ; Ye and Hu, 2021 ; Mendoza and Jimenez, 2022 ). In idiopathic pulmonary fibrosis, nintedanib and pirfenidone are successful therapeutics which are clinically available. These are identified based on their anti-fibrotic effects in in vitro studies ( Lehmann et al. , 2018 ; Amati et al. , 2023 ). Metformin also showed potential in pre-clinical fibrosis research in idiopathic pulmonary fibrosis ( Kheirollahi et al. , 2019 ). The repurposing of these compounds for treatment of endometriosis seems promising based on the commonalities in etiology. On the other hand, the interaction with nerves seems to be rather unique for endometriotic fibrosis as compared to other fibrotic diseases. There is evidence about the involvement of neuropeptides in fibrotic development in other diseases. For example, substance P is described as having a pro-fibrotic effect in several other diseases, like myocardial and idiopathic pulmonary fibrosis, via its receptor neurokinin 1, probably by enhancing TGF-β release and oxidative stress ( Peng et al. , 2019 ; Słoniecka and Danielson, 2019 ). Both pro- and anti-fibrotic effects of CGRP are described in the literature, whereas in endometriosis only pro-fibrotic effects are known ( Li et al. , 2020 ; Kayalar and Oztay, 2022 ). However, the close histo-anatomical relationship between fibrosis and nerves is not described in other fibrotic diseases. Interestingly, endometriosis is unique within the group of fibrotic diseases because in endometriosis pain is a central symptom, which is not the case in most other fibrotic diseases such as, for example, idiopathic pulmonary fibrosis, systemic sclerosis or liver cirrhosis. In most other fibrotic diseases, symptoms occur as a result of organ dysfunction due to tissue stiffness. The pain-related findings in this review also answer the question in our introduction of whether fibrosis is a favorable or unfavorable event in endometriosis. Based on this systematic review, we can consider fibrosis as an unfavorable outcome, as the extent of fibrosis correlated strongly with more severe dysmenorrhea in humans and more pronounced pain behavior in animal studies ( Odagiri et al. , 2009 ; Yan et al. , 2019b ). In contrast, anti-angiogenic therapy leading to an increase of fibrosis at an early stage of lesion development might show beneficial effects because in this case, the early fibrosis formation may hinder further lesion growth and cyclical bleeding of the lesions ( Liu et al. , 2015 ). The systematic methodology of this review has several strengths. First, to our knowledge, this is the first review about fibrosis in endometriosis with a comprehensive systematic approach. The results of both cellular and molecular processes and clinically orientated studies are included in this review. With this approach, we are able to link aetiologic studies to clinically orientated research. In this unique way, all aspects of the relevance of fibrosis in endometriosis have been brought together. Second, in the systematic risk of bias assessment, we used different validated tools. The most suitable risk of bias tool for the observational studies is the validated MINORS tool and for the animal studies, this is the validated SYRCLE tool ( Slim et al. , 2003 ; Hooijmans et al. , 2014 ). For the in vitro experimental studies, the most suitable tool is the ROBINS-I tool ( Sterne et al. , 2016 ). We used different tools for different types of studies in order to assess the risk of bias in this comprehensive review in the most reliable way possible. The in vitro design of the experimental studies lack a specific validated assessment tool, so therefore we additionally used the non-validated risk of bias assessment formulated by Post et al. (2020) . This systematic assessment for risk of bias helps to value all the evidence presented in this review. This systematic review has its limitations, too. A significant amount of information presented in this review is based on animal studies. This can be seen as a limitation, as animal models for endometriosis face a number of drawbacks. Most animal models lack a human-like menstrual cycle as well as spontaneous development of endometriosis, which complicates the interpretation of the results of these studies. Therefore, the results of animal studies and potential therapeutics described in this review are not directly useable in current practice in humans. Next, we excluded some studies not presented in English, as we were unsure if we were able to read and interpret them correctly. From a few articles, we were not able to retrieve a full-text version, so these were also excluded. These studies might have provided us with additional information. A question not fully answered in this review is what the effect of newly developed anti-fibrotic therapies on already-established endometriosis would be. Most intervention studies are designed in such a way that their therapies are predominantly showing a preventive effect on fibrogenesis. At the moment, efforts to regress fibrosis are underexplored. Some animal experiments try to capture the difference between regression and prevention of fibrosis in their study design by varying the moment of starting their intervention. Some studies were indeed able to show a decrease in fully developed fibrosis. However, this situation is difficult to compare with human subjects but is extremely relevant. Generally, there is a substantial delay in the diagnosis of endometriosis and therefore, future therapies must ideally be able to not only stop fibrosis but also to resolve already established fibrotic tissue. More research is required to bridge the gap between knowledge about the etiology of fibrosis in endometriosis, the current clinical care, and possible therapeutic targets. Based on the pathways identified to be relevant for fibrosis in endometriosis, the similarities between endometriosis and other fibrotic diseases seem very relevant to explore. As research in endometriosis is still in a developing phase relative to its huge societal implications, similarities between broadly extensively studied other diseases including systemic sclerosis and idiopathic pulmonary fibrosis can be extremely useful to explore. Additionally, it might be insightful to investigate which exact mechanisms from which of these diseases align best with pathways of fibrosis in endometriosis. Another issue highlighted by this review is the lack of studies in human subjects. Already several years ago the relevance of fibrosis in endometriosis was stressed almost simultaneously by two research groups ( Guo, 2018 ; Vigano et al. , 2018 ). Since then, many pre-clinical potential therapeutics have been described, but none of them have been tested in clinical trials regarding their effect on fibrosis in endometriosis. Partially, this may be due to the current gap between endometriosis models and the human in vivo situation. Most pre-clinical work is performed in isolated cells or in rodents, both far from being representative for the clinical situation. An adequate pre-clinical endometriosis model bridging the gap from cells to humans is highly necessary. Fortunately, the number of studies directed at other pre-clinical models for both eutopic and ectopic endometrium is increasing. Such a pre-clinical model should fulfill several requirements ( Gołąbek-Grenda and Olejnik 2022 ). First, it should be as close to the human in vivo situation as possible, thus preferably active immune cells, endometriosis-related hormones and cytokines, and interaction with ECM should be present. Second, the fibrotic environment should be preserved in order to assess the effect of anti-fibrotic agents on fibrosis in endometriosis. Moreover, a suitable model ideally should be reproducible among different research groups and cost-effective. Currently, endometriosis organoids are nearing these fulfillments, recapitulating 3D structure and cell–cell interactions ( Boretto et al. , 2019 ; Esfandiari et al. , 2021 ). The recent establishment of a successful eutopic endometrium model will also contribute to developments in endometriosis research ( Ahn et al. , 2021 ). With continuing developments in the field of ex vivo tissue culture and organ-on-a-chip systems, useful endometriosis models seem to be reachable in the near future.