Table 2.

Experimental studies with human-derived material.

Reference	Sample size	Intervention	Assay methods	Results	Conclusion	Relevance to fibrosis
MULTIPLE ENDOMETRIOSIS SYBTYPES
Matsuzaki and Darcha, 2013; Involvement of the Wnt/B-catenin signaling pathway in cellular and molecular mechanisms of fibrosis in endometriosis (Matsuzaki and Darcha, 2013)	40 OMA and DE, not specified. 40 eutopic endometrium, 30 healthy endometrium	siRNA knockdown of β-catenin; Tcf/β-catenin antagonists (PKF 115–584, 6.25 µM and CGP049090, 6.25 µM), with or without TGF-β1 (5 ng/ml). Wnt3a stimulation	RT-qPCR	α-SMA and collagen I mRNA expression not altered by β-catenin siRNA in stromal cells. TGF-β1 increased α-SMA and collagen I expression, effect attenuated by β-catenin siRNA, both in ectopic and eutopic cells. α-SMA and collagen I expression decreased by treatment with PKF 115-584 and CGP049090 in ectopic and eutopic stromal cells. Treatment attenuated TGF-β1 dependent increase	Wnt/β-catenin activation may be involved in fibrogenesis in endometriosis	Wnt/β-catenin signaling promotes fibrosis, potential therapeutic target
Matsuzaki et al., 2020; Dose-dependent pro- or anti-fibrotic response of endometriotic stromal cells to interleukin (IL)-1β and tumor necrosis factor α (Matsuzaki et al., 2020)	36 OMA and DE, not specified. 16 eutopic endometrium, 8 healthy endometrium	IL-1β (1–10 pg/ml) or TNF-α (10–1000 pg/ml). With or without TGF-β1 (5 ng/ml)	WB, RT-qPCR, IF	Fibrotic marker expression is higher in ectopic versus eutopic cells. In eutopic cells, no effect of IL-1β or TNF-α. Fibrotic marker expression increased after low-dose IL-1β or TNF-α but decreased after high doses.	Low-grade inflammation stimulates a fibrotic phenotype, whereas high-grade inflammation inactivates a fibrotic phenotype in endometriotic stromal cells	Fibrogenesis reacts differently on high- and low-grade inflammatory stimulus
Shao and Wei, 2018; FOXP1 enhances fibrosis via activating Wnt/B-catenin signaling pathway in endometriosis (Shao and Wei, 2018)	6 OMA and DE, not specified, 6 eutopic endometrium, 6 healthy endometrium	siRNA knockdown of Forkhead box protein 1 (FOXP1); Wnt signaling inhibitor AVX939	WB, RT-qPCR	Fibrotic markers, β-catenin, and FOXP1 expression are higher in ectopic versus eutopic cells. siRNA knockdown of FOXP1 decreased fibrotic markers expression, β-catenin acetylation, and Wnt signaling. Wnt signaling inhibition attenuated effects of FOXP1 knockdown	FOXP1 is upregulated in endometriotic stromal cells and stimulates fibrosis via Wnt/β-catenin signaling pathways	Wnt/β-catenin signaling has an important role in fibrogenesis
OVARIAN ENDOMETRIOTIC CYSTS
Hirakawa et al., 2019; β-catenin signaling inhibitors ICG-001 and C-82 improve fibrosis in preclinical models of endometriosis (Hirakawa et al., 2019)	11 OMA, 6 healthy endometrium	CREB binding protein (CBP)/β-catenin signaling inhibitors ICG-001 or C-82, concentrations 0–200 µM	WB, RT-qPCR	α-SMA expression higher in ectopic stromal cells than eutopic cells (mRNA and protein). ICG-001 and C-82 treatment downregulated α-SMA mRNA expression but not protein expression, decreased viability and proliferation and increased apoptosis	CBP/β-catenin is an important signaling pathway in endometriosis and a potential therapeutic target	β-catenin signaling has an important role in fibrogenesis
Huang et al., 2022b; Tetramethylpyrazine (TMP) retards the progression and fibrogenesis of endometriosis (Huang et al., 2022b)	5 OMA	Activated platelets; 0, 25, or 100 µM TMP	WB, RT-qPCR	Activated platelets induced FMT-like morphological changes in stromal cells. TMP treatment abolished this effect. TMP dose dependently suppressed of α-SMA, CCN2, and collagen I RNA expression and TGF-β1, α-SMA, p-Smad3, and collagen I protein expression. Treatment attenuated increase of contractility and reduced collagen production	TMP treatment inhibits platelet-induced myofibroblast activation in stromal cells resulting in reduced contractility and collagen production	TMP treatment has anti-fibrotic effect via inhibition of myofibroblast activation induced by platelets
Mohankumar et al., 2020; Bis-indole-derived nuclear receptor 4A1 (NR4A1) ligands as inhibitors of endometriosis (Mohankumar et al., 2020)	2 OMA, experiments in triplicate	DIM-C-pPhOH and DIM-C-pPhOH-3Cl-5-OCH3 in various concentrations. Knockdown with siNR4A1	WB, RT-qPCR, IF	NR4A1 knockdown decreased expression of BCL-2 family and α-SMA, increased Bax, caspase 3, and induced apoptosis. DIM-C-pPhOH and DIM-C-pPHOH-3-Cl-5-OCH3 decreased expression of fibrotic markers and BCL-2 family, induced apoptosis	NR4A1 is a pro-endometriotic transcription factor and inhibition with Bis-indole-derived antagonist is promising as a new non-hormonal therapy	NR4A1 is a pro-endometriotic factor and inhibition results in decreased expression of fibrotic factors
Muraoka et al., 2023; Fusobacterium infection facilitates the development of endometriosis through the phenotypic transition of endometrial fibroblasts (Muraoka et al., 2023)	4 OMA, 4 eutopic endometrium, 4 healthy endometrium	TAGLN vector, pcDNA3.4-TAGLN, siRNA targeting TAGLN, F. nucleatum co-culture	IHC, RT-qPCR, scRNA-seq, FISH	OMA fibroblast increased expression of transgelin (TAGLN), α-SMA, vimentin. TAGLN siRNA decreased proliferation and contractility, increased by TAGLN stimulation and IL-6. TGF-β upregulated TAGLN expression, abolished by Smad2/3 inhibitor SB431542. F. Nucleatum co-culture with THP1-derived macrophages stimulated M2 differentiation and elevated TAGLN expression	TGF-β promotes myofibroblastic transition, marked by TAGLN expression. TGF-β signaling can be activated by F. nucleatum infection, plays a role in pathogenesis. Antibiotic treatment can be a potential therapeutic target	Fusobacterium infection in endometrium triggers myofibroblast activation and thereby attributes to endometriotic lesion establishment
Nagai et al., 2020; Focal adhesion kinase-mediated sequences, including cell adhesion, inflammatory response and fibrosis as a therapeutic target in endometriosis (Nagai et al., 2020)	1 OMA, 1 eutopic endometrium, 1 healthy endometrium	5 µM FAK inhibitor, 20 µM MEK inhibitor, 15 µM JNK inhibitor	WB, ELISA	FAK and MCP1 expression was upregulated in endometriosis. TGF-β1 increased α-SMA expression, FAK inhibition attenuated this effect. Co-culture of U937 (macrophage cell line) upregulated TGF-β1 expression, effect attenuated by FAK inhibition	FAK mediated development of endometriotic lesions is a potential therapeutic target	FAK has a stimulating effect in fibrosis in endometriosis
Nasu et al., 2010; Heparin is a promising agent for the treatment of endometriosis-associated fibrosis (Nasu et al., 2010)	9 OMA	Heparin sodium 1–100 µg/ml	WB	Heparin treatment decreased protein expression of α-SMA, RhoA, Rock I and II, and collagen gel contraction	Heparin inhibited Rho/Rock signaling and fibrotic markers, which suggests that the Rho/Rock pathway is the mechanism of action of heparin in influencing myofibroblastic transformation	Heparin has anti-fibrotic properties via inhibition of Rho/Rock signaling
Shi et al., 2017; Transforming growth factor β1 from endometriomas promotes fibrosis in surrounding ovarian tissues via Smad2/3 signaling (Shi et al., 2017)	3 OMA	TGF-β1 (10 ng/ml)	WB, RT-qPCR	Smad signaling pathway markers upregulated directly after TGF β1 stimulation, fibrotic markers increased	Endometriotic cyst cells produce TGF-β1 leading to fibrosis and adhesions to ovarian tissue via TGF-β1/Smad signaling pathways	Smad pathway is a driver of fibrosis
Tsuno et al., 2011; Fasudil inhibits the proliferation and contractility and induces cell cycle arrest and apoptosis of human endometriotic stromal cells: a promising agent for the treatment of endometriosis (Tsuno et al., 2011)	8 OMA	Fasudil (ROCK inhibitor) 100 µM	WB	Fasudil reduced α-SMA, ROCK I and II but not RhoA expression. BCL-2 family expression strongly reduced by fasudil, leading to increased apoptosis. Collagen gel contractility and myofibroblastic differentiation were reduced	Fasudil inhibits cell proliferation, induces cell cycle arrest and apoptosis by down-regulating BCL-2, inhibits collagen contractility and the myofibroblastic transformation, via Rho/ROCK mediated signaling	Fasudil has potential anti-fibrotic properties via ROCK signaling inhibition and apoptosis induction via BCL-2 signaling
Tsuno et al., 2009; Decidualization attenuates the contractility of eutopic and ectopic endometrial stromal cells: implications for hormone therapy of endometriosis (Tsuno et al., 2009)	12 OMA, 8 eutopic endometrium, 9 healthy endometrium	In vitro decidualization by db-cAMP and medroxy-progesteron acetate (MPA) or dienogest	WB, ELISA	RhoA, ROCK I and II, and α-SMA expression and collagen gel contraction reduced after in vitro decidualization by both protocols	Decidualization inhibits the contractility of stromal cells by downregulation of collagen I receptor and Rho-ROCK pathways; inhibits differentiation to myofibroblasts	Contractility and myofibroblastic transformation is attenuated by decidualization, which could be of importance for hormonal interventions
Wang et al., 2023; PIM2 promotes the development of ovarian endometriosis by enhancing glycolysis and fibrosis (Wang et al., 2023)	50 OMA, 50 eutopic endometrium, 50 healthy endometrium	Flag-PIM2, PIM2 inhibitor SMI-4a, PIM2 siRNA PKM2 inhibitor 3K	WB, IHC	PIM2 (proviral insertion in murine lymphomas 2) was upregulated in OMA and positively correlated with HK2, PKM2, SMH (smooth muscle myosin heavy chain), Desmin and α-SMA. Flag-PIM2 increased expression of Desmin, SMH and α-SMA, siRNA knockdown decreased this expression. PKM2 inhibitor abolished stimulating effect	PIM2 promotes glycolysis and fibrogenesis via enhancing PKM2 expression	PIM2 promotes fibrosis in endometriosis via PKM2, SMI-4a is a potential anti-fibrotic target
Wu et al., 2018; Exosomal miR-214 from endometrial stromal cells inhibits endometriosis fibrosis (Wu et al., 2018)	24 OMA, 24 eutopic endometrium, 24 healthy endometrium	miRNA-214 with or without TGF-β stimulation	WB, RT-qPCR, ISH, IF	Expression of α-SMA, CTGF, collagen A1 was increased in OMA. Expression increased in all cells after TGFβ stimulation. Expression in OMA was reduced after miRNA-214 treatment. miRNA-214 attenuated effect of TGF-β stimulation in all cells	miRNA-214 is downregulated in endometriosis, upregulation is a potential therapeutic strategy for endometriosis	The downregulation of miRNA-214 in endometriosis may drive fibrosis via CTGF, upregulation is a potential therapeutic strategy
Yan et al., 2019b; Neuropeptides substance P and calcitonin gene-related peptide accelerate the development and fibrogenesis of endometriosis (Yan et al., 2019b)	8 OMA	Substance P (SP), calcitonin gene-related protein (CGRP), aprepitant, CGRP fragment 8-37	WB, RT-qPCR, IHC	SP or CGRP treatment induced expression of α-SMA, collagen A1, and markers for myofibroblastic differentiation. Aprepitant and/or CGRP fragment 8-37 (as receptor antagonists) blocked these effects	Sensory nerves have an important role in promoting fibrogenesis. SP, CGRP and their receptors stimulate EMT, FMT, and SMM. Anatomical link between DE and multiple nerve plexus could explain higher fibromuscular content in DE	Colocalization of nerves and fibrosis and fibrosis-stimulating effect of neuropeptides implies contribution of fibrosis to pain in endometriosis
Yoshino et al., 2020; Relaxin-2 may suppress endometriosis by reducing fibrosis, scar formation, and inflammation (Yoshino et al., 2020)	6 OMA	Relaxin-2 100 ng/ml	WB, IHC, RT-qPCR	Relaxin-2 treatment reduced collagen and interleukin-8 expression and collagen gel contraction but did not affect α-SMA and CTGF expression. Protein kinase A inhibition by H89 attenuated effect of relaxin treatment	Relaxin-2 treatment may reduce fibrosis, scar forming, and inflammation in endometriosis	Relaxin-2 reduced formation of collagen but did not affect myofibroblast differentiation, anti-fibrotic properties thereby unclear
Yuge et al., 2007; Collagen gel contractility is enhanced in human endometriotic stromal cells: a possible mechanism underlying the pathogenesis of endometriosis-associated fibrosis (Yuge et al., 2007)	10 OMA, 8 healthy endometrium	Y-27632 (ROCK inhibitor) 0.1–100 µM	WB, ELISA	Expression of RhoA, ROCK I and II, and α-SMA and collagen gel contraction was elevated in OMA cells. Y-27632 reduced fibrotic marker expression and decreased collagen gel contraction	ROCK pathway overexpression and successful ROCK inhibition suggest that ROCK mediated myofibroblastic differentiation is responsible for the collagen contraction in endometriosis	Rho/ROCK inhibition is a potential anti-fibrotic therapeutic strategy
Zeng et al., 2018; NR4A1 is involved in fibrogenesis in endometriosis (Zeng et al., 2018)	23 OMA, 15 healthy endometrium	NR4A1 siRNA knockdown, Csn-β1, TGF-β1, MK2206	WB, RT-qPCR, IHC	NR4A1 siRNA combined with TGF-β1 increased α-SMA, FN, COL1A1, CTGF expression. Csn-β1 decreased TGF-β1-dependent NR4A1 phosphorylation and decreased α-SMA, FN, COL1A1, and CTGF expression	NR4A1 can regulate fibrogenesis in endometriosis in a TGF-β1 dependent manner	NR4A1 has anti-fibrotic properties, phosphorylated NR4A1 has pro-fibrotic properties, both acting via AKT and TGF- β1 signaling
Zhang et al., 2016a; Platelets drive smooth muscle metaplasia and fibrogenesis in endometriosis through epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation (Zhang et al., 2016a)	17 OMA	A83-01, co-culture with platelets, with or without activation with thrombin or thrombin alone	WB, IHC, RT-qPCR, ELISA	Expression of markers for EMT, FMT, fibrosis and Smad signaling, and collagen gel contraction increased after co-culture with activated platelets. TGF-β1 inhibition with A83-01 attenuated these effects	Activated platelets promote EMT, FMT, SMM via TGF-β1 and Smad signaling pathway, leading to fibrosis in endometriosis. Platelet-targeted therapy could therefore be a promising therapeutic strategy	Platelets stimulate fibrogenesis via TGF-β
Zhang et al., 2021; Downregulation of exosomal miR-214-3p targeting CCN2 contributes to endometriosis fibrosis and the role of exosomes in the horizontal transfer of miR-214-3p (Zhang et al., 2021)	OMA, eutopic endometrium and healthy endometrium stromal cell line	miR-214-3p mimics, miR-214-3p inhibitors	WB, IHC, RT-qPCR	Expression of α-SMA, CCN2, and collagen A1 elevated in OMA, miRNA-214-3p transfection decreased CCN2 expression and fibrotic marker, miRNA-214-3p inhibition increased CCN2	miRNA-214-3p is downregulated, causing CCN2 increase in endometriosis. miR-214-3p has the potential to stop fibrosis progression via CCN2 signaling. Exosomes are a potential miRNA drug carrier	miRNA-214 downregulation in endometriosis stimulates fibrosis, miRNA therapy inhibits CCN2 to reduce fibrosis. Exosomes have the potential as RNA-based therapy carriers
Zhang et al., 2022; Ferroptosis induced by iron overload promotes fibrosis in ovarian endometriosis and is related to subpopulations of endometrial stromal cells (Zhang et al., 2022)	38 OMA, 38 eutopic endometrium	Ferric ammonium citrate (FAC), erastin; Ferrostatin-1, deferoxamine mesylate	WB, IHC, electron microscopy, iron quantification, HE	Iron deposits and iron ion levels increased in OMA versus eutopic tissue. ROS and markers for ferroptosis 4-NHE, MDA, PTGS2, and NOX1 were increased in OMA. FAC treatment induced ferroptosis and upregulated α-SMA and COL1, effect attenuated by ferroptosis inhibitor ferrostatin-1. Erastin-induced ferroptosis but not fibrotic marker expression	Ferroptosis is induced in endometriosis by increased iron concentration. FAC treatment simulates effects of ferroptosis and caused increased fibrotic marker expression	Iron accumulation in endometriosis can trigger ferroptosis and subsequentially fibrogenesis
Zhang et al., 2023b; Flavonoids quercetin and kaempferol are NR4A1 antagonists and suppress endometriosis in female mice (Zhang, Mohankumar, et al., 2023b)	OMA epithelial and stromal cell line	Quercetin 25-100 µM and kaempferol 25-150 µM; NR4A1 siRNA	WB, IF	siNR4A1, quercetin, and kaempferol all inhibited endometriotic, but not normal endometrial cell proliferation. Quercetin and kaempferol suppressed mTOR signaling. siNR4A1, quercetin and kaempferol inhibited the expression of α-SMA, CTGF, COL1A1, and FN in epithelial cells but not stromal cells	NR4A1 has a central role in fibrogenesis and inhibition with quercetin and kaempferol are promising therapeutic targets	NR4A1 has a pro-fibrotic effect, but the discrepancy between the anti-fibrotic effect on epithelial and stromal cells of its inhibitors quercetin and kaempferol needs more research
DEEP ENDOMETRIOSIS
González-Foruria et al., 2017; Dysregulation of the ADAM17/Notch signaling pathways in endometriosis: from oxidative stress to fibrosis (González-Foruria et al., 2017)	8 DE, 8 eutopic endometrium, 8 healthy endometrium, 202 PF	100 µM DAPT or 2,3 µM FLI-06 (y-secretase inhibitors). H2O2 increasing concentrations 0-40 µM, cell culture supernatants or ADAM17 purified protein 0,01 µg/ml	Picrosirius red stain, WB	Notch cleavage inhibition (DAPT or FLI-6) reduced α-SMA and collagen I levels in DIE, not in control. ADAM17 or supernatant increased cleaved Notch and α-SMA, with or without H2O2	Increased oxidative stress hyperactivates the ADAM17/Notch signaling pathway and consequently increased expression of fibrotic markers α-SMA and Collagen I	Oxidative stress promotes fibrogenesis and FMT through ADAM17/Notch signaling
Leconte et al., 2010; Antiproliferative effects of cannabinoid agonists on deep infiltrating endometriosis (Leconte et al., 2010)	14 DE, 14 eutopic endometrium, 12 healthy endometrium	WIN 55212-2 0,3 to 40 µM	WB	WIN55212-2 decreased proliferation, ROS production, and (p)AKT, with no effect on (p)ERK and α-SMA	Cannabinoid agonist inhibits Akt signaling and decreases DE stromal cell proliferation and ROS production. No significant effect on α-SMA expression	Cannabinoid agonist treatment does not lead to significantly reduced α-SMA expression
Matsuzaki et al., 2023; Interleukin (IL)-10 is not anti-fibrotic but pro-fibrotic in endometriosis: IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression (Matsuzaki et al., 2023)	54 DE, 30 healthy endometrium	TGF-β; IL-6, soluble IL-6 receptor (sIL-6R), IL-10	IF, WB	IL-10 increased col 1 expression, attenuated by STAT3 siRNA. IL-10 increased α-SMA positive cells and collagen contraction, but not col 1 positive cells, attenuated by STAT3 siRNA. Strongest pro-fibrotic effect of IL-10 if administration after TGF-β or IL-6/sIL-6R stimulation, milder effect if administration before	IL-10 is pro-fibrotic via STAT3 activation in endometriosis as it promotes myofibroblast proliferation and collagen expression	IL-10, known as anti-inflammatory cytokine has pro-fibrotic effects in endometriosis, highlighting the complex inflammatory interaction in endometriosis
Matsuzaki et al., 2022; Persistent activation of signal transducer and activator of transcription 3 via interleukin (IL)-6 trans-signaling is involved in fibrosis of endometriosis (Matsuzaki et al., 2022)	36 DE, 24 eutopic endometrium, 32 healthy endometrium	STAT3 siRNA; IL-6, soluble IL-6 receptor (sIL-6R), TGF-β, S3I-201 (STAT3 inhibitor), NF-κB inhibitor BMS-345541	IF, WB	IL-6 or sIL-6R no effect in healthy, but increased COL1 in endometriotic cells. STAT3 siRNA and S3I-201 decreased COLl1 expression in endometriotic cells. TGF-β and/or IL-6/sIL-6R increased α-SMA positive cells. STAT3 siRNA had no effect, whereas S3I-201 decreased COL1 positive cells, both decreased α-SMA positive cells	Dysregulated STAT3 activation stimulates fibrogenesis via IL-6 and soluble IL-6 receptor signaling in endometriosis	STAT3/IL-6 dysregulation promotes fibrogenesis in endometriosis
Matsuzaki et al., 2016; Soft matrices inhibit cell proliferation and inactivate the fibrotic phenotype of deep endometriotic stromal cells in vitro (Matsuzaki et al., 2016)	40 DE, 40 eutopic endometrium, 23 healthy endometrium	Culture cells on top of 2, 4, 8, 16, 30 kPa stiffness gel, with or without TGF-β 5 ng/mL	IHC, RT-qPCR	In DE cells proliferation, α-SMA and collagen increased on stiffer matrix, strongest with TFG-β, also without. Both patient and healthy eutopic cells showed only α-SMA and collagen expression on high stiffness matrix with TGF-β stimulation	Soft matrix inhibited cell proliferation and decreased fibrotic markers. Stiff matrix increased fibrotic markers. This implies that DE cells react to stiff environment	Stiffness of fibrosis stimulates further fibrotic changes, causing a positive feedback loop
Matsuzaki and Darcha, 2014; Antifibrotic properties of epigallocatechin-3-gallate in endometriosis (Matsuzaki and Darcha, 2014)	45 DE, 10 healthy endometrium	Epigallocatechin-3-gallate (EGCG) and N-acetyl-L-cysteine (NAC), with or without TGF-β 5 ng/ml	WB, RT-qPCR, IF	EGCG treatment decreased fibrotic markers in DE and healthy cells, and attenuated TGF-β-dependent increase of these markers. NAC treatment decreased α-SMA, but did not affect other fibrotic markers in healthy cells, with no effect in ectopic cells. Immunofluorescence showed decrease of α-SMA positive cells after EGCG treatment, no effect of NAC	Epigallocatechin-3-gallate is a potential anti-fibrotic drug candidate	Epigallocatechin-3-gallate as a potential treatment for endometriosis decreases fibrotic markers

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.