Molecular and Cellular Pathogenesis of Endometriosis Notes
Endometriosis: Molecular and Cellular Pathogenesis
Abstract
Studies indicate molecular and cellular differences between endometriotic lesions and eutopic endometrium.
Eutopic endometrium in patients with endometriosis differs from that in those without.
Aberrant non-steroid signaling pathways influence endometriosis pathogenesis, potentially via crosstalk with estrogen-mediated mechanisms.
Non-steroid pathways may offer therapeutic targets that do not affect estrogen levels.
The article describes molecular and cellular features of endometriotic lesions, focusing on the WNT/β-signaling pathway.
Exosomes and microRNAs are discussed regarding their potential role in endometriosis pathogenesis.
1. Introduction
Endometriosis is a gynecological condition characterized by endometrial-like glands and stroma outside the uterus, resulting in cyclic proliferation and breakdown.
Internal bleeding leads to local inflammatory reactions, scar tissue formation, and adhesions.
Symptoms include dysmenorrhea, dyspareunia, and/or pelvic pain, which compromise the quality of life.
Prevalence is 5-10% in menstruating women and up to 35% in infertile women, but is difficult to determine due to asymptomatic cases or misdiagnosis.
Three forms of endometriosis occur in the pelvic cavity: peritoneal, ovarian, and deeply infiltrating lesions.
Lesions are described as white, red, and black based on morphology and appearance.
Red lesions are highly vascularized and active.
White and yellow-brown lesions are latent stages with fibrosis and devascularization.
Black lesions contain intraluminal debris from tissue breakdown and healing during scarification of red lesions.
In rare cases, endometriosis occurs extraperitoneally in sites like the colon, kidney, liver, pancreas, and lungs.
1.1. Current Theories on the Pathogenesis of Endometriosis
The pathogenesis of endometriosis is still poorly understood and controversial.
Proposed theories include:
Implantation theory
Metaplasia theory
Induction theory
Endometriosis disease theory
Endometriosis as a stem cell based condition
Laux-Biehlmann et al. proposed that inflammatory processes and activation of peripheral nerve endings in response to menstrual debris from retrograde menstruation contribute to endometriosis development and pain.
The implantation theory suggests that small, early lesions establish and grow due to retrograde transport of viable menstrual endometrial cells.
These cells attach to the peritoneum, proliferate, differentiate, and invade underlying tissue.
Dispersion of endometrial cells via the lymphatic system may cause lesions at distant locations.
Prerequisites of the implantation theory:
Occurrence of retrograde menstruation
Presence of viable endometrial cells in the retrograde reflux
Adhesive capacity of shed endometrial cells onto the peritoneum, alongside proliferation and implantation
Peritoneal fluid (PF) contains macrophage secretions, ovarian exudate, tubal fluid, plasma transudate, and refluxed endometrial material.
The dynamic exchange of fluid in the pelvic cavity may explain the anatomical distribution of endometriotic lesions.
Endometriosis is observed in only a subgroup of women, even though PF contains endometrial tissue in up to 59% of patients.
Prolonged and heavier menstrual flow may increase retrograde reflux material in women with endometriosis.
Restricted endometriosis development may be due to a permissive peritoneal environment.
Early endometriotic foci development depends on location, infiltration depth, hormones, cytokines, growth factors, and other factors in peritoneal or ovarian fluid or the bloodstream.
Eutopic and ectopic endometrial cell proliferation is enhanced in the presence of PF and follicular fluid from women with endometriosis.
Tumor necrosis factor-α (TNF-α) increases proliferative potential, as well as other cytokines and steroid hormones.
Estrogen-mediated signaling and TNF-α are required for apoptosis evasion and enhanced proliferation of ectopic lesions in an animal model.
Microtrauma of the uterus or peritoneal surfaces, followed by inflammatory responses and repair mechanisms, may generate a permissive environment for endometriosis induction.
Leyendecker and colleagues proposed a tissue injury and repair mechanism (TIAR) to explain the pathophysiology of adenomyosis and endometriosis development.
Women with endometriosis or adenomyosis display alterations in dysperi- and hyperstalsis waves, which might contribute to more trauma.
Altered uterine peristalsis could cause dislocation of more basal endometrium, leading to a greater number of stem cell-like cells in the retrograde reflux.
Eutopic endometrium from women with endometriosis displays reduced decidualization capacity, indicating that more undifferentiated cells are flushed retrogradely.
Microtrauma could cause exposure of extracellular matrix components (ECM) in the peritoneal cavity, promoting adhesion and proliferation of endometrial stromal cells.
Surgery may aggravate endometriosis development or progression via repair processes under the TIAR concept.
The coelomic metaplasia theory suggests that Müllerian-type epithelium can transform into endometrial tissue.
This could explain endometriosis in women without retrograde menstruation or in men undergoing estrogen treatment for prostatic carcinoma or suffering from Persistent Mullerian Duct Syndrome (PMDS).
Women with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, who have Müllerian duct defects, can develop endometriosis despite the absence of menstruation.
Endometriosis could develop through metaplasia due to aberrant activation of genes normally active during embryonic development of the female genital tract.
The embryonic rest theory suggests that developmentally misplaced Müllerian/endometrial tissue could be stimulated to undergo metaplasia.
Displaced embryonic epithelial remnants or ectopic endometrial-like glands can be found along the fetal female reproductive tract, serving as a possible source for endometriotic lesions.
The induction theory combines the implantation and coelomic metaplasia theories, suggesting that unknown substances from shed endometrium induce undifferentiated mesenchyma to form endometrial-like tissue.
The endometriotic disease theory (EDT) suggests that step-wise progression from temporary lesions to severe forms is due to cellular modifications resulting from epigenetic or genetic alterations.
Cystic ovarian endometriosis is clonal in origin.
Some endometriotic cells are invasive in vitro, associated with the loss of E-cadherin expression.
There is increasing evidence of a germline predisposition to endometriosis.
Familial clustering of endometriosis in humans and rhesus monkeys has been reported.
Increased prevalence among first-degree relatives of women with endometriosis has been observed.
The age at onset of symptoms is similar in affected, non-twin sisters, and there is concordance in monozygotic twins.
Environmental factors, such as chronic exposure to dioxins, might also play a role in disease etiology.
Endometriosis is likely to be a complex genetic trait in which multiple genes interact with each other and the environment.
The endometrium is a highly regenerative tissue containing cells with stem cell characteristics.
Freshly isolated endometrial epithelial and stromal cells contain a rare population of cells with clonogenic activity, visualized as colony-forming units (CFUs).
CFUs in the endometrial stromal cell fractions are comparable to mesenchymal stem cells (MSC) in their multilineage differentiation potential.
Enrichment of these endometrial MSC-like cells (eMSCs) is possible by their co-expression of the perivascular cell markers CD146 and PDGF-Rb.
The clonogenicity of endometrial epithelial and stromal cells shows a non-significant trend depending on the menstrual cycle stage, with increased clonogenicity in the proliferative stage for stromal cells and in the secretory stage for epithelial cells.
CFUs can also be detected in noncycling endometrium.
Retrograde misplaced MSCs in the pelvic cavity could be a critical factor in establishing an early endometriotic lesion.
Menstrual blood contains cells with plasticity, namely Endometrial Regenerative Cells (ERC).
ERCs resemble MSC in their appearance, growth properties, and differentiation potential, but contrast to MSCs, they express matrix-metalloproteases (MMP-3 and MMP-10), the angiogenic factor ANG-2 and cytokines (GM-CSF, PDGF-BB) as revealed by proteome analysis.
Menstrual blood-derived MSCs (MenSCs or MMCs) are typical fibroblast-like and similar to bone marrow-derived MSCs.
MenSCs display higher clonogenicity, proliferation, and migration rates than bone marrow-derived MSCs, and higher angiogenic potential both in vitro and in an animal model.
MenSCs can participate in repair processes in a rat model of Myocardial Infarction and in the restoration of impaired cardiac function, differentiating into MenSCs-derived cardiomyocytes at the transplantation site.
MenSCs can exert antimicrobial and immunomodulatory properties and secrete tissue regenerative factors in the cecal ligation and puncture (CLP) mouse sepsis model.
The immunomodulatory capacities of MenSCs depend on the animal model system.
Menstrual blood contains cells with plasticity, which are a novel source for cell-based replacement therapies.
Retrograde menstruation can transport cells with a stem-cell-like phenotype into the pelvic cavity, and possibly more than one cell type with putative stem/progenitor cell properties exists.,
Research into menstrual blood derived cells with plasticity is still at an early stage.
A standardized approach to isolate and characterize the stem cell-like cells in menstrual blood is of importance to decipher their role in the pathogenesis of endometriosis.
1.2. Pathogenesis of Endometriotic Lesions
Peritoneal, ovarian, and rectovaginal lesions may have discrete pathologies and etiologies.
Peritoneal lesions are most likely due to implantation of retrograde refluxed menstrual endometrium.
Ovarian endometriosis formation (chocolate cysts) might be attributable to:
Inversion and progressive invagination of the ovarian cortex after accumulation of menstrual debris from superficial implants.
Secondary involvement of functional ovarian cysts by endometrial implants on the ovarian surface.
Metaplasia of the coelomic epithelium covering the ovary.
Deeply infiltrating endometriosis of the rectovaginal septum could be:
A natural evolution of peritoneal endometriosis of the pouch of Douglas due to secondary infiltration.
An adenomyotic nodule originating by metaplasia of Müllerian/embryonic remnants in the rectovaginal septum.
A permissive peritoneal environment for the onset and progression of endometriotic lesions might also be associated with altered function of immune-related cells and local pelvic inflammatory processes, helping evasion of clearance by the immune system.
Reduced macrophage-mediated cytolysis in women with endometriosis and altered leukocyte populations within endometriotic lesions are observed.
These altered leukocytes may secrete abnormal levels of local and systemic proinflammatory cytokines and growth factors with growth-promoting and angiogenic properties.
Altered secretion of immune factors, formation of autoantibodies, and impaired immune recognition and clearance of ectopic endometrial cells facilitate the onset and progression of endometriosis.
The gut microbiome could be crucial in the pathogenesis of endometriosis by aberrant priming of immune responses.
An altered immune response is a key factor in evasion of apoptosis of endometrial cells at ectopic sites.
1.3. Classification Systems of Endometriosis
Classification systems include those by Acosta et al., Kistner et al., the American Fertility Classification, and the revised American Society for Reproductive Medicine classification of endometriosis.
These classifications divide endometriosis into stages of increasing severity with involvement of the ovaries and adhesion formation.
The number, size, and location of endometrial implants, plaques, endometriomas, and/or adhesions are charted with a score system to determine disease stages.
The common goal is to predict the chance for conception after treatment based on disease severity.
The rASRM classification is commonly used in investigative studies.
It has been shown to provide a good and reproducible tool in staging endometriosis both during surgery and by a blinded reviewer using visual documentation.
The rASRM classification system is prone to observational error and is not as effective in predicting pregnancy.
A limitation of the rASRM staging system is the scoring of only intraperitoneal endometriosis, thereby underrepresenting other manifestations such as extraperitoneal lesions
in the bowl or bladder.New options to classify and score endometriosis are under investigation to reflect the multifaceted aspects of endometriosis and its impact on fertility.
The endometriosis fertility index (EFI) assesses pregnancy outcomes after endometriosis surgery, considering the reproductive potential of the fallopian tubes, fimbria, and ovaries.
The ENZIAN-score is a staging system based on tumor grading systems, which takes the localization and severity of deep infiltrating and retroperitoneal lesions into account.
Clinical assessment of the practicality and reproducibility of the ENZIAN-score revealed that it is helpful but requires further adaptations.
One critical aspect is the occurrence of duplicate scoring of lesion between rASRM and ENZIAN staging systems; thus, ENZIAN is not a complementation of the rASRM system in its current version.
Revisions simplified the scoring system and enhanced its benefit for staging deep infiltrating retroperitoneal endometriosis, but it still lacks poor international acceptance.
1.4. Local Microenvironment: Driving Factor in the Onset and Progression of Endometriotic Lesions
Endometriotic lesions are composed of glandular epithelial cells surrounded by stromal cells at the ectopic site.
Glandular epithelium is cytokeratin positive and includes E-cadherin positive and E-cadherin negative cells.
Endometriotic stromal cells express mesenchymal markers such as vimentin and THY-1 and can be distinguished from surrounding fibroblasts by expression of the membrane metallo-endopeptidase CD10.
Differences in the composition of the extracellular matrix (ECM) surrounding endometriotic glands and stroma are reported, influencing the functional responses of endometriotic stromal cells.
The local microenvironment plays a pivotal role in the onset and progression of endometriotic lesions.
Misplaced endometrial cells need to respond to local stimuli in PF, evade immune detection, and adhere to the host tissue surface.
Once an ectopic lesion is formed, crosstalk between stromal and epithelial cells, paracrine signaling, hormonal responsiveness, and angiogenesis are required for persistence at the ectopic site.
Hull et al. identified key pathways active in the molecular interactions between ectopic endometrial tissue and its site of attachment:
Cellular injury (ubiquitin/proteasome)
Inflammation (NFκB)
Tissue remodeling (TGF-β)
Cellular proliferation (KRAS)
A proteomic study of peritoneal endometriotic stromal cells revealed extensive metabolic reprogramming and acquisition of cancer-like changes.
This is reflected in increased cellular invasiveness and adhesiveness, reduced apoptotic potential and altered immune function.
The local microenvironment can alter gene expression through epigenetic changes (DNA methylation, histone modifications, miRNA).
MicroRNAs play a role in endometriosis progression.
Endometriosis-associated molecular networks are regulated by miRNA at the posttranscriptional level.
Differential expressed miRNAs and altered DNA methylation patterns occur during endometriosis onset and/or progression.
Endometriosis is estrogen-dependent and progesterone-resistant, affecting promotor regions.
Endometriotic cells display chromosomal anomalies and instability that could alter gene expression.
Cells release extracellular vesicles (EVs) such as exosomes and microvesicles.
EVs are an alternative source for intercellular communication, containing miRNAs or enzymes, and modulate cellular responses.
Endometrial epithelial cell-derived exosomes contain miRNAs with target genes in signaling pathways connected to successful embryo-endometrial crosstalk during implantation.
Ectonucleotidase-containing exosomes in aspirates from endometriomas could contribute to endometriosis progression and local suppression of immune responses by regulating extracellular ATP and rising extracellular adenosine levels.
Endometriotic stromal cell derived exosomes could exert enhanced angiogenic effects.
Modified miRNA expression profiles in endometrial stromal cells from women with endometriosis include miRNAs involved in angiogenesis mediated by peritoneal fluid (PF).
Endometrial exosomes could be flushed retrograde into the pelvic cavity or be shed there by menstrual cells and influence the fate of ectopic cells enabling the establishment of a sufficient blood supply in order to grow and survive at the ectopic site.
Endometrial exosomes from women with endometriosis might also play a role in endometriosis manifestation as a disease.
The onset and progression of endometriosis could be a combination of several steps and factors.
Seeding endometrial tissue displays intrinsic (epi)genetic, biochemical, and structural changes.
Shed endometrial-derived exosomes could prime the soil for attachment at ectopic sites by retrograde flushed exosomes and local modulation of cells and tissue via intercellular communication.
Retrograde transported menstrual cells could attach to this primed soil and form temporary lesions.
Released exosomes of ectopic endometrial cells could facilitate immune evasion, enhance proliferation, invasion, and angiogenesis of the lesion.
Intercellular communication mediated via exosomes could be a missing link between different theories on the pathogenesis of endometriosis. E.g. :
Exosomes could induce metaplasia of cells at ectopic sites theories
Aid in tissue remodeling after injury (TIAR concept).
Exosomes could exert morphoregulatory function by altering signal transduction pathways.
1.5. Non-Steroid Signaling: The WNT Connection in Endometriosis
The establishment and progression of endometriosis requires aberrant biological processes in ectopic endometrium compared to eutopic endometrium, concerning :
Responsiveness of ectopic cells to signaling peptides
Adhesion to and invasion into the peritoneum
Morphogenesis and development
Dysregulation of apoptosis
Angiogenesis in the ectopic endometrium.
A subpopulation of endometriotic cells exhibits stem cell characteristics and/or plasticity, which supports recurrence of the disease.
Numerous studies revealed differences in the transcriptome of eutopic and ectopic endometrium.
Comparative analyses of gene expression patterns discovered many dysregulated genes assigned to pathways such as:
Cell cycle
Adherence and tight junctions
MAP kinase
TGF-β
WNT
Jak-STAT
mTOR signaling pathways
Cytokine-cytokine receptor interactions appeared dysregulated.
The indicated pathways may contribute to the pathogenesis of endometriosis by integrating different signals and activities.
Some effectors of signaling pathways possibly involved in the pathogenesis of endometriosis, particularly kinases, may serve as potential targets for non-steroid therapeutics in the future.
Studies have searched for interactions in the context of the WNT/β-catenin signal pathway.
The canonical WNT/β-catenin signal pathway, stimulated by individual WNT ligands binding to the frizzled and LRP5/6 receptors, is a key regulatory system in biology and pathophysiology.
It is essential for the development of multicellular organisms and for the homeostasis of many regenerating tissues.
Prenatal and postnatal processes are orchestrated by WNT signaling, in particular those which depend on the proper renewal of somatic stem cells, such as endometrium, mammary gland, blood vessels and intestine.
Dysregulated components of the WNT/β-catenin pathway often contribute to formation and/or progression of different types of tumors and other human diseases.
The multifunctional protein β-catenin is the central signal-transducing molecule of the WNT pathway.
It is a direct binding partner of the intercellular adhesion and metastasis suppressor protein E-cadherin, exerting a morphoregulatory function.
It is important for the structure and stabilization of the adherence junctions (AJ) and thus for formation of functional epithelial tissues.
Disruption of AJs by stimulation of epithelial mesenchymal transition (EMT) through signaling of for example receptor tyrosine kinases (RTK; e.g. EGF receptors) induces loss of epithelial cell architecture, driving cells to become more motile and invade surrounding tissue.
Cytoplasmic ß-catenin not binding to E-cadherin underlies constant degradation, highly controlled by different proteins and a cascade of phosphorylation events at β- catenin itself.
Phosphorylation of β - catenin through kinase GSK3β finally initiates its degradation, conversely its inhibitation leads to stabilization of β- catenin allowing its transcriptional activation.
Stabilized β -catenin can reach the nucleus and interact with members of the transcription factor family TCF-LEF.
This complex regulates transcription of its target genes such as involved in proliferation, tissue development and architecture as well as angiogenesis.
Xiong et al. hypothesized a link between estrogen and β-catenin in the pathogenesis of endometriosis.
Estradiol (E2) treatment of human endometrial stromal cells (HESC) from patients with endometriosis enhances the level of β -catenin, its nuclear localization as well as the cells’ invasiveness.
Downregulation of β-catenin in HESCs decreases invasiveness.
Implantation of human endometrium into the pelvic cavity of immune-compromised NOD-SCID mice under E2 injection led to upregulation of vascular vascular endothelial growth factor (VEGF) and MMP-9 as well as the formation of adhesive and invasive endometriotic lesions. Downregulation of β-catenin prevented such lesions and repressed VEGF and MMP-9 expression. These data imply that E2 might accelerate disease progression by upregulating β -catenin and thus it target genes possibly under conditions of abnormal estrogen levels.
Zang et al. addressed whether neovascularization of endometriotic lesions is enhanced by upregulation of VEGF through E2.
VEGF, E2 and canonical WNT signal transduction depend on each other mechanistically in the pathogenesis of endometriosis.
E2 enhances the level of β-catenin protein possibly through binding of the estrogen receptor α (ERα) to ERE sites in the β - catenin promoter thereby stimulating its transcription.
Nuclear β -catenin target the TCF-LEF binding sites in the VEGF gene promotor finally enhancing the expression of VEGF mRNA.
de Mattos et al. studied components of the WNT/β-catenin pathway in a rat model of peritoneal endometriosis.
They identified decreased levels of GSK3β and E-cadherin as well as a higher amount of nuclear β-catenin in endometriotic lesions.
Decrease in GSK3β leads to stabilization of β -catenin, which then exhibits elevated expression and presence in the nucleus as a consequence enhance the expression of β -catenin target genes which include VEGF (required for angiogenesis).
E-cadherin mRNA is decreased in ectopic endometrium may be due to the fact that it contains more stromal than epithelial cells or the fact of complete or partial EMT taking place resulting in the downregulation of E-cadherin changing the molecular composition and functional features of the epithelial cells in the ectopic endometrial lesions.
de Mattos et al. also found that elevated levels of WNT4 and WNT7b in the ectopic lesions.
Gaetje et al. showing increased WNT4 and WNT7a levels in human endometriotic lesions.
WNT ligands such as WNT4 act as regulators of cell proliferation and differentiation, influencing such processes also in pathophysiological events like endometriosis or tumor development.
Inhibition of the WNT/β-catenin pathway reduces cell migration, invasion and matrix metalloproteinase expression (necessary for invasion).
Matsuzaki and Darcha showed inhibitory effects of the small molecule PKF 115-584 on cell migration and invasion in epithelial and stromal cells in vitro from patients with endometriosis prepared at the menstrual phase.
Inhibition of TCF-LEF/β-catenin-mediated effects and target gene expression was weaker in patients without endometriosis.
Conclusion
The WNT/β-catenin pathway is a remunerative target for novel therapeutics.
The molecular hallmarks of endometriosis comprise a hormone-dependent (estrogen-dependence, progesterone resistance) and inflammatory condition with a (epi)genetic predisposition driven most likely by cells with plasticity.
Newly discovered biological concepts of general relevance (e.g. exosomes and miRNAs) are also relevant for the pathogenesis of endometriosis.
Intercellular communication mediated by exosomes could be viewed as a novel mechanistic tool to orchestrate cell fate by modulating signaling pathways.
A challenge in endometriosis research will be the assessment of non-steroid signaling pathways as targets for novel therapeutics to treat endometriosis. This might be a chance to replace E2-depletion therapies to minimize adverse side effects such as early menopause.
Endometriosis affects the reproductive system in several significant ways:
Infertility: Up to 35% of women with endometriosis experience infertility. Endometrial lesions can interfere with normal reproductive functions, including ovulation and the transport of sperm.
Pain Symptoms: Women often suffer from dysmenorrhea (painful periods), dyspareunia (pain during intercourse), and pelvic pain, which can severely affect their quality of life and hinder efforts to conceive.
Hormonal Influence: The ectopic endometrial tissue responds to hormonal changes during the menstrual cycle, causing inflammation and pain, further complicating reproductive issues.
Scar Tissue: Endometriosis can lead to the formation of scar tissue (adhesions), which may obstruct the fallopian tubes and distort pelvic anatomy, negatively impacting the reproductive process.
Altered Ovarian Function: The development of ovarian endometriomas (chocolate cysts) can disrupt normal ovarian function and hormonal balance, influencing fertility potential.
The article aims to explain how endometriosis develops at a molecular and cellular level. It focuses on the differences between normal endometrial tissue and the tissue found in endometriosis. The article discusses how certain signaling pathways, which don’t involve hormones, might be targeted for new treatments. Additionally, it looks at how the WNT/β-signaling pathway and small molecules called exosomes and microRNAs may play roles in the disease. Overall, it highlights how endometriosis affects women's health, especially concerning infertility and related symptoms such as pain.