Endometriosis is a common, chronic inflammatory disease defined by the presence of extrauterine endometrial tissue. The aetiology of endometriosis is complex and multifactorial, where several not fully confirmed theories describe its pathogenesis. This review examines existing theories on the initiation and propagation of different types of endometriotic lesions, as well as critically appraises the myriad of biologically relevant evidence that support or oppose each of the proposed theories. The current literature suggests that stem cells, dysfunctional immune response, genetic predisposition, and aberrant peritoneal environment may all be involved in the establishment and propagation of endometriotic lesions. An orchestrated scientific and clinical effort is needed to consider all factors involved in the pathogenesis of this multifaceted disease and to propose novel therapeutic targets to reach effective treatments for this distressing condition.

The endometrium is the primary target organ for the ‘female’ sex steroid hormone estrogen, which exerts effects in the endometrium via two main classical estrogen receptor (ER) isoforms, ERα and ERβ. The main function of the endometrium, embryo implantation, appears unperturbed in ERβ knockout mice, which has led researchers to disregard other potentially important functional roles that ERβ may have in endometrium. This review focuses on ERβ in the human endometrium and its protective role from the undesired effects of ERα. We conducted a systematic search using PubMed and Ovid for publications between January 1996 and February 2014. All studies that examined ERβ expression or function in non-pregnant endometrium or cells derived from the endometrium were considered, including human and animal studies. Studies of the basic function of ERβ isoforms in restraining ERα-mediated cell-specific trophic/mitotic responses to estrogen in other tissues has allowed appreciation of the important potential role of ERβ in the regulation of cell fate in the human endometrium. Our current understanding of ERβ expression and function in endometrium is, however, incomplete. ERβ is dynamically expressed in healthy premenstrual endometrium, persists in post-menopausal atrophic endometrium and may play an important role in endometrial disease. All endometrial cell types express ERβ and aberrations in ERβ expression have been reported in almost all benign and malignant endometrial proliferative disease. The collective evidence suggests that ERβ has an important role in normal endometrial function and also in most, if not all, benign and malignant endometrial diseases. However, the conduct of studies of endometrial ERβ expression needs to be standardized: agreement is needed regarding the most appropriate control tissue for endometrial cancer studies as well as development of standardized methods for the quantification of ERβ immunohistochemical data, similar to those scoring systems employed for other hormonally regulated tissues such as breast cancer, since these data may have direct clinical implications in guiding therapy.

About 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients. CTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn’s post-test was used for comparisons between groups. Statistical significance was set at p < 0.05. EpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis. Our results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.

The human endometrium is a highly regenerative organ undergoing over 400 cycles of shedding and regeneration over a woman’s lifetime. Menstrual shedding and the subsequent repair of the functional layer of the endometrium is a process unique to humans and higher-order primates. This massive regenerative capacity is thought to have a stem cell basis, with human endometrial stromal stem cells having already been extensively studied. Studies on endometrial epithelial stem cells are sparse, and the current belief is that the endometrial epithelial stem cells reside in the terminal ends of the basalis glands at the endometrial/myometrial interface. Since almost all endometrial pathologies are thought to originate from aberrations in stem cells that regularly regenerate the functionalis layer, expansion of our current understanding of stem cells is necessary in order for curative treatment strategies to be developed. This review critically appraises the postulated markers in order to identify endometrial stem cells. It also examines the current evidence supporting the existence of epithelial stem cells in the human endometrium that are likely to be involved both in glandular regeneration and in the pathogenesis of endometrial proliferative diseases such as endometriosis and endometrial cancer.

STUDY QUESTION: What is the role of iron in the pathophysiology of endometriosis?SUMMARY ANSWER: Iron excess is demonstrated wherever endometriotic tissues are found and is associated with oxidative stress, an inflammatory micro-environment, and cell damage; the iron-mediated oxidative stress is independently linked to subfertility, symptom severity, and malignant transformation.WHAT IS KNOWN ALREADY: Iron is found in excess in endometriotic tissues, and multiple mechanisms have been studied and posited to explain this. It is clear that iron excess plays a vital role in promoting oxidative stress and cell damage. The evidence base is large, but no comprehensive reviews exist to summarize our understanding and highlight the overarching themes to further our understanding and suggest future directions of study for the field.STUDY DESIGN, SIZE, DURATION: This systematic review with a thematic analysis retrieved studies from the PubMed, Embase, Web of Science, and Cochrane Library databases and searches were conducted from inception through to August 2022. Human and animal studies published in the English language were included and identified using a combination of exploded MeSH terms (‘Iron’ and ‘Endometriosis’) and free-text search terms (‘Iron’, ‘Ferric’, ‘Ferrous’, ‘Endometriosis’, ‘Endometrioma’).PARTICIPANTS/MATERIALS, SETTING, METHODS: This review was reported in accordance with the PRISMA guidelines. All studies reporting original data concerning the role of iron or iron complexes in the pathophysiology of endometriosis were included. Studies that did not report original data or provided a review of the field were excluded. Bias analysis was completed for each included study by using the Newcastle–Ottawa scoring system.MAIN RESULTS AND THE ROLE OF CHANCE: There were 776 records identified and these were screened down to 53 studies which met the eligibility criteria, including 6 animal and 47 human studies, with 3556 individual participants. Iron excess is demonstrated in various tissues and fluids, including ovarian endometriomas, ovarian follicles, ectopic endometriotic lesions, and peritoneal fluid. Markers of oxidative stress are strongly associated with high iron levels, and aberrant expression of iron-transport proteins has been demonstrated. Abnormal resistance to ferroptosis is likely. Iron-mediated oxidative stress is responsible for a pro-inflammatory micro-environment and is linked to subfertility, symptom severity, and, possibly, malignant transformation.LIMITATIONS, REASONS FOR CAUTION: A minority of the included studies were of objectively low quality with a high risk of bias and may lead to misleading conclusions. Additionally, multiple studies failed to appropriately characterize the included patients by known confounding variables, such as menstrual cycle phase, which may introduce bias to the findings.WIDER IMPLICATIONS OF THE FINDINGS: Current literature depicts a central role of aberrant iron mechanics and subsequent oxidative stress in endometriosis. It is likely that iron excess is at least partly responsible for the persistence and proliferation of ectopic endometriotic lesions. As such, iron mechanics represent an attractive target for novel therapeutics, including iron chelators or effectors of the iron-oxidative stress pathway. There are significant gaps in our current understanding, and this review highlights and recommends several topics for further research. These include the role of iron chelation, resistance to ferroptosis, the relationship between iron excess and localized hypoxia, systemic iron pathophysiology in endometriosis, and the role of oxidative stress in malignant transformation.STUDY FUNDING/COMPETING INTEREST(S): J.W. and S.G.P. are supported by clinical fellowships at Liverpool University Hospital NHS Foundation trust. No additional funding was requested or required for the completion of this work. C.J.H. is supported by a Wellbeing of Women project grant (RG2137). D.K.H. is supported by a Wellbeing of Women project grant (RG2137) and an MRC clinical research training fellowship (MR/V007238/1). The authors have no conflicts of interest to declare.REGISTRATION NUMBER: A protocol was prospectively registered with the PROSPERO database in August 2021 (CRD42021272818).