Role of autophagy and ferroptosis in the development of endometriotic cysts (Review)

Endometriosis is an estrogen-dependent condition affecting 10% of women in their reproductive life ( 1 ). It is characterized by the growth of endometrial-like tissues at sites outside of the uterus and is associated with pelvic pain and infertility. The pathogenesis of endometriosis is still unclear, but a number of factors are known, including retrograde menstruation, coelomic metaplasia, induction theory and hormonal, immunologic determinant, stem cell and genetic/epigenetic factors ( 2 ). According to Sampson's theory, endometriotic lesions are originated from the shed endometrial tissues of the uterus through retrograde menstrual dissemination, move and attach to the ectopic sites and then form endometriotic lesions ( 3 ). The retrograde endometrium encounters a number of challenges in the peritoneal cavity, including mechanical damage, abundant free iron and high oxidative levels, lack of oxygen, or immune attack ( 4 ). Periodic hemorrhage from ectopic endometriotic lesions is a hallmark of endometriosis and causes persistent iron overload ( 5 ). Despite a number of difficulties, endometriotic tissues may overcome these hurdles in the microenvironment by using mechanisms to escape from cell death. Retrograde menstruation occurs in as a ≥90% of menstruating women, but the incidence rate for endometriosis is only 10% ( 6 ). Thus, only a fraction of endometriotic cells may escape cell death in the harsh environment of intraperitoneal iron overload and hypoxia. This suggests that there must be other genetic and environmental factors that determine the onset and progression of this disease, such as mechanisms that prevent cell death ( 4 ). Regulated cell death (RCD) is characterized by specific signaling cascades orchestrated by diverse biomolecules ( 7 ). RCD is further classified into apoptotic and non-apoptotic subcategories [i.e., autophagy (mitophagy), ferroptosis, necroptosis, pyroptosis, anoikis and cuproptosis] ( 7 ). Autophagy, defined as self-degradation, is a process related to nonapoptotic cell death induced by a large number of intracellular/extracellular stimuli ( 8 ). Damaged proteins and organelles are removed and recycled by autophagy ( 9 ). This process plays a pivotal role in maintaining quality control and cellular homeostasis. On the other hand, ferroptosis is defined as a reactive oxygen species (ROS)-dependent cell death related to iron accumulation and lipid peroxidation, which is different from other forms of cell death ( 10 ). We recently reported that autophagy is dynamically regulated by various intrinsic [e.g., phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mTOR) and extrinsic (e.g., hypoxia and oxidative stress)] pathways, effectively attenuating the induction of apoptosis and promoting the survival of endometriotic cells ( 11 ). The periodic and repeated bleeding in endometriotic lesions is thought to trigger iron-dependent cell death known as ferroptosis. However, endometriotic cells may have acquired a potential mechanism to escape cell death in the harsh environment of intraperitoneal iron overload, hypoxia and nutrient deprivation by regulating autophagy and ferroptosis. The present review summarized the current understanding of the mechanisms underlying autophagy and ferroptosis in endometriosis and discussed spatiotemporal orchestration of ferroptosis-mediated cell death regulation.

Autophagy and ferroptosis have been shown to be induced or conversely suppressed in humans and preclinical animal models, but the reasons for such discrepancies and their mechanisms are still not fully understood. The present review discussed the role of autophagy and ferroptosis by dividing the development of endometriosis into four stages: Lesion initiation, induction of cell death, escape from cell death and formation of established lesions. Assuming that endometriosis evolves from retrograde endometrium, it was estimated how autophagy and ferroptosis change as endometriosis progresses ( Fig. 5 ). Endometrial tissues originating from retrograde menstruation encounter environmental challenges, e.g., iron overload, oxidative stress and hypoxia caused by periodic bleeding ( Fig. 5 ① ) ( 4 , 46 ). Several lines of evidence indicate that iron has a crucial role in the proliferation of endometriotic cells. Ding et al ( 62 ) found that in comparison with control mice, endometriotic lesions in the iron-overload model mice were larger and more numerous. Under iron overload or hypoxic conditions, autophagy plays a positive role in scavenging ROS, preserving mitochondrial integrity, counteracting metabolic insults, avoiding apoptosis and protecting cells ( 10 ). Endometrial cells derived from retrograde menstruation may be able to survive even under harsh conditions, possibly through activation of autophagy-dependent quality control mechanisms ( 101 ). In endometriosis, ferroptosis can occur through two pathways, the activation of the autophagy pathway controlled by iron and HIF-1 ( 28 , 29 ) and the expression of ferritinophagy-related proteins such as DMT1 ( 59 ). Indeed, the levels of both iron and MDA, a secondary product of free radical lipid peroxidation, are increased in the peritoneal fluid and ovaries, whereas expression of anti-ferroptosis-related proteins (e.g., GPX4 and GSH) are decreased, highlighting the importance of ferroptosis ( 62 , 102 ). Moreover, increased ferroptosis is found to enhance the expression of pro-angiogenic factors vascular endothelial growth factor and IL-8 ( 103 , 104 ). Ferroptosis has been shown to contribute to development and maintenance of subsequent endometriotic lesions through modulating the interaction between inflammation and angiogenesis ( 101 , 103 ). Additionally, it has been reported that ferric ammonium citrate-induced ferroptosis may promote fibrosis process in endometriosis ( 102 ). Autophagy is essential for establishing early lesions by counteracting the ferroptosis-mediated deleterious effects. Only a rare fraction of cells are likely able to survive in harsh environments mediated by iron/hypoxia-dependent oxidative stress. It is easy to understand that an excessive ferroptosis induces the regression of endometriotic lesions via lipid peroxidation-driven membrane destruction ( Fig. 5 ② ). Indeed, erastin, a ferroptosis inducer, inhibits endometriotic lesion growth through iron accumulation and decreased FPN expression in a mouse model of endometriosis ( 45 ). Perhaps, excessive ROS induces autophagic cell death, accelerates iron-dependent ferroptosis and amplifies the process of lipid peroxidation and the extent of membrane rupture ( 105 ). Autophagy also plays a role in eliminating damaged proteins and subcellular organelles to sustain cell viability, while unrecoverable damage can trigger cell death ( 106 ). By contrast, autophagy removes damaged intracellular components, such as cellular proteins, lipids, nucleic acids and mitochondria and can inhibit ferroptosis ( Fig. 5 ③ ). Nrf2-dependent autophagy activation may inhibit ferroptosis via enhancing the defense system against oxidative stress ( 65 ). Only a fraction of endometrial cells may receive survival signals and acquire a ferroptosis-resistant phenotype. On the other hand, intrinsic modulators (e.g., estrogen, PI3K/AKT/mTOR, AMPK, p53 and Beclin1) can inhibit autophagy through the expression of various ATG proteins ( Fig. 5 ④ ). mTOR inhibitors have been reported to confer therapeutic efficacy against endometriosis through activation of autophagy ( 34 ). Furthermore, downregulation of the expression of NCOA4 and VDAC2/3 and upregulation of the expression of xCT/GPX4 induces the suppression of ferroptosis and causes further progression of endometriotic lesions.

The present review conducted a narrative review of the targeted literature that focused on autophagy and ferroptosis in endometriosis. These mechanisms are well studied in cancer cells and the present review first drew information from these studies. It then summarized the roles of molecules regulating autophagy and ferroptosis that have been reported so far in endometriosis. Electronic databases including PubMed ( https://pubmed.ncbi.nlm.nih.gov/ ) and Google Scholar ( https://scholar.google.jp/ ) were searched for literature published up to the October 31, 2023, combining the following keywords: 'Autophagy', 'ferroptosis', 'regulated cell death', 'survival', and 'endometriosis'. The search terms were combined using the Boolean operators And OR ( Table I ). Additionally, a manual reference search of published articles was conducted. Included studies comprised original research publications in English and reference lists from review articles. Duplicated studies, literature irrelevant to the research topic and non-English publications were excluded. The flowchart depicted in Fig. 1 outlines the study selection process, detailing both inclusion and exclusion criteria. The initial phase involves identifying records through electronic database searches, manual searches and the reference lists of relevant articles and reviews. Titles and abstracts underwent a preliminary screening. After duplicates were removed, these titles and abstracts were reviewed to discard non-relevant studies. The final phase of eligibility involved analyzing the full-text articles, excluding any from which detailed data could not be obtained. The authors independently evaluated the articles to determine their suitability for inclusion or exclusion before reviewing the full texts. The properties of the identified molecules were searched in the National Library of Medicine database ( https://www.ncbi.nlm.nih.gov/ ).

Induction of autophagy and ferroptosis may contribute to the survival and proliferation of the retrograde tissues originating from the endometrium, whereas suppression of autophagy and ferroptosis may play an important role in the progression of endometriotic lesions. The key molecular actors (e.g., estrogen, PI3K/AKT/mTOR, NCOA4, VDAC, xCT, GPX4, Nrf2, or p53) may switch ferroptosis on and off. Hence, in light of our prior report indicating that endometriotic cells may possess a potential anti-apoptotic capability to survive in changing environments ( 11 ), a timely fine-tuning of autophagy and ferroptosis levels could regulate the development and progression of endometriosis. In conclusion, autophagy and ferroptosis play a dual role in the initiation and progression of endometriosis through multiple mechanisms regulated by intrinsic factors (e.g., estrogen and the PI3K/AKT/mTOR pathway) or extrinsic stressors (e.g., iron overload, oxidative stress and hypoxia).