{"paper_id":"4b7df82e-6c6f-4fa6-b07d-be054ad369cf","body_text":"Erythrocytes carried into the peritoneal cavity by menstrual reflux and/or bleeding lesions\nare known to be inducers of oxidative stress ( Van\nLangendonckt  et al. , 2002a , b ;  Defrère  et al. ,\n2006 ,  2008 ,  2011 ;  Lousse  et\nal. , 2009 ,  2012 ). Indeed,\nerythrocytes are likely to release pro-oxidant and proinflammatory factors like Hb and its\nhighly toxic by-products heme and iron into the peritoneal environment ( Van Langendonckt  et al. ,\n2002a , b ) ( Fig. 1 ). Unless they are properly chelated, free iron and heme\nbecome key players in the formation of deleterious ROS ( Van Langendonckt  et al. , 2002a , b ;  Agarwal\n et al. , 2005 ). Several  in vitro  studies ( Defrère  et al. , 2006 ;  Lousse  et al. , 2009 ) have\ndemonstrated the involvement of iron overload in the proliferation of endometriotic lesions\ninduced in murine models. This strongly suggests that iron is implicated in endometriosis\ndevelopment in women, as demonstrated by the presence of iron-loaded macrophages in\nperitoneal endometriotic lesions in affected individuals ( Van Langendonckt  et al. , 2002a , b ) ( Fig. 2 ). Iron conglomerates containing hemosiderin, another form of iron storage\nfound in cases of iron overload, have also been witnessed in endometriotic lesions ( Van Langendonckt  et al. , 2002b ).\nIndeed, erythrocytes reside in the peritoneal cavity of most (90%) menstruating women, so\nwhy do some individuals develop endometriotic lesions and others not? One hypothesis states\nthat peritoneal protective mechanisms are swamped by menstrual reflux in some patients,\neither because of its abundance or due to defective scavenging systems ( Donnez  et al. , 2016 ;  Van Langendonckt  et al. ,\n2002a , b ). A key defense mechanism to\ncounteract the effects of hemorrhage is mediated by haptoglobin (Hp), which is able to bind\nto extracellular Hb, thereby attenuating its oxidative and inflammatory potential ( Donnez  et al. , 2016 ).\nErythrocytes carried into the peritoneal cavity by menstrual reflux, bleeding\nendometrial lesions or hemoperitoneum, hemoglobin and its highly toxic by-products\n(heme and iron), and macrophages, inducing oxidative stress . Activated\nmacrophages are also able to deliver various inflammatory molecules and trigger\noxidative stress. CO, carbon monoxide; HO, heme oxygenase; NO, nitric oxide; NOS, nitric\noxide synthase.\nIron-overloaded macrophages in an endometriotic lesion identified by Prussian blue\nstaining . Activated macrophages are highly engaged in erythrocyte degradation,\nas suggested by the presence of numerous iron-loaded macrophages in peritoneal fluid and\nlesions from endometriosis patients (from  Van\nLangendonckt  et al. , 2002b , with permission).\nFor more than 20 years now, we have been claiming that iron plays a crucial role in\nendometriosis ( Van Langendonckt  et\nal. , 2002a , b ) and\nadvocating use of iron chelators, since they were shown to prevent initiation and\nprogression of the disease in murine models ( Defrère\n et al. , 2011 ). Despite our findings, iron chelators were never\ndeveloped in clinical research for treatment of endometriosis, but the role of highly toxic\nHb by-products like iron was highlighted in two recent reviews published by  Wyatt  et al.  (2023)  and  Vercellini  et al.  (2024) .\n\nNeutrophils, macrophages, natural killer (NK) cells, and dendritic cells are cell\npopulations of the innate immune system predominantly involved in endometriosis pathogenesis\n( Kapoor  et al. , 2021 ;\n Gajbhiye, 2023 ). Macrophages are immune\ncells charged with detecting foreign elements in the system and subsequently destroying\nthem. Iron metabolism and the role of macrophages in the pelvic cavity in endometriosis\npathology are graphically postulated in  Fig. 3 .\nActivated macrophages recruited inside the pelvic cavity are deeply engaged in degradation\nof erythrocytes, as indicated by numerous iron-loaded macrophages in the peritoneal fluid\nfrom both endometriosis patients and mice intraperitoneally injected with erythrocytes.\nMacrophages typically phagocytose senescent erythrocytes or endocytose the Hb–Hp complex. Hb\nand heme degradation by heme oxygenase (HO) release iron, which is then incorporated into\nferritin inside macrophages or sent back to the iron transporter transferrin via peritoneal\nfluid.\nEndometrial cell and macrophage interaction in the pelvic cavity .\nErythrocytes and endometrial cells are carried into the pelvic cavity by retrograde\nmenstruation and phagocytosed by peritoneal macrophages. Heme digestion by HO-1 releases\niron, which is either stored in the form of ferritin and hemosiderin or released to bind\nto transferrin. Endometrial cells with adhesive characteristics start to invade the\nmesothelium and trigger inflammatory signals that recruit more peritoneal macrophages.\nLocal inflammation and increased levels of ROS contribute to acquisition of a\nproliferative phenotype and proangiogenic features crucial to endometriotic lesion\ndevelopment (adapted from  Cacciottola  et\nal. , 2021 ). HO-1, heme oxygenase-1; ROS, reactive oxygen\nspecies.\nA number of studies have emphasized the involvement of peritoneal macrophages in iron\nmetabolism ( Van Langendonckt  et\nal. , 2002a , b ;  Taylor  et al. , 2021 ). Cellular\niron storage within ferritin hampers the ability of iron to generate free radicals and\nthereby confers an antioxidant effect. However, ongoing delivery of iron to macrophages may\noverwhelm the capacity of ferritin to store and sequester the metal, causing oxidative\ninjury to cells ( Van Langendonckt  et\nal. , 2002a , b ). We\nhypothesized in 2016 that the iron detoxification system could be progressively overwhelmed\nduring the menstrual cycle in endometriosis patients, leading to abnormal macrophage\nactivation ( Donnez  et al. ,\n2016 ). By releasing cytokines that trigger other cells, activated macrophages\ninitiate the process of inflammation. In this way, iron overload induces oxidative\nstress.\n\nHO-1 is a heme-degrading enzyme strongly upregulated by heme. It protects cells from\nheme-generated oxidative stress by producing beneficial molecules that deliver unique\nprotective and antioxidant effects, including carbon monoxide, bilirubin, and biliverdin\n( Van Langendonckt  et al. ,\n2002a , b ;  Donnez  et al. , 2016 ). HO-1 induction is also\nassociated with increased ferritin synthesis, free iron scavenging, and ensuing protection\nagainst any negative repercussions.\nHowever, in endometriosis, inducible HO-1 shows weak expression by macrophages and\nmesothelial cells, which make up the majority of cells in the peritoneal cavity, and there\nis no concomitant upturn in peritoneal fluid levels of bilirubin, its final byproduct. All\nthis strongly suggests that detoxifying systems, while present, might be insufficient to\nmetabolize Hb in the case of endometriosis ( Donnez\n et al. , 2016 ) or peritoneal hemoperitoneum, as in the series\nreported by  Chaggar  et al. \n(2024) .\n\nIn the uterine environment, the function of all immune cells, including macrophages, NK\ncells, and T cells, is regulated by associated increases in levels of proinflammatory\nmediators ( Cacciottola  et al. ,\n2021 ;  Kapoor  et al. ,\n2021 ;  Taylor  et al. ,\n2021 ;  Nazri  et al. ,\n2023 ;  Oală  et al. ,\n2024 ). Proinflammatory pathways prevent apoptotic pathways from clearing debris, so\nthese unwanted cells may travel and adhere to distant sites.\nMacrophages are able to deliver various inflammatory molecules that are responsible for\nboth initiation and progression of endometriosis ( Taylor  et al. , 2021 ;  Dolmans and Donnez, 2022 ;  Donnez and\nCacciottola, 2022 ;  Ni and Li, 2024 )\n( Fig. 3 ). They are also known for their\nwide-ranging functional and phenotypic alterations ( Nazri  et al. , 2020 ;  Dolmans and Donnez, 2022 ). These changes are governed by stimuli like oxidative\nstress, tissue damage, and hormones, leading to activation of different pathways of\nproliferation, migration, and invasion ( Agarwal\n et al. , 2005 ;  Donnez\n et al. , 2016 ).\nMacrophage migration inhibitory factor is an inflammatory cytokine that assumes a critical\nfunction in the early development of endometriosis ( Chekini  et al. , 2021 ). It recruits macrophages into endometriotic\nlesions and helps them proliferate by release of proinflammatory cytokines and other growth\nfactors ( Cacciottola  et al. ,\n2021 ).  Stratopoulou  et al. \n(2023)  investigated the role of M2 macrophages in endometrial invasiveness in\nadenomyosis. They found that accumulation of M2 macrophages enhances the invasion capacity\nof endometrial cells. In their model, M2 macrophage infiltration was sufficient to promote\nthe disease and its progression. They raised the possibility of collective cell migration\n(CCM) involvement in the invasion process of myometrium by endometrium. CCM was also\ndemonstrated in a baboon model of endometriosis, mimicking the invasion process seen in\nendometriosis ( Donnez  et al. ,\n2015 ;  Orellana  et al. ,\n2017 ).\nAs several papers ( Stratopoulou  et\nal. , 2021 ;  Donnez  et\nal. , 2024 ) have indeed confirmed common pathogenic features in both deep\nendometriosis and adenomyosis, namely excessive macrophage accumulation, fibrosis, and\nirregular angiogenesis, why not go further and extrapolate that infiltration by activated\nmacrophages is pivotal to invasion by endometrial cells in both diseases?\n\nROS are intermediaries produced by normal oxygen metabolism, but are known to have\ndeleterious effects ( Agarwal  et al. ,\n2005 ). To protect themselves, cells have developed a wide range of antioxidant\nsystems to limit ROS production, inactivate the molecules, and repair cell damage. In\nhealthy individuals, ROS and antioxidants are in balance. However, when the balance is\ntipped toward an overabundance of ROS, oxidative stress ensues and can impact the\nreproductive lifespan of women ( Donnez  et\nal. , 2016 ;  Cacciottola  et\nal. , 2021 ). Oxidative stress occurs when the balance between ROS\nproduction and antioxidant defense is disrupted due to either inadequate antioxidant\nprotection or excess production of ROS. Various lines of evidence support the role of\noxidants in the development of endometriosis, since endometriotic cells show higher\nendogenous oxidative stress levels, elevated ROS production, and alterations to ROS\ndetoxification pathways ( Donnez  et\nal. , 2016 ).\nFirst of all, Hb, heme, and iron derivatives are generated from hemolysis of erythrocytes\nabnormally accumulating in endometriotic lesions. Second, the ability to survive the\noxidative activity of these derivatives appears to be conducive to endometriotic cell\ngrowth. Lower levels of apoptosis observed in lesions suggest that aberrant adenomyotic and\nendometriotic cells may survive and contribute to progression of the disease ( d’Argent  et al. , 2023 ). Finally,\nendometriotic lesions residing in their unique microenvironment may display significant\nindividual differences in terms of degree of responsiveness to free radicals or antioxidant\ndefenses ( Donnez  et al. ,\n2016 ). Investigating the mechanisms underlying oxidative stress associated with\nendometriosis may well prove fruitful for determining the specific pathways responsible for\ninitiation and progression of the disease ( Kapoor\n et al. , 2021 ;  Dolmans\nand Donnez, 2022 ).\n\nSmall extracellular vesicles (sEVs) (<200 nm) are cell-derived vesicles containing\nmicroRNAs (miRNAs) that regulate post-transcriptional gene expression. In 2020,  Nazri  et al.  (2020) \ncharacterized exosomes found in peritoneal fluid from endometriosis patients. In a very\nrecent paper,  Zipponi  et al. \n(2024)  proved the feasibility of  in vitro  culture of the\nendometrioma wall and managed to isolate and examine secreted exosomes. Analysis of miRNA\nexosome content and predicted target genes may well prove to be a promising starting point\nfor a better understanding of endometriosis pathogenesis, addressing the potential influence\nof miRNA expression in sEVs secreted by lesions and macrophages from women with the disease.\nCharacterization of exosomes opens up brand new avenues for diagnosis and investigation of\nendometriosis ( Nazri  et al. ,\n2023 ;  Zipponi  et al. ,\n2024 ).\n\nThere is no doubt that the pathogenesis of endometriosis is multifactorial. It is also\nclear that iron overload, delivery of inflammatory molecules by activated macrophages, and\noxidative stress create a favorable environment for endometrial cells to implant, progress,\nand metastasize to other locations. Iron overload in the pelvic cavity and its consequences\n(activation of macrophages and oxidative stress) could potentially be the link explaining\nthe high incidence of endometriosis after hemoperitoneum, as reported in the current issue\nof  Human Reproduction Open  by  Chaggar  et al.  (2024) .","source_license":"CC0","license_restricted":false}