Dysregulated autophagy in endometriosis: molecular mechanisms, controversies, and clinical implications

Autophagy appears to play an important role in the pathophysiology of endometriosis (Fig.  4 A), influencing processes such as cell survival, immune evasion, cell homeostasis, oxidative stress response, and lesion persistence or progression (Fig.  4 B). Accumulating evidence indicates that autophagy is not uniformly activated or suppressed in endometriosis but is instead dynamically regulated, depending on the cellular context, lesion location, and microenvironmental cues such as hypoxia, iron overload, hormonal regulation, and inflammatory signaling. Fig. 4 Retrograde menstruation and estrogen-driven hypoxia-induced immune and cellular dysregulation in endometriosis. A  Schematic representation of retrograde menstruation and lesion establishment. Viable endometrial fragments entering the peritoneal cavity encounter hypoxia, elevated estrogen, oxidative stress, and iron overload, which together drive immune dysfunction, EMT, altered adhesion, angiogenesis, and ferroptosis. These changes create a permissive niche for ectopic lesion survival and growth, with autophagy dysregulation contributing to disease progression—ESCs, endometrial stromal cells; EPCs, endometrial epithelial cells. B  Autophagy dysregulation is increasingly recognized as a central mechanism in endometriosis. Hypoxia and iron overload activate autophagy in endometriotic lesions via PARP1/SIRT1 and DIRAS3. Elevated estrogen levels disrupt immune balance by downregulating HCK and upregulating CXCR4/CXCL12, impairing macrophage and NK cell function. Autophagy promotes lesion persistence by enhancing migration, invasion, EMT, and resistance to ferroptosis. Figure created with BioRender.com Retrograde menstruation and estrogen-driven hypoxia-induced immune and cellular dysregulation in endometriosis. A  Schematic representation of retrograde menstruation and lesion establishment. Viable endometrial fragments entering the peritoneal cavity encounter hypoxia, elevated estrogen, oxidative stress, and iron overload, which together drive immune dysfunction, EMT, altered adhesion, angiogenesis, and ferroptosis. These changes create a permissive niche for ectopic lesion survival and growth, with autophagy dysregulation contributing to disease progression—ESCs, endometrial stromal cells; EPCs, endometrial epithelial cells. B  Autophagy dysregulation is increasingly recognized as a central mechanism in endometriosis. Hypoxia and iron overload activate autophagy in endometriotic lesions via PARP1/SIRT1 and DIRAS3. Elevated estrogen levels disrupt immune balance by downregulating HCK and upregulating CXCR4/CXCL12, impairing macrophage and NK cell function. Autophagy promotes lesion persistence by enhancing migration, invasion, EMT, and resistance to ferroptosis. Figure created with BioRender.com From a therapeutic standpoint, targeting autophagy presents both opportunities and challenges. While pharmacological modulation of autophagy (e.g., via mTOR inhibitors or autophagy inducers) holds promise, the dual role of autophagy in promoting both cell survival and cell death necessitates a nuanced approach. Future therapies may benefit from more personalized strategies that consider lesion type, stage, molecular profile, and autophagic status of the patient. More detailed studies focusing on the molecular mechanisms of autophagy in endometriosis are essential to uncover new therapeutic targets. In fact, the integration of single-cell transcriptomics, proteomics, and functional imaging will be essential to unravel the heterogeneity of autophagy regulation in endometriosis. These tools may help identify biomarkers for early diagnosis and novel therapeutic targets, ultimately improving outcomes for patients with this complex and often debilitating disease. Identifying reliable biomarkers and understanding individual variations in autophagy activity in endometriosis could aid in diagnosis and monitoring treatment responses. In addition, clinical trials are needed to evaluate the efficacy and safety of autophagy-modulating therapies in endometriosis patients.

Endometriosis is a common gynecologic disease affecting approximately 10–15% of all women of reproductive age and 20–50% of all infertile women [ 1 , 2 ]. It is characterized by functional uterine endometrial glands and stroma-like tissue outside the uterine cavity, including the ovaries, pelvic peritoneum, and rectovaginal septum [ 3 , 4 ]. Endometriosis is a chronic inflammatory estrogen-dependent disease associated with a wide range of symptoms, such as chronic pelvic pain, dysmenorrhea, and infertility, as well as fatigue, painful sex, and depression, among others, which significantly impact the quality of life [ 5 , 6 ]. One of the challenges patients face is the delayed diagnosis, which often occurs 8–10 years after symptom onset [ 7 ]. Furthermore, there is currently no cure to prevent or fully eradicate endometriosis, although treatments and therapies exist to help reduce symptoms [ 8 , 9 ]. The etiology of this disease remains unclear. Among the proposed hypotheses, the theory of retrograde menstruation proposed by Sampson is the most widely accepted, as retrograde menstruation occurs in approximately 90% of women [ 10 ]. According to this theory, menstrual debris flows anterogradely to the vagina and through the fallopian tubes into the peritoneal cavity during menstruation. Viable endometrial cells may then settle in dependent areas of the peritoneum, where they attach, implant, proliferate and ultimately give rise to endometriosis. Nowadays, endometriosis is recognized as a polygenic and multifactorial disease [ 11 , 12 ]. Although it is a benign disease, it shares common biological behaviors with cancer, such as migration, proliferation, and invasion, highlighting its complexity and potential implications for human health [ 13 ]. Among the biological processes that may contribute to these behaviors, autophagy, a highly conserved cellular mechanism responsible for the degradation and recycling of intracellular components, has recently gained attention [ 14 – 16 ]. A PubMed search using the terms “endometriosis”, “autophagy”, and “autophagy in endometriosis” reveals an appreciable increase in scientific studies in both areas, with the role of autophagy in endometriosis being particularly prominent since 2018. The earliest PubMed citation for endometriosis dates to 1927 ( n = 2), while the first for autophagy appeared thirty-six years later ( n = 1). Although the first citation linking autophagy and endometriosis was in 1986, more in-depth studies did not emerge until 2009. Notably, 2018 marked the year with the highest number of publications on this topic ( n = 22) (data updated as of January 25, 2025). Despite this growing body of research, many fundamental questions remain unanswered. This persistent gap in understanding is largely because, although endometriosis is a long-standing condition, scientific knowledge regarding its underlying mechanisms remains relatively limited. In our previous review, we discussed the role of autophagy in both normal and pathological endometrium, particularly in the context of endometrial cancer [ 17 ]. In this current work, we aim to provide an overview of the current insights into the role of autophagy in endometriosis. Autophagy is a highly conserved catabolic process crucial for maintaining cellular homeostasis by regulating lysosomal degradation and recycling various cellular components. It is typically activated in response to different cellular stress conditions and functions primarily as a survival mechanism. Based on the nature of the cargo and the pathway through which it is delivered to the lysosome, autophagy can be classified into three different categories: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) [ 18 – 20 ]. The most extensively studied form of autophagy is macroautophagy. This process is regulated by the highly conserved autophagy-related genes (ATG) [ 21 ] and involves several sequential steps: initiation, expansion, closure, and fusion with the endolysosomal system [ 22 ]. An emerging consensus in mammalian cells explains that the autophagosome membrane originates from endoplasmic reticulum (ER) membranes. However, several cellular compartments, such as the Golgi, endosomes, mitochondria, and the plasma membrane, also contribute to the expansion of the budding autophagosome [ 22 ]. The initiation process consists of the transmission of the signal to the membrane of origin, from which the primordial autophagy structures emerge, expand, and close. This step requires the activation of two ubiquitin-like conjugating systems essential for the formation of the phagophore and the subsequent elongation of the autophagosomal membrane [ 23 – 25 ]. The first of these complexes entails the conjugation of the ubiquitin-like protein ATG12 to ATG5 through the sequential activation of an E1-like (ATG7) and an E2-like (ATG10) protein [ 26 , 27 ]. The covalently formed ATG5-ATG12 complex then interacts with ATG16L1 at the membrane of the nascent phagophore, and the complex remains there until the phagophore is fully formed (Fig. 1 A) [ 28 – 30 ]. In mammals, the second system (Fig. 1 B) is associated with a group of Atg8(yeast)-like proteins, such as LC3 (microtubule-associated protein light chain 3), GABARAP (gamma-aminobutyric acid receptor-associated protein), and GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), with LC3 being the most representative and well-studied member [ 31 ]. In the case of LC3, the autophagy-specific cysteine protease ATG4B cleaves the pro-form of LC3 at the C-terminus, generating a truncated LC3-I form that exposes a glycine residue susceptible to being lipidated [ 26 – 31 ]. Under conditions of increased autophagy flux, this residue is conjugated to phosphatidyl ethanolamine (PE) [ 32 ], resulting in the generation of the highly hydrophobic LC3-II form. This process is regulated by the activation of the E1-like and E2-like enzymes, ATG7 and ATG3, respectively. Fig. 1 Schematic representation of the two ubiquitin-like conjugation systems required for phagophore expansion. A  ATG12 is conjugated to ATG5 by ATG7 (an E1-like protein) and ATG10 (an E2-like protein), forming the ATG12-ATG5-ATG16L1 complex. B  LC3 is cleaved by the protease ATG4B to LC3-I, activated by ATG7, transferred to s ATG3 (E2-like enzyme), and conjugated to phosphatidylethanolamine (PE) by the ATG12–ATG5–ATG16L1 complex, generating the lipidated LC3-II form. LC3-II integrates into the autophagosomal membrane and is essential for its elongation and closure —figure created with BioRender.com Schematic representation of the two ubiquitin-like conjugation systems required for phagophore expansion. A  ATG12 is conjugated to ATG5 by ATG7 (an E1-like protein) and ATG10 (an E2-like protein), forming the ATG12-ATG5-ATG16L1 complex. B  LC3 is cleaved by the protease ATG4B to LC3-I, activated by ATG7, transferred to s ATG3 (E2-like enzyme), and conjugated to phosphatidylethanolamine (PE) by the ATG12–ATG5–ATG16L1 complex, generating the lipidated LC3-II form. LC3-II integrates into the autophagosomal membrane and is essential for its elongation and closure —figure created with BioRender.com Once the phagophore is completed and the ATG5-ATG12-ATG16L1 complex is removed, LC3-II is involved in the expansion of the autophagosomal membrane. During this process, LC3-II also acts as a cargo receptor through its LC3-interacting region (LIR) [ 32 ], facilitating cargo recruitment into the expanding autophagosome. As the membrane elongates and encloses the cargo, the closure process begins. The membrane completes its seals in this step, forming a fully enclosed vesicle. The autophagosome is now a sealed structure, ready to undergo fusion with the lysosome. During this process, LC3-II is further removed from the outer autophagosome membrane by ATG4B before being degraded within the autolysosomes by lysosomal hydrolases [ 33 ]. The fusion of the autophagosome with the lysosome results in the formation of the autolysosome, where lysosomal enzymes degrade the engulfed cargo. Beyond the activation of the two essential ubiquitin-like conjugating systems, additional regulatory layers are required to trigger the autophagy process fully. Importantly, this complex regulation underscores autophagy as a sophisticated and intricate cellular process. As described above, autophagy is controlled by the ATGs [ 21 ], which are hierarchically recruited to the omegasome in mammals. However, the activity of these ATGs is not only determined by their inherent properties but can also be influenced by intracellular nutrients, growth factors (such as the mammalian target of rapamycin or mTOR), energy status, and hypoxia [ 23 , 34 – 36 ]. The energy status influences autophagy through a key signaling pathway involving the enzyme AMP-activated protein kinase (AMPK) [ 37 ]. When the cell is under energy stress (low ATP/ADP ratio), AMPK is activated, which in turn signals the mammalian target of rapamycin complex 1 (mTORC1). Additionally, growth factors such as insulin also stimulate mTORC1 through their receptor, and it triggers the canonical signaling pathway, which inhibits autophagy [ 38 ]. One of the classical regulators of autophagy is mTORC1 [ 39 ]. Under normal nutrient conditions, mTORC1 is active and inhibits autophagy by phosphorylating ULK1 (unc-51-like autophagy activating kinase), which blocks its interaction with AMPK as well as by phosphorylating ATG13 [ 40 , 41 ]. ULK1 is a central component of the autophagy machinery and plays a role in autophagosome biogenesis by forming the ULK complexes. These complexes comprise multiple members, including ULK1 and ULK2, ATG13, ATG101, and the FAK family kinase-interacting protein of 200 kDa (FIP200) [ 42 , 43 ] (Fig. 2 A). Their role in autophagy execution involves, for example, phosphorylating key substrates such as AMBRA1 [ 44 ] or BECN1 (also known as Beclin-1) [ 45 ], which results in increased activity of the BECN1-ATG14-VPS34-VPS15 class III autophagy-specific class III phosphatidylinositol 3-kinase (PI3K) core complexes [ 46 ] and promotes nucleation. Fig. 2 Autophagy regulation by mTORC1. A  Under normal nutrient conditions, mTORC1 is active and inhibits autophagy by phosphorylating ULK1 and ATG13, preventing ULK1 complex formation and its interaction with AMPK. As a result, the autophagy initiation machinery remains inactive. B  Starvation conditions activate AMPK, which phosphorylates TSC2 and ULK1, leading to mTORC1 inactivation. ULK1 can form the ULK complex with ATG13, FIP200 and ATG101 —figure created with BioRender.com Autophagy regulation by mTORC1. A  Under normal nutrient conditions, mTORC1 is active and inhibits autophagy by phosphorylating ULK1 and ATG13, preventing ULK1 complex formation and its interaction with AMPK. As a result, the autophagy initiation machinery remains inactive. B  Starvation conditions activate AMPK, which phosphorylates TSC2 and ULK1, leading to mTORC1 inactivation. ULK1 can form the ULK complex with ATG13, FIP200 and ATG101 —figure created with BioRender.com On the other hand, nutrient starvation triggers the inactivation of mTORC1 activity by AMPK-dependent phosphorylation of upstream TSC2 [ 47 ] or by phosphorylating ULK1 [ 48 – 50 ], which relieves the inhibitory signals from mTORC1 and restores the pro-autophagy function of ULK complexes. Free from mTORC1, the kinase ULK1 phosphorylates other components such as ATG13 and FIP200 (within the ULK complex) [ 51 ], and the complex translocates to discrete locations on the ER previously marked by ATG9 [ 52 ] (Fig. 2 B). ATG9 seems to participate in early and later stages of autophagy and has been described to carry lipids and vesicles to expand the membranes through an ULK1 activity-dependent manner [ 53 ]. Subsequently, the PI3K complexes are recruited, generating ER domains or structures called the omegasome [ 54 ], and initiating the autophagy process. Whereas the composition of the autophagy-specific class III PI3K complexes can differ depending on the precise combination of their members, it is broadly accepted that the main components are BECN1, the sensor of membrane curvature ATG14, phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3K3C3, best known as VPS34), and phosphoinositide-3-kinase regulatory subunit (4 PI3KR4, also known as VPS15) [ 55 ]. Additional members such as AMBRA1, BIF-1, UVRAG, or Rubicon fine-tune the precise molecular function of these complexes [ 56 – 59 ]. VPS34 produces PtdIns [ 3 ]P(phosphatidylinositol‑3‑phosphate) and participates in the expansion of autophagosomal membranes until closure by recruiting PtdIns [ 3 ]P-binding ATG proteins [ 60 ], such as WD-repeat domain phosphoinositide-interacting proteins (WIPI) and the double FYVE-containing protein 1 (DFCP1). WD-repeat domain- phosphoinositide interacting 2 (WIPI2) and ATG2 are involved in ATG9 recycling [ 61 ]. PtdIns [ 3 ]P is also produced in the endosome by the class III PI3K complex, which contains an ultraviolet irradiation resistance-associated gene (UVRAG) instead of ATG14 [ 62 ]. In recent years, accumulating data has pointed to autophagy as a key cellular pathway involved in the progression of endometriosis. However, its precise role remains controversial, with studies reporting both upregulation and downregulation of autophagic activity in endometriosis [ 63 ]. The principal findings regarding the status of autophagy in the progression of endometriosis, along with the proposed pharmacological compounds used for its treatment (including their mechanism of action, effects on autophagy modulation, and main biological effects) are summarized in Tables 1 and 2 , and 3 . Table 1 Autophagy downregulation in endometriosis (EMS) Autophagy upregulation in endometriosis progression References Model Effects on autophagy and associated molecules Global effects Role of autophagy Liu H et al., Reproduction 2017 [ 65 ] Tissue level: normal endometrium from patients with tubal infertility, eutopic endometrium from patients with ovarian endometriosis, and ectopic endometrium from ovarian endometriotic cysts (stage III or IV) High expression of HIF-1α and LC3 protein expression Autophagy increased in the ectopic endometrium from patients with endometriosis compared to the normal and eutopic endometrium Promotion of invasion and metastasis In vitro: Hypoxia-induced autophagy in cultured human endometrial stromal cells (ESCs) Increased LC3-II, BECN1, HIF-1α, and autophagosomes accumulation Hypoxia treatment induced autophagy in ESCs, dependent on HIF-1α, enhanced migration and invasion Xu T-X et al., Eur Rev Med Pharmacol Sci. 2016 [ 67 ] Tissue level: Ectopic endometrial tissues obtained from patients with ovarian endometriosis and eutopic endometrial tissue obtained from sterile women with tubal infertility Elevated expression of HIF-1α Elevated expression of HIF-1α in the ectopic endometriotic tissue was associated with increased miR-210 expression Cell survival In vitro: hypoxia treatment in ovarian endometriotic cell line CRL-7566 Increased LC3-II, reduced p62 Hypoxia treatment increased HIF-1α and miR-210 expression. Hypoxia enhanced autophagy via the Bcl2/BECN1 Liu H et al., Biol Reprod 2018 [ 69 ] Tissue level: normal endometrium tissue samples obtained from patients with tubal infertility, eutopic endometrium of endometriosis and ovarian endometriotic cyst tissue samples (ectopic endometrium) Increased HIF-1α, LC3 Autophagy upregulated in ectopic tissue Promotion of migration and invasion through EMT induction In vitro: hypoxia treatment in primary endometrial epithelial cells and Ishikawa endometrial cancer cell line Increased expression levels of HIF-1α, BECN1, LC3-II, and autophagosomes formation Hypoxia enhanced HIF-1α-dependent autophagy Chen ML et al., Cytokine 2023 [ 70 ] In vitro: hypoxia treatment in ovarian endometriotic cell line CRL-7566 Increased miR-150-5p expression levels Hypoxia treatment upregulated miR-150-5p expression in CRL-7566 cells, leading to increased autophagy and EMT. Knocking down miR-150-5p suppressed hypoxia-induced autophagy, migration, and EMT Promotion of migration and invasion through EMT induction Liu H et al., J Cell Mol Med 2018 [ 73 ] Tissue level: healthy control group, eutopic endometrium, and ectopic endometrium from ovarian endometriosis patients (stage III or IV endometriosis) Increased protein expression of HIF-1 and LC3-II total accumulation lncRNA-MALAT1 RNA expression and autophagy were increased in ovarian endometriosis tissues compared to normal and eutopic endometrium. lncRNA-MALAT1expression level correlated positively with HIF-1α Cell survival In vitro: hypoxia conditions, ESCs Increased levels of HIF-1α, BECN1, and LC3B-II protein expression, and autophagic flux were observed with the GFP-RFP-LC3 construct (more red puncta) Hypoxia-induced lncRNA-MALAT1 and autophagy. lncRNA-MALAT1 upregulation depended on HIF-1α signaling. Knockdown of lncRNA-MALAT1 suppressed hypoxia-induced autophagy Huang J et al., Front Endocrinol 2021 [ 74 ] In vitro: ESCs from ectopic endometrium, shRNA against HMGB-1. Cells were subjected to hypoxia Under hypoxia conditions, autophagy-related marker protein levels (BECN1 and ATG13) decreased when knocking down HMGB-1 HMGB-1 promoted the upregulation of autophagy in endometriosis Potential role in promoting endometriosis Huang J et al., Int Immunopharmacol 2024 [ 75 ] In vitro: overexpressed HMGB1 in ESCs. These cells were isolated from the ectopic endometrium of patients with endometriosis Under hypoxia conditions, the increased protein levels of BECN1, ATG13 and LC3-II BECN1, ATG13 and LC3-II levels were enhanced in endometriotic ectopic endometrium. Potential role in promoting endometriosis In vivo: Hmgb1-deficient (Hmgb1fl/f) mice Decreased protein level of BECN1, Atg13 and LC3-II BECN1, Atg13 and LC3-II were significantly decreased in Hmgb1 mouse ESCs compared to WT, under hypoxic conditions. Hmgb1deficiency inhibited autophagy Louwe PA et al., Nat Commun 2021 [ 78 ] In vivo: mouse model of endometriosis Inflammation-driven changes in macrophage phenotype suggest a potential role for autophagy in macrophage differentiation and function Elevated numbers of macrophages in peritoneal fluid and endometriotic lesions. Macrophages exhibit inflammation-driven phenotypes Autophagy may contribute to macrophage plasticity and functional adaptation in response to inflammation Xu H et al., Biosci Rep (2019) [ 81 ] In vivo: mouse model of endometriosis Overexpression of EPHA3 inhibited mTOR signaling and increased Atg3, LC3-II/LC3-I, and BECN1 Inhibition of mTOR signaling led to increased macrophage autophagy and apoptosis, reducing inflammation and fibrosis Autophagy contributed to macrophage apoptosis and immune regulation Huang Y et al., Front Immunol (2022) [ 82 ] In vitro: cultured peritoneal macrophages from endometriosis patients and MST1-knockout macrophages MST1 deficiency led to MAPK overactivation, promoted M2 macrophage polarization, and reduced phagocytosis. MST1-knockout macrophages secreted IL-10, which enhanced autophagy in ectopic ESCs Increased M2 macrophages contribute to immune tolerance and ectopic tissue survival, while IL-10-driven autophagy may support ectopic endometrial cell persistence Facilitating survival and adaptation within the peritoneal environment Lei ST et al., Cell Prolif (2024) [ 83 ] Tissue level: Endometriosis peritoneal fluid (PF) was obtained from patients with endometriosis. Then, the PF cells were collected to isolate the macrophages for in vitro experiments Decreased expression of HCK, impaired macrophage phagocytosis, and upregulated autophagy Increased number and weight of ectopic lesions due to macrophage dysfunction Promotes macrophage phagocytosis and endometriosis progression Zhou Y et al., Toxicology 2022 [ 84 ] In vitro: eutopic ESCs from endometriosis or adenomyosis patients Autophagy was functionally activated in response to iron overload, confirmed by LC3. SIRT1 silencing mitigated iron overload-induced PARP1 downregulation and autophagy activation Autophagy played a protective role by reducing ROS accumulation and counteracting iron overload-induced apoptosis. PARP1/SIRT1 signaling was implicated in the regulation of autophagy under iron overload conditions Cell survival Dong X et al., Mol Hum Reprod 2023 [ 85 ] In vivo: mouse model of endometriosis Iron overload increases autophagy: BECN1 and LC3-II Lesion development is enhanced by iron overload and autophagy activation; autophagy inhibitors suppress lesion growth Autophagy-dependent ferroptosis: Iron-induced ferroptosis is promoted by autophagy, influencing lesion growth in vivo Yotova I et al., Reprod Biomed Online 2023 [ 88 ] Tissue level: normal endometrium and endometriosis lesion samples Autophagy induction (serum starvation): upregulation of DIRAS3 and decreased LC3I/LC3II ratio DIRAS3 facilitated autophagy to promote cell survival, which may contribute to lesion persistence and disease progression. Autophagy is induced by DIRAS3, helping cells survive under stress conditions In vitro: 12Z human endometriosis epithelial cell line; DIRAS3 siRNA knockdown Allavena G et al., Fertil Steril 2015 [ 89 ] Tissue level: normal endometrium (tubal sterilization) and paired eutopic/ectopic tissues from women with ovarian endometriotic cysts Increased LC3B-II protein levels and LC3-II/LC3-I ratio, decreased p62 expression, and induction of LC3B , ATG14 , BECN1 , ATG7 genes Ovarian endometriomas showed increased autophagy compared with eutopic or normal endometrium, along with decreased p53, slight Bcl-2 downregulation, and increased HO-1. Potential role as a cell survival factor Ding Y et al., Translational Research 2021 [ 90 ] Tissue level: ovarian granulosa cells (GCs) of patients with endometriosis Increased BECN1 and LC3-II/LC3-I ratio GCs from ovarian endometriomas showed increased BECN1 and autophagy, which may support progesterone biosynthesis via lysosome-mediated LDL degradation Not assessed  Zhou Y et al., Reproduction 2021 [ 91 ] Tissue level: Endometrial tissues from patients with endometriosis or adenomyosis Increased LC3B-II levels Endometrial tissues from endometriosis/adenomyosis showed decreased Indian Hedgehog signaling, associated with increased autophagy and enhanced cell survival Cell survival Li H et al., Gynecol Endocrinol 2023 [ 92 ] Tissue level: Endometrial tissues from patients with endometriosis were collected during the proliferative phase Increased BECN1 and LC3 levels; low expression of p62, GPX4, and p53 High levels of BECN1, LC3, and reduced p62, GPX4, and p53 suggested autophagy and ferroptosis involvement in patients with endometriosis Induces ferroptosis In vivo: endometriosis rat model through allograft endometrial transplantation Rapamycin increased expression of autophagy proteins (BECN1, LC3), and si-Atg5 reduced expression of autophagy proteins Autophagy-induced ferroptosis played a role in the progression of the disease. Rapamycin induced ferroptosis and oxidative stress, while si-Atg5 inhibited these effects In vitro: Primary endometriosis cells from the rat model and cultured with Rapamycin (autophagy inducer), with si-Atg5 (autophagy inhibitor), and with si-Atg5 plus Erastin (ferroptosis inducer) Rapamycin increased autophagy and ferroptosis, while si-Atg5 inhibited autophagy and ferroptosis Zhu S et al., Int Immunopharmacol 2023 [ 93 ] Tissue level: ectopic and eutopic endometrium from patients with endometriosis High expression of LC3 and lower expression of p62 in the ectopic endometrium compared to the eutopic endometrium Increased cGAS-STING and autophagy expression suggest a role in EMS progression Not assessed In vivo: rat model of endometriosis by endometrial auto-transplantation Increased cGAS-STING and autophagy expression suggest a role in EMS progression. STING antagonist reduced ectopic lesion size by inhibiting autophagy, suggesting a potential therapeutic effect Not assessed In vitro: Primary human ESCs that were transfected with lentivirus to overexpress STING Increased expression of BECN1 and LC3 and decreased expression of p62 Overexpression of STING promoted autophagy and the migration and invasion of ESCs Promotes cellular motility and migration/invasion Yin B et al., Reprod Sci 2018 [ 94 ] In vivo: mouse models of endometriosis with caloric restriction applied before or after lesion induction Decreased expression of IGF-1, p-Akt, ER-b, and mTOR and increased expression of p-AMPK, SIRT1, and LC3 Caloric restriction reduced lesion growth and fibrosis in mice, with suppressed PI3K/Akt/mTOR and CREB signaling and increased autophagy in endometriotic lesions Potential cell death mechanism Zheng J et al., Reprod Sci, 2018 [ 96 ] Tissue level: normal eutopic endometrium and paired eutopic/ectopic tissues from women with ovarian endometriosis Increased LC3-II expression, LC3-II/LC3-I ratio, BECN1 levels Ovarian endometriomas showed increased autophagy and reduced HOXA10 (mRNA/protein) compared with eutopic and normal endometrium. HOXA10 correlated negatively with autophagy, while serum CA125 correlated positively with autophagy and negatively with HOXA10 Not assessed Abbreviations: Akt (AKT serine/threonine kinase), AMPK (AMP-activated protein kinase), ATG5 (Autophagy related 5), ATG7 (Autophagy related 7), ATG13 (Autophagy related 13), ATG14 (Autophagy related 14), Bcl-2 (BCL2 apoptosis regulator), BECN1 (Beclin 1), CA-125 (Cancer antigen 125), CR (Caloric restriction), CREB (cAMP response element-binding protein), DIRAS3 (DIRAS family GTPase 3), EMT (Epithelial-mesenchymal transition), EPHA3 (Ephrin type-A receptor 3), ER- α (Estrogen receptor alpha), ER-β (Estrogen receptor beta), ESCs (Endometrial stromal cells), GCs (Granulosa cells), GPX4 (Glutathione peroxidase 4), HCK (Hematopoietic cellular kinase), HIF-1α (Hypoxia-inducible factor 1-alpha), HMGB-1 (High mobility group box 1), HO-1 (Heme oxygenase 1), HOXA10 (Homeobox A10), IGF-1 (Insulin-like growth factor 1), IL-10 (Interleukin 10), LC3 (Microtubule-associated protein light chain 3), LDL (Low-density lipoprotein), lncRNA (Long non-coding RNA), MALAT1 (Metastasis associated lung adenocarcinoma transcript 1), MAPK (Mitogen-activated protein kinase), MESCs (Mouse endometrial stromal cells), MST1 (Macrophage stimulating 1), mTOR (Mechanistic target of rapamycin kinase), P4 (Progesterone), p53 (Tumor protein p53) , p62/SQSTM1 (Sequestosome 1), PARP1 (Poly(ADP-ribose) polymerase 1), PF (Peritoneal fluid), ROS (Reactive oxygen species), shRNA (Short hairpin RNA), siRNA (Small interfering RNA), SIRT1 (Sirtuin 1), and STING (Stimulator of interferon genes) Autophagy downregulation in endometriosis (EMS) Tissue level: Endometriosis peritoneal fluid (PF) was obtained from patients with endometriosis. Then, the PF cells were collected to isolate the macrophages for in vitro experiments Abbreviations: Akt (AKT serine/threonine kinase), AMPK (AMP-activated protein kinase), ATG5 (Autophagy related 5), ATG7 (Autophagy related 7), ATG13 (Autophagy related 13), ATG14 (Autophagy related 14), Bcl-2 (BCL2 apoptosis regulator), BECN1 (Beclin 1), CA-125 (Cancer antigen 125), CR (Caloric restriction), CREB (cAMP response element-binding protein), DIRAS3 (DIRAS family GTPase 3), EMT (Epithelial-mesenchymal transition), EPHA3 (Ephrin type-A receptor 3), ER- α (Estrogen receptor alpha), ER-β (Estrogen receptor beta), ESCs (Endometrial stromal cells), GCs (Granulosa cells), GPX4 (Glutathione peroxidase 4), HCK (Hematopoietic cellular kinase), HIF-1α (Hypoxia-inducible factor 1-alpha), HMGB-1 (High mobility group box 1), HO-1 (Heme oxygenase 1), HOXA10 (Homeobox A10), IGF-1 (Insulin-like growth factor 1), IL-10 (Interleukin 10), LC3 (Microtubule-associated protein light chain 3), LDL (Low-density lipoprotein), lncRNA (Long non-coding RNA), MALAT1 (Metastasis associated lung adenocarcinoma transcript 1), MAPK (Mitogen-activated protein kinase), MESCs (Mouse endometrial stromal cells), MST1 (Macrophage stimulating 1), mTOR (Mechanistic target of rapamycin kinase), P4 (Progesterone), p53 (Tumor protein p53) , p62/SQSTM1 (Sequestosome 1), PARP1 (Poly(ADP-ribose) polymerase 1), PF (Peritoneal fluid), ROS (Reactive oxygen species), shRNA (Short hairpin RNA), siRNA (Small interfering RNA), SIRT1 (Sirtuin 1), and STING (Stimulator of interferon genes) Table 2 Autophagy downregulation in endometriosis (EMS) Autophagy downregulation in endometriosis progression References Model Effects on autophagy Global effects Role of autophagy Mei J et al., Autophagy 2018 [ 97 ] In vitro: ESCs isolated from normal endometrium, eutopic endometrium and ectopic lesion from patient with endometriosis Decreased LC3B and BECN1 Decreased autophagic levels in ectopic isolated ESCs compared to normal ESCs; the same effects were seen after stimulation with 17-β estradiol Promotion of cytotoxic activity of NK cells Zhang L et al., Int J Fertil Steril 2015 [ 98 ] Tissue level: normal endometrium and paired eutopic/ectopic ovarian endometrium from women with endometriosis (proliferative phase) Decreased BECN1 mRNA and protein levels Ectopic and eutopic endometrium from endometriosis showed reduced autophagy vs. normal tissue. Serum CA125 was elevated and negatively correlated with BECN1 in eutopic endometrium Not assessed Ren Y et al., Arch Gynecol Obstet 2010 [ 99 ] Tissue level: normal endometrium from adenomyosis-free patients and eutopic endometrium from patients with adenomyosis Decreased BECN1 mRNA and protein levels In adenomyosis, eutopic endometrium showed decreased autophagy at tissue and in cultured stromal cells vs. normal, with serum CA125 negatively correlated with BECN1 Not assessed In vitro: ESCs from eutopic and normal endometrium Sui X et al., Exp Ther Med 2018 [ 100 ] Liquid biopsies: peripheral venous blood and peritoneal fluid collected from patients without and with endometriosis Decreased LC3 and BECN1 protein expression Autophagic markers were found decreased in endometrial tissues and liquid biopsies from patients with endometriosis, while MMP-2 expression was increased Not assessed Tissue level: small endometrial tissue from control patients and patients presenting endometriosis before surgery (proliferative phase) Decreased LC3 and BECN1 mRNA expression Choi J et al., Mol Hum Reprod 2014 [ 101 ] Tissue level: normal eutopic endometrial tissues were obtained from premenopausal women, ectopic endometriotic tissues were obtained from ovarian endometriotic cysts. Endometrial tissues were classified by menstrual cycle phase: early proliferative, mid-to-late proliferative, early secretory, mid-secretory and late-secretory phase Constant expression levels of phosphorylated p70S6K, cleaved caspase-3 and LC3-II through menstrual cycle In normal eutopic tissues, autophagy increased with mTOR inhibition and correlated with apoptosis; in ectopic tissues, mTOR activity remained high with reduced autophagy and apoptosis Cell death Decreased LC3-II, increased phosphorylated p70S6K in EETs In vitro: Normal endometrial stromal cells (NESCs) and endometriotic cyst stromal cells (ECSCs) in the proliferative phase were obtained from eutopic endometrial tissues and ovarian endometriotic tissues, respectively Higher levels of mTOR activity, decreased LC3-II, and autophagosomes formation in ECSCs In NESCs, autophagy was induced by mTOR inhibition during secretory/menstrual phases and correlated with apoptosis; in ECSCs, higher mTOR activity after hormone withdrawal led to reduced autophagy and apoptosis Pei T et al., Mol Cell Endocrinol 2019 [ 103 ] In vitro: eutopic ESCs from women with or without endometriosis Increased YAP and mTOR levels, and decreased LC3-II/I ratio Decreased autophagy in eutopic ESCs, associated with an increased YAP expression, suggesting a role for YAP in regulating the mTOR-autophagy signaling in endometriosis Not assessed Mei J et al., Hum Reprod 2015 [ 104 ] In vitro: Eutopic and ectopic secretory phase ESCs vs. normal secretory phase ESCs Decreased LC3-II, BECN1 and PR. Increased p62, Erα, CXCR4, CXCL12 and NF-κB activation Estrogen signaling inhibited autophagy Autophagy suppression supported ESC survival in endometriosis Luo X et al., Biomed Res 2018 [ 105 ] Tissue samples: normal eutopic endometrium and ectopic tissues from ovarian endometriotic cysts (stage III–IV) Increased p62 and a decreased LC3-II levels Advanced-stage ovarian endometriotic cysts showed reduced autophagy vs. normal endometrium Anti-proliferative effects, inhibition of cell invasiveness In vitro: endometrial CRL-7566 Cells Low autophagy enhanced endometrial cell invasiveness via fascin-1, a Wnt/β-catenin target Jamali N et al., Reprod Sci 2021 [ 106 ] In vivo: rat model of endometriosis Decreased BECN1 and Atg5 , and increased of mTOR in ectopic endometrium Reduced endometrial implant size, decreased TNF-α and 17β-estradiol levels, decreased oxidative stress Not assessed Siracusa R et al., Int J Mol Sci 2021 [ 107 ] In vivo: rat model of endometriosis Decreased BECN1 and LC3-II, and increased of AKT and mTOR Decreased BECN1 and LC3-II, and increased of AKT and mTOR Not assessed Kong Z R et al., BMC Women’s Health 2022 [ 108 ] Tissue level: endometrial tissue from women with endometriosis, collected during the proliferative phase Decreased BECN1 and LC3 Decreased autophagy markers correlated with advanced stages of endometriosis Not assessed Abbreviations: Akt (AKT serine/threonine kinase), ATG5 (Autophagy related 5), BECN1 (Beclin 1), CA125 (Cancer antigen 125), CXCL12 (C-X-C motif ligand 12), CXCR4 ( C-X-C motif chemokine receptor 4), ECSCs (Endometriotic cyst stromal cells), EETs (Ectopic endometriotic tissues), ER-α (Estrogen receptor alpha), ESCs (Endometrial stromal cells), LC3 (Microtubule-associated protein light chain 3), MMP-2 (Matrix metalloproteinase-2), mTOR (Mechanistic target of rapamycin kinase), NEETs (Normal eutopic endometrial tissues), NESCs (Normal endometrial stromal cells), NK (Natural killer), NK-kß (Nuclear factor kappa-light-chain-enhancer of activated B cells), p70S6K (Ribosomal protein S6 kinase beta-1), p62/SQSTM1 (Sequestosome 1), PR (Progesterone receptor), TNF-α (Tumor necrosis factor α), Wnt (Wingless/Integrated), and YAP (Yes-associated protein) Autophagy downregulation in endometriosis (EMS) In vitro: ESCs isolated from normal endometrium, eutopic endometrium and ectopic lesion from patient with endometriosis Abbreviations: Akt (AKT serine/threonine kinase), ATG5 (Autophagy related 5), BECN1 (Beclin 1), CA125 (Cancer antigen 125), CXCL12 (C-X-C motif ligand 12), CXCR4 ( C-X-C motif chemokine receptor 4), ECSCs (Endometriotic cyst stromal cells), EETs (Ectopic endometriotic tissues), ER-α (Estrogen receptor alpha), ESCs (Endometrial stromal cells), LC3 (Microtubule-associated protein light chain 3), MMP-2 (Matrix metalloproteinase-2), mTOR (Mechanistic target of rapamycin kinase), NEETs (Normal eutopic endometrial tissues), NESCs (Normal endometrial stromal cells), NK (Natural killer), NK-kß (Nuclear factor kappa-light-chain-enhancer of activated B cells), p70S6K (Ribosomal protein S6 kinase beta-1), p62/SQSTM1 (Sequestosome 1), PR (Progesterone receptor), TNF-α (Tumor necrosis factor α), Wnt (Wingless/Integrated), and YAP (Yes-associated protein) Table 3 Targeting autophagy in endometriosis (EMS) References Model Drug Effects on autophagy and related molecules Global effects Role of autophagy Ruiz A et al., Cell Death Dis 2016 [ 114 ] In vitro: human endometriotic and endometrial stromal cells and primary human endometriotic cells isolated from peritoneal and ovarian lesions from two independent patients. HCQ (hydroxychloroquine) Increased of LC3-II HCQ treatment affected the survival of human endometrial and endometriotic cells. Protein levels of p21 (a cyclin-dependent kinase inhibitor involved in cell cycle arrest) markedly increased with siRNA targeting ATG7 and to a lesser degree with LC3B and BECN1 Cell survival In vivo models: mouse models (C57BL/6, Balb/c) of endometriosis treated with HCQ or PBS Increased mRNA expression of ATG5 and ATG3 in lesions from HCQ-treated mice relative to PBS-treated mice HCQ reduced autophagy., HCQ treatment increased the levels of IP-10 and macrophages in the peritoneal cavity Matsuzaki et al., Br J Pharmacol. 2018 [ 115 ] In vitro: isolated deep endometriotic stromal cells (DES), endometrial stromal (EES) and epithelial (EEE) cells of patients with endometriosis and endometrial stromal cells of patients without endometriosis (NEES). CQ alone, CQ + MK2206, or CQ + MK2206 + U012 - MK2206 + CQ reduced cell growth and regrowth in EES and decreased implant size more effectively than either drug alone, though some cell regrowth was observed after treatment discontinuation Cell survival In vivo: mouse model of endometriosis treated with vehicle, CQ, MK2206, or MK2206 + CQ LC3 expression increased in endometriotic implants treated with CQ alone, MK2206 alone, or MK2206 + CQ. p62 expression increased with CQ alone, no significant difference with MK2206 alone, MK2206 + CQ, or vehicle Lu H et al., Arch Gynecol Obstet 2018 [ 116 ] In vitro: Proliferative ESCs were obtained from endometriosis patients RA (retinoic acid) Increase LC3-II, BECN1, and ATG3 mRNA expression and decrease p62 expression. Similar patterns were described at the protein level for p62 and LC3-II, and an increase in GFP-puncta RA treatment induced autophagy in proliferative ESCs, and increased autophagic flux, but this effect was reduced by treatment with CQ. Increased proliferation in proliferative ESCs was suppressed after BECN1 knockdown Anti-proliferative effect Choi J et al., 2014 Mol Hum Reprod [ 101 ] In vitro: ECSCs in the proliferative phase obtained from ovarian endometriotic tissues Rapamycin Decreased expression of phosphorylation of p70S6K and S6K increased the expression of LC3-II and punctuated LC3-II structures accumulated in the cytoplasm Rapamycin induced autophagy and promoted apoptosis in ECSCs; 3-MA reversed this effect, indicating apoptosis was autophagy-dependent Pro-apoptotic effect Luo X et al., Biomed Res 2018 [ 105 ] In vitro: endometrial CRL-7566 Cells Rapamycin Increased number of LC3-positive autophagic puncta in the rapamycin-treated group Rapamycin suppressed proliferation and clonogenicity of CRL-7566 cells, decreased filopodia size and invasive capabilities Anti-proliferative effect, inhibition of clonogenicity and cell invasion Yu J-J et al., Reproduction 2016 [ 119 ] In vitro: ESCs isolated from control and ectopic endometrial tissues Rapamycin Decreased expression of IL15 Rapamycin decreased IL receptor levels in ESCs; reduced autophagy may increase IL-15 sensitivity, promoting cell survival, proliferation, invasiveness, and suppressing NK cell cytotoxicity Promotion cytotoxic activity of NK cells Mei J et al., Autophagy 2018 [ 97 ] In vitro: ESCs from control endometrium, eutopic endometrium and ectopic lesion from patients with endometriosis. Co-culture with human NK cells isolated from peripheral blood mononuclear cells (PBMCs) or peritoneal fluid (PF) Rapamycin Increased secretion levels of CXCL8 and IL23A in supernatants of Rap-ESC restriction in the ratio of FCGR3- NK cells in co-culture decreased HCK expression Autophagy inhibition in ESCs (3-MA or siATG5) decreased HCK expression and expanded FCGR3⁻ NK cells, whereas rapamycin increased HCK and limited this effect Promotion cytotoxic activity of NK cells In vivo: Endometriosis-like lesions induced in mice and treated with 3-MA or rapamycin Increased mRNA levels of LC3B, BECN1 and HCK Rapamycin increased LC3B, BECN1, and HCK, whereas 3-MA reduced autophagy, promoted lesion growth, and expanded FCGR3⁻ NK cells D’Amico R et al., Antioxidants 2022 [ 123 ] In vivo: rat model of endometriosis Açai Berry (an Amazonian fruit) Increased autophagy: inhibition of PI3K, AKT, and ERK1/2 phosphorylation; mTOR inactivation; ATG1 dephosphorylation, activating ATG1/ULK1 complex; recruitment of Ambra1/BECN1 and Atg9; increased LC3II expression Reduced lesion size, restored oxidative balance (increased NRF2, NQO-1, HO-1), restored apoptosis (reduced BCL-2, increased BAX) Promotes autophagy and mitophagy while restoring oxidative stress and apoptosis Liu H et al., Reproduction 2017 [ 65 ] In vitro: Hypoxia-induced autophagy in cultured human ESCs 3-MA (3-methyladenine) Inhibited autophagy by blocking autophagosome formation; reduced LC3-II expression Reduced hypoxia-induced migration and invasion of ESCs Inhibition of autophagy impaired the pro-invasive effect of hypoxia CQ (chloroquine) Chen ML et al., Cytokines 2023 [ 70 ] In vitro: hypoxia treatment in ovarian endometriotic cell line CRL-7566 3-MA (3-methyladenine) Inhibited autophagy with 3-MA suppressed hypoxia-induced autophagy Inhibited autophagy with 3-MA suppressed hypoxia-induced autophagy, migration, and EMT Inhibition of autophagy by suppressing hypoxia, migration and EMT Ham J et al., Biomed Pharmacother 2024 [ 125 ] In vitro: End1/E6E7 and VK2/E6E7 cell lines. Each cell type was derived from the epithelial cells of the endocervix and vaginal mucosa Transcrocetin Impaired autophagic flux (confirmed by pre-treatment with CQ) Suppressed cell proliferation and caused G1-phase cell cycle arrest via p21 upregulation; induced oxidative stress and disrupted mitochondrial bioenergetics Transcrocetin-induced oxidative stress and mitochondrial dysfunction impaired autophagy Choi J et al., Fertil Steril. 2015 [ 129 ] In vitro: Estrogen-treated ECSCs were dissociated and purified from ovarian endometriotic tissue Dienogest (progestin) Upregulation of LC3-II, increased autophagosomes formation, decreased phosphorylation of AKT, ERK1/2, and S6K Treatment with dienogest in estrogen-treated ECSCs induced autophagy. Same effects were observed after incubation with AKT and ERK1/2 inhibitors. Dienogest treatment also promoted apoptosis in ECSCs (cleaved caspase-3). The addition of 3-MA reverted dienogest-induced autophagy and apoptosis Apoptosis Cornillie FJ et al., Int J Gynecol Pathol 1986 [ 130 ] Tissue level: endometriotic implants in infertile patients with endometriosis, before and after treatment with antiprogesterone steroid ethylnorgestrienone Gestrinone R2323 (antiprogesterone steroid ethylnorgestrienone) Autophagic lysosomes Most patients showed undifferentiated endometriotic tissues, with epithelial involution and increased autophagic lysosomes Anti-proliferative effect Borahay MA, et al. Obstet Gynecol 2013 [ 131 ] In vitro: CRL-7566 endometrial cell line MIS (müllerian inhibiting substance) Increase LC3-II and BECN1 expression MIS treatment in the CRL-7566 cell line induced autophagy inhibited proliferation-induced apoptosis and increased cleaved caspase-9 and cell-cycle arrest, as evidenced by incremented expression of p27 CDK-inhibitor Apoptosis Zhang B, et al. Cell Death Dis 2018 [ 132 ] In vitro: human ESCs obtained from the endometrium of healthy control women and ectopic ESCs from ectopic lesions of patients with endometriosis. NK cells isolated from PBMCs were used in co-cultures PPD (protopanaxidiol) Increased protein expression of LC3B-II, BECN1, and downregulated p62 expression. Increased mRNA expression of ATG12 , ATG5 , ATG4C , ATG9B , ATG4A , ATG3 , MAP1LC3B , BECN1 , GABARAPL2 , RAB24 , WIPI1 , BNPI3 , EIF2AK3 , RB1 , DRAM2 , PIK3CG , and decreased mRNA levels of ESR1 , SQSTM1 and TGFB1 PPD and related compounds reduced ectopic ESC viability, promoted autophagy, reversed estrogen’s inhibitory effect on autophagy, increased pro-apoptotic signaling, and enhanced NK cell cytotoxicity Anti-proliferative effect, apoptosis, promotion of NK cells cytotoxic activity In vivo: mouse model of endometriosis treated with PPD, PPT, G-Rg3, G-Rh2, EsA, E2, or 3-MA at varying doses to evaluate effects on lesion development and autophagy Decrease in ERα and the increase in PRα in ectopic lesions PPD, PPT, G-Rg3, G-Rh2, and EsA reduced the number and weight of ectopic lesions, with PPD showing the strongest effect. PPD also counteracted the lesion-promoting effects of E2 Lin YK, et al., Front Endocrinol 2022 [ 134 ] In vivo: mice model of endometriosis. Normal endometrial stromal cells (NESCs) and ectopic ESCs with or without SCM-198 treatment SCM-198 Increased autophagy by inhibiting TNF-α-activated aromatase-estrogen-ERα signaling and increasing PRB expression Reduced ectopic lesion growth, promoted ectopic ESC apoptosis Apoptosis Abbreviations: 3-MA (3-Methyladenine), AKT (AKT serine/threonine kinase), ATG1 (Autophagy related 1), ATG3 (Autophagy related 3), ATG4 (Autophagy related 4), ATG5 (Autophagy related 5), ATG7 (Autophagy related 7), ATG9 (Autophagy related 9), ATG12 (Autophagy related 12), BAX (Bcl2 associated X protein), BCL-2 (BCL2 apoptosis regulator), BNIP3 (BCL2/adenovirus E1B 19kDa protein interacting protein 3), CQ (Chloroquine), CXCL8 ( C-X-C motif chemokine 8), DES (Deep endometriotic stromal cells), DRAM2 (DNA damage regulated autophagy modulator 2), E2 (Estradiol), ECSCs (Endometriotic cyst stromal cells), EEE (Endometrial epithelial cells), EIF2AK3 (Eukaryotic translation initiation factor 2-alpha kinase 3), EMS (Endometriosis), ERK1/2 (Extracellular signal-regulated kinase 1/2), ERα (Estrogen receptor α), ESCs (Endometrial stromal cells), ESR1 (Estrogen receptor 1), EMT (Epithelial-mesenchymal transition), FCGR3 (Fc gamma receptor III), GABARAPL2 (GABA Type A receptor-associated protein like 2), G-Rg3 / G-Rh2 (Ginsenoside Rg3 / Ginsenoside Rh2), GFP (Green fluorescent protein), HCK (Hematopoietic cellular kinase), HCQ (Hydroxychloroquine), HO-1 (Heme oxygenase 1), IL15 (Interleukin 15), IP-10 (10 kDa interferon-γ-induced protein), IP-10 (Interferon gamma-induced protein 10), LC3 (Microtubule-associated protein light chain 3), MIS (Müllerian inhibiting substance), mTOR (Mechanistic target of rapymicin), NEES (Endometrial stromal cells of patients without endometriosis), NESCs (Normal endometrial stromal cells), NK (Natural killer), NQO-1 (NADPH quinone dehydrogenase 1), NRF2 (Nuclear factor erythroid 2-related factor 2), PARP (poly(ADP-ribose) polymerase 1), PBMCs (Peripheral blood mononuclear cells), PBS (Phosphate-buffered saline), PF (Peritoneal fluid), PI (Propidium iodide), PI3K (Autophagy-specific class III phosphatidylinositol 3-kinase), PPD (Protopanaxadiol), PPT (Protopanaxatriol), PR (Progesterone receptor), RA (Retinoic acid), Rap (Rapamycin), RB1 (Retinoblastoma protein), S6K/p70S6K (Ribosomal protein S6 kinase beta-1), siRNA (Small interfering RNA), SQSTM1/p62 (Sequestosome 1), TNFα (Tumor necrosis factor α), TGFß1 (Transforming growth factor beta 1), and ULK1 (Unc-51 like autophagy activating kinase 1), WIPI1 (WD repeat domain phosphoinositide interacting 1) Targeting autophagy in endometriosis (EMS) In vitro: isolated deep endometriotic stromal cells (DES), endometrial stromal (EES) and epithelial (EEE) cells of patients with endometriosis and endometrial stromal cells of patients without endometriosis (NEES). CQ alone, CQ + MK2206, or CQ + MK2206 + U012 LC3 expression increased in endometriotic implants treated with CQ alone, MK2206 alone, or MK2206 + CQ. p62 expression increased with CQ alone, no significant difference with MK2206 alone, MK2206 + CQ, or vehicle Increased secretion levels of CXCL8 and IL23A in supernatants of Rap-ESC restriction in the ratio of FCGR3- NK cells in co-culture decreased HCK expression Abbreviations: 3-MA (3-Methyladenine), AKT (AKT serine/threonine kinase), ATG1 (Autophagy related 1), ATG3 (Autophagy related 3), ATG4 (Autophagy related 4), ATG5 (Autophagy related 5), ATG7 (Autophagy related 7), ATG9 (Autophagy related 9), ATG12 (Autophagy related 12), BAX (Bcl2 associated X protein), BCL-2 (BCL2 apoptosis regulator), BNIP3 (BCL2/adenovirus E1B 19kDa protein interacting protein 3), CQ (Chloroquine), CXCL8 ( C-X-C motif chemokine 8), DES (Deep endometriotic stromal cells), DRAM2 (DNA damage regulated autophagy modulator 2), E2 (Estradiol), ECSCs (Endometriotic cyst stromal cells), EEE (Endometrial epithelial cells), EIF2AK3 (Eukaryotic translation initiation factor 2-alpha kinase 3), EMS (Endometriosis), ERK1/2 (Extracellular signal-regulated kinase 1/2), ERα (Estrogen receptor α), ESCs (Endometrial stromal cells), ESR1 (Estrogen receptor 1), EMT (Epithelial-mesenchymal transition), FCGR3 (Fc gamma receptor III), GABARAPL2 (GABA Type A receptor-associated protein like 2), G-Rg3 / G-Rh2 (Ginsenoside Rg3 / Ginsenoside Rh2), GFP (Green fluorescent protein), HCK (Hematopoietic cellular kinase), HCQ (Hydroxychloroquine), HO-1 (Heme oxygenase 1), IL15 (Interleukin 15), IP-10 (10 kDa interferon-γ-induced protein), IP-10 (Interferon gamma-induced protein 10), LC3 (Microtubule-associated protein light chain 3), MIS (Müllerian inhibiting substance), mTOR (Mechanistic target of rapymicin), NEES (Endometrial stromal cells of patients without endometriosis), NESCs (Normal endometrial stromal cells), NK (Natural killer), NQO-1 (NADPH quinone dehydrogenase 1), NRF2 (Nuclear factor erythroid 2-related factor 2), PARP (poly(ADP-ribose) polymerase 1), PBMCs (Peripheral blood mononuclear cells), PBS (Phosphate-buffered saline), PF (Peritoneal fluid), PI (Propidium iodide), PI3K (Autophagy-specific class III phosphatidylinositol 3-kinase), PPD (Protopanaxadiol), PPT (Protopanaxatriol), PR (Progesterone receptor), RA (Retinoic acid), Rap (Rapamycin), RB1 (Retinoblastoma protein), S6K/p70S6K (Ribosomal protein S6 kinase beta-1), siRNA (Small interfering RNA), SQSTM1/p62 (Sequestosome 1), TNFα (Tumor necrosis factor α), TGFß1 (Transforming growth factor beta 1), and ULK1 (Unc-51 like autophagy activating kinase 1), WIPI1 (WD repeat domain phosphoinositide interacting 1) Multiple factors, including the microenvironment, autophagy regulators (such as signaling pathways and associated proteins), cellular stress and survival responses, or genetic factors, contribute to the upregulation of autophagy in endometriosis progression. The following subsections will explore the key drivers of autophagy upregulation in endometriosis and their implications for disease progression (Table  1 ). The hypoxic microenvironment is described as one of the first stresses that may be faced by shed endometrial cells that retrograde to the pelvic cavity [ 64 ]. Although its precise role in endometriosis is not fully understood, several studies have suggested a potential involvement of hypoxia in the progression of the disease in recent years. HIF-1α (Hypoxia-inducible factor 1-alpha) has been reported to be upregulated in ectopic endometrium and may contribute to the invasive behavior of human endometrial stromal cells (ESCs) [ 65 ]. Under hypoxic conditions, HIF-1α is stabilized and activates BNIP3 (BCL2 Interacting Protein 3). This protein interacts with the anti-apoptotic factors BCL-XL and BCL2, enabling the association with BECN1, resulting in autophagy induction [ 66 ]. Liu H and colleagues have shown that HIF-1α and autophagy-related protein LC3 expression levels are upregulated in ectopic endometriotic tissue compared with eutopic endometrium in patients with endometriosis and with normal endometrium from healthy donors [ 65 ]. As expected, hypoxia treatment induced autophagy in ESCs in a time-dependent manner, resulting in increased accumulation of autophagosomes and upregulation of HIF-1α, BECN1, and LC3 protein expression in a HIF-1α-dependent manner. In addition, hypoxia treatment also enhanced the migration and invasion ability of ESCs, whereas these effects were inhibited after HIF-1α knockdown. These results suggest that HIF-1α may promote ESCs invasion and metastasis in endometriosis by upregulating autophagy. Another study reported elevated HIF-1α expression in the ectopic endometriotic tissue compared with eutopic endometrium from the same patients, which was associated with increased miR-210 expression [ 67 ]. According to the authors, hypoxia treatment appeared to increase HIF-1α and miR-210 expression in the ovarian endometriotic cell line CRL-7566. The study further suggested that miR-210 may promote autophagy under hypoxic conditions by increasing LC3-II and decreasing p62/SQSTM1 levels, a marker of autophagic flux. Knockdown of miR-210 was associated with reduced autophagy. miR-210 may contribute to the pathological development of endometriosis by enhancing cell survival and promoting autophagy via regulation of the BCL2/BECN1 axis. Hypoxia has also been implicated in promoting epithelial-mesenchymal transition (EMT), a critical cell migration and invasion process, in endometriosis [ 68 ]. Liu H et al. reported that, in addition to increased HIF-1α and LC3 expression levels, the mesenchymal cell marker vimentin appeared to be significantly upregulated in ectopic endometriotic tissue compared with the eutopic endometrium from the same patients [ 69 ]. The study further indicated that hypoxia enhances autophagy activity in Ishikawa cells, and this process is dependent on HIF-1α. Subsequently, BECN1 siRNA in hypoxia-treated Ishikawa cells showed suppression of hypoxia-induced EMT and mesenchymal capabilities. Similarly, a recent study reported that hypoxia treatment appears to upregulate miR-150-5p expression in CRL-7566 ovarian endometriosis cells, which was associated with post-transcriptional suppression of PDCD4 (Programmed cell death 4), increased NF-κB (Nuclear factor-kappa B) signaling, autophagy and EMT [ 70 ]. Knocking down miR-150-5p suppressed hypoxia-induced autophagy, migration, and EMT. Taken together, these findings suggest that autophagy may play an important role in promoting the migratory and invasive potential of endometrial cells through the induction of EMT. The involvement of long non-coding RNAs (lncRNAs) in hypoxia-driven autophagy is gaining attention [ 71 , 72 ]. In a separate study, Liu H et al. reported that autophagy seems to contribute to the pathogenesis of ovarian endometriosis, associated with elevated expression of HIF-1α and lncRNA-MALAT1 during the pathological process [ 73 ]. The RNA expression of HIF-1α-dependent lncRNA-MALAT1 was increased in endometriotic tissue compared with both eutopic endometrium from the same patients and normal endometrium from healthy donors, accompanied by increased HIF-1α protein expression and LC3-II accumulation. Interestingly, knockdown of lncRNA-MALAT1 appeared to reduce hypoxia-induced autophagy, suggesting a pro-survival role for autophagy in ESCs during endometriosis and its regulation by lncRNA-MALAT1. Beyond HIF-1α, several factors play significant roles in regulating autophagy under hypoxic conditions, further emphasizing the complexity of the disease microenvironment. Among these factors, HMGB1 (High mobility group box 1) has been implicated in endometriosis and in the upregulation of autophagy within this pathology [ 74 ]. In this study, HMGB1 knockdown appeared to decrease autophagy-related marker protein levels, such as BECN1 and ATG13, in ESCs under hypoxic conditions. Recently, the same group observed higher levels of autophagy-related genes in an HMGB1-ESCs overexpression cell line under hypoxic conditions [ 75 ]. HMGB1 expression was significantly higher in ectopic endometriotic tissue than in eutopic tissue from the same patients and normal endometrium from healthy donors. Autophagy-related markers BECN1, ATG13, and LC3I/II were also upregulated in ectopic tissue. Furthermore, studies in Hmgb1 -deficient mice showed a decrease in implant lesions, inflammation, and downregulation of autophagy-related genes, suggesting a possible role for HMGB1 in inducing autophagy in endometriosis. Macrophages are highly plastic immune cells that can polarize into two main phenotypes: M1, which are classically activated and pro-inflammatory, or M2, which are alternatively activated and promote anti-inflammatory responses and tissue repair. More specifically, M1 macrophages are predominantly found in the eutopic endometrium, where they promote increased inflammation. Conversely, M2 macrophages are enriched in ectopic lesions, where they may facilitate angiogenesis, immune evasion, and disease progression [ 76 ]. In endometriosis, macrophages play a multifaceted and sometimes contradictory role, depending on the context. Moreover, recent studies have linked macrophage behavior to autophagy [ 77 ]. In a mouse endometrial model, the peritoneal immune microenvironment appeared to be significantly altered [ 78 ]. These individuals exhibit elevated numbers of macrophages in both peritoneal fluid and endometriotic lesions. Within this environment, distinct macrophage phenotypes may promote lesion progression or exert a protective effect (Fig. 3 ). Fig. 3 Autophagy-driven dysfunction of macrophages and NK cells contributes to immune evasion and lesion persistence in endometriosis. A  EPHA3 overexpression in macrophages inhibited mTORC1 signaling, downregulated Bcl-2, and upregulated Atg3, LC3-II/I, and BECN1.MST1-deficient macrophages, regulated by miR-887-5p, secreted IL-10, which induced autophagy in ectopic ESCs, while MST1 loss also activated p38 MAPK, promoting an anti-inflammatory, tissue-repairing macrophage phenotype. In endometriosis patients, HCK deficiency in macrophages—driven by estrogen and hypoxia—impaired phagocytosis and enhanced ectopic lesion growth. This dysfunction was associated with altered glutamine metabolism and c-FOS/c-JUN signaling. B  Estrogen-mediated suppression of autophagy in human ESCs downregulated HCK expression and led to increased secretion of CXCL8/IL-8 and IL-23 A. These changes upregulated a subset of FCGR3⁻ NK cells, characterized by impaired cytotoxic activity. E2, estrogen; ER, estrogen receptor; ESCs, endometrial stromal cells. Figure created with BioRender.com Autophagy-driven dysfunction of macrophages and NK cells contributes to immune evasion and lesion persistence in endometriosis. A  EPHA3 overexpression in macrophages inhibited mTORC1 signaling, downregulated Bcl-2, and upregulated Atg3, LC3-II/I, and BECN1.MST1-deficient macrophages, regulated by miR-887-5p, secreted IL-10, which induced autophagy in ectopic ESCs, while MST1 loss also activated p38 MAPK, promoting an anti-inflammatory, tissue-repairing macrophage phenotype. In endometriosis patients, HCK deficiency in macrophages—driven by estrogen and hypoxia—impaired phagocytosis and enhanced ectopic lesion growth. This dysfunction was associated with altered glutamine metabolism and c-FOS/c-JUN signaling. B  Estrogen-mediated suppression of autophagy in human ESCs downregulated HCK expression and led to increased secretion of CXCL8/IL-8 and IL-23 A. These changes upregulated a subset of FCGR3⁻ NK cells, characterized by impaired cytotoxic activity. E2, estrogen; ER, estrogen receptor; ESCs, endometrial stromal cells. Figure created with BioRender.com The immune microenvironment plays a critical role in the pathophysiology of multiple diseases, including endometriosis [ 79 , 80 ]. Several studies have shown that autophagy can be modulated by the microenvironment in this disease, with macrophages being identified as a promising regulator. Xu et al. observed that overexpression of EPHAE3 (Erythropoietin-producing hepatocellular carcinoma A3) appeared to inhibit the activation of the mTORC1 signaling pathway, downregulate bcl-2 expression, upregulate the expression of Atg3, LC3-II/LC3-I, and BECN1, and seemed to enhance autophagy of macrophages in an endometriosis mouse model [ 81 ]. Another study highlighted the contribution of macrophages to the immune microenvironment in endometriosis [ 82 ]. The authors demonstrated that MST1 (Macrophage Stimulating 1)-deficient macrophages, through miR-887-5p and IL-10 secretion, may promote autophagy in ectopic endometrial cells, potentially facilitating their survival and proliferation. MST1 is a serine/threonine kinase involved in immune response regulation. Its deficiency in ectopic tissues impairs macrophage function, leading to increased autophagic activity. Loss of MST1 promoted p38 MAPK activation, driving macrophage polarization toward an anti-inflammatory, tissue-repairing phenotype. These findings suggest a role for MST1-p38 MAPK signaling in macrophage reprogramming. A recent study reported that hematopoietic cellular kinase (HCK) deficiency in macrophages from endometriosis patients, driven by high estrogen levels and exacerbated by hypoxic conditions, appeared to impair phagocytosis and seemed to promote the growth of ectopic lesions. This dysfunction appeared to be linked to altered glutamine metabolism, -c-FOS/c‐JUN signaling and upregulated autophagy, suggesting that excessive autophagy and impaired macrophages phagocytosis may contribute to the pathogenesis of endometriosis [ 83 ]. As described previously, endometrial cells are exposed to several stress conditions, including hypoxia, oxidative stress, and inflammatory signals. These conditions activate cellular stress responses that significantly contribute to the pathogenesis of endometriosis. Key molecules involved in these responses, including those regulating autophagy, have been implicated in the progression and maintenance of the disease. PARP1 and SIRT1 have been identified as key regulators of cellular response to iron overload and oxidative stress in eutopic endometrial stromal cells ESCs [ 84 ]. By enhancing autophagic activity, PARP1 and SIRT1 may promote the survival and adaptation of ESCs within the iron-rich microenvironment characteristic of endometriotic lesions. Iron overload suppressed PARP1 expression while upregulating SIRT1, which, in turn, activated autophagy (increased LC3B-II accumulation, BECN1, and increased autophagic flux), possibly through deacetylation of core autophagic proteins. Activated autophagy may decrease oxidative stress and prevent apoptosis, suggesting a protective role, thereby facilitating the development and progression of endometriotic lesions. Ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, has emerged in recent years as a key mechanism in the pathophysiology of ovarian endometriosis, where localized iron overload is a hallmark feature. In fact, excess iron accumulation in ectopic ESCs has been reported to increase reactive oxygen species (ROS), promote lipid peroxidation, and induce mitochondrial alterations, ultimately compromising cell viability. These effects appear to be reversed by treatment with ferroptosis inhibitors, suggesting the involvement of ferroptotic pathways [ 85 ]. Interestingly, iron overload also stimulates autophagy, as shown by increased expression of BECN1 and LC3-II. Inhibition of autophagy reduces ferroptosis, suggesting that in this context, ferroptosis may be at least partially dependent on the autophagic process. In vivo studies using a mouse model of endometriosis support these findings, indicating that both iron overload and autophagy may influence lesion development. Despite the presence of a pro-ferroptotic environment, ectopic lesions exhibit a degree of resistance to ferroptosis. This resistance may be mediated by activation of the ATF4-xCT pathway, wherein ATF4 upregulated the expression of the cystine/glutamate antiporter (xCT), protecting cells from oxidative damage. DIRAS3, a member of the RAS family, has been described as a tumor suppressor and an autophagy modulator [ 86 , 87 ]. Yotova et al. recently reported that DIRAS3 expression was elevated in ectopic endometriosis lesions compared with eutopic and control endometrium [ 88 ]. In the same study, experiments in an endometriotic epithelial cell line demonstrated that DIRAS3 expression further increased under serum starvation, a condition that induces autophagy. Moreover, DIRAS3 was shown to co-localize with the autophagy marker LC3-II, suggesting a direct involvement in autophagosome formation. Knockdown of DIRAS3 was found to reduce LC3 puncta and inhibit LC3-I to LC3-II conversion, indicating that it may be important for autophagy initiation. These findings indicate that DIRAS3 may promote autophagy in endometriotic epithelial cells, helping them adapt to stress conditions, such as iron overload and oxidative stress. Several autophagy regulators have been investigated in relation to the progression of endometriosis. Allavena G et al. reported that autophagy is upregulated in ovarian endometriomas compared with eutopic and normal endometrium, as indicated by higher LC3B-II protein levels, reduced p62/SQSTM1 expression, and induction of MAP1LC3B , ATG14 , BECN1 , and ATG7 genes [ 89 ]. The significant decrease in p53 expression and the upregulation of HO-1 (Heme oxygenase 1) observed in the study, in ovarian endometriomas compared to normal endometrium, suggest that impaired apoptosis and persistent oxidative stress may favor autophagy stimulation. Another study reported increased BECN1 levels and LC3-II/LC3-I ratio in granulosa cells (GCs) from patients with endometriosis [ 90 ]. Additionally, mRNA levels of key autophagy-regulated genes, including BECN1 , ATG13 , and MAP1LC3B , were significantly upregulated in patients with endometriosis. These results suggest that autophagy is activated in GCs and may contribute to ovarian endometriosis development. Zhou et al. observed the downregulation of several components of the Indian Hedgehog pathway, known to regulate autophagy, while LC3B-II protein expression was higher in endometriotic tissues [ 91 ]. Similarly, a recent study found increased expression of the autophagy-related proteins BENC1 and LC3 in endometriosis patients [ 92 ]. Together, these findings reinforce the notion that dysregulated autophagy, driven by specific molecular pathways, may play a pivotal role in the pathogenesis and persistence of endometriotic lesions. Apart from autophagy-regulator factors, a recent study described the implication of the autophagy cGAS-STING pathway in endometriosis [ 93 ]. The authors observed that STING overexpression may promote autophagy, as well as enhance the migration and invasion capabilities of ESCs. In line with these results, STING inhibition was found to reduce autophagy in an in vivo rat endometrial model of endometriosis. In addition to pathway regulators, nutritional conditions can also influence autophagy. Yin B et al. reported that caloric restriction (CR) reduced lesion growth in a mouse model of endometriosis [ 94 ]. The effect of CR was associated with reduced PI3K/AKT/mTOR and CREB signaling and increased expression of p-AMPK (phospho-AMP-activated protein kinase), SIRT1, and LC3, suggesting that CR may induce autophagy in endometriotic lesions. Several other factors may contribute to the dysregulation observed in this disease. HOXA10 (Homeobox A10) is a transcription factor essential for embryonic and adult uterine development, and its expression decreases in endometriosis [ 95 ]. A recent study by Zhen J et al. suggested that decreased HOXA10 expression may be responsible for the increased autophagy observed in endometriotic tissues [ 96 ]. Ovarian endometriomas showed higher LC3-II, an increased LC3-II/LC3-I ratio, and elevated BECN1 levels, whereas HOXA10 expression was decreased compared with eutopic endometrium. Interestingly, serum CA125 levels, a factor highly present in endometriosis that increases with elevated disease staging (I-IV), positively correlated with LC3-II protein levels while showing an inverse correlation with HOXA10, suggesting a potential association between HOXA10 deficiency and autophagy activation. Autophagy has been increasingly associated with endometriosis, although its precise role in lesion survival and progression remains to be clarified. Contrary to previous results, several factors, including the microenvironment, autophagy regulators, cellular stress and survival responses, and genetic factors, have been described to contribute to the downregulation of autophagy in endometriosis (Table  2 ). This apparent controversy highlights the complexity of our understanding of the specific role of autophagy in this disease. Considering both direct and indirect effects and autophagy’s context-specific effects, a comprehensive perspective is essential to unravel its role in endometriosis. Endometriosis is characterized by a dysregulation of the immune response, with several immune factors contributing to a specific microenvironment associated with the development of the disease [ 79 , 80 ]. A study reported that impaired Natural Killer (NK) cell cytotoxic activity appears to be associated with the dysfunctional immune environment in endometriosis [ 97 ]. The study revealed that autophagy suppression in ESCs, regulated by estrogen, leads to decreased autophagy levels and the downregulation of hematopoietic cell kinase (HCK). This process results in increased secretion of a pro-inflammatory chemokine involved in neutrophil recruitment (CXCL8/IL8) and a cytokine that promotes the differentiation of T-cells and plays a role in inflammation (IL23A) by ESCs. These changes favored the expansion of FCGR3⁻ NK cells, a subset with impaired cytotoxicity, thereby contributing to immune dysfunction in endometriosis. These findings suggest that the estrogen-autophagy-HCK axis could be involved in NK cell dysfunction in endometriosis, contrasting with the autophagy upregulation observed in macrophages. The regulation of autophagy in endometriosis remains one of the most controversial aspects, as studies have reported both upregulation and downregulation of autophagic activity within endometriotic lesions. A study reported that both ectopic and eutopic endometrium from patients with endometriosis exhibit reduced autophagic capacity, reflected by significantly lower BECN1 expression compared with normal endometrium [ 98 ]. In the same study, serum CA125 levels were significantly increased in patients with endometriosis and were negatively correlated with BECN1 protein expression in eutopic endometrium. In adenomyosis, a condition characterized by the invasion of endometrial tissue into the uterine muscle, BECN1 mRNA and protein expression levels were also significantly decreased in cultured ESCs of eutopic endometrium compared with normal endometrium [ 99 ]. Another study [ 100 ] reported that in both serum and peritoneal fluid from patients with endometriosis, MMP-2 expression was increased compared with women without endometriosis. In contrast, expression of MAP1LC3B and BECN1 was significantly decreased. Choi J et al. investigated whether autophagy and mTOR activity are induced differently during the menstrual cycle in normal eutopic endometrial tissues and ectopic endometriotic tissues, which were obtained from ovarian endometriotic cysts [ 101 ]. In normal endometrium, increased autophagy correlates with decreased mTOR activity (as measured by levels of p70S6K) and increased cleaved caspase-3 throughout the menstrual cycle. In contrast, ectopic endometriotic tissues showed no cyclic changes, with lower LC3-II and cleaved caspase-3 levels but higher phosphorylated p70S6K. These results suggest that altered mTOR activation in endometriotic tissue could be associated with reduced autophagy and apoptosis, which may favor lesion persistence. Some factors involved in stress and survival response signaling pathways may also contribute to the downregulation of autophagy in endometriosis. Among these regulators, the Hippo-YAP pathway, a critical regulator of cell proliferation and survival, has been implicated as a potential contributor to autophagy downregulation [ 102 ]. Pei et al. reported that the survival Hippo-YAP pathway appeared to modulate autophagy in this disease [ 103 ]. Specifically, the study reported upregulation of YAP and mTOR expression accompanied by decreased LC3-II/LC3-I ratio in eutopic ESCs compared with normal endometrium. Furthermore, YAP knockdown restored the LC3-II/LC3-I ratio, suggesting that the Hippo-YAP signaling pathway may contribute to autophagy downregulation in endometriosis. Endometriosis is a gynecological disorder influenced by hormones, which play a pivotal role in its development and regulation. Mei J et al. described another additional mechanism involved in the downregulation of autophagy in endometriosis [ 104 ]. The authors observed an upregulation of the ERα (Estrogen receptor alpha) and p62/SQSTM1, alongside a downregulation of LC3-II, BECN1, and the progesterone receptor (PR) in both eutopic and ectopic secretory phase ESCs compared with normal secretory phase ESCs. The study also reported that CXCR4 (C-X-C motif chemokine receptor type 4) and CXCL12 (C-X-C motif chemokine 12) secretion were higher in eutopic and ectopic ESCs and strongly negatively correlated with autophagic levels. The authors observed that estrogen promoted the survival of ESCs via sequential CXCL12/CXCR4 upregulation and autophagy inhibition through activation of NF-κB. In a follow-up study, the same group confirmed that ectopic ESCs from patients with endometriosis had reduced autophagy, as observed by the decrease in LC3B and BECN1 expression, and that similar effects were induced by 17-β estradiol [ 101 ]. Luo X et al. observed that autophagy levels were significantly lower in endometriosis tissue samples from ovarian endometriotic cysts in an advanced-stage of the disease compared with normal endometrium [ 105 ]. The reduction in autophagy levels was characterized by an increase in p62/SQSTM1 and a decrease in LC3-II levels. In addition, the authors demonstrated that the low levels of autophagy promoted the invasiveness of endometrial cells at least partially through the expression of fascin-1 (filopodial protein fascin-1), a target gene of the Wnt/𝛽-catenin signal-transduction pathway. Consistent findings were observed in animal models. In a rat model of endometriosis, BENC1 and Atg5 expression were downregulated in the ectopic endometrial tissues compared with the eutopic tissue, while an increase in mTOR gene expression was observed [ 106 ]. Another rat study similarly showed increased p-AKT and mTOR expression levels and decreased Benc1 [ 107 ]. A recent study reported that the autophagy markers BECN1 and LC3 expression were significantly lower in patients with endometriosis compared with normal endometrium [ 108 ]. This downregulation also correlated negatively with the clinical stage of the disease, suggesting that a reduction in autophagy activity may be associated with more severe forms of the disease. As previously mentioned, there is currently no cure for endometriosis; however, treatments are available to help reduce symptoms. Long-term treatment of patients with endometriosis includes repetitive cycles of drug therapy, surgical therapy, or both. Moreover, existing treatments are often associated with failures, side effects, and a high incidence of recurrence after medical treatment [ 109 – 111 ]. These challenges underscore the need to develop novel therapeutic strategies or improve existing ones. As discussed in this review, one promising approach is targeting autophagy, given the increasing evidence of its involvement in endometriosis. Several drugs and compounds are currently under investigation for their ability to regulate autophagy in endometriosis, with mechanisms ranging from mTOR modulation to direct inhibition or induction of autophagic processes (Table 3 ). In normal physiological conditions, epithelial cells typically undergo anoikis (programmed cell death) upon detachment from the extracellular matrix [ 112 , 113 ]. In endometriosis, autophagy may support the survival of shed endometrial cells by preventing anoikis and facilitating the establishment and progression of ectopic lesions. A study by Ruiz A. et al. reported that the in vitro survival ability of human endometriotic and endometrial stromal cells derived from a uterine myoma was disrupted when treated with hydroxychloroquine (HCQ), an autophagic flux inhibitor [ 114 ]. Moreover, in a mouse model of endometriosis, HCQ treatment before implantation significantly reduced the number of ectopic lesions. Additionally, it increased peritoneal macrophages levels and increased chemokine 10 kDa interferon-γ-induced protein (IP-10). Moreover, mRNA levels of autophagic markers, including Atg5 and Atg3 , were increased in lesions from HCQ-treated mice compared with those in PBS-treated mice. Interestingly, LC3B protein expression was also increased in the eutopic endometrium of endometriosis-induced mice compared with controls. These results suggest that HCQ could represent a potential therapeutic agent for women with endometriosis, targeting the microenvironment to inhibit lesion growth. In this context, Matsuzaki et al. explored the combination of MK2206 (an AKT inhibitor) and chloroquine (CQ) [ 115 ]. Their results indicated that this combination significantly inhibited cell growth and regrowth of deep endometriotic stromal cells (DESCs) in vitro and reduced the size of endometriotic implants in a mouse xenograft model of endometriosis. In contrast, neither drug alone significantly altered the size of the implants compared to vehicle controls. The authors concluded that this combinatory treatment should not be evaluated in future clinical trials, as CQ primarily affected endometrial stromal cells rather than DESCs, and MK2206 alone was not effective in reducing implant size or inducing apoptosis in the xenograft model of endometriosis. In another study, Lu X et al. studied the role of retinoic acid (RA) in autophagy-mediated endometriosis [ 116 ]. RA treatment appeared to promote autophagy in proliferative ESCs. The authors observed an increase in MAP1LC3B , BECN1 , and ATG3 mRNA expression and a decrease in p62/SQSTM1 expression. Moreover, RA exposure increased the autophagic flux, as observed by an increase in GFP-puncta compared to control cells. These effects were reduced by treatment with CQ. The effect of rapamycin, a specific mTOR inhibitor that induces autophagy in mammalian cells [ 117 , 118 ], was tested in endometriosis. Choi et al. described that rapamycin treatment produced a pro-apoptotic effect in proliferative endometriotic cyst stromal cells (ECSCs) obtained from ovarian endometriotic tissues [ 101 ]. Rapamycin treatment was associated with activation of autophagy in ECSCs, as evidenced by decreased phosphorylation of p70S6K and S6K and increased LC3-II expression. These changes were accompanied by elevated levels of cleaved caspase-3, and cleaved PARP, as well as increased cell death. Interestingly, adding 3-methyladenine (3-MA), a PIK3 inhibitor, reversed these effects, suggesting that mTOR inhibition may promote apoptosis of endometriotic cell by through autophagy. Additionally, Luo X et al. demonstrated that rapamycin treatment induced autophagy, as evidenced by an increased number of LC3-positive autophagic puncta, and suppressed cell proliferation and clonogenicity in CRL-7566 endometrial cells [ 105 ]. Another study reported a decrease in the expression levels of IL receptors (IL15Rα and IL2Rβ) in rapamycin-treated ESCs [ 119 ]. IL-15 was found to be highly expressed in ectopic ESCs and ectopic endometrium from patients with endometriosis compared with normal ESCs and endometrium from healthy donors, respectively. The authors suggested that decreased autophagy in ESCs in endometriosis may enhance their sensitivity to IL-15 by upregulating IL-15 receptor expression. Functionally, IL-15 inhibited apoptosis and promoted cell viability, cell proliferation and invasiveness in ESCs. In addition, IL-15 was also suggested to facilitate immune evasion by suppressing the cytotoxic activity of NK cells, creating a defective immune environment in the ectopic milieu and favoring endometriosis progression. Consistent with this hypothesis, in a follow-up study, the same group identified an association between decreased autophagy in endometriosis and the accumulation of FCGR3- NK cells along disease progression [ 97 ]. Co-cultures of ESCs and NK cells showed an increase in FCGR3- NK cells and alterations in immune markers, with 3-MA treatment enhancing these effects while limiting those of rapamycin. Silencing ATG5 in ESCs similarly promoted FCGR3- NK cell differentiation and reduced NK cytotoxicity. In a mouse model, exposure to 3-MAresulted in larger ectopic lesions and higher levels of FCGR3- NK cells. The study also highlighted the role of HCK in regulating NK differentiation, with decreased HCK levels stimulating CXCL8 and IL23A secretion, further contributing to a dysfunctional immune environment. These findings suggest that autophagy inhibition in ESCs may accelerate disease progression and that strategies to enhance NK cell cytotoxicity could represent a promising immunotherapeutic approach for endometriosis. Rapamycin has shown promising results in treating several diseases [ 120 – 122 ], including endometriosis, but its success is still limited by challenges such as side effects, dose optimization, and other factors. In this context, different strategies to modulate mTOR activity are being explored. For example, a recent study suggested that plant-based modulation of mTOR activity could be a promising strategy for targeting endometriosis in a rat model [ 123 ]. In this study, the administration of Açai Berry, an Amazonian fruit, inhibited PI3K, AKT, and ERK1/2 phosphorylation, thereby inactivating mTOR and promoting autophagy. It also enhanced the activity of the ATG1/ULK1 complex, facilitating autophagosome nucleation through BECN1 and Atg9 recruitment and increased LC3II expression. These findings highlight the potential of plant-based strategies, including Açai Berry, to complement existing treatments and address the limitations of rapamycin and other therapeutic agents. Given the role of hypoxia in endometriosis progression, targeting hypoxia-induced autophagy may offer a therapeutic advantage. The study of Liu et al. [ 69 ] reported that autophagy inhibition by using 3-MA and CQ appeared to reduce hypoxia-induced migration and invasion of ESCs. Similarly, the study of Chen et al. [ 70 ] found that autophagy inhibition with 3-MA seemed to suppress hypoxia-induced autophagy, migration, and EMT in CRL-7566 ovarian endometriosis cells in vitro. Transcrocetin, a potent antioxidant and antiproliferative carotenoid [ 124 ], has been suggested to have potential in targeting autophagy for endometriosis treatment [ 125 ]. In epithelial cells of the endocervix and vaginal mucosa, transcrocetin was found to inhibit cell proliferation, induce G1-phase arrest, and disrupt mitochondrial function, including impairing autophagic flux. These effects, combined with oxidative stress, suggest that transcrocetin could represent a promising non-hormonal therapeutic candidate that modulates autophagy in endometriosis. Typically, endometriosis is characterized by increased estradiol production and consequent proliferation of endometriotic tissues, together with a resistance to progesterone [ 126 , 127 ]. Thus, strategies for endometriosis treatment are based on decreasing estradiol levels or increasing the response to progesterone. Gonadotropin-releasing hormone (GnRH) agonists, such as GnRHa, are commonly used in endometriosis treatment, but their prolonged use can lead to severe hypoestrogenism. Alternatively, progestins offer a treatment option with fewer side effects and lower serum estrogen levels [ 128 ]. Further observations indicate that progesterone treatment does not affect LC3-II levels, autophagosome formation, or the phosphorylation status of AKT, ERK1/2, or S6K in estrogen-treated ECSCs [ 129 ]. However, treatment with the progestin dienogest in estrogen-treated ECSCs was found to promote autophagy, as evidenced by increased LC3-II expression and autophagosome formation. Moreover, the decreased phosphorylation of AKT, ERK1/2, and S6K was also observed after exposure to dienogest. Similar effects were observed when ECSCs were treated with AKT and ERK1/2 inhibitors, which were associated with increased LC3-II expression and reduction in S6K phosphorylation. Furthermore, dienogest treatment appeared to enhance apoptosis in ECSCs, as seen by increased cleaved caspase-3. The addition of 3-MA was found to reverse dienogest-induced autophagy and apoptosis. These findings suggest that dienogest may induce autophagy in endometriotic cells by inhibiting AKT, ERK1/2 and mTOR activities, thereby potentially contributing to increased apoptosis. In a study by Cornillie et al. (1896), the effects of the antiprogesterone steroid ethylnorgestrienone (R2323) treatment were examined in infertile patients with endometriosis. The authors observed regressive alterations and cellular degeneration in several endometriotic foci, including increased autophagic lysosomes [ 130 ]. Despite these changes, no significant difference in the extent of the disease was observed according to the American Fertility Society classification system. In contrast to the effects observed with antiprogesterone treatments, Borahay et al. studied the effect of Müllerian inhibiting substance (MIS), a 140-kDa homodimeric glycoprotein member of the transforming growth factor-beta (TGF-𝛽) superfamily, in endometriosis [ 131 ]. MIS treatment in the CRL-7566 endometriosis cell line was found to inhibit proliferation and appeared to induce apoptosis, increased cleaved caspase-9, and cell-cycle arrest, as evidenced by increased expression of p27 CDK-inhibitor. In addition, MIS treatment was associated with increase autophagic activity, reflected by a significant increase in LC3-II and BECN1 expression. Altogether, these results suggest that MIS may represent a potential candidate for future therapeutic approaches targeting endometriosis, although further preclinical and clinical validation is required. The effect of protopanaxadiol (PPD) and protopanaxatriol (PPT), two metabolites of ginsenosides with anti-cancer properties, has also been studied in the context of endometriosis [ 132 ]. PPD, PPT, the ginsenosides G-Rg3 and G-Rh2, significantly reduced the viability of ectopic ESCs. The authors reported that PPD appeared to limit the progression of endometriosis, potentially by counteracting the inhibitory effects of estrogen on autophagy and by enhancing NK cell cytotoxicity. PPD treatment downregulated the expression of ERα and upregulated the expression of PR in ectopic ESCs and in an intraperitoneal endometriosis mouse model. Additionally, PPD was found to induce autophagy in ectopic ESCs, as indicated by increased LC3B-II, BECN1 expression, and downregulated p62/SQSTM1 expression, effectively reversing the inhibitory effects of estrogen on autophagy. PPD also enhanced the transcription of several autophagy-related genes, including ATGs and MAP1L3CB , while decreasing ESR1 and TGF -𝛽 mRNA levels, in ectopic ESCs. Furthermore, PPD treatment led to a higher ratio of pro-apoptotic molecules (BAX/BAK) to anti-apoptotic molecules (BCL2/BCL-XL) and increased NK cell cytotoxic activity. The authors also explored the interrelation between estradiol (E2) and PPD and found that E2 treatment increased the number and weight of mouse ectopic lesions. In contrast, PPD reduced both the number and weight of lesions and mitigated the stimulatory effect of E2 on the growth of ectopic lesions in mice. These results suggest that PPD could represent a putative therapeutic strategy to treat endometriosis. Building upon these findings with hormonal modulators, SCM-198, a synthetic form of leonurine [ 133 ], has also been investigated as a potential therapeutic approach in endometriosis. A study by Lin et al. [ 134 ] reported that increased ERα signaling, and decreased progesterone receptor isoform B (PRB) expression synergistically led to a hypo-autophagy state in ectopic endometrial stromal cells, inhibiting apoptosis. The high expression of TNF-α in ectopic ESCs seemed to enhance the anti-apoptotic effects mediated by low autophagy through activation of the aromatase-estrogen-ERα signaling pathway. SCM-198 was found to reduce the growth of ectopic lesions in EMS mice and promote the apoptosis of ectopic ESCs both in vivo and in vitro. This apoptotic effect appeared to involve upregulation of autophagy, potentially through inhibition of TNF-α-activated aromatase-estrogen-ERα signaling and by increasing PRB expression. The study suggests that SCM-198 may be an effective therapeutic strategy for regulating the inflammation-endocrine-autophagy axis in endometriosis. Despite growing interest in the role of autophagy in endometriosis, several limitations continue to hinder progress in the field. A major concern is the heterogeneity in study designs, particularly regarding the menstrual cycle phase during sample collection. Autophagy is hormonally regulated and varies across the cycle, yet many studies do not control for or report this variable, introducing significant biological noise [ 135 ]. Specifically, autophagic activity in normal endometrium is low in the proliferative phase but rises markedly during the late secretory phase, peaking at menstruation with progesterone withdrawal [ 136 ]. Markers like LC3-II reflect this cyclicity, paralleling apoptosis and tissue remodeling [ 137 ]. However, there is a notable lack of direct and detailed studies investigating the modulation of autophagy-related genes on a gene-by-gene basis throughout the different phases of the human menstrual cycle in the endometrium. While autophagic activity and markers such as LC3-II have been shown to vary with the menstrual phases, comprehensive gene-specific expression analyses remain scarce. This gap highlights the need for further research to elucidate the precise molecular regulation of autophagy during normal menstrual cyclicity. Failure to consider these physiological variations at the time of sample collection can lead to apparent contradictions across studies and limit the ability to draw meaningful conclusions. Additionally, small and inconsistent sample sizes are commonly used, which is an important factor limiting statistical power and reproducibility. This is especially problematic in studies using patient-derived tissues, where variability in lesion type and location further complicates interpretation. Most studies do not provide detailed information on lesion characteristics, such as whether they are superficial, ovarian, or deep infiltrating, despite the likelihood that these subtypes exhibit distinct biological behaviors. Another major limitation is the lack of hormonal profiling in study participants. This omission is significant because estrogen and progesterone are key regulators of autophagy, and their fluctuating levels across the menstrual cycle profoundly influence cellular homeostasis. The degree of progesterone resistance varies among patients and lesion types, introducing additional heterogeneity. A further source of variability arises from differences in tissue selection and control groups. Some studies compare ectopic lesions to eutopic endometrium from the same patient, while others use endometrium from women without endometriosis as controls. These approaches are not equivalent, as the eutopic endometrium of affected women often exhibits molecular alterations, including progesterone resistance and inflammatory changes, which may influence autophagy independently of lesion formation. Additionally, control groups frequently include women with infertility or other gynecological conditions, which can introduce confounding factors related to hormonal status, immune function, or metabolic stress. The lack of standardized criteria for selecting normal controls difficulties results interpretation and may account for some of the discrepancies observed across studies. Another critical gap is the lack of longitudinal studies. Most current research is cross-sectional, capturing autophagy-related changes at a single time point. This limits our understanding of how autophagy evolves in response to disease progression or therapeutic intervention. Furthermore, the interplay between autophagy and other cellular processes, such as oxidative stress, inflammation, or cell survival, remains incompletely understood. Dissecting their relation is crucial for identifying precise therapeutic targets and avoiding unintended consequences of pathway modulation. Regarding the study of immune responses in murine models of endometriosis, it is important to acknowledge their limitations, more particularly the variability in macrophage polarization observed in the in vivo model. Differences in genetic background can significantly influence the balance between M1- and M2-like phenotypes, thereby affecting lesion development and inflammatory profiles. Moreover, the classical M1/M2 dichotomy does not fully describe the functional plasticity of macrophages observed in vivo, where hybrid or intermediate states are common. These factors complicate the extrapolation of findings to human disease and highlight the need for complementary approaches using patient-derived samples and high-resolution immune profiling techniques. Finally, while autophagy-targeting therapies have shown promise in preclinical models, their clinical translation remains limited. The efficacy, safety, and long-term outcomes of such interventions are largely unexplored.