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Endometriosis (EM) is a common gynecologic condition that often leads to infertility in women of reproductive age. Cell adhesion molecule 2 (CADM2) is involved in maintaining cell adhesion and polarity, as well as suppressing tumors. However, the role and mechanism of CADM2 in endometriosis is unclear. Therefore, this study evaluated the expression levels of CADM2 and epithelial-mesenchymal transition (EMT)-related marker proteins (E-cadherin, α-SMA, and N-cadherin). Compared to normal endometrial tissue, CADM2 was expressed at low levels in ectopic endometrial tissue from patients with EM. We performed clone formation assays, wound healing assays, and Transwell cell invasion assays to investigate the effects of CADM2 on the biological behavior of endometriosis epithelial cells (11Z) and ectopic endometrial stromal cells (EESCs). The growth, migration, and invasion abilities of these cells were significantly inhibited by overexpression of CADM2. The results were reversed after the knockdown of CADM2. Finally, western blotting (WB) was utilized to detect the effect of CADM2 on EMT in endometriosis cells. CADM2 inhibited EMT in endometriosis cells. In conclusion, our study suggests that CADM2 is a negative regulator of endometriosis development and may inhibit endometriosis development by suppressing EMT., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Aims: Current evidence suggests that there is no completely effective method for endometriosis (EMS) without trauma due to diverse adverse effects. Reliable evidence illustrates that inhibiting ferroptosis is a potential strategy for EMS. We sufficiently verified that the expression of endogenous protein PDZ and LIM domain 3 (PDLIM3) was significantly increased in EMS., Methods: PDLIM3 knockdown reduced primary ectopic endometrial stromal cells' (EESCs) viability and migration, and elevated ferroptosis signaling indicators including Fe 2+ , malondialdehyde (MDA), and reactive oxygen species (ROS) in EESCs., Results: Mechanistic studies revealed that inhibition of PDLIM3 accelerated glioma-associated oncogene-1 (Gli1) degradation and further deactivated Hedgehog signaling. Gli1 inhibitor, GANT61, abrogated the impact of PDLIM3 deletion on EESC growth, migration, and ferroptosis. In vivo experiments suggested that PDLIM3 reduction repressed the growth of endometrial lesions. Likewise, repression of PDLIM3 promoted ferroptosis and attenuated Hedgehog signaling in endometrial lesions., Conclusions: Collectively, silencing of PDLIM3 facilitates ferroptosis in EMS by inducing Gli1 degradation and blocking Hedgehog signaling. It may provide an alternative strategy for developing therapeutic agents of EMS in the future., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Autophagy has emerged as an important process of cell metabolism. With continuous in-depth research on autophagy, TFEB has been a key transcription factor regulating autophagy levels in recent years. Studies have established that TFEB regulates autophagy and apoptosis in various diseases. However, the relationship between TFEB and the pathogenesis of endometriosis remains unclear. This study aimed to investigate the effect of TFEB on the mechanism of endometriosis progression. The results showed that TFEB and autophagy-related protein LC3 are highly expressed in ectopic endometrium of patients with endometriosis, overexpression of TFEB in cultured human endometrial stromal cells (HESCs) by lentivirus not only promoted autophagy but also inhibited apoptosis. In addition, the migration and invasion ability of HESCs were enhanced by TFEB overexpression. Furthermore, inhibiting autophagy with specific inhibitors can attenuate migration and invasion of HESCs induced by TFEB. The rat models of endometriosis show that TFEB knockdown can suppress lesion growth in vivo. Our results suggest that autophagy may be involved in the progression mechanism of endometriosis, and the mechanism of autophagy disorder in endometriosis is probably related to TFEB. TFEB may be a key molecule in promoting endometriosis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Endometriosis, a common chronic gynecological disease, refers to the presence and proliferation of endometrial tissue in locations other than the uterine cavity. Approximately 6 to 10% of the population of women of childbearing age are known to have endometriosis; the most common clinical signs are pelvic pain and infertility. Although endometriosis is a benign disease, it exhibits some typical features of malignant tumors, such as proliferation, invasion, metastasis, and recurrence. Endometriosis is considered a chronic, inflammatory, and estrogen-dependent disease, and multiple factors contribute to its occurrence and development. In recent years, increasing attention has been given to the role of apoptosis in the pathogenesis of this disease. Some researchers believe that spontaneous apoptosis of the endometrium is critical in maintaining its normal structure and function, and abnormal apoptosis can promote the occurrence and development of endometriosis. Inflammation is another likely process in the pathogenesis of endometriosis. Inflammation mediates the adhesion, proliferation, differentiation, and invasion of ectopic lesions of endometriosis, primarily by regulating the function of immune cells and increasing the level of proinflammatory cytokines in body fluids. The ultimate initiators of apoptosis and inflammatory cell death (pyroptosis) are the caspase family proteases. In this article, we review the progress in recent years in caspase function as well as the possible role of these enzymes in the pathogenesis of endometriosis, indicating potential treatment strategies., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Ovarian endometriosis is a common gynecological disease, and one of its most significant symptoms is infertility. In patients with endometriosis, defects in endometrial decidualization lead to impaired endometrial receptivity and embryo implantation, thus affecting early pregnancy and women's desire to have children. However, the mechanisms underlying the development of endometriosis and its associated defective decidualization are unclear. We find that NEK2 expression is increased in the ectopic and eutopic endometrium of patients with endometriosis. Meanwhile, NEK2 interacts with FOXO1 and phosphorylates FOXO1 at Ser184, inhibiting the stability of the FOXO1 protein. Importantly, NEK2-mediated phosphorylation of FOXO1 at Ser184 promotes cell proliferation, migration, invasion and impairs decidualization. Furthermore, INH1, an inhibitor of NEK2, inhibits the growth of ectopic lesions in mouse models of endometriosis and promotes endometrial decidualization in mouse models of artificially induced decidualization. Taken together, these findings indicate that NEK2 regulates the development of endometriosis and associated disorders of decidualization through the phosphorylation of FOXO1, providing a new therapeutic target for its treatment., (© 2024. The Author(s).)

Purpose: Endometriosis is an estrogen-dependent inflammatory disease and one of the most common gynecological diseases in women of reproductive age. The aim of the review was to explore the relationship between the chromatin regulatory factors and endometriosis., Methods: By searching for literature on chromatin regulators and endometriosis in PuMed. Finally, 98 documents were selected., Results: Chromatin regulators (CRs) are essential epigenetic regulatory factors that can regulate chromatin structure changes and are usually divided into three categories: DNA methylation compounds, histone modification compounds, and chromatin remodeling complexes. Noncoding RNAs are also chromatin regulators and can form heterochromatin by binding to protein complexes. Chromatin regulators cause abnormal gene expression by regulating chromatin structure, thereby affecting the occurrence and development of endometriosis., Conclusion: This review summarizes the participation of chromatin regulators in the mechanisms of endometriosis, and these changes in related chromatin regulators provide a comprehensive reference for diagnosis and treatment of endometriosis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Endometriosis is an estrogen-dependent disorder of menstruating primates where tissues similar to the inner lining of the uterus exist "ectopically" outside of the uterus. The ectopic endometrium, like the endometrium within the uterus, expresses estrogen receptors (ER) and progesterone receptors (PR) and undergoes hormone-dependent cell proliferation and bleeding each menstrual cycle. The goal of this study was to conduct abdominopelvic positron emission tomography (PET) scans with computed tomography (CT) imaging of rhesus macaques (Macaca mulatta) using radiotracers that target ER and PR [16α-[ 18 F]fluoroestradiol (FES) and 12-[ 18 F]fluoro-furanyl-nor-progesterone (FFNP)] in individuals with and without endometriosis. We also aimed to determine if menstrual cycle phase and/or the presence of endometriosis affected the uptake of these radiotracers., Procedures: Rhesus macaques with either clinically diagnosed endometriosis (n = 6) or no endometriosis (n = 4) underwent PET/CT scans with FES. A subset of the animals also underwent PET/CT scans with FFNP. Standard uptake values corrected for body weight (SUVs) were obtained for each radiotracer in target and background tissues (e.g., intestinal). We performed repeated measure analysis of variance tests to determine how uterine and background uptake differed with scan time, phase of the menstrual cycle, and disease state., Results: Abdominopelvic PET/CT could not resolve small, individual endometriotic lesions. However, macaques with endometriosis displayed higher uterine uptake compared to those without the disorder. Radiotracer uptake differed by menstrual cycle phase with increased uterine uptake of both radiotracers in the proliferative phase of the menstrual cycle. Background intestinal uptake of FFNP increased over time after infusion, but only during the proliferative phase., Conclusions: PET/CT with FES and FFNP support the concept that ER and PR levels are altered in individuals with endometriosis. This highlights the impact of the disease on typical reproductive tract function and may provide a novel pathway for the identification of individuals with endometriosis., (© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Introduction: The microbial community plays a crucial role in the pathological microenvironment. However, the structure of the microbial community within endometriotic lesions and its impact on the microenvironment is still limited., Methods: All 55 tissue samples, including ovarian ectopic (OEMs) and normal (NE) endometrium, were subjected to 16S rRNA sequencing, metabolomic and proteomic analysis., Results: We found the abundance of Tuzzerella is significantly lower in OEMs compared to NE tissue (p < 0.01). We selected samples from these two groups that exhibited the most pronounced difference in Tuzzerella abundance for further metabolomic and proteomic analysis. Our findings indicated that endometriotic lesions were associated with a decrease in L-Glutamine levels. However, proteomic analysis revealed a significant upregulation of proteins related to the complement pathway, including C3, C7, C1S, CLU, and A2M. Subsequent metabolic and protein correlation predictions demonstrated a negative regulation between L-Glutamine and C7. In vitro experiments further confirmed that high concentrations of Glutamine significantly inhibit C7 protein expression. Additionally, immune cell infiltration analysis, multiplex immunofluorescence, and multifactorial testing demonstrated a positive correlation between C7 expression and the infiltration of regulatory T cells (Tregs) in ectopic lesions, while L-Glutamine was found to negatively regulate the expression of chemotactic factors for Tregs., Conclusion: In this study, we found a clear multi-omics pathway alteration, "Tuzzerella (microbe)-L-Glutamine (metabolite)-C7 (protein)," which affects the infiltration of Tregs in endometriotic lesions. Our findings provide insights into endometriosis classification and personalized treatment strategies based on microbial structures., (© 2024. The Author(s).)

NADPH oxidase 4 (NOX4) plays an important role in the regulation of oxidative stress, which is associated with endometriosis. This study aims to investigate the effects of NOX4 in endometriosis and its molecular mechanisms. Clinical specimens were collected, and human endometrial stromal cells (HESCs) were isolated. The knockdown of NOX4 cell lines was established on the HESCs and induced by peritoneal fluid. The levels of NOX4 were determined by using immunohistochemistry (IHC) staining, western blotting, and qPCR, respectively. The levels of oxidative stress markers were determined by using western blotting and ELISAs, respectively. The correlation of NOX4 and oxidative stress markers was analyzed by the Pearson correlation coefficient. The levels of NOX4 were dramatically elevated in the ectopic endometrium. Besides, oxidative stress biomarkers were also dysregulated in the ectopic endometrium as compared to the normal endometrium. Pearson's correlation coefficient analysis revealed a relationship between NOX4 and oxidative stress biomarkers in the ectopic endometrium. NOX4 modulated the expressions of oxidative stress markers in endometrial stromal cells stimulated by the peritoneal fluid from endometriosis. The effects of NOX4 on endometriosis are in part by its regulatory effects against oxidative stress., (© 2023. The Author(s), under exclusive licence to Institute of Plant Genetics Polish Academy of Sciences.)

In recent years, the matrisome, a set of proteins that make up the extracellular matrix (ECM) or are closely involved in ECM behavior, has been shown to have great importance for characterizing and understanding disease pathogenesis and progression. The matrisome is especially critical for examining diseases characterized by extensive tissue remodeling. Endometriosis is characterized by the extrauterine growth of endometrial tissue, making it an ideal condition to study through the lens of matrisome gene expression. While large gene expression datasets have become more available and gene dysregulation in endometriosis has been the target of several studies, the gene expression profile of the matrisome specifically in endometriosis has not been well characterized. In our study, we explored four Gene Expression Omnibus (GEO) DNA microarray datasets containing eutopic endometrium of people with and without endometriosis. After batch correction, menstrual cycle phase accounted for 53% of variance and disease accounted for 23%; thus, the data were separated by menstrual cycle phase before performing differential expression analysis, statistical and machine learning modeling, and enrichment analysis. We established that matrisome gene expression alone can effectively differentiate endometriosis samples from healthy ones, demonstrating the potential of matrisome gene expression for diagnostic applications. Furthermore, we identified specific matrisome genes and gene networks whose expression can distinguish endometriosis stages I/II from III/IV. Taken together, these findings may aid in developing future in vitro models of disease, offer insights into novel treatment strategies, and advance diagnostic tools for this underserved patient population., (© 2023. The Author(s).)

Endometriosis is an immune chronic inflammatory disease, and there are currently no more effective drugs for treating endometriosis due to its unknown etiology. Salbutamol is a β2-adrenergic receptor (β2AR) agonist commonly used to treat asthma by selectively activating β2 receptors on airway smooth muscle and leukocytes, exerting bronchial dilation and synergistic anti-inflammatory effects. In recent years, β2AR agonists have been used in endometriosis studies, and we speculate that salbutamol may have a therapeutic effect on endometriosis. The purpose of this research was to explore the therapeutic effect of salbutamol on endometriosis mice. The mouse endometriosis model was established and treated with different doses of salbutamol. Endometrial lesions were harvested for pathological diagnosis, immunohistochemistry (IHC), Masson staining, and toluidine blue analysis. We found that the number and size of endometriotic lesions were all significantly decreased after 3 weeks of treatment with different doses of salbutamol on endometriosis model mice (P < 0.05). After Salbutamol treatment, the amount of mast cells (toluidine blue) and macrophages (F4/80) in the lesions as well as the expressions of interleukin (IL)-1β, tumor necrosis factor (TNF)-ɑ, platelet-derived growth factor subunit B (PDGFB), CD31, transforming growth factor (TGF)-β, Masson staining, BCL2, TUBB3, substance P (SP), and nerve growth factor (NGF) were significantly reduced (P < 0.05). These results suggested that salbutamol could effectively treat endometriosis in mice by reducing immune inflammatory cells and factors, angiogenesis, and fibrosis, increasing apoptosis of endometriotic lesions, and decreasing neurogenesis., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis is a condition characterized by increased oxidative stress and chronic inflammation, which can be treated with progestins and other progesterone receptor ligands. However, some patients are refractory to this treatment and the reason is uncertain. Here we investigated the effects of the selective progesterone receptor modulator ulipristal acetate (UPA) on proliferation, reactive oxygen species (ROS), and proinflammatory cytokine production by endometriotic cells and endometrial cells from women with histologically proven endometriosis (n = 22) and endometriosis-free controls (n = 6). Epithelial and stromal cells were isolated and treated in triplicate for 24 h with 1 μM, 10 μM, or 100 μM UPA. Cells were tested for proliferation and ROS production, while cell supernatants were assayed for interleukin (IL)-6, C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor (TNF)-α concentrations. Proliferation, ROS production, and IL-6 and CCL2 secretion were increased in non-stimulated epithelial and stromal cells from endometriotic lesions compared to endometrial cells from endometriosis patients and controls. UPA induced a dose-dependent increase of cell proliferation only in endometriosis, while enhancing ROS production by all cell types evaluated. UPA reduced CCL2 production in controls but failed to do that in endometriosis, whereas TNF-α was undetectable. We conclude that treatment of endometriotic cells with UPA stimulated in vitro proliferation and ROS production and failed to revert the proinflammatory cytokine excess that characterized these cells, unravelling possible mechanisms of drug resistance in the treatment of endometriosis., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis (EMS) is a common benign gynecological disease affecting women of reproductive age. It is characterized by abnormal growth of endometrial tissue outside the uterine cavity, resulting in chronic pelvic pain and infertility. Endometrial physiological and pathological processes are intimately connected to autophagy. Mitophagy is an essential selective mode that protects cells from metabolic stress and hypoxia. Mitochondrial autophagy mediated by prohibitin 2 (PHB2) is dependent on the PRKN/Parkin pathway and is involved in numerous human diseases. Uncertainty remains as to whether mitophagy regulation by PHB2 contributes to the occurrence and progression of EMS. This study aims to investigate the mechanism underlying the role of PHB2 in EMS. This study detected the protein and mRNA expression of PHB2 in ectopic and normal endometrial tissues of ovarian EMS, in addition to ectopic endometrial cell line 12Z and endometrial stromal cell line KC02-44D for gene overexpression or knockdown. Cell function experiments and mitochondrial function experiments were conducted to investigate the role of PHB2 in the endometrium. Bioinformatic analysis and experiments were also used to investigate the upstream transcription factors that influence PHB2 expression. PHB2 was downregulated in ectopic endometrium, and PHB2 overexpression inhibited cell proliferation, migration, and invasion and promoted apoptosis. The upregulation of mitophagy markers, including Parkin and LC3II/I, and the downregulation of autophagy degradation markers P62 and TOMM20 in EMS suggest that PHB2 may contribute to cell proliferation, migration, invasion, and apoptosis via PRKN/Parkin-mediated mitophagy. Analysis and validation of bioinformatics data revealed that the transcription factor GABPA binds directly to the PHB2 promoter region and controls the transcriptional expression of PHB2. This study investigated the role of PHB2 in the onset of EMS. It inhibits EMS growth via PRKN/Parkin-mediated mitophagy, and GABPA controls the transcriptional disorder of PHB2. This study's findings suggest a novel method for investigating the clinical potential of PHB2 in EMS., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis is a hormone-dependent disease associated with impaired immunoregulation. In our recent study, we have characterized the trascriptomic transformation of eutopic endometrium from patients with minimal/mild endometriosis and controls across the menstrual cycle. However, the regulatory mechanism of altered immune microenvironment in eutopic endometrial stromal cells (ESCs) remains unclear. Here, we want to explore the regulation of immune cell to progesterone resistance and endometrial receptivity in the eutopic ESCs by cytokine (TGF-β1), and to understand the effect of TGF-β1 on the decidualization of the eutopic ESCs. Primary culture of eutopic ESCs was performed to explore the effects of TGF-β1 on the expression of Smad and progesterone receptor (PR) and the in vitro decidualization. Additionally, co-immunoprecipitation (Co-IP) was used to explore the direct interaction between Smad and PR. We found an attenuate expression of PRB protein (p=0.026) after using TGF-β1 in eutopic ESCs, although the difference of PRA before and after treatment was not significant (p=0.678). Similarly, the results of qRT-PCR showed that the mRNA level of PR (p<0.001), PRB (p=0.003) and HOXA10 (p<0.001) decreased significantly after TGF-β1 treatment, but that increased (p<0.023, for all) after SB431542 treatment in the eutopic ESCs. Moreover, TGF-β1 has a negative effect on the in vitro decidualization of eutopic ESCs (p=0.003). And the group with treatment of both TGF-β1 and SB435142 in eutopic ESCs showed significant decidual-like changes with increased prolactin level (p=0.01). We did not observe any physical interaction between the PR and p-Smad3/Smad3 proteins by using Co-IP. By activating TGF-β/Smad signaling in eutopic ESCs, elevated TGF-β1 from CD45+ immune cells could attenuate expression of PR, and further decrease endometrial receptivity., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis is a chronic disease associated with a disrupted oxidative balance and chronic inflammation. In this study, we investigated the role of glutathione S-transferase Mu class 4 (GSTM4) in endometriosis and determined whether 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX) regulates GSTM4 expression to affect cellular functions and oxidative stress. GSTM4 expression was detected by immunohistochemistry in endometrium from 15 endometriosis patients and 15 healthy controls. Western blotting was used to detect the expression of GSTM4, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-9 (MMP-9), Survivin, B-cell lymphoma-extra-large (Bcl-XL), Bax, kelch-like ECH-associated protein 1 (Keap1), and nuclear factor-erythroid 2-related factor 2 (Nrf2) in primary endometrial stromal cells with endometriosis (EESC) and normal endometrial stromal cells (NESC). The effects of NBDHEX on cell proliferation, migration, and invasion were evaluated using Cell Counting Kit-8 (CCK8) and Transwell assays. Apoptosis was detected by flow cytometry. The expression of GSTM4 was significantly increased in endometrium from endometriosis patients. Upon NBDHEX treatment, ESC exhibited reduced proliferation, migration and invasion abilities, and increased apoptosis. NBDHEX decreased the expression of endometriosis prognostic markers (PCNA and MMP-9) and anti-apoptotic proteins (Survivin and Bcl-xl), while it increased the expression of the apoptotic protein Bax. It had no effect on Keap1 expression, and it decreased the expression of Nrf2. The effect of siRNA-mediated knockdown of GSTM4 was similar to that of suppressing GSTM4 expression with NBDHEX treatment. These results indicate that GSTM4 is highly expressed in endometriosis and its expression is inhibited by NBDHEX. Decreased expression of GSTM4 inhibits cell growth, migration, and invasion, and negatively regulates Nrf2 to affect oxidative stress-induced apoptosis. Our results suggest that GSTM4 may play a role in ameliorating the progression of endometriosis. NBDHEX may have therapeutic potential in the treatment of endometriosis., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

MicroRNAs (miRs) play an important role in the pathophysiology of endometriosis; however, the role of miR-210 in endometriosis remains unclear. This study explores the role of miR-210 and its targets, IGFBP3 and COL8A1, in ectopic lesion growth and development. Matched eutopic (EuE) and ectopic (EcE) endometrial samples were obtained for analysis from baboons and women with endometriosis. Immortalized human ectopic endometriotic epithelial cells (12Z cells) were utilized for functional assays. Endometriosis was experimentally induced in female baboons (n = 5). Human matched endometrial and endometriotic tissues were obtained from women (n = 9, 18-45 years old) with regular menstrual cycles. Quantitative reverse transcript polymerase chain reaction (RT-qPCR) analysis was performed for in vivo characterization of miR-210, IGFBP3, and COL8A1. In situ hybridization and immunohistochemical analysis were performed for cell-specific localization. Immortalized endometriotic epithelial cell lines (12Z) were utilized for in vitro functional assays. MiR-210 expression was decreased in EcE, while IGFBP3 and COL8A1 expression was increased in EcE. MiR-210 was expressed in the glandular epithelium of EuE but attenuated in those of EcE. IGFBP3 and COL8A1 were expressed in the glandular epithelium of EuE and were increased compared to EcE. MiR-210 overexpression in 12Z cells suppressed IGFBP3 expression and attenuated cell proliferation and migration. MiR-210 repression and subsequent unopposed IGFBP3 expression may contribute to endometriotic lesion development by increasing cell proliferation and migration., (© 2023. The Author(s).)

Impairment of decidualization of eutopic human endometrial stromal cells (hESCs) may cause an increase in cell survival of endometrial tissue in the peritoneal cavity constituting a precondition for endometriosis development. Decidualization is a physiological process involving progesterone action and cAMP signaling. We here evaluated the effect of 8-Br-cAMP, the adenylate cyclase activator forskolin and of the progestin progesterone and medroxyprogesterone acetate (MPA) alone and in combination on decidualization induction using prolactin ELISA, and on cell size, cell granularity, and cell survival via flow cytometry in hESCs of patients with and without endometriosis. While progestins alone did not induce functional decidualization in hESCs, 8-Br-cAMP and forskolin induced decidualization in hESCs from both cohorts, whereas the induction of FOXO1 transcription and prolactin secretion by forskolin was significantly lower than by 8-Br-cAMP. 8-Br-cAMP- and forskolin-induced prolactin secretion was significantly enhanced by MPA, but not by progesterone. Decidualization entailed a decrease in cell size and in cell granularity. In general, hESCs from women with mild (ASRM I/II) as well as severe (ASRM III/IV) endometriosis in trend displayed a higher granularity, whereas mainly hESCs from severe endometriosis showed a stronger resistance to the induction of cell death after decidualization induction. In both cohorts, the amount of the decidual marker protein prolactin rather exhibited an anti-proportional correlation to cell death induction during six day treatment. This study contributes to widen our understanding of the connection of decidualization and cell death in endometriosis., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis is an estrogen-dependent inflammatory disease characterized by the growth of endometrial-like tissues containing endometrial stromal cells and glandular epithelium outside the uterine cavity. An insufficient response to progesterone contributes to disease progression and systemic inflammation during the pathogenesis of endometriosis. Patients with endometriosis usually experience painful symptoms, dysmenorrhea, and infertility, which contribute to a significant reduction in their quality of life. To determine the possible molecular mechanisms of endometriosis and explore novel therapeutic targets, we derived primary human ovarian endometriotic stromal cells (hOESCs) from a patient of reproductive age with ovarian endometriosis. In this study, we successfully established immortalized human ovarian endometriotic stromal cell lines (ihOESCs) using primary stromal cells obtained from endometriotic lesions to overcome short lifespan and growth inhibition. Immortalization of hOESCs with human telomerase reverse transcriptase (hTERT) transfection led to cells that maintained a proliferative state under passage culture conditions without mutagenesis during cellular senescence. The morphology and karyotype of ihOESCs were unchanged compared with those of hOESCs. Moreover, ihOESCs were continuously positive for vimentin and negative for E-cadherin expression. Following decidual stimuli and inflammatory responses, both hOESCs and ihOESCs sensitively express decidualization markers and proinflammatory cytokines. Collectively, we characterized ihOESCs to maintain their phenotypic and functional properties with a longer lifespan and normal physiological responses than those of hOESCs. These immortalized cells could aid in a detailed understanding of the pathological mechanisms of endometriosis., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis is a common gynecological disorder characterized by the presence of the endometrial glands and the stroma outside the uterine cavity. The disease affects reproductive function and quality of life in women of reproductive age. Endometriosis is similar to tumors in some characteristics, such as glycolysis. PIM2 can promote the development of tumors, but the mechanism of PIM2 in endometriosis is still unclear. Therefore, our goal is to study the mechanism of PIM2 in endometriosis. Through immunohistochemistry, we found PIM2, HK2, PKM2, SMH (smooth muscle myosin heavy chain), Desmin, and α-SMA (α-smooth muscle actin) were strongly expressed in the ovarian endometriosis. In endometriotic cells, PIM2 enhanced glycolysis and fibrosis via upregulating the expression of PKM2. Moreover, the PIM2 inhibitor SMI-4a inhibited the development of endometriosis. And we established a PIM2 knockout mouse model of endometriosis to demonstrate the role of PIM2 in vivo. In summary, our study indicates that PIM2 promotes the development of endometriosis. PIM2 may serve as a promising therapeutic target for endometriosis., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis (EMs) is a systemic and chronic disease with cancer-like feature, namely, distant implantation, which caused heavy healthy burden of nearly 200 million females. LncRNAs have been proved as new modulators in epithelial-mesenchymal transition (EMT) and EMs. Quantitative real-time PCR was conducted to measure the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (Linc-ROR), and miR-204-5p in ectopic endometrium (n = 25), eutopic endometrium (n = 20), and natural control endometrium (n = 22). Overexpression of Linc-ROR, knockdown or overexpression of miR-204-5p in End1/E6E7 and Ishikawa cells, was conducted to detect the function of Linc-ROR and miR-204-5p in EMs. Furthermore, luciferase reports were used to confirm the combination of Linc-ROR and miR-204-5p and the combination between miR-204-5p and SMAD4. Cell-Counting Kit-8, EdU assay, transwell assays, and Western blotting were used to detect the function of Linc-ROR and miR-204-5p in EMs cancer-like behaviors and EMT process. Linc-ROR was up-regulated in ectopic endometrium. Overexpressed Linc-ROR promotes cell proliferation, invasion, and EMT process. Linc-ROR regulated the EMT process, cellular proliferation, and invasion of EMs via binding to miR-204-5p. In addition, overexpression of Linc-ROR up-regulated SMAD4, a target protein of miR-204-5p, with which regulated EMT process and cancer-like behaviors in EMs together. Linc-ROR/miR-204-5p/SMAD4 axis plays a vital role in regulation EMT process in EMs, which might become a novel therapeutic targets and powerful biomarkers in EMs therapy., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis (ENDO) is a chronic estrogen-dependent gynecological condition that affects reproductive-age women, causing pelvic pain, infertility, and increased risk for ovarian cancer. Diabetes mellitus (DM) is a metabolic disease with significant morbidity and mortality and rising incidence worldwide. The occurrence of DM among ENDO patients remains understudied, despite commonalities in these conditions' immune, inflammatory, and metabolic dysfunctions. This pilot study evaluated whether a subset of women with ENDO manifests DM co-morbidity and if so, whether DM promotes ENDO status. Archived ectopic lesions obtained at ENDO surgery from non-diabetic (ENDO-N; n = 11) and diabetic (ENDO-DM; n = 15) patients were identified by a search of an electronic health database. Retrieved samples were analyzed by immunohistochemistry for markers of proliferation (Ki67, PTEN), steroid receptor signaling (ESR, PGR) and macrophage infiltration (CD68). Immunostaining data were expressed as percentages of immune-positive cells in lesion stroma and epithelium. In lesion stroma, the percentages of nuclear immune-positive cells were higher for ESR2 and lower for PGR-T, in ENDO-DM than ENDO-N patients. The percentages of nuclear immune-positive cells for ESR1 and PTEN tended to be higher and lower, respectively, in ENDO-DM than ENDO-N groups. In lesion glandular epithelium, the percentages of nuclear immune-positive cells were higher for ESR1 and ESR2, in ENDO-DM than ENDO-N groups. ENDO-N lesions had lower percentages of stromal CD68 immune-positive cells than ENDO-DM Type 1 lesions. Findings demonstrate DM in a subset of women with ENDO, which was associated with significant changes in lesion stromal and epithelial nuclear steroid hormone receptor levels, suggestive of disease progression., (© 2023. The Author(s).)

The signal transducer and activator of transcription (STAT) pathway, which regulates cell proliferation and immunity, has been implicated in chronic inflammatory diseases such as rheumatoid arthritis. However, few reports have described the effects of STAT inhibitors on endometriosis, another chronic inflammatory disease. Here, we investigated the intraperitoneal microenvironment and the effects of a STAT inhibitor in a mouse model of endometriosis. In the treatment group, a STAT3 inhibitor (Stattic®, 80 mg/kg) was orally administered three times per week; control animals received orally dosed phosphate-buffered saline. Endometriosis-like lesions and peritoneal lavage fluid were collected before and 1, 2, and 3 weeks after STAT3 inhibitor administration was initiated. The lesion area was significantly increased in both groups after the first week. However, in the treatment group, the lesion areas were significantly reduced at weeks 2 and 3 compared with week 1. Transforming growth factor (TGF)-β messenger RNA (mRNA) levels in ascites cells were significantly lower at weeks 1 and 2 than at week 0. Interleukin (IL)-6 mRNA levels were significantly higher at week 1 than at week 0 but were significantly lower at weeks 2 and 3 than at week 1. Thus, STAT inhibitors appeared to reduce the extent of endometriosis in this mouse model, and may also inhibit the IL-6 signaling pathway and reduce TGF-β levels. This study suggests that STAT inhibitors warrant further exploration for use in the treatment of endometriosis., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis (EMS) is an estrogen-dependent disease. However, little is known about the regulation of estrogen, a potential therapeutic target, in EMS, which remains very poorly managed in the clinic. We hypothesized that microRNAs (miRNAs) can be exploited therapeutically to regulate transcription factor 21 (TCF21) and steroidogenic factor-1 (SF-1) gene expression. In our study, paired eutopic and ectopic endometrial samples were obtained from women with EMS and processed by a standard protocol to obtain human endometrial stromal cells (EMs) for in vitro studies. We found that miR-92a-3p levels were decreased in ectopic endometrium and ectopic stromal cells (ESCs) compared with paired eutopic lesions. miR-92a-3p overexpression significantly suppressed the proliferation and migration of ESCs, whereas a decreased level of miR-92a-3p generated the opposite results. Next, we identified TCF21 as a candidate target gene of miR-92a-3p. In vitro cell experiments showed that miR-92a-3p negatively regulated the expression of TCF21 and its downstream target gene SF-1. Moreover, cell proliferation and invasion ability decreased after the silencing of SF-1 and increased after SF-1 overexpression. We also observed that silencing SF-1 while inhibiting miR-92a-3p partially blocked the increase in cell proliferation and invasion ability caused by miR-92a-3p knockdown while overexpressing both SF-1 and miR-92a-3p mitigated the impairment in cell proliferation and invasion ability caused by miR-92a-3p overexpression. Our results may provide a novel potential therapeutic target for the treatment of EMS., (© 2023. The Author(s).)

Endometriosis (EMs) is a life-long endocrine disorder and a common cause for female infertility and pelvic pain. The key characteristics of eutopic endometrium of EMs patients are high proliferative and migratory potentials. Cuproptosis is a recently identified copper- and-mitochondrial-dependent regulated cell death. Regretfully, its role in EMs remains unclear. In this study, Kyoto Encyclopedia of Genes and Genomes analyses of differentially expressed genes (DEGs) indicated strong activation of the PI3K-Akt-mTOR pathway and biological process analysis reported positive regulation of kinase activity. Next, we screened 11 cuproptosis-related DEGs and found all of them were downregulated in the EMs group, which indicated the suppression of cuproptosis in EMs. One key cuproptosis-related gene, PDHA1, was selected via support vector machine, random forest algorithm and lasso regularization to build a risk-scoring model, which was tested in both internal and external validations. In conclusion, the downregulation and kinase activity of PDHA1 may function with the PI3K-Akt-mTOR pathway in some way, which could suppress the cuproptosis level and account for the cancer-like pathology in EMs., (© 2022. The Author(s).)

Endometriosis is a chronic gynecological disease in women of childbearing age, which leads to infertility with risk of endometrial and ovarian cancer. The pathogenesis of endometriosis is poorly understood, and cure/treatment for it is not available, except for symptomatic treatment. The recurrence rate of endometriosis is high. SLP-2 is an inner mitochondrial membrane protein whose participation has been explained in cases of endometrial stromal cell growth, differentiation and migration, but its role in endometriosis is yet to be understood. Previous studies have found altered expression of stomatin-like protein 2 (SLP-2) in the serum of endometriotic patients. Therefore, we have studied the possible role of SLP-2 in the development of endometriosis. We found the ubiquitous and high expression of SLP-2 in the endometriotic tissue of both human endometriosis patients and rat endometriosis model. SLP-2 is seen in the glandular epithelial cells and stromal cells in the eutopic/normal or non-endometriosis group endometrium from human subjects. Finding high expression levels of SLP-2 in endometriotic tissue and ovarian cystic cells derived from endometriosis patients, we explored the possible role of SLP-2 in the cell aggregation, colonization, migration, and invasion in the human endometriotic cells associated with the progression of the endometriosis. Transient silencing of SLP-2 by its siRNA hinders endometriotic cells, aggregation, migration, and invasion into the extracellular matrix, which confirms SLP-2 involvement in endometriotic disease onset and progression. This study unravels the ubiquitous expression of SLP-2 in the human ectopic endometrial tissue and its role in the endometriotic cell migration, colonization, aggregation, and invasion leading to endometriosis progression., (© 2022. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Endometriosis is a chronic inflammatory condition affecting women of reproductive age. A relevant feature of endometriosis is the presence of fibrotic tissue inside and around the lesions, thus contributing to the classic endometriosis-related symptoms, pain, and infertility. The molecular mechanisms responsible for the development of fibrosis in endometriosis are not yet defined. The present review aimed to examine the biological mechanisms and signalling pathways involved in fibrogenesis of endometriotic lesions, highlighting the difference between deep infiltrating and ovarian endometriosis. The main cell types involved in the development of fibrosis are platelets, myofibroblasts, macrophages, and sensory nerve fibers. Members of the transforming growth factor (TGF) -β family, as well as the receptor Notch, or the bioactive sphingolipid sphingosine 1-phosphate (S1P), play a role in the development of tissue fibrosis, resulting in their metabolism and/or their signalling pathways altered in endometriotic lesions. It is relevant the knowledge of the molecular mechanisms that guide and support fibrosis in endometriosis, to identify new drug targets and provide new therapeutic approaches to patients., (© 2022. The Author(s).)

Oocyte-secreted growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are critical paracrine regulators of female fertility. Recent studies demonstrated that serum concentrations are associated with the number of oocytes retrieved during IVF, and therefore potential clinical use as biomarkers. However, it is unknown if the presence of endometriosis affects serum GDF9 or BMP15. An exploratory case-control study was prospectively performed on 60 women who underwent laparoscopy between April 2017 and August 2018 at two hospitals. GDF9 and BMP15 were measured by validated immunoassays in pre-operative serum samples. Data were analysed relative to laparoscopic assessment of endometriosis and staging. There were 35 women with confirmed laparoscopic diagnosis of endometriosis and 25 controls with no evidence of endometriosis at laparoscopy. GDF9 was detectable in 40% of controls and 48% of cases. There was no difference in median GDF9 concentrations between controls (20.0 pg/ml, range 20.0-2504 pg/ml) and cases (20.0 pg/ml, range 20.0-2963 pg/ml). BMP15 was detectable in 48% of controls and 58% of cases, with no difference in median concentrations between controls (26.5 pg/ml, range 24.0-1499 pg/ml) and cases (24.0 pg/ml, range 24.0-796 pg/ml). Furthermore, there were no significant differences in the proportion of detectable samples or concentrations of GDF9 or BMP15 with differing severities of endometriosis. In conclusion, serum concentrations of oocyte-secreted factors, GDF9 and BMP15 did not differ between control patients and patients with endometriosis. For clinical application in reproductive medicine, GDF9 and BMP15 serum biomarker quantitation is unlikely to be aberrant in the presence of endometriosis., (© 2022. The Author(s).)

Endometriosis is a chronic gynecologic disorder characterized by abnormal growth of endometrium-like tissues in the ectopic regions of the pelvic peritoneum. The pathophysiology of endometriosis is not completely understood; however, excessive endometrial cell proliferation together with resistance to apoptosis facilitates the migration, implantation, and survival of endometrial cells in the distant sites. Endoplasmic reticulum (ER) stress response (also called unfolded protein response) is a cellular defense mechanism triggered by ER stress. When severe enough, the so-called response initiates cell suicide, i.e., apoptosis. Therefore, therapeutic induction of ER stress in endometriotic cells could promote apoptosis and contribute to the management of disease. In this review, we discuss the pathogenic role of ER stress in endometriosis and the most recent findings regarding the induction of ER stress in connection with endometriosis., (© 2023. The Author(s), under exclusive licence to Cell Stress Society International.)

The inflammasomes are a family of recently described multi-protein cytoplasmic sensors that orchestrate the inflammatory response and participate in a variety of inflammatory conditions. We hypothesized that the activation of pyrin domain‑containing protein 3 (NLRP3) inflammasome by granulosa cells (hGCs) may be activated in women with endometriosis and influence oocyte maturation and IVF outcomes. We performed a cross-sectional study to investigate the NLRP3 inflammasome status in follicular fluid (FF) and in hGCs from 44 women undergoing controlled ovarian stimulation for IVF/ICSI. Study subjects were divided into two groups according to the infertility etiology: group with tubal or male factor (control, n = 22) vs. group with endometriosis (n = 22). The FF IL-1beta and IL-18 levels in the endometriosis group were significantly higher than those in the non-endometriosis group, i.e., 5010 pg/mL and 2738 pg/mL, respectively (p < 0.05). No correlation was found between clinical pregnancy and live birth rate and analyzed inflammasome component levels (p > 0.05). In addition, the hGCs from endometriosis women demonstrated high expression of NLRP3 inflammasome at both protein and mRNA levels. Higher expression of inflammasome components within the ovary compartment may result from the exaggerated inflammatory state associated with endometriosis and thus impact the fertility of these women., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intraperitoneal adhesions complicate over half of abdominal-pelvic surgeries with immediate, short, and long-term sequelae of major healthcare concern. The pathogenesis of adhesion development is similar to the pathogenesis of wound healing in all tissues, which if unchecked result in production of fibrotic conditions. Given the similarities, we explore the published literature to highlight the similarities in the pathogenesis of intra-abdominal adhesion development (IPAD) and other fibrotic diseases such as keloids, endometriosis, uterine fibroids, bronchopulmonary dysplasia, and pulmonary, intraperitoneal, and retroperitoneal fibrosis. Following a literature search using PubMed database for all relevant English language articles up to November 2020, we reviewed relevant articles addressing the genetic and epidemiological similarities and differences in the pathogenesis and pathobiology of fibrotic diseases. We found genetic and epidemiological similarities and differences between the pathobiology of postoperative IPAD and other diseases that involve altered fibroblast-derived cells. We also found several genes and single nucleotide polymorphisms that are up- or downregulated and whose products directly or indirectly increase the propensity for postoperative adhesion development and other fibrotic diseases. An understanding of the similarities in pathophysiology of adhesion development and other fibrotic diseases contributes to a greater understanding of IPAD and these disease processes. At a very fundamental level, blocking changes in the expression or function of genes necessary for the transformation of normal to altered fibroblasts may curtail adhesion formation and other fibrotic disease since this is a prerequisite for their development. Similarly, applying measures to induce apoptosis of altered fibroblast may do the same; however, apoptosis should be at a desired level to simultaneously ameliorate development of fibrotic diseases while allowing for normal healing. Scientists may use such information to develop pharmacologic interventions for those most at risk for developing these fibrotic conditions., (© 2021. Society for Reproductive Investigation.)

Endometriosis is a disease defined by the presence of abnormal endometrium at ectopic sites, causing pain and infertility in 10% of women. Mutations in the chromatin remodeling protein ARID1A (AT-rich interactive domain-containing protein 1A) have been identified in endometriosis, particularly in the more severe deep infiltrating endometriosis and ovarian endometrioma subtypes. ARID1A has been shown to regulate chromatin at binding sites of the Activator Protein 1 (AP-1) transcription factor, and AP-1 expression has been shown in multiple endometriosis models. Here, we describe a role for AP-1 subunit JUNB in promoting invasive phenotypes in endometriosis. Through a series of knockdown experiments in the 12Z endometriosis cell line, we show that JUNB expression in endometriosis promotes the expression of epithelial-to-mesenchymal transition genes co-regulated by ARID1A including transcription factors SNAI1 and SNAI2, cell adhesion molecules ICAM1 and VCAM1, and extracellular matrix remodelers LOX and LOXL2. In highly invasive ARID1A-deficient endometriotic cells, co-knockdown of JUNB is sufficient to suppress invasion. These results suggest that AP-1 plays an important role in the progression of invasive endometriosis, and that therapeutic inhibition of AP-1 could prevent the occurrence of deep infiltrating endometriosis., (© 2022. Society for Reproductive Investigation.)

Adenomyosis is an estrogen-dependent gynecological disorder. The abnormal migration and invasion of the eutopic endometrium is thought to be the primary role in the pathogenesis of adenomyosis. However, the exact underlying mechanism remains unclear. This study investigated involvement of serum and glucocorticoid-regulated kinase 1 (SGK1) in the pathogenesis of adenomyosis. The SGK1 expression level was higher in the eutopic endometrium of adenomyosis. Upregulation of SGK1 can promote the migration, invasion of human stromal endometrial cells (HESC). Through RNA sequencing and other technical methods, we found that SGK1 regulates the expression of the important downstream molecule Lysophosphatidic acid receptor 2 (LPAR2), and ultimately regulates the expression level of functional proteins such as matrix metalloproteinase 2 and matrix metalloproteinase 9, which are related to migration and invasion. Then, we found that 17β-estradiol (E 2 ) upregulated the expression of SGK1 in endometrial cells in a dose-dependent manner. Furthermore, SGK1 shRNA significantly suppressed the migration and invasion induced by E 2 in endometrial cells, as well as the related factors. Our study revealed the possible role of SGK1 in the migration and invasion in the development of adenomyosis., (© 2022. Society for Reproductive Investigation.)

The prevalence of endometriosis is approximately 10% in women of reproductive age, and 30-50% of women with endometriosis are infertile. Metformin has been reported to inhibit the growth of ectopic lesions in endometriosis. However, its effect on the eutopic endometrium of endometriosis is unknown. This study aimed to identify whether metformin affects endometrial receptivity in infertile women with minimal/mild endometriosis. We enrolled 10 infertile women who were diagnosed with minimal/mild endometriosis through laparoscopy. Paired endometrial tissues of the secretory phase from participants were collected during surgery and after 2 months of metformin treatment (n = 5) or no medical treatment (n = 5). Protein expression profiles of the paired endometrium were detected by proteomics and compared using the self-control method (2 months later vs. in surgery). Proteomics data revealed six proteins associated with endometrial receptivity among the significantly upregulated proteins after metformin treatment (fold change > 1.5, P < 0.05). Insulin-like growth factor binding protein 7 (IGFBP-7) showed the most robust increase in these six endometrial receptivity-related proteins (fold change: 8.668, P < 0.05), while there was no significant change in the controls (fold change: 1.906, P > 0.05). The upregulation of IGFBP-7 has been validated through target proteomics, immunohistochemistry, and further demonstrated in endometriosis mouse models induced by autotransplantation. This study revealed that metformin upregulated the expression of IGFBP-7 in the endometrium of human and mouse models of endometriosis. Metformin potentially affects endometrial receptivity of minimal/mild endometriosis by improving the expression of the endometrial receptivity marker IGFBP-7., (© 2022. Society for Reproductive Investigation.)

Emerging data indicates an association between endometriosis and subclinical atherosclerosis, with women with endometriosis at a higher risk for cardiovascular disease later in life. Inflammation is proposed to play a central role in the pathophysiology of both diseases and elevated levels of systemic pro-inflammatory cytokines including macrophage migration inhibitory factor (MIF), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) are well documented. However, a thorough understanding on the mediators and mechanisms which contribute to altered cytokine expression in both diseases remain poorly understood. MicroRNAs (miRNAs) are important post-transcriptional regulators of inflammatory pathways and numerous studies have reported altered circulating levels of miRNAs in both endometriosis and atherosclerosis. Potential contribution of miRNA-mediated inflammatory cascades common to the pathophysiology of both diseases has not been evaluated but could offer insight into common pathways and early manifestation relevant to both diseases which may help understand cause and effect. In this review, we discuss and summarize differentially expressed inflammatory circulating miRNAs in endometriosis subjects, compare this profile to that of circulating levels associated with atherosclerosis when possible, and then discuss mechanistic studies focusing on these miRNAs in relevant cell, tissue, and animal models. We conclude by discussing the potential utility of targeting the relevant miRNAs in the MIF-IL-6-TNF-α pathway as therapeutic options and offer insight into future studies which will help us better understand not only the role of these miRNAs in the pathophysiology of both endometriosis and atherosclerosis but also commonality between both diseases., (© 2022. Society for Reproductive Investigation.)

This aim of this study was to investigate whether macrophages protect endometriotic cells from oxidative injury and to elucidate the underlying mechanisms of any protection. Endometriotic cells cultured with or without differentiated macrophages (dTHP-1 cells) were treated with hydrogen peroxide (H 2 O 2 ) or methemoglobin, a major component of hemoglobin species in endometriotic cyst fluid. Co-culture experiments, microarray analysis, screening and validation of differentially expressed genes (DEGs), cell proliferation and viability assays, and experiments using a specific inhibitor were conducted to investigate the functional cross-talk between endometriotic cells and macrophages. Microarray analysis revealed that endometriotic cells co-cultured with dTHP-1 differentially express several genes compared with monoculture. Quantitative enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis identified TGF-β1 as a promising candidate gene expressed in endometriotic cells co-cultured with dTHP-1 cells. TGF-β1 stimulated the expression of heme oxygenase-1 (HO-1) in dTHP-1 cells. HO-1 expression was increased in dTHP-1 cells co-cultured with endometriotic cells compared with the dTHP-1 monoculture. Both H 2 O 2 and methemoglobin upregulated the expression of the HO-1 protein in the dTHP-1 monoculture; moreover, co-culture with endometriotic cells further enhanced HO-1 production. The co-culture with dTHP-1 protected endometriotic cells against oxidative injury. Blockade of HO-1 abolished the protective effects of macrophages. In an oxidative stress environment, TGF-β1 produced by endometriotic cells may protect against oxidative injury through the upregulation of macrophage-derived HO-1. The cross-talk between endometriotic cells and macrophages may contribute to the progression and pathogenesis of endometriosis., (© 2022. Society for Reproductive Investigation.)

Endometriosis is a disease that involves dysfunction of mitochondria, imbalance of proliferation, and apoptosis. Coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) is a major mitochondrial protein which could regulate the mitochondrial function and apoptosis in various tumor cells, promote migration and then lead to tumor progression. This study aimed to explore the role of CHCHD2 on endometriosis. We investigated the expression of CHCHD2 in ectopic and eutopic endometrium tissues of patients with endometriosis and normal endometrium tissues. Furthermore, CHCHD2 was downregulated to explore the corresponding change of mitochondrial function and morphology, mitochondrial-mediated apoptosis pathway, and proliferation and migration of ectopic endometrial stromal cells. Our results demonstrated that the mRNA and protein expression levels of CHCHD2 were significantly increased in eutopic and ectopic endometrium tissues compared with the normal endometrium tissues. The knockdown of CHCHD2 could cause mitochondrial dysfunction, including the opening of mitochondrial permeability transition pore, loss of mitochondrial membrane potential and the release of cytochrome c, and morphological damage. In addition, CHCHD2 down-expression could also lead to inhibition of cell proliferation, decrease of migration ability, and aggravation of mitochondrial-mediated apoptosis. Together, these findings suggest that increased expression of CHCHD2 in endometriotic tissues may contribute to the pathogenesis of endometriosis via regulating mitochondrial function and apoptosis, and CHCHD2 may be a potential target for interrupting the development of endometriosis., (© 2022. Society for Reproductive Investigation.)

Endometriosis is a benign tumor that affect 6-10% women of reproductive age. To date, it is suggested that the aberrant microRNA (miRNA) expressions play important roles in the pathogenesis of endometriosis. Reviewing the literature, we found nine overexpressed miRNAs, which were thoroughly investigated in the context of endometriotic tissues and cells. Most of the overexpressed miRNAs induced endometriosis-specific characteristics including inhibition of apoptosis and decidualization, upregulation of fibrogenesis, invasion, migration, cell proliferation, attachment to extracellular matrix, inflammation, and angiogenesis in the endometriotic cells. Then, we found that the downstream target molecules of these miRNAs, such as early growth response protein-1, extracellular signal-regulated kinase, matrix metallopeptidase 1, signal transducer and activator of transcription 3, cyclooxygenase-2, phosphoinositide 3-kinase, AKT, mammalian target of rapamycin, and vascular endothelial growth factor-A are promising for the therapeutic targets of endometriosis. Recent findings suggest that complex molecular mechanisms leading to development and progression of endometriosis by miRNAs may exist in endometriosis. The meticulous balance between tumorigenic miRNAs and tumoristatic miRNAs may destine the natural course and response to the surgical, medical, and hormonal treatments of this disease. Further investigations into endometriosis-associated miRNAs may elucidate the pathogenesis of endometriosis and help to develop novel therapeutics., (© 2021. The Author(s) under exclusive licence to The Japanese Society for Clinical Molecular Morphology.)

Autophagy is a conserved fundamental cellular process with a primary function of catabolizing harmful or surplus cellular contents such as protein aggregates, dysfunctional/long-lived organelles, intracellular pathogens, and storage nutrients. An increasing body of evidence reveals that basal autophagy is essential for maintaining endometrial homeostasis and mediating endometrial-specific functions, including menstrual cycle, embryo implantation, and decidualization. However, perturbed levels of autophagy can lead to severe endometrial pathologies, including endometriosis, endometrial hyperplasia, endometrial cancer, adenomyosis, and leiomyoma. This review highlights the most recent findings on the activity, regulation, and function of autophagy in endometrium physiology and pathology. Understanding the mechanistic roles of autophagy in endometrium homeostasis and disease is key to developing novel therapeutic strategies for endometrium-related infertility and malignancies., (© 2021. Society for Reproductive Investigation.)

The development of more efficacious, non-hormonal therapeutics for endometriosis is still an unmet medical need begging to be fulfilled. Growing evidence indicates that endometriotic lesions are wounds undergoing repeated tissue injury and repair, and, as such, platelets play an important role in lesional progression. Tetramethylpyrazine (TMP), a compound derived from a herb that has been used for thousands of years to combat "blood stasis" in traditional Chinese medicine, is a prescription drug in China for the treatment of cerebrovascular disorders. We tested the hypothesis that TMP can decelerate lesional progression through arresting epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), and fibrogenesis. We found in our in vitro experiments that TMP treatment suppresses platelet-induced EMT, FMT, cellular contractility, and collagen production in a concentration-dependent manner. We also showed that in a mouse model of endometriosis, treatment with TMP significantly reduced lesion weight and the extent of lesional fibrosis and improved hyperalgesia, mostly likely through the reduction of lesional aggregation of platelets and the lesional expression of markers of EMT, FMT, and fibrogenesis. In light of our results and in view of its excellent safety profiles, TMP appears to be a promising drug candidate for treating endometriosis., (© 2021. The Author(s).)

The cause of endometriosis, which is characterized by the existence of functional endometrial tissue outside the uterine cavity, is poorly understood. Seminal plasma (SP) is rich in multiple cytokines that may promote endometrial tissue survival. Here, we evaluated the effect of SP on growth of endometrial mesenchymal stem cells (MSCs) from women with endometriosis (E-MSCs) and women without endometriosis (NE-MSCs). Proliferation, cell foci formation, cell cycle progression, and growth marker expression of E- and NE-MSCs were promoted by SP. These effects may be mediated through activation of transforming growth factor beta 1 (TGF-β1), Akt, and p42/44 signaling, which enhances CDK2 and CDK6 expression and accelerates cell cycle progression. Xenografts exposed to SP exhibited a three-fold increase in volume and four-fold increase in weight after 14 days. Our findings demonstrate that TGF-β1 in SP may promote endometrial tissue survival which will allow us to understand the pathogenesis and develop novel approaches for prevention and therapies of endometriosis., (© 2021. Society for Reproductive Investigation.)

Endometriosis is a common, estrogen-dependent benign tumor that affect 3-10% women of reproductive age, and is characterized by the ectopic growth of endometrial tissue, which is found primarily in the rectovaginal septum, ovaries, and pelvic peritoneum. To date, accumulating evidence suggests that various epigenetic aberrations, including the expression of aberrant microRNAs (miRNAs), play definite roles in the pathogenesis of endometriosis. This review summarizes the recent findings on the aberrantly repressed miRNAs, as well as their potential roles regarding the pathogenesis of endometriosis., (© 2021. The Japanese Society for Clinical Molecular Morphology.)

The key relationship between Sampson's theory and the presence of mesenchymal stem cells in the menstrual flow (MenSCs), as well as the changes in post-transcriptional regulatory processes as actors in the etiopathogenesis of endometriosis, are poorly understood. No study to date has investigated the imbalance of miRNAs in MenSCs related to the disease. Thus, through literature and in silico analyses, we selected four predicted miRNAs as regulators of EGR1, SNAI1, NR4A1, NR4A2, ID1, LAMC3, and FOSB involved in pathways of apoptosis, angiogenesis, response to steroid hormones, migration, differentiation, and cell proliferation. These genes are frequently overexpressed in the endometriosis condition in our group studies. They were the trigger for the miRNAs search. Therefore, a case-control study was conducted with MenSCs of women with and without endometriosis (ten samples per group). Crossing information obtained from the STRING, PubMed, miRPathDB, miRWalk, and DIANA TOOLS databases, we chose to explore the expression of miR-21-5p, miR-100-5p, miR-143-3p, and miR-200b-3p by RT-qPCR. We found an upregulation of the miR-200b-3p in endometriosis MenSCs (P = 0.0207), with a 7.93-fold change (ratio of geometric means) compared to control. Overexpression of miR-200b has been associated with increased cell proliferation, stemness, and accentuated mesenchymal-epithelial transition process in eutopic endometrium of endometriosis. We believe that dysregulated miR-200b-3p may establish primary changes in the MenSCs, thus favoring tissue implantation at the ectopic site., (© 2022. Society for Reproductive Investigation.)

Endometriosis is a common gynecological disease associated with infertility, and it represents an economic burden worldwide. However, the molecular mechanisms underlying endometriosis development have not yet been fully elucidated. Here, we aimed to identify reliable key genes and the related regulatory network that may be involved in endometriosis. Differentially expressed genes (DEGs) were identified through integrated analysis of four expression datasets of endometriosis from Gene Expression Omnibus. Gene functional analysis and protein-protein interaction network construction were performed to reveal the potential function of DEGs. Subsequently, candidate hub genes were defined and validated in GSE105764 dataset, and the associated regulatory networks were constructed. Additionally, GSE120103 dataset was applied to identify the differential expression between the infertile and fertile groups of patients with stage IV endometriosis. Finally, real-time quantitative polymerase chain reaction analysis was performed to identify the differential expression of hub genes in the collected clinical specimens. Robust rank aggregation integrated analysis determined 158 DEGs. Epithelial cell differentiation was the most significantly enriched biological process, and leukocyte transendothelial migration was the most significantly enriched pathway. Eight hub genes including CLDN3, CLDN5, CLDN7, CLDN11, HOXC8, HOXC6, HOXB6, and HOXB7 were identified, and most of these were validated as abnormally expressed genes in both the infertile group and patients with endometriosis. Transcriptional factors and microRNAs related to these genes were identified. Altogether, our integrated analysis identified critical gene signatures, involved pathways, and regulatory networks, which could provide clinically significant insights into the molecular mechanisms underlying endometriosis and its related infertility., (© 2021. Society for Reproductive Investigation.)

Endometriosis is a gynecological disease affecting 6-10% of women of reproductive age. In addition to gynecologic symptoms, endometriosis is associated with various systemic effects, including inflammation, altered body weight, and behavioral changes. Previous murine studies demonstrate that endometriosis is causally inked to increased pain sensitization, behavioral changes, and low body mass index (BMI). One possible cellular target that may mediate some of these findings is the hypocretin/orexin neurons. This neuronal system plays a role in regulating wakefulness/sleep cycles, pain perception, and appetite. We hypothesize that endometriosis alters activity level of the hypocretin/orexin (Hcrt) neuronal system. Mice underwent endometriosis induction surgeries (endo) or sham surgeries (sham) for the development of the experimental model. Immunocytochemistry was performed on harvested samples from the lateral hypothalamus, and activation levels of Hcrt cells were examined by quantifying the expression of phosphorylation of cAMP-responsive element binding protein (CREB) in these cells after an acute stress in sham and endo mice. Mice with endometriosis had greater Hcrt neurons activation than sham mice. Mice with endometriosis fed with high fat diet showed a lower fat/body weight and fat/lean tissue ratio compared to mice without endometriosis. There was no significant difference in food intake between sham and endometriosis mice. These results demonstrate that endometriosis is associated with low body mass and increased hypocretin/orexin activity, which could be implicated in the behavioral changes and to differences in body composition., (© 2021. Society for Reproductive Investigation.)

Evidence to date supports regulatory T cell (Treg) alterations in endometriosis; however, the relationship remains unclear, and Tregs have not previously been investigated with respect to infertility in endometriosis. This prospective cross-sectional cohort study details circulating and endometrial tissue-specific disturbances in Tregs and broader gated populations in women of reproductive age with and without endometriosis (n = 57 and 29, respectively) using flow cytometry and immunohistochemistry. Participants were characterised by menstrual cycle phase, r-ASRM endometriosis disease stage and fertility status.In the endometrium of women with endometriosis, endometrial Tregs and CD4+ lymphocyte proportions did not change between the proliferative and secretory phases, while in women without the disease, they significantly decreased (p = 0.045 and p = 0.039, respectively). In women with endometriosis, endometrial Tregs were lower than in women without endometriosis overall (p = 0.050 as a proportion of all CD45+ immune cells). We have shown for the first time that proportions of CD4+ lymphocytes (p = 0.021), overall lymphocytes (p = 0.034) and non-granulocytes (p = 0.027) were significantly decreased in the endometrium of women with moderate-severe (r-ASRM stages III and IV) compared to minimal-mild (r-ASRM stages I and II) endometriosis. During the secretory phase, circulating Treg proportions were significantly increased in infertile compared to fertile women (p = 0.049). This study confirms differences in endometrial Tregs in women with endometriosis, with blunting of normal menstrual cyclical variations, reduced proportions during the proliferative phase and disease stage-specific relationships., (© 2021. Society for Reproductive Investigation.)

It is suggested that aberrantly expressed microRNAs are involved in the pathogenesis of endometriosis. Our previous study demonstrated that expression of the microRNA hsa-miR-199a-3p is attenuated in human endometriotic cyst stromal cells (ECSCs). The current study aimed to define the roles of hsa-miR-199a-3p in the development of endometriosis. ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometrioma and normal endometrial tissues, respectively. We evaluated the effect of transfected hsa-miR-199a-3p on the migration, invasion, and contractility of ECSCs using Transwell migration assays, in vitro wound healing assays, Transwell invasion assays, and collagen gel contraction assays. We also examined the downstream target of hsa-miR-199a-3p with an online public database search and luciferase reporter assay. Expression of hsa-miR-199a-3p in ECSCs was significantly lower than that in NESCs, whereas the expression of p21-activated kinase 4 (PAK4) mRNA was significantly higher. Transfection of hsa-miR-199a-3p inhibited the migration, invasion, and contractility of ECSCs via inhibition of PAK4 mRNA expression. PAK4 was confirmed to be the direct target of hsa-miR-199a-3p. Transfection of PAK4 small interfering RNA and the PAK4 inhibitor PF-3758309 also inhibited ECSC migration, invasion, and contractility. These findings suggest that hsa-miR-199a-3p may act as a tumor suppressor in endometriosis development. Attenuation of hsa-miR-199a-3p expression was favorable for ECSCs to acquire the highly invasive, motile, and contractile characteristics of endometriotic cells and is involved in the development of endometriosis. Accordingly, PAK4 inhibitors may be promising for the treatment of endometriosis., (© 2021. Society for Reproductive Investigation.)

Endometriosis is an estrogen-dependent disease. Several researches have reported the dysregulated circular RNAs (circRNAs) in endometriosis, whereas the functions of circRNAs are largely unknown. This study aims to explore the role and mechanism of circ&#95;0075503 in migration and invasion of eutopic endometrial stromal cells. 30 paired ectopic and eutopic endometrium tissues were collected from patients with endometriosis. And primary endometrial stromal cells (ESCs) were stimulated with estradiol (E2) to establish the in vitro cellular model of endometriosis. The levels of circ&#95;0075503, miR-15a-5p and Krüppel-like factor 12 (KLF12) were measured by quantitative reverse transcription polymerase chain reaction or western blot assays. Cell viability, migration and invasion were examined via 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, transwell assay or western blot assays. The target relationship between miR-15a-5p and circ&#95;0075503 or KLF12 was analyzed by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. Circ&#95;0075503 expression was elevated in ectopic endometrium and ectopic ESCs. Down-regulation of circ&#95;0075503 suppressed E2-induced promotion of cell viability, migration and invasion in eutopic ESCs. Circ&#95;0075503 could act as a sponge for miR-15a-5p, and KLF12 was targeted by miR-15a-5p. Inhibition of miR-15a-5p reversed the effects of circ&#95;0075503 knockdown on E2-treated ESCs migration and invasion. Besides, miR-15a-5p repressed E2-induced promotion effects on cell migration and invasion via targeting KLF12. Circ&#95;0075503 could regulate KLF12 expression by sponging miR-15a-5p. Knockdown of circ&#95;0075503 inhibited E2-induced enhancement of cell migration and invasion in eutopic ESCs by regulating miR-15a-5p/KLF12 axis, indicating a novel target for the treatment of endometriosis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The vitamin D receptor (VDR) and aryl hydrocarbon receptor (AHR) are two nuclear receptors that exert their effects by binding with ligands and forming a molecular complex. These complexes translocate to the nucleus and activate the expression of a series of genes which have a response element to VDR or AHR. Both receptors have been identified in the pathogenesis of endometriosis, a common disease characterized by the formation of endometrium-like tissue in ectopic zones. Despite numerous therapies, there is no definitive cure for endometriosis at the pharmacological level. Our study aims to describe the location and the expression of VDR and AHR at the protein level. For this purpose, an evaluation was performed using tissue from the three normal phases of the endometrium (proliferative, early, and late secretory) and in endometriosis by immunohistochemistry, using anti-VDR and anti-AHR antibodies. We demonstrate that in the nuclei of glandular cells in endometriosis, the expression of VDR and AHR is mutually exclusive-when the expression of one receptor is high, the other one is low-suggesting a possible target in the treatment of endometriosis. We also identify a significant change in the expression of glandular cytoplasmic AHR between the proliferative and late secretory endometrium., (© 2021. The Author(s).)

Cell membrane ion channels have important roles in cell migration during cancer development and metastasis. Although endometriosis is a benign gynecological disease, some migration and invasion characteristics of endometriosis are similar to those of cancer. However, only a few studies have examined cell membrane ion channels and their associations with endometriosis. This study aimed to investigate the effects of these ion channels on development of endometriosis. A total of 39 women who underwent laparoscopic ovarian cyst enucleation were included in the study population. Eutopic endometrium or ectopic endometrium tissues were obtained from each patient based on allocation to an endometriosis group (n=21) or a control group (n=18). Quantitative real-time PCR (qRT-PCR) and western blot analyses were performed to quantify NKCC1, NKCC2, and CLCN3 mRNA expression and protein concentrations. SiRNA transfection and migration assays of the endometrial stromal cells were performed to test the effects of the ion channels on the migration ability. The qRT-PCR and western blot analyses revealed significantly elevated mRNA expression and protein expression of NKCC1, NKCC2, and CLCN3 in the ectopic endometrial tissue from the patients with endometriosis (p < 0.05). Migration assay of siRNA transfected cells suggested a decreased migratory potential of the endometrial stromal cells (p < 0.001). The magnitudes of expression of NKCC1, NKCC2, and CLCN3 were positively correlated with endometrioma size. The increased expression of NKCC1, NKCC2, and CLCN3 in endometriosis offers opportunities to understand mechanisms of endometriosis and develop novel therapeutic approaches., (© 2021. Society for Reproductive Investigation.)

Endometriosis causes infertility and the alterations in endometrial receptivity. Pinopodia in eutopic endometrial epithelium may have significant implications in the endometriosis-associated infertility. The aim of this study is to ascertain whether the surgical interventions to remove endometrioid ovarian cysts (EOCs) can improve endometrial receptivity. The study included 172 patients of reproductive age with EOC, who underwent laparoscopic cystectomy. Aspiration endometrial biopsy was performed at 6 and 12 months after the surgery during the proliferation and secretion phases. Histopathology analysis included H&E staining and IHC. Morphometric studies were performed on endometrial biopsies collected during the proliferation phase of 28 patients, and the secretion phase of 12 patients. The expression of IHC markers for estrogen receptors (ER) and progesterone receptors (PR) and the percentage of cells containing pinopodia were determined. A significant increase in the ER and PR expression was observed in the epithelium during the "middle stage, proliferation phase" and in the stroma and glands during "middle stage, secretion phase". A delay in endometrial secretory transformation and statistically significant decrease in the number of pinopodia was observed on the apical surface of the cells. These structural and functional alterations were observed both at 6 and 12 months after cystectomy. The endometriosis-associated infertility after surgical intervention of EOC could be due to the extensive expression of ER and PR during the proliferation and secretion phases, as well as the delayed secretory transformation and impaired formation of pinopodia in the eutopic endometrium in the patients at 6 and 12 months after surgery., (© 2021. Society for Reproductive Investigation.)