Endometrial Research Articles

Assessment of the Role of Endometrial Receptivity Analysis in Enhancing Assisted Reproductive Technology Outcomes for Advanced-Age Patients.

In contemporary society, socially active women are increasingly planning their fertility for later in life. The fertility outcomes for advanced-age patients, even with egg donation, are often suboptimal due to endometrial aging. Recurrent implantation failure (RIF) is one of the core problems for assisted reproductive technology (ART), especially for advanced-age patients. High-quality, euploid embryos and synchronization between the embryonic stage and the uterine endometrial lining are crucial for positive outcomes. The study aims to improve ART outcomes with personalized embryo transfer (pET) according to endometrial receptivity analysis (ERA) in advanced-age patients with challenging reproductive histories, and RIF by utilizing, donor oocytes andpreimplantation genetic testing for aneuploidy (PGT-A) for embryo testing. A randomized, controlled observational follow-up study was conducted from2020 to 2023. After obtaining informed consent, 320 patients with RIF were selected. Patients were allocated into the study group and control group 1 based on consistent application of randomization principles, while control group 2 was selected separately. The study group included patients undergoing PGT-A and ERA, aged 35-45 years, with a mean age of 40.5±3.7 years. Control group 1 comprised patients undergoing PGT-A, aged 35-45 years, with a mean age of 40±4.2 years. Control group 2 consisted of patients undergoing PGT-A and ERA, aged less than 35 years, with a mean age of 31.6±2.2 years. Results suggestthat ERA may improve implantation and pregnancy outcomes in advanced-age patients, particularly those withRIFs. The pregnancy rate was significantly higher in the study group (77.9%), compared to control group 1 (57.6%) (p=0.0007), and no significant difference compared to control group 2(77.3%) (p=0.94). The implantation rate was higher in the study group (54.1%) than in control group 1 (39.4%) (p=0.0009), and there wasno significant difference between the study group and control group 2(50%, p=0.87). The live birth rate was also higher in the study group (71.3%), compared to control group 1 (39.4%) (p<0.0001). There were no significant differences between the study group and control group 2 (65.9%, p=0.50). pETguided by ERAsignificantly improves pregnancy, implantation, and live birth rates in advanced-age patients with challenging reproductive histories. pET providesART outcomes with no significant difference between advanced-age patients and younger patients with pET guided by ERA.

Adipokine Modulation in Endometrial Hyperplasia by Polyunsaturated Fatty Acids

Background Obesity is associated with a higher prevalence of endometrial hyperplasia, thereby increasing the risk of endometrial and ovarian cancers. The precise mechanisms linking obesity to endometrial hyperplasia remain unclear, but dysregulation of adipose tissue homeostasis is known to play a significant role. Hypertrophied adipocytes in obese individuals secrete various bioactive substances, including cytokines, growth factors, hormones, and metabolites. Additionally, hyperplastic adipocytes exhibit enhanced aromatase activity, leading to increased estrogen synthesis, which further promotes the development of endometrial hyperplasia. Purpose The purpose of this study is to explore the anti-inflammatory and anti-proliferative activities of the poly unsaturated fatty acids. Methodology An extensive literature survey has been performed to identify the role of adipokines and elevated endogenous estrogen levels in activating cell survival signaling pathways, such as PI3K/Akt/mTOR, MEK/ERK1, and JAK–STAT in endometrial cells and their possible role in Endometrial Hyperplasia. Further, the possible beneficial anti-inflammatory and anti-proliferative effects of polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA) were explored. Results Numerous studies suggest the beneficial role of dietary fats, such as EPA, DHA, and AA in modulating the growth of endometrium in obesity-induced endometrial hyperplasia. PUFAs can activate adenosine monophosphate-activated protein kinase (AMPK), which inhibits gluconeogenesis and lipogenesis. It also phosphorylates acetyl-CoA, leading to a decrease in malonyl-CoA, which inhibits mitochondrial CPT1. Additionally, AMPK activation promotes β-oxidation, and PPAR-γ mechanisms by down regulating the NF-kB pathway involved in endometrial hyperplasia. Conclusion This review sheds light on the potential of PUFAs in mitigating estrogen synthesis, adipokine secretion, and endogenous aromatase activity in obesity induced endometrial hyperplasia. Furthermore, it critically evaluates the role and mechanisms of PUFAs in attenuating obesity-associated endometrial hyperplasia and reducing the risk of ovarian cancer.

Statement of Retraction: miR-96-5p regulated TGF-β/SMAD signaling pathway and suppressed endometrial cell viability and migration via targeting TGFBR1

Statement of Retraction: miR-96-5p regulated TGF-β/SMAD signaling pathway and suppressed endometrial cell viability and migration via targeting TGFBR1

The association between endometrial polyps and insulin resistance from the expression of PI3K and AKT proteins perspective

BackgroundInsulin resistance (IR) induces hyperinsulinemia, which activates downstream signaling pathways such as the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway, ultimately leading to abnormal proliferation and apoptosis of endometrial cells. This is thought to be a key pathogenic mechanism underlying the development of endometrial polyps (EP). This study aims to investigate the relationship between IR and the development of EP, the expression levels of downstream signaling molecules, including PI3K and AKT, and related laboratory parameters were examined.MethodsA total of 100 patients who visited the gynecology outpatient clinic of Zhongda Hospital affiliated with Southeast University from May 2021 to March 2023 and were diagnosed with abnormal endometrial echoes by vaginal ultrasound and underwent hysteroscopic diagnostic curettage were enrolled in this study. General data and relevant hematological indicators were compared, and intraoperative specimens were obtained for pathological examination. Possible factors influencing the development of endometrial polyps were analyzed using Pearson correlation analysis and logistic regression analysis.ResultsIn terms of body mass index, waist circumference, fasting insulin, insulin resistance index, serum total testosterone, and free testosterone index, women of childbearing age in the endometrial polyp group had higher values than those in the non-polyp group, while sex hormone-binding globulin in the endometrial polyp group was lower than that in the non-polyp group, and the differences were statistically significant (P < 0.05). The expression scores and mRNA expression levels of PI3K and AKT proteins were higher in the EP group than in the non-EP group (p < 0.05). Pearson correlation analysis showed a positive correlation between HOMA-IR and the expression scores of PI3K and AKT proteins (p < 0.01).ConclusionsInsulin resistance and abnormal activation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway may be potential pathogenic mechanisms for the development of endometrial polyps.

New insights into molecular mechanisms underlying malignant transformation of endometriosis: BANCR promotes miR-612/CPNE3 pathway activity

Research questionDoes LncRNA BANCR promote the malignant transformation of endometriosis by activating the miR-612/CPNE3 pathway? DesignThe expression patterns of BANCR, miR-612 and CPNE3 in normal endometrium, eutopic endometrium from endometriosis, eutopic endometrium or malignant tissues from endometriosis-associated ovarian cancer. On the basis of primary normal endometrial stromal cells (NESC) and eutopic endometrial stromal cells (EESC), the regulatory relationships between BANCR, miR-612 and CPNE3, and the potential mechanisms that promote the malignant transformation of endometriosis, were elucidated in vitro and in vivo. ResultsThe expression levels of BANCR and CPNE3 were lowest in normal endometrium, significantly increased in eutopic endometrium (P < 0.05) and was significantly increased in eutopic endometrium (P < 0.05). During the malignant transformation of endometriosis, the expression levels of BANCR and CPNE3 were significantly upregulated (P < 0.05), whereas those of miR-612 were significantly downregulated (P < 0.05). miRNA-612 was found to target BANCR and CPNE3. The overexpression and knockdown of BANCR in NESC and EESC upregulated and downregulated the expression of CPNE3 and promoted or prevented cell proliferation and migration, respectively; these effects were reversed by miR-612 mimics and inhibitor. These changes were all statistically significant (P < 0.05). In-vivo experiments revealed that BANCR significantly increased the survival of subcutaneous endometrial cells by regulating miR-612/CPNE3 (P < 0.05). ConclusionThe expression of BANCR gradually increased with the progression of endometriosis during malignant transformation, and promoted the proliferation and migration of endometrial cells via the miR-612/CPNE3 pathway. BANCR may represent a novel target for monitoring the malignant transformation of endometriosis.

Restoration of functional endometrium in an intrauterine adhesion rat model with endometrial stromal cells transplantation

BackgroundIntrauterine adhesion (IUA) as a prevalent gynecological disease is developed from infection or trauma. However, therapeutic strategies to repair damaged endometrium are relatively limited. Emerging studies have shed light on the crucial role of endometrial stromal cells (EnSCs) in the process of uterine endometrial regeneration. EnSCs isolated from the uterine endometrium have similar characteristics to mesenchymal stem cells (MSCs). However, it is still unknown whether EnSCs could be used as donor cells to treat IUA. The aim of this study was to evaluate the potential efficacy of EnSCs in treating rat IUA.MethodsHuman EnSCs were isolated from the endometrial tissue of healthy female donors and subjected to extensive expansion and culture in vitro. Immunofluorescence, flow cytometry, cell proliferation assay, trilineage differentiation experiment, and decidualization assay were used to characterize the biological properties of EnSCs. We evaluated the immunoregulatory potential of EnSCs by analyzing their secreted cytokines and conducting bulk RNA sequencing after IFN-γ treatment. After EnSCs were transplanted into the uterine muscle layer in IUA rats, their therapeutic effects and underlying mechanisms were analyzed using histological analysis, Q-PCR, fertility and pregnancy outcome assay, and transcriptome analysis.ResultsWe successfully isolated EnSCs from the endometrium of human donors and largely expanded in vitro. EnSCs exhibited characteristics of mesenchymal stem cells and retained responsiveness to sex hormones. Following IFN-γ stimulation, EnSCs upregulated the anti-inflammatory cytokines and activated immunosuppressive molecules. Xenogeneic transplantation of EnSCs successfully repaired injured endometrium and significantly restored the pregnancy rate in IUA rats. Mechanistically, the therapeutic effects of EnSCs on IUA endometrium functioned through anti-inflammation, anti-fibrosis and the secretion of regeneration factor.ConclusionsDue to their large expansion ability, immunoregulatory properties, and great potential in treating IUA, EnSCs, as a valuable source of donor cells, could offer a potential treatment avenue for injury-induced IUA.

Establishment of an Embryo Implantation Model In Vitro.

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa)preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-β-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.

In Vitro Human Endometrial Cells and In Vivo Rat Model Studies Suggest That Ulipristal Acetate Impacts Endometrial Compatibility for Embryo Implantation

Ulipristal acetate (UPA) and levonorgestrel are used as emergency hormonal contraceptives. Although both are highly effective in preventing pregnancy, UPA shows efficacy even when taken up to 120 h after unprotected sexual intercourse. To investigate whether the mechanism of UPA's contraceptive action involves post-fertilization effects. In vitro and in vivo studies using cultured human endometrial cells and a pre-clinical rat model. Endometrial cells treated with UPA showed changes in the expression of receptivity gene markers and a significant decrease in trophoblast spheroids attached to the cultured cells. In addition, administration of UPA to female unmated rats decreased the expression of implantation-related genes in the endometrium and inhibited the number of implantation sites in the mated group compared to the non-treated group. These results support that UPA as an emergency contraceptive might have post-fertilization effects that may affect embryo implantation.

Androgen-responsive FOXP4 is a target for endometrial carcinoma

Although low estrogen is considered to suppress uterine endometrial carcinoma, the most cases occur in the postmenopausal stage. After menopause, the production of androgen level also declines. Therefore, to resolve the above enigma, we hypothesize that the postmenopausal decline of androgen is a trigger of its progression. In the present study, to validate this hypothesis, we examine the pathological roles of androgen/AR by analyzing clinical data, culturing endometrioid cancer cell lines, and using murine models. Clinical data show that androgen receptor (AR) expression and serum dihydrotestosterone (DHT) are associated with lower disease-free survival (DFS). DHT suppresses malignant behaviors in AR-transfected human endometrial cancer cells (ECC). In ovariectomized Ptenff/PRcre/+ mice, DHT decreases the proliferation of spontaneously developed murine ECC. In AR-transfected human ECC and Ptenff/PRcre/+ mice, DHT suppresses FOXP4 expression. FOXP4-overexpressed human ECC increases, while FOXP4-knocked-down ECC shows decreased malignant behaviors. DHT/AR-mediated ECC suppression is restored by FOXP4 overexpression. The high FOXP4 expression is significantly correlated with low postoperative DFS. These findings indicate that the androgen/AR system suppresses the malignant activity of endometrial carcinoma and that downstream FOXP4 is another target molecule. These findings will also impact developments in clinical approaches to elderly health.

Endometrial epithelial cell organoids as tools for studying the CD39 family of enzymes and for validating enzyme inhibitors.

Extracellular adenosine triphosphate (ATP) conducts a complex dynamic system of broadly represented cell signaling. Ectonucleotidases are the enzymes with nucleotide hydrolytic ability that regulate ATP levels in physiological and pathological conditions, thus playing a key role in the so-called purinergic signaling. Altered ectonucleotidase expression has been reported in cancer, and the ectonucleoside triphosphate diphosphohydrolase (NTPDase) family of enzymes, with its best-known form NTPDase1 (CD39), is targeted in cancer immunotherapy. The tandem of enzymes CD39-CD73 is responsible for the generation of immunosuppressive adenosine in the tumor microenvironment, and inhibition strategies are of great interest. Organoids have emerged as very convenient models for the study of tumors since they are three-dimensional cultures that retain many of the features of tissue. The present study aims to contribute to improving the methodology and the molecular tools needed for the study of ectonucleotidases in healthy and disease conditions. The study, performed in an endometrial cancer cell model, could be extended to other types of tumors and pathologies in which the purinergic system is involved. We generated organoids from endometrial cancer cells overexpressing NTPDase2 (CD39L1) and NTPDase3 (CD39L3) as fusion proteins with EGFP, and we performed functional assays by adapting in situ cytochemistry protocols. This allowed us to simultaneously detect enzyme activity and protein expression and to demonstrate that organoids can be used to test ectonucleotidase inhibitors-a result that can be used to develop new cancer treatment options.

USP22 is required for human endometrial stromal cell proliferation and decidualization by deubiquitinating FoxM1

Despite significant advances in assisted reproductive technology (ART), recurrent implantation failure (RIF) still occurs in some patients. Poor endometrial receptivity and abnormal human endometrial stromal cell (HESC) proliferation and decidualization have been identified as the major causes. Ubiquitin-specific protease 22 (USP22) has been reported to participate in the decidualization of endometrial stromal cells in mice. However, the role of USP22 in HESC function and RIF development remains unknown. In this study, clinical endometrial tissue samples were gathered to investigate the involvement of USP22 in RIF, and HESCs were utilized to examine the molecular mechanisms of USP22 and Forkhead box M1 (FoxM1). The findings indicated a high expression of USP22 in the secretory phase of the endometrium. Knockdown of USP22 led to a notable reduction in the proliferation and decidualization of HESCs, along with a decrease in FoxM1 expression, while overexpression of USP22 yielded opposite results. Furthermore, USP22 was found to deubiquitinate FoxM1 in HESCs. Moreover, both USP22 and FoxM1 were downregulated in the endometria of patients with RIF. In conclusion, these results suggest that USP22 may have a significant impact on HESCs proliferation and decidualization through its interaction with FoxM1, potentially contributing to the underlying mechanisms of RIF pathogenesis.

Overview of crosstalk between stromal and epithelial cells in the pathogenesis of adenomyosis and shared features with deep endometriotic nodules.

Since the first description of adenomyosis more than 150 years ago, multiple hypotheses have attempted to explain its pathogenesis. Indeed, research over recent years has greatly enhanced our knowledge of the underlying causes. This has opened up avenues for the development of strategies for both disease prevention and treatment of its main symptoms, such as pelvic pain, heavy menstrual bleeding, and infertility. However, the current means are still largely ineffective, so it is vital that we shed light on the pathways involved. Dysregulated mechanisms and aberrant protein expression have been identified as contributing factors in interactions between endometrial epithelial and stromal cells, ultimately leading to the growth of adenomyotic lesions. These include collective cell migration, epithelial-to-mesenchymal transition, hormonal influence, and signaling from non-coding RNAs and extracellular vesicles. We provide a concise summary of the latest insights into the crosstalk between glands and stroma in ectopic adenomyotic lesion formation. While there is an abundance of literature on similarities between adenomyosis and deep endometriosis, there are insufficient data on the cytochemical, molecular, and pathogenetic mechanisms of these two disorders. However, various shared features, including alterations of cell adhesion molecules, abnormal hormone regulation, and the presence of cancer-driving mutations and epigenetic modifications, have been identified. Nevertheless, the pathogenic mechanisms that contribute to the cause and development of these enigmatic diseases have not been fully elucidated yet.

Interleukin-1β induces and accelerates human endometrial stromal cell senescence and impairs decidualization via the c-Jun N-terminal kinase pathway

As the mean age of first-time mothers increases in the industrialized world, inquiries into causes of human reproductive senescence have followed. Rates of ovulatory dysfunction and oocyte aneuploidy parallel chronological age, but poor reproductive outcomes in women older than 35 years are also attributed to endometrial senescence. The current studies, using primary human endometrial stromal cell (ESC) cultures as an in vitro model for endometrial aging, characterize the proinflammatory cytokine, IL-1β-mediated and passage number-dependent effects on ESC phenotype. ESC senescence was accelerated by incubation with IL-1β, which was monitored by RNA sequencing, ELISA, immunocytochemistry and Western blotting. Senescence associated secreted phenotype (SASP) proteins, IL-1β, IL-6, IL-8, TNF-α, MMP3, CCL2, CCL5, and other senescence-associated biomarkers of DNA damage (p16, p21, HMGB1, phospho-γ-histone 2 A.X) were noted to increase directly in response to 0.1 nM IL-1β stimulation. Production of the corresponding SASP proteins increased further following extended cell passage. Using enzyme inhibitors and siRNA interference, these effects of IL-1β were found to be mediated via the c-Jun N-terminal kinase (JNK) signaling pathway. Hormone-induced ESC decidualization, classical morphological and biochemical endocrine responses to estradiol, progesterone and cAMP stimulation (prolactin, IGFBP-1, IL-11 and VEGF), were attenuated pari passu with prolonged ESC passaging. The kinetics of differentiation responses varied in a biomarker-specific manner, with IGFBP-1 and VEGF secretion showing the largest and smallest reductions, with respect to cell passage number. ESC hormone responsiveness was most robust when limited to the first six cell passages. Hence, investigation of ESC cultures as a decidualization model should respect this limitation of cell aging. The results support the hypotheses that “inflammaging” contributes to endometrial senescence, disruption of decidualization and impairment of fecundity. IL-1β and the JNK signaling pathway are pathogenetic targets amenable to pharmacological correction or mitigation with the potential to reduce endometrial stromal senescence and enhance uterine receptivity.

Estrogen-induced chromatin looping changes identify a subset of functional regulatory elements.

Transcriptional enhancers can regulate individual or multiple genes through long-range three-dimensional (3D) genome interactions, and these interactions are commonly altered in cancer. Yet, the functional relationship between changes in 3D interactions associated with regulatory regions and differential gene expression appears context-dependent. In this study, we used HiChiP to capture changes in 3D genome interactions between active regulatory regions of endometrial cancer cells in response to estrogen treatment and uncovered significant differential long-range interactions that are strongly enriched for estrogen receptor α (ER) bound sites (ERBS). The ERBS anchoring differential loops with either a gene's promoter or distal regions were correlated with larger transcriptional responses to estrogen compared to ERBS not involved in differential interactions. To functionally test this observation, CRISPR-based Enhancer-i was used to deactivate specific ERBS, which revealed a wide range of effects on the transcriptional response to estrogen. However, these effects are only subtly and not significantly stronger for ERBS in differential loops. In addition, we observed an enrichment of 3D interactions between the promoters of estrogen up-regulated genes and found that looped promoters can work together cooperatively. Overall, our work suggests that changes in 3D genome structure upon estrogen treatment identify some functionally important regulatory regions; however, these changes aren't required for a transcriptional response to E2 in endometrial cancer cells.

Blockage of the NLRP3 inflammasome by MCC950 inhibits migration and invasion in adenomyosis

Does the NOD-like receptor protein 3 (NLRP3) inflammasome have an effect in adenomyosis? Fresh-frozen endometrial tissues and paraffin specimens were obtained from endometrial tissues from patients with adenomyosis and controls. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were applied to assess expression of the NLRP3 inflammasome components. Primary eutopic endometrial stromal cells were isolated from the uteri of patients with adenomyosis. After NLRP3 was knocked down using small interfering RNA, proliferation, invasion and epithelial-mesenchymal transition (EMT) were evaluated using EdU, CCK8, transwell assays and western blot. Importantly, a mouse model of adenomyosis was established to evaluate the effects of the NLRP3 inhibitor MCC950 on the formation of adenomyosis. Expression of the NLRP3 inflammasome components was elevated in the ectopic or eutopic endometrium of patients with adenomyosis. NLRP3 knockdown inhibited migration, invasion and EMT in endometrial cells and primary endometrial cells (P < 0.0001). MCC950, which blocks the NLRP3 inflammasome, reduced migration and invasion of endometrial cells (P < 0.01) and primary endometrial cells (P < 0.0001) considerably. Importantly, in the mouse model of adenomyosis, MCC950 had a mitigating effect on the severity of adenomyosis (P < 0.01). NLRP3 was found to enhance migration, invasion and EMT of human endometrial cells in adenomyosis. Notably, the NLRP3 inhibitor MCC950 reduced migration and invasion of endometrial cells effectively. Furthermore, in the mouse model of adenomyosis, MCC950 exhibited a therapeutic effect by alleviating the severity of adenomyosis.

Chromatin modifiers in endometriosis pathogenesis

Introduction. It was revealed that various epigenetic abnormalities may play an important role in the endometriosis pathogenesis. The regulation of chromatin structure is carried out mainly by chromatin modifiers (CMs), which stimulate generation of genomic regions with different functional structures and thus change the patterns or levels of gene expression by exerting expected biological functions and causing epigenetic changes.Aim: to consider СМs role in endometriosis pathogenesis and their regulation mechanism assessing current publications.Materials and Methods. The search was conducted in the databases PubMed, Scopus, Web of Science, Google Scholar and eLibrary. Keywords and phrases in Russian and English related to the research topic were used as follows: "endometriosis", "chromatin modifiers", "histone acetylation", "DNA methylation", "microRNA", "endometriosis", "chromatin modifiers", "histone acetylation", "DNA methylation", "microRNA". The evaluation of articles was carried out in accordance with PRISMA recommendations.Results. Chromatin modifiers control differentiation, growth and development, aging and cell death by interacting with various functional chromatin elements. They can cause abnormal gene expression by regulating chromatin structure affecting emergence and development of endometriosis. DNA methylation determines cell types, controls gene expression and genome stability. Abnormal DNA methylation in gene promoter regions necessary for normal endometrial response affects endometriosis development. DNA methyltransferase (DNMT) inhibitors reduce the methylation of human homeobox A10 (HOXA10) and progesterone receptor (PR) genes and potentiate their expression in endometrial cells, improving endometrial susceptibility and inhibiting cell cycle progression. Abnormal histone modifications in endometrial cells may facilitate or hinder the access of transcription mechanisms to chromatin DNA. Histone deacetylase inhibitors effectively eliminate the effects of abnormal histone modifications in endometriosis cells and prevent endometriosis progression. The expression of non-coding RNAs and chromatin remodeling complexes also alters chromatin structure being involved in arising endometriosis and is associated with infertility by promoting proliferation, invasion and migration of endometriod cells.Conclusion. Chromatin modifiers play a key role in developing endometriosis by controlling gene expression and chromatin structure. Understanding underlying mechanisms provides valuable information for diagnostics and development of new approaches to treat endometriosis.

The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

ObjectivesInterleukin 33 (IL-33) is a crucial inflammatory factor that functions as an alarm signal in endometriosis (EMs). Epithelial-mesenchymal transition (EMT), a process related to inflammatory signals, intracellular reactive oxygen species (ROS) production, and lipid peroxidation, have been proposed as potential mechanisms that contribute to the development and progression of EMs. IL-33 is highly upregulated in the ectopic milieu. Moreover, ectopic endometrial cells constitutively express interleukin-33 receptor ST2 (IL-33R). However, the role of IL-33/ST2 in the EMT of EMs remains largely unknown. In this study, we aimed to mechanistically determine the role of IL-33/ST2 in EMs-associated fibrosis.Materials and methodsWe established a non-lethal oxidative stress model to explore the conditions that trigger IL-33 induction. We performed α-smooth muscle actin (α-SMA) protein detection, cell counting kit-8 (CCK-8) assays, and scratch assays to analyze the impact of IL-33 on primary endometrial stromal cells (ESCs) proliferation and invasion. Clinical samples from patients with or without EMs were subjected to immunohistochemical (IHC) and and immunofluorescence(IF) staining to assess the clinical relevance of IL-33 receptor ST2 and EMT-related proteins. Furthermore, we used the ectopic human endometrial epithelial cell line 12Z and normal human epithelial cell line EEC to evaluate the effects of IL-33 on Wnt/β-catenin signaling. The effect of IL-33 on EMT-associated fibrosis was validated in vivo by intraperitoneal injections of IL-33 and antiST2.ResultsWe observed that ectopic milieu, characterized by ROS, TGF-β1, and high level of estrogen, triggers the secretion of IL-33 from ectopic ESCs. Ectopic endometrial lesions exhibited higher level of fibrotic characteristics and ST2 expression than that in the normal endometrium. Exogenous recombinant human (rhIL-33) enhanced ESC migration and survival. Similarly, 12Z cells displayed a higher degree of EMT characteristics with elevated expression of CCN4 and Fra-1, downstream target genes of the WNT/β-catenin pathway, than that observed in EECs. Conversely, blocking IL-33 with neutralizing antibodies, knocking down ST2 or β-catenin with siRNA, and β-catenin dephosphorylation abolished its effects on EMT promotion. In vivo validation demonstrated that IL-33 significantly promotes EMs-related fibrosis through the activation of Wnt/β-catenin signaling.ConclusionOur data strongly support the vital role of the IL-33/ST2 pathway in EMs-associated fibrosis and emphasize the importance of the EMT in the pathophysiology of fibrosis. Targeting the IL-33/ST2/Wnt/β-catenin axis may hold promise as a feasible therapeutic approach for controlling fibrosis in EMs.

Abstract A021: Hunting for synthetic lethal partners in DNA damage response-altered endometrial cancer

Abstract DNA damage response (DDR) genes are frequently altered in cancer, which may lead to protein loss-of-function (LoF) and result in particular DDR defects that can be therapeutically targeted via the concept of synthetic lethality (SL). There is a high prevalence of DDR alterations in endometrial cancer, a gynecologic malignancy with increased incidence and mortality in recent decades, and an unmet clinical need for a wider range of treatment options to reduce adverse effects and combat therapy resistance. In this study, a large-scale, agnostic bioinformatic screening approach was employed to discover novel potential SL targets specific to DDR LoF biomarkers in endometrial cancer for subsequent experimental validation in vitro. SL analysis was performed with the in-house Target / Biomarker / Lineage (TBL) bioinformatic pipeline (Division of Cancer Therapeutics, The Institute of Cancer Research) using datasets obtained from the Cancer Dependency Map (DepMap). The TBL pipeline statistically analyzed cancer cell line target gene dependency probabilities, inferred from Project Achilles and Project Score genome-wide CRISPR-Cas9 dropout screens, and integrated this with mutation and copy number variation genomic datasets. Potential interactions were filtered for target-biomarker mutual exclusivity using patient data from The Cancer Genome Atlas (TCGA). The ability of the TBL pipeline to identify known SL interactions was demonstrated through in silico identification of ARID1B/ARID1A in ovarian cancer and SMARCA2/SMARCA4 in lung cancer as significant hits. In addition, the identification of WRN/ARID1A as a significant hit in uterine cancer, given that ARID1A loss has been significantly associated with microsatellite instability (MSI) in endometrial cancer, is consistent with previous findings of WRN helicase as a SL partner with MSI in endometrial cancer. Analysis and downstream refinement of endometrial cancer significant hits led to the identification of seven final candidate target-biomarker gene pairs. These putative SL partners comprise novel potential endometrial cancer targets, with various functions ranging from nucleotide biosynthesis to epigenetic remodeling, and include novel potential and established LoF biomarkers relevant to endometrial cancer biology, namely DNA mismatch repair. In vitro validation of the final candidates by genetic or pharmacologic approaches to assess target gene dependency in endometrial cancer cell lines with altered or wildtype biomarker status will be discussed. These experiments will provide key experimental data to validate our bioinformatic approach to SL target-biomarker discovery, and may identify novel SL partners involving DDR LoF alterations in endometrial cancer that could lead to the development of new molecular targeted therapies and improved treatment options for this patient subpopulation. Citation Format: Julie Zhou, Konstantinos Mitsopoulos, Olivia Rossanese. Hunting for synthetic lethal partners in DNA damage response-altered endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A021.

Dysregulated RNA editing of EIF2AK2 in polycystic ovary syndrome: clinical relevance and functional implications

BackgroundPolycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive ages. Our previous study has implicated a possible link between RNA editing and PCOS, yet the actual role of RNA editing, its association with clinical features, and the underlying mechanisms remain unclear.MethodsTen RNA-Seq datasets containing 269 samples of multiple tissue types, including granulosa cells, T helper cells, placenta, oocyte, endometrial stromal cells, endometrium, and adipose tissues, were retrieved from public databases. Peripheral blood samples were collected from twelve PCOS and ten controls and subjected to RNA-Seq. Transcriptome-wide RNA-Seq data analysis was conducted to identify differential RNA editing (DRE) between PCOS and controls. The functional significance of DRE was evaluated by luciferase reporter assays and overexpression in human HEK293T cells. Dehydroepiandrosterone and lipopolysaccharide were used to stimulate human KGN granulosa cells to evaluate gene expression.ResultsRNA editing dysregulations across multiple tissues were found to be associated with PCOS in public datasets. Peripheral blood transcriptome analysis revealed 798 DRE events associated with PCOS. Through weighted gene co-expression network analysis, our results revealed a set of hub DRE events in PCOS blood. A DRE event in the eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2:chr2:37,100,559) was associated with PCOS clinical features such as luteinizing hormone (LH) and the ratio of LH over follicle-stimulating hormone. Luciferase assays, overexpression, and knockout of RNA editing enzyme adenosine deaminase RNA specific (ADAR) showed that the ADAR-mediated editing cis-regulated EIF2AK2 expression. EIAF2AK2 showed a higher expression after dehydroepiandrosterone and lipopolysaccharide stimulation, triggering changes in the downstrean MAPK pathway.ConclusionsOur study presented the first evidence of cross-tissue RNA editing dysregulation in PCOS and its clinical associations. The dysregulation of RNA editing mediated by ADAR and the disrupted target EIF2AK2 may contribute to PCOS development via the MPAK pathway, underlining such epigenetic mechanisms in the disease.

Chronic endometritis. Local prooxidant, antioxidant and immunometabolic disorders. An open-label, prospective, randomized study

Aim. To determine local features of lipoperoxidation processes, antioxidant defense, state of matrix metalloproteinases in chronic endometritis. Materials and methods. Eighty women aged 29.33±6.71 (min-max [24–36]) with histologically and immunohistochemically confirmed chronic endometritis and a history of reproductive losses were examined (group 1). The control group consisted of 30 patients without chronic endometritis confirmed by the results of a histochemical study and no history of reproductive losses, suffering from infertility of tubal-peritoneal origin at the age of 30.11±6.9 years (min-max [24–37]; group 2). All 130 patients underwent transvaginal echography of the pelvic organs, and histological and immunohistochemical examination of endometrial samples was performed. The activity of lipid peroxidation factors was analyzed: primary, secondary and final products of lipid peroxidation in heptane and isopropanolol fractions of uterine cavity aspirate, enzymes of the antioxidant system: superoxide dismutase, catalase, glutathione peroxidase, deoxyribonuclease I (DNase), by spectrophotometric method, remodeling factors – matrix metalloproteinases MMP-2, -9 by flow fluorimetry. Results. In patients of group 1, according to transvaginal echography of the pelvic organs, M-echo was 6.29±1.22 mm, in group 2 – 9.10±1.15 mm. In chronic endometritis, disruption of pro-oxidant and activity of antioxidant factors in the endometrium in women with chronic endometritis and a history of reproductive losses. In the heptane fraction of the phenolic extract, the concentration of primary products of lipid peroxidation (diene conjugates) was 12.98% higher than in the control, the content of secondary products of lipid peroxidation (ketodienes) was higher by 15.45%, conjugated trienes by 18.67% higher than the values in the control group, which indicates increased lipid peroxidation of endometrial cell membranes during CE. In the isopropanol phase, the products of lipid peroxidation of phospholipid acyl residues are detected. In the isopropanol fraction of the lipid extract, the amount of diene conjugates was higher, ketodienes by 24.32% and conjugated trienes by 26.14%. An increase in the activity of the enzymes superoxide dismutase by 23.98%, catalase by 14.34%, glutathione peroxidase by 12.39%, and DNase by 2.9 times was recorded compared to the indicators of group 2. Conclusion. Chronic endometritis is accompanied by changes in the system of pro-oxidant and antioxidant factors, the content of tissue remodeling products. The pathogenetic changes underlying oxidative stress in the endometrium may be increased leukocyte infiltration, imbalance of primary, secondary and end products of lipid peroxidation, enzymes superoxide dismutase, catalase, glutathione peroxidase, deoxyribonuclease I (DNase), expression of matrix metalloproteinases-2, -9.