Transcriptome analysis of eutopic endometrium in adenomyosis after GnRH agonist treatment

Background Adenomyosis is a chronic gynecological disease characterized by invasion of the uterine endometrium into the muscle layer. In assisted reproductive technology (ART), gonadotropin-releasing hormone agonist (GnRHa) is often used to improve pregnancy rates in patients with adenomyosis, but the underlying mechanisms are poorly understood. Methods Eutopic endometrial specimens were collected from patients with adenomyosis before and after GnRHa treatment in the midsecretory phase. RNA sequencing (RNA-Seq) of these specimens was performed for transcriptome analysis. The differentially expressed genes (DEGs) of interest were confirmed by real-time PCR and immunohistochemistry. Results A total of 132 DEGs were identified in the endometrium of patients with adenomyosis after GnRHa treatment compared with the control group. Bioinformatics analysis predicted that immune system-associated signal transduction changed significantly after GnRHa treatment. Chemokine (C-C motif) ligand 21 (CCL21) was found to be highly expressed in the eutopic endometrium after GnRHa treatment, which may be involved in the improvement of endometrial receptivity in adenomyosis. Conclusion This study suggests that molecular regulation related to immune system-associated signal transduction is an important mechanism of GnRHa treatment in adenomyosis. Immunoreactive CCL21 is thought to regulate inflammatory events and participate in endometrial receptivity in adenomyosis.

Dysregulation of X-Ray Repair Cross Complementing 4 Expression in the Eutopic Endometrium of Women with Endometriosis

Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of double-stranded DNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Towards this, we focused on the X-Ray Repair Cross-Complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (p<0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the GEO database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in-vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AFX and 8-hydroxy-2’-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (ThESCs) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AFX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.

N-Acyl Dopamines Induce Apoptosis in Endometrial Stromal Cells from Patients with Endometriosis

Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N-acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N-arachidonoyldopamine, N-docosahexaenoyldopamine, and N-oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.

The expression of BECN1, LC3B, and BCL2 genes in eutopic endometrium of patients with adenomyosis: A cross-sectional study

Introduction: Both autophagy and apoptosis play a role in the cyclic remodeling of the endometrium. The abnormal regulation of genes and signaling pathways in the eutopic endometrium plays a role in the abnormal migration and implantation in adenomyosis. Objective: The present study investigates the mRNA expression of autophagy and apoptosis-related genes BECN1, LC3B, and BCL2 in the eutopic endometrium of patients with adenomyosis compared with healthy premenopausal women. Materials and methods: The present work was a cross-sectional study conducted between July 2018 and April 2019. The participants were 32 premenopausal women who attended the surgery for adenomyosis and other benign gynecological conditions. The participants were divided into two groups, with 16 women in the adenomyosis group and 16 healthy women in the control group. Endometrial tissues were collected during the proliferative menstrual phase for a quantitative real-time polymerase chain reaction. Results: The mRNA expression of BECN1, LC3B, and BCL2 were normalized by geometric mean mRNA expression of actin and GAPDH. There was no significant difference in mRNA expression for all three genes when comparing the control and adenomyosis groups. Conclusions: The mRNA expressions of autophagy-related genes BECN1 and LC3B and anti-apoptosis-related gene BCL2 were not significantly different in the eutopic endometrium of patients with adenomyosis compared with healthy premenopausal women during the proliferative menstrual phase.

Mesenchymal Stromal Cells Isolated from Ectopic but Not Eutopic Endometrium Display Pronounced Immunomodulatory Activity In Vitro

A comparative analysis of the cell surface markers and immunological properties of cell cultures originating from normal endometrium and endometrioid heterotopias of women with extragenital endometriosis was carried out. Both types of cell cultures expressed surface molecules typical of mesenchymal stromal cells and did not express hematopoietic and epithelial markers. Despite similar phenotype, the mesenchymal stromal cells derived from the two sources had different immunomodulation capacities: the cells of endometrioid heterotopias but not eutopic endometrium could suppress dendritic cell differentiation from monocytes as well as lymphocyte proliferation in allogeneic co-cultures. A comparative multiplex analysis of the secretomes revealed a significant increase in the secretion of pro-inflammatory mediators, including IL6, IFN-γ, and several chemokines associated with inflammation by the stromal cells of ectopic lesions. The results demonstrate that the stromal cells of endometrioid heterotopias display enhanced pro-inflammatory and immunosuppressive activities, which most likely impact the pathogenesis and progression of the disease.

Expression of BDNF and TrKB in Patients With Endometriosis and Their Correlation With Dysmenorrhea

Background: Brain-derived neurotrophic factor (BDNF) has been recognized as a regulator in the formation and maintenance of chronic pain in various chronic disorders. BDNF together with its high-affinity tyrosine kinase type B (TrkB) receptor were found to be extensively expressed in mammalian female reproductive system. However, BDNF and TrkB expression in different stages of endometriosis, and the correlation between their expression in ectopic lesions and endometriosis pain remains unclear.Methods: This study enrolled sixty-two women underwent laparoscopic surgery. Forty-six women diagnosed as ovarian endometrioma, were recruited in the study group. Sixteen women diagnosed as ovarian benign tumors were recruited in the control group. Samples from eutopic endometrium and ovarian endometriotic lesions were obtained at laparoscopic surgery. The message RNA (mRNA) level of BDNF and TrKB was detected by real-time PCR, while the protein level was detected by immunohistochemical staining for eutopic and ectopic endometrium in both groups. Dysmenorrhea was assessed by the visual analogue scale (VAS) before the surgery.Results: The expression of BDNF and TrKB were higher in ovarian endometriotic lesions than those in eutopic endometrium and normal endometrium (P<0.05), and there was no cyclical change. While their expression in eutopic endometrium were higher than those in the normal endometrium (P<0.05). The expression of BDNF and TrKB in ovarian endometriotic lesions stage IV were higher than those in stage III and II (P<0.05). Their expression in stage III were higher than those in stage II but there were no significance (P>0.05). Furthermore, the correlation between the mRNA expression of BDNF, TrKB in eutopic endometrium, and dysmenorrhea VAS score revealed that r=0.52 and 0.56, respectively (P<0.05). The correlation between BDNF and TrKB in both eutopic and ectopic endometrium were revealed that r=0.82 and 0.66, respectively (P<0.05).Conclusions: BDNF and TrKB may play essential roles in promoting disease progression during the development of endometriosis, and are closely related to dysmenorrhea caused by endometriosis.

Single cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues.

Endometriosis is characterized by growth of endometrial-like tissue outside of the uterus affecting many women in their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, limiting diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared to matched eutopic endometrium, control endometrium, and organoids derived from these tissues, generating data on over 100,000 cells across 12 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell unique to the peritoneal lesions with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesions microenvironments, and a novel progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment representing the first comprehensive cell atlas of the disease, essential information for advancing therapeutics and diagnostics.

Single cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues.

Endometriosis is characterized by growth of endometrial-like tissue outside of the uterus affecting many women in their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, limiting diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared to matched eutopic endometrium, control endometrium, and organoids derived from these tissues, generating data on over 100,000 cells across 12 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell unique to the peritoneal lesions with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesions microenvironments, and a novel progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment representing the first comprehensive cell atlas of the disease, essential information for advancing therapeutics and diagnostics.

Low Expression of STING Promoted Endometrial Stromal Cells Invasion and Migration Via STING/IRF3/IFNb1 Pathway in Endometriosis Eutopic Endometrium

Background: Recent studies have confirmed that endometriosis is a chronic inflammatory disease. In our previous work, we found that STING (stimulator of interferon genes) was differentially expressed in eutopic endometrium and controlled endometrium by proteomics.Method: we used the 11 pairs of samples to verify STING expression by WB and IHC experiments. We detected cells proliferation by EdU assays, cells invasion and migration by Transwell assays. The effect of signaling pathway in HESC was detected by WB and Elisa expreriments.Results: STING was significantly lower expressed in eutopic endometrium of endometriosis, while IHC results showed that STING was expressed in both stroma and glandular epithelium of normal endometrium, but in endometriosis, STING was mainly expressed in the stroma of eutopic endometrium, and mainly in glandular epithelium of ectopic endometrium. Further study on the role of STING on endometrial stromal cells showed that low expression of STING could promote the HESC proliferation by EdU experiments, invasion and migration by Transwell experiments. The effect of STING/IRF3/IFNb1 signaling pathway in HESC with low expression of STING was also reduced, mainly showed the decreased expression of phosphorylated IRF3 and TBK1, and the decreased secretion of IFNb1. In order to further study the effect of IFNb1, secreted by STING/ IRF3/IFNb1 signaling pathway, on stromal cells, we added exogenous IFNb1 to the HESC with low expression of STING, and found that IFNb1 could reverse the invasion and migration function of stromal cells, but little effect on cell proliferation.Conclusions: We clarified that STING expressed mainly in stromal tissues and lower in endometriosis eutopic endometrium compared to normal endometrium. Low expressed STING promoted stromal cells invasion and migration via STING/IRF3/IFNb1 signaling pathway.

Single-cell transcriptomic analysis of endometriosis provides insights into fibroblast fates and immune cell heterogeneity

Background Endometriosis is an oestrogen-dependent disease with an unclear aetiology and pathogenesis affecting 6–10% of the global female population, predominantly those of reproductive age. Herein, we profile the transcriptomes of approximately 55,000 single cells from three groups including ectopic endometrium, eutopic endometrium from women with endometriosis, and eutopic endometrium from healthy women to create a single-cell transcriptome atlas of endometriosis. Results We have identified 9 cell types and performed single-cell analysis of fibroblasts, and determined a potential developmental trajectory associated with endometriosis. We also identified fibroblast subpopulations related to endometriosis development and found that StAR played an important role in this process. Moreover, T cells in endometriosis were less activated or inflammatory with decreased effector CD8 + T cells, while the composition ratio of natural killer cells decreased and the percentage of monocytes/macrophages increased in endometriosis cysts. In addition, the effectiveness of immune cells in endometriosis lesions, eutopic endometrium from women with endometriosis, and eutopic endometrium from healthy women was distinct. Cell–cell interaction analyses highlighted the imbalanced immune environment in endometriosis lesions and immune cells in endometriosis could promote the development of the disease. Conclusion Our study provided a systematic characterisation of endometriosis and insights into the aetiology and pathology of endometriosis.