[Estrogen, estrogen receptor and miR-21 in adenomyosis: their pathogenic roles and regulatory interactions].

OBJECTIVE: To explore the pathogenic roles of miR-21, estrogen (E2), and estrogen receptor (ER) in adenomyosis. METHODS: We examined the expression levels of miR-21 in specimens of adenomyotic tissue and benign cervical lesions using qRT-PCR. In primary cultures of cells isolated from the adenomyosis lesions, the effect of ICI82780 (an ER inhibitor) on miR-21 expression levels prior to E2 activation or after E2 deprivation were examined with qRT-PCR. We further assessed the effects of a miR-21 mimic or an inhibitor on proliferation, apoptosis, migration and autophagy of the cells. RESULTS: The expression level of miR-21 was significantly higher in adenomyosis tissues than in normal myometrium (P < 0.05). In the cells isolated from adenomyosis lesions, miR-21 expression level was significantly higher in E2 activation group than in ER inhibition + E2 activation group and the control group (P < 0.05); miR-21 expression level was significantly lower in cells in E2 deprivation+ER inhibition group than in E2 deprivation group and the control group (P < 0.05). The adenomyosis cells transfected with miR-21 inhibitor showed inhibited proliferation and migration, expansion of mitochondrial endoplasmic reticulum, increased lysosomes, presence of autophagosomes, and increased cell apoptosis, while transfection of the cells with the miR-21 mimic produced the opposite effects. CONCLUSION: MiR-21 plays an important role in promoting proliferation, migration, and antiapoptosis in adenomyosis cells by altering the cell ultrastructure, which may contribute to early pathogenesis of the disease. In addition to binding with E2, ER can also regulate miR-21 through other pathways to participate in the pathogenesis of adenomyosis, thus having a stronger regulatory effect on miR-21 than E2.

Extracellular vesicle-encapsulated miR-25-3p promotes epithelial-mesenchymal transition and migration of endometrial epithelial cells by inducing macrophage polarization.

The pathogenesis of adenomyosis is closely related to the epithelial-mesenchymal transition and macrophages. MicroRNAs have been extensively investigated in relation to the epithelial-mesenchymal transition in a range of malignancies. However, there is a paucity of research on extracellular vesicles derived from the eutopic endometrium of adenomyosis and their encapsulated microRNAs. In this study, we investigated the role of microRNA-25-3p derived from extracellular vesicles in inducing macrophage polarization and promoting the epithelial-mesenchymal transition in endometrial epithelial cells of patients with adenomyosis and controls. We obtained eutopic endometrial samples and isolated extracellular vesicles from the culture supernatant of primary endometrial cells. Real-time quantitative PCR analysis demonstrated that microRNA-25-3p was highly expressed in extracellular vesicles, as well as in macrophages stimulated by extracellular vesicles from eutopic endometrium of adenomyosis; and macrophages transfected with microRNA-25-3p exhibited elevated levels of M2 markers, while displaying reduced levels of M1 markers. After co-culture with the above polarized macrophages, endometrial epithelial cells expressed higher levels of N-cadherin and Vimentin, and lower protein levels of E-cadherin and Cytokeratin 7. It was revealed that microRNA-25-3p encapsulated in extracellular vesicles from eutopic endometrial cells could induce macrophage polarization toward M2, and the polarized macrophages promote epithelial-mesenchymal transition in epithelial cells. However, in vitro experiments revealed no significant disparity in the migratory capacity of endometrial epithelial cells between the adenomyosis group and the control group. Furthermore, it was observed that microRNA-25-3p-stimulated polarized macrophages also facilitated the epithelial-mesenchymal transition and migration of endometrial epithelial cells within the control group. Thus, the significance of microRNA-25-3p-induced polarized macrophages in promoting the development of adenomyosis is unclear, and macrophage infiltration alone may be adequate for this process. We emphasize the specificity of the local eutopic endometrial microenvironment and postulate its potential significance in the pathogenesis of adenomyosis.

The pathogenesis of endometriosis and adenomyosis: insights from single-cell RNA sequencing†.

Endometriosis and adenomyosis are two similar gynecological diseases that are characterized by ectopic implantation and the growth of the endometrial tissue. Previous studies have reported that they share a common pathophysiology in some respects, such as a similar cellular composition and resistance to the progestogen of lesions, but their underlying mechanisms remain elusive. Emerging single-cell ribonucleic acid sequencing (scRNA-seq) technologies allow for the dissection of single-cell transcriptome mapping to reveal the etiology of diseases at the level of the individual cell. In this review, we summarized the published findings in research on scRNA-seq regarding the cellular components and molecular profiles of diverse lesions. They show that epithelial cell clusters may be the vital progenitors of endometriosis and adenomyosis. Subclusters of stromal cells, such as endometrial mesenchymal stem cells and fibroblasts, are also involved in the occurrence of endometriosis and adenomyosis, respectively. Moreover, CD8+ T cells, natural killer cells, and macrophages exhibit a deficiency in clearing the ectopic endometrial cells in the immune microenvironment of endometriosis. It seems that the immune responses are activated in adenomyosis. Understanding the immune characteristics of adenomyosis still needs further exploration. Finally, we discuss the application of findings from scRNA-seq for clinical diagnosis and treatment. This review provides fresh insights into the pathogenesis of endometriosis and adenomyosis as well as the therapeutic targets at the cellular level.

Transcriptome analysis of adenomyosis eutopic endometrium reveals molecular mechanisms involved in adenomyosis-related implantation failure and pregnancy disorders.

BACKGROUND: Women with adenomyosis are characterized by having defective decidualization, impaired endometrial receptivity and/or embryo-maternal communication, and implantation failure. However, the molecular mechanisms underlying adenomyosis-related infertility remain unknown, mainly because of the restricted accessibility and the difficult preservation of endometrial tissue in vitro. We have recently shown that adenomyosis patient-derived endometrial organoids, maintain disease-specific features while differentiated into mid-secretory and gestational endometrial phase, overcoming these research barriers and providing a robust platform to study adenomyosis pathogenesis and the associated molecular dysregulation related to implantation and pregnancy disorders. For this reason, we aim to characterize the dysregulated mechanisms in the mid-secretory and gestational endometrium of patients with adenomyosis by RNA-sequencing. METHODS: Endometrial organoids were derived from endometrial biopsies collected in the proliferative phase of women with adenomyosis (ADENO) or healthy oocyte donors (CONTROL) (n = 15/group) and differentiated into mid-secretory (-SECorg) and gestational (-GESTorg) phases in vitro. Following RNA-sequencing, the significantly differentially expressed genes (DEGs) (FDR < 0.05) were identified and selected for subsequent functional enrichment analysis and QIAGEN Ingenuity Pathway Analysis (IPA). Statistical differences in gene expression were evaluated with the Student's t-test or Wilcoxon test. RESULTS: We identified 1,430 DEGs in ADENO-SECorg and 1,999 DEGs in ADENO-GESTorg. In ADENO-SECorg, upregulated genes included OLFM1, FXYD5, and RUNX2, which are involved in impaired endometrial receptivity and implantation failure, while downregulated genes included RRM2, SOSTDC1, and CHAC2 implicated in recurrent implantation failure. In ADENO-GESTorg, upregulated CXCL14 and CYP24A1 and downregulated PGR were related to pregnancy loss. IPA predicted a significant inhibition of ID1 signaling, histamine degradation, and activation of HMGB1 and Senescence pathways, which are related to implantation failure. Alternatively, IPA predicted an inhibition of D-myo-inositol biosynthesis and VEGF signaling, and upregulation of Rho pathway, which are related to pregnancy loss and preeclampsia. CONCLUSIONS: Identifying dysregulated molecular mechanisms in mid-secretory and gestational endometrium of adenomyosis women contributes to the understanding of adenomyosis-related implantation failure and/or pregnancy disorders revealing potential therapeutic targets. Following experimental validation of our transcriptomic and in silico findings, our differentiated adenomyosis patient-derived organoids have the potential to provide a reliable platform for drug discovery, development, and personalized drug screening for affected patients.

Activation of HAND2-FGFR signaling pathway by lncRNA HAND2-AS1 in adenomyosis†.

Heart and neural crest derivatives expressed transcript 2 (HAND2) is a critical mediator of progesterone action in endometrial stromal cells. Silencing of Hand2 expression in mouse uterus leads to an unopposed FGFR-mediated action that causes female mice infertility. To investigate the involvement of HAND2-FGFR signaling in pathogenesis of adenomyosis, immunohistochemistry, in situ hybridization, and quantitative real-time PCR were employed to assess gene expression in the normal endometrium, the paired eutopic endometrium and ectopic lesions obtained from women with adenomyosis. DNA methylation in the regions of HAND2 promoter and the first exon was also monitored in these samples. Our results revealed that HAND2 expression were dramatically reduced, but FGF9 expression and FGFR-ERK1/2-mediated MAPK signaling pathway were enhanced in the eutopic endometrium and ectopic lesions of patients with adenomyosis compared to the normal controls. Interestingly, expression of HAND2-AS1, a long noncoding RNA that resides adjacent to HAND2 in genome, was also reduced in adenomyosis. DNA methylation analysis revealed that the bidirectional promoter between HAND2 and HAND2-AS1, and the first exon of HAND2 gene was heavily methylated in the eutopic endometrium and the ectopic lesions of adenomyosis. To investigate the regulation of gene expression by HAND2-AS1, HAND2-AS1 expression was silenced in human endometrial stromal cells. In contrast to the downregulation of HAND2 in response to HAND2-AS1 silencing, FGF9 expression was augmented significantly. Endometrial stromal cells lacking HAND2-AS1 exhibited enhanced proliferation and migration potentials. Collectively, our studies revealed a new molecular mechanism by which HAND2-AS1 is involved in the pathogenesis of adenomyosis via modulating HAND2-FGFR-mediated signaling.

Dysregulated expression of GATA2 and GATA6 transcription factors in adenomyosis: implications for impaired endometrial receptivity.

OBJECTIVE: To study the effect of adenomyosis on the localized expression of the GATA binding proteins 2 and 6 (GATA2 and GATA6) zinc-finger transcription factors that are involved in proliferation of hematopoietic and endocrine cell lineages, cell differentiation, and organogenesis, potentially leading to impaired endometrial implantation. DESIGN: Laboratory based experimental study. SETTING: Academic hospital and laboratory. PATIENTS: Human endometrial stromal cells (HESCs) of reproductive age patients, 18-45 years of age, with adenomyosis were compared with patients with no pathology and leiomyomatous uteri as controls (n = 4 in each group, respectively). Additionally, midsecretory phase endometrial sections were obtained from patients with adenomyosis and control patients with leiomyoma (n = 8 in each group, respectively). INTERVENTIONS: GATA2 and GATA6 immunohistochemistry and H-SCORE were performed on the midsecretory phase endometrial sections from adenomyosis and leiomyoma control patients (n = 8 each, respectively). Control and adenomyosis patient HESC cultures were treated with placebo or 10-8 M estradiol (E2), or decidualization media (EMC) containing 10-8 M E2, 10-7 M medroxyprogesterone acetate, and 5 × 10-5 M cAMP for 6 and 10 days. Additionally, control HESC cultures (n = 4) were transfected with scrambled small interfering RNA (siRNA) (control) or GATA2-specific siRNAs for 6 days while adenomyosis HESC cultures (n = 4) were transfected with human GATA2 expression vectors to silence or induce GATA2 overexpression. MAIN OUTCOME MEASURES: Immunohistochemistry was performed to obtain GATA2 and GATA6 H-SCORES in adenomyosis vs. control patient endometrial tissue. Expression of GATA2, GATA6, insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), progesterone receptor (PGR), estrogen receptor 1 (ESR1), leukemia inhibitory factor (LIF), and Interleukin receptor 11 (IL11R) messenger RNA (mRNA) levels were analyzed using by qPCR with normalization to ACTB. Silencing and overexpression experiments also had the corresponding mRNA levels of the above factors analyzed. Western blot analysis was performed on isolated proteins from transfection experiments. RESULTS: Immunohistochemistry revealed an overall fourfold lower GATA2 and fourfold higher GATA6 H-SCORE level in the endometrial stromal cells of patients with adenomyosis vs. controls. Decidual induction with EMC resulted in significantly lower GATA2, PGR, PRL and IGFBP1 mRNA levels in HESC cultures from patients with adenomyosis patient vs. controls. Leukemia inhibitory factor and IL11R mRNA levels were also significantly dysregulated in adenomyosis HESCs compared with controls. . Silencing of GATA2 expression in control HESCs induced an adenomyosis-like state with significant reductions in GATA2, increases in GATA6 and accompanying aberrations in PGR, PRL, ESR1 and LIF levels. Conversely, GATA2 overexpression via vector in adenomyosis HESCs caused partial restoration of the defective decidual response with significant increases in GATA2, PGR, PRL and LIF expression. CONCLUSION: In-vivo and in-vitro experiment results demonstrate that there is an overall inverse relationship between endometrial GATA2 and GATA6 levels in patients with adenomyosis who have diminished GATA2 levels and concurrently elevated GATA6 levels. Additionally, lower GATA2 and higher GATA6 levels, together with aberrant levels of important receptors and implantation factors, such as ESR1, PGR, IGFBP1, PRL, LIF, and IL11R mRNA in HESCs from patients with adenomyosis or GATA2-silenced control HESCs, support impaired decidualization. These effects were partially restored with GATA2 overexpression in adenomyosis HESCs, demonstrating a potential therapeutic target.

Expression of high-mobility group box-1 in eutopic/ectopic endometrium and correlations with inflammation-related factors in adenomyosis.

OBJECTIVE: To investigate the expression of HMGB1 and toll-like receptor 4 (TLR4) in adenomyosis eutopic/ectopic endometrium. METHODS: Twenty patients with adenomyosis and 20 controls, all undergoing laparoscopy, were recruited from September 2015 to July 2016. Samples were collected from the endometrium without adenomyosis (CE), the eutopic endometrium with adenomyosis (EuE), and the ectopic endometrium with adenomyosis (EE). The mRNA and protein expression of HMGB1 and TLR4, and interleukin-6 (IL-6) and interleukin-8 (IL-8) RNA expression levels were measured. RESULTS: The average age of the adenomyosis women was 43.4 ± 5.3 years; their BMI was 23.3 ± 2.3 kg/m2. The control group included women aged 38.8 ± 9.8 years, with BMI 22.2 ± 3.4 kg/m2. The mRNA expression levels of HMGB1, TLR4, IL-6, and IL-8 in the EE and EuE groups were higher than those in the CE group (p < .01), and those in the EE group were higher than those in the EuE group (p < .01). The protein expression levels of HMGB1 and TLR4 in the EE and EuE groups were higher than those in the CE group (p < .01); they were higher in the EE group than the ones in the EuE group (p < .01). HMGB1 mRNA was significantly positively correlated with TLR4 in EuE and EC patients (r = 0.538 and r = 0.916, p < .01), as well as with IL-6 (r = 0.470 and r = 0.976, p < .01) and IL-8 (r = 0.574 and r = 0.650, p < .01). CONCLUSIONS: The overexpression of HMGB1 and TLR4 in EuE and EE is positively correlated with IL-6 and IL-8 expression. The HMGB1 signaling-mediated immune-inflammatory system might be involved in the development of adenomyosis.

Abnormal activation of the Wnt3a/ß-catenin signaling pathway promotes the expression of T-box transcription factor 3(TBX3) and the epithelial-mesenchymal transition pathway to mediate the occurrence of adenomyosis.

BACKGROUND: T-box transcription factor 3(TBX3) is a transcription factor that can regulate cell proliferation, apoptosis, invasion, and migration in different tumor cells; however, its role in adenomyosis (ADM) has not been previously studied. Some of ADM's pathophysiological characteristics are similar to those of malignant tumors (e.g., abnormal proliferation, migration, and invasion). METHODS AND RESULTS: We hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced Institute of Cancer research (ICR) mice to establish ADM disease model. The study procedure included western blotting and immunohistochemistry to analyze protein levels; additionally, we used intraperitoneal injection of Wnt/ß-catenin pathway inhibitor XAV-939 to study the relationship between TBX3 and Wnt/ß-catenin pathway as well as Anti-proliferation cell nuclear antigen( PCNA) and TUNEL to detect cell proliferation and apoptosis, respectively. TBX3 overexpression and epithelial-to-mesenchymal transition (EMT) in ADM mice was found to be associated with activation of the Wnt3a/ß-catenin pathway. Treatment with XAV-939 in ADM mice led to the inhibition of both TBX3 and EMT; moreover, abnormal cell proliferation was suppressed, the depth of invasion of endometrium cells was limited. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells. CONCLUSION: These findings suggest that TBX3 may play an important role in the development of ADM. The expression of TBX3 in ADM was regulated by the Wnt3a/ß-catenin pathway. The activation of the Wnt3a/ß-catenin pathway in ADM promoted TBX3 expression and induced the occurrence of EMT, thus promoting cell proliferation and inhibiting apoptosis, ultimately accelerating the development of ADM. The study provides a reference for the diagnosis of ADM.

Study on differentially expressed genes and participating pathways of ectopic endometrium in adenomyosis patients with different data sets.

BACKGROUND: Adenomyosis is a benign uterine disease and affected patients present with symptoms such as menorrhagia, chronic pelvic pain, abnormal uterine bleeding, and infertility. However, the specific mechanisms by which adenomyosis occurs need to be further studied. OBJECTIVE: Dataset of adenomyosis from our hospital and a public database were analyzed using bioinformatics. Corresponding differentially expressed genes (DEGs) and gene enrichment were detected to explore potential genetic adenomyosis targets. METHODS: Clinical data on adenomyosis were accessed based on the pathological specimens of patients with adenomyosis obtained from the Shengjing Hospital. R software was used to screen for DEGs, and volcano and cluster maps were drawn. Adenomyosis datasets (GSE74373) were downloaded from the GEO database. GEO2R online tool was used to screen for DEGs between adenomyosis and normal controls. Genes with P < 0.01 and |logFC| >1 were selected as DEGs. DAVID software was used for functional and pathway enrichment analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on common DEGs to obtain descriptions of the genes. The online database STRING was used for interaction gene retrieval. Moreover, Cytoscape software was used to construct a protein-protein interaction (PPI) network map for common DEGs to visualize potential gene interactions and screen the hub genes. RESULTS: A total of 845 DEGs were identified in the dataset obtained from Shengjing Hospital. A total of 175 genes were downregulated, and 670 genes were upregulated. In the GSE74373 database, 1679 genes were differentially expressed, 916 genes were downregulated, and 763 genes were upregulated. A total of 40 downregulated and 148 upregulated common DEGs showed potential gene interactions. The top ten upregulated hub genes were CDH1, EPCAM, CLDN7, ESRP1, RAB25, SPINT1, PKP3, TJP3, GRHL2, and CDKN2A. CONCLUSION: Genes involved in tight junction may be key in the development of adenomyosis and may provide a potential treatment strategy for adenomyosis.

Comparing gene expression in deep infiltrating endometriosis with adenomyosis uteri: evidence for dysregulation of oncogene pathways.

BACKGROUND: The pathogenesis of deep infiltrating endometriosis (DIE) is poorly understood. It is considered a benign disease but has histologic features of malignancy, such as local invasion or gene mutations. Moreover, it is not clear whether its invasive potential is comparable to that of adenomyosis uteri (FA), or whether it has a different biological background. Therefore, the aim of this study was to molecularly characterize the gene expression signatures of both diseases in order to gain insight into the common or different underlying pathomechanisms and to provide clues to pathomechanisms of tumor development based on these diseases. METHODS: In this study, we analyzed formalin-fixed and paraffin-embedded tissue samples from two independent cohorts. One cohort involved 7 female patients with histologically confirmed FA, the other cohort 19 female patients with histologically confirmed DIE. The epithelium of both entities was microdissected in a laser-guided fashion and RNA was extracted. We analyzed the expression of 770 genes using the nCounter expression assay human PanCancer (Nanostring Technology). RESULTS: In total, 162 genes were identified to be significantly down-regulated (n = 46) or up-regulated (n = 116) in DIE (for log2-fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05) compared to FA. Gene ontology and KEGG pathway analysis of increased gene expression in DIE compared to FA revealed significant overlap with genes upregulated in the PI3K pathway and focal adhesion signaling pathway as well as other solid cancer pathways. In FA, on the other hand, genes of the RAS pathway showed significant expression compared to DIE. CONCLUSION: DIE and FA differ significantly at the RNA expression level: in DIE the most expressed genes were those belonging to the PI3K pathway, and in FA those belonging to the RAS pathway.

Endometriosis and adenomyosis: shared pathophysiology.

Endometriosis and adenomyosis are closely related disorders. Their pathophysiologies are extremely similar. Both tissues originate from the eutopically located intracavitary endometrium. Oligoclones of endometrial glandular epithelial cells with somatic mutations and attached stromal cells may give rise to endometriosis if they travel to peritoneal surfaces or the ovary via retrograde menstruation and/or may be entrapped in the myometrium to give rise to adenomyosis. In both instances, the endometrial cell populations possess survival and growth capabilities conferred by somatic epithelial mutations and epigenetic abnormalities in stromal cells. Activating mutations of KRAS are the most commonly found genetic variant in endometriotic epithelial cells, whereas the adenomyotic epithelial cells almost exclusively bear KRAS mutations. Epigenetic abnormalities in the stromal cells of endometriosis and adenomyosis are very similar and involve an abnormal expression pattern of nuclear receptors, including the steroid receptors. These epigenetic defects give rise to excessive local estrogen biosynthesis by aromatase and abnormal estrogen action via estrogen receptor-ß. Deficient progesterone receptor expression results in progesterone resistance in both endometriosis and adenomyosis.

Adenomyosis: single-cell transcriptomic analysis reveals a paracrine mesenchymal-epithelial interaction involving the WNT/SFRP pathway.

OBJECTIVE: To assess the cellular and molecular landscape of adenomyosis. DESIGN: Single-cell analysis of genome-wide messenger RNA (mRNA) expression (single-cell RNA sequencing) of matched tissues of endometrium, adenomyosis, and myometrium using relatively large numbers of viable cells. SETTING: Not applicable. PATIENT(S): Patients (n = 3, age range 40-44 years) undergoing hysterectomy for diffuse adenomyosis. MAIN OUTCOME MEASURE(S): Definition of the molecular landscape of matched adenomyotic, endometrial and myometrial tissues from the same uterus using single-cell RNA sequencing and comparison of distinct cell types in these tissues to identify disease-specific cell populations, abnormal gene expression and pathway activation, and mesenchymal-epithelial interactions. RESULT(S): The largest cell population in the endometrium was composed of closely clustered fibroblast groups, which comprise 36% of all cells and seem to originate from pericyte progenitors differentiating to estrogen/progesterone receptor-expressing endometrial stromal- cells. In contrast, the entire fibroblast population in adenomyosis comprised a larger (50%) portion of all cells and was not linked to any pericyte progenitors. Adenomyotic fibroblasts eventually differentiate into extracellular matrix protein-expressing fibroblasts and smooth muscle cells. Hierarchical clustering of mRNA expression revealed a unique adenomyotic fibroblast population that clustered transcriptomically with endometrial fibroblasts, suggestive of an endometrial stromal cell population serving as progenitors of adenomyosis. Four other adenomyotic fibroblast clusters with disease-specific transcriptomes were distinct from those of endometrial or myometrial fibroblasts. The mRNA levels of the natural WNT inhibitors, named, secreted frizzled-related proteins 1, 2, and 4, were higher in these 4 adenomyotic fibroblast clusters than in endometrial fibroblast clusters. Moreover, we found that multiple WNTs, which originate from fibroblasts and target ciliated and unciliated epithelial cells and endothelial cells, constitute a critical paracrine signaling network in adenomyotic tissue. Compared with endometrial tissue, unciliated and ciliated epithelial cells in adenomyosis comprised a significantly smaller portion of this tissue and exhibited molecular evidence of progesterone resistance and diminished regulation of estrogen signaling. CONCLUSION(S): We found a high degree of heterogeneity in fibroblast-like cells in the adenomyotic uterus. The WNT signaling involving differential expression of secreted frizzled-related proteins, which act as decoy receptors for WNTs, in adenomyotic fibroblasts may have a key role in the pathophysiology of this disease.

Cytochrome-c-oxidase and ATPsynthase content increases in the endometrium of the patients with adenomyosis.

BACKGROUND: Adenomyosis is characterized by overgrowth of endometrial glands and stroma in the myometrium and is associated with reduced apoptosis. One of the key participants in one of the pathways of apoptosis is cytochrome c, whose expression can be regulated by actin-binding proteins involved in the formation of structures that provide cell motility. The aim of the study was to determine the content of actin-binding proteins, cytochrome c, and terminal members of the mitochondrial respiratory chain in endometrial biopsies of patients with adenomyosis and the control group. METHODS AND RESULTS: The content of all studied proteins was determined by Western blotting, and the mRNA content of the corresponding genes was determined by quantitative RT-PCR. The relative content of alpha-actinin1 and mRNA of the gene encoding it in biopsy specimens from patients with adenomyosis was higher than in controls by 86 and 84% (p < 0.05), respectively. The relative content of alpha-actinin4 did not change, as did cytochrome c. The content of cytochrome-c-oxidase and ATPsynthase in the group with adenomyosis exceeded the control level by 270 and 121% (p < 0.05), respectively, but the relative content of mRNA of these genes did not change, which may indicate a change in regulation at the level of protein synthesis. CONCLUSION: The results may indicate a local increase in the synthesis of ATP in pathological endometrial cells, which indicates the possible effectiveness of local application of H+-ATP synthase inhibitors (for example, macrolide antibiotic) to reduce the severity of clinical symptoms of adenomyosis.

Targeted next-generation sequencing for the detection of cancer-associated somatic mutations in adenomyosis.

Adenomyosis is a condition characterised by the invasion of endometrial tissues into the uterine myometrium, the molecular pathogenesis of which remains incompletely elucidated. Lesion profiling with next-generation sequencing (NGS) can lead to the identification of previously unanticipated causative genes and the detection of therapeutically actionable genetic changes. Using an NGS panel that included 275 cancer susceptibility genes, this study examined the occurrence and frequency of somatic mutations in adenomyotic tissue specimens collected from 17 women. Extracted DNA was enriched using targeted formalin-fixed paraffin-embedded tissue cores prior to the identification of lesion-specific variants. The results revealed that KRAS and AT-rich interactive domain 1A (ARID1A) were the two most frequently mutated genes (mutation frequencies: 24% and 12%, respectively). Notably, endometrial atypical hyperplasia did not involve adenomyotic areas. We also identified, for the first time, two potentially pathogenic mutations in the F-box/WD repeat-containing protein 7 (FBXW7) and cohesin subunit SA-2 (STAG2) genes. These findings indicate that mutations in the KRAS, ARID1A, FBXW7 and STAG2 genes may play a critical role in the pathogenesis of adenomyosis. Additional studies are needed to assess whether the utilisation of oncogenic driver mutations can inform the surveillance of patients with adenomyosis who had not undergone hysterectomy.Impact statementWhat is already known on this subject? Although somatic point mutations in the KRAS oncogene have been recently detected in adenomyosis, the molecular underpinnings of this condition remains incompletely elucidated. Lesion profiling with next-generation sequencing (NGS) can lead to the identification of previously unanticipated causative genes and the detection of therapeutically actionable genetic changes.What do the results of this study add? The results of NGS revealed that KRAS and AT-rich interactive domain 1A (ARID1A) were the two most frequently mutated genes (mutation frequencies: 24% and 12%, respectively). We also identified, for the first time, two potentially pathogenic mutations in the F-box/WD repeat-containing protein 7 (FBXW7) and cohesin subunit SA-2 (STAG2) genes.What are the implications of these findings for clinical practice and/or further research? The utilisation of oncogenic driver mutations has the potential to inform the surveillance of patients with adenomyosis who had not undergone hysterectomy.

Adjustment of progesterone administration after endometrial transcriptomic analysis does not improve reproductive outcomes in women with adenomyosis.

RESEARCH QUESTION: Do patients with adenomyosis present a dysregulated endometrial receptivity that can be reversed with personalized embryo transfer (PET) by transcriptomic-based progesterone adjustment, improving IVF outcomes? DESIGN: A multicentre, retrospective, cohort study that transcriptomically analysed the endometrial receptivity of the endometrium in patients with adenomyosis (n = 81) and healthy women (n = 231). Subsequently, implantation, biochemical and clinical miscarriage, and live birth rates between adenomyosis patients with one previous implantation failure using donor oocytes who received (n = 59) or did not receive (n = 66) PET based on endometrial receptivity, were observed to evaluate if adjusted progesterone improves reproductive outcomes of adenomyosis patients. RESULTS: Patients with adenomyosis significantly presented an altered endometrial receptivity (non-receptive) compared with healthy patients (53.1% versus 37.2%, P = 0.0179), elevating the risk of adenomyosis patients having a non-receptive endometrium 42.59% higher (95% CI 41.50 to 44.45). No significant differences were found in implantation (62.7% versus 78.8%, P = 0.0514), biochemical (13.5% versus 3.9%, P = 0.1223) and clinical (10.8% versus 15.4%, P = 0.7543) miscarriage, or live birth rates (75.7% versus 80.8%, P = 0.6066), in patients with PET compared with those without PET. CONCLUSIONS: Women with adenomyosis presented an altered expression of genes involved in decidualization, and a higher rate of non-receptive endometrial statuses than controls. Although progesterone is indispensable for implantation, adjusting progesterone before PET, using endometrial transcriptomic signatures, does not improve IVF outcomes in patients with adenomyosis. Other molecular mechanisms beyond progesterone regulation may be involved in implantation failure.

Are Adenomyosis and Endometriosis Phenotypes of the Same Disease Process?

In recent literature reviews, we concluded that the possibility that endometrial molecular aberrations are the sole or a necessary determinant of endometriosis and the Tissue Injury and Repair (TIAR) theory are yet to be convincingly proven. Here, we critically examine the theory that adenomyosis and endometriosis represent different phenotypes of a single disease. A common etiopathology for adenomyosis and endometriosis has been suggested because both conditions entail the presence of endometrial tissue at locations other than the lining of the uterus. There are wide differences in reported disease incidence and prevalence and, consequently, in estimates of the coexistence of both conditions. There are some similarities but also differences in their clinical features and predisposing factors. Each condition has a range of subtypes. These differences alone pose the question of whether subtypes of endometriosis and adenomyosis have different etiopathologies, and, in turn, this raises the question of whether they all share a common etiology. It is debatable whether the recognized differences between the eutopic endometrium in adenomyosis and endometriosis compared to those in unaffected women are the cause or the effect of the disease. The finding of common mutations, particularly of KRAS, lend support to the notion of shared predisposing factors, but this alone is insufficient evidence of causation.

Circ_0061140 Facilitates Adenomyosis Progression by Upregulating LIN28B in vitro.

OBJECTIVES: The aim of our study was to explore the role of circular RNA_0061140 (circ_0061140) in adenomyosis progression and its associated mechanism. DESIGN: We first analyzed the expression pattern of circ_0061140 in endometrial tissues of adenomyosis patients (n = 27) and uterine fibroid patients (n = 15). Loss-of-function experiments were conducted to analyze the biological roles of circ_0061140 in regulating the viability, apoptosis, proliferation, migration, and invasion of endometrial epithelial cells. The downstream microRNA (miRNA)/messenger RNA (mRNA) axis of circ_0061140 was predicted by bioinformatics tool Starbase, and its working mechanism was verified by rescue experiments. METHODS: Cell viability, apoptosis, proliferation, invasion, and migration were assessed by cell counting kit-8 assay, flow cytometry analysis, 5-ethynyl-2'-deoxyuridine assay, transwell assay, and scratch test. The binding relationship between miR-141-3p and circ_0061140 or lin-28 homolog B (LIN28B) was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Circ_0061140 expression was upregulated in adenomyosis patients. Circ_0061140 knockdown suppressed the viability, proliferation, invasion, and migration and triggered the apoptosis of endometrial epithelial cells. Circ_0061140 served as a miRNA sponge for miR-141-3p, and miR-141-3p silencing partly reversed circ_0061140 knockdown-induced effects in endometrial epithelial cells. miR-141-3p directly interacted with LIN28B mRNA. LIN28B overexpression partly diminished miR-141-3p overexpression-mediated influences in endometrial epithelial cells. Circ_0061140 knockdown downregulated LIN28B expression by elevating miR-141-3p level in endometrial epithelial cells. LIMITATIONS: The functional verification of circ_0061140/miR-141-3p/LIN28B axis was merely conducted in vitro. CONCLUSION: Circ_0061140 contributed to adenomyosis progression by binding to miR-141-3p to induce LIN28B expression in vitro.

Decreased expression of GRIM-19 induces autophagy through the AMPK/ULK1 signaling pathway during adenomyosis†.

The processes underlying adenomyosis are similar to those of tumor metastasis, and it is defined as progressive invasion by the endometrium and the subsequent creation of ectopic lesions. GRIM-19 regulates cell death via the mitochondrial respiratory chain. Stress following oxygen deprivation can induce tumor cell autophagy, leading to cell invasion and migration. Here, we revealed that GRIM-19 negatively regulates autophagy, and, at least in adenomyosis, decreased expression of GRIM-19 is accompanied by an increased level of autophagy and 5'-adenosine monophosphate-activated protein kinase-Unc-51 like autophagy activating kinase 1 (AMPK-ULK1) activation. Upregulation of GRIM-19 expression in human primary endometrial cells and ISHIKAWA cells inhibits autophagy via the AMPK-ULK1 pathway and helps control cell invasion and migration. In addition, we also identified increased expression of AMPK and ULK1, and higher levels of autophagy in the uterine tissues of GRIM-19+/- mice. Importantly, the function of the GRIM-19-AMPK-ULK1 axis in regulating autophagy in adenomyosis is similar to that of tumor tissues, which may help elucidate the regulation of adenomyosis tumor-like behavior, and is expected to help identify novel targets for the diagnosis and treatment of adenomyosis.

Spatiotemporal expression pattern of Progesterone Receptor Component (PGRMC) 1 in endometrium from patients with or without endometriosis or adenomyosis.

The endometrium plays a crucial role in reproduction and, in humans, is cyclically remodeled under hormonal control. Estradiol favors tissue proliferation whereas progesterone inhibits tissue growth and induces morphological changes. Endometriosis is often associated with fertility issues and with exacerbated estrogen and reduced progesterone concentration or response in the eutopic endometrium. However, underlying mechanisms remain unclear. Progesterone Receptor Membrane Component (PGRMC) 1 is a protein able to modulate progesterone response and its murine knockout reduced fertility. However, the precise spatiotemporal pattern of PGRMC1 expression in the human endometrium is still poorly characterized. We investigated variations of eutopic endometrial PGRMC1 expression by combining RT-qPCR, immunofluorescence and in situ hybridization. We found that PGRMC1 expression progressively increases during the proliferative phase and decreases during the secretory phase. However, immunolabeling and identification of mRNA-containing cells were regularly heterogeneous in samples, according to tissue depth, with a gradient extending from the surface epithelium towards the basalis. There was no significant difference in PGRMC1 mRNA amounts between patients with or without endometriosis or adenomyosis, for any phase of the menstrual cycle, but cells with strong or moderate PGRMC1 immunolabeling were reduced during the proliferative phase in endometriotic patients. In conclusion, although the cyclical variation of PGRMC1 expression globally follows fluctuation of ovarian steroids, further work is required to precisely characterize hormonal control and identify the additional levels of regulation responsible for local adjustment of PGRMC1 concentration. This is particularly important in the light of recent studies emphasizing the correlation between adequate PGRMC1 amounts and fertility.

RNA-seq reveals co-dysregulated circular RNAs in the adenomyosis eutopic endometrium and endometrial-myometrial interface.

BACKGROUND: Uterine adenomyosis is associated with chronic pelvic pain, abnormal uterine bleeding, and infertility. The pathogenesis of adenomyosis is still unclear. Circular RNAs (circRNAs) have been implicated in several benign diseases and malignant tumors. We aimed to explore the co-dysregulated circular RNA profile in the eutopic endometrium and endometrial-myometrial interface (EMI) of adenomyosis. METHODS: Total RNA was extracted from the eutopic endometrium and EMI of 5 patients with adenomyosis and 3 patients without adenomyosis. Next-generation sequencing was performed to identify the circRNA expression profile of the two tissue types. Bioinformatics analysis was performed to predict circRNA-binding miRNAs and miRNA-binding mRNAs and construct ceRNA networks, and functional enrichment analysis was performed to predict the biological functions of circRNAs. RESULTS: Among the adenomyosis patients, 760 circRNAs were significantly upregulated and 119 circRNAs were significantly downregulated in the EMI of adenomyosis, while 47 circRNAs were significantly upregulated and 17 circRNAs were significantly downregulated in the eutopic endometrium of adenomyosis. We identified hsa_circ_0002144 and hsa_circ_0005806 as co-upregulated and hsa_circ_0079536 and hsa_circ_0024766 as co-downregulated in the eutopic endometrium and EMI. Bioinformatics analysis was performed to construct a ceRNA network of codifferentially expressed circRNAs. The MAPK signaling pathway is the most important signaling pathway involved in the function of the ceRNA network. CONCLUSIONS: Co-dysregulated circRNAs were present in the eutopic endometrium and EMI of adenomyosis. MiRNA binding sites were observed for all of these circRNAs and found to regulate gene expression. Co-dysregulated circRNAs may induce the eutopic endometrial invagination process through the MAPK signaling pathway and promote the progression of adenomyosis.