Project description:BackgroundWomen 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.MethodsEndometrial 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.ResultsWe 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.ConclusionsIdentifying 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.

Project description:BackgroundAdenomyosis (AM) is a common benign chronic gynaecological disorder; however, the precise pathogenesis of adenomyosis is still poorly understood. Single-cell RNA sequencing (scRNA-seq) can uncover rare subpopulations, explore genetic and functional heterogeneity, and reveal the uniqueness of each cell. It provides us a new approach to reveal biological issues from a more detailed and microscopic perspective. Here, we utilize this revolutionary technology to identify the changes of gene expression patterns between ectopic lesions and the eutopic endometrium at the single-cell level and explore a potential novel pathogenesis of AM.MethodsA control endometrium (sample with leiomyoma excluding endometrial disorders, n = 1), eutopic endometrium and ectopic lesion (from a patient with adenomyosis, n = 1) samples were analysed by scRNA-seq, and additional leiomyoma (n = 3) and adenomyosis (n = 3) samples were used to confirm colocalization and vasculogenic mimicry (VM) formation. Protein colocalization was visualized by immunofluorescence, and CD34-periodic acid-Schiff (PAS) double staining was used to assess the formation of VM.ResultsThe scRNA-seq results suggest that cancer-, cell motility- and inflammation- (CMI) associated terms, cell proliferation and angiogenesis play important roles in the progression of AM. Moreover, the colocalization of EPCAM and PECAM1 increased significantly in the ectopic endometrium group (P < 0.05), cell subpopulation with high copy number variation (CNV) levels possessing tumour-like features existed in the ectopic lesion sample, and VNN1- and EPCAM-positive cell subcluster displayed active cell motility in endometrial epithelial cells. Furthermore, during the transformation of epithelial cells to endothelial cells, we observed the significant accumulation of VM formation (positively stained with PAS but not CD34, P < 0.05) in ectopic lesions.ConclusionsIn the present study, our results support the theory of adenomyosis derived from the invasion and migration of the endometrium. Moreover, cell subcluster with high CNV level and tumour-associated characteristics is identified. Furthermore, epithelial-endothelial transition (EET) and the formation of VM in tumours, the latter of which facilitates the blood supply and plays an important role in maintaining cell growth, were also confirmed to occur in AM. These results indicated that the inhibition of EET and VM formation may be a potential strategy for AM management.

Project description:Abstract: Objective: Adenomyosis is a clinical disorder defined by the presence of endometrial glands and stroma within the myometrium, the pathogenesis of which is poorly understood. We postulate that dysregulation of genes and pathways in eutopic endometrium may predispose to ectopic implantation. No study, to our knowledge, has examined the global transcriptome of isolated eutopic endometrium from women with clinically significant adenomyosis. Design: Laboratory-based study with full IRB approval and consents. Material and Methods: Endometrial sampling was performed on hysterectomy specimens (proliferative phase) from symptomatic women with pathologically-confirmed diffuse adenomyosis (n=3). Controls (n=5) were normo-ovulatory subjects without adenomyosis. All subjects were free from leiomyoma, endometriosis, and hormonal exposures. Isolated purified total RNA was subjected to microarray analysis using the Gene 1.0 ST Affymetrix platform. Data were analyzed with GeneSpring and Ingenuity Pathway analysis. Validation of several genes was undertaken by QRT-PCR. Results: Comparison of transcriptomes of proliferative endometrium from women with and without adenomyosis revealed 140 up-regulated and 884 down-regulated genes in samples from women with adenomyosis compared to controls. Highly differentially expressed genes include those involved in regulation of apoptopsis, steroid hormone responsiveness, and proteins involved in extracellular matrix remodeling, as well as microRNAs of unknown significance. Affected canonical pathways included eukaryotic initiation factor 2 signaling, oxidative phosphorylation, mitochondrial dysfunction, estrogen receptor signaling, and mTOR signaling. Conclusions: The eutopic endometrium in patients with adenomyosis has fundamental abnormalities that may predispose to invasion and survival beyond the myometrial interface. Key Words: adenomyosis, endometrium, microarray, microRNA, endometriosis, apoptosis, signaling. Abstract: Objective: Adenomyosis is a clinical disorder defined by the presence of endometrial glands and stroma within the myometrium, the pathogenesis of which is poorly understood. We postulate that dysregulation of genes and pathways in eutopic endometrium may predispose to ectopic implantation. No study, to our knowledge, has examined the global transcriptome of isolated eutopic endometrium from women with clinically significant adenomyosis. Design: Laboratory-based study with full IRB approval and consents. Material and Methods: Endometrial sampling was performed on hysterectomy specimens (proliferative phase) from symptomatic women with pathologically-confirmed diffuse adenomyosis (n=3). Controls (n=5) were normo-ovulatory subjects without adenomyosis. All subjects were free from leiomyoma, endometriosis, and hormonal exposures. Isolated purified total RNA was subjected to microarray analysis using the Gene 1.0 ST Affymetrix platform. Data were analyzed with GeneSpring and Ingenuity Pathway analysis. Validation of several genes was undertaken by QRT-PCR. Results: Comparison of transcriptomes of proliferative endometrium from women with and without adenomyosis revealed 140 up-regulated and 884 down-regulated genes in samples from women with adenomyosis compared to controls. Highly differentially expressed genes include those involved in regulation of apoptopsis, steroid hormone responsiveness, and proteins involved in extracellular matrix remodeling, as well as microRNAs of unknown significance. Affected canonical pathways included eukaryotic initiation factor 2 signaling, oxidative phosphorylation, mitochondrial dysfunction, estrogen receptor signaling, and mTOR signaling. Conclusions: The eutopic endometrium in patients with adenomyosis has fundamental abnormalities that may predispose to invasion and survival beyond the myometrial interface. Key Words: adenomyosis, endometrium, microarray, microRNA, endometriosis, apoptosis, signaling. A total of 8 samples were used and analyzed by disease state. Endometrial samples from hysterectomy specimens in proliferative phase of menstrual cycle from symptomatic women with pathologically-confirmed diffuse adenomyosis were compared with endometrial samples from normo-ovulatory healthy subjects with no endometrial or uterine pathology.

Project description:Abstract: Objective: Adenomyosis is a clinical disorder defined by the presence of endometrial glands and stroma within the myometrium, the pathogenesis of which is poorly understood. We postulate that dysregulation of genes and pathways in eutopic endometrium may predispose to ectopic implantation. No study, to our knowledge, has examined the global transcriptome of isolated eutopic endometrium from women with clinically significant adenomyosis. Design: Laboratory-based study with full IRB approval and consents. Material and Methods: Endometrial sampling was performed on hysterectomy specimens (proliferative phase) from symptomatic women with pathologically-confirmed diffuse adenomyosis (n=3). Controls (n=5) were normo-ovulatory subjects without adenomyosis. All subjects were free from leiomyoma, endometriosis, and hormonal exposures. Isolated purified total RNA was subjected to microarray analysis using the Gene 1.0 ST Affymetrix platform. Data were analyzed with GeneSpring and Ingenuity Pathway analysis. Validation of several genes was undertaken by QRT-PCR. Results: Comparison of transcriptomes of proliferative endometrium from women with and without adenomyosis revealed 140 up-regulated and 884 down-regulated genes in samples from women with adenomyosis compared to controls. Highly differentially expressed genes include those involved in regulation of apoptopsis, steroid hormone responsiveness, and proteins involved in extracellular matrix remodeling, as well as microRNAs of unknown significance. Affected canonical pathways included eukaryotic initiation factor 2 signaling, oxidative phosphorylation, mitochondrial dysfunction, estrogen receptor signaling, and mTOR signaling. Conclusions: The eutopic endometrium in patients with adenomyosis has fundamental abnormalities that may predispose to invasion and survival beyond the myometrial interface. Key Words: adenomyosis, endometrium, microarray, microRNA, endometriosis, apoptosis, signaling. Abstract: Objective: Adenomyosis is a clinical disorder defined by the presence of endometrial glands and stroma within the myometrium, the pathogenesis of which is poorly understood. We postulate that dysregulation of genes and pathways in eutopic endometrium may predispose to ectopic implantation. No study, to our knowledge, has examined the global transcriptome of isolated eutopic endometrium from women with clinically significant adenomyosis. Design: Laboratory-based study with full IRB approval and consents. Material and Methods: Endometrial sampling was performed on hysterectomy specimens (proliferative phase) from symptomatic women with pathologically-confirmed diffuse adenomyosis (n=3). Controls (n=5) were normo-ovulatory subjects without adenomyosis. All subjects were free from leiomyoma, endometriosis, and hormonal exposures. Isolated purified total RNA was subjected to microarray analysis using the Gene 1.0 ST Affymetrix platform. Data were analyzed with GeneSpring and Ingenuity Pathway analysis. Validation of several genes was undertaken by QRT-PCR. Results: Comparison of transcriptomes of proliferative endometrium from women with and without adenomyosis revealed 140 up-regulated and 884 down-regulated genes in samples from women with adenomyosis compared to controls. Highly differentially expressed genes include those involved in regulation of apoptopsis, steroid hormone responsiveness, and proteins involved in extracellular matrix remodeling, as well as microRNAs of unknown significance. Affected canonical pathways included eukaryotic initiation factor 2 signaling, oxidative phosphorylation, mitochondrial dysfunction, estrogen receptor signaling, and mTOR signaling. Conclusions: The eutopic endometrium in patients with adenomyosis has fundamental abnormalities that may predispose to invasion and survival beyond the myometrial interface. Key Words: adenomyosis, endometrium, microarray, microRNA, endometriosis, apoptosis, signaling.

Project description:Adenomyosis is a prevalent, estrogen-dependent uterine disorder wherein endometrial cells are abnormally present in the myometrium and are surrounded by hyperplastic/hypertrophic smooth muscle. Its etiology is unclear, although endometrial cell invasion into the myometrium has been postulated. RNA methylation, particularly N6-methyladenosine (m6A), plays an important role in regulating various physiological processes and invasive disorders. The goal of this in silico and lab-based experimental study was to explore a possible role for m6A in adenomyosis. Gene expression profiles of both the endometrium and myometrium of women with adenomyosis (cases) and without disease (controls) were obtained from the publicly available Gene Expression Omnibus (GEO) database. In the endometrium, STRING database analysis revealed that METTL3 functions as a "hub" gene of m6A RNA methylation regulators, and the genes involved in m6A regulation, including METTL3, FTO, ZC3H13, and YTHDC1 expression, were significantly decreased in cases versus controls. Functional, co-expression, and correlational analyses of endometrium from cases versus controls revealed decreased total m6A levels, induced by METTL3, and the downstream elevated insulin-like growth factor-1(IGF1) and D-Dopachrome Tautomerase (DDT), with the latter two having known functions in epithelial proliferation and cell migration, which are important processes in the pathogenesis of adenomyosis in endometrium. m6A RNA methylation regulators, including RBM15/15B, ALKBH5, FTO, YTHDF1/2, KIAA1429, HNRNPC, METTL3, ZC3H13, and YTHDC2, were also differentially expressed in the myometrium from cases versus controls. We validated decreased total m6A levels and differential expression of m6A RNA methylation regulators in the myometrium of patients with adenomyosis using qRT-PCR, immunohistochemistry and tissues available from our biorepository. Possible target genes, including cadherin 3(CDH3), sodium channelβ-subunit 4 (SCN4B), and placenta-specific protein 8 (PLAC8), which are involved in cell adhesion, muscle contraction and immune response in the myometrium of adenomyosis patients were also validated. Thus, through extensive public database mining and validation of select genes, this study, for the first time, implicates m6A and its methylation regulators in the pathogenesis of adenomyosis. Follow on functional studies are anticipated to elucidate mechanisms involving m6A and its regulators and down-stream effectors in the pathogenesis of this enigmatic reproductive disorder and potentially identify druggable targets to control its associated symptoms.

Project description: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. 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 the top 20 were 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. 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. In conclusion, 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.

Project description:Objective: Bromocriptine treatment has been shown to reduce menstrual bleeding and pain in women with adenomyosis in a pilot clinical trial. The underlying mechanism contributing to the treatment effect is however unknown. This study was to explore the effect of bromocriptine on the proliferation and migration properties of the endometrium in women with adenomyosis, by assessing cellular and molecular changes after six months of vaginal bromocriptine treatment. Methods: Endometrial specimens were collected during the proliferative phase from women with adenomyosis (n=6) before (baseline) and after six months of vaginal bromocriptine treatment. Immunohistochemistry was used to determine changes in the protein expression of Ki67 in the endometrium. Primary endometrial stromal cells isolated at baseline were expanded in vitro and exposed to different doses of bromocriptine to determine the optimal half-maximum inhibitory concentration (IC50) using CellTiter-Blue® Cell Viability Assay. Cell proliferation was assessed by bromodeoxyuridine ELISA assay and Ki67 gene expression was checked by real-time PCR. The migratory ability of endometrial stromal cells was determined by wound healing and transwell migration assays. Small RNA sequencing was applied on tissues collected from women with adenomyosis before and after bromocriptine treatment to identify differentially expressed micro RNAs. Bioinformatic methods were used for target gene prediction and the identification of biological pathways. Results: Vaginal bromocriptine treatment reduced the Ki67 protein expression in the endometrium of women with adenomyosis and did not change the prolactin mRNA expression and protein concentration of prolactin in endometrial tissues. Bromocriptine significantly inhibited the proliferative and migrative abilities of endometrial stromal cells derived from women with adenomyosis in vitro. Moreover, small RNA sequencing revealed 27 differentially expressed micro RNAs (miRNAs) between the endometrium of women with adenomyosis before and after six months of vaginal bromocriptine treatment. KEGG pathway analysis on targeted genes of 27 miRNAs showed that several signaling pathways associated with cell proliferation and apoptosis were enriched after treatment. Conclusion: Bromocriptine treatment exhibits an anti-proliferative effect in the endometrium of women with adenomyosis in vivo and in vitro. Bromocriptine might inhibit the proliferation of endometrial tissue in adenomyosis in part through the regulation of dysregulated microRNAs and proliferation-associated signaling pathways.

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Transcriptome sequencing of adenomyosis eutopic endometrium: A new insight into its pathophysiology.

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