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Endometriosis is a sex hormone-dependent, painful disease that affects 10-15% of women worldwide with no definitive cure, and current treatments are not always effective. This limitation is mainly due to gaps in our knowledge about the mechanisms involved in the pathogenesis of endometriosis at the cellular and molecular levels. Hormonal dysregulation appears to be responsible for inflammation, angiogenesis, endometrial non-receptivity, embryo implantation failure and infertility in women with endometriosis. Although correlative evidence about possible causes of hormonal dysregulations exists, the functional mechanisms remain unknown. Reliable research models of endometriosis are needed to investigate the exact mechanisms that underlie hormone disruptions. This Commentary discusses the available in-vivo and in-vitro systems for studying endometriosis. The authors emphasize the recently developed human endometriosis organoids as cutting-edge and innovative research models for endometriosis investigations, discuss their advantages and describe challenges that must be addressed to yield a reliable in-vitro model of human endometriosis. Moreover, it discusses microfluidic technology to address the present challenges for producing advanced endometriosis organoids and how to benefit from CRISPR technology to improve our knowledge about disturbed hormonal function in patients with endometriosis., (Copyright © 2022. Published by Elsevier Ltd.)

Objective: Endometriosis is an estrogen-dependent disease characterized by the presence of endometriotic tissue outside the uterine cavity, the condition that immunological factors play important roles in its pathogenesis. Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine that triggers dendritic cell-mediated T helper2 inflammatory responses. TSLP receptor, or cytokine receptor- like factor 2 (CRLF2), forms a functional heterodimeric complex with IL-7 receptor alpha (IL-7Rα) to bind with TSLP. The present study aimed to elucidate the expression and epigenetic alterations of TSLP gene parallel to TSLP receptor and IL-10 genes expression in endometrial tissues of patients with endometriosis compared to controls., Materials & Methods: In this case-control study, 45 women with and without endometriosis was enrolled. The relative expression of TSLP, TSLPR and IL-10 genes were examined using qPCR. Chromatin Immunoprecipitation (ChIP) was also used to monitor epigenetic marks of methylation and acetylation on lysine 9 of histone H3 (H3K9me/ac) and DNA methylation in TSLP promoter., Results and Conclusion: TSLP, TSLPR and IL-10 genes were overexpressed in ectopic endometriotic lesions compared to controls. In ectopic samples, significant H3K9 hyper-acetylation and hypo-methylation parallel to DNA hypo-methylation were detected in TSLP promoter compared to eutopic and control groups (p < 0.05). These epigenetic changes were aligned with TSLP gene expression profile. These data collectively identify TSLP and TSLPR as candidate genes critically involved in development of endometriosis beyond their role in promoting Th2 immune responses. In addition, acetylation and methylation of H3K9 may have effective roles in TSLP dysregulation in endometriosis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Endometriosis, as chronic estrogen-dependent disease, is defined by the presence of endometrial-like tissue outside the uterus. Proliferation of endometrial tissue and neoangiogenesis are critical factors in development of endometriosis. Hence, vascular endothelial growth factor (VEGF) as well as insulin-like growth factor 1 and 2 (IGF1, 2) may be involved as inducers of cellular proliferation or neoangiogenesis. Imprinted long noncoding RNA H19 (lncRNA H19) has been suggested to be involved in pathogenesis of endometriosis via regulation of cellular proliferation and differentiation. Epigenetic aberrations appear to play an important role in its pathogenesis. The present study was designed to elucidate VEGF, IGF1, IGF2 and H19 lncRNA genes expression and epigenetic alterations of differentially methylated region (DMR) of H19 (H19-DMR) regulatory region in endometrial tissues of patients with endometriosis, in comparison with control women., Methods: In this case-control study, 24 women with and without endometriosis were studied for the relative expression of VEGF, IGF1, IGF2 and H19 lncRNA genes using real-time polymerase chain reaction (PCR) technique. Occupancy of the MeCP2 on DMR region of H19 gene was assessed using chromatin immunoprecipitation (ChIP), followed by real-time PCR., Results: Genes expression profile of H19, IGF1 and IGF2 was decreased in eutopic and ectopic endometrial tissues of endometriosis group, compared to the control tissues. Decreased expression of H19 in ectopic samples was significant in comparison with the controls (P < 0.05). Gene expression of VEGF was increased in eutopic tissues of endometriosis group, compared to control group. Whereas its expression level was lower in ectopic lesions versus eutopic and control endometrial samples. ChIP analysis revealed significant and nearly significant hypomethylation of H19-DMR region II in eutopic and ectopic samples, compared to the control group respectively. This epigenetic change was aligned with expression of IGF2. While methylation of H19-DMR region I was not significantly different between the eutopic, ectopic and control endometrial samples., Conclusion: These data showed that VEGF, IGF1, IGF2 and H19 lncRNA genes expression and epigenetic alterations of H19 lncRNA have dynamic role in the pathogenesis of endometriosis, specifically in the way that hypomethylation of H19-DMR region II can be involved in IGF2 dysregulation in endometriosis., (© 2022. The Author(s).)

Objective: Toll-like receptors (TLRs, as members of the innate immune system) are expressed in the human endometrium and their aberrant regulation and expression are involved in the pathogenesis of endometrial diseases. This study is aimed at evaluation of TLR3 signaling pathway genes and its genetic changes in endometriosis patients., Materials and Methods: Blood samples were collected from 83 endometriosis patients and 93 healthy fertile women and PCR was performed in blood-derived DNA for detection of SNP of TLR3. Also, ectopic (EC) and eutopic (EU) endometrial biopsies were obtained from endometriosis patients (n = 20), as well as endometrium from healthy women (n = 16, CE). Q-PCR was performed for determination of mRNA expression level of TLR3 signaling pathway genes (TLR3, TICAM, NF-kB1A, CXCL10, IRF3, IFN-B1, IL-6 and IL-8). Also, serum protein levels of TLR3, IFN-β, IL-6 and IL-8 were determined using ELISA., Results: The mRNA expression levels of TLR3, NF-kB1A, IFN-B1, IRF3, TICAM1, IL-6 and IL-8 were significantly higher in EU compared to ectopic ones and also compared to CE. SNPs frequency (rs3775291 and rs3775290) was not significantly different between patients and controls. Serum protein levels of TLR3, IFN-β, IL-6 and IL-8 were significantly increased in endometriosis patients., Conclusion: Significant changes were observed in the expression of IL-6 and IL-8 cytokines and other genes in TLR3 cascade in diseased EU, demonstrating that EU similarly to EC is in an intensive inflammatory state. These fundamental alterations in the concept of immune response in EU may lead to its activation, escapes from apoptosis, and displaced implantation of the endometrium., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Endometriosis is major gynecological disease that affects over 10% of women worldwide and 30%-50% of these women have pelvic pain, abnormal uterine bleeding and infertility. The cause of endometriosis is unknown and there is no definite cure mainly because of our limited knowledge about its pathophysiology at the cellular and molecular levels. Therefore, demystifying the molecular mechanisms that underlie endometriosis is essential to develop advanced therapies for this disease. In this regard, HOX genes are remarkable because of their critical role in endometrial development and receptivity during implantation, which is attributed to their ability to mediate some of the sex steroid functions during the reproductive period. Access to the expression profiles of these genes would provide the necessary information to uncover new genes for endometriosis and assist with disease diagnosis and treatment. In this study we demonstrate an altered expression pattern for the HOX clusters (A-D) and their cofactors in both eutopic and ectopic conditions compared to control tissue biopsies. Remarkably, most of the intensive changes occurred in eutopic samples from endometriosis patients compared to control tissue biopsies. Pathway analysis revealed the involvement of differentially expressed genes in cancer that correlate with an association between endometriosis and cancer. Our results suggest critical roles for the HOX cluster and their cofactors in endometriosis pathophysiology., (© 2021. The Author(s).)

Research Question: Do human endometriosis organoids recapitulate aberrant progesterone signalling in the disease to serve as advanced experimental models for uncovering epigenetic mechanisms involved in attenuated progesterone response in endometriosis?, Design: Initially, the organoids were established from acquired biopsies (women with and without endometriosis) and characterized by morphological, histological and immunostaining analyses., Results: A panel of endometriosis-related genes showed a pattern of expressions in cytochrome c oxidase subunit II (COX2), matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase-3 (TIMP3), transforming growth factor beta 1 (TGF-β1), and zinc finger E-box binding homeobox 1 (ZEB1), and a contradictory expression pattern for cadherin (CDH1), POU class 5 homeobox 1 (POU5F1; also known as OCT4), and Nanog homeobox (NANOG) in the endometriosis organoids that is concordant with published research. These endometriosis organoids failed to upregulate 17β-Hydroxysteroid dehydrogenase 2 (17HSDβ2), progestogen associated endometrial protein (PAEP), secreted phosphoprotein 1 (SPP1), and leukaemia inhibitory factor (LIF) in response to progesterone at the level observed in control endometrium organoids. Progesterone receptor B (PRB) gene expression significantly decreased in both eutopic and ectopic organoids compared with control endometrium organoids. DNA hypermethylation, as an epigenetic mechanism for suppression of transcription, was detected at the PRB promoter in the eutopic, but not ectopic, organoids. Therefore, other epigenetic mechanisms, such as histone modifications and microRNAs, may be responsible for PRB downregulation in ectopic organoids., Conclusions: Endometriosis organoids are powerful preclinical models that can be used to investigate the molecular mechanisms involved in endometriosis-associated progesterone resistance., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: To evaluate and compare the methylation pattern of Human Homeobox (HOX) clusters (A-D) and HOX cofactors in normal, eutopic, and ectopic endometrial tissues with ectopic and eutopic endometriosis organoids as advanced preclinical research models., Design: A chromatin immunoprecipitation (ChIP) array containing 84 genes was used to analyze methylation levels of HOX clusters (A-D) and HOX cofactors in normal, eutopic, and ectopic endometrial biopsy specimens as well as ectopic and eutopic endometriosis organoids., Setting: Reproductive biomedicine and cell science research centers., Patient(s): Nine healthy women without endometriosis (control) and 16 women diagnosed with endometriosis., Intervention(s): Ectopic endometrial lesions were obtained using a laparoscopic procedure, and eutopic and control endometrium biopsy specimens were obtained using pipelle sampling., Main Outcome Measure(s): Methylation levels of HOX clusters (A-D) and HOX cofactors in eutopic and ectopic endometrial biopsy specimens, as well as eutopic and ectopic endometriosis organoids and normal endometrium., Result(s): Most HOX clusters (A-D) and HOX cofactors showed methylation alterations in ectopic/eutopic endometrial tissues and ectopic/eutopic endometriosis organoids compared with normal endometrium. These methylation alterations had the same pattern in ectopic/eutopic tissue biopsy specimens and ectopic/eutopic endometriosis organoids in most genes. A contrariwise methylation pattern was observed in 28 of 84 genes in the ectopic/eutopic tissue biopsy specimens and ectopic/eutopic endometriosis organoids., Conclusion(s): Because a conserved pattern of methylation alterations in endometriosis tissues and organoids was observed for most of the investigated genes (56 of 84), it can be concluded that endometriosis organoids maintain epigenetic changes. Therefore, our study suggests endometriosis organoids as a novel preclinical model to determine the epigenetic mechanisms that underlie endometriosis., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: Endometriosis is a common complex gynecological disorder that may result in infertility. Macrophage migration inhibitory factor (MIF) is a key pro-inflammatory cytokine that is overexpressed in endometriosis tissues. However, hitherto, no study tested the possible relevancy at genetic level. The aim of this study was to evaluate MIF polymorphisms and possible associations between haplotype of the gene and endometrioma., Study Design: In this experiment, 115 patients with confirmed endometrioma and 120 of women who were not diagnosed with endometrioma were recruited for this case-control genetic association study. The coding region of MIF was resequenced to detect variations of potential significance. Restriction fragment length polymorphism was used to type the -173 G/C (rs755622) promoter Single nucleotide polymorphism (SNP). Haplotype analyses were then undertaken to assess the effect of genetic variations., Results: We detected one functional SNP in promoter (rs755622) and non-functional mutations across the gene including (rs2096525, rs182012324, rs33958703 and rs2070766) in our samples. However, haplotype analysis showed a significant association between MIF and endometrioma where a single haplotype CC carrying only the minor allele at -173 G/C was significantly over-represented in the patients group (P = 0.007) and remained significant even after correction for (Bonferroni adjusted P = 0.028)., Conclusion: We report a strong linkage between a novel MIF haplotype and endometrioma. This association is consistent with expression data at both transcript and protein levels suggesting the -173C/G promoter as a critical factor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

Purpose: Endometriosis is a gynecological disease that causes the uterine lining to appear in other organs outside the uterus. As DNA methylation has an important role in this disorder, its profiling can reveal new information to improve the diagnosis and treatment of endometriosis patients., Methods: We conducted a genome-wide methylation profiling of ectopic and eutopic endometrial tissues from women with and without endometriosis using Infinium Human Methylation 450K BeadChip arrays. DNA methylation samples were collected from nine ectopic and nine eutopic endometrial tissues of endometriosis and six endometrial tissues of healthy controls., Results: Correlation heatmaps and the principal component analysis divided the samples into two clusters, one consisting of all ectopic samples and the other consisting of both eutopic and control samples unexpectedly without segregation between them. The assay identified a group of methylated genes that were overrepresented in biological processes, including abnormality in signaling, development, and adhesion of cells. Pathway analysis revealed disruption in HTLV infection pathways, PI3K-Akt, oxytocin, and relaxin signaling. Moreover, we found eutopic lesions are strongly associated with autoimmune disease., Conclusions: Our results confirmed the role of DNA methylation alternations in endometriosis development and pathogenesis. Our finding suggests aberrant DNA methylation can activate several signaling pathways including PI3k-AKT signaling, relaxin, and oxytocin which are associated with the pathogenesis of endometriosis.

Background: A predominant difference between endometrial and normal cells is higher proliferation rate in the former cells which is benign. The genes of inhibitor of differentiation (ID) family play a major role in cell proliferation regulation which might be targeted by the nuclear transcription factor Y (NF-Y) for subsequent epigenetic modifications through the CCAAT box regulatory region. The present study was designed to investigate the epigenetic role of NF-Y on ID gene family in endometrial tissue of patients with endometriosis., Materials & Methods: In this case-control study, 20 patients with endometriosis and 20 normal women were examined for the relative expression of the NF-YA, NF-YB, NF-YC and ID genes by real-time PCR during the proliferative phase. The occupancy of NF-Y on CCAAT box region of ID genes was investigated using chromatin immunoprecipitation (ChIP) followed by real-time PCR., Results: The NF-YA was over-expressed in eutopic endometrium during the proliferative phase. Although the expression level of NF-YB and NF-YC were unchanged in eutopic samples, they were remarkably higher in ectopic group (P<0.05). The ID2 and ID3 genes were up-regulated in ectopic and eutopic tissues, however ID1 and ID4 genes were down-regulated in these samples (P<0.05). The ChIP analysis revealed significant enrichment of NF-Y on regulatory regions of ID2,3 genes in eutopic group, but reduced binding level of NF-Y to the ID1,3 promoters in ectopic specimens (P<0.05)., Conclusion: The ability of NF-Y to regulate ID genes via CCAAT box region suggests the possible role of NF-Y transcription factor in epigenetic changes in endometrial tissues which may open novel avenues in finding new therapeutic strategies.

Endometriosis, which has been considered an epigenetic disease, is a prevalent gynecological disorder worldwide. With an emphasis on changes in the HOXA10 gene expression of the endometrium of women with endometriosis, the aim of this study was to investigate HOXA10 gene expression and its correlation with the epigenetic characteristics of the specific promoter region of the gene in the eutopic and ectopic endometrium of women with endometriosis. Thirty-six patients and 21 healthy fertile women were recruited as participants of this study. In this study group, chromatin immunoprecipitation and real-time polymerase chain reaction technique were performed to quantify the epigenetic profile of HOXA10, parallel to its expression. During the secretory phase in eutopic tissues, reduction in HOXA10 gene expression was identified along with lower acetylation and higher methylation of H3K9 as well as higher incorporation of MeCP2 on the HOXA10 gene promoter. In contrast with control group, studies of ectopic endometriotic lesions in the secretory phase demonstrate a correlation between induction of HOXA10 gene and higher levels of H3K9ac, H3K27me3, and H3K4me3 in the promoter region of the HOXA10 gene. Further distinctions from the control group were revealed in the proliferative phase of the ectopic endometrium, where upregulation of HOXA10 coincided with lower incorporation of MeCP2 and higher levels of H3K4me3 in the promoter region. Since it is well known that aberrant expression of HOXA10 is involved in pathogenesis of the endometrium, our data emphasized the epigenetic role of this gene aberration related to clinical pathophysiology of endometriosis.

Purpose: The purpose of the present study was to investigate the epigenetic mechanisms responsible for the aberrant aromatase expression (CYP19A1) in Cumulus Cells (CCs) of infertile endometriosis patients., Method: Cumulus cells were obtained from 24 infertile patients with and without endometriosis who underwent ovarian stimulation for intracytoplasmic sperm injection. Expression of CYP19A1 gene was quantified using Reverse Transcription Q-PCR. DNA methylation, histone modifications, and binding of Estrogen Receptor, ERβ to regulatory DNA sequences of CYP19A1 gene were evaluated by Chromatin ImmunoPrecipitation (ChIP) assay., Results: CYP19A1 gene expression in CCs of endometriosis patients was significantly lower than the control group (P = 0.04). Higher incorporation of MeCP2 (as a marker of DNA methylation) on PII and PI.4 promoters, and hypoacetylation at H3K9 in PII and hypermethylation at H3K9 in PI.4 were observed in CYP19A1 gene in endometriosis patients (P < 0.05). Moreover, a decreased level of ERβ binding to PII and an increased level of its binding to PI.3 and PI.4 promoters of CYP19A1 were observed in endometriosis patients when compared to control., Conclusion: Significant reduction of CYP19A1 gene expression in CCs of endometriosis patients may be the result of epigenetic alterations in its regulatory regions, either by DNA methylation or histone modifications. These epigenetic changes along with differential binding of ERβ (as a transcription factor) in CYP19A1 promoters may impair follicular steroidogenesis, leading to poor Oocyte and embryo condition in endometriosis patients.

Objective: To evaluate the expression of MIF, CD74, and COX-2 in normal, ectopic, and eutopic endometrium during the menstrual cycle and to assess MIF level in peripheral blood., Design: The expressions of MIF, CD74, and COX-2 in normal, ectopic, and eutopic endometrium were evaluated with the use of real-time polymerase chain reaction. MIF protein in peripheral blood samples was checked with the use of ELISA., Setting: Reproductive biomedicine research center., Patient(s): Sixteen normal women and 20 women with endometriosis., Intervention(s): Ectopic biopsies were obtained with the use of laparoscopic procedure, and eutopic and control biopsies were obtained with the use of Pipelle. Peripheral blood samples were collected before laparoscopy., Main Outcome Measure(s): The expression of MIF, CD74, and COX-2 in normal, ectopic and eutopic endometrium during the menstrual cycle and the expression level of MIF in peripheral blood samples., Result(s): Relative mRNA expression of MIF, CD74, and COX-2 were significantly higher in ectopic endometrium than in eutopic and control endometrium. Also, there were significant differences in expression of these genes in normal, ectopic, and eutopic endometrium during the menstrual cycle. Moreover, women with endometriosis had significantly higher circulating levels of MIF compared with control subjects., Conclusion(s): Dynamic expression of MIF, CD74, and COX-2 during the menstrual cycle could play an essential role in reproduction, inflammation, and endometrium reconstruction. A higher expression of these genes in ectopic endometrium can be considered as a molecular biomarker for endometriosis development and pathophysiology. Also, a high level of MIF in blood serum can act as a biomarker in the diagnosis of endometriosis., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: Endometriosis is a common gynecological and inflammatory disorder. Macrophage migration inhibitory factor (MIF) is a key pro-inflammatory cytokine that is secreted by accumulated active macrophages in ectopic endometrial tissues. Two promoter polymorphisms of MIF [-794(CATT)5-8 /-173G/C] were identified to susceptibility and severity of several immune and inflammatory diseases. We aimed to evaluate the possible association between MIF promoter polymorphisms and susceptibly to endometriosis and its corolation with mRNA level. Materials and Methods: This case-control study was performed in Royan Institute from 2015 to 2017. Polymorphisms were evaluated in 106 endometriosis patients and 110 controls. For 17 endometrioma tissues, gene expression studies were conducted during secretory phase of menstrual cycle. Restriction fragment length polymorphism (RFLP) analysis was performed to determine -173G/C polymorphism and -794(CATT)5-8 were detected by sequencing. Quantitative polymerase chain reaction (Q-PCR) was carried out to determine MIF expression level. Results: Homozygote of CATT7 was observed only in endometriosis whilst we did not detect the significant allele and genotype variation in both groups. The homozygotes for -(CATT)5-8 and -173G/C polymorphisms were obtained to estimate the haplotype frequencies. Significantly higher haplotype frequencies were observed for CATT5/G in controls [global P value=0.044]. Additionally, the CATT5/C and CATT7/G haplotypes were not detected in any groups. Expression level of mRNA in ectopic tissue of endometriosis patients with CATT6,7/CC haplotype, were significantly higher compared to other haplotypes including CATT5,5/GG (2.91 fold, P=0.007), CATT5,5/GC (2.48 fold, P=0.047) and CATT6,6/GG (2.08 fold, P=0.046). Conclusion: We report, for the first time, a strong linkage between the decreased repetition of CATT and G allele in control and CATT6/C and CATT7/C haplotypes in endometriosis patients. Increased MIF expression is affected by genetic variants in the MIF promoter in ectopic endometrial tissues. This promoter haplotype might play an important role in the development and establishment of endometriosis. [ABSTRACT FROM AUTHOR]

Background: Gelatinases degrade extracellular matrix (ECM) components to allow for physiological remodeling and contribute to pathological tissue destruction in endometriosis. It is known that the function of gelatinases is resistant to suppression by progesterone in endometriosis. The ability of progesterone to impact gene expression depends on the progesterone receptor-A/-B (PR-A/PR-B) ratio. An imbalanced PR-A/PR-B ratio in endometriotic tissue may be the result of the differential expression of MMP-2 and MMP-9, which could be important in the etiology and pathogenesis of the disease. Hence, we decided to study the association of PR-A/PR-B ratio and gelatinases expression in endometriosis. Materials and Methods: In this prospective case-control study, we enrolled 40 women, 20 in the case group who were diagnosed with stage III/IV endometriosis and 20 normal subjects without endometriosis (controls) who referred to Royan Institute, Tehran, Iran during 2013-2014. We obtained 60 tissue samples [ectopic (n=20), eutopic (n=20), and normal endometrium (n=20)]. RNA was extracted from the tissue samples in order to analyze PR-A, PR-B, MMP-2, and MMP-9 mRNA levels through real-time polymerase chain reaction (PCR). Results: There was significantly lower expression of the PR-B isoform in ectopic tissues compared to the control (P=0.002) and eutopic endometrium (P=0.006) tissues. PR-A expression was higher, but not significantly so, in the same ectopic and eutopic endometrium tissues compared to the control tissues (P=0.643). There was significant overexpression of MMP-9 in ectopic samples compared to control (P=0.014) and eutopic endometrium (P=0.012) samples. The PR-A/PR-B ratio was not significantly higher in either eutopic or ectopic samples compared to the control samples (P=0.305). Conclusion: Our findings support an altered PR-B expression in endometriosis, which may be associated with MMP-9 overexpression. This finding can be important for disease pathogenesis. [ABSTRACT FROM AUTHOR]

Although many genes have been identified using high throughput technologies in endometriosis (ES), only a small number of individual genes have been analyzed functionally. This is due to the complexity of the disease that has different stages and is affected by various genetic and environmental factors. Many genes are upregulated or downregulated at each stage of the disease, thus making it difficult to identify key genes. In addition, little is known about the differences between the different stages of the disease. We assumed that the study of the identified genes in ES at a system-level can help to better understand the molecular mechanism of the disease at different stages of the development. We used publicly available microarray data containing archived endometrial samples from women with minimal/mild endometriosis (MMES), mild/severe endometriosis (MSES) and without endometriosis. Using weighted gene co-expression analysis (WGCNA), functional modules were derived from normal endometrium (NEM) as the reference sample. Subsequently, we tested whether the topology or connectivity pattern of the modules was preserved in MMES and/or MSES. Common and specific hub genes were identified in non-preserved modules. Accordingly, hub genes were detected in the non-preserved modules at each stage. We identified sixteen co-expression modules. Of the 16 modules, nine were non-preserved in both MMES and MSES whereas five were preserved in NEM, MMES, and MSES. Importantly, two non-preserved modules were found in either MMES or MSES, highlighting differences between the two stages of the disease. Analyzing the hub genes in the non-preserved modules showed that they mostly lost or gained their centrality in NEM after developing the disease into MMES and MSES. The same scenario was observed, when the severeness of the disease switched from MMES to MSES. Interestingly, the expression analysis of the new selected gene candidates including CC2D2A, AEBP1, HOXB6, IER3, and STX18 as well as IGF-1, CYP11A1 and MMP-2 could validate such shifts between different stages. The overrepresented gene ontology (GO) terms were enriched in specific modules, such as genetic disposition, estrogen dependence, progesterone resistance and inflammation, which are known as endometriosis hallmarks. Some modules uncovered novel co-expressed gene clusters that were not previously discovered. [ABSTRACT FROM AUTHOR]

Objective: Endometriosis, as a common inflammatory chronic disease is characterized by endometrial tissue growth outside the uterine cavity. It was reported that lipopolysaccharides (LPS) activate a transcription factor called LPSinduced tumor necrosis factor-alpha (LITAF) in macrophages, which induced transcription of cytokine genes such as tumor necrosis factor alpha (TNF-α). B-cell lymphoma 6 protein (BCL6) is a transcription factor which expression was increased in endometrial tissues of infertile women with endometriosis. In addition, it was shown that mRNA and protein of LITAF and BCL6 were inversely related in mature B lymphocytes and B-Cell lymphomas. Accordingly, we investigated gene expression of LITAF, BCL6 and TNF-α in eutopic and ectopic endometrial tissues of women with endometriosis compared to the controls. Materials and Methods: In this case-control study, 10 women with endometriosis (endometriosis group) and 10 women without endometriosis (control group) enrolled after diagnostic laparoscopy. Real-time polymerase chain reaction (PCR) technique was used to quantitatively analyze gene expression. One-Way ANOVA was used for data analysis. Results: This study showed that LITAF gene expression was significantly higher in ectopic endometrial tissues compared to the control samples. Expression level of BCL6 gene was significantly increased in the ectopic tissues of women with endometriosis compared to the control and eutopic samples. Although TNF-ɑ gene expression was increased in the ectopic lesions compared to the eutopic and control endometrial samples, these differences were not significant. Conclusion: The results suggested that over-expression of these inflammatory genes in ectopic endometrial lesions can be considered as a molecular scenario in pathophysiology of endometriosis by induction of inflammatory cascades and cellular proliferation. [ABSTRACT FROM AUTHOR]

Background: Endometriosis are defined as a progesterone-resistance disease. Two progesterone receptor (PR) isoforms, namely PR-A and PR-B, mediate the special effects of progesterone. One of the most effective polymorphism in the promoter region of PGR is the +331G/A. Objective: The differential expression level of PR isoforms due to +331G/A polymorphism may be able to influence the function of progesterone and reduce the susceptibility of endometriosis. Materials and Methods: This analytic, case-control study was carried out at Royan Institute, Tehran, Iran. Whole-blood samples were collected from 98 infertile women undergoing laparoscopy for endometriosis and 102 healthy fertile women. After DNA extraction, genotype frequencies were determined by polymerase chain reactionrestriction fragment length polymorphism. Then, RNA was extracted from the selected eutopic tissue samples of endometriosis patients. Analysis of PR-A and PR-B mRNA expressions were performed using Real-time polymerase chain reaction. Results: The frequency distribution of GG, GA genotypes in +331G/A polymorphism was 98.04%, 1.96% in the patients and 97.96%, 2.04% in the control groups, respectively (p = 0.968). Although our data did not show any significant association with +331G/A in the patient and control groups, we were able to demonstrate significantly higher expression level of PR-B and no significant lower expression level of PR-A isoforms in patients by favoring GA to GG genotypes (p = 0.017, p = 0.731, respectively). Conclusion: Our findings show that patients with GA genotypes had a higher expression level of PR-B compared to patients with GG genotypes. [ABSTRACT FROM AUTHOR]

Background: Endometriosis is a common, chronic inflammatory disease which is defined as an overgrowth of endometrial tissue outside the uterine cavity. The etiology of this disease is complex and multifactorial but there is a strong evidence that supports the presence of endometrial stem cells and their possible involvement in endometriosis. Objective: In this study, we analyzed the mRNA expression of REX-1 sternness gene and reconsidered three other sternness genes SOX-2, NANOG, OCT-4 in women with endometriosis compared to normal endometrium. Materials and Methods: Ten ectopic and ten eutopic tissue samples along with 23 normal endometrium specimens were recruited in this study. The expression levels of OCT-4, NANOG, SOX-2, and REX-1 genes were evaluated by the quantitative real-time polymerase chain reaction. Results: The transcription levels of OCT-4, NANOG, and SOX-2 mRNA were significantly increased in ectopic lesions compared with eutopic and control group (p = 0.041, p = 0.035, p = 0.048), although the REX-1 mRNA increase was not significant between endometriosis and control groups. Also, there were differences in the expression level of these genes in normal endometrium during the menstrual cycles (p = 0.031, p = 0.047, p = 0.031). Conclusion: Based on our data, we confirm the dynamic role of sternness genes in proliferation and growth of normal endometrium during the menstrual cycle and conclude that differential expression levels of these genes may contribute to the pathophysiology of endometriosis. [ABSTRACT FROM AUTHOR]

Background: Endometriosis is a prevalent gynecological disease, with limited known etiology and more researches are required to identify its etiology. In this manner, there is no evidence for expression and function of 3'HOX genes in 4 clusters in the limb and pelvic organs such as the uterus and its disorders (Genes in the HOXA-D clusters are subdivided into 13 paralogous groups). Objective: This study designed to investigate the expression profile of 5 paralogous (1-5) in four clusters of HOX genes (A, B, C, and D) in ectopic and eutopic tissues of women with endometriosis compared to the normal endometrium. Materials and Methods: Samples were obtained from thirty patients (15 with and 15 without endometriosis) of reproductive age with normal menstrual cycles. The same patient provided both eutopic and ectopic tissues and control women were laparoscopically checked for the absence of endometriosis. The expression profile of these HOX genes was investigated by quantitative real-time polymerase chain reaction technique. Results: We observed significant up-regulation of some members of HOXC and D clusters (HOXD1, HOXD3, HOXC4 and HOXC5) in ectopic and eutopic tissues vs. control. Also, our data showed significant down-regulation of all of HOXA and HOXB paralogous except HOXA1 in ectopic tissues versus control. Conclusion: Our data showed specific cluster dependent modulation of the HOX genes expression in endometriosis (over-expression of some HOX genes in cluster C and D and down-regulation of HOX genes in cluster A and B) in ectopic and eutopic tissues compare to control group. Therefore, it is possible that change of expression level of these genes in endometrium plays a role in the pathogenesis of endometriosis. [ABSTRACT FROM AUTHOR]

Endometriosis is a benign gynecological disease that is manifested by the presence and growth of endometrial cells and glands outside the uterine. Active angiogenesis, migration, and invasion of endometrial tissue outside the uterine are critical for the development of endometriosis and lead to the survival and growth of endometriotic lesions. Metformin, as an anti-diabetic agent, represents anti-angiogenic property. Here, we performed a study using human normal endometrial stromal cells (N-ESCs) from healthy endometrial tissue and human eutopic endometrial stromal cells (EU-ESCs) and ectopic endometrial stromal cells (ECT-ESCs) from endometriosis patients. ESCs were cultured and treated with different concentrations of Metformin (0–20 mmol/l) for 72 h to evaluate Metformin effect on cell viability, proliferation, migration was measured by methyl thiazolyl tetrazolium (MTT) assay and scratch test respectively as well as expression of angiogenesis and migration markers. The Metformin reduced cell migration, and proliferation of endometriotic stromal cells in a time and concentration dependently manner. Furthermore, Metformin attenuated the expression of angiogenic and inflammatory genes in human endometriotic stromal cells. The direct anti-proliferative effect on ECT-ESCs combined with the effects of Metformin on inflammatory and angiogenesis-related genes expression supports its therapeutic potential for endometriosis. Metformin could be used as an effective adjuvant in endometriosis treatment. • Metformin reduced cell migration, and proliferation of endometriotic stromal cells. • Metformin attenuated the expression of angiogenic and inflammatory genes in human endometriotic stromal cells. • Metformin could be used as an effective adjuvant in endometriosis treatment. [ABSTRACT FROM AUTHOR]