Higher Oxidative Stress in Endometriotic Lesions Upregulates Senescence-Associated p16ink4a and ß-Galactosidase in Stromal Cells.

Endometriosis affects a significant proportion of women worldwide; however, no definitive cure for this disease has been discovered to date. Oxidative stress promotes endometriotic lesion maintenance in the peritoneal cavity in women. Furthermore, there is evidence of the mitogen-activated protein kinase (MAPK) signaling pathway and senescence involvement in the physiopathogenesis of endometriosis. Reactive oxygen species (ROS) cause oxidative damage and are expected to trigger senescence in the endometrium while also causing alterations in MAPK signaling. However, the role of ROS in the senescence-associated phenotype in endometriosis remains unknown. In this context, this study attempted to delineate the pathways linking ROS to senescence in endometrial and endometriotic lesions of healthy individuals and those with endometriosis. Our results indicate a higher presence of ROS in endometriotic lesions, and the upregulation of MAPK. Furthermore, we show that endometriotic lesions in stromal cells stimulated with hydrogen peroxide develop more senescence traits than eutopic and non-endometriosis endometrium. Overall, endometriotic cells respond differently to extracellular distress. Our contribution to further research in this field contributed to the roadmap of endometriosis' search for alternative treatments.

Downregulation of DROSHA: Could It Affect miRNA Biogenesis in Endometriotic Menstrual Blood Mesenchymal Stem Cells?

Menstrual blood mesenchymal stem cells (MenSCs) have gained prominence in the endometriosis scientific community, given their multifunctional roles in regenerative medicine as a noninvasive source for future clinical applications. In addition, changes in post-transcriptional regulation via miRNAs have been explored in endometriotic MenSCs with a role in modulating proliferation, angiogenesis, differentiation, stemness, self-renewal, and the mesenchymal-epithelial transition process. In this sense, homeostasis of the miRNA biosynthesis pathway is essential for several cellular processes and is related to the self-renewal and differentiation of progenitor cells. However, no studies have investigated the miRNA biogenesis pathway in endometriotic MenSCs. In this study, we profiled the expression of eight central genes for the miRNA biosynthesis pathway under experimental conditions involving a two-dimensional culture of MenSCs obtained from healthy women (n = 10) and women with endometriosis (n = 10) using RT-qPCR and reported a two-fold decrease in DROSHA expression in the disease. In addition, miR-128-3p, miR-27a-3p, miR-27b-3p, miR-181a-5p, miR-181b-5p, miR-452-3p, miR-216a-5p, miR-216b-5p, and miR-93-5p, which have been associated with endometriosis, were identified through in silico analyses as negative regulators of DROSHA. Because DROSHA is essential for miRNA maturation, our findings may justify the identification of different profiles of miRNAs with DROSHA-dependent biogenesis in endometriosis.

Altered Differential Expression of Genes and microRNAs Related to Adhesion and Apoptosis Pathways in Patients with Different Phenotypes of Endometriosis.

We aim to investigate the expression of genes (MAPK1 and CAPN2) and microRNAs (miR-30a-5p, miR-7-5p, miR-143-3p, and miR-93-5p) involved in adhesion and apoptosis pathways in superficial peritoneal endometriosis (SE), deep infiltrating endometriosis (DE), and ovarian endometrioma (OE), and to evaluate whether these lesions share the same pathophysiological mechanisms. We used samples of SE (n = 10), DE (n = 10), and OE (n = 10), and endometrial biopsies of these respective patients affected with endometriosis under treatment at a tertiary University Hospital. Endometrial biopsies collected in the tubal ligation procedure from women without endometriosis comprised the control group (n = 10). Quantitative real-time polymerase chain reaction was performed. The expression of MAPK1 (p < 0.0001), miR-93-5p (p = 0.0168), and miR-7-5p (p = 0.0006) was significantly lower in the SE group than in the DE and OE groups. The expression of miR-30a (p = 0.0018) and miR-93 (p = 0.0052) was significantly upregulated in the eutopic endometrium of women with endometriosis compared to the controls. MiR-143 (p = 0.0225) expression also showed a statistical difference between the eutopic endometrium of women with endometriosis and the control group. In summary, SE showed lower pro-survival gene expression and miRNAs involved in this pathway, indicating that this phenotype has a different pathophysiological mechanism compared to DE and OE.

Altered p16Ink4a, IL-1ß, and Lamin b1 Protein Expression Suggest Cellular Senescence in Deep Endometriotic Lesions.

Endometriosis causes immunological and cellular alterations. Endometriosis lesions have lower levels of lamin b1 than the endometrium. Moreover, high levels of pro-inflammatory markers are observed in the peritoneal fluid, follicular fluid, and serum in endometriosis lesions. Thus, we hypothesized that the accumulation of senescent cells in endometriosis tissues would facilitate endometriosis maintenance in an inflammatory microenvironment. To study senescent cell markers and the senescence-associated secretory phenotype (SASP) in endometriosis lesions, we conducted a cross-sectional study with 27 patients undergoing video laparoscopy for endometriosis resection and 19 patients without endometriosis. Endometriosis lesions were collected from patients with endometriosis, while eutopic endometrium was collected from patients both with and without endometriosis. Tissues were evaluated for senescence markers (p16Ink4a, lamin b1, and IL-1ß) and interleukin concentrations. The expression of p16Ink4a increased in lesions compared to that in eutopic endometrium from endometriosis patients in the secretory phase. In the proliferative phase, lesions exhibited lower lamin b1 expression but higher IL-4 expression than the eutopic endometrium. Further, IL-1ß levels were higher in the lesions than in the eutopic endometrium in both the secretory and proliferative phases. We believe that our findings may provide targets for better therapeutic interventions to alleviate the symptoms of endometriosis.

What Do the Transcriptome and Proteome of Menstrual Blood-Derived Mesenchymal Stem Cells Tell Us about Endometriosis?

Given the importance of menstrual blood in the pathogenesis of endometriosis and the multifunctional roles of menstrual mesenchymal stem cells (MenSCs) in regenerative medicine, this issue has gained prominence in the scientific community. Moreover, recent reviews highlight how robust the integrated assessment of omics data are for endometriosis. To our knowledge, no study has applied the multi-omics approaches to endometriosis MenSCs. This is a case-control study at a university-affiliated hospital. MenSCs transcriptome and proteome data were obtained by RNA-seq and UHPLC-MS/MS detection. Among the differentially expressed proteins and genes, we emphasize ATF3, ID1, ID3, FOSB, SNAI1, NR4A1, EGR1, LAMC3, and ZFP36 genes and MT2A, TYMP, COL1A1, COL6A2, and NID2 proteins that were already reported in the endometriosis. Our functional enrichment analysis reveals integrated modulating signaling pathways such as epithelial-mesenchymal transition (↑) and PI3K signaling via AKT to mTORC1 (↓ in proteome), mTORC1 signaling, TGF beta signaling, TNFA signaling via NFkB, IL6 STAT3 signaling, and response to hypoxia via HIF1A targets (↑ in transcriptome). Our findings highlight primary changes in the endometriosis MenSCs, suggesting that the chronic inflammatory endometrial microenvironment can modulate these cells, providing opportunities for endometriosis etiopathogenesis. Moreover, they identify challenges for future research leveraging knowledge for regenerative and precision medicine in endometriosis.

Altered transcriptome in cumulus cells of infertile women with advanced endometriosis with and without endometrioma.

RESEARCH QUESTION: Is the transcriptome of cumulus cells of infertile women with advanced endometriosis (EIII/IV), with and without endometrioma, altered? DESIGN: In this prospective case-control study, next-generation RNA sequencing was used to compare the transcript profile of cumulus cells among infertile patients undergoing ovarian stimulation for intracytoplasmic sperm injection with EIII/IV, with (n = 9) and without endometrioma (n = 9), and controls (n = 9). An in-silico enrichment analysis was conducted to establish the possibly altered pathways in cumulus cells of patients with endometriosis. RESULTS: Most of the differentially expressed genes (DEG) were found when cumulus cells from women with EIII/IV with endometrioma were compared with controls (DEG, n = 461). In women with EIII/IV without endometrioma, only 66 DEG were verified compared with controls. The enrichment analysis showed that some DEG in cumulus cells of endometriosis are involved in important pathways for the oocyte competence acquisition, such as oxidative phosphorylation, metabolism, mitochondrial function, acetylation and steroid biosynthesis. No DEG were found when cumulus cells from women with EIII/IV with and without endometrioma were compared. CONCLUSION: RNA sequencing results suggest that cumulus cells of infertile women with EIII/IV have an altered transcriptome, regardless of endometrioma. The present findings offer a better understanding of the genes and molecular mechanisms that may be involved in endometriosis-related infertility, mostly in the oocyte competence acquisition process.

miR-532-3p: a possible altered miRNA in cumulus cells of infertile women with advanced endometriosis.

RESEARCH QUESTION: Is the profile of microRNA (miRNA) altered in cumulus cells of infertile women with early (EI/II) and advanced (EIII/IV) endometriosis? DESIGN: In this prospective case-control study, a miRNA profile including 754 targets was evaluated in samples of cumulus cells from infertile women with endometriosis (5 EI/II, 5 EIII/IV) and infertile controls (5, male and/or tubal factor) undergoing ovarian stimulation for intracytoplasmic sperm injection, using TaqMan® Array Human MicroRNA Cards A and B. The groups were compared with Kruskal-Wallis test, followed by Benjamini-Hochberg correction and Dunn's post hoc test. An in silico enrichment analysis was performed to list the possibly altered pathways in which the altered miRNA target genes are involved. RESULTS: Only the miRNA miR-532-3p showed significant differences among the analysed groups, being down-regulated in the EIII/IV group compared with the infertile control group, as well as compared with the EI/II group. The enrichment analysis showed that some genes regulated by this miRNA are involved in important pathways for the acquisition of oocyte competence, such as the oxytocin, calcium, Wnt, FoxO, ErbB and Ras signalling pathways, as well as the oocyte meiosis pathway. CONCLUSION: The present findings bring new perspectives to understanding the follicular microenvironment of infertile women with different stages of endometriosis. It is suggested that the dysregulation of miR-532-3p may be a potential mechanism involved in the aetiopathogenesis of endometriosis-related infertility. Further studies are needed to evaluate these pathways in cumulus cells of infertile women with the disease, as well as their impact on the acquisition of oocyte competence.

Peritoneal fluid of women with endometriosis reduces SOD1 in bovine oocytes in vitro maturation.

Studies have demonstrated oxidative stress in peritoneal fluid (PF) from women with endometriosis and the importance of enzymatic antioxidant machinery to avoid oocyte oxidative damage. Considering that PF constantly surrounds the ovaries and has direct contact with the oocyte at ovulation, we wonder if PF from women with endometriosis may affect antioxidant enzyme gene expression. Thus, the present study aims to evaluate the PF impact from infertile women with minimal and mild endometriosis and from fertile control women without endometriosis on SOD1, CAT, GSR gene's expression in experimental bovine oocytes matured in vitro. Samples of PF were obtained from women who underwent videolaparoscopy-7 infertile with EI/II and 7 fertile without endometriosis. Immature bovine oocytes underwent in vitro maturation in the absence of PF and in the presence of three concentrations (1, 5 and 10%) of PF from fertile and from infertile women with EI/II. After 22 to 24 h of IVM, oocytes were denuded and stored for analysis of SOD1, CAT and GSR by real-time polymerase chain reaction. Oocyte SOD1 expression was significantly lower in the 10% endometriosis group (0.67 ± 0.32) when compared with no-peritoneal fluid (1.05 ± 0.24, p < 0.008) and 10% control groups (1.06 ± 0.22, p < 0.006). These findings raise the possibility of a deleterious influence of PF from women with EI/II on the oocyte, not only after ovulation but also during the maturation process, which could contribute to worsening oocyte quality, being one of the mechanisms related to infertility in patients with endometriosis.

PTGS2 down-regulation in cumulus cells of infertile women with endometriosis.

A deleterious effect of endometriosis on oocyte quality has been proposed. Evidence suggests that cumulus cells could be used as indirect biomarkers of oocyte quality. The PTGS2 gene, which encodes cyclooxygenase 2 (COX-2), is deregulated in endometriotic lesions and plays a crucial role in the acquisition of oocyte competence. To date, research evaluating PTGS2 expression in cumulus cells of infertile patients with endometriosis has not been conducted. The aim this study was to compare the expression levels of PTGS2 in cumulus cells of infertile women, with and without endometriosis, undergoing ovarian stimulation for intracytoplasmic sperm injection (ICSI). Therefore, a case-control study compared PTGS2 gene expression in the cumulus cells of 38 infertile patients with endometriosis and 40 without, using real-time polymerase chain reaction. For the first time, decreased expression of PTGS2 was found in cumulus cells of infertile women with endometriosis compared with controls (7.2 ± 10.5 versus 12.4 ± 15.7), which might be related to reduced levels of COX-2 in the cumulus cells of women with the disease. Consequently, we hypothesize that lower transcript levels of PTGS2 in cumulus cells may be involved in the impairment of oocyte quality, suggesting a possible mechanism involved in disease-related infertility.

Down-regulation of the CYP19A1 gene in cumulus cells of infertile women with endometriosis.

Aromatase plays a fundamental role in the establishment of oocyte quality, which might be compromised in infertile women with endometriosis. The expression of the CYP19A1 gene (that encodes aromatase) was compared in cumulus cells and oestradiol concentrations in the follicular fluid of infertile women with and without endometriosis submitted to ovarian stimulation for intracytoplasmic sperm injection. Cumulus cells were isolated and the expression of the CYP19A1 was quantitated through real-time polymerase chain reaction. Oestradiol concentrations in follicular fluid were measured by chemiluminescence immunoassay. A lower expression of the CYP19A1 in the cumulus cells of infertile women with endometriosis was observed compared with controls (0.17 ± 0.13 and 0.56 ± 0.12, respectively), and no significant difference in the follicular fluid oestradiol concentrations was observed between groups. Our results show reduced expression of the CYP19A1 in cumulus cells of infertile women with endometriosis, which may play a role in the pathogenesis of endometriosis-related infertility.

Endometriosis and physical exercises: a systematic review.

Regular physical exercise seems to have protective effects against diseases that involve inflammatory processes since it induces an increase in the systemic levels of cytokines with anti-inflammatory and antioxidant properties and also acts by reducing estrogen levels. Evidence has suggested that the symptoms associated with endometriosis result from a local inflammatory peritoneal reaction caused by ectopic endometrial implants. Thus, the objective of the present review was to assess the relationship between physical exercise and the prevalence and/or improvement of the symptoms associated with endometriosis. To this end, data available in PubMed (1985-2012) were surveyed using the terms "endometriosis and physical exercises", "endometriosis and life style and physical exercises" in the English language literature. Only 6 of the 935 articles detected were included in the study. These studies tried establish a possible relationship between the practice of physical exercise and the prevalence of endometriosis. The data available are inconclusive regarding the benefits of physical exercise as a risk factor for the disease and no data exist about the potential impact of exercise on the course of the endometriosis. In addition, randomized studies are necessary.

Identification of a rare copy number polymorphic gain at 3q12.2 with candidate genes for familial endometriosis.

Endometriosis is a complex disease that affects 10-15% of women of reproductive age. Familial studies show that relatives of affected patients have a higher risk of developing the disease, implicating a genetic role for this disorder. Little is known about the impact of germline genomic copy number variant (CNV) polymorphisms on the heredity of the disease. In this study, we describe a rare CNV identified in two sisters with familial endometriosis, which contain genes that may increase the susceptibility and progression of this disease. We investigated the presence of CNVs from the endometrium and blood of the sisters with endometriosis and normal endometrium of five women as controls without the disease using array-CGH through the Agilent 2x400K platform. We excluded common CNVs that were present in the database of genomic variation. We identified, in both sisters, a rare CNV gain affecting 113kb at band 3q12.2 involving two candidate genes: ADGRG7 and TFG. The CNV gain was validated by qPCR. ADGRG7 is located at 3q12.2 and encodes a G protein-coupled receptor influencing the NF-kappaß pathway. TFG participates in chromosomal translocations associated with hematologic tumor and soft tissue sarcomas, and is also involved in the NF-kappa B pathway. The CNV gain in this family provides a new candidate genetic marker for future familial endometriosis studies. Additional longitudinal studies of affected families must confirm any associations between this rare CNV gain and genes involved in the NF-kappaß pathway in predisposition to endometriosis.

Caldesmon: new insights for diagnosing endometriosis.

Considerable effort has been invested in searching for less invasive methods of diagnosing endometriosis. Previous studies have indicated altered levels of the CALD1 gene (encoding the protein caldesmon) in endometriosis. The aims of our study were to investigate whether average CALD1 expression and caldesmon protein levels are differentially altered in the endometrium and endometriotic lesions and to evaluate the performance of the CALD1 gene and caldesmon protein as potential biomarkers for endometriosis. Paired biopsies of endometrial tissue (eutopic endometrium) and endometriotic lesions (ectopic endometrium) were obtained from patients with endometriosis to evaluate CALD1 gene expression and caldesmon protein levels by real-time PCR and Western blot analysis, respectively. In addition, immunostaining for caldesmon to determine cellular localization was also performed. Endometrium from women without endometriosis was used as a control. Increased CALD1 expression and caldesmon levels were detected in the endometriotic lesions. The electrophoretic profile of caldesmon by Western blot analysis was clearly different between the control group (endometrium of women without endometriosis) and the group of women with endometriosis (eutopic endometrium and endometriotic lesions). Caldesmon expression as determined by immunostaining showed no variation among the cell types in endometriotic lesions and eutopic endometrium. Stromal cells marked positively in eutopic endometrium from control patients and in the endometriotic lesions. The presence of caldesmon in the endometrium of patients with and without endometriosis permitted diagnoses with 95% sensitivity (specificity 100%) and 100% sensitivity (specificity 100%) for the disease and for minimal to mild endometriosis in the proliferative phase of the menstrual cycle, respectively. In the secretory phase, minimal to mild endometriosis was detected with 90% sensitivity and 93.3% specificity. Caldesmon is a possible predictor of endometrial dysregulation in patients with endometriosis. A potential limitation of our study is the fact that other endometrial diseases were not excluded, and therefore prospective studies are needed to confirm the potential of caldesmon as a biomarker exclusively for endometriosis.

Site-Specific Regulation of Sulfatase and Aromatase Pathways for Estrogen Production in Endometriosis.

Endometriosis is a highly prevalent gynecological disease characterized by lesions in different sites. Regulation of specific estrogen pathways may favor the formation of distinct microenvironments and the progression of endometriosis. However, no study has simultaneously evaluated the gene and protein regulation of the main estrogen-synthesizing enzymes in endometriosis. Thus, our goals were to study the relationship between gene and protein expression of aromatase (CYP19A1 or ARO), steroid sulfatase (STS), and hydroxysteroid 17-beta dehydrogenase (HSD17B1) in superficial (SUP), ovarian (OMA), and deep infiltrating (DIE) endometriotic lesion sites as well as in the eutopic endometrium of patients with (EE) and without (control) endometriosis in the same and large cohort of patients. The site-specific expression of these enzymes within different cells (glandular and stromal components) was also explored. The study included 108 patients surgically diagnosed with endometriosis who provided biopsies of EE and endometriotic lesions and 16 disease-free patients who collected normal endometrium tissue. Our results showed that CYP19A1 was detected in all endometriosis tissues and was in higher levels than in control. Unique patterns of the STS and HSD17B1 levels showed that they were most closely regulated in all tissues, with manifestation at greater levels in DIE compared to the other endometriotic lesion sites, OMA and SUP. Gene and protein expression of ARO, STS, and HSD17B1 occurred at different rates in endometriotic sites or EE. The distinctive levels of these estrogen-synthesizing enzymes in each endometriotic site support the hypothesis of a tissue microenvironment that can both influence and be influenced by the expression of different estrogenic pathways, locally affecting the availability of estrogen needed for maintenance and progression of endometriotic lesions.

Transcriptomic analysis of cumulus cells shows altered pathways in patients with minimal and mild endometriosis.

Endometriosis is a chronic inflammatory disorder that is highly associated with infertility. This association seems to be related to oocyte impairment, mainly in the initial stages of endometriosis (minimal and mild), where no distortions or adhesions are present. Nonetheless, invasive oocyte analyses are not routinely feasible; thus, indirect assessment of oocyte quality is highly desirable, and, in this context, cumulus cells (CCs) may be more suitable targets of analysis. CCs are crucial in oocyte development and could be used as an index of oocyte quality. Therefore, this prospective case-control study aimed to shed light on the infertility mechanisms of endometriosis I/II by analyzing the CCs' mRNA transcription profile (women with endometriosis I/II, n = 9) compared to controls (women with tubal abnormalities or male factor, n = 9). The transcriptomic analyses of CCs from patients with minimal and mild endometriosis revealed 26 differentially expressed genes compared to the controls. The enrichment analysis evidenced some altered molecular processes: Cytokine-cytokine receptor interactions, Chemokine signaling, TNF signaling, NOD-like receptor signaling, NF-kappa B signaling, and inflammatory response. With the exception of CXCL12, all enriched genes were downregulated in CCs from patients with endometriosis. These findings provide a significant achievement in the field of reproductive biology, directing future studies to discover biomarkers of oocyte quality in endometriosis.

Overexpression of miR-200b-3p in Menstrual Blood-Derived Mesenchymal Stem Cells from Endometriosis Women.

The key relationship between Sampson's theory and the presence of mesenchymal stem cells in the menstrual flow (MenSCs), as well as the changes in post-transcriptional regulatory processes as actors in the etiopathogenesis of endometriosis, are poorly understood. No study to date has investigated the imbalance of miRNAs in MenSCs related to the disease. Thus, through literature and in silico analyses, we selected four predicted miRNAs as regulators of EGR1, SNAI1, NR4A1, NR4A2, ID1, LAMC3, and FOSB involved in pathways of apoptosis, angiogenesis, response to steroid hormones, migration, differentiation, and cell proliferation. These genes are frequently overexpressed in the endometriosis condition in our group studies. They were the trigger for the miRNAs search. Therefore, a case-control study was conducted with MenSCs of women with and without endometriosis (ten samples per group). Crossing information obtained from the STRING, PubMed, miRPathDB, miRWalk, and DIANA TOOLS databases, we chose to explore the expression of miR-21-5p, miR-100-5p, miR-143-3p, and miR-200b-3p by RT-qPCR. We found an upregulation of the miR-200b-3p in endometriosis MenSCs (P = 0.0207), with a 7.93-fold change (ratio of geometric means) compared to control. Overexpression of miR-200b has been associated with increased cell proliferation, stemness, and accentuated mesenchymal-epithelial transition process in eutopic endometrium of endometriosis. We believe that dysregulated miR-200b-3p may establish primary changes in the MenSCs, thus favoring tissue implantation at the ectopic site.

Eutopic endometrium from women with endometriosis and chlamydial endometritis share immunological cell types and DNA repair imbalance: A transcriptome meta-analytical perspective.

The aim of this study was to identify the key similarities between the eutopic endometrium of women with endometriosis and chlamydia-induced endometritis taking into account tissue microenvironment heterogeneity, transcript gene profile, and enriched pathways. A meta-analysis of whole transcriptome microarrays was performed using publicly available data, including samples containing both glandular and stromal endometrial components. Control samples were obtained from women without any reported pathological condition. Only samples obtained during the proliferative menstrual phase were included. Cellular tissue heterogeneity was predicted using a method that integrates gene set enrichment and deconvolution approaches. The batch effect was estimated by principal variant component analysis and removed using an empirical Bayes method. Differentially expressed genes were identified using an adjusted p-value < 0.05 and fold change = 1.5. The protein-protein interaction network was built using the STRING database and interaction score over 400. The Molecular Signatures Database was used to analyse the functional enrichment analysis. Both conditions showed similarities in cell types in the microenvironment, particularly CD4+ and CD8+ Tem cells, NKT cells, Th2 cells, basophils, and eosinophils. With regards to the regulation of cellular senescence and DNA integrity/damage checkpoint, which are commonly enriched pathways, 21 genes were down-regulated and directly related to DNA repair. Compared to the endometriosis samples, some chlamydial endometritis samples presented a lack of enriched immune pathways. Our results suggest that both conditions show similar distributions of microenvironment cell types, the downregulation of genes involved in DNA repair and cell cycle control, and pathways involved in immune response evasion.

Identification of suitable reference genes for mesenchymal stem cells from menstrual blood of women with endometriosis.

It has been suggested that menstrual blood-derived mesenchymal stem/stromal cells (MenMSCs) are associated with the etiopathogenesis of endometriosis and considerable effort has been invested in searching for target genes and deciphering associated molecular pathways. However, reference gene stability for proper reproducible normalization in the analyses of the expression data validation is still unexplored in this experimental context. Therefore, in this exploratory study, we used stringent case and control selection criteria and collected menstrual blood from women with a laparoscopic diagnosis of advanced endometriosis and from fertile women without endometriosis. We tested for the first time the stability of 32 candidate reference genes to achieve increased accuracy and reliable results in the quantification of gene expression and direct future experiments using reverse transcription-quantitative PCR (RT-qPCR) in MenMSCs for endometriosis studies. Using the RefFinder web tool, we recommend the EIF2B1 and POP4 reference genes for the normalization of RT-qPCR data in study designs similar to ours. Furthermore, we suggest avoiding the commonly used GAPDH and ACTB reference genes as they are unstable. This high-visibility study is capable of directing different experimental designs as MenMSCs are derived from a minimally invasive tissue source with multifunctional roles in regenerative medicine.

Transcriptome meta-analysis reveals differences of immune profile between eutopic endometrium from stage I-II and III-IV endometriosis independently of hormonal milieu.

Eutopic endometrium appears to be crucial for endometriosis development. Despite of the evident importance, data regarding the cellular microenvironment remain unclear. Our objective was to explore the tissue microenvironment heterogeneity, transcripts, and pathways that are enriched in all phases of the menstrual cycle by analysing publicly deposited data derived from whole transcriptome microarrays of eutopic endometria of women with and without endometriosis. A meta-analysis of the transcriptome microarrays was performed using raw data available from a public database. Eligibility criteria included eutopic endometrium samples from women with endometriosis and healthy controls without any pathological condition reported the presence of an adequately reported normal menstrual phase, and samples containing both glandular and stromal components. Raw data were processed using a robust multiarray average method to provide background correction, normalisation, and summarisation. The batch effect was estimated by principal variant component analysis and removed using an empirical Bayes method. Cellular tissue heterogeneity was inferred using the xCell package. Differentially expressed genes were identified based on a 5% adjusted p value and a 2.0-fold change. Pathways were identified by functional enrichment based on the Molecular Signatures Database, a p value of < 5%, and an FDR q value of ≤ 25%. Genes that were more frequently found in pathways were identified using leading edge analysis. In a manner independent of cycle phase, the subpopulations of activated dendritic cells, CD4 T effector memory phenotype cells, eosinophils, macrophages M1, and natural killer T cells (NKT) were all higher in stage I-II endometriosis compared to those in healthy controls. The subpopulations of M2 macrophages and natural killer T cells were elevated in eutopic endometriums from women with stage III-IV endometriosis, and smooth muscle cells were always more prevalent in healthy eutopic endometriums. Among the differently expressed genes, FOS, FOSB, JUNB, and EGR1 were the most frequently mapped within the interaction networks, and this was independent of stage and cycle phase. The enriched pathways were directly related to immune surveillance, stem cell self-renewal, and epithelial mesenchymal transition. PI3K AKT mTOR, TGF signalling, and interferon alpha/gamma responses were enriched exclusively in stage III-IV endometriosis. The cellular microenvironments and immune cell profiles were different between eutopic endometriums from women with stage I-II and stage III-IV endometriosis, and these differences were independent of the hormonal milieu. Specifically, a pro-inflammatory profile was predominant in stage I-II endometriosis, and M1-M2 polarization into eutopic endometrium may be crucial for the progression of the disease. The higher prevalence of NKT cells in eutopic endometriums from women with endometriosis that was independent of cycle phase or staging suggested a sustained stress and/or damage to these eutopic endometriums. Based on this, the results of this meta-analysis are important for identifying challenges and opportunities for future research.