Gene expression profiles separate endometriosis lesion subtypes and indicate a sensitivity of endometrioma to estrogen suppressive treatments through elevated ESR2 expression.

BACKGROUND: Endometriosis is a common, gynaecological disease characterised by the presence of endometrial-like cells growing outside the uterus. Lesions appear at multiple locations, present with variation in appearance, size and depth of invasion. Despite hormones being the recommended first-line treatment, their efficacy, success and side effects vary widely amongst study populations. Current, hormonal medication for endometriosis is designed to suppress systemic oestrogen. Whether these hormones can influence the lesions themselves is not yet clear. Evidence of hormone receptor expression in endometriotic lesions and their ability to respond is conflicting. A variation in their expression, activation of transcriptional co-regulators and the potential to respond may contribute to their variation in patient outcomes. Identifying patients who would benefit from hormonal treatments remain an important goal in endometriosis research. METHODS: Using gene expression data from endometriosis lesions including endometrioma (OMA, n = 28), superficial peritoneal lesions (SUP, n = 72) and deeply infiltrating lesions (DIE, n = 78), we performed principal component analysis, differential gene expression and gene correlation analyses to assess the impact of menstrual stage, lesion subtype and hormonal treatment on the gene expression. RESULTS: The gene expression profiles did not vary based on menstrual stage, but could distinguish lesion subtypes with OMA significantly differentiating from both SUP and DIE. Additionally, the effect of oestrogen suppression medication altered the gene expression profile in OMA, while such effect was not observed in SUP or DIE. Analysis of the target receptors for hormonal medication indicated ESR2 was differentially expressed in OMA and that genes that correlated with ESR2 varied significantly between medicated and non-medicated OMA samples. CONCLUSIONS: Our results demonstrate of the different lesion types OMA present with strongest response to hormonal treatment directly through ESR2. The data suggests that there may be the potential to target treatment options to individual patients based on pre-surgical diagnoses.

New concepts on the etiology of endometriosis.

Endometriosis is a serious, chronic disorder where endometrial tissue grows outside the uterus, causing severe pelvic pain and infertility. It affects 11% of women. Endometriosis is a multifactorial disorder of unclear etiology, although retrograde menstruation plays a major role. It has a genetic component with over 40 genetic risk factors mapped, although their mechanism of action is still emerging. New evidence suggests a role for retrograde menstruation of endometrial stem/progenitor cells, now that identifying markers of these cells are available. Recent lineage tracing and tissue clearing microscopy and 3D reconstruction has provided new understanding of endometrial glandular structure, particularly the horizontal orientation and interconnection of basalis glands. New sequencing technologies, particularly whole genome DNA sequencing are revealing somatic mutations, including in cancer driver genes, in normal and eutopic endometrium of patients with endometriosis, as well as ectopic endometriotic lesions. Methylome sequencing is offering insight into the regulation of genes and the role of the environmental factors. Single cell RNA sequencing reveals the transcriptome of individual endometrial cells, shedding new light on the diversity and range of cellular subpopulations of the major cell types present in the endometrium and in endometriotic lesions. New endometrial epithelial organoid cultures replicating glandular epithelium are providing tractable models for studying endometriosis. Organoids derived from menstrual fluid offer a non-invasive source of endometrial tissue and a new avenue for testing drugs and developing personalized medicine for treating endometriosis. These new approaches are rapidly advancing our understanding of endometriosis etiology.

Risk factors for non-response and discontinuation of Dienogest in endometriosis patients: A cohort study.

INTRODUCTION: Progestins are commonly prescribed first-line drugs for endometriosis. High rates of non-response and intolerance to these drugs have been previously reported. However, no study to date has investigated the characteristics and comorbidities of patients taking progestins in relation to treatment outcomes, so identifying which patients will respond to or tolerate the treatment is currently impossible. The purpose of this study, therefore, was to identify risk factors for non-response and discontinuation of Dienogest (DNG) in women with endometriosis. MATERIAL AND METHODS: This is a retrospective cohort study including women currently taking, or newly prescribed, DNG for endometriosis-associated pain presenting in the Endometriosis Clinic of the University Hospital of Bern between January 2017 and May 2018. Women with initiation of treatment directly after surgery for endometriosis were excluded. For all participants the symptoms and comorbidities were documented. Effectiveness, tolerability and discontinuation of DNG were the primary end points. Univariate and multivariate binary logistic regression models were carried out to identify risk factors for non-response, intolerance and discontinuation of DNG. RESULTS: A sufficient or excellent treatment response was reported by 85/125 (68%) participants. Genital bleeding during the DNG treatment was negatively (OR 0.185, 95% CI 0.056-0.610, P = .006) and rASRM endometriosis stages III and IV were positively (OR 3.876, 95% CI 1.202-12.498, P = .023) correlated with the DNG response. When accounting for exclusively pretreatment factors, primary dysmenorrhea (OR 0.236, 95% CI 0.090-0.615, P = .003) and suspicion of adenomyosis (OR 0.347, 95% CI 0.135-0.894, P = .028) were inversely correlated with DNG response, and the latter was also correlated with treatment discontinuation (OR 3.189, 95% CI 1.247-8.153, P = .015). CONCLUSIONS: Genital bleeding during the DNG treatment and low rASRM stages are independent risk factors for DNG non-response. Before treatment initiation, primary dysmenorrhea and suspicion of adenomyosis correlate with DNG non-response. The results could assist the clinician first to provide detailed information to women before treatment initiation, second to identify and possibly modify in-therapy factors correlated to treatment effectiveness and lastly to switch treatment on time if needed.

Tissue specific regulation of transcription in endometrium and association with disease.

STUDY QUESTION: Are genetic effects on endometrial gene expression tissue specific and/or associated with reproductive traits and diseases? SUMMARY ANSWER: Analyses of RNA-sequence data and individual genotype data from the endometrium identified novel and disease associated, genetic mechanisms regulating gene expression in the endometrium and showed evidence that these mechanisms are shared across biologically similar tissues. WHAT IS KNOWN ALREADY: The endometrium is a complex tissue vital for female reproduction and is a hypothesized source of cells initiating endometriosis. Understanding genetic regulation specific to, and shared between, tissue types can aid the identification of genes involved in complex genetic diseases. STUDY DESIGN, SIZE, DURATION: RNA-sequence and genotype data from 206 individuals was analysed and results were compared with large publicly available datasets. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA-sequencing and genotype data from 206 endometrial samples was used to identify the influence of genetic variants on gene expression, via expression quantitative trait loci (eQTL) analysis and to compare these endometrial eQTLs with those in other tissues. To investigate the association between endometrial gene expression regulation and reproductive traits and diseases, we conducted a tissue enrichment analysis, transcriptome-wide association study (TWAS) and summary data-based Mendelian randomisation (SMR) analyses. Transcriptomic data was used to test differential gene expression between women with and without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE: A tissue enrichment analysis with endometriosis genome-wide association study summary statistics showed that genes surrounding endometriosis risk loci were significantly enriched in reproductive tissues. A total of 444 sentinel cis-eQTLs (P < 2.57 × 10-9) and 30 trans-eQTLs (P < 4.65 × 10-13) were detected, including 327 novel cis-eQTLs in endometrium. A large proportion (85%) of endometrial eQTLs are present in other tissues. Genetic effects on endometrial gene expression were highly correlated with the genetic effects on reproductive (e.g. uterus, ovary) and digestive tissues (e.g. salivary gland, stomach), supporting a shared genetic regulation of gene expression in biologically similar tissues. The TWAS analysis indicated that gene expression at 39 loci is associated with endometriosis, including five known endometriosis risk loci. SMR analyses identified potential target genes pleiotropically or causally associated with reproductive traits and diseases including endometriosis. However, without taking account of genetic variants, a direct comparison between women with and without endometriosis showed no significant difference in endometrial gene expression. LARGE SCALE DATA: The eQTL dataset generated in this study is available at http://reproductivegenomics.com.au/shiny/endo_eqtl_rna/. Additional datasets supporting the conclusions of this article are included within the article and the supplementary information files, or are available on reasonable request. LIMITATIONS, REASONS FOR CAUTION: Data are derived from fresh tissue samples and expression levels are an average of expression from different cell types within the endometrium. Subtle cell-specifc expression changes may not be detected and differences in cell composition between samples and across the menstrual cycle will contribute to sample variability. Power to detect tissue specific eQTLs and differences between women with and without endometriosis was limited by the sample size in this study. The statistical approaches used in this study identify the likely gene targets for specific genetic risk factors, but not the functional mechanism by which changes in gene expression may influence disease risk. WIDER IMPLICATIONS OF THE FINDINGS: Our results identify novel genetic variants that regulate gene expression in endometrium and the majority of these are shared across tissues. This allows analysis with large publicly available datasets to identify targets for female reproductive traits and diseases. Much larger studies will be required to identify genetic regulation of gene expression that will be specific to endometrium. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Health and Medical Research Council (NHMRC) under project grants GNT1026033, GNT1049472, GNT1046880, GNT1050208, GNT1105321, GNT1083405 and GNT1107258. G.W.M is supported by a NHMRC Fellowship (GNT1078399). J.Y is supported by an ARC Fellowship (FT180100186). There are no competing interests.

Early-stage endometrial cancer, CTNNB1 mutations, and the relation between lymphovascular space invasion and recurrence.

INTRODUCTION: Women diagnosed with early-stage (FIGO 1) endometrial cancer, grade 1 and 2 can have a good prognosis. Most of these women can be treated successfully with a hysterectomy and bilateral salpingo-oophorectomy and without the additional adjuvant treatment that is accompanied by more risks for complications. However, when recurrence does occur, the consequences can be dire. Accurate decisions must therefore be made by surgeons to avoid either under- or over-treatment. Risk and patient stratification for tailoring treatment still need further improvement. Both histopathology and genetic variants could be integrated into the decision process if relevant factors were identified. MATERIAL AND METHODS: Morphological features and the presence of selected genetic mutations in isolated malignant endometrial epithelial cells from these tumors were analyzed in a strictly defined cohort of FIGO 1, grade 1 and 2 low-risk endometrial cancer. Their presence in this cohort, their relation to recurrence, and the association between histopathological features and mutations were determined. This analysis was performed using archival formalin-fixed paraffin-embedded tissue, complete re-evaluation of histopathological features, laser capture microdissection of epithelial cells, and a polymerase chain reaction-based mutational screening assay. RESULTS: Twenty-one women with recurrence, after initial identification as low-risk endometrial cancer, were compared with 20 matched control women. The histological marker of lymphovascular invasion was significantly associated with recurrence. There was also a very high prevalence of mutations in CTNNB1 gene, occurring in 50% of this cohort. PTEN mutations were also observed in 27.8% of cases and PIK3CA mutations in 22.2%; none of these mutations were significantly related to recurrence. CONCLUSIONS: This study supports the importance of lymphovascular space invasion to identify women with significant risk for recurrence in initially low-risk, early-stage endometrial cancer. It also identifies CTNNB1 as a significant mutation in early-stage disease, and although it may not represent a marker for recurrence its high prevalence in early stage disease could have relevance for both pathogenesis and early treatment.

The association between progestins, nuclear receptors expression and inflammation in endometrial stromal cells from women with endometriosis.

Endometriosis is an inflammatory disease and nuclear receptors play a crucial role in mediating the inflammatory response. In endometrial stromal cells (ESC), nuclear receptors expression can be influenced by the local environment. Progestins are first-line, on-label treatments of endometriosis that may have direct effects on endometriotic lesions through these nuclear receptors. Therefore, we investigated whether there was an association between nuclear receptors expression and the influence of progestins on inflammatory cytokines production in a preliminary, in vitro study with primary cultures. ESC from endometrial biopsies of six subjects with histologically confirmed endometriosis were treated for 6 h with medium alone or with TNF-α (10 or 100 ng/ml) in the presence of dienogest (DNG), medroxyprogesterone acetate (MPA) and norethisterone acetate (NETA) 10-5 M. The progestin-mediated change in IL6, IL8 and MCP-1 mRNA transcription was measured, as was the PRA, PRB, GR, AR and MCR protein expression. The change (medium versus TNF-α 10 ng/ml and medium versus TNF-α 100 ng/ml) in IL6 mRNA transcription was positively associated with the change in PRB, but not PRA with both DNG and NETA treatment. The change in IL8 mRNA was negatively associated with AR expression in the presence of NETA. The change in MCP-1 mRNA expression was positively associated with GR expression and negatively associated with MCR after MPA treatment. The associations between the change in cytokines mRNA expression and nuclear receptors protein expression in response to progestins activity may indirectly suggest different activities of these compounds at a local level worthy of further investigations.

Laparoscopic management of ectopic pregnancies: a comparison between interstitial and "more distal" tubal pregnancies.

BACKGROUND/AIMS: Laparoscopy is an established, safe, and feasible management option for tubal pregnancies, even in women with significant hemoperitoneum. In case of interstitial pregnancy, however, a laparoscopic surgical approach is still a matter of debate. The objective of this study is to evaluate the safety and feasibility of a laparoscopic approach to interstitial pregnancies. METHODS: A total of 92 women with ectopic pregnancy who underwent a surgical management from April 2009 to August 2015 were reviewed. Clinical and surgical outcomes of confirmed interstitial pregnancies (n = 10) (IP group) were compared with those of "more distal" tubal pregnancies (n = 79) (TP group). RESULTS: Although there were no differences between the two groups in gestational age, ß-hCG values were significantly higher in the IP group (p = 0.005). All patients with IP were treated by laparoscopic wedge resection. The rate of surgical complications (p = 0.413) and subsequent MTX treatment (p = 0.531) were not significantly different between groups. Operating room (OR) time (p = 0.007) was higher in the IP than in the TP group. After stratification for the presence of hemoperitoneum this difference remained, with patients in the IP group having longer OR time (p = 0.034) and additionally higher intra-operative blood loss (EBL) (p = 0.013). On the other hand, in the absence of hemoperitoneum no differences between the two groups were observed. CONCLUSIONS: In experienced hands, the laparoscopic management of interstitial pregnancies seems to be as safe and feasible as that of other tubal pregnancies. However, it could be technically more challenging, especially in case of hemoperitoneum.

TNFα-induced IKKß complex activation influences epithelial, but not stromal cell survival in endometriosis.

STUDY QUESTION: Can the activity of the IκB kinase (IKKß) complex in endometriotic cells contribute to endometriotic lesion survival? SUMMARY ANSWER: There is a constitutive activity of the IKKß catalytic complex in peritoneal and deeply infiltrating lesions that can influence epithelial, but not stromal cell viability. WHAT IS KNOWN ALREADY: Endometriotic lesions exist in an inflammatory microenvironment with higher local concentrations of cytokines, such as tumour necrosis factor α (TNFα). TNFα stimulates the activation of the IKKß complex, an important nodal point in multiple signalling pathways that influence gene transcription, proliferation and apoptosis. However, few data on the regulation of IKKß in endometriotic tissue are currently available. STUDY DESIGN, SIZE, DURATION: A retrospective analysis of endometriotic tissue from peritoneal, ovarian and deeply infiltrating lesions from 37 women. PARTICIPANTS/MATERIALS, SETTING, METHODS: Basal and activated (phosphorylated) IKKß concentrations were analysed by western blotting and immunohistochemistry. The relationship between the expression and activation of these proteins and peritoneal fluid (TNFα) concentrations, measured via ELISA, was examined. A subsequent in vitro analysis of TNFα treatment on the activation of IKKß and the effect on epithelial and stromal cell viability by its inhibition with PS1145 was also performed. MAIN RESULTS AND ROLE OF CHANCE: Levels of the phosphorylated IKKß complex in endometriotic lesions had a significant positive correlation with peritoneal fluid TNFα concentrations. Phosphorylated IKKß complex was more prevalent in peritoneal and deeply infiltrating endometriosis lesions compared with ovarian lesions. IKKß was present in both epithelial and stromal cells in all lesions but active IKKß was limited to epithelial cells. TNFα stimulated an increased expression of phosphorylated IKKß and the inhibition of this kinase with PS1145 significantly influenced ectopic epithelial cells viability but not eutopic epithelial cells, or endometrial stromal cells. LIMITATIONS, REASONS FOR CAUTION: In vitro analysis on epithelial cells was performed with immortalized cell lines and not primary cell cultures and only low sample numbers were available for the study. WIDER IMPLICATIONS OF THE FINDINGS: The regulation of aberrant signalling pathways represents a promising yet relatively unexplored area of endometriosis progression. The IKKß complex is activated by inflammation and is critical nodal point of numerous downstream kinase-signalling pathways, including NFκB (nuclear factor κB), mTOR (mammalian target of rapamycin) and BAD (Bcl2-antagonist of cell death). This study shows a significant relationship between peritoneal fluid TNFα and IKKß activation in epithelial cells that will have significant consequences for the continued survival of these cells at ectopic locations through the regulation of downstream pathways. LARGE SCALE DATA: None. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Swiss National Science Foundation (Grant Number 320030_140774). The authors have no conflict of interest to declare.

Does dienogest influence the inflammatory response of endometriotic cells? A systematic review.

OBJECTIVE AND DESIGN: A systematic review of all literature was done to assess the ability of the progestin dienogest (DNG) to influence the inflammatory response of endometriotic cells. MAIN OUTCOME MEASURES: In vitro and in vivo studies report an influence of DNG on the inflammatory response in eutopic or ectopic endometrial tissue (animal or human). RESULTS: After strict inclusion criteria were satisfied, 15 studies were identified that reported a DNG influence on the inflammatory response in endometrial tissue. These studies identified a modulation of prostaglandin (PG) production and metabolism (PGE2, PGE2 synthase, cyclo-oxygenase-2 and microsomal PGE synthase-1), pro-inflammatory cytokine and chemokine production [interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-α, monocyte chemoattractant protein-1 and stromal cell-derived factor-1], growth factor biosynthesis (vascular endothelial growth factor and nerve growth factor) and signaling kinases, responsible for the control of inflammation. Evidence supports a progesterone receptor-mediated inhibition of the inflammatory response in PR-expressing epithelial cells. It also indicated that DNG inhibited the inflammatory response in stromal cells, however, whether this was via a PR-mediated mechanism is not clear. CONCLUSIONS: DNG has a significant effect on the inflammatory microenvironment of endometriotic lesions that may contribute to its clinical efficacy. A better understanding of the specific anti-inflammatory activity of DNG and whether this contributes to its clinical efficacy can help develop treatments that focus on the inhibition of inflammation while minimizing hormonal modulation.

A Comparison of Radiocolloid and Indocyanine Green Fluorescence Imaging, Sentinel Lymph Node Mapping in Patients with Cervical Cancer Undergoing Laparoscopic Surgery.

BACKGROUND AND PURPOSE: (99)TC combined with blue-dye mapping is considered the best sentinel lymph node (SLN) mapping technique in cervical cancer. Indocyanine green (ICG) with near infrared fluorescence imaging has been introduced as a new methodology for SLN mapping. The aim of this study was to compare these two techniques in the laparoscopic treatment of cervical cancer. METHODS: Medical records of patients undergoing laparoscopic SLN mapping for cervical cancer with either (99)Tc and patent blue dye (Group 1) or ICG (Group 2) from April 2008 until August 2012 were reviewed. Sensitivity, specificity, and overall and bilateral detection rates were calculated and compared. RESULTS: Fifty-eight patients were included in the study-36 patients in Group 1 and 22 patients in Group 2. Median tumor diameter was 25 and 29 mm, and mean SLN count was 2.1 and 3.7, for Groups 1 and 2, respectively. Mean non-SLN (NSLN) count was 39 for both groups. SLNs were ninefold more likely to be affected by metastatic disease compared with NSLNs (p < 0.005). Sensitivity and specificity were both 100 %. Overall detection rates were 83 and 95.5 % (p = nonsignificant), and bilateral detection rates were 61 and 95.5 % (p < 0.005), for Groups 1 and 2, respectively. In 75 % of cases, SLNs were located along the external or internal iliac nodal basins. CONCLUSIONS: ICG SLN mapping in cervical cancer provides high overall and bilateral detection rates that compare favorably with the current standard of care.

Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF.

BACKGROUND: The study was designed to compare the effect of in vitro FSH stimulation on the hormone production and gene expression profile of granulosa cells (GCs) isolated from single naturally matured follicles obtained from natural cycle in vitro fertilization (NC-IVF) with granulosa cells obtained from conventional gonadotropin-stimulated IVF (c-IVF). METHODS: Lutein granulosa cells from the dominant follicle were isolated and cultured in absence or presence of recombinant FSH. The cultures were run for 48 h and six days. Messenger RNA (mRNA) expressions of anti-Müllerian hormone (AMH) and FSH receptor were measured by quantitative polymerase chain reaction (qPCR). AMH protein and progesterone concentration (P4) in cultured supernatant were measured by ELISA and RIA. RESULTS: Our results showed that the mRNA expression of AMH was significantly higher in GCs from NC- than from c-IVF on day 6 after treatment with FSH (1 IU/mL). The FSH stimulation increased the concentration of AMH in the culture supernatant of GCs from NC-IVF compared with cells from c-IVF. In the culture medium, the AMH level was correlated significantly and positively to progesterone concentration. CONCLUSIONS: Differences in the levels of AMH and progesterone released into the medium by cultured GC as well as in AMH gene expression were observed between GCs obtained under natural and stimulated IVF protocols. The results suggest that artificial gonadotropin stimulation may have an effect on the intra-follicular metabolism. A significant positive correlation between AMH and progesterone may suggest progesterone as a factor influencing AMH secretion.

Detection of the pan neuronal marker PGP9.5 by immuno-histochemistry and quantitative PCR in eutopic endometrium from women with and without endometriosis.

PURPOSE: To assess endometrial gene as well as protein expression of neuroendocrine and supposedly endometriosis-associated product PGP9.5 and pain symptoms in women with endometriosis and controls undergoing laparoscopy, using molecular biological and immuno-histochemical approaches in the same patients. METHODS: Biopsy of eutopic endometrium from 29 patients by sharp curettage, and preparation of paraffin blocks. Determination of PGP9.5 gene expression and protein abundance using qPCR and immuno-histochemistry. RESULTS: qPCR; The PGP9.5 mRNA expression level between women with (N = 16) and without (N = 13) endometriosis was not different, regardless of pain symptoms or menstrual cycle phase. PGP9.5 expression was higher in women who reported pain compared to those who did not; however, this association was not statistically significant. The expression of PGP9.5 mRNA was higher in women with endometriosis and pain during the proliferative than in the secretory phase (P = 0.03). Furthermore, in the first half of the cycle, the abundance of the PGP9.5 transcript was also significantly higher in endometriosis patients compared to those without (P = 0.03). Immuno-histochemistry; Thirteen of the 16 endometriosis patients showed positive PGP9.5 immuno-reactivity in the endometrium, whereas no such signal was observed in women without endometriosis. The absolute number of nerve fibres per mm(2) in women with endometriosis was similar, regardless of the pain symptoms. CONCLUSIONS: PGP9.5 mRNA expression is increased in the proliferative phase of endometriotic women with pain. The presence of nerve fibres was demonstrated by a PGP9.5 protein signal in immuno-histochemistry and restricted to patients with endometriosis. Based on these results, however, there did not appear to be a direct association between the gene expression and protein abundance in women with and without endometriosis or those that experienced pain.

Laparoscopic management of bowel endometriosis: resection margins as a predictor of recurrence.

OBJECTIVE: To evaluate possible predictive factors for recurrence after laparoscopic segmental bowel resection for bowel endometriosis. DESIGN: Cohort study. SETTING: Academic tertiary referral center. METHODS: 95 symptomatic women with bowel endometriosis who underwent laparoscopic segmental bowel resection at the Endometriosis clinic, University of Berne, between 2002 and 2012 were enrolled. Since 14 women were lost to follow-up, 81 formed the final cohort. Clinical and histological characteristics were examined as possible predictive factors for disease recurrence. MAIN OUTCOME MEASURES: Recurrence, defined as a subsequent operation due to recurrent endometriosis-associated pain with a histologically confirmed endometriotic lesion. RESULTS: Recurrence was observed in 13 (16%) patients. Variables that were significantly associated to recurrence by the Cox regression analysis were positive bowel resection margins (hazard ratio 6.5, 95% confidence interval 1.8-23.5, p = 0.005), age <31 years (hazard ratio 5.6, 95% confidence interval 1.7-18.6, p = 0.005) and body mass index ≥23 kg/m(2) (hazard ratio 11.0, 95% confidence interval 2.7-44.6, p = 0.001). CONCLUSIONS: Positive bowel resection margins as well as age <31 years and body mass index ≥23 kg/m(2) appear to be independent predictors of disease recurrence.

Enhanced inflammatory activity of endometriotic lesions from the rectovaginal septum.

Endometriosis is characterised by the growth of ectopic lesions at multiple locations outside the uterine cavity and may be considered a collection of distinct but related conditions. The exact aetiology of endometriosis is still not clear although a role for inflammation is increasingly accepted. We therefore investigated the inflammatory activity of eutopic tissue and that of the matching ectopic lesions from different locations by measuring the genetic expression of inflammatory chemokines and cytokines. The gene expression in matching eutopic and ectopic tissue was compared, as was the gene expression in lesions from different locations. A significantly higher mRNA expression of the chemokines ENA-78 and RANTES and the cytokines IL-6 and TNF α was observed in endometriotic lesions of the rectovaginal septum (RVS) compared to that of matching eutopic tissue. Comparisons across lesion locations showed a significantly higher expression of IL-6 and TNF α in the RVS compared to lesions from either the ovaries or the peritoneum. These results show that the production of some inflammatory chemokines and cytokines is significantly increased in the ectopic endometrial tissue compared to matching eutopic tissue. Furthermore, IL-6 and TNF α are produced in significantly higher quantities in RVS lesions compared to other lesions.

Global Analysis of Transcription Start Sites and Enhancers in Endometrial Stromal Cells and Differences Associated with Endometriosis.

Identifying tissue-specific molecular signatures of active regulatory elements is critical to understanding gene regulatory mechanisms. In this study, transcription start sites (TSS) and enhancers were identified using Cap analysis of gene expression (CAGE) across endometrial stromal cell (ESC) samples obtained from women with (n = 4) and without endometriosis (n = 4). ESC TSSs and enhancers were compared to those reported in other tissue and cell types in FANTOM5 and were integrated with RNA-seq and ATAC-seq data from the same samples for regulatory activity and network analyses. CAGE tag count differences between women with and without endometriosis were statistically tested and tags within close proximity to genetic variants associated with endometriosis risk were identified. Over 90% of tag clusters mapping to promoters were observed in cells and tissues in FANTOM5. However, some potential cell-type-specific promoters and enhancers were also observed. Regions of open chromatin identified using ATAC-seq provided further evidence of the active transcriptional regions identified by CAGE. Despite the small sample number, there was evidence of differences associated with endometriosis at 210 consensus clusters, including IGFBP5, CALD1 and OXTR. ESC TSSs were also located within loci associated with endometriosis risk from genome-wide association studies. This study provides novel evidence of transcriptional differences in endometrial stromal cells associated with endometriosis and provides a valuable cell-type specific resource of active TSSs and enhancers in endometrial stromal cells.

Computer-Aided Histopathological Characterisation of Endometriosis Lesions.

Endometriosis is a common gynaecological condition characterised by the growth of endometrial tissue outside the uterus and is associated with pain and infertility. Currently, the gold standard for endometriosis diagnosis is laparoscopic excision and histological identification of endometrial epithelial and stromal cells. There is, however, currently no known association between the histological appearance, size, morphology, or subtype of endometriosis and disease prognosis. In this study, we used histopathological software to identify and quantify the number of endometrial epithelial and stromal cells within excised endometriotic lesions and assess the relationship between the cell contents and lesion subtypes. Prior to surgery for suspected endometriosis, patients provided menstrual and abdominal pain and dyspareunia scores. Endometriotic lesions removed during laparoscopic surgery were collected and prepared for immunohistochemistry from 26 patients. Endometrial epithelial and stromal cells were identified with Cytokeratin and CD10 antibodies, respectively. Whole slide sections were digitised and the QuPath software was trained to automatically detect and count epithelial and stromal cells across the whole section. Using this classifier, we identified a significantly larger number of strongly labelled CD10 stromal cells (p = 0.0477) in deeply infiltrating lesions (99,970 ± 2962) compared to superficial lesions (2456 ± 859). We found the ratio of epithelial to stromal cells was inverted in deeply infiltrating endometriosis lesions compared to superficial peritoneal and endometrioma lesions and we subsequently identified a correlation between total endometrial cells and abdominal pain (p = 0.0005) when counted via the automated software. Incorporating histological software into current standard diagnostic pipelines may improve endometriosis diagnosis and provide prognostic information in regards to severity and symptoms and eventually provide the potential to personalise adjuvant treatment decisions.

Gene expression of the endocannabinoid system in endometrium through menstrual cycle.

Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to investigate the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated t test, corrected for multiple testing with Bonferroni's method. A total of 40 of the 70 genes were present in > 90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.

Altered differentiation of endometrial mesenchymal stromal fibroblasts is associated with endometriosis susceptibility.

Cellular development is tightly regulated as mature cells with aberrant functions may initiate pathogenic processes. The endometrium is a highly regenerative tissue, shedding and regenerating each month. Endometrial stromal fibroblasts are regenerated each cycle from mesenchymal stem cells and play a pivotal role in endometriosis, a disease characterised by endometrial cells that grow outside the uterus. Why the cells of some women are more capable of developing into endometriosis lesions is not clear. Using isolated, purified and cultured endometrial cells of mesenchymal origin from 19 women with (n = 10) and without (n = 9) endometriosis we analysed the transcriptome of 33,758 individual cells and compared these to clinical characteristics and in vitro growth profiles. We show purified mesenchymal cell cultures include a mix of mesenchymal stem cells and two endometrial stromal fibroblast subtypes with distinct transcriptomic signatures indicative of varied progression through the differentiation processes. The fibroblast subgroup characterised by incomplete differentiation was predominantly (81%) derived from women with endometriosis and exhibited an altered in vitro growth profile. These results uncover an inherent difference in endometrial cells of women with endometriosis and highlight the relevance of cellular differentiation and its potential to contribute to disease susceptibility.

Protocol for the Endometriosis Research Queensland Study (ERQS): an integrated cohort study approach to improve diagnosis and stratify treatment.

INTRODUCTION: Endometriosis is a common gynaecological disease associated with pelvic pain and subfertility. There are no non-invasive diagnostic tests, medical management requires suppression of oestrogens and surgical removal is associated with risk. Endometriosis is a complex genetic disease with variants in at least 27 genetic regions associated with susceptibility. Previous research has implicated a variety of biological mechanisms in multiple cell types. Endometrial and endometriotic epithelial cells acquire somatic mutations at frequency higher than expected in normal tissue. Stromal cells have altered adhesive capacity and immune cells show altered cytotoxicity. Understanding the functional consequences of these genetic variants on each cell type requires the collection of patient symptoms, clinical and genetic data and disease-relevant tissue in an integrated program. METHODS AND ANALYSIS: The aims of this study are to collect tissue associated with endometriosis, chart the genetic architecture related to endometriosis in this tissue, isolate and propagate patient-specific cellular models, understand the functional consequence of these genetic variants and how they interact with environmental factors in pathogenesis and treatment response.We will collect patient information from online questionnaires prior to surgery and at 6 and 12 months postsurgery. Treating physicians will document detailed surgical data. During surgery, we will collect blood, peritoneal fluid, endometrium and endometriotic tissue. Tissue will be used to isolate and propagate in vitro models of individual cells. Genome wide genotyping and gene expression data will be generated. Somatic mutations will be identified via whole genome sequencing. ETHICS AND DISSEMINATION: The study has been approved and will be monitored by the Metro North Human Research Ethics committee (HREC) and research activities at the University of Queensland (UQ) will be overseen by the UQ HREC with annual reports submitted. Research results will be published in peer-reviewed journals and presented at conferences were appropriate. This study involves human participants and was approved by RBWH Human Research Ethics Committee; HREC/2019/QRBW/56763.The University of Queensland; 2017002744. Participants gave informed consent to participate in the study before taking part.

Lessons from implementing the Australian National Action Plan for Endometriosis.

Abstract: Endometriosis is a common yet under-recognised chronic disease with one in nine (more than 830,000) women and those assigned female at birth diagnosed with endometriosis by the age of 44 years in Australia. In 2018, Australia was the first country to develop a roadmap and blueprint to tackle endometriosis in a nationwide, coordinated manner. This blueprint is outlined in the National Action Plan for Endometriosis (NAPE), created from a partnership between government, endometriosis experts and advocacy groups. The NAPE aims to improve patient outcomes in the areas of awareness and education, clinical management and care and research. As researchers and clinicians are working to improve the lives of those with endometriosis, we discuss our experiences since the launch of the plan to highlight areas of consideration by other countries when developing research priorities and clinical plans. Historically, major barriers for those with endometriosis have been twofold; first, obtaining a diagnosis and secondly, effective symptom management post-diagnosis. In recent years, there have been calls to move away from the historically accepted 'gold-standard' surgical diagnosis and single-provider specialist care. As there are currently no reliable biomarkers for endometriosis diagnosis, specialist endometriosis scans and MRI incorporating artificial intelligence offer a novel method of visualisation and promising affordable non-invasive diagnostic tool incorporating well-established technologies. The recognised challenges of ongoing pain and symptom management, a holistic interdisciplinary care approach and access to a chronic disease management plan, could lead to improved patient outcomes while reducing healthcare costs. Lay summary: Endometriosis is a chronic disease where tissue like the lining of the uterus is found in other locations around the body. For the 830,000 people living with endometriosis in Australia, this often results in an immense burden on all aspects of daily life. In 2018, Australia was the first country to introduce a roadmap and blueprint to tackle endometriosis in a nationwide coordinated manner with the National Action Plan for Endometriosis. This plan was created as a partnership between government, endometriosis experts and advocacy groups. There are several other countries who are now considering similar plans to address the burden of endometriosis. As researchers and clinicians are working to improve the lives of those with endometriosis, we share our experiences and discuss areas that should be considered when developing these national plans, including diagnostic pathways without the need for surgery, and building new centres of expertise in Endometriosis and Pelvic Pain.