Extensive heterogeneity in the expression of steroid receptors in superficial peritoneal endometriotic lesions.

RESEARCH QUESTION: Is the expression of steroid hormone receptors (oestrogen receptor-α and progesterone receptor A/B) and proliferative markers (Bcl-2 and Ki67) uniform among superficial peritoneal endometriotic lesions? DESIGN: A retrospective cohort study of 24 patients with surgically and histologically confirmed endometriosis. Immunofluorescence was used to determine the proportion of oestrogen receptor-α (ERα), progesterone receptor A/B, Bcl-2 and Ki67 positive cells in 271 endometriotic lesions (defined as endometriotic gland profile/s within an individual region of CD10 stromal immunostaining from a single biopsy) from 67 endometriotic biopsies from 24 patients. Data were analysed to examine associations related to menstrual cycle stage, lesion location and gland morphology. RESULTS: Oestrogen receptor-α and progesterone receptor A/B expression in superficial peritoneal endometriotic lesions was extremely heterogeneous. Bcl-2 immunostaining in endometriotic lesions was also variable, whereas Ki67 immunostaining was minimal. Menstrual cycle stage associations were limited in steroid hormone receptor and Bcl-2 expression in lesions. Patterns in progesterone receptor A/B and Bcl-2 immunostaining were associated with lesion location. Bcl-2 was differentially expressed, based on lesion gland morphology. CONCLUSIONS: These data demonstrate considerable diversity in the expression of steroid hormone receptors and Bcl-2 between lesions, even within an individual patient.

Distribution of smooth muscle actin and collagen in superficial peritoneal endometriotic lesions varies from the surrounding microenvironment.

RESEARCH QUESTION: Do different subtypes of superficial peritoneal endometriotic lesions exist, based on the presence and morphology of smooth muscle, collagen fibres and immune cell populations? DESIGN: A retrospective cohort study of 24 patients, from across the menstrual cycle, with surgically and histologically confirmed endometriosis. Immunofluorescence was used to delineate the CD10 stromal area of lesions (n = 271 lesions from 67 endometriotic biopsies), and then smooth muscle actin (SMA) positive tissue and immune cell populations (CD45+ and CD68+) were quantified within and adjacent to these lesions. Second harmonic generation microscopy was used to evaluate the presence and morphology of type-1 collagen fibres within and surrounding lesions. RESULTS: Overall, immune cell numbers and the area of SMA and collagen within endometriotic lesions tended to be low, but a spectrum of presentations significantly varied, particularly in the adjacent tissue microenvironment, based on lesion locations, the morphology of endometriotic gland profiles, or both. Lesions in which collagen fibres formed well aligned capsules around the CD10+ stromal border were identified compared with lesions in which collagen fibre distribution was random. Considerable inter- and intra-patient variability in the morphology of SMA and collagen was observed within and surrounding lesions. CONCLUSION: These data demonstrate considerable diversity in the presence of immune cells and morphology of SMA and collagen within, but even more so, surrounding endometriotic lesions, even within individual patients. This heterogeneity, especially within individual patients, presents a challenge to incorporating these cell and tissue types into any new endometriosis classification systems or prognostic approaches.

Cyclic processes in the uterine tubes, endometrium, myometrium, and cervix: pathways and perturbations.

This review leads the 2023 Call for Papers in MHR: 'Cyclical function of the female reproductive tract' and will outline the complex and fascinating changes that take place in the reproductive tract during the menstrual cycle. We will also explore associated reproductive tract abnormalities that impact or are impacted by the menstrual cycle. Between menarche and menopause, women and people who menstruate living in high-income countries can expect to experience ∼450 menstrual cycles. The primary function of the menstrual cycle is to prepare the reproductive system for pregnancy in the event of fertilization. In the absence of pregnancy, ovarian hormone levels fall, triggering the end of the menstrual cycle and onset of menstruation. We have chosen to exclude the ovaries and focus on the other structures that make up the reproductive tract: uterine tubes, endometrium, myometrium, and cervix, which also functionally change in response to fluctuations in ovarian hormone production across the menstrual cycle. This inaugural paper for the 2023 MHR special collection will discuss our current understanding of the normal physiological processes involved in uterine cyclicity (limited specifically to the uterine tubes, endometrium, myometrium, and cervix) in humans, and other mammals where relevant. We will emphasize where knowledge gaps exist and highlight the impact that reproductive tract and uterine cycle perturbations have on health and fertility.

Evidence for the role of multidisciplinary team care in people with pelvic pain and endometriosis: A systematic review.

BACKGROUND: Endometriosis is a chronic, inflammatory condition characterised by the presence of endometrial-like tissue outside the uterine cavity. Given the multi-system nature of the disease and the potential for significant negative impact on quality of life, there has been a long-standing recognition of the need for multidisciplinary care for people with endometriosis. However, there is paucity to the data supporting this approach, and much of the evidence is anecdotal. AIM: This systematic review aims to describe recent evidence-based models and patient-centred perspectives of multidisciplinary care for endometriosis, to improve understanding of the role of an integrated, multidisciplinary team in effectively addressing patients' care needs. MATERIALS AND METHODS: PubMed, Medline, Embase and Web of Science were searched for relevant articles published between 1 January 2010 to 7 July 2022. RESULTS: Nineteen studies met the inclusion and exclusion criteria and pinpointed a multidisciplinary team consisting of gynaecologists, pain specialists, nurses, physiotherapists, psychologists, sex therapists, nutritionists, complementary medicine practitioners, and social workers to be most commonly utilised in holistically managing people with pelvic pain and endometriosis. Furthermore, patient perspectives on care highlighted the need for reliable information, respect and validation of experiences or preferences, discussion of long-term treatment plans and social and emotional supports. CONCLUSION: The trend for multidisciplinary team care for people with endometriosis is growing. Further consumer-driven clinical studies and outcome evaluations need to be conducted to determine the effect of multidisciplinary care on improvements to quality of life for people living with endometriosis and or pelvic pain.

Elucidating the role of long intergenic non-coding RNA 339 in human endometrium and endometriosis.

Endometriosis is a complex disease, influenced by genetic factors. Genetic markers associated with endometriosis exist at chromosome 1p36.12 and lead to altered expression of the long intergenic non-coding RNA 339 (LINC00339), however, the role of LINC00339 in endometriosis pathophysiology remains unknown. The aim of this work was to characterize the expression patterns of LINC00339 mRNA in endometrium and endometriotic lesions in situ and to determine the functional role of LINC00339 in human endometrium. We employed RNA-sequencing (RNA-seq), quantitative RT-PCR and in situ hybridization to investigate the abundance of LINC00339 transcripts in endometrium and endometrial cell lines and to describe the pattern and localization of LINC00339 expression in endometrium and endometriotic lesions. LINC00339 mRNA expression was manipulated (overexpressed and silenced) in endometrial stromal cell lines and RNA-seq data from overexpression models were analysed using online bioinformatics platforms (STRING and Ingenuity Pathway Analysis) to determine functional processes. We demonstrated the expression of LINC00339 in endometriotic lesions for the first time; we found LINC00339 expression was restricted to the lesion foci and absent in surrounding non-lesion tissue. Furthermore, manipulation of LINC00339 expression in endometrial stromal cell lines significantly impacted the expression of genes involved in immune defence pathways. These studies identify a novel mechanism for LINC00339 activity in endometrium and endometriosis, paving the way for future work, which is essential for understanding the pathogenesis of endometriosis.

Comparing endometriotic lesions with eutopic endometrium: time to shift focus?

Endometriosis is a heterogeneous disease in terms of patient symptoms, treatment responsiveness and the presentation of endometriotic lesions. This article explores the histological features of endometriotic lesions, highlighting their sometimes underappreciated heterogeneity. We note the variability in evidence for and against the menstrual cycle responsiveness of lesions and consider the utility of drawing parallels between endometriotic lesions and eutopic endometrium. We ask whether histopathologic features beyond just the presence/absence of endometrial-like glands and/or stroma could help improve disease stratification. At the same time, we acknowledge the desire of many clinicians and patients to avoid invasive surgery thereby limiting the ability to histologically phenotype lesions. The ability to derive clinically useful histological information from endometriotic lesions, in association with patient data, would be invaluable to clinicians to help improve treatment options in such a diverse group of patients. However, in suggesting that a shift in focus may enable the development of a better patient stratification system, we recognise that our wish for a single comprehensive stratification system may be beyond reach for a disease of such diverse presentation.

Associations between body mass index and the surgical phenotype and location of endometriotic lesions.

RESEARCH QUESTION: Is there a relationship between body mass index (BMI) and endometriotic lesions, specifically surgical phenotype and lesion location? DESIGN: An observational retrospective cohort study at the Royal Women's Hospital, Melbourne, Australia, including 471 histologically confirmed endometriosis patients. Statistical analyses included multivariate logistic regression and multivariate modelling, correcting for multiple testing. Outcomes were the presence or absence of surgically classified lesion phenotypes, as per revised American Society for Reproductive Medicine criteria including superficial or deep, peritoneal or ovarian, and adhesions (Study I); and lesions at specific anatomical locations (including pelvic side wall, uterosacral ligament, pouch of Douglas, ovarian, uterovesical fold, bladder, and pararectal endometriosis) (Study II). RESULTS: In Study I, patients with higher BMI were more likely to have superficial peritoneal lesions (odds ratio [OR] 1.070, 95% confidence interval [CI] 1.004-1.144; P = 0.044), and less likely to have deep ovarian lesions (OR 0.928, 95% CI 0.864-0.993; P = 0.034). In Study II, patients with higher BMI were less likely to have uterovesical fold lesions (OR 0.927, 95% CI 0.867-0.985; P = 0.021) or anterior compartment lesions (OR 0.940, 95% CI 0.888-0.989; P = 0.023). After correcting for multiple testing, the relationship between BMI and lesion phenotypes did not persist (P > 0.01). CONCLUSIONS: This analysis does not conclusively support an influence of BMI on endometriotic lesion phenotype based on surgical classification or location. Further investigation of the physiological disturbances underlying BMI and the promotion of endometriotic lesion phenotypes and their location is warranted, but any effect is likely to be small.

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.

Obesity does not alter endometrial gene expression in women with endometriosis.

RESEARCH QUESTION: Does obesity affect endometrial gene expression in women with endometriosis, specifically women with stage I disease? DESIGN: Differential gene expression analysis was conducted on endometrium from women with and without endometriosis (n = 169). Women were diagnosed after surgical visualization and staged according to the revised American Society for Reproductive Medicine (stage I-IV). Women were grouped by body mass index (BMI) (kg/m2) as underweight, normal, pre-obese or obese. After accounting for menstrual cycle stage, endometrial gene expression was analysed by BMI (continuous and grouped) in women with endometriosis, and in non-endometriosis controls. RESULTS: No significant interaction effect was found between BMI and endometriosis status on endometrial gene expression. We have previously reported that obese women with endometriosis have a reduced incidence of stage I disease; however, stratifying our analysis into stage I endometriosis versus combined II, III and IV endometriosis failed to reveal any differentially expressed endometrial genes between normal, pre-obese and obese patients. CONCLUSIONS: Despite obesity having deleterious effects on endometrial gene expression in other gynaecological pathologies, e.g. endometrial cancer and polycystic ovary syndrome, our results do not support an association between BMI and altered endometrial gene expression in women with or without endometriosis.

The Association of Sonographic Evidence of Adenomyosis with Severe Endometriosis and Gene Expression in Eutopic Endometrium.

STUDY OBJECTIVE: To examine the presence of sonographic evidence of adenomyosis (SEOA) in patients undergoing laparoscopic surgery for the investigation of endometriosis and to assess if there is an association between SEOA and endometriosis severity. Using gene expression analysis, we also aimed to determine if gene expression in eutopic endometria differed in patients with and without adenomyosis. DESIGN: A prospective study (Canadian Task Force classification II-2). SETTING: A tertiary medical center. PATIENTS: Reproductive-age women who underwent laparoscopic surgery after presenting to a pelvic pain-focused gynecology clinic. INTERVENTIONS: Endometrial tissue, detailed patient questionnaires, pathology, and surgical notes were collected. Sonographic data from tertiary ultrasounds performed up to 12 months before surgery were retrospectively added (n = 234, researchers blinded to surgical and pathological findings). Gene array data from endometrial biopsies (n = 41) were used to analyze differential gene expression; patients were divided into 2 groups according to the presence or absence of SEOA. MEASUREMENTS AND MAIN RESULTS: Of the 588 patients recruited, 234 (40%) had an available pelvic scan and were included in this study. The average age of the included women was 30.6 years, with 35% having SEOA. Patients with SEOA were 5.4 years older (p = .02). There was no significant difference in the rates of endometriosis between groups; however, patients with SEOA were more likely to have stage IV endometriosis (41% vs 9.8%, p <.001). Patients with SEOA were also more likely to have other markers of severe endometriosis such as endometriomas and deep infiltrating endometriosis (p <.001). No significant difference was observed in endometrial gene expression between adenomyosis cases and controls after adjusting for menstrual c`ycle phases and the presence/absence of endometriosis. CONCLUSION: Sonographic features of adenomyosis may be included as a component of the clinical assessment when attempting to predict the presence of severe endometriosis. No differences in gene expression were observed. Further research is needed to characterize uterine adenomyosis and to explore molecular pathways involved in its pathogenesis.

Endometriosis risk alleles at 1p36.12 act through inverse regulation of CDC42 and LINC00339.

Genome-wide association studies (GWAS) have identified markers within the WNT4 region on chromosome 1p36.12 showing consistent and strong association with increasing endometriosis risk. Fine mapping using sequence and imputed genotype data has revealed strong candidates for the causal SNPs within these critical regions; however, the molecular pathogenesis of these SNPs is currently unknown. We used gene expression data collected from whole blood from 862 individuals and endometrial tissue from 136 individuals from independent populations of European descent to examine the mechanism underlying endometriosis susceptibility. Association mapping results from 7,090 individuals (2,594 cases and 4,496 controls) supported rs3820282 as the SNP with the strongest association for endometriosis risk (P = 1.84 × 10−5, OR = 1.244 (1.126-1.375)). SNP rs3820282 is a significant eQTL in whole blood decreasing expression of LINC00339 (also known as HSPC157) and increasing expression of CDC42 (P = 2.0 ×10−54 and 4.5x10−4 respectively). The largest effects were for two LINC00339 probes (P = 2.0 ×10−54; 1.0 × 10−34). The eQTL for LINC00339 was also observed in endometrial tissue (P = 2.4 ×10−8) with the same direction of effect for both whole blood and endometrial tissue. There was no evidence for eQTL effects for WNT4. Chromatin conformation capture provides evidence for risk SNPs interacting with the promoters of both LINC00339 and CDC4 and luciferase reporter assays suggest the risk SNP rs12038474 is located in a transcriptional silencer for CDC42 and the risk allele increases expression of CDC42. However, no effect of rs3820282 was observed in the LINC00339 expression in Ishikawa cells. Taken together, our results suggest that SNPs increasing endometriosis risk in this region act through CDC42, but further functional studies are required to rule out inverse regulation of both LINC00339 and CDC42.

The genetic regulation of transcription in human endometrial tissue.

Study question: Do genetic effects regulate gene expression in human endometrium? Summary answer: This study demonstrated strong genetic effects on endometrial gene expression and some evidence for genetic regulation of gene expression in a menstrual cycle stage-specific manner. What is known already: Genetic effects on expression levels for many genes are tissue specific. Endometrial gene expression varies across menstrual cycle stages and between individuals, but there are limited data on genetic control of expression in endometrium. Study design, size, duration: We analysed genome-wide genotype and gene expression data to map cis expression quantitative trait loci (eQTL) in endometrium. Participants/materials, setting, methods: We recruited 123 women of European ancestry. DNA samples from blood were genotyped on Illumina HumanCoreExome chips. Total RNA was extracted from endometrial tissues. Whole-transcriptome profiles were characterized using Illumina Human HT-12 v4.0 Expression Beadchips. We performed eQTL mapping with ~8 000 000 genotyped and imputed single nucleotide polymorphisms (SNPs) and 12 329 genes. Main results and the role of chance: We identified a total of 18 595 cis SNP-probe associations at a study-wide level of significance (P < 1 × 10-7), which correspond to independent eQTLs for 198 unique genes. The eQTLs with the largest effect in endometrial tissue were rs4902335 for CHURC1 (P = 1.05 × 10-32) and rs147253019 for ZP3 (P = 8.22 × 10-30). We further performed a context-specific eQTL analysis to investigate if genetic effects on gene expression regulation act in a menstrual cycle-specific manner. Interestingly, five cis-eQTLs were identified with a significant stage-by-genotype interaction. The strongest stage interaction was the eQTL for C10ORF33 (PYROXD2) with SNP rs2296438 (P = 2.0 × 10-4), where we observe a 2-fold difference in the average expression levels of heterozygous samples depending on the stage of the menstrual cycle. Large scale data: The summary eQTL results are publicly available to browse or download. Limitations, reasons for caution: A limitation of the present study was the relatively modest sample size. It was not powered to identify trans-eQTLs and larger sample sizes will also be needed to provide better power to detect cis-eQTLs and cycle stage-specific effects, given the substantial changes in expression across the menstrual cycle for many genes. Wider implications of the findings: Identification of endometrial eQTLs provides a platform for better understanding genetic effects on endometriosis risk and other endometrial-related pathologies. Study funding/competing interest(s): Funding for this work was provided by NHMRC Project Grants GNT1026033, GNT1049472, GNT1046880, GNT1050208, GNT1105321 and APP1083405. There are no competing interests.

Endometrial vezatin and its association with endometriosis risk.

STUDY QUESTION: Do endometriosis risk-associated single nucleotide polymorphisms (SNPs) found at the 12q22 locus have effects on vezatin ( ITALIC! VEZT) expression? SUMMARY ANSWER: The original genome-wide association study (GWAS) SNP (rs10859871), and other newly identified association signals, demonstrate strong evidence for ITALIC! cis-expression quantitative trait loci (eQTL) effects on ITALIC! VEZT expression. WHAT IS KNOWN ALREADY: GWAS have identified several disease-risk loci (SNPs) associated with endometriosis. The SNP rs10859871 is located within the ITALIC! VEZT gene. ITALIC! VEZT expression is altered in the endometrium of endometriosis patients and is an excellent candidate for having a causal role in endometriosis. Most of the SNPs identified from GWAS are not located within the coding region of the genome. However, they are likely to have an effect on the regulation of gene expression. Genetic variants that affect levels of gene expression are called expression quantitative trait loci (eQTL). STUDY DESIGN, SIZE, DURATION: Samples for genotyping and ITALIC! VEZT variant screening were drawn from women recruited for genetic studies in Australia/New Zealand and women undergoing surgery in a tertiary care centre. Coding variants for ITALIC! VEZT were screened in blood from 100 unrelated individuals (endometriosis-dense families) from the QIMR Berghofer Medical Research Institute dataset. SNPs at the 12q22 locus were imputed and reanalysed for their association with endometriosis. Reanalysis of endometriosis risk-association was performed on a final combined Australian dataset of 2594 cases and 4496 controls. Gene expression was performed on 136 endometrial samples. eQTL analysis in whole blood was performed on 862 individuals from the Brisbane Systems Genetics Study. Endometrial tissue-specific eQTL analysis was performed on 122 samples (eutopic endometrium) collected following laparoscopic surgery. VEZT protein expression studies employed ITALIC! n = 56 (western blotting) and ITALIC! n = 42 (immunohistochemistry) endometrial samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: The women recruited for this study provided blood and/or endometrial tissue samples in a hospital setting. Genomic DNA was screened for common and coding variants. SNPs of interest in the 12q22 region were genotyped using Agena MassARRAY technology or Taqman SNP genotyping assay. Gene expression profiles from RNA extracted from blood and endometrial tissue samples were generated using Illumina whole-genome expression chips (Human HT-12 v4.0). Whole protein extracted from endometrium was used for VEZT western blots, and paraffin sections of endometrium were employed for VEZT immunohistochemistry semi-quantitative analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 11 coding variants of ITALIC! VEZT (including one novel variant) were identified from an endometriosis-dense cohort. Polymorphic coding and imputed SNPs were combined with previous GWAS data to reanalyse the endometriosis risk association of the 12q22 region. The disease association signal at 12q22 was due to coding variants in ITALIC! VEZT or ITALIC! FGD6 (FYVE, RhoGEF and PH domain-containing 6) and SNPs with the strongest signals were either intronic or intergenic. We found strong evidence for ITALIC! VEZT cis-eQTLs with the sentinel SNP (rs10859871) in blood and endometrium, where the endometriosis risk allele (C) was associated with an increase in ITALIC! VEZT expression. We could not demonstrate this genotype-specific effect on VEZT protein expression in endometrium. However, we did observe a menstrual cycle stage specific increase in VEZT protein expression in endometrial glands, specific to the secretory phase ( ITALIC! P = 2.0 × 10(-4)). LIMITATIONS, REASONS FOR CAUTION: In comparison to the blood sample datasets, the study numbers of endometrial tissues were substantially reduced. Protein studies failed to complement RNA results, also likely a reflection of the low study numbers in these experiments. ITALIC! In silico prediction tools used in this investigation are typically based on cell lines different to our tissues of interest, thus any functional annotations drawn from these approaches should be considered carefully. Therefore, functional studies on VEZT and related pathway components are still warranted to unequivocally implicate a causal role for VEZT in endometriosis pathophysiology. WIDER IMPLICATIONS OF THE FINDINGS: GWAS have proven to be very valuable tools for deciphering complex diseases. Endometriosis is a text-book example of a complex disease, involving genetic, lifestyle and environmental influences. Our focused investigation of the 12q22 region validates an association with increased endometriosis risk. Endometriosis risk SNPs (including rs10859871) located within this locus demonstrated evidence for ITALIC! cis-eQTLs on ITALIC! VEZT expression. By examining women who possess an enhanced genetic risk of developing endometriosis, we have identified an effect on ITALIC! VEZT expression and therefore a potential gene/gene pathway in endometriosis disease establishment and development. STUDY FUNDING/COMPETING INTERESTS: Funding for this work was provided by NHMRC Project Grants GNT1012245, GNT1026033, GNT1049472 and GNT1046880. G.W.M. is supported by the NHMRC Fellowship scheme (GNT1078399). S.J.H.-C. is supported by the J.N. Peters Bequest Fellowship. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Differences in the cellular composition of small versus large uterine fibroids.

Uterine fibroids are clonally derived from a single cell; however, despite being monoclonal, the cellular phenotypes that make up uterine fibroids are heterogeneous consisting of predominantly smooth muscle cells (SMC) and fibroblasts. This raises the question as to when clonal cell differentiation occurs during fibroid development, and does this information provide clues about possible mechanisms regulating the growth process that leads to fibroids of symptom-causing size? This study investigated the differences in the cellular composition of fibroids by immunohistochemistry (IHC). A tissue microarray (n = 21 hysterectomy cases) was used for the investigation of large uterine fibroids and normal myometrium. An investigation of small fibroids (≤ 5mm) used a separate group of samples (n = 7 hysterectomy cases, total of n = 17 fibroids). A panel of cell phenotypic markers was selected based on our previous in situ investigations and included aldehyde dehydrogenase 1 (ALDH1A1) and vimentin for different fibroblast sub-populations, smooth muscle actin (SMA) as a marker for SMCs, CD31 for endothelial cells and CD45 for leucocytes. Proliferating cell nuclear antigen (PCNA) was also studied to identify proliferating cells. The cellular composition of small fibroids differs significantly from large fibroids. Small fibroids are more cellular (increased cells/mm(2)) than large fibroids, have more blood vessels and also have a higher ratio of SMC to fibroblasts than large fibroids. Large fibroids have more cell proliferation (measured by PCNA) and fewer leucocytes (measured by CD45) than adjacent myometrium, whereas small fibroids are less proliferative and have similar number of leucocytes to myometrium. Different cellular composition between fibroids of different sizes may provide important clues as to the mechanisms that drive fibroid growth.

Functional evaluation of genetic variants associated with endometriosis near GREB1.

STUDY QUESTION: Do DNA variants in the growth regulation by estrogen in breast cancer 1 (GREB1) region regulate endometrial GREB1 expression and increase the risk of developing endometriosis in women? SUMMARY ANSWER: We identified new single nucleotide polymorphisms (SNPs) with strong association with endometriosis at the GREB1 locus although we did not detect altered GREB1 expression in endometriosis patients with defined genotypes. WHAT IS ALREADY KNOWN: Genome-wide association studies have identified the GREB1 region on chromosome 2p25.1 for increasing endometriosis risk. The differential expression of GREB1 has also been reported by others in association with endometriosis disease phenotype. STUDY DESIGN, SIZE, DURATION: Fine mapping studies comprehensively evaluated SNPs within the GREB1 region in a large-scale data set (>2500 cases and >4000 controls). Publicly available bioinformatics tools were employed to functionally annotate SNPs showing the strongest association signal with endometriosis risk. Endometrial GREB1 mRNA and protein expression was studied with respect to phases of the menstrual cycle (n = 2-45 per cycle stage) and expression quantitative trait loci (eQTL) analysis for significant SNPs were undertaken for GREB1 [mRNA (n = 94) and protein (n = 44) in endometrium]. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants in this study are females who provided blood and/or endometrial tissue samples in a hospital setting. The key SNPs were genotyped using Sequenom MassARRAY. The functional roles and regulatory annotations for identified SNPs are predicted by various publicly available bioinformatics tools. Endometrial GREB1 expression work employed qRT-PCR, western blotting and immunohistochemistry studies. MAIN RESULTS AND THE ROLE OF CHANCE: Fine mapping results identified a number of SNPs showing stronger association (0.004 < P < 0.032) with endometriosis risk than the original GWAS SNP (rs13394619) (P = 0.034). Some of these SNPs were predicted to have functional roles, for example, interaction with transcription factor motifs. The haplotype (a combination of alleles) formed by the risk alleles from two common SNPs showed significant association (P = 0.026) with endometriosis and epistasis analysis showed no evidence for interaction between the two SNPs, suggesting an additive effect of SNPs on endometriosis risk. In normal human endometrium, GREB1 protein expression was altered depending on the cycle stage (significantly different in late proliferative versus late secretory, P < 0.05) and cell type (glandular epithelium, not stromal cells). However, GREB1 expression in endometriosis cases versus controls and eQTL analyses did not reveal any significant changes. LIMITATIONS, REASONS FOR CAUTION: In silico prediction tools are generally based on cell lines different to our tissue and disease of interest. Functional annotations drawn from these analyses should be considered with this limitation in mind. We identified cell-specific and hormone-specific changes in GREB1 protein expression. The lack of a significant difference observed following our GREB1 expression studies may be the result of moderate power on mixed cell populations in the endometrial tissue samples. WIDER IMPLICATIONS OF THE FINDINGS: This study further implicates the GREB1 region on chromosome 2p25.1 and the GREB1 gene with involvement in endometriosis risk. More detailed functional studies are required to determine the role of the novel GREB1 transcripts in endometriosis pathophysiology. STUDY FUNDING/COMPETING INTERESTS: Funding for this work was provided by NHMRC Project Grants APP1012245, APP1026033, APP1049472 and APP1046880. There are no competing interests.

Clonality of smooth muscle and fibroblast cell populations isolated from human fibroid and myometrial tissues.

Uterine fibroids are conventionally defined as clonally derived benign tumours from the proliferation of a single smooth muscle cell (SMC). We have previously identified fibroblast-like cells in fibroids, the presence of which raises the question as to whether all cells within the fibroid have the same clonal origin. The first aim of this study was to develop a fluorescence-activated cell sorting (FACS)-based method to isolate different cell types from human myometrium and fibroid tissues. Secondly, we aimed to use X chromosome inactivation analysis to determine the clonality of cell subpopulations isolated from myometrial and fibroid tissues. Human myometrium and fibroid tissues were collected from women undergoing hysterectomy. Immunohistochemistry (IHC) and flow cytometry confirmed that in addition to SMCs, fibroblasts constitute a significant proportion of cells in human myometrium and fibroid tissues. FACS based on CD90 and ALDH1 reliably separated cells into three myometrial and four fibroid subpopulations: SMCs, vascular smooth muscle cells and two fibroblast subsets. Clonality was first determined by X chromosome inactivation using the classic DNA methylation-sensitive HUMARA assay. Data from this assay were highly variable, with only a quarter of samples meeting the definition of clonal fibroid and non-clonal myometrium. However, using an RNA-based X chromosome inactivation HUMARA assay, we were able to demonstrate clonality of all cellular constituents of most fibroids. Our results confirm that most fibroids are derived from a single cell, and for the first time demonstrates that these clonal cells differentiate into fibroblast and SMC subpopulation as the fibroid grows.

Common fibroid-associated genes are differentially expressed in phenotypically dissimilar cell populations isolated from within human fibroids and myometrium.

Uterine fibroids are a prevalent gynaecological condition in reproductive-aged women and are the commonest reason for hysterectomy. The cellular composition of clonal fibroids are heterogeneous, with phenotypically dissimilar cells that include smooth muscle cells (SMC), vascular SMC (VSMC) and fibroblasts. The aim of our study was to investigate genes that are commonly differentially expressed between fibroid and myometrial whole tissues in phenotypically different sub-populations of cells isolated from fibroid and myometrium. Genes to be investigated by fluorescence-activated cell sorting, quantitative real-time PCR and immunocytochemistry include transforming growth factor ß (TGFB) and retinoic acid (RA) signalling families and steroid hormone receptors. We hypothesised that each cell population isolated from fibroid and myometrium would differ in the expression of fibroid-associated genes. We demonstrated that phenotypically different cellular constituents of uterine fibroids differentially express cellular RA-binding protein 2 (CRABP2), progesterone receptor B (PRB) and TGFB receptor 2 mRNA in fibroid-derived cells of VSMC and SMC phenotype. CRABP2 mRNA was also differentially expressed in fibroblasts and VSMC sub-populations from within clonal fibroid tumours. We conclude that differential regulation of RA, TGFB and PR pathway transcription occurs in fibroid-associated SMC and -fibroblasts and that investigation of paracrine interactions between different cell types within the fibroid microenvironment provides an important new paradigm for understanding the pathophysiology of this common disease.

The relationship between sleep disturbances and endometriosis: A systematic review.

OBJECTIVE: Endometriosis is associated with a range of symptoms that can negatively impact a person's quality of life. While pain and infertility have received at lot of attention, sleep disturbances in individuals with endometriosis has been overlooked in both clinical practice and research. Therefore, the primary aim of this systematic review was to gather evidence from the current literature to illustrate the association between sleep disturbances and endometriosis. STUDY DESIGN: A literature search was conducted using three electronic databases (OVID EMBASE, MEDLINE, and Web of Science). Observational studies, published in English, that involved participants aged 18 years or older that compared sleep outcomes between endometriosis patients and those without a history of endometriosis were included. The quality of each study was assessed using the Joanna Briggs Institute critical appraisal tools. RESULTS: Nine studies (six case-control and three cross-sectional) were included in this review; 7 with low risk of bias and 2 with moderate risk of bias. The studies demonstrated heterogeneity in the assessment of sleep disturbances. However, 7 studies reported a significant positive association between endometriosis and sleep disturbances. Moreover, this impact on sleep was further complicated by the complex interaction between pain, fatigue and quality of life. CONCLUSION: Current studies suggest an association between sleep disturbances and endometriosis, which may provide a blueprint for future clinical recommendations to screen and treat sleep disturbances in individuals with endometriosis to improve their quality of life. Future studies should aim to standardise the methods of assessing sleep disturbances and explore potential contributing factors.

Positron emission tomography in the evaluation of endometriosis: A systematic review.

Despite the profound impact of endometriosis worldwide, delays in diagnosis and suboptimal surveillance techniques are well-recognised issues. Case studies have reported incidental uptake of 18F-FDG PET tracer in endometriotic lesions. However, the utility of PET imaging as a non-invasive diagnostic tool for endometriosis is currently unclear. The purpose of this systematic review was to summarise the existing evidence and determine the value of available PET scanning techniques in the detection and monitoring of endometriosis. MEDLINE, EMBASE, CENTRAL, SCOPUS and Web of Science were searched from conception to 05/03/23. Eligible studies included participants with a history of known or suspected endometriosis who underwent a PET scan for any indication. All PET tracers and protocols were eligible. Outcomes included correlation of PET tracer uptake with the presence of endometriosis seen at laparoscopy or confirmed on histology, sensitivity of tracer uptake, specificity of tracer uptake, site of lesions with tracer uptake, stage of lesions with tracer uptake, SUVmax of endometriosis lesions and adverse reactions to PET imaging. The protocol for this review was registered with PROSPERO (ID: CRD42023405260). Eight studies describing 110 participants were eligible for inclusion. Six studies assessed 18F-FDG with combined PET-CT, one study assessed 18F-FDG PET alone, and the remaining study assessed PET-CT with an alternative tracer, 68Ga-DOTATATE. For 18F-FDG imaging, the correlation of PET avidity with lesions or sites of endometriosis ranged from 0-55 %. Pre-operative 68Ga-DOTATATE PET-CT detected endometriosis in 33 % of cases. All included studies were cohort studies, six were assessed to have low risk of bias, one with moderate risk and one with high risk of bias. Overall, 18F-FDG PET scanning does not appear to consistently identify endometriotic lesions, and therefore its reliability and usefulness in endometriosis diagnosis is limited. The utility of 68Ga-DOTATATE PET-CT remains uncertain. Findings are constrained by limited available evidence reporting outcomes of PET imaging for endometriosis. Other existing PET tracers with biological plausibility in the detection or monitoring of endometriosis warrant further investigation.

Noninvasive diagnostic imaging for endometriosis part 2: a systematic review of recent developments in magnetic resonance imaging, nuclear medicine and computed tomography.

Endometriosis affects 1 in 9 women, taking 6.4 years to diagnose using conventional laparoscopy. Non-invasive imaging enables timelier diagnosis, reducing diagnostic delay, risk and expense of surgery. This review updates literature exploring the diagnostic value of specialist endometriosis magnetic resonance imaging (eMRI), nuclear medicine (NM) and computed tomography (CT). Searching after the 2016 IDEA consensus, 6192 publications were identified, with 27 studies focused on imaging for endometriosis. eMRI was the subject of 14 papers, NM and CT, 11, and artificial intelligence (AI) utilizing eMRI, 2. eMRI papers describe diagnostic accuracy for endometriosis, methodologies, and innovations. Advantages of eMRI include its: ability to diagnose endometriosis in those unable to tolerate transvaginal endometriosis ultrasound (eTVUS); a panoramic pelvic view, easy translation to surgical fields; identification of hyperintense iron in endometriotic lesions; and ability to identify super-pelvic lesions. Sequence standardization means eMRI is less operator-dependent than eTVUS, but higher costs limit its role to a secondary diagnostic modality. eMRI for deep and ovarian endometriosis has sensitivities of 91-93.5% and specificities of 86-87.5% making it reliable for surgical mapping and diagnosis. Superficial lesions too small for detection in larger capture sequences, means a negative eMRI doesn't exclude endometriosis. Combined with thin sequence capture and improved reader expertise, eMRI is poised for rapid adoption into clinical practice. NM labeling is diagnostically limited in absence of suitable unique marker for endometrial-like tissue. CT studies expose the reproductively aged to radiation. AI diagnostic tools, combining independent eMRI and eTVUS endometriosis markers, may result in powerful capability. Broader eMRI use, will optimize standards and protocols. Reporting systems correlating to surgical anatomy will facilitate interdisciplinary preoperative dialogues. eMRI endometriosis diagnosis should reduce repeat surgeries with mental and physical health benefits for patients. There is potential for early eMRI diagnoses to prevent chronic pain syndromes and protect fertility outcomes.