Endometriosis is associated with chronic pelvic pain, largely due to immune dysregulation within the peritoneal cavity. The activation status of peritoneal immune cells is not well understood, and comparisons with systemic immune cells may provide insights for diagnosing and treating inflammation and pain in endometriosis. To investigate immune cell activation and inhibition status in peritoneal fluid and blood in endometriosis patients using full-spectrum flow cytometry. This study included patients undergoing laparoscopy for diagnosis or treatment of peritoneal endometriosis or for unrelated conditions; peritoneal fluid was collected from n = 6 endometriosis patients and n = 8 controls, and matched blood from n = 5 endometriosis patients and n = 7 controls. Immune cells were analysed using a 20-marker full-spectrum flow cytometry panel. Data were analysed for statistical significance using the Kruskal-Wallis or Mann-Whitney U test, with a p value below 0.05 considered significant. The main differences between endometriosis and control samples were found in lymphoid populations in peritoneal fluid and myeloid populations in blood. Contrary to our expectations, the expression of PD-1 on peritoneal fluid NK and T cell populations was significantly lower in endometriosis than in controls (p < 0.05). The significant decrease in immune checkpoint PD-1 expression represents a novel immunopathological feature of endometriosis and highlights potential therapeutic targets for managing inflammation and pain through immune checkpoint modulation.

We aimed to assess the landscape of endometriosis randomised-controlled trials (RCTs) between 1990 and 2024 by characterising RCTs by management type, primary outcome, country of origin, trial registration, sample size and minimum detectable difference based upon standard RCT power targets of 80

ABSTRACT Endometriosis, a chronic debilitating disease affects 1 in 7–10 girls and women, who have symptoms of severe chronic pain and subfertility and significantly impacts the overall quality of life. Currently, no effective early diagnostic methods are available for early stages of endometriosis. We used menstrual fluid‐derived small extracellular vesicles (MF‐sEVs) from women with self‐reported endometriosis (laparoscopically diagnosed, n = 8) and self‐reported without endometriosis and no painful periods (n = 9). MF‐sEVs were separated using differential ultracentrifugation and characterised using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western Blot, flow cytometry, mass‐proteomics analysis and functional assays. Spherical‐shaped sEVs were identified with a median diameter of ∼120 nm, expressing sEV marker proteins. The MF‐sEV proteins were classified as endometrial origin. Over 5000 proteins were identified, ∼77% of which were decreased whilst only 22 proteins (largely comprising immunoglobulins) were increased in endometriosis/MF‐sEVs compared to control/MF‐sEVs. Decreased proteins were involved in nitrogen compound metabolism, immune response, intracellular signal transduction, regulation of programmed cell death, maintenance of cell polarity and actin cytoskeleton organisation. Flow cytometry demonstrated a significant increase in CD86 expression (immune activation marker) in endometriosis/MF‐sEVs. Mesothelial cells showed a significant decrease in cellular resistance and junctional protein expression. MF‐sEVs are possible contributors to the pathogenesis of endometriosis and may have the potential for early detection of the disease.

Background: Endometriosis is characterised by chronic inflammation in the peritoneal cavity causing acute and chronic pelvic pain, largely explained by dysregulation in the immune environment within peritoneal fluid. The activation status of the peritoneal immune cells is unclear. In addition, a comparison with the status of the systemic immune system is desirable to explore avenues of diagnosis and treatment of endometriosis-related inflammation and pain. Objective(s): To investigate the immune environment in endometriosis in peritoneal fluid and blood by full-spectrum flow cytometry with a focus on activation and inhibition of immune cells. Study Design: This was an observational study in patients undergoing laparoscopy for diagnosis or treatment of peritoneal endometriosis or for unrelated conditions; PF was collected from n=5 endometriosis patients and n=4 controls, blood from n=4 endometriosis patients and n=3 controls. Data were analysed for statistical significance using ANOVA, the Kruskal-Wallis or Mann-Whitney U test, with a p-value below 0.05 considered significant. Results: We observed a prevailing of myeloid immune cells in the peritoneal fluid as opposed to lymphoid cells in the blood. The main differences between endometriosis and control samples, however, were found in the smaller compartments, i.e., in lymphoid populations in peritoneal fluid and myeloid populations in blood. PD-1 levels in peritoneal fluid endometriosis samples were significantly lower than in controls (p<0.05). Conclusion(s): The immune checkpoint PD-1 could be a new angle of treating endometriosis-related inflammation and pain in women suffering from this chronic and intractable condition. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Study question Can extracellular vesicles serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound? Summary answer Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes. What is known already Endometriosis is characterised by intraperitoneal lesions of endometrial tissue. Although ultrasound detection of severe endometriosis is possible, the diagnosis of peritoneal lesions currently relies on invasive laparoscopy, which leads to a delay of 8-10 years. We have previously shown that peritoneal fluid (PF) of women with endometriosis contains endometriosis specific EVs. Traced in peripheral blood (PB), EVs could serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound. Study design, size, duration Women 18-45 years of age undergoing laparoscopic surgery for endometriosis or unrelated conditions were invited to participate in our study (HREC 20-159A/HREC 20-882A). Following informed consent, we collected PF and PB samples from n = 101 participants (n = 47 controls, n = 54 cases). Exclusion criteria were pregnancy, malignancy, and menopause. Participants/materials, setting, methods We processed PF and PB samples by differential ultracentrifugation and validated the presence of EVs by nanoparticle tracking analysis (NTA), Western blotting and transmission electron microscopy (TEM). EVs were analysed by untargeted, label-based quantitative proteomics using Tandem Mass Tags (TMT). Data analysis was performed using Proteome Discoverer v2.4 (Thermo Fisher) followed by statistical analysis using R. Candidate biomarker proteins were tested by traditional and automated Western blotting (WES, ProteinSimple). Main results and the role of chance We ascertained the identity of EVs by TEM and NTA (mode size 121.8 ± 18.0 nm (blood, n = 5) and 155.9 ± 37.2 (PF, n = 6)), and signals for syntenin and ALIX in immunoblots as well as expression of CD9, CD63 and CD81 in capture bead-flow cytometry. Proteomic analysis identified 9145 protein groups across all sample groups, with 602 significantly regulated between comparisons (adjusted P-value <0.05). In PF, 533 proteins changed significantly in abundance (245 up/288 down), while in blood, only four proteins changed in abundance. We tested the presence/absence of two of these as candidate markers on PB EV protein preparations from n = 32 samples to date; our best marker showed a sensitivity of 67% and a specificity of 90%; upon exclusion of severe endometriosis cases, sensitivity improved to 80%. Limitations, reasons for caution Because of the changes in vesicle composition and amounts with the menstrual cycle, we have to group the samples by menstrual cycle phase and disease severity. This leaves relatively small numbers per group per condition. Another limitation concerns the relatively small amount of sample liquid and thus, EVs available for analysis. Wider implications of the findings Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes. A non-invasive clinical test would allow for earlier diagnosis and thus treatment of endometriosis. It would also allow patients to initiate fertility treatment early. Trial registration number not applicable

Endometriosis is defined by the presence of extrauterine endometrial-like tissue, which can cause pain and infertility in 10% of reproductive-age women. To date, the pathogenesis is poorly understood resulting in significant diagnostic delays and poor therapeutic outcomes in many women. Small extracellular vesicles (sEVs) (<200 nm) are cell-derived vesicles containing molecules that can influence gene expression and behaviour in target cells. One such cargo are microRNAs (miRNAs), which are short, non-coding RNAs mostly 19-25 nucleotides in length that regulate post-transcriptional gene expression. This mini-review focuses on the role of sEV-miRNAs, which are conceivably better biomarkers for endometriosis than free miRNAs, which reflect the true pathophysiological state in the body, as sEV-encapsulated miRNAs are protected from degradation compared to free miRNA and provide direct cell-to-cell communication via sEV surface proteins. sEV-miRNAs have been implicated in the immunomodulation of macrophages, the proliferation, migration and invasion of endometrial cells, and angiogenesis, all hallmarks of endometriosis. The diagnostic potential of sEV-miRNA was investigated in one study that reported the sensitivity and specificity of two sEV-miRNAs (hsa-miR-22-3p and hsa-miR-320a-3p) in distinguishing endometriosis from non-endometriosis cases. Only three studies have explored the therapeutic potential of sEV-miRNAs in vivo in mice-two looked into the role of sEV-hsa-miR-214-3p in decreasing fibrosis, and one investigated sEV-hsa-miR-30c-5p in suppressing the invasive and migratory potential of endometriotic lesions. While early results are encouraging, studies need to further address the potential influence of factors such as the menstrual cycle as well as the location and extent of endometriotic lesions on miRNA expression in sEVs. Given these findings, and extrapolating from other conditions such as cancer, diabetes, and pre-eclampsia, sEV-miRNAs could present an attractive and urgently needed future diagnostic and therapeutic target for millions of women suffering from endometriosis. However, research in this area is hampered by lack of adherence to the International Society for Extracellular Vesicles 2018 guideline in separating and characterising sEVs, as well as the World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project protocols.

Uterine Fibroids, or leiomyomata, affect millions of women world-wide, with a high incidence of 75% within women of reproductive age. In ~30% of patients, uterine fibroids cause menorrhagia, or heavy menstrual bleeding, and more than half of the patients experience symptoms such as heavy menstrual bleeding, pelvic pain, or infertility. Treatment is symptomatic with limited options including hysterectomy as the most radical solution. The genetic foundations of uterine fibroid growth have been traced to somatic driver mutations (MED12, HMGA2, FH−/−, and COL4A5-A6). These also lead to downstream expression of angiogenic factors including IGF-1 and IGF-2, as opposed to the VEGF-driven mechanism found in the angiogenesis of hypoxic tumors. The resulting vasculature supplying the fibroid with nutrients and oxygen is highly irregular. Of particular interest is the formation of a pseudocapsule around intramural fibroids, a unique structure within tumor angiogenesis. These aberrations in vascular architecture and network could explain the heavy menstrual bleeding observed. However, other theories have been proposed such as venous trunks, or venous lakes caused by the blocking of normal blood flow by uterine fibroids, or the increased local action of vasoactive growth factors. Here, we review and discuss the evidence for the various hypotheses proposed.

Abstract Study question Can an endometriosis-specific protein signature in small extracellular vesicles (sEV) from peritoneal fluid (PF) be utilised as a non-invasive biomarker of the condition? Summary answer Yes, potentially. We found differences in the concentrations and protein cargo of PF-derived sEV between controls and endometriosis samples, most notably in CD44 expression. What is known already Endometriosis, defined as endometrial-like tissue outside the uterus, causes pain and/or subfertility in 10% of reproductive age women. The cause is unknown, resulting in inadequate diagnostic methods and treatment options. There is no clinically relevant biomarker for endometriosis yet. Small extracellular vesicles (sEV), produced by virtually every cell, have been described in diseases such as cancer, diabetes, and pre-eclampsia, and could similarly be important in endometriosis. We previously identified sEV in PF of women with endometriosis, and here investigated the protein cargo of PF sEV as biomarker of the disease. Study design, size, duration PF samples were obtained from participants in the ENDOX study, Endometriosis CaRe Centre, Nuffield Department of Women’s and Reproductive Health, University of Oxford (REC ref. 09/H0604/58) according to WERF EPHect standards. Women between 18-49 years of age (n = 63) who had undergone diagnostic laparoscopy were classified according to cycle phase (proliferative/secretory/menstrual) and severity of endometriosis (ASRM stages I+II or stages III+IV). Exclusion criteria were hormonal treatment, malignancy, pregnancy, breastfeeding, and inability to understand the consent form. Participants/materials, setting, methods The participant groups were control proliferative, n = 7; control secretory, n = 9; control menstrual, n = 3; StI+II proliferative, n = 8; StI+II secretory, n = 10; St1+II menstrual, n = 7; StIII+IV proliferative, n = 5; StIII+IV secretory, n = 11; StIII+IV menstrual, n = 3. 1 mL PF was centrifuged to remove cells, debris, and microvesicles. sEV were isolated using size exclusion chromatography (SEC) and analysed by nanoparticle tracking analysis (NTA), immunoblotting, and mass spectrometry (LC-MS/MS). Main results and the role of chance We confirmed the presence of exosomes in PF from women at different stages of endometriosis and from disease-free patients at different menstrual cycle phases by NTA, immunoblotting and mass spectrometry. Enriched sEV were positive for ALIX, CD9, and syntenin. The mode size of PF particles from women with endometriosis was 115 ± 15.5 nm, whereas in non-endometriotic women it was 95 ± 17.3 nm (n.s.). sEV concentrations were higher in endometriosis compared to controls, and highest in stage III-IV endometriosis, followed by stage I-II endometriosis and controls, irrespective of menstrual cycle phase (P = 0.0210). sEV concentration in stage III-IV endometriosis decreased consistent with a transition from proliferative to secretory phase. Likewise, PF-derived sEV numbers within stage I-II endometriosis samples increased, as these samples transitioned from proliferative to secretory cycle phases. Proteomic analysis showed distinct distribution patterns of proteins within endometriosis PF-derived sEVs compared to controls. Consistent with earlier studies, we found CD44 as an sEV protein uniquely within the endometriosis population and contributing significantly to the separation of endometriosis and control samples by the highest variable importance projection (VIP) score in our data set. Limitations, reasons for caution The main limitation of this study is the small number of samples across the different groups, and the limited amount of PF per sample. Wider implications of the findings PF-derived sEV differ between endometriosis and control patients. Concentrations vary regardless of cycle phase and disease stage, and this difference appears to be reflected in the proteomics analysis. The presence of CD44 within sEV could help diagnose endometriosis. Trial registration number not applicable

Abstract Study question Can we identify lesion-derived endometriosis-specific sEV cargo in peritoneal fluid of women with endometriosis, and detect the same endometriosis-specific cargo in sEV from peripheral blood? Summary answer Endometriosis-specific sEV are present in PF. Identification in peripheral blood is challenging due to low abundance in comparison to blood-borne sEV. What is known already Endometriosis is characterised by intraperitoneal lesions of endometrial tissue. Although ultrasound detection of severe endometriosis is possible, the final diagnosis currently relies on invasive laparoscopy as the gold standard, which leads to a delay of 8-10 years after women first present to their GPs with symptoms. We have previously shown that the peritoneal fluid (PF) of women with endometriosis contains endometriosis-specific sEV. If these can be traced in blood samples, endometriosis-specific sEV could serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound. Study design, size, duration This is an observational pilot study of 29 patients in total. From March 2021 until August 2021, we collected PF samples from 11 controls and 16 endometriosis cases and blood samples from 12 controls and 11 endometriosis cases. Of these, 19 were paired samples (blood and PF from the same participant). Participants/materials, setting, methods Women >18 years of age undergoing laparoscopic surgery for endometriosis or unrelated conditions were invited to participate (HREC 08078B/HREC 10148B). Exclusion criteria were pregnancy, malignancy, and menopause. Purified, validated sEV were analysed by label based quantitative proteomics Tandem Mass Tag (TMT). Data analysis was performed using Proteome Discoverer v2.4 (Thermo Fisher), statistical analysis using the R (LIMMA), with a protein false discovery rate of 1% and a quantitative threshold of an adjusted p-value <0.05. Main results and the role of chance We ascertained the identity of sEV by TEM and NTA (mode size 121.8 ± 18.0 nm (blood, n = 5) and 155.9 ± 37.2 (PF, n = 6)), and signals for syntenin and ALIX in immunoblots as well as expression of CD9, CD63 and CD81 in capture bead-flow cytometry. Proteomics analysis identified 9145 proteins groups across all sample groups, of these proteins 5,429 are consistently quantifiable, with 602 significantly different between comparisons (adjusted P-value <0.05). PCA showed separation of samples by type rather than batch. Fractionated analysis by SPS-MS3 methodology identified 7064 protein groups, with 3408 proteins consistently quantified across sample groups. Of these, 602 were found to differ significantly across all comparisons. In PF, 533 proteins changed significantly in abundance (245 up and 288 down), while in blood, four proteins changed in abundance. Limitations, reasons for caution The main limitation of the study is in its small sample size, which meant that we could not stratify data according to endometriosis stage. Due to limited amount of protein within sEV samples, not all assays could be done on all samples. Wider implications of the findings Our findings suggest that our methodology makes it feasible to define a protein signature of endometriosis based on sEV cargoes drawn from blood samples. This would represent a non-invasive biomarker of endometriosis and could help diagnose the condition, or rule out endometriosis as the origin of symptoms in the future. Trial registration number not applicable

Metastatic tumour progression is facilitated by tumour associated macrophages (TAMs) that enforce pro-tumour mechanisms and suppress immunity. In pulmonary metastases, it is unclear whether TAMs comprise tissue resident or infiltrating, recruited macrophages; and the different expression patterns of these TAMs are not well established. Using the mouse melanoma B16F10 model of experimental pulmonary metastasis, we show that infiltrating macrophages (IM) change their gene expression from an early pro-inflammatory to a later tumour promoting profile as the lesions grow. In contrast, resident alveolar macrophages (AM) maintain expression of crucial pro-inflammatory/anti-tumour genes with time. During metastatic growth, the pool of macrophages, which initially contains mainly alveolar macrophages, increasingly consists of infiltrating macrophages potentially facilitating metastasis progression. Blocking chemokine receptor mediated macrophage infiltration in the lung revealed a prominent role for CCR2 in Ly6C+ pro-inflammatory monocyte/macrophage recruitment during metastasis progression, while inhibition of CCR2 signalling led to increased metastatic colony burden. CCR1 blockade, in contrast, suppressed late phase pro-tumour MR+Ly6C- monocyte/macrophage infiltration accompanied by expansion of the alveolar macrophage compartment and accumulation of NK cells, leading to reduced metastatic burden. These data indicate that IM has greater plasticity and higher phenotypic responsiveness to tumour challenge than AM. A considerable difference is also confirmed between CCR1 and CCR2 with regard to the recruited IM subsets, with CCR1 presenting a potential therapeutic target in pulmonary metastasis from melanoma.

Uterine Fibroids (leiomyomata) and endometriosis affect millions of women world-wide. Although aetiology and natural history of the conditions are markedly different, symptoms can overlap and make differential diagnoses necessary, often using invasive methods such as laparoscopy. Considerable comorbidity exists between the two conditions and needs to be taken into account when treating fibroids and/or endometriosis. The genetic foundations of both uterine fibroids and endometriosis remain to be fully understood but recent evidence suggest common underpinnings. Here, we discuss the comorbidity of uterine fibroids and endometriosis and the implications for diagnosis, treatment and risks.

Genetic analyses in humans and macaques followed by cellular and mouse studies demonstrate NPSR1 as a nonhormonal drug target in endometriosis.

Introduction Millions of women suffer from the consequences of endometriosis and uterine fibroids, with fibroids the cause for over 50% of hysterectomies in the USA, and direct costs for their treatment estimated at between US$4 and US$9 billion. Endometriosis commonly affects millions of women worldwide predominantly during reproductive age, with severe menstrual and non-menstrual pain and subfertility the main symptoms. Due to the ‘unhappy triad’ of endometriosis—lack of awareness, lack of clinically relevant biomarkers and the unspecific nature of symptoms—women wait on average for 8–12 years before the definitive endometriosis diagnosis is made. Treatment options for both conditions are not satisfactory at the moment, especially with a view to preserving fertility for the women and families affected. In the Fibroids and Endometriosis Oxford (FENOX) study, we combine the investigation of fibroids and endometriosis, and plan to collect high-quality tissue samples and medical data of participants over a time frame of 5 years after surgical intervention. Methods and analysis Biological samples such as blood, saliva, urine, fat, peritoneal fluid and—if found—endometrial tissue or fibroids as well as detailed clinical and intraoperative data will be collected from women undergoing surgery and participating in the study after informed consent. We plan to recruit up to 1200 participants per disease arm (ie, endometriosis and uterine fibroids) over 5 years. Participants will fill in detailed and validated questionnaires on their medical history and quality of life, with follow-ups for 5 years. Enrolment started on 2 April 2018, and FENOX will close on 31 March 2028. We will analyse the biological samples using state-of-the-art molecular biology methods and correlate the findings with the medical records and questionnaire data. Ethics and dissemination The findings will be published in high-ranking journals in the field and presented at national and international conferences. Trial registration number ISRCTN13560263.

Background Endometriosis is a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis. Methods We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients. Results Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69(+) T cell subsets is increased in endometriosis when compared to control patient samples. On these CD69(+) cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69(+) and CD69(-) populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play roles in endometriosis. Conclusions This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets.

Endometriosis is a common gynaecological disease of women in reproductive age, and is thought to arise from retrograde menstruation and implantation of endometrial tissue, mostly into the peritoneal cavity. The condition is characterized by a chronic, unresolved inflammatory process thereby contributing to pain as cardinal symptom in endometriosis. Elevated reactive oxygen species (ROS) and oxidative stress have been postulated as factors in endometriosis pathogenesis. We here set out for a systematic study to identify novel mechanisms and pathways relating to oxidative stress in ectopic peritoneal lesions. Using combined proteomic and transcriptomic approaches, we identified novel targets including upregulated pro-oxidative enzymes, such as amine oxidase 3/vascular adhesion protein 1 (AOC3/VAP1) as well as downregulated protective factors, in particular alkenal reductase PTGR1 and methionine sulfoxide reductase. Consistent with an altered ROS landscape, we observed hemoglobin / iron overload, ROS production and lipid peroxidation in ectopic lesions. ROS-derived 4-hydroxy-2-nonenal induced interleukin IL-8 release from monocytes. Notably, AOC3 inhibitors provoked analgesic effects in inflammatory pain models in vivo, suggesting potential translational applicability.

Objective: To demonstrate the feasibility of studying exosomes directly from peritoneal fluid, we isolated exosomes from endometriosis patient samples and from controls, and characterized their cargo. Design: Case-control experimental study. Setting: Academic clinical center. Patient (s): Women with and without endometriosis who underwent laparoscopic surgery (n = 28 in total). Intervention (s): None. Main Outcome Measure (s): Concentration of exosomes within peritoneal fluid and protein content of the isolated exosomes. Result (s): Peritoneal fluid samples were pooled according to the cycle phase and disease stage to form six experimental groups, from which the exosomes were isolated. Exosomes were successfully isolated from peritoneal fluid in all the study groups. The concentration varied with cycle phase and disease stage. Proteomic analysis showed specific proteins in the exosomes derived from endometriosis patients that were absent in the controls. Five proteins were found exclusively in the endometriosis groups: PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin alpha-chain. Conclusion (s): Exosomes are present in peritoneal fluid. The characterization of endometriosis-specific exosomes opens up new avenues for the diagnosis and investigation of endometriosis. Copyright (C) 2019 The Authors. Published by Elsevier Inc. on behalf of the American Society for Reproductive Medicine.