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.

Functional changes of immune cells: signal of immune tolerance of the ectopic lesions in endometriosis?

RESEARCH QUESTION: What is the potential role of immune cells and their inflammatory cytokines in the pathogenesis, development and establishment of endometriosis? DESIGN: Peritoneal fluid from 59 women (43 with endometriosis and 16 controls) who had undergone laparoscopic surgery was analysed. Changes in the population of innate and adaptive immune cells, cytokines, chemokines and growth factor expression were measured by flow cytometry, Luminex Technology and enzyme-linked immunosorbent assay. RESULTS: No differences were found in the frequencies of the innate and adaptive immune cells between women with and without endometriosis. In the peritoneal fluid of women with endometriosis, IL-1ß, IL-1RN, IL-2, IL-4, IL-8, IL-10, IL-12 (p70), IL-17α, FGF2, G-CSF, MCP-1, MIP-1α and TNF-α were significantly increased compared with controls. A correlation between IL-2, MCP-1, MIP-1α, TNF-α and the severity of endometriosis was observed. The concentration of neopterin, a possible biomarker for this disease, was increased in women with endometriosis compared with controls. CONCLUSIONS: The functional activity of immune cells seemed to be reduced despite their numbers remaining unchanged. The data indicate that a shift of TH cytokine profile occurs, which increases the TH1-TH2 ratio. This is driven by the increased levels of the cytokines (TNF-α and IL-2) in women with severe endometriosis.

Exosomes and their cargo are important regulators of cell function in endometriosis.

Endometriosis is a chronic oestrogen-dependent gynaecological disorder characterized by non-menstrual pelvic pain, infertility and the extrauterine growth of endometrial-like glands and stroma. It has been noted that the eutopic endometrium of women with endometriosis is functionally distinct from that of women without endometriosis. Moreover, ectopic endometrial implants are functionally different from the eutopic endometrium of women with endometriosis. However, the mechanisms directing these differences are ill-defined. It is proposed here that small membrane-bound extracellular vesicles called exosomes are important vehicles in the protection and transport of signalling molecules central to the dysregulation of endometrial function in women with endometriosis. Therefore, a critical review of the literature linking exosomes and their cargo to the pathobiology of endometriosis was conducted. Circulating peritoneal fluid and endometrial cell exosomes contained long non-coding RNA, miRNA and proteins involved in histone modification, angiogenesis and immune modulation that differed significantly in women with endometriosis compared with controls. Moreover, experimental evidence supports a role for exosomes and their cargo in angiogenesis, neurogenesis, immune modulation and endometrial stromal cell invasion. It is therefore suggested that exosomes play an important role in the pathophysiology of endometriosis.

Early maternal separation accelerates the progression of endometriosis in adult mice.

BACKGROUND: A large body of research highlights the importance of early-life environmental impact on the health outcome in adulthood. However, whether early-life adversity (ELA) has any impact on the development of endometriosis is completely unclear. In this study, we tested the hypothesis that ELA, as manifested by neonatal separation, can accelerate the progression of endometriosis in mouse through activation of the adrenergic receptor ß2 (ADRB2) signaling pathway, leading to increased angiogenesis and progression of endometriotic lesions. METHODS: Eight female Balb/C mice, in late pregnancy, were used used for this study, which later gave birth to 22 female newborn pubs. Eleven additional female Balb/C mice were also used as donors of uterine tissues. The 22 newborn pubs were randomly divided into 2 equal-sized groups, maternal separation (MS) and no separation (NS). Pubs in the MS group were separated from their dams for 3 h/day from postnatal day (PND) 1 to 21, while those in the NS control remained in the home cage with their dams. In adulthood (8-week old), 3 mice in each group were randomly selected to undergo a battery of behavior tests. The remaining 8 mice in each group were induced with endometriosis by intraperitoneal injection of uterine fragments from donor mice. Four weeks after the induction, all mice were sacrificed and their endometriotic lesions were excised for quantification and then prepared for immunohistochemistry analysis. RESULTS: We confirmed that MS during infancy resulted in anxiety and depression-like behaviors as previously reported. We also found that in MS mice the lesion weight was increased by over 2 folds and generalized hyperalgesia was also significantly increased as compared with NS mice. Immunostaining analysis demonstrated that MS accelerated the development of endometriosis likely through decreased dopamine receptor D2 (DRD2) expression and activation of the ADRB2/cAMP-response element binding protein (CREB) signaling pathway, leading to increased angiogenesis and progression of endometriotic lesions. CONCLUSIONS: Exposure of female mouse pups to ELA such as MS during their infancy period accelerates the progression of endometriosis, possibly through altered neuronal wiring and hyperactivity of the hypothalamic-pituitary-adrenal axis.

Oestrogen receptors and hypoxia inducible factor 1 alpha expression in abdominal wall endometriosis.

RESEARCH QUESTION: What are the protein levels and localization of oestrogen receptors (including ERa, ERb and G protein-coupled oestrogen receptor [GPER]) and hypoxia-inducible factor-1alpha (HIF-1a) in normal control endometrium (COEM) and ectopic endometrium from abdominal wall endometriosis (AWE). DESIGN: AWE (n = 20) were obtained during surgery; COEM (n = 40) were collected by curettage. All tissues were obtained during the proliferative or secretory phase. Formalin-fixed paraffin-embedded tissues were used for immunohistochemical study for oestrogen receptors and HIF-1a proteins. RESULT(S): The expression of oestrogen receptors and HIF-1a in AWE differed from that in the corresponding menstrual cycle phase of COEM. Compared with COEM, ERa and HIF-1a were decreased whereas ERb and GPER were increased in AWE. The greatest difference was in GPER, with increased protein expression in both the cytoplasm and nucleus of endometrial epithelial and stromal cells, as well as a distinct change in localization from cytoplasmic expression to nuclear and cytoplasmic expression, compared with COEM. CONCLUSIONS: Our data suggest that the expression changes of oestrogen receptors and HIF-1a, especially GPER, are associated with AWE, which may provide new clues to understanding the cause of endometriosis.

The follicular fluid metabolome differs according to the endometriosis phenotype.

RESEARCH QUESTION: Is there a follicular fluid-specific metabolic profile in deep infiltrating endometriosis (DIE) depending on the presence of an associated ovarian endometrioma (OMA) that could lead to the identification of biomarkers for diagnosis and prognosis of the disease? DESIGN: In this prospective cohort study, proton nuclear magnetic resonance (1H-NMR) experiments were carried out on 50 follicular fluid samples from patients presenting with DIE, associated or not associated with an OMA, and 29 follicular fluid samples from patients with infertility caused by a tubal obstruction. RESULTS: Concentrations of glucose, citrate, creatine and amino acids such as tyrosine and alanine were lower in women with DIE than control participants, whereas concentrations of lactate, pyruvate, lipids and ketone bodies were higher. Metabolic analysis revealed enhanced concentrations of glycerol and ketone bodies in patients with OMA, indicative of an activation of lipolysis followed by beta-oxidation. Concentrations of lactate and pyruvate were increased in patients without OMA, whereas the concentration of glucose was decreased, highlighting activation of the anaerobic glycolysis pathway. Differences in concentrations of amino acids such as threonine and glutamine were also statistically relevant in discriminating between the presence or absence of OMA. CONCLUSIONS: Results indicate a mitochondrial dysregulation in endometriosis phenotypes, with a modified balance between anaerobic glycolysis and beta-oxidation in OMA phenotypes that could affect the fertility of women with endometriosis. As the composition of the follicular fluid has been shown to be correlated with oocyte development and outcome of implantation after fertilization, these findings may help explain the high level of infertility in these patients.

Dopamine agonists as genital endometriosis target therapy.

OBJECTIVE: The present study was to find a pathogenic evidence for dopamine agonist application in patients with endometriosis associated pain syndrome. PATIENTS AND TECHNIQUE: The study involved 227 patients of reproductive age with histologically confirmed genital endometriosis (GE) of I-III degree according to ASRM classification. The control group included 12 women with no laparoscope detected gynecologic pathology. The levels of prolactin (PRL), peripheral blood (PB), and peritoneal fluid (PF) were evaluated by chemiluminescence immune assay. The pain syndrome was measured by McGill visual analogue scale. Statistica10 program (StatSoft, Inc., Tulsa, OK) was applied for obtained data processing. RESULTS: A correlation was established between GE rate and levels of PRL and PB (Rs = 0.28, p < .05) as well as a correlation of PRL in PB and PF (Rs = 0.29, p < .05). Patients receiving cabergoline combined with hormone therapy standard schemes manifested considerable pain syndrome relief. CONCLUSIONS: PRL involvement in GE pathogenesis and more intense therapeutic impact on pain syndrome in case of combined administration of dopamine and standard hormone therapy prove cabergoline application in clinical practice.

Cytokine and growth factor profile in endometriosis: a multiplex analysis of peritoneal fluid to assess diagnostic utility.

We aimed to assay cytokines and growth factors in peritoneal fluid samples from women with and without endometriosis to understand the inflammatory milieu, and assess their potential diagnostic utility. This cross-sectional study conducted at a tertiary care hospital included 54 women, aged 20-45 years, with regular menstrual history and undergoing diagnostic/therapeutic laparoscopy for infertility and/or pain. Peritoneal fluid samples were collected after insertion of trocar & laparoscope but prior to other surgical intervention. A multiplex immunoassay of 27 cytokines and growth factors was performed. The concentration of FGF2 and CSF3 were significantly lower in women with endometriosis than without endometriosis (p = .043 and .003, respectively). The levels of CCL2 and IL1RN were significantly higher in moderate-severe than in minimal-mild endometriosis (p = .038 and .043, respectively). Phase-specific comparison revealed that in proliferative phase, the levels of CSF2 and CSF3 were lower in women with endometriosis than without the disease (p = .047 and .013, respectively). The ROC curve analysis provided a cutoff value 0.78 and 0.76 for FGF2 and CSF3, respectively. Cytokines and growth factors such as FGF2, CSF3, CSF2, CCL2 and IL1RN seem to contribute to the pathogenesis of endometriosis and may have a potential utility for the diagnosis of endometriosis.

Reprogramming of Mesothelial-Mesenchymal Transition in Chronic Peritoneal Diseases by Estrogen Receptor Modulation and TGF-ß1 Inhibition.

In chronic peritoneal diseases, mesothelial-mesenchymal transition is determined by cues from the extracellular environment rather than just the cellular genome. The transformation of peritoneal mesothelial cells and other host cells into myofibroblasts is mediated by cell membrane receptors, Transforming Growth Factor ß1 (TGF-ß1), Src and Hypoxia-inducible factor (HIF). This article provides a narrative review of the reprogramming of mesothelial mesenchymal transition in chronic peritoneal diseases, drawing on the similarities in pathophysiology between encapsulating peritoneal sclerosis and peritoneal metastasis, with a particular focus on TGF-ß1 signaling and estrogen receptor modulators. Estrogen receptors act at the cell membrane/cytosol as tyrosine kinases that can phosphorylate Src, in a similar way to other receptor tyrosine kinases; or can activate the estrogen response element via nuclear translocation. Tamoxifen can modulate estrogen membrane receptors, and has been shown to be a potent inhibitor of mesothelial-mesenchymal transition (MMT), peritoneal mesothelial cell migration, stromal fibrosis, and neoangiogenesis in the treatment of encapsulating peritoneal sclerosis, with a known side effect and safety profile. The ability of tamoxifen to inhibit the transduction pathways of TGF-ß1 and HIF and achieve a quiescent peritoneal stroma makes it a potential candidate for use in cancer treatments. This is relevant to tumors that spread to the peritoneum, particularly those with mesenchymal phenotypes, such as colorectal CMS4 and MSS/EMT gastric cancers, and pancreatic cancer with its desmoplastic stroma. Morphological changes observed during mesothelial mesenchymal transition can be treated with estrogen receptor modulation and TGF-ß1 inhibition, which may enable the regression of encapsulating peritoneal sclerosis and peritoneal metastasis.

Interleukin-1ß inhibits estrogen receptor-α, progesterone receptors A and B and biomarkers of human endometrial stromal cell differentiation: implications for endometriosis.

Human blastocyst nidation in the uterus and successful pregnancy require coordinated endometrial expression of estrogen receptor (ER)-α, progesterone receptors (PR)-A and -B and the gap junction protein, connexin (Cx)43. Our prior work established that inflammation associated with conditions of reduced fecundity, particularly endometriosis, can perturb eutopic decidual function. In the current studies, we have modeled endometrial decidualization in primary human endometrial stromal cell cultures derived from normal controls (NESC) and from the eutopic endometria of women with endometriosis (EESC) to test the hypothesis that a proinflammatory cytokine, interleukin (IL)-1ß, can disrupt stromal cell differentiation. The cells were grown under a standard protocol with hormones (10 nM 17ß-estradiol, 100 nM progesterone and 0.5 mM dibutyryl cAMP) for up to 7 days in the absence or presence of IL-1ß. Time-course experiments showed that IL-1ß compromised decidual function in both NESC and EESC, which was accompanied by rapid phosphorylation of ER-α, PR and Cx43 and their cellular depletion. Inhibition of the extracellular signal-regulated kinase (ERK)1/2 pathway by a selective pharmacological blocker (PD98059) or siRNA interference, or the addition of hormones themselves, blocked the phosphorylation of ERK mediators; increased the production of steroid receptors, Cx43, prolactin, insulin-like growth factor binding protein-1 (IGFBP)-1 and vascular endothelial growth factor (VEGF) and accelerated the differentiation. The results indicate that inhibition of IL-1ß can enhance decidualization in NESC and EESC in vitro. Strategies to interfere with this pathway might be implemented as an in vivo approach to enhance fertility in women with endometriosis and, potentially, other inflammatory pathologies.

Elevated heme impairs macrophage phagocytosis in endometriosis.

Endometriosis (EMS) is a chronic inflammatory disease characterized by the presence of extrauterine endometrial tissues. It has been previously reported that the refluxed blood containing viable endometrial tissues and the defective elimination of peritoneal macrophages in the pelvic cavity may involve in EMS pathogenesis. However, the mechanism by which macrophages exhibit attenuated phagocytic capability in EMS remains undetermined. Herein, we found that heme, the byproduct of lysed erythrocytes, accumulated abnormally in the peritoneal fluid (PF) of patients with EMS (14.22 µmol/L, 95% confidence interval (CI): 12.54-16.71), compared with the EMS-free group (9.517 µmol/L, 95% CI: 8.891-10.1053). This abnormal accumulation was not associated with the color of PF, phase of the menstrual cycle or severity of the disease. The reduced phagocytic ability of peritoneal macrophages (pMφs) was observed in the EMS group. Consistently, a high-concentration (30 µmol/L) heme treatment impaired EMS-pMφs phagocytosis more than a low-concentration (10 µmol/L) heme treatment. A similar phenomenon was observed in the EMS-free control pMφs (Ctrl-pMφs) and the CD14+ peripheral monocytes (CD14+ Mos). These results indicated that a high heme concentration exhibits a negative effect on macrophage phagocytosis, which supplements the mechanism of impaired scavenger function of pMφs in EMS.

20(S)-Ginsenoside Rg3-loaded electrospun membranes to prevent postoperative peritoneal adhesion.

Postoperative peritoneal adhesions are one of the most common surgical complications. In this study, we developed a 20(S)-ginsenoside Rg3-loaded methoxy poly (ethylene glycol)-block-poly(L-lactide-co-glycolide) (mPEG-b-PLGA) electrospun membrane (PEM/Rg3) that could not only serve as a physical barrier, but also as a drug delivery system that releases 20(S)-ginsenoside Rg3 constantly to prevent postoperative peritoneal adhesions. The characteristics of PEM/Rg3, including scanning electron microscopy, water contact angle, and mechanical analyses, were assessed. Degradation and drug release assays of PEM/Rg3 were performed. The anti-adhesion efficacy of PEM/Rg3 was evaluated in an abdomen-cecum mouse model. The adhesion scores, adhesion areas, hematoxylin and eosin (H&E) staining, immunofluorescence, and western blotting were assessed. The 20(S)-ginsenoside Rg3 loaded mPEG-b-PLGA electrospun fibers were successfully fabricated. The fibers were smooth, with no obvious drug crystals. PEM/Rg3 membranes were biodegradable and could be degraded gradually to release 20(S)-Ginsenoside Rg3 constantly from the membranes. The animal study showed that PEM/Rg3 exhibited an excellent adhesion prevention ability when compared with the control group, the PEM group, and polylactic acid (PLA) commercial membrane (Surgiwrap™) group. Immunofluorescence and western blotting studies showed that PEM/Rg3 inhibited the expressions of interleukin 1 (IL-1), interleukin 6 (IL-6), and reactive oxygen species modulator-1 (ROMO1). The 20(S)-ginsenoside Rg3-loaded mPEG-b-PLGA electrospun membranes exhibited satisfactory anti-adhesion efficacy by inhibiting inflammatory responses and oxidative stress. This composite represents a promising strategy to prevent postoperative peritoneal adhesions.

CCR5/CCR5 ligand-induced myeloid-derived suppressor cells are related to the progression of endometriosis.

RESEARCH QUESTION: Immunological disorders have been reported to promote the progression of endometriosis. Several recent studies have shown that myeloid-derived suppressor cells (MDSC) drive the progression of endometriosis. The aim of this case-control study was to test whether CCR5 and its ligands drive MDSC accumulation and play a role in the progression of endometriosis. DESIGN: Thirty-six endometriosis patients and 20 controls were recruited. All subjects underwent laparoscopy. An ELISA kit was used to define CCR5 ligands in plasma and peritoneal fluid from endometriosis patients; flow cytometry was then used to characterize CCR5+MDSC in peripheral blood and peritoneal fluid. RESULTS: Data showed that endometriosis patients displayed a significantly higher production of plasma CCL3 (P = 0.046) and peritoneal fluid CCL3/5 (P = 0.042/0.036) compared with those from the uterine leiomyoma group. Furthermore, the concentrations of peritoneal fluid CCL5 were elevated in late stage patients compared with those from the uterine leiomyoma group. Accumulation of blood CCR5+Mo-MDSC was detected in endometriosis patients compared with those from both the ovarian dermoid cysts and uterine leiomyoma groups. Endometriosis patients also showed an elevation of CCR5+MDSC and CCR5+Mo-MDSC in peritoneal fluid samples compared with uterine leiomyoma samples. It was also found that enrichment of CCR5+MDSC (r = 0.6807; P < 0.0001) and CCR5+Mo-MDSC (r = 0.6893; P < 0.0001) were correlated with enhanced production of CCL5 in peritoneal fluid from endometriosis patients. CONCLUSIONS: This study showed that CCR5 and its ligands could drive the progression of endometriosis by enhancing the accumulation of MDSC. These findings might produce a promising treatment that targets CCR5+MDSC for endometriosis patients.

Expression of the TFDP1 gene in the endometrium of women with deep infiltrating endometriosis.

The field of endometriosis etiopathogenesis aims to identify the origin of disease in endometrial disorders. Changes in gene and protein expression related to cell adhesion, collagenases, and, mainly, cell cycle regulators have been identified. We set out to analyze the expression of the transcription factor DP-1 (TFDP1) gene, which encodes a protein that controls the G1/S phase passage of the cell cycle, in the endometrium of women with deep infiltrating endometriosis (DIE). Samples of endometrium from both endometriosis-affected women and healthy women were collected, cultured and maintained at the Cell Bank of the Pelvic Pain and Endometriosis Unit of the Federal University of Sao Paulo. This study analyzed five samples from the endometrium cell culture of healthy patients (i.e. no pelvic disease, as determined by means of laparoscopic tubal ligation) and six samples from women diagnosed with DIE. Samples were evaluated for TFDP1 gene expression by real-time PCR. We observed a downregulation of TFDP1 in the endometrium cells of women with DIE when compared to the control (a fold-change of -2.05, p value=.011). The TFDP1 gene is part of the cell cycle pathway, but its function is not yet clear. Additional studies are necessary to clarify the function of TFDP1 in endometriosis etiopathogenesis.

The modulating effects of Resveratrol on the expression of MMP-2 and MMP-9 in endometriosis women: a randomized exploratory trial.

Endometriosis is an inflammatory disease; the hallmark of inflammation is over-activation of matrix metalloproteinases (MMPs). The regulatory effects of Resveratrol on MMPs were formerly depicted in other cell lines. This study aimed at investigating the effects of Resveratrol on expression of MMP-2 and -9 in endometriosis patients. This trial was carried out on endometriosis patients (n = 34) who were randomly divided into treatment (i = 17) and control (n = 17) groups. Alongside the routine protocol, the control and treatment groups took placebo and Resveratrol (400 mg), respectively, for 12-14 weeks. Endometrial tissue and fluid as well as blood sampling from both groups were done before and after the intervention. The level of mRNA and protein of both MMP-2 and -9 reduced in the endometrium of treatment group following intervention. Also, the serum and the endometrial fluid concentration of them lowered within the treatment group. Moreover, the serum and endometrial fluid levels of MMP-2 as well as MMP-9 were also diminished following the surgical removal of endometritic lesions. We showed that Resveratrol can modify the inflammation process in the endometrium of women with endometriosis at least in the level of MMP-2 and -9 expressions. The therapeutic potency of Resveratrol in endometriosis needs more clinical studies.

Resveratrol reduces the expression of insulin-like growth factor-1 and hepatocyte growth factor in stromal cells of women with endometriosis compared with nonendometriotic women.

Resveratrol, a phytoalexin polyphenol, has antiproliferative, antiangiogenic, anti-inflammatory, and antioxidant properties. The present study has assessed the effect of resveratrol treatment on the expression of insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) in endometrial stromal cells (ESCs) from women with and without endometriosis. Endometrial tissues were obtained from 40 endometriotic patients and 15 nonendometriotic control women. After the enzymatic digestion, 13 eutopic ESCs (EuESCs), 8 ectopic ESCs (EESCs), and 11 control ESCs (CESCs) were treated with resveratrol (100 µM) for 6, 24, and 48 hr. The gene and protein expressions of IGF-1 and HGF were measured using real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods, respectively. Results showed that resveratrol treatment decreased significantly the gene expression of IGF-1 and HGF in EuESCs, EESCs, and CESCs (p < 0.05). The effect of resveratrol treatment on the reduction of IGF-1 gene expression was statistically more noticeable in EESCs compared with CESCs (p < 0.05). Also, in the case of HGF gene expression, the reducing effect of resveratrol treatment was statistically more considerable in EESCs compared with EuESCs and CESCs (p < 0.05 and p < 0.01, respectively). The IGF-1 and HGF protein production decreased significantly in EuESCs and EESCs (p < 0.05) but not in CESCs. These findings suggest that resveratrol treatment could reduce the expression of IGF-1 and HGF in ESCs especially in EESCs, which play a pivotal role in disease progression.

Developmental endothelial locus-1 prevents development of peritoneal adhesions in mice.

Postoperative peritoneal adhesions, fibrous bands formed in the peritoneal cavity following surgery, represent a common, challenging and costly problem faced by surgeons and patients, for which effective therapeutic options are lacking. Since aberrant inflammation is one of the key mechanisms underlying peritoneal adhesion formation, here we set out to study the role of developmental endothelial locus-1 (Del-1), which has been recently identified as an endogenous inhibitor of inflammation, in the formation of postoperative peritoneal adhesions using a mouse model of peritoneal adhesions induced by ischemic buttons. Del-1-deficient mice had a higher incidence of adhesions, and their adhesions had higher quality and tenacity scores. Del-1 deficiency also led to enhanced inflammation mediators and collagen production. Finally, Del-1 supplementation decreased the incidence and severity of postoperative peritoneal adhesions. Taken together, these results indicate a protective role for Del-1 in postoperative peritoneal adhesion formation.

MicroRNA expression pattern differs depending on endometriosis lesion type.

MicroRNA (miRNA), noncoding segments of RNA involved in post-transcriptional regulation of protein expression are differentially expressed in eutopic endometrium of women with and without endometriosis compared to endometriotic lesions. However, endometriotic lesion types are known to be biochemically distinct and therefore hypothesized that miRNAs are differentially expressed in endometriomas compared to peritoneal and deep-infiltrating lesions. Therefore, endometrial biopsies and ectopic implants from women (n = 38) undergoing laparoscopic surgery for chronic pelvic pain were collected. Samples of endometriomas, peritoneal or deep-infiltrating lesions were selected from our tissue bank for study participants who exclusively had only one lesion type noted on their surgical report. Quantitative real-time polymerase chain reaction for miR-9, miR-21, miR-424, miR-10a, miR-10b, and miR-204 was performed. miR-204 expression was significantly lower (P = 0.0016) in the eutopic endometrium of women with endometriosis compared to controls. Relative expression of miR-21, miR-424, and miR-10b differed significantly (P < 0.05) across endometriotic lesion types. Finally, all miRNAs isolated from endometriomas, peritoneal and deep-infiltrating lesions studied were differentially expressed compared to matched eutopic endometrium samples. We therefore conclude that miRNA expression in the eutopic endometrium from women with endometriosis differs from symptomatic controls. Moreover, miRNA expression pattern is dependent on the endometriotic lesion type studied. We suggest that identification of different miRNA expression patterns for endometriomas, peritoneal and deep-infiltrating lesions could contribute to individualized patient care for women with endometriosis.

Secreted frizzled-related protein 2 (SFRP2) expression promotes lesion proliferation via canonical WNT signaling and indicates lesion borders in extraovarian endometriosis.

STUDY QUESTION: What is the role of SFRP2 in endometriosis? SUMMARY ANSWER: SFRP2 acts as a canonical WNT/CTNNB1 signaling agonist in endometriosis, regulating endometriosis lesion growth and indicating endometriosis lesion borders together with CTNNB1 (also known as beta catenin). WHAT IS KNOWN ALREADY: Endometriosis is a common, chronic disease that affects women of reproductive age, causing pain and infertility, and has significant economic impact on national health systems. Despite extensive research, the pathogenesis of endometriosis is poorly understood, and targeted medical treatments are lacking. WNT signaling is dysregulated in various human diseases, but its role in extraovarian endometriosis has not been fully elucidated. STUDY DESIGN, SIZE, DURATION: We evaluated the significance of WNT signaling, and especially secreted frizzled-related protein 2 (SFRP2), in extraovarian endometriosis, including peritoneal and deep lesions. The study design was based on a cohort of clinical samples collected by laparoscopy or curettage and questionnaire data from healthy controls and endometriosis patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Global gene expression analysis in human endometrium (n = 104) and endometriosis (n = 177) specimens from 47 healthy controls and 103 endometriosis patients was followed by bioinformatics and supportive qPCR analyses. Immunohistochemistry, Western blotting, primary cell culture and siRNA knockdown approaches were used to validate the findings. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 220 WNT signaling and CTNNB1 target genes analysed, 184 genes showed differential expression in extraovarian endometriosis (P < 0.05) compared with endometrium tissue, including SFRP2 and CTNNB1. Menstrual cycle-dependent regulation of WNT genes observed in the endometrium was lost in endometriosis lesions, as shown by hierarchical clustering. Immunohistochemical analysis indicated that SFRP2 and CTNNB1 are novel endometriosis lesion border markers, complementing immunostaining for the known marker CD10 (also known as MME). SFRP2 and CTNNB1 localized similarly in both the epithelium and stroma of extraovarian endometriosis tissue, and interestingly, both also indicated an additional distant lesion border, suggesting that WNT signaling is altered in the endometriosis stroma beyond the primary border indicated by the known marker CD10. SFRP2 expression was positively associated with pain symptoms experienced by patients (P < 0.05), and functional loss of SFRP2 in extraovarian endometriosis primary cell cultures resulted in decreased cell proliferation (P < 0.05) associated with reduced CTNNB1 protein expression (P = 0.05). LIMITATIONS REASONS FOR CAUTION: SFRP2 and CTNNB1 improved extraovarian endometriosis lesion border detection in a relatively small cohort (n = 20), although larger studies with different endometriosis subtypes in variable cycle phases and under hormonal medication are required. WIDER IMPLICATIONS OF THE FINDINGS: The highly expressed SFRP2 and CTNNB1 improve endometriosis lesion border detection, which can have clinical implications for better visualization of endometriosis lesions over CD10. Furthermore, SFRP2 acts as a canonical WNT/CTNNB1 signaling agonist in endometriosis and positively regulates endometriosis lesion growth, suggesting that the WNT pathway may be an important therapeutic target for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Academy of Finland and by Tekes: Finnish Funding Agency for Innovation. The authors have no conflict of interest to declare.

Intraperitoneal administration of activated protein C prevents postsurgical adhesion band formation.

Postsurgical peritoneal adhesion bands are the most important causes of intestinal obstruction, pelvic pain, and female infertility. In this study, we used a mouse model of adhesion and compared the protective effect of activated protein C (APC) to that of the Food and Drug Administration-approved antiadhesion agent, sodium hyaluronate/carboxymethylcellulose (Seprafilm) by intraperitoneal administration of either APC or Seprafilm to experimental animals. Pathological adhesion bands were graded on day 7, and peritoneal fluid concentrations of tissue plasminogen activator (tPA), d-dimer, thrombin-antithrombin complex, and cytokines (IL-1ß, IL-6, interferon-γ, tumor necrosis factor-α, transforming growth factor-ß1) were evaluated. Inflammation scores were also measured based on histologic data obtained from peritoneal tissues. Relative to Seprafilm, intraperitoneal administration of human APC led to significantly higher reduction of postsurgical adhesion bands. Moreover, a markedly lower inflammation score was obtained in the adhesive tissues of the APC-treated group, which correlated with significantly reduced peritoneal concentrations of proinflammatory cytokines and an elevated tPA level. Further studies using variants of human APC with or without protease-activated receptor 1 (PAR1) signaling function and mutant mice deficient for either endothelial protein C receptor (EPCR) or PAR1 revealed that the EPCR-dependent signaling activity of APC is primarily responsible for its protective activity in this model. These results suggest APC has therapeutic potential for preventing postsurgical adhesion bands.