Correlation between altered central pain processing and concentration of peritoneal fluid inflammatory cytokines in endometriosis patients with chronic pelvic pain

Translational research has not yet elucidated whether alterations in central pain processes are related to peripheral inflammatory processes in chronic pain patients. We tested the hypothesis that the concentration of cytokines in the peritoneal fluid of endometriosis patients with chronic pain correlate with parameters of hyperexcitability of the nociceptive system. The concentrations of 15 peritoneal fluid cytokines were measured in 11 patients with chronic pelvic pain and a diagnosis of endometriosis. Six parameters assessing central pain processes were recorded. Positive correlations between concentration of some cytokines in the peritoneal fluid and amplification of central pain processing were found. The results suggest that inflammatory mechanisms may be important in the pathophysiology of altered central pain processes and that cytokines produced in the environment of endometriosis could act as mediators between the peripheral lesion and changes in central nociceptive processes.

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

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

Phospholipase A2 group IIA is elevated in endometriomas but not in peritoneal fluid and serum of ovarian endometriosis patients.

Our previous gene expression analysis identified phospholipase A2 group IIA (PLA2G2A) as a potential biomarker of ovarian endometriosis. The aim of this study was to evaluate PLA2G2A mRNA and protein levels in tissue samples (endometriomas and normal endometrium) and in serum and peritoneal fluid of ovarian endometriosis patients and control women. One-hundred and sixteen women were included in this study: the case group included 70 ovarian endometriosis patients, and the control group included 38 healthy women and 8 patients with benign ovarian cysts. We observed 41.6-fold greater PLA2G2A mRNA levels in endometrioma tissue, compared to normal endometrium tissue. Using Western blotting, PLA2G2A was detected in all samples of endometriomas, but not in normal endometrium, and immunohistochemistry showed PLA2G2A-specific staining in epithelial cells of endometrioma paraffin sections. However, there were no significant differences in PLA2G2A levels between cases and controls according to ELISA of peritoneal fluid (6.0 ± 4.4 ng/ml, 6.6 ± 4.3 ng/ml; p = 0.5240) and serum (2.9 ± 2.1 ng/ml, 3.1 ± 2.2 ng/ml; p = 0.7989). Our data indicate that PLA2G2A is implicated in the pathophysiology of ovarian endometriosis, but that it cannot be used as a diagnostic biomarker.

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

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

Tumour border configuration in colorectal cancer: proposal for an alternative scoring system based on the percentage of infiltrating margin.

AIMS Information on tumour border configuration (TBC) in colorectal cancer (CRC) is currently not included in most pathology reports, owing to lack of reproducibility and/or established evaluation systems. The aim of this study was to investigate whether an alternative scoring system based on the percentage of the infiltrating component may represent a reliable method for assessing TBC. METHODS AND RESULTS Two hundred and fifteen CRCs with complete clinicopathological data were evaluated by two independent observers, both 'traditionally' by assigning the tumours into pushing/infiltrating/mixed categories, and alternatively by scoring the percentage of infiltrating margin. With the pushing/infiltrating/mixed pattern method, interobserver agreement (IOA) was moderate (κ = 0.58), whereas with the percentage of infiltrating margins method, IOA was excellent (intraclass correlation coefficient of 0.86). A higher percentage of infiltrating margin correlated with adverse features such as higher grade (P = 0.0025), higher pT (P = 0.0007), pN (P = 0.0001) and pM classification (P = 0.0063), high-grade tumour budding (P < 0.0001), lymphatic invasion (P < 0.0001), vascular invasion (P = 0.0032), and shorter survival (P = 0.0008), and was significantly associated with an increased probability of lymph node metastasis (P < 0.001). CONCLUSIONS Information on TBC gives additional prognostic value to pathology reports on CRC. The novel proposed scoring system, by using the percentage of infiltrating margin, outperforms the 'traditional' way of reporting TBC. Additionally, it is reproducible and simple to apply, and can therefore be easily integrated into daily diagnostic practice.

Minimally invasive surgery when treating endometriosis has a positive effect on health and on quality of work life of affected women

STUDY QUESTION: What is the effect of the minimally invasive surgical treatment of endometriosis on health and on quality of work life (e.g. working performance) of affected women? SUMMARY ANSWER: Absence from work, performance loss and the general negative impact of endometriosis on the job are reduced significantly by the laparoscopic surgery. WHAT IS KNOWN ALREADY: The benefits of surgery overall and of the laparoscopic method in particular for treating endometriosis have been described before. However, previous studies focus on medical benchmarks without including the patient's perspective in a quantitative manner. STUDY DESIGN, SIZE, DURATION: A retrospective questionnaire-based survey covering 211 women with endometriosis and a history of specific laparoscopic surgery in a Swiss university hospital, tertiary care center. Data were returned anonymously and were collected from the beginning of 2012 until March 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women diagnosed with endometriosis and with at least one specific laparoscopic surgery in the past were enrolled in the study. The study investigated the effect of the minimally invasive surgery on health and on quality of work life of affected women. Questions used were obtained from the World Endometriosis Research Foundation (WERF) Global Study on Women's Health (GSWH) instrument. The questionnaire was shortened and adapted for the purpose of the present study. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 587 women invited to participate in the study, 232 (232/587 = 40%) returned the questionnaires. Twenty-one questionnaires were excluded due to incomplete data and 211 sets (211/587 = 36%) were included in the study. Our data show that 62% (n = 130) of the study population declared endometriosis as influencing the job during the period prior to surgery, compared with 28% after surgery (P < 0.001). The mean (maximal) absence from work due to endometriosis was reduced from 2.0 (4.9) to 0.5 (1.4) hours per week (P < 0.001). The mean (maximal) loss in working performance after the surgery averaged out at 5.7% (12.6%) compared with 17.5% (30.5%) before this treatment (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: The mediocre response rate of the study weakens the representativeness of the investigated population. Considering the anonymous setting a non-responder investigation was not performed. A bias due to selection, information and negativity effects within a retrospective survey cannot be excluded, although study-sensitive questions were provided in multiple ways. The absence of a control group (sham group; e.g. patients undergoing specific diagnostic laparoscopy without treatment) is a further limitation of the study. WIDER IMPLICATIONS OF THE FINDINGS: Our study shows that indicated minimally invasive surgery has a clear positive effect on the wellbeing and working performance of women suffering from moderate to severe endometriosis. Furthermore, national net savings in indirect costs with the present number of surgeries is estimated to be €10.7 million per year. In an idealized setting (i.e. without any diagnosis delay) this figure could be more than doubled. STUDY FUNDING/COMPETING INTERESTS: The study was performed on behalf of the University Hospital of Bern (Inselspital) as one of the leading Swiss tertiary care centers. The authors do not declare any competing interests.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer

BACKGROUND The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. OBJECTIVE To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. METHODS IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. RESULTS IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. CONCLUSIONS Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

H19 lncRNA alters stromal cell growth via IGF signaling in the endometrium of women with endometriosis

Endometriosis affects approximately 15% of reproductive aged women and is associated with chronic pelvic pain and infertility. However, the molecular mechanisms by which endometriosis impacts fertility are poorly understood. The developmentally regulated, imprinted H19 long noncoding RNA (lncRNA) functions to reduce the bioavailability of microRNA let-7 by acting as a molecular sponge. Here we report that H19 expression is significantly decreased in the eutopic endometrium of women with endometriosis as compared to normal controls. We show that decreased H19 increases let-7 activity, which in turn inhibits Igf1r expression at the post-transcriptional level, thereby contributing to reduced proliferation of endometrial stromal cells. We propose that perturbation of this newly identified H19/Let-7/IGF1R regulatory pathway may contribute to impaired endometrial preparation and receptivity for pregnancy in women with endometriosis. Our finding represents the first example of a lncRNA-based mechanism in endometriosis and its associated infertility, thus holding potential in the development of novel therapeutics for women with endometriosis and infertility.

Comparison of ovarian cancer markers in endometriosis favours HE4 over CA125

Endometriosis is a gynaecological condition with an associated chronic inflammatory response. The ectopic growth of 'lesions', consisting of endometrial cells outside the uterine cavity, stimulates an inflammatory response initiating the activation of macrophages, and resulting in increased cytokine and growth factor concentrations in the peritoneal fluid (PF). Endometriosis‑associated inflammation is chronic and long lasting. In patients with endometriosis, the risk of developing ovarian cancer within 10 years, particularly of the endometrioid or clear cell subtype, is increased 2.5‑4 times. Endometriosis creates a peritoneal environment that exposes the affected endometriotic and the normal ovarian surface epithelial cells to agents that have been suggested to be involved in the pathogenesis of cancer. Concentrations of several cytokines and growth factors were increased in the PF of patients with endometriosis. The ovarian cancer marker, CA125, was one such growth factor; however, this remains to be confirmed. Human epididymis protein 4 (HE4) was detected at high concentrations in patients with ovarian cancer and was identified as the best biomarker for the detection of ovarian cancer. The present study determined the levels of HE4 and CA125 in the peritoneal fluid of 258 patients with and 100 control individuals without endometriosis attending the Department of Obstetrics and Gynaecology, University of Berne (Berne, Switzerland) between 2007 and 2014. The cases were subdivided into groups without hormonal treatment (n=107), or treated with combined oral contraceptives (n=45), continuous gestagens (n=56) or GnRH agonists (n=50). Both of these markers were significantly increased in the non‑treated endometriosis samples compared with the control group. Hormone treatment with either of the three agents mentioned resulted in the concentration of CA125 returning to the control levels and the concentration of HE4 decreasing to below the control levels. CA125, however not HE4, significantly differed between the proliferative and secretory cycle phases. Since HE4 is sensitive to hormonal treatment and robust towards menstrual cycle variation, HE4 is potentially superior to CA125 as an endometriosis marker and therefore has greater potential as a marker for the identification of women at risk of developing ovarian cancer.

Kinase signalling pathways in endometriosis: potential targets for non-hormonal therapeutics.

BACKGROUND Endometriosis, the growth of endometrial tissue outside the uterine cavity, is associated with chronic pelvic pain, subfertility and an increased risk of ovarian cancer. Current treatments include the surgical removal of the lesions or the induction of a hypoestrogenic state. However, a reappearance of the lesion after surgery is common and a hypoestrogenic state is less than optimal for women of reproductive age. Additional approaches are required. Endometriosis lesions exist in a unique microenvironment characterized by increased concentrations of hormones, inflammation, oxidative stress and iron. This environment influences cell survival through the binding of membrane receptors and a subsequent cascading activation of intracellular kinases that stimulate a cellular response. Many of these kinase signalling pathways are constitutively activated in endometriosis. These pathways are being investigated as therapeutic targets in other diseases and thus may also represent a target for endometriosis treatment. METHODS To identify relevant English language studies published up to 2015 on kinase signalling pathways in endometriosis, we searched the Pubmed database using the following search terms in various combinations; 'endometriosis', 'inflammation', 'oxidative stress', 'iron', 'kinase', 'NF kappa', 'mTOR', 'MAPK' 'p38', 'JNK', 'ERK' 'estrogen' and progesterone'. Further citing references were identified using the Scopus database and finally current clinical trials were searched on the clinicaltrials.gov trial registry. RESULTS The current literature on intracellular kinases activated by the endometriotic environment can be summarized into three main pathways that could be targeted for treatments: the canonical IKKβ/NFκB pathway, the MAPK pathways (ERK1/2, p38 and JNK) and the PI3K/AKT/mTOR pathway. A number of pharmaceutical compounds that target these pathways have been successfully trialled in in vitro and animal models of endometriosis, although they have not yet proceeded to clinical trials. The current generation of kinase inhibitors carry a potential for adverse side effects. CONCLUSIONS Kinase signalling pathways represent viable targets for endometriosis treatment. At present, however, further improvements in clinical efficacy and the profile of adverse effects are required before these compounds can be useful for long-term endometriosis treatment. A better understanding of the molecular activity of these kinases, including the specific extracellular compounds that lead to their activation in endometriotic cells specifically should facilitate their improvement and could potentially lead to new, non-hormonal treatments of endometriosis.

Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis.

Endometriosis is a gynecologic disease that is characterized by nonspecific symptoms and invasive diagnostics. To date, there is no adequate noninvasive method for the diagnosis of endometriosis. Although more than 100 potential biomarkers have been investigated in blood and/or peritoneal fluid, none of these has proven useful in clinical practice. The aim to find a suitable panel of biomarkers that would allow noninvasive diagnosis thus remains of interest. We evaluated the concentrations of 16 cytokines and other secretory proteins in serum and peritoneal fluid of 58 women with ovarian endometriosis (cases) and 40 healthy women undergoing sterilization or patients with benign ovarian cysts (controls) using multiplexed double fluorescence-based immunometric assay platform and enzyme-linked immunosorbent assay. Significantly higher concentrations of glycodelin-A were shown in serum, and significantly higher levels of glycodelin-A, IL-6, and IL-8, and lower levels of leptin were measured in the peritoneal fluid of cases versus controls. In serum, the best performance was shown by models that included the ratio of leptin/glycodelin-A and the ratio of ficolin 2/glycodelin-A, whereas in the peritoneal fluid the best models included the ratio of biglycan/leptin, regulated on activation normal T-cell expressed and secreted/IL-6 and ficolin-2/glycodelin-A, and IL-8 per milligram of total protein, all in combination with age. The models using serum and peritoneal fluid distinguished between ovarian endometriosis patients and controls regardless of the menstrual cycle phase with relatively high sensitivity (72.5% to 84.2%), specificity (78.4% to 91.2%), and area under the curve (0.85 to 0.90).

Tumor infiltrating lymphocytes in ovarian cancer.

Several improvements in ovarian cancer treatment have been achieved in recent years, both in surgery and in combination chemotherapy with targeting. However, ovarian tumors remain the women's cancers with highest mortality rates. In this scenario, a pivotal role has been endorsed to the immunological environment and to the immunological mechanisms involved in ovarian cancer behavior. Recent evidence suggests a loss of the critical balance between immune-activating and immune-suppressing mechanisms when oncogenesis and cancer progression occur. Ovarian cancer generates a mechanism to escape the immune system by producing a highly suppressive environment. Immune-activated tumor infiltrating lymphocytes (TILs) in ovarian tumor tissue testify that the immune system is the trigger in this neoplasm. The TIL mileau has been demonstrated to be associated with better prognosis, more chemosensitivity, and more cases of optimal residual tumor achieved during primary cytoreduction. Nowadays, scientists are focusing attention on new immunologically effective tumor biomarkers in order to optimize selection of patients for recruitment in clinical trials and to identify relationships of these biomarkers with responses to immunotherapeutics. Assessing this point of view, TILs might be considered as a potent predictive immunotherapy biomarker.