Evaluation of a panel of 28 biomarkers for the non-invasive diagnosis of endometriosis.

BACKGROUND: At present, the only way to conclusively diagnose endometriosis is laparoscopic inspection, preferably with histological confirmation. This contributes to the delay in the diagnosis of endometriosis which is 6-11 years. So far non-invasive diagnostic approaches such as ultrasound (US), MRI or blood tests do not have sufficient diagnostic power. Our aim was to develop and validate a non-invasive diagnostic test with a high sensitivity (80% or more) for symptomatic endometriosis patients, without US evidence of endometriosis, since this is the group most in need of a non-invasive test. METHODS: A total of 28 inflammatory and non-inflammatory plasma biomarkers were measured in 353 EDTA plasma samples collected at surgery from 121 controls without endometriosis at laparoscopy and from 232 women with endometriosis (minimal-mild n = 148; moderate-severe n = 84), including 175 women without preoperative US evidence of endometriosis. Surgery was done during menstrual (n = 83), follicular (n = 135) and luteal (n = 135) phases of the menstrual cycle. For analysis, the data were randomly divided into an independent training (n = 235) and a test (n = 118) data set. Statistical analysis was done using univariate and multivariate (logistic regression and least squares support vector machines (LS-SVM) approaches in training- and test data set separately to validate our findings. RESULTS: In the training set, two models of four biomarkers (Model 1: annexin V, VEGF, CA-125 and glycodelin; Model 2: annexin V, VEGF, CA-125 and sICAM-1) analysed in plasma, obtained during the menstrual phase, could predict US-negative endometriosis with a high sensitivity (81-90%) and an acceptable specificity (68-81%). The same two models predicted US-negative endometriosis in the independent validation test set with a high sensitivity (82%) and an acceptable specificity (63-75%). CONCLUSIONS: In plasma samples obtained during menstruation, multivariate analysis of four biomarkers (annexin V, VEGF, CA-125 and sICAM-1/or glycodelin) enabled the diagnosis of endometriosis undetectable by US with a sensitivity of 81-90% and a specificity of 63-81% in independent training- and test data set. The next step is to apply these models for preoperative prediction of endometriosis in an independent set of patients with infertility and/or pain without US evidence of endometriosis, scheduled for laparoscopy.

Non-invasive diagnosis of endometriosis based on a combined analysis of six plasma biomarkers.

BACKGROUND: Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. The aim of this study was to evaluate the combined performance of six potential plasma biomarkers in the diagnosis of endometriosis. METHODS: This case-control study was conducted in 294 infertile women, consisting of 93 women with a normal pelvis and 201 women with endometriosis. We measured plasma concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha, high-sensitivity C-reactive protein (hsCRP), and cancer antigens CA-125 and CA-19-9. Analyses were done using the Kruskal-Wallis test, Mann-Whitney test, receiver operator characteristic, stepwise logistic regression and least squares support vector machines (LSSVM). RESULTS: Plasma levels of IL-6, IL-8 and CA-125 were increased in all women with endometriosis and in those with minimal-mild endometriosis, compared with controls. In women with moderate-severe endometriosis, plasma levels of IL-6, IL-8 and CA-125, but also of hsCRP, were significantly higher than in controls. Using stepwise logistic regression, moderate-severe endometriosis was diagnosed with a sensitivity of 100% (specificity 84%) and minimal-mild endometriosis was detected with a sensitivity of 87% (specificity 71%) during the secretory phase. Using LSSVM analysis, minimal-mild endometriosis was diagnosed with a sensitivity of 94% (specificity 61%) during the secretory phase and with a sensitivity of 92% (specificity 63%) during the menstrual phase. CONCLUSIONS: Advanced statistical analysis of a panel of six selected plasma biomarkers on samples obtained during the secretory phase or during menstruation allows the diagnosis of both minimal-mild and moderate-severe endometriosis with high sensitivity and clinically acceptable specificity.

Non-steroidal targets in the diagnosis and treatment of endometriosis.

Endometriosis, a chronic gynecologic disease frequently resulting in chronic pelvic pain, severe dysmenorrhoea, and subfertility, is defined as the presence of endometrial tissue at extrauterine locations, most commonly on the peritoneum and ovaries. Conclusive diagnosis requires laparoscopic surgery followed by histological confirmation. The treatment options -at present- are limited to hormonal therapies and/or surgical ablation of the lesions, and are characterized by high recurrence rates, significant side-effects and limited duration of administration. The pathogenesis of endometriosis is still unclear and numerous immunological and inflammatory factors have been suggested to be involved in the development of the disease, including interleukin (IL)-1, IL-2, IL-6, IL-8, IL-12, tumour necrosis factor -alpha (TNF-alpha), regulated on activation, normal T-Cell expressed and secreted (RANTES) and its receptor cognate chemokine receptor 1 (CCR1), peroxisome proliferator activated receptors (PPARs), matrix metalloproteinases (MMPs) and cyclooxygenase (COX). Another crucial mechanism in endometriosis is the vascularisation of the endometriotic lesions, with a key role for vascular endothelial growth factor (VEGF). Recently, protease activated receptors (PARs), mitogen-activated protein kinases (MAPKs) and tyrosine kinases have also been associated with the pathophysiology of endometriosis. The aim of this article is to discuss molecules that have recently been found to have connections with the pathogenesis of endometriosis, as potential targets to develop new methods for noninvasive diagnosis and for novel medical management of this disease. This review also critically addresses how these molecules can be tested in basic, preclinical and clinical research, the status of this research and the importance of potential side effects.

Global gene analysis of late secretory phase, eutopic endometrium does not provide the basis for a minimally invasive test of endometriosis.

BACKGROUND: Endometriosis occurs in 10% of women and is currently diagnosed by invasive laparoscopic testing. We tested the hypothesis that endometrial gene expression in late secretory phase endometrium differs between patients with and without endometriosis. METHODS: Ten patients with laparoscopically proven endometriosis (minimal/mild n = 5 and moderate/severe n = 5) and six controls, underwent endometrial biopsy in the late secretory phase (Day 23 onwards). Microarray interrogation of eutopic endometrial gene expression was performed. RESULTS: Microarray data were obtained for all control samples and eight samples from the endometriosis patients (n = 4 minimal/mild, n = 4 moderate/severe disease). Eight genes were identified as up-regulated and one gene was down-regulated in all endometriotic samples (more than 1.75-fold, P < 0.01). Real-time PCR analysis of protocadherin-17 (PCDH17), protein tyrosine phosphatase, receptor type, R (PTPRR) and interleukin-6 signal transducer (IL6ST) expression validated the microarray findings. CONCLUSIONS: Expression of very few transcripts differs, in late secretory eutopic endometrium, between controls and patients with endometriosis. The median fold changes of these genes are small. No transcripts were identified that could discriminate between minimal/mild and moderate/severe endometriosis. Therefore, interrogation of the late secretory endometrial transcriptome is not likely to form the basis of a minimally invasive diagnostic test for endometriosis.

Endometriosis, recurrent miscarriage and implantation failure: is there an immunological link?

Endometriosis is more frequently diagnosed in patients with infertility than in a normal population. The goal of this paper is to give an overview of the clinical and fundamental evidence for a possible link between endometriosis and (recurrent) miscarriage or implantation failure after treatment with assisted reproductive technology. According to the literature, there is insufficient evidence for an association between endometriosis and (recurrent) miscarriage, but there is, however, epidemiological evidence to support the link between endometriosis and recurrent implantation failure after assisted reproduction. This can possibly be explained by alterations in humoral and cell-mediated immunity in women with endometriosis. Humoral immunological changes include increased formation of antibodies against endometrial antigens, anti-laminin-1 auto-antibodies and other auto-immune antibodies (e.g. antiphospholipid). Cell-mediated immunological changes include alterations in peritoneal and follicular fluid immune cells and cytokines. The possible negative effect of these immunological changes on folliculogenesis, ovulation, oocyte quality, early embryonic development and implantation in women with endometriosis suggests that infertility in endometriosis patients may be related to alterations within the follicle or oocyte, resulting in embryos with decreased ability to implant.

Treatment with anti-TNF monoclonal antibody (c5N) reduces the extent of induced endometriosis in the baboon.

BACKGROUND: Inflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha), are important in the pathogenesis of endometriosis. We assessed the efficacy of anti-TNF monoclonal antibody (mAb, c5N), known to prevent induced endometriosis in baboons, in reducing established endometriosis in baboons. METHODS: This prospective, randomized, blinded, controlled study was conducted in baboons at the Institute of Primate Research (IPR), Nairobi, Kenya. Endometriosis was induced in 18 adult female baboons (Papio anubis) with regular menstrual cycles and a normal pelvis; the extent of endometriosis was documented by videolaparoscopy 25 days later. The baboons were then randomly assigned to receive a single infusion of either placebo (n=7, 5 ml/kg) or c5N (n=11, 5 mg/kg). Follow-up laparoscopy was performed 25 days later to document any differences in the number, surface area and estimated volume of lesions between the two groups and between the first and the second laparoscopies in each group. Representative biopsies of at least one endometriotic lesion per baboon were obtained at the final laparoscopy. RESULTS: Significant reductions in total surface area, estimated total volume of endometriotic lesions and both number and surface area of red lesions were observed after treatment with c5N, but not after placebo treatment, when compared to the initial laparoscopy. Conversely, a significant increase in the number of typical and red lesions was observed after placebo treatment when compared to the initial laparoscopy. Neither c5N nor placebo treatment affected the menstrual cycle. CONCLUSION: In baboons with induced endometriosis, anti-TNF-mAb (c5N) treatment significantly reduced the extent of endometriosis, mainly due to reducing both the number and surface area of red lesions. These findings suggest that anti-TNF-mAb therapy may have therapeutic potential for active peritoneal endometriosis.

Synergism between beta 2-adrenoceptor agonists and subtype-selective alpha 1A-adrenoceptor antagonists in the tocolytic effect on pregnant rat uterus in vitro.

1. Despite great efforts in recent decades, premature birth is still a leading cause of perinatal morbidity and mortality. beta2-Adrenoceptor agonists are frequently used as tocolytics, although their use is rather controversial. Previous animal studies have revealed that blockade of alpha1A-adrenoceptors results in relaxation of the pregnant rat myometrium. 2. The aim of the present study was to investigate the uterus relaxant effect of the beta2-adrenoceptor agonists (terbutaline, ritodrin) applied together with the subtype-selective alpha1A-adrenoceptor antagonists (WB 4101, 5-methylurapidil) in an in vitro rat model. The main objective of the experiments was to clarify whether there was an additive or a potentiating synergism between the two drug classes. 3. Myometrial rings were taken from female, 22-day pregnant (end-term) Sprague-Dawley rats. Electrical field stimulation (EFS) was used to elicit rhythmical contractions. Non-cumulative concentration-response curves were constructed to the beta2-adrenoceptor agonists and the alpha1A-adrenoceptor antagonists alone and to beta2-adrenoceptor agonists co-administered with the alpha1A-adrenoceptor antagonists. 4. Both groups of drugs inhibited EFS-induced contractions in a dose-dependent way. Administering the beta2-adrenoceptor agonists in combination with the alpha1A-adrenoceptor antagonists resulted in a significant decrease in the EC50 and an increase in the maximal contraction inhibiting effect. 5. The potentiating synergism that has been revealed between beta2-adrenoceptor agonists and alpha1A-adrenoceptor antagonists in the uterus relaxant effect may be of great clinical importance because it could improve the efficacy of therapy of preterm delivery.