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.
BACKGROUND
An early semi-invasive diagnosis of endometriosis has the potential to allow early treatment and minimize disease progression but no such test is available at present. Our aim was to ...perform a combined mRNA microarray and proteomic analysis on the same eutopic endometrium sample obtained from patients with and without endometriosis.
METHODS
mRNA and protein fractions were extracted from 49 endometrial biopsies obtained from women with laparoscopically proven presence (n= 31) or absence (n= 18) of endometriosis during the early luteal (n= 27) or menstrual phase (n= 22) and analyzed using microarray and proteomic surface enhanced laser desorption ionization-time of flight mass spectrometry, respectively. Proteomic data were analyzed using a least squares-support vector machines (LS-SVM) model built on 70% (training set) and 30% of the samples (test set).
RESULTS
mRNA analysis of eutopic endometrium did not show any differentially expressed genes in women with endometriosis when compared with controls, regardless of endometriosis stage or cycle phase. mRNA was differentially expressed (P< 0.05) in women with (925 genes) and without endometriosis (1087 genes) during the menstrual phase when compared with the early luteal phase. Proteomic analysis based on five peptide peaks 2072 mass/charge (m/z); 2973 m/z; 3623 m/z; 3680 m/z and 21133 m/z using an LS-SVM model applied on the luteal phase endometrium training set allowed the diagnosis of endometriosis (sensitivity, 91; 95% confidence interval (CI): 74–98; specificity, 80; 95% CI: 66–97 and positive predictive value, 87.9%; negative predictive value, 84.8%) in the test set.
CONCLUSION
mRNA expression of eutopic endometrium was comparable in women with and without endometriosis but different in menstrual endometrium when compared with luteal endometrium in women with endometriosis. Proteomic analysis of luteal phase endometrium allowed the diagnosis of endometriosis with high sensitivity and specificity in training and test sets. A potential limitation of our study is the fact that our control group included women with a normal pelvis as well as women with concurrent pelvic disease (e.g. fibroids, benign ovarian cysts, hydrosalpinges), which may have contributed to the comparable mRNA expression profile in the eutopic endometrium of women with endometriosis and controls.
BACKGROUND The aim of our study was to test the hypothesis that multiple-sensory small-diameter nerve fibres are present in a higher density in endometrium from patients with endometriosis when ...compared with women with a normal pelvis, enabling the development of a semi-invasive diagnostic test for minimal–mild endometriosis. METHODS Secretory phase endometrium samples (n = 40), obtained from women with laparoscopically/histologically confirmed minimal–mild endometriosis (n = 20) and from women with a normal pelvis (n = 20) were selected from the biobank at the Leuven University Fertility Centre. Immunohistochemistry was performed to localize neural markers for sensory C, Aδ, adrenergic and cholinergic nerve fibres in the functional layer of the endometrium. Sections were immunostained with anti-human protein gene product 9.5 (PGP9.5), anti-neurofilament protein, anti-substance P (SP), anti-vasoactive intestinal peptide (VIP), anti-neuropeptide Y and anti-calcitonine gene-related polypeptide. Statistical analysis was done using the Mann–Whitney U-test, receiver operator characteristic analysis, stepwise logistic regression and least-squares support vector machines. RESULTS The density of small nerve fibres was ∼14 times higher in endometrium from patients with minimal–mild endometriosis (1.96 ± 2.73) when compared with women with a normal pelvis (0.14 ± 0.46, P < 0.0001). CONCLUSIONS The combined analysis of neural markers PGP9.5, VIP and SP could predict the presence of minimal–mild endometriosis with 95% sensitivity, 100% specificity and 97.5% accuracy. To confirm our findings, prospective studies are required.
The administration of toxin-specific therapy in snake envenoming is predicated on improved diagnostic techniques capable of detecting specific venom toxins. Various serological tests have been used ...in detecting snakebite envenoming. Comparatively, enzyme-linked immunosorbent assay (ELISA) has been shown to offer a wider practical application. We report an inhibition ELISA for detecting three-finger toxin (3FTx) proteins in venoms of African spitting cobras. The optimized assay detected 3FTxs in
(including other
sp.) venoms, spiked samples, and venom-challenged mice samples. In venoms of
sp., the assay showed inhibition, implying the detection of 3FTxs, but showed little or no inhibition in non-
sp. In mice-spiked samples, one-way ANOVA results showed that the observed inhibition was not statistically significant between spiked samples and negative control (
-value = 0.164). Similarly, the observed differences in inhibition between venom-challenged and negative control samples were not statistically significant (
-value = 0.9109). At an LOD of 0.01 µg/mL, the assay was able to confirm the presence of 3FTxs in the samples. Our results show a proof of concept for the use of an inhibition ELISA model as a tool for detecting 3FTxs in the venoms of African spitting cobra snakes.
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.
Endometriosis, defined as the ectopic presence of endometrial-like cells, is associated with infertility and pelvic pain in women. Whereas pathogenesis and spontaneous evolution of endometriosis are ...still poorly understood, recurrences after surgical therapy or after medical treatment are common. Spontaneous endometriosis occurs only in women and in nonhuman primates (NHPs). Inbred rhesus monkeys kept in colonies offer an attractive preclinical model to study the inheritance of spontaneous endometriosis. Baboons with spontaneous or induced endometriosis appear to be the best NHP model to study pathogenesis, pathophysiology, spontaneous evolution and new medical treatment options. In baboons, induction of endometriosis after intrapelvic injection of menstrual endometrium leads to biological changes in peritoneal cavity and in endometrium. This induction process may allows the study of cause-effect relationships which may lead to the discovery of new biomarkers for the development of new non-invasive diagnostic tests and drugs that may prevent or treat endometriosis.
Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known ...to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.
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.
Neutralization of lethality in mice model at the preclinical level has been established by the World Health Organization as the gold standard for the evaluation of antivenom efficacy. The assessment ...of the neutralization profiles of antivenoms helps to discern the efficacy or otherwise of these antivenoms at neutralizing the toxic effects induced by medically significant snake venoms. However, for many antivenoms, information on their preclinical efficacy remains limited. Therefore, to strengthen global efforts at reducing the impact of snakebite envenoming, the provision of information on the preclinical efficacy of antivenoms, especially in parts of the world where antivenom availability and accessibility is problematic, including sub-Saharan Africa is crucial. This study presents the lethal and toxic activities of N. ashei venom and the neutralizing capacity of two commonly used commercial antivenoms in Kenya; VINS™ and Inoserp™. Median lethal dose (LD50), minimum necrotizing dose (MND) and minimum edema-forming dose (MED) of N. ashei venom as well as the neutralization of these effects were evaluated in mice. The LD50 of N. ashei venom was found to be 4.67 (3.34–6.54) mg/kg while MND and MED were 11.00 μg and 0.80 μg respectively. Both VINS™ and Inoserp™ antivenoms demonstrated capacity to neutralize the lethal and toxic effects induced by Naja ashei venom albeit at varying efficacies. Our results thus confirm the toxic effects of N. ashei venom as previously observed with other Naja sp. venoms and also underscore the relevance of para-specific neutralizing capacity of antivenoms in the design of antivenoms.
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•Naja ashei venom induces cytotoxic effects as previously observed with other cobras.•VINS™ and Inoserp™ antivenoms abrogate lethal effects induced by N. ashei venom.•Para-specific neutralization of venom remains crucial in the design of antivenoms.
STUDY QUESTION
Is it possible to replicate the previously identified genetic association of four single-nucleotide polymorphisms (SNPs), rs12700667, rs7798431, rs1250248 and rs7521902, with ...endometriosis in a Caucasian population?
SUMMARY ANSWER
A borderline association was observed for rs1250248 and endometriosis (P = 0.049). However, we could not replicate the other previously identified endometriosis-associated SNPs (rs12700667, rs7798431 and rs7521902) in the same population.
WHAT IS KNOWN ALREADY
Endometriosis is considered a complex disease, influenced by several genetic and environmental factors, as well as interactions between them. Previous studies have found genetic associations with endometriosis for SNPs at the 7p15 and 2q35 loci in a Caucasian population.
STUDY DESIGN, SIZE, DURATION
Allele frequencies of SNPs were investigated in patients with endometriosis and controls.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Blood samples and peritoneal biopsies were taken from a Caucasian female population consisting of 1129 patients with endometriosis and 831 controls. DNA was extracted for genotyping. The study was performed at a University hospital and research laboratories.
MAIN RESULTS AND THE ROLE OF CHANCE
A weak association with endometriosis (all stages) was observed for rs1250248 (P = 0.049). No significant associations were observed for the SNPs rs12700667, rs7798431 and rs7521902. A non-significant trend towards the association of rs1250248 with moderate/severe endometriosis was observed (odds ratio 1.18, 95% confidence interval 0.97–1.44).
LIMITATIONS, REASONS FOR CAUTION
The inability to confirm all previous findings may result from differences between populations and type II errors.
WIDER IMPLICATIONS OF THE FINDINGS
Our result demonstrates the difficulty of identifying common genetic variants in complex diseases.
STUDY FUNDING/COMPETING INTEREST(S)
This study was supported by grants from the Karolinska Institutet and Stockholm City County/Karolinska Institutet (ALF), Stockholm, Sweden, Swedish Medical Research Council (K2007-54X-14212-06-3, K2010-54X-14212-09-3), Stockholm, Sweden, Leuven University Research Council (Onderzoeksraad KU Leuven), the Leuven University Hospitals Clinical Research Foundation (Klinisch onderzoeksfonds) and by the National Scientific Foundation (Fonds voor Wetenschappelijk Onderzoek, FWO). The authors have no conflict of interest.
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