Alexandra Vodolazkaia

Density of small diameter sensory nerve fibres in endometrium: a semi-invasive diagnostic test for minimal to mild endometriosis.

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, Adelta, 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 approximately 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.

Nonhuman primate models for translational research in endometriosis.

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.

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.

Biomarkers of endometriosis

A noninvasive test for endometriosis would be useful for the early detection of endometriosis in symptomatic women who have pelvic pain and/or subfertility with normal ultrasound results. This would include nearly all cases of minimal-to-mild endometriosis, some cases of moderate-to-severe endometriosis without a clearly visible ovarian endometrioma, and cases with pelvic adhesions and/or other pelvic pathology that might benefit from surgery to improve pelvic pain and/or subfertility. This overview discusses the diagnostic performance of noninvasive or semi-invasive tests for endometriosis, including panels of known peripheral blood biomarkers, protein/peptide markers discovered by proteomics, miRNA, and endometrial nerve fiber density. Tests with high sensitivity and acceptable specificity have been developed; some have been validated in independent populations and are therefore promising. To make real progress, international agreement on biobank development is needed for standard operating procedures for the collection, treatment, storage, and analysis of tissue samples and for detailed clinical phenotyping of these samples. Furthermore, it is necessary to validate the diagnostic accuracy of any promising test prospectively in an independent symptomatic patient population with subfertility and/or pain without clear ultrasound evidence of endometriosis and with a clinical indication for surgery, divided into cases with laparoscopically and histologically confirmed endometriosis and controls with laparoscopically confirmed absence of endometriosis.

Combined mRNA microarray and proteomic analysis of eutopic endometrium of women with and without endometriosis

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.

Endometriosis and autoimmune disease: association of susceptibility to moderate/severe endometriosis with CCL21 and HLA-DRB1.

This study investigates the association of rheumatoid arthritis-associated single nucleotide polymorphisms in endometriosis. We found an association of CCL21 (rs2812378) and HLA-DRB1 (rs660895) with moderate to severe endometriosis.

Proteomics analysis of plasma for early diagnosis of endometriosis

OBJECTIVE: To test the hypothesis that differential surface-enhanced laser desorption/ionization time-of-flight mass spectrometry protein or peptide expression in plasma can be used in infertile women with or without pelvic pain to predict the presence of laparoscopically and histologically confirmed endometriosis, especially in the subpopulation with a normal preoperative gynecologic ultrasound examination. METHODS: Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry analysis was performed on 254 plasma samples obtained from 89 women without endometriosis and 165 women with endometriosis (histologically confirmed) undergoing laparoscopies for infertility with or without pelvic pain. Data were analyzed using least squares support vector machines and were divided randomly (100 times) into a training data set (70%) and a test data set (30%). RESULTS: Minimal-to-mild endometriosis was best predicted (sensitivity 75%, 95% confidence interval [CI] 63–89; specificity 86%, 95% CI 71–94; positive predictive value 83.6%, negative predictive value 78.3%) using a model based on five peptide and protein peaks (range 4.898–14.698 m/z) in menstrual phase samples. Moderate-to-severe endometriosis was best predicted (sensitivity 98%, 95% CI 84–100; specificity 81%, 95% CI 67–92; positive predictive value 74.4%, negative predictive value 98.6%) using a model based on five other peptide and protein peaks (range 2.189–7.457 m/z) in luteal phase samples. The peak with the highest intensity (2.189 m/z) was identified as a fibrinogen &bgr;-chain peptide. Ultrasonography-negative endometriosis was best predicted (sensitivity 88%, 95% CI 73–100; specificity 84%, 95% CI 71–96) using a model based on five peptide peaks (range 2.058–42.065 m/z) in menstrual phase samples. CONCLUSION: A noninvasive test using proteomic analysis of plasma samples obtained during the menstrual phase enabled the diagnosis of endometriosis undetectable by ultrasonography with high sensitivity and specificity. LEVEL OF EVIDENCE: II

Replication of endometriosis-associated single-nucleotide polymorphisms from genome-wide association studies in a Caucasian population

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.

A high sensitivity assay is more accurate than a classical assay for the measurement of plasma CRP levels in endometriosis

BackgroundEndometriosis is associated with chronic subclinical inflammation. C-reactive protein (CRP), a marker of inflammation, could serve as a biomarker of endometriosis. We tested the hypothesis that a high sensitivity CRP assay (hsCRP) is more accurate than a classical CRP assay in the detection of subclinical inflammation in plasma of women with endometriosis.MethodsCRP levels were measured by hsCRP and classical CRP assays in plasma of 204 women with endometriosis and 91 women without endometriosis. Both assays were compared with respect to their value for the diagnosis of endometriosis.ResultsThe number of plasma samples with detectable CRP was significantly higher (100%) using the hsCRP assay when compared to the classical CRP assay (42.7%) (p < 0.0001). Significantly increased CRP plasma levels were found in women with endometriosis when compared with controls when the hsCRP assay was used in samples obtained during the luteal phase (p = 0.008). The highest discriminative ability for the diagnosis of endometriosis was also obtained using the hsCRP assay during the luteal phase, especially for moderate -severe endometriosis. At a cut-off level of hsCRP > 0.71 mg/L, moderate-severe stages were diagnosed with 80.7% sensitivity and 63.9% specificity during the luteal phase. Using a similar cut-off value for CRP analyzed by the classical method, moderate-severe endometriosis was diagnosed with lower sensitivity (67.7%, p = 0.06) and comparable specificity (63.9%).ConclusionsThe hsCRP assay was superior to the classical CRP assay for the detection of low CRP levels and for revealing subclinical inflammation in plasma of women with endometriosis.

Analysis of common variations in tumor-suppressor genes on chr1p36 among Caucasian women with endometriosis

OBJECTIVE Epidemiological data indicate that endometriosis increases the risk of epithelial ovarian cancer (EOC), but the mechanism of cancer transition is unknown. Results from genome-wide association studies (GWAS) and transcriptome sequencing have demonstrated that genes located in the 1p36 region are important in both endometriosis and endometriosis-associated cancer development. Therefore, we tested the hypothesis that SNPs in two tumor-suppressor genes (CHD5 and ARID1A) in the 1p36 region are associated with endometriosis. METHODS Allele frequencies of SNPs were investigated in 1685 Caucasian women consisting of 947 women with endometriosis and 738 controls. Peripheral blood samples were retrieved, DNA extracted and allelic frequencies of SNPs in two tumor-suppressor genes (CHD5 and ARID1A) were analyzed using TaqMan Open Array technique. RESULTS Associations were observed for 3 SNPs in the CHD5 gene: rs1883603 (OR 1.31, 95% CI 1.00-1.71), rs9434741 (OR 1.41, 95% CI 1.16-1.71) and rs17436816 (OR 1.24, 95% CI 1.02-1.50). After correction for multiple comparisons, rs9434741 (CHD5) remained significantly associated with endometriosis (p<0.01). No associations were detected for ARID1A. CONCLUSIONS In this Caucasian population, endometriosis seems to be associated with the tumor-suppressor gene CHD5. Our findings support recent data, suggesting that the 1p36 region plays an important role in endometrios. To validate these data, replication in an independent population is warranted.