Technical Verification and Assessment of Independent Validation of Biomarker Models for Endometriosis.

There is a great need for a noninvasive diagnosis for endometriosis. Several biomarkers and biomarker panels have been proposed. Biomarker models consisting of CA-125, VEGF, Annexin V, and glycodelin/sICAM-1 were previously developed by our group. The objective of our current study was to assess the impact of technical and biological variability on the performance of those previously developed prediction models in a technical verification and a validation setting. The technical verification cohort consisted of peripheral blood plasma samples from a subset of the patients included in the original study of Vodolazkaia et al. (99 women with and 37 women without endometriosis). The validation study was done in plasma samples of an independent patient cohort (170 women with and 86 women without endometriosis). Single immunoassays were used for CA-125, VEGF-A, sICAM-1, Annexin V, and glycodelin. Statistical analyses were done using univariate and multivariate (logistic regression) approaches. The previously reported prediction models for endometriosis had a low performance in both the technical verification and validation setting. New prediction models were developed, which included CA-125, Annexin V, and sICAM-1, but CA-125 was the only marker that was retained in the models across the technical verification and validation study. Overall, successful validation of a biomarker model depends on several factors such as patient selection, collection methods, assay selection/handling, stability of the marker, and statistical analysis and interpretation. There is a need for standardized studies in large, well-defined patient cohorts with robust assay methodologies.

Evaluation of Total, Active, and Specific Myeloperoxidase Levels in Women with and without Endometriosis.

Myeloperoxidase (MPO) is a proinflammatory enzyme and a marker for neutrophil activation and oxidative stress. Since oxidative stress and inflammation are linked to the pathogenesis of endometriosis, we hypothesized that the total, active, and specific (active/total) MPO levels were significantly different in plasma of women with and without endometriosis. Samples were selected from our biobank from women with endometriosis (n = 212) and controls without endometriosis (n = 121) across the menstrual cycle. Total MPO plasma levels were measured by immunoassay and MPO activity by enzymatic assay. Total and active MPO levels did not differ significantly among endometriosis cases and controls, whereas the specific MPO activity was significantly lower in women with endometriosis than that in controls (p = 0.0159). After the subdivision of control patients into women with a normal pelvis and women with other benign gynecological disorders, a significant difference was observed only between women with endometriosis and women with other benign gynecological disorders (p = 0.0266). In conclusion, systemic MPO levels may not be suited as a single biomarker for endometriosis. Our data support the involvement of MPO in other gynecological disorders but do not provide any evidence for an association with endometriosis.

The Presence of Endometrial Cells in Peritoneal Fluid of Women With and Without Endometriosis.

To reinforce Sampson's theory of retrograde menstruation in the pathogenesis of endometriosis, proof should be provided that during menstruation endometrial cells are present in peritoneal fluid (PF). We hypothesize that the prevalence of PF samples containing endometrial cells is higher in patients with endometriosis than in controls without endometriosis during menstruation. We selected from our biobank PF samples of 17 reproductive-age women with (n = 9) or without (n = 8) endometriosis who had received a diagnostic laparoscopy for investigation of pain/infertility. Peritoneal fluid had been collected during laparoscopy in the menstrual phase of the cycle, centrifuged, and the resulting pellet was stored at -80°C. About 5-µm sections of frozen PF pellets were stained using the Dako Envision Flex system with primary antibodies against epithelial cell adhesion molecule (Ep-CAM; endometrial epithelial cells), CD10 (endometrial stromal cells), prekeratin (epithelial/mesothelial cells), vimentin (endometrial/mesothelial/immune cells), calretinin (mesothelial cells), and CD68 (macrophages). The PF cells positive for Ep-CAM were detected in 5 of 9 patients with endometriosis and 6 of 8 controls ( P = .62). CD10 stained positively in 6 of the 9 patients with endometriosis and 3 of the 8 controls ( P = .35). Calretinin and prekeratin staining showed the presence of mesothelial cells in all pellets. Vimentin stained approximately 100% of the PF cells. CD68+ macrophages represented >50% of cells in all pellets. The prevalence of PF samples containing endometrial epithelial and stromal cells was not higher in patients with endometriosis than in controls without endometriosis during menstruation. Our findings question the relevance of endometrial cells in PF for the pathogenesis of endometriosis and support the importance of other mechanisms such as immune dysfunction and/or endometrial stem cells.

Laparoscopic Surgery: A New Technique to Induce Endometriosis in a Mouse Model.

BACKGROUND: This prospective pilot study was designed to induce endometriosis in a mouse model using laparoscopy, a less invasive and more precise approach than laparotomy. We aimed to achieve a peritoneal implant rate of at least 50% by varying both duration of anesthesia and intra-abdominal insufflation pressure. METHODS: Female BALB/cANnCrl mice in metestrus or diestrus were used as donors (n = 5) or recipients (n = 20) of uterine transplant tissue. Each recipient mouse was laparoscopically inoculated with 10 uterine pieces (range: 10-12) from donor mice into the abdominal cavity. Before starting the study, recipient mice were randomly assigned to 4 groups with variable duration of anesthesia (ketamine/xylazine or pentobarbital) and variable intra-abdominal pressure (5 or 15 mm Hg). One week after laparoscopy, endometriosis incidence and peritoneal implant take rate were documented visually during laparotomy. The retrieved lesions were histologically analyzed. RESULTS: Laparoscopic inoculation of uterine pieces in recipient mice resulted in an endometriosis incidence of 100% (20/20 animals) and an individual peritoneal implant take rate of 60% (121/206), ranging from 17% (2/12) till 83% (10/12), without differences between the 4 subgroups, and with a histological confirmation rate of 92% (58/63). CONCLUSIONS: To the best our knowledge, this is the first report showing that endometriosis can be induced by laparoscopic surgery in rodents, with a 100% incidence and a median peritoneal implant take rate of 60%. This laparoscopic model offers important advantages over traditional laparotomy models that are limited by surgery-associated trauma and/or adhesion formation.

Dysregulation of Lysyl Oxidase Expression in Lesions and Endometrium of Women With Endometriosis.

UNLABELLED: Lysyl oxidases (LOXs) are enzymes involved in collagen deposition, extracellular membrane remodeling, and invasive/metastatic potential. Previous studies reveal an association of LOXs and endometriosis. We aimed to identify the mechanisms activated by upregulation of lysyl oxidases (LOX) in endometriotic cells and tissues. We hypothesized that LOX plays a role in endometriosis by promoting invasiveness and epithelial to mesenchymal transition (EMT). METHODS: The LOX protein expression levels were measured by immunohistochemistry in lesions and endometrium on a tissue microarray (TMA) and in endometrial biopsies from patients and controls during the window of implantation (WOI). Estradiol regulation of LOX expression was determined by quantitative polymerase chain reaction (qPCR). Proliferation, invasion, and migration assays were performed in epithelial (endometrial epithelial cell), endometrial (human endometrial stromal cell), and endometriotic cell lines (ECL and 12Z). Pathway-focused multiplex qPCR was used to determine transcriptome changes due to LOX overexpression. RESULTS: LOX protein was differentially expressed in ovarian versus peritoneal lesions. During WOI, LOX levels were higher in luminal epithelium of patients with endometriosis-associated infertility compared to controls. Invasive epithelial cell lines expressed higher levels of LOX than noninvasive ones. Transfection of LOX into noninvasive epithelial cells increased their migration in an LOX inhibitor-sensitive manner. Overexpression of LOX did not fully induce EMT but the expression of genes related to fibrosis and extracellular matrix remodeling were dysregulated. CONCLUSIONS: This study documents that expression of LOX is differentially regulated in endometriotic lesions and endometrium. A role for LOX in mediating proliferation, migration, and invasion of endometrial and endometriotic cells was observed, which may be implicated in the establishment and progression of endometriotic lesions.

Cross-species behavioural genetics: A starting point for unravelling the neurobiology of human psychiatric disorders.

Identifying the genetic and neurobiological mechanisms underlying certain behavioural traits is an important strategy to understand the aetiology of various psychiatric disorders and to find potential new treatment possibilities. It has proven a great challenge to develop paradigms that allow translational research for behavioural phenotypes that are relevant for disorders across the psychiatric spectrum. Recently, there has been increasing attention for studies that implement rodent behavioural paradigms in the home cage to assess the association between genetic backgrounds and behavioural traits. The application of interspecies genetics to unravel these traits has revealed novel insights in the genetic mechanisms that are encoding phenotypes relevant to biological processes underlying psychiatric disorders. By means of two examples, namely the stress-induced hyperthermia paradigm and the home cage environment, this review aims to show that by using individual genetic variations with phenotypes obtained from mice and across categories of neuropsychiatric disorders, novel insights in the neurobiological trajectory of psychiatric disorders can be obtained.

The autonomic stress-induced hyperthermia response is not enhanced by several anxiogenic drugs.

While anxiety models are often based on locomotor activity responses, the stress-induced hyperthermia (SIH) paradigm uses the autonomic stress response by measuring body temperature. The effects of putative anxiogenic compounds in the SIH paradigm are inconclusive in mice and have not been examined in rats. Furthermore, it has been suggested that drug-induced effects on body temperature could be dependent on locomotor activity levels. Therefore, the effects of three anxiogenic substances, yohimbine (an α(2) receptor antagonist), mCPP (a 5HT(2C) receptor agonist) and FG-7142 (a GABA(A) receptor inverse agonist acting at the benzodiazepine site) on the stress-induced body temperature and locomotor activity response were studied in rats using novel cage stress. All anxiogenic compounds resulted in hypothermia. In contrast, FG-7142 and yohimbine increased locomotor activity levels, whereas mCPP reduced locomotor activity levels. The lack of an increased body temperature response of anxiogenic compounds indicates that the anxiogenic capacity of a drug does not necessarily yield increased autonomic stress responsivity. Moreover, the present study shows that a drug-induced decreased body temperature can be accompanied by increased locomotor activity, suggesting that both parameters represent independent parameters of the stress response.