Biomarker Panel in Ovarian Torsion

The overall goal of the project is to find a panel of novel biomarkers for early detection of ovarian torsion.

The specifc aims of the current study are:

1. Screen for changes in blood protein biomarkers concentrations specifc for patients with ovarian torsion. The biomarkers are: D-Dimer (21.7-5,280 ng/ml), FABP4/A-FABP (0.637-155 ng/ml), GM-CSF (0.012-2.98 ng/ml), ICAM-1/CD54 (6.9-1674 ng/ml), IFN-γ (0.058-14.2 ng/ml), IL-1α (0.005-1.27 ng/ml), IL-6 (0.005-1.2 ng/ml), IL-10 (0.005-1.2 ng/ml), IL-17/IL-17A (0.012-3.1 ng/ml), Park7/DJ-1 (0.26- 63.1 ng/ml), TNF-α (0.01-2.4 ng/ml), TSLP (0.003-0.7 ng/ml), VCAM-1/CD106 (7.8-1891 ng/ml) and VEGF (0.008-2.4 ng/ml).
2. Compare the amount of oxidative stress defense proteins SOD1, Catalase, and Thioredoxin amount in the peritoneal fluid wash between patients with or without confirmed ovarian torsion. To our knowledge, this is the first study that specifically evaluates the expression of oxidation stress defense proteins in ovarian torsion. The results will enable us to decide whether to include oxidation stress biomarkers within the panel.
3. Study population methodology and techniques

A prospective, 12-24 months study.

20 Patients with confirmed ovarian torsion will be enrolled.

The patients will complete a questionnaire prior to and following the procedure

3.5ml blood samples will be obtained immediately before laparoscopy, postoperative day, and four weeks after laparoscopy during the follow-up.

The peritoneal fluid wash will be collected during the laparoscopy from 14 patients (out of the 20 enrolled) that will provide additional written consent specific for the procedure.

20 Patients suspected of ovarian torsion with no ovarian torsion, as confirmed by laparoscopy, will be enrolled.

The patients will complete a questionnaire prior to and following the procedure

5ml blood samples will be obtained immediately before laparoscopy and four weeks after laparoscopy during the follow-up.

The peritoneal fluid wash will be collected during the laparoscopy from 14 patients (out of the 20 enrolled) that will provide additional written consent specific for the procedure.

20 Healthy controls will be enrolled and provide a 5ml blood sample.

• biomakers for ovarian torsion (Diagnostic test): Blood biomarkers analysis using multiplex ELISA: The Luminex system is a magnetic bead-based multiplex ELISA that enable to measure simultaneously multiple proteins within the same sample. Human magnetic Luminex assay will be used for the following analytes: D-Dimer (21.7-5,280 ng/ml), FABP4/A-FABP (0.637-155 ng/ml), GM-CSF (0.012-2.98 ng/ml), ICAM-1/CD54 (6.9-1674 ng/ml), IFN-γ (0.058-14.2 ng/ml), IL-1α (0.005-1.27 ng/ml), IL-6 (0.005-1.2 ng/ml), IL-10 (0.005-1.2 ng/ml), IL-17/IL-17A (0.012-3.1 ng/ml), Park7/DJ-1 (0.26- 63.1 ng/ml), TNF-α (0.01-2.4 ng/ml), TSLP (0.003-0.7 ng/ml), VCAM-1/CD106 (7.8-1891 ng/ml) and VEGF (0.008-2.4 ng/ml). Plasma samples stored at -70°C will be sent to accredited lab at BioTest (Israel) for performing the multiplex ELISA readout assay test using MagPix system.
• peritoneal fluid proteins analysis (Diagnostic test): Western blot for peritoneal fluid proteins analysis We will look for the relative abundance of proteins involved in the protection of cells against oxidative stress and the regulation of reactive oxygen species (ROS) using western blot. Superoxide dismutase 1 (SOD1, 16kDa) scavenges superoxides (O-2) within cells. Catalase (60kDa) is hydrogen peroxide (H2O2) scavenging enzyme regulate ROS concentrations within cells by reducing H202 into O2 and water. Thioredoxin (12kDa) facilitates the reduction of proteins via cysteine thiol-disulfide exchange. Alpha smooth muscle actin (42kDa) will be used as a loading control. These four proteins will be resolved by western blot given their different molecular weights using western blot cocktail antibodies (abcam # ab179843).

Primary outcomes

• preoperative biochemical test -BIOMARKER PANEL FOR OVARIAN TORSION [2 years]: Screen for elevation in blood protein biomarkers concentrations specifc for patients with ovarian torsion. The biomarkers are: D-Dimer (21.7-5,280 ng/ml), FABP4/A-FABP (0.637-155 ng/ml), GM-CSF (0.012-2.98 ng/ml), ICAM-1/CD54 (6.9-1674 ng/ml), IFN-γ (0.058-14.2 ng/ml), IL-1α (0.005-1.27 ng/ml), IL-6 (0.005-1.2 ng/ml), IL-10 (0.005-1.2 ng/ml), IL-17/IL-17A (0.012-3.1 ng/ml), Park7/DJ-1 (0.26- 63.1 ng/ml), TNF-α (0.01-2.4 ng/ml), TSLP (0.003-0.7 ng/ml), VCAM-1/CD106 (7.8-1891 ng/ml) and VEGF (0.008-2.4 ng/ml).

Sex: Female, ages: 12 Years to 50 Years, accepts healthy volunteers

Inclusion Criteria:

* Female patients between 12 and 50 years who were admitted to the gynecological emergency room at Assuta medical center with signs and symptoms suggestive of ovarian torsion
* Patients that will undergo laparoscopy for the diagnosis of ovarian torsion
* Able to provide informed consent for themselves or have a legal guardian that can provide informed consent (in cases of patients under 18 years old)

Exclusion criteria:

* Patients that are unable to provide informed consent

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• Batel Hamou, MD, Principal investigator, Assuta Ashdod University Hospital