Background: Extracellular vesicles (EVs) are membrane-bound vesicles found in all biological fluids, containing various regulatory molecules, including microRNAs (miRNAs). It has been suggested that the EVs in human follicular fluid (the fluid surrounding the oocyte within the ovary) play a crucial role in oocyte development through these miRNAs. However, several methods exist for isolating these EVs from follicular fluid. Before further research can be conducted on the pathophysiology and potential diagnostic and therapeutic applications of these EVs and their content, the optimal isolation technique must be determined for future studies. Each method has its advantages and disadvantages, and the purity and efficiency may vary between species and different biological fluids. There are only a limited number of studies comparing the various techniques for application in human follicular fluid.

Objective: This study will compare the three most commonly used techniques for isolating EVs: size-based, sedimentation-based (differential ultracentrifugation), and buoyancy-based isolation techniques. Methodology: In this prospective study, follicular fluid will be collected from four patients undergoing transvaginal oocyte retrieval following ovarian stimulation at Ghent University Hospital. Patients over 40 years of age or with endometriosis, adenomyosis, or PCOS (polycystic ovarian syndrome) will not be included in this study. During the oocyte retrieval procedure, follicular fluid will be collected from 2-4 oocytes per patient. Since follicular fluid is not used in the patient's treatment (residual material), it does not affect the treatment. After collection, the three isolation techniques will be applied to each sample. The techniques will be compared in terms of purity (absence of contamination) and efficiency (EV concentration) using transmission electron microscopy, nanoparticle tracking analysis, and western blotting.