Optimization of the Isolation Protocol for Extracellular Vesicles (EVs) From Human Follicular Fluid

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.

Study Overview

• Status: Active, not recruiting
• Conditions: Extracellular Vesicles
• Intervention / Treatment: Other: Differential ultracentrifugation; Other: OptiPrep™ density gradient ultracentrifugation; Other: Size-exclusion chromatography; Other: Combined techniques

• Study Type: Interventional
• Enrollment (Estimated): 4
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Belgium
  • Ghent, Belgium, 9000
    • Ghent University Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

• Patients planned for an oocyte retrieval at Ghent University Hospital.

Exclusion Criteria:

• Diagnosis of endometriosis/adenomyosis
• Diagnosis of PCOS (Polycystic Ovary Syndrome)
• Macroscopic blood contamination at oocyte retrieval

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: N/A
• Interventional Model: Single Group Assignment
• Masking: None (Open Label)

Number of Arms

1

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Intervention group	Other: Differential ultracentrifugation; Differential ultracentrifugation; Other: OptiPrep™ density gradient ultracentrifugation; OptiPrep™ density gradient ultracentrifugation; Other: Size-exclusion chromatography; Size-exclusion chromatography; Other: Combined techniques; Combined techniques

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Purety	Purety of EV isolation	3 months

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Efficiency	Efficiency in terms of number of particles	3 months
RNA efficiency	Efficiency in terms of RIN scoring	6 months

Collaborators and Investigators

• Sponsor: University Hospital, Ghent
• Collaborators: University Ghent

Study record dates

Study Major Dates

• Study Start (Actual): July 22, 2025
• Primary Completion (Estimated): December 1, 2026
• Study Completion (Estimated): December 1, 2026

Study Registration Dates

• First Submitted: April 23, 2025
• First Submitted That Met QC Criteria: April 23, 2025
• First Posted (Actual): May 1, 2025

Study Record Updates

• Last Update Posted (Actual): June 2, 2026
• Last Update Submitted That Met QC Criteria: June 1, 2026
• Last Verified: June 1, 2026

More Information

Terms related to this study

• Keywords: Isolation; Extracellular vesicles
• Additional Relevant MeSH Terms: Pathological Conditions, Anatomical; Skin Diseases; Skin Diseases, Vesiculobullous; Blister; Investigative Techniques; Chemistry Techniques, Analytical; Chromatography; Chromatography, Liquid; Chromatography, Gel
• Other Study ID Numbers: ONZ-2024-0419; G096624N (Other Grant/Funding Number: FWO)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No