Investigating the impact of extracellular vesicle addition during IVM on the fertilization rate of equine oocytes following ICSI.

Overview

• This study investigated whether adding extracellular vesicles (EVs) from small ovarian follicles during the in vitro maturation (IVM) of equine oocytes improves fertilization rates after intracytoplasmic sperm injection (ICSI).
• Results demonstrated that EV supplementation increased fertilization success and reduced oocyte degeneration, suggesting EVs could enhance equine IVF outcomes.

Background and Problem

• In vitro embryo production (IVEP) in horses is challenged by several factors, including:
  • No reliable superovulation method to obtain many oocytes.
  • Limited supply of cumulus oocyte complexes (COCs) for culture.
  • Low efficiency of in vitro maturation (IVM) and fertilization stages.
• Extracellular vesicles (EVs) are small nanoparticles found in ovarian follicular fluid that mediate cellular communication and may influence oocyte development.
• Previous research proposes EVs could be beneficial supplements during IVM to improve oocyte quality and fertilization success.

Objective and Hypothesis

• The study aimed to test if EVs isolated from small (<20 mm) ovarian follicles could enhance:
  • Oocyte maturation quality.
  • Fertilization rates following ICSI in mares.

Methods

• Sample Collection and EV Isolation:
  • Follicular fluid was collected postmortem from equine ovaries.
  • EVs were isolated from the fluid using standard procedures and characterized by:
    • Nanoparticle tracking analysis (NTA) to measure EV size distribution.
    • Flow cytometry to detect specific surface markers (CD81 and CD63), common to EV populations.
    • Transmission electron microscopy to visualize typical EV morphology (disk-shaped particles).
• In Vitro Maturation (IVM):
  • COCs were cultured for IVM with or without EV supplementation (200 µg EV protein/ml).
  • EV uptake by COCs was confirmed using fluorescently labeled EVs and confocal microscopy, showing internalization during IVM.
• Fertilization and Embryo Culture:
  • Following IVM, only oocytes that extruded the first polar body (~37%) were selected for ICSI.
  • Fertilization rates, degeneration, and cleavage were assessed after ICSI during culture using a time-lapse monitoring system.

Key Findings

• Confirmation of EV Uptake:
  • Fluorescent microscopy showed EVs were internalized by COCs during IVM, indicating direct interaction.
• EV Characterization:
  • EVs were mainly submicron in size.
  • Markers CD81 and CD63 were present on a subset of EVs, confirming their identity.
  • Electron microscopy revealed characteristic EV morphology.
• Impact on Fertilization:
  • The EV-treated oocytes had a significantly higher fertilization rate post-ICSI (34.7%) compared to controls (20.2%), with statistical significance (P < 0.05).
  • There was reduced degeneration in EV-treated oocytes.
  • Cleavage efficiency tended to improve in the EV group (P < 0.1), indicating better early embryo development potential.
• Limitations:
  • Despite improvements, early embryonic arrest occurred commonly in both treated and control groups, pointing to other barriers in equine IVEP.

Conclusions and Implications

• Follicular fluid-derived EVs from small ovarian follicles can be internalized by equine COCs during IVM and positively influence fertilization outcomes.
• EV supplementation decreases oocyte degeneration and promotes cleavage efficiency, contributing to more successful ICSI fertilizations.
• These findings suggest EVs could become a valuable addition to optimize current equine in vitro embryo production protocols.
• Future research is needed to overcome embryonic arrest and further enhance equine embryogenesis in vitro.