Day-1: Maturation IVM

Below is an introduction provided by Dr. Lotte Stroebech of Stroebech Media, offering key protocols and considerations for handling and transporting oocytes during Ovum Pick-Up (OPU) and in vitro maturation (IVM). This overview highlights the importance of maintaining optimal temperatures, ensuring proper media equilibration and warming, and following best practices for aspiration and transport. By adhering to these guidelines, laboratories can help safeguard oocyte quality and maximize outcomes in the IVF process.

Oocyte aspiration by OPU or SH Ovary Collection

Media for equilibration and warming:
OPU Medium, WASH Medium, IVM Medium
Or H-IVM Medium in case the COC’s are to be matured during transportation.
Do never hold oocytes in the OPU medium longer than necessary and max 30 minutes.
If your IVF laboratory is not within 30 minutes reach, start the maturation in vials at the OPU site.

The H-IVM Medium is HEPES buffered and does not require CO2 equilibration and only needs warming to 38.8°C prior to use.
The H-IVM Medium can also be used in a CO2 gas atmosphere as it contains bicarbonate.

In the IVF laboratory
• Warm the WASH Medium
• Prepare and pre-equilibrate IVM Medium in 4WP wells or in maturation transportation vials
• Prepare a 35 mm petri dish (SH) or separate 4WP’s (OPU) with IVM Medium for rinsing the COC’s prior to transfer to the IVM wells/vials
• Warm bottles of 0.9% saline for washing of the ovaries (31-33°C)
• Add 150 μl of Heparin to 50 ml conical tubes

If the tubes have a bad smell, take off the lid under a laminar flow hood and let air out 2 hours before adding Heparin

Ovum Pick Up (OPU)

Preheat OPU Medium to 37°C.

Make sure that the aspiration tube warmer is not too hot, it should not be more than 37°C, however, it is often much warmer. Place a thermometer inside a tube with oil in the tube warmer to verify the temperature.

Transport the oocytes to the laboratory preferably after each OPU. Keep the oocytes warm at all times.
Pour OPU content into a filtered dish and wash with OPU Medium until content is clear. Make sure the dish/filter does not dry out at any time. Search for oocytes and wash them through the WASH Medium to get rid of debris.
Rinse once in IVM Medium prior to transferring oocytes into final maturation, as quickly as possible.
No more than 20 oocytes per vial if maturation takes place during transportation. Place vials at 38.8°C in a preheated shipper. Validate the shipper has the correct- and a stable temperature.

Oocytes should be surrounded by many layers of cumulus cells. If many are naked: adjust vacuum pump pressure and check size of the aspiration tubes. Too narrow tubes will strip the cumulus cells off or, more likely, too wide tubes will also strip the oocytes due to turbulence.