Prepare the washing solution
10 ml 10× PBS (final concentration 1× v/v)
90 ml Sterile water
0.1 g PVA (final concentration: 1% w/v)
All reagents are mixed in a hot water bath or in a pot adding boiling water to dissolve the PVA. Shake the bottle frequently to avoid the PVA stick to the bottom of the bottle. Repeat the process until the PVA is dissolved. The washing solution can be stored at room temperature for several months in the dark.
The day before oocyte retrieval:
1. Prepare the label for the collection tube.
2. If possible, UV-light treat the collection tubes, pipettes, box of 20 µl tips and washing solution overnight to decrease risk of contamination.
At the day of oocyte retrieval:
3. If cumulus cells are also being collected, first remove the cumulus cells from the cumulus-oocyte-complex (COC) mechanically with two syringe needles (size: 18G) as if they were a knife and fork and keep the oocyte in the incubator until step 5.
4. Prepare a petri dish with one drop of 50 µl hyaluronidase and three drops of washing solution. Cover the drops with oil.
5. Transfer the COC to the drop of hyaluronidase to remove the cumulus cells. Use the pipette with a denudation pipette (size: 145 µm) to ‘mix’ the COC with the hyaluronidase. Wash the oocyte in the three sequential washing solution drops and assess stage (GV, MI, MII) and morphology under the ICSI microscope (or light microscope with 400× magnification).
6. Prepare a petri dish with one drop of 25 µl Tyrode's solution and two drops of washing solution.
7. Transfer the oocyte to the Tyrode’s to remove zona pellucida. Use the denudation pipette (size: 145 µm) to speed up the process by pipetting the oocyte up and down.
8. The zona-free oocyte is washed twice by pipetting up and down in the washing solution. The oocyte is transferred with 2 µl washing solution in a denudation pipette (size: 145 µm) to the bottom of the UV irradiated 0.2 ml collection tube.
9. Flash freeze the collection tube in liquid nitrogen and store at -80 °C until analysis. The oocyte is lysed by the flash freezing and can be used for omics research, but not for biopsies.