Simplified MII Oocytes Vitrification Protocols

The following protocols and tips are for the vitrification of embryos from 2PN (two-pronuclear zygote) to blastocyst using our Vitrification Freeze Kit (Irvine Scientific, Catalog # 90133), Cryolock™(Catalog # CL-R-CT) or CryoTip® (Catalog # 40709) or HSV Security Vitrification Straws (Catalog # 25246-25251).

Protocols

ALL PROCEDURES MUST BE PERFORMED AT ROOM TEMPERATURE (22 - 27°C).

NOTE: As referenced in clinical literature, we recommend oocyte vitrification to be performed within 2 hours from time of oocyte retrieval1.

Have all necessary materials, tools and equipment ready and easily accessible before starting procedure.

Simplified MII Oocyte Vitrification with CryoTip

As an added precaution during the preparation procedure, carefully examine each CryoTip outside of the package. Prior to use, all CryoTips should be examined under a suitable magnification (40x power) for possible damage (such as tip breakages or cracks) that may have occurred during transport.

1. Aseptically dispense:

• one (1) 20 μL drop of H and
• three (3) 20 μL drops of ES (ES1, ES2 and ES3)

NOTE: ES1 and ES2 should be in close proximity to H (but not touching).

2. Place MII oocyte(s) (2 maximum), into H and expose undisturbed for 1 minute.

3. Merge ES1 with H. Allow spontaneous mixing for 2 minutes.

Use tip of transfer pipette to move ES1 towards H until drops merge.

4. Then merge ES2 with H+ES1. Allow spontaneous mixing for 2 minutes.

5. Transfer oocyte(s) from merged drop to ES3 and expose undisturbed for 6-10 minutes.

6. During the 6-10 minute exposure, aseptically dispense one (1) 50 μL drop of VS.

NOTE: Visually observe re-expansion (equilibration) of oocyte(s) to at least 80% of original volume.

7. Transfer oocyte(s) from ES3 to VS for 30 seconds before loading.

8. Gently pipette oocyte(s) once within VS drop to ensure complete rinse in VS.

NOTE: To minimize floating, after 10 seconds pipette the specimen(s) to the bottom of the VS drop.

9. Load, seal and plunge CryoTip® within 80 seconds, within 110 seconds after exposure to VS drop.

10. Refer to CryoTip Loading Protocol diagram and product insert for detailed loading instructions.

Simplified MII Oocyte Vitrification with HSV Device

1. Aseptically dispense:

• one (1) 20 μL drop of H and
• three (3) 20 μL drops of ES (ES1, ES2 and ES3)

NOTE: ES1 and ES2 should be in close proximity to H (but not touching).

2. Place MII oocyte(s) (2 maximum), into H and expose undisturbed for 1 minute.

3. Merge ES1 with H. Allow spontaneous mixing for 2 minutes. 

Use tip of transfer pipette to move ES1 towards H until drops merge.

4. Then merge ES2 with H+ES1. Allow spontaneous mixing for 2 minutes.

5. Transfer oocyte(s) from merged drop to ES3, and expose undisturbed for 6-10 minutes.

6. During above 6-10 minute exposure, aseptically dispense one (1) 50 μL drop of VS.

7. Transfer oocyte(s) from ES3 to VS for 30 seconds before loading.

8. Gently pipette oocyte(s) once within VS drop to ensure complete rinse in VS.

NOTE: To minimize floating, after 10 seconds pipette the specimen(s) to the bottom of the VS drop.

9. Load, seal and plunge HSV Device (per device instructions) within 80 seconds, not to exceed 110 seconds after exposure to VS.

10. Refer to HSV Device Loading Protocol diagram and product insert for detailed loading instructions.

 

Tips for Oocyte Vitrification

  • All procedures are to be done at ROOM TEMPERATURE (22-27°C). DO NOT USE HEATED STAGE.
  • Have all necessary material, tools and equipment ready and easily accessible before starting procedure.
  • CryoTips should be pre-labeled with patient information, and assembled with connector and syringe or pipette (for loading), prior to starting procedure. To protect the finely pulled tip from damage, keep it covered with metal cover sleeve until ready to load specimen(s).
  • HSV Device should be pre-labeled with patient information and the thin end of the blue plastic insertion device should be connected to the colored end of the handling rod.
  • Where possible, select only the best quality MII oocytes for vitrification.
  • The recommended CryoTip or HSV Device capacity is a MAXIMUM of 2 specimens.
  • Process only as many specimen(s) as will be loaded per CryoTip or HSV Device at one time.
  • Minimize exposure of specimens to light during equilibration in ES and VS solutions.
  • Transfer specimens between drops using a minimal volume of medium.
  • The timing for exposure to VS is CRITICAL:
    • Maintain microscopic visualization of specimen(s) by adjusting focus as needed during rapid exposure to VS (specimens will float in the drop).
    • Keep transfer pipette tip close to drop for quick manipulations.
    • Load, seal and plunge the CryoTip within 80 seconds, not to exceed 110 seconds after initial exposure to VS.

Related Protocols

Simplified Embryo Vitrification Protocols

Simplified Embryo and Oocyte Warming Protocols

 

1. Song WY, Sun YP, Jin HX, et al. Chinese Journal of Obstetrics and Gynecology 2010; 45(8):578-82