Homo sapiens (oocytes)

(Cryopreservation of Supernumerary Oocytes in IVF/ICSI Cycles)


The study from which this protocol is derived aimed to investigate cryopreservation of oocytes in patients refusing embryo cryopreservation, patients from whom no sperm can be retrieved and patients with enough oocytes to yield a number of fresh and cryopreserved embryos to transfer. The authors found that “Although a low implantation rate was observed and a higher abortion rate than in fresh cycles, our results show that in sibling oocytes, the process of cryopreservation apparently does not affect the fertilization and cleavage rate.”


•  IVF medium (B2 INRA Medium, Laboratoire CCD)

•  Hyaluronidase (Sigma)

•  Fine bore glass pipettes

•  Propanediol (Sigma Aldrich Srl)

•  Sucrose (Sigma Aldrich Srl)

•  Serum protein supplement (Pacific Andrology)

•  Plastic straws (Paillettes Cristal)

•  Automated biological vertical freezer

•  Liquid nitrogen


1.  Transfer cumulus-oocyte complexes to IVF medium at 37°C in an atmosphere of 5% CO2 in air.

2.  Perform complete removal of the cumulus and corona cells using hyaluronidase (80 IU/ml) and mechanical disruption with fine bore glass pipettes.

3.  Examine all oocytes for the presence of the polar body.

4.  Include only metaphase II oocytes.

5.  No more than 3 hours after retrieval, equilibrate eggs in cryoprotectant by stepwise addition of the cryoprotectant to a final concentration of 1.5 mol/l 1.2-propanediol plus 0.3mol/l sucrose and a serum protein supplement.

6.  Load oocytes into plastic straws.

7.  Transfer into an automated biological vertical freezer.

8.  Set the initial temperature to 23°C.

9.  Then slowly reduce to -8°C at a rate of -2°C/min.

10.  Induce ice nucleation manually at -8°C.

11.  Cool the straws slowly to -30°C at a rate at -0.3°C/min and then rapidly to -150°C at a rate of -50°C/min.

12.  After 5 minutes of temperature stabilization, transfer the straws into liquid nitrogen.

13.  Thaw oocytes by plunging the straws into 30°C water.

14.  Perform stepwise dilution of the cryoprotectants.

15.  Transfer thawed eggs to IVF medium at 37OC in an atmosphere of 5% CO2 in air.


P.E.Levi Setti, E.Albani, P.V.Novara, A.Cesana and G.Morreale. Cryopreservation of supernumerary oocytes in IVF/ICSI cycles. Human reproduction. 2005 Oct;21(2):370-375

Added by: Doug Rogers on September 30, 2012

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This is a brilliant protocol!

by Doug Rogers @ about about 5 years ago.

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