Improved survival rate
after cryopreservation of human fresh and aged unfertilized oocytes using a
specially developed oocyte cryopreservation regime.
DS Yang, KL Winslow and PL Blohm. Florida
Institute for Reproductive Medicine,
Objectives: Most reported oocyte cryopreservation regimes using 1,2-propanediol (PROH) and sucrose are identical or very
similar to the embryo cryopreservation protocols (protocol E) used in human
assisted reproduction laboratories. The oocytes cryopreserved with these
protocols often have poor survival after thawing. Lack of sufficient shrinkage
when metaphase II (MII) oocytes exposed to embryo freezing solution containing
1.5M PROH for equilibrium was observed during our initial experiment with cryopreservation
of unfertilized oocytes. Based on the physiology of oocyte and its response to
freezing/thawing solutions a special oocyte cryopreservation regime (protocol
O) was developed in our lab. This study compares the survival of cryopreserved
human oocytes using protocol E and protocol O.
Design: Prospective comparative study.
Materials and Method: A total of 111 MII oocytes were used
in this study. These include: 1) 15 MII oocytes developed from germinal vesicle
(GV) and metaphase I (MI) stage in vitro 24/48 hours after oocyte retrieval
(group A); 2) 81 unfertilized oocytes 24/48 hours after insemination (group B);
3) 15 fresh oocytes from 3 oocytes donation cycles (group C). Only MII
unfertilized cumulus free oocytes were used in this study. With protocol E, 53
oocytes (8, 39 and 6 from group A, B and C, respectively) were pre-equilibrated
in 1.5M PROH, and 1.5M PROH containing 0.1M sucrose for 5 minutes each at 220C
before loaded in straws. Fifty-eight oocytes (7, 42 and 9 from group A, B and
C, respectively) were pre-equilibrated in 1.5M PROH, and 1.5M PROH containing
0.2M sucrose for 5 minutes each at 370C with protocol O. Controlled cooling
rate to -70C was at -20C/minute and -30C /minute with protocol E and protocol O
respectively before seeding and further cooling. During equilibrium with 1.5M
PROH oocytes were observed for morphology change. After thawing, cryoprotectants were removed by stepwise dilution. Oocytes
survived the freezing/thawing procedures were cultured in HTF with 10% serum
for 2 hours to further assess their viability. Fisher's exact test was applied
to analyze the results.
Results: Significant oocyte shrinkage was
observed on the oocytes equilibrated in 1.5M PROH at 370C. Eighteen of the
oocytes (34%, 18/53) cryopreserved in protocol E survived freezing/thawing and
2 hour culture (2, 15 and 1 oocyte from group A, B and C, respectively). The
survival rate of the oocytes cryopreserved in protocol O was significantly
better than in protocol E (P<0.001). Forty-one oocytes (71%, 41/58) in
protocol O survived freezing/thawing and 2 hour culture (5, 30 and 6 oocytes
from group A, B and C, respectively).
Conclusions: The oocyte cryopreservation regime
(protocol O) used in this study achieved superior cryosurvival
rate after freezing/thawing of human mature oocytes. Equilibrium at 370C
improves water and PROH permeability of the cytoplasm membrane of unfertilized
oocytes. Increased concentration of sucrose in freezing solution, through
removal of more intracytoplasmic water, further reduces ice formation in
oocytes. More rapid cooling to subzero temperature may minimize oocyte toxicity
of the cryoprotectants. Further clinical study is
needed on the fertilization and implantation of cryopreserved fresh human
oocyte after thawing.