A twin pregnancy after microinjection of human cryopreserved oocyte with a specially developed oocyte cryopreservation regime.

DS Yang, PL Blohm, KL Winslow and L Cramer. Florida Institute for Reproductive Medicine, Jacksonville, Florida.

Objectives: Based on the physiology of the human unfertilized mature oocyte and its morphological change in response to freezing solution (1.5M PROH) during equilibrium, a human oocyte cryopreservation regime was developed. A significantly improved cryosurvival rate with human fresh and aged unfertilized oocytes has been achieved with this regime when it was compared with traditional embryo cryopreservation regime. This report describes our first attempt of fertilization and transfer of cryopreserved human oocytes at Florida Institute for Reproductive Medicine, which has resulted in a twin pregnancy.

Design: A case report.

Materials and Method: Thirty-two metaphase II (MII) oocytes were retrieved from a 28 year old donor after controlled superovulation with GnRH analogue, FSH and hCG. Because a second recipient was not available, 6 MII oocytes were cryopreserved with our oocyte freezing protocol (n=3, protocol O) and embryo freezing protocol (n=3, protocol E) after denude of cumulus and corona. Nine months later, a couple from our oocyte donation program consented to receive these donated cryopreserved oocytes. With protocol E, the oocytes were pre-equilibrated in 1.5M PROH, and 1.5M PROH containing 0.1M sucrose for 5 minutes each at 220C before being loaded into straws. The oocytes were pre-equilibrated in 1.5M PROH and 1.5M PROH containing 0.2M sucrose for 5 minutes each at 370C with protocol O. Her endometrium was prepared by transdermal administration of estradiol followed by intramuscular injections of progesterone started on her cycle day 13. All 6 oocytes were thawed on day 14 and the survived oocytes were fertilized by intracytoplasmic sperm injection (ICSI) using her husband's sperm. An embryo transfer was carried out on day 16 of her cycle.

Results: None of the 3 oocytes cryopreserved with protocol E survived after thaw, however, 2 of the 3 oocytes cryopreserved with protocol O survived freezing and thawing procedures. After ICSI, both oocytes achieved normal fertilization and cleavage. Two grade one 4-cell embryos were transferred into her uterus 2 days after thaw. A serum hCG test was positive (194 mIU/ml) 13 days after the embryo transfer. Ultrasonographies at 6 and 8 weeks of gestation showed a healthy twin pregnancy and it is ongoing at the time of this reporting.

Conclusions: It is suggested from this case study that equilibrium at high temperature and increased concentration of sucrose in freezing solution may improve cryosurvival and embryo development of the human mature oocyte. More clinical study is needed to assess the efficiency and safety of the procedure.