Human and mouse oocytes were cryopreserved by a slow freeze, rapid thaw method, using propanediol (PROH) as the cryoprotectant. A simulated cryopreservation was also included in the study to detect the level of damage attributable to the PROH alone. Comparison of the mouse and human oocytes cryopreserved by the same method showed opposing results, with a poor morphological survival rate of 4% observed for mouse oocytes and a subsequent normal fertilization rate of 0%. In 171 cryopreserved human oocytes a higher survival rate of 64% was achieved, and this showed more similarity to the mouse pronuclear oocytes survival of 53%. A comparison of human oocytes, cryopreserved within the cumulus and denuded of cumulus and corona prior to cryopreservation, demonstrated a higher survival rate in the denuded oocytes of 69% compared to 48%. A delay prior to cryopreservation of 1 or > or = 2 days had no effect on the immediate survival of oocytes, but culture for a further 24 h after thawing reduced survival, with the day 1 oocytes exhibiting the most dramatic reduction in survival (28%). Using a lectin binding method, abundant cortical granules were observed in all cryopreserved oocytes analysed. The meiotic spindle and chromosomes were examined in cryopreserved oocytes using fluorescence microscopy and 60% of the surviving oocytes had a normal spindle and chromosome configuration.

Survival following cryopreservation of fresh and aged human oocytes by the propanediol (PROH) procedure was observed in 51 and 73% of oocytes respectively, immediately after thawing. This survival was reduced in both types of oocytes at the time of insemination (3-4 h) to 41% in fresh and 61% in aged oocytes. Insemination of the cryopreserved and control oocytes with spermatozoa from one donor resulted in total fertilization rates similar to our in-vitro fertilization (IVF) rate for non-male factor patients. The normal fertilization rate for fresh cryopreserved oocytes was slightly lower (46%) than the rate for IVF oocytes (59%) (P < 0.05), while the abnormal fertilization rates were not significantly different (16 and 15% respectively). In contrast, a reduction in the normal fertilization rate was observed for the aged cryopreserved oocytes (13%) compared to the IVF rate (P < 0.001). Associated with this was an increase in the abnormal fertilization rate for the aged cryopreserved oocytes, which was significantly higher (47%) than the IVF rate (15%) (P < 0.001). Significant differences in the total and normal fertilization rates were observed between cryopreserved oocytes obtained from cohorts with < or = 27 (total: 84%, normal: 68%) and > 27 oocytes (total: 55%, normal: 33%) (P < 0.05). Fertilized oocytes and oocytes with abnormal or absent spindles were examined for chromosomal loss and no stray chromosomes were observed in any of these cryopreserved oocytes (n = 137). In the cryopreserved oocytes which had undergone normal fertilization, four scorable karyotypes were achieved and in all of these two sets of 23 chromosomes were observed.

This study reports the subsequent embryo development of cryopreserved mature human oocytes following insemination or intracytoplasmic sperm injection (ICSI). Metaphase II oocytes were cryopreserved using a slow freezing-rapid thawing procedure employing the cryoprotectant 1,2-propanediol. The study was conducted at two centres. The normal insemination of cryopreserved oocytes was undertaken in one centre, and ICSI of cryopreserved oocytes in the other. Both methods resulted in a 50% normal fertilization rate. A low rate of abnormal fertilization was observed in the inseminated group of oocytes (5%) compared with 21% for the ICSI oocytes; this was not significantly different. Embryo development was assessed daily for 7 days. All normal fertilized cryopreserved oocytes in both groups cleaved on day 2, with a similar appearance to in-vitro fertilization and ICSI embryos. In the normal inseminated oocytes, there was a significant decrease in the number of embryos cleaving on day 3 (33%) compared with the development of ICSI oocytes, with a subsequent gradual reduction over days 4 and 5 (22 and 11% respectively) resulting in one early blastocyst on day 7 (11%). In contrast, all ICSI-generated embryos continued to cleave on day 3, with a gradual reduction over subsequent days (day 4, 86%; day 5, 57%; day 6, 43%; day 7, 29%). By day 7, two of the blastocysts had started to hatch, resulting in a 66% hatching rate of blastocysts formed from ICSI of cryopreserved oocytes. This is the first study to show normal development to the hatching blastocyst stage following ICSI of cryopreserved human oocytes.

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