Preovulatory human oocytes were cooled to 0 degrees C at 1 degree C/min, with or without the cryoprotectant dimethyl sulphoxide (DMSO), to assess the effects of cooling on the meiotic spindles and on oocyte structure. Batches of oocytes, cultured for 3-9 h, were held at 0 degrees C for 20 or 60 min and then fixed for transmission electron microscopy (TEM) either at 0 or 8 degrees C. Control oocytes were not cooled and were fixed at 22 or 37 degrees C for comparison. TEM revealed that 80% of the oocytes were at metaphase II, while 20% were at metaphase I and most had resumed meiosis recently. Control oocytes had more or less barrel-shaped meiotic spindles composed of microtubules (MT), some associated with chromosomes at kinetochores. Both metaphase I and II spindles were disassembled when cooled and fixed at 0 degrees C, with or without DMSO, due to extensive depolymerization of MT. The few MT that survived were found at the poles or were bundled together or were associated with chromosomes. Kinetochores were not prominent. Some oocytes cooled with DMSO and fixed at 0 or 8 degrees C showed evidence of MT, but the spindles were still disorganized and were abnormal in structure. Chromosomes tended to clump together or were dislocated in the cortical ooplasm in cooled oocytes, but widespread scattering was not observed. This was particularly evident in the absence of DMSO. Elements of the endoplasmic reticulum, Golgi, mitochondria and the cytosol were also adversely affected in some of the cooled oocytes and their surrounding cumulus cells. The results show that meiotic spindles are very sensitive to simple cooling and that DMSO does not provide substantial stabilization of the meiotic spindle even at 0 degrees C. The findings are discussed with reference to recent work on frozen human and mouse oocytes.

Studies on the cryopreservation of 162 four-cell and eight-cell human embryos indicate that morphological survival and pregnancies can be achieved by specific techniques of freezing and thawing. Survival rates are highest when embryos are cooled at 0.3 degrees C/min to -80 degrees C in the presence of 1.5 M dimethylsulfoxide (DMSO) and thawed at +8 degrees C/min from -80 to +4 degrees C. Morphological survival of four-cell and eight-cell human embryos after freezing and thawing is not affected by irregularites in blastomere size or the presence of small cytoplasmic fragments. Light and electron microscopic examination of fixed specimens indicates a good correlation between the appearance of frozen-thawed embryos at the dissecting microscope level and the extent of cryoinjury. Sixty-eight of 136 four-cell and eight-cell embryos (50%) survived with half or more of their blastomeres intact when cooled to low temperatures and thawed at the rates described. The transfer of these 68 embryos into 45 patients resulted in nine pregnancies.

Embryo cryopreservation has been studied at Monash University since 1981 and has been available to patients since mid-1983. Of approximately 1200 patients' cycles of in vitro fertilization (IVF), 445 have had excess embryos which they requested to be frozen. To date 205 patients have requested thawing of their embryos and 144 have had frozen-thawed embryos replaced in utero, resulting in 16 pregnancies. Four of these pregnancies aborted, four are ongoing, and eight deliveries have resulted, including one stillbirth at 26 weeks and one set of twins. Analysis of the data collected to date shows that patients with three or more embryos frozen have a significantly higher pregnancy rate than patients with one or two embryos frozen (23 versus 4%, respectively). Embryo viability, but not embryo survival, following freeze-thawing is related to the degree of embryonic fragmentation and the cell stage at freezing. Eight-cell embryos had a significantly higher viability than other cleavage stages. Those resulting in pregnancy tended to be the faster-dividing eight-cell embryos and were undamaged after freezing and thawing. However, when considering all cleavage stages, there was little effect of freezing damage on embryo viability, providing that at least 50% of the cell complement of embryos were intact and the zona pellucida was undamaged. Nor was there any marked effect of the age of embryos postinsemination. It is also possible that patients who were pregnant following the initial embryo replacement on the cycle of IVF treatment are more likely to conceive following replacement of their frozen-thawed embryos.

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