Birth after transfer of cryopreserved embryos.

A normal baby girl was delivered after the successful transfer of two thawed embryos which had been frozen for eight weeks. Embryo cryopreservation may improve the chances of conception by women who are undergoing in-vitro fertilization (IVF), reduce the risks of multiple pregnancies in association with IVF, and offer the chance of conception to women with serious gynaecological disease which threatens ovarian function.

Deep-freezing and transfer of human embryos.

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.

Cryopreservation of human embryos.

Studies on the cryopreservation of ninety-seven 4-cell and 8-cell human embryos indicate that morphologic survival can be achieved by means of two different cryoprotectants and two different freezing procedures. To date, pregnancies can be achieved after freezing and thawing of 8-cell human embryos cooled at 0.3 degree C per minute to -80 degrees C in the presence of 1.5 molar dimethyl sulfoxide (DMSO) and thawed at +8 degrees C per minute from -80 degrees C to +4 degrees C. By this procedure, 27 of 47 (57%) embryos frozen survived with 50% or more of their blastomeres intact. The transfer of these 27 embryos to 22 patients resulted in five pregnancies (22%). Morphologic survival of 4-cell and 8-cell human embryos after freezing and thawing is not affected by slight irregularities 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.

Effect of delayed insemination on in-vitro fertilization, culture and transfer of human embryos.

Oocytes were obtained from patients with tubal infertility at fixed times after the onset of the endogenous LH rise or hCG injection, and were inseminated immediately after recovery or after periods of 4-4 1/2, 5-5 1/2 and 6-6 1/2 h in culture in vitro. Delayed insemination resulted in a marked increase in the proportion of oocytes that were fertilized and developed to normal embryos and maximum rates occurred after 5-5 1/2 h in culture (0-1/2 h, 26%; 4-4 1/2 h, 50%; 5-5 1/2 h, 89%; 6-6 1/2 h, 69%). The range and mean (+/- s.d.) intervals from insemination for the pronuclear and early cleavage stages were 27-43 (35.6 +/- 4.4) h for 2-cell stages, 36-65 (45.7 +/- 8.3) h for 4-cell stages, 45-73 (54.3 +/- 12.6) h for 8-cell stages and 68-85 h for the 16-cell stage. In 7/50 patients receiving 1 or 2 embryos at the 2-, 4- and 8-cell stages, fetal development was normal and 2 women had twin pregnancies (36% success compared with 8% for single embryos). All pregnancies were from the groups in which insemination was delayed for 5-6 1/2 h. It is concluded that a short period of culture in vitro may allow the completion of oocyte maturation, and improve the results of in-vitro fertilization.

The use of fluorescein diacetate to assess embryo viability in the mouse.

Preimplantation mouse embryos that were exposed to fluorescein diacetate (FDA) accumulated intracellular fluorescein and fluoresced brightly under ultraviolet (u.v.) light. The rate at which intracellular fluorescein was lost from the cells was measured at 37, 28 and 4 degrees C and the rate decreased as the storage temperature decreased. The rate at which intracellular fluorescein accumulated increased as FDA concentration increased until a maximum rate was attained. The ability to accumulate intracellular fluorescein could be removed by heating embryos at 56 degrees C for 30 min or by damaging the cell membrane. Cells grown under inadequate culture conditions lost the ability to accumulate intracellular fluorescein. Exposure of 2-cell mouse embryos to FDA and u.v. light did not alter the rate of blastocyst formation in vitro, and exposure of blastocysts to FDA and u.v. light did not alter the rate of implantation or post-implantation development in vivo.