The widespread use of clomiphene citrate and exogenous gonadotrophins for in vitro fertilization (IVF) in human frequently results in the production of multiple embryos. Replacement of more than two embryos increases pregnancy rate but may result in multiple pregnancies with increased pre- and post-natal abnormality. Preservation of embryos for a limited time allows fewer embryos to be replaced on several different occasions and thus the problems of multiple pregnancy can be minimized, the effectiveness of a single IVF procedure increased and embryo replacement in adverse maternal conditions avoided. Preimplantation embryos have been successfully cryopreserved in many animal species. The sensitivity of embryos to cooling and freezing varies between species and stages of embryo development. We report here the cryopreservation procedures that allow a high survival rate of four- and eight-cell human embryos and the establishment of a pregnancy following the freezing and storage of an eight-cell embryo for 4 months in liquid nitrogen. The pregnancy terminated at 24 weeks' gestation due to development of a septic Streptomyces agalactiae chorion amnionitis after premature membrane rupture.

Summary. 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\ p=n-\ 4\ m=1/ 2\ , 5\ p=n-\ 5\ m=1/ 2\ and 6\ p=n-\ 6\ m=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\ p=n-\ 5\ m=1/ 2\ h in culture ( 0\ p=n-\ \ m=1/ 2\ h, 26%; 4\ p=n-\ 4\ m=1/ 2\ h, 50%; 5\ p=n-\ 5\ m=1/ 2\ h, 89%; 6\ p=n-\ 6\ m=1/ 2\ h, 69%). The range and mean (\m=+-\s.d.) intervals from insemination for the pronuclear and early cleavage stages were 27\p=n-\43(35\m=.\6\m=+-\4\m=.\4)h for 2-cell stages, 36\p=n-\65 (45\m=.\7\ m=+-\ 8\m=.\3) h for 4-cell stages, 45\p=n-\73 (54\m=.\3\ m=+-\ 12\m=.\6) h for 8-cell stages and 68\p=n-\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\ p=n-\ 6\ m=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.

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A hatched human blastocyst obtained after in-vitro fertilization and culture was examined by transmission electron microscopy and the ultrastructural features compared with hatched mouse and bovine blastocysts. The human blastocyst contained a continuous layer of trophoblast cells with apical junctional complexes, an inner cell mass and the beginning of a primitive endoderm layer. Certain ultrastructural features were common to the blastocysts of all 3 species; these included characteristic junction regions between adjacent trophoblast cells, an abundance of microvilli on the external surfaces of the blastocysts and the presence of well developed mitochondria and numerous ribosomes in the trophoblast cells. The features that were dissimilar included the extent of development of the endoderm layer, the appearance of the inner cell mass and the nature and extent of vesicular inclusions in the trophoblast cells.