Follicular growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice.

OBJECTIVE: To investigate follicle growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice. STUDY DESIGN: Fresh or frozen-thawed human ovarian cortex was grafted subcutaneously or under the kidney capsule of 43 mice (35 nude mice and eight SCID mice), 14 of which were non-stimulated controls, 21 injected intra-peritoneally with gonadotrophins during 2 weeks and eight injected during 3 months. Follicle count was compared by Chi-square. RESULTS: Proportions of primordial follicles were significantly lower in grafts than in the tissue before transplantation in gonadotrophin-stimulated mice (37% versus 79%), but not in non-stimulated mice (51% versus 74%). Proportions of primary and secondary follicles were increased after transplantation indicating early follicular growth. One antral follicle was observed in a graft in a mouse stimulated for 3 months. CONCLUSION: Primordial follicles in fresh or frozen-thawed human ovarian cortex transplanted under the kidney capsule or subcutaneously can grow and are responsive to hormonal stimulation. CONDENSATION: Primordial follicles in fresh and cryopreserved human ovarian cortical grafts can initiate growth after transplantation to immunodeficient mice

Biological aspects of testicular sperm extraction.

Since the first reports of successful pregnancies after treatment with intra cytoplasmic sperm injection (ICSI) in humans, intensive investigations focused on the use of testicular spermatozoa and immature sperm cells in the treatment of azoopsermic patients. Several studies explore the technical development of the preparation, isolation and cryo storage of testicular germ cells. Other studies focus on ICSI itself and try to identify the biochemical and biophysical processes involved in fertilisation after injection of a testicular sperm cell into a human oocyte. Indications for azoospermic patients to whom this first line treatment can be offered are becoming more defined. But one of the major concerns is of course the safety of the technique, especially, for the health and reproductive life of the babies born after application of ICSI with testicular germ cells. An evaluation of ICSI with testicular germ cells is presented in this manuscript.

Slow and ultrarapid freezing of fully grown germinal vesicle-stage mouse oocytes: optimization of survival rate outweighed by defective blastocyst formation.

PURPOSE: The cryopreservation of mature metaphase II-stage mouse oocytes is associated with decreased fertilizability, spindle damage, and increased polyploidy. Therefore, we investigated the outcome of cryopreservation of immature germinal vesicle-stage mouse oocytes. METHODS: Oocytes were punctured from Graafian follicles in primed F1 hybrid mice and were then released into maturation medium containing the meiotic inhibitor dibutyryl cyclic AMP. Both slow and ultrarapid freezing protocols with dimethyl sulfoxide, 1,2-proponediol, or a mixture of both agents were tested. We recorded morphological survival rates, in vitro maturation rates, and two-cell and blastocyst formation rates. Each group of frozen oocytes was compared with both unfrozen germinal vesicle-stage oocytes and metaphase II-stage oocytes. RESULTS: An optimal cryosurvival rate of 78% was reached after ultrarapid freezing with 3 M dimethyl sulfoxide followed by one-step dilution, but a decreased rate of two-cell formation was observed. Freezing with a combination of dimethyl sulfoxide and 1,2-propanediol did not improve this fertilization-decreasing effect. Very low cryosurvival rates after freezing with 1,2-propanediol indicated its inappropriateness for ultrarapid freezing of immature oocytes. The rates of in vitro maturation were equivalent for frozen-thawed and freshly collected germinal vesicle-stage oocytes, independent of the freezing protocol used. We report, nevertheless, as a general characteristic for both slow and ultrarapid freezing of fully grown germinal vesicle-stage oocytes, that the in vitro development up to the blastocyst stage is inhibited despite full nuclear maturation. CONCLUSION: We report that cryopreservation of immature germinal vesicle-stage oocytes is invariably associated with a low developmental capacity after fertilization. The rate of in vitro nuclear maturation did not equate with developmental competence. This in turn suggests the importance of cytoplasmic maturation for embryonic development.