CRYOPRESERVATION OF OOCYTES AND EMBRYOS - USE OF A MOUSE MODEL TO INVESTIGATE EFFECTS UPON ZONA HARDNESS AND FORMULATE TREATMENT STRATEGIESIN AN IN-VITRO FERTILIZATION PROGRAM

Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F-1 animals, Zonae pellucidae were exposed to alpha- chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg /ml bovine serum albumin upon a heated stage (37 degrees C) and were o bserved constantly through an inverted microscope, The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was r ecorded, A dose-dependent response in dissolution time was clearly see n, with 1% alpha-chymotrypsin being chosen as the routine working solu tion, Cryopreservation of 2-cell mouse embryos using propanediol did n ot cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s), Zona hardening was not suspected to occur after the f reezing of human embryos as there was no difference in implantation ra tes per embryo for in-vitro fertilization (IVF) and intracytoplasmic s perm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7 %); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/358 (7.9%); ICSI : 18/112 (16.1%)], Conversely, significant hardening of the zonae of m ature oocytes was seen following cryopreservation (747 +/- 393 s) comp ared with freshly ovulated oocytes (151 +/- 68 s), It is concluded tha t (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservat ion of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdi ssection of the zona in such cases generally unwarranted.