CRYOPRESERVATION OF SINGLE HUMAN SPERMATOZOA

A procedure is described that allows cryopreservation and efficient po st-thaw recovery of either a single or a small group of human spermato zoa, This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The m ethod involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positionin g them in straws between two small air bubbles prior to freezing, Sper matozoa from poor specimens were cryopreserved and their fertilizing a bility after thawing was compared with that of fresh spermatozoa from fertile men, Human eggs used for fertilization testing were either 1 d ay old or in-vitro matured, Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and pr epared for intracytoplasmic sperm injection, The feasibility of cryopr eserving a single spermatozoon was assessed, Fifteen motile spermatozo a were frozen in 15 zonae, of which 14 were recorered after thawing. T en were injected into spare eggs, of which eight became fertilized, Sp ermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa, The recove ry and fertilization rates with spermatozoa frozen in animal zonae mer e 87 and 78% respectively, The fertilization rate was marginally highe r (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently, The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.