Cryopreservation of single human spermatozoa

A procedure is described that allows cryopreservation and efficientpost-thaw recovery of either a single or a small group of humanspermatozoa. This is achieved by injecting them into cell-free human, mouseor hamster zonae pellucidae before the addition of cryoprotectant. Themethod involves a combination of physical micromanipulation procedures andglycerol-mediated cryoprotection. Zonae were tracked by positioning them instraws between two small air bubbles prior to freezing. Spermatozoa frompoor specimens were cryopreserved and their fertilizing ability afterthawing was compared with that of fresh spermatozoa from fertile men. Humaneggs used for fertilization testing were either 1 day old or in-vitromatured. Only 2% of the frozen zonae were lost and >75% of spermatozoacryopreserved in this manner were recovered and prepared forintracytoplasmic sperm injection. The feasibility of cryopreserving asingle spermatozoon was assessed. Fifteen motile spermatozoa were frozen in15 zonae, of which 14 were recovered after thawing. Ten were injected intospare eggs, of which eight became fertilized. Spermatozoa recoveredmechanically from human zonae fertilized the same proportion of oocytes asfresh fertile control spermatozoa. The recovery and fertilization rateswith spermatozoa frozen in animal zonae were 87 and 78% respectively. Thefertilization rate was marginally higher (P < 0.05) than that forspermatozoa frozen in human zonae, perhaps because the latter may haveacrosome reacted more frequently. The zona pellucida appears to be anideally suited sterile vehicle for storage of single spermatozoa.