Cryopreservation of individually selected sperm: methodology and case report of a clinical pregnancy

OBJECTIVE: To describe a new technique for freezing individually isolated spermatazoa from testicular biopsies, epididymal aspirates and oligospermic semen samples METHODS: Samples were evaluated for the presence of motile sperm before cryopreservation. Motile or twitching sperm were isolated with an ICSI needle for single sperm cryopreservation. Selected sperm were loaded on the High Security Straw (HSV; Irvine Scientific; Irvine,CA), in ~0.5 µl of fluid to facilitate recovery. The sample was also frozen using conventional methodology in cryovials (100–1000 µl aliquots). In both freezing techniques, the samples were slow cooled. Test-yolk buffer-glycerol (Irvine) was used as the cryoprotectant. Test-thaws were performed to assess sperm recovery and motility. RESULTS: Six men with azoospermia had single sperm cryopreservation, as well as freezing aliquots of their testicular or epididymal sperm in traditional cryovials. In addition, two men with oligospermia also had individual sperm selected and frozen. In all 8 cases, the ~0.5 µl of fluid containing sperm was quite easily unloaded from the HSV straw during thawing. The percent sperm recovery ranged from 33% to 100%. Motility was evident in all but one sample. In six cases, the sperm were used for intracytoplasmic sperm injection of mature oocytes. Fertilization occurred in all but one case. In this study, we report the first clinical pregnancy with this technique. This pregnancy was remarkable in that a single motile sperm identified and selected in the initial testicular preparation was successfully frozen. We were able to subsequently recover this sperm, fertilize an oocyte and the resultant embryo gave rise to a live birth. The methodology described in this preliminary report offers a new modality for sequestering small numbers of sperm. It may be particularly useful in cases involving severe impairment of spermatogenesis, where extensive screening may be necessary to find a few viable sperm.