Objectives: Conventional sperm freezing procedures need the addition of a relatively large volume of cryoprotectant. The dilution of extremely poor sperm suspensions from ejaculate or testicular tissue may make the recovery of viable spermatozoa difficult at the moment of the intracytoplasmic sperm injection (ICSI) procedure. The cryopreservation of a few spermatozoa in empty zonae pellucidae is an interesting solution for crypto-azoospermic infertile men. We have modified this procedure by filling empty human zonae with TEST Yolk Buffer, an optimal cryoprotective medium, in order to analyse the number of zonae lost after thawing, the number of recovered spermatozoa per zona after thawing, and the sperm motility rate before freezing and after thawing.
Materials and methods: Fifty empty human zonae pellucidae previously filled with TEST Yolk Buffer were injected with 750 motile spermatozoa from ten infertile men (15 spermatozoa per zona). Sterile straws containing two zonae each were frozen following a two-phase protocol.
Results: All of the zonae and 445/750 spermatozoa (59%) were recovered. The mean number (+ SD) of spermatozoa per zona was 8.9 +/- 1.9 (range: 5-12). The recovery rate of motile spermatozoa was 73% (327/445), with a mean number of motile spermatozoa per zona of 6.5 +/- 1.7 (range: 3-10).
Conclusions: The cryopreservation of a small number of motile spermatozoa within empty zonae pellucidae using TEST Yolk Buffer as a freezing medium is possible without any major loss of spermatozoa and with an appreciable maintenance of sperm motility. This procedure seems to avoid: i) uncertain sperm retrieval after a laborious and time-consuming search on the day of oocyte aspiration; ii) the need for a repeated testicular biopsy; and iii) the need for heterologous insemination or oocyte cryopreservation (11).