Laser-assisted cryopreservation of single human spermatozoa in cell-free zona pellucida

Improved procedure for efficient cryopreservation of single human spermatozoa in cell-free human zona pellucida is reported. We used a diode laser system for efficient and precise creation of a single hole into the zona pellucida of a degenerated or immature human oocyte. This allowed the extraction of the cytoplasm using a micropipette with a diameter of 10-15 microns. Through the same opening, human spermatozoa were inserted into the empty zona. We used motile and laser immobilized spermatozoa. Immobilized sperm were obtained by a single laser irradiation delivered in the vicinity of the sperm tail prior to insertion. This new immobilization procedure was shown to have no deleterious effect on membrane integrity and sperm viability. Following sperm transfer into the zona, the laser-drilled hole was closed with an oil droplet which was expelled from the micropipette during withdrawal to avoid loss of spermatozoa. This facilitated detection of the otherwise translucent zona during the cryopreservation procedure. After thawing, all cryopreserved zonae (20/20) could be successfully retrieved. Spermatozoa were recovered from the zona pellucida through the hole used for insertion. The rate of sperm recovery for initially motile spermatozoa was 80% vs. 92% for laser immobilized spermatozoa. Sperm viability was 81% and 84%, respectively, detected by a Hoechst stain. This technique makes cryopreservation of single human spermatozoa easy and feasible and appears beneficial for couples with severe male infertility and for those facing repeated surgical sperm extraction.