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

Improved procedure for efficient cryopreservation of single human spermatoz oa in cell-free human zona pellucida is reported. We used a diode laser sys tem for efficient and precise creation of a single hole into the zona pellu cida of a degenerated or immature human oocyte. This allowed the extraction of the cytoplasm using a micropipette with a diameter of 10-15 mu m. Throu gh the same opening, human spermatozoa were inserted into the empty zona. W e used motile and laser immobilized spermatozoa. Immobilized sperm were obt ained by a single laser irradiation delivered in the vicinity of the sperm tail prior to insertion. This new immobilization procedure was shown to hav e no deleterious effect on membrane integrity and sperm viability. Followin g sperm transfer into the zona, the laser-drilled hole was closed with an o il droplet which was expelled from the micropipette during withdrawal to av oid loss of spermatozoa. This facilitated detection of the otherwise transl ucent zona during the cryopreservation procedure. After thawing, all cryopr eserved zonae (20/20) could be successfully retrieved. Spermatozoa were rec overed from the zona pellucida through the hole used for insertion. The rat e of sperm recovery for initially motile spermatozoa was 80% vs. 92% for la ser immobilized spermatozoa. Sperm viability was 81% and 84%, respectively, detected by a Hoechst stain. This technique makes cryopreservation of sing le human spermatozoa easy and feasible and appears beneficial for couples w ith severe male infertility and for those facing repeated surgical sperm ex traction.