Simple vitrification for small numbers of human spermatozoa

Abstract

Conventional freezing procedures and containers are not appropriate for spermatozoa from the testis because of their low number and poor in-situ motility, and various types of container have been utilized to freeze small numbers of spermatozoa. This study tried to develop a vitrification method for small numbers of spermatozoa using the Cell Sleeper, which is a closed type of cell-cryopreservation container. The container with spermatozoa were cooled in liquid nitrogen vapour and then stored in a cryotank. Sperm motility parameters improved significantly (P < 0.05) by vitrification in oil-free droplets rather than in droplets covered with oil. After vitrification of five spermatozoa per container, all spermatozoa were recovered and the viable sperm rate was significantly higher when spermatozoa were vitrified in a 3.5-ll droplet rather than in 0.5 ll (72.0% versus 38.0%; P < 0.01). Recovery, motility and viability rates of vitrified–warmed spermatozoa were similar between the Cell Sleeper and the CryoTop groups. In conclusion, the Cell Sleeper is a highly effective tool for the cryopreservation of small numbers of spermatozoa and limited cells can be vitrified quickly and simply without significant loss.

Effect of sperm storage and selection techniques on sperm parameters

“…Biological carriers include empty zona and Volovox globator algae. Nonbiological carriers include straws, mini straws, copper loop cryoloop, calcium alginate beads, ICSI pipette microdroplets, agarose gel microspheres [AbdelHafez et al 2009], and sleeper cells [Endo et al 2012]. …”

“…Recent improvements in the volume of a sample that can be vitrified in a single container have made it a good alternative to conventional freezing [Isachenko et al 2011]. The development of closed systems for vitrification using sleeper cells has decreased the risk of cross contamination [Endo et al 2012]. Thus, vitrification has the potential to become the preferred technique used for sperm cryopreservation in the near future.…”

“…Newer methods of storing sperm in high security straws provide an alternative to cryoloop since it is a closed system. Vitrification using sleeper cells is another technique that can be used to preserve a small number of sperm [Endo et al 2012]. The advantage here is that it becomes a closed system.…”

Small-volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop

“…Until recently, Endo et al . (, ,b) reported that a small number of human spermatozoa can be successful vitrified using Cryotop. This indicated that Cryotop may be also a suitable carrier to cryopreserve human spermatozoa.…”

On-Chip Cryopreservation: A Novel Method for Ultra-Rapid Cryoprotectant-Free Cryopreservation of Small Amounts of Human Spermatozoa

“…However, it is arguable whether the spermatozoa would be infected with bacteria or pathogens mediated by liquid nitrogen, which have leaked into the PDMS chip during storage in a cryotank. According to Endo [24] and Vajta [25], the risk of contamination of germ plasma during cryopreservation and cryobanking in IVF units is small and no report mentions LN2 as a probable vehicle for disease transmission in assisted reproduction treatment. Our study failed to detect the contaminants in the cryotanks, which have been managed in a clean room at 23–28°C with air conditioning for over 5 years of continuous use (data not shown).…”