Resistance of human spermatozoa to cryoinjury in repeated cycles of thaw-refreezing

Abstract

Objective: To study the resistance of human spermatozoa to cryoinjury in repeated cycles of thaw-refreezing by using the fast liquid nitrogen vapor method. Materials and Methods: Semen specimens were obtained from sixteen normal and oligozoospermic individuals who required disposal at the sperm bank. Five of them had testicular cancer. Specimens were thawed and an aliquot was removed for analysis. The remaining specimens were refrozen without removing the cryomedia. Repeated freeze-thaw cycles were performed until no motile sperm were observed. Sperm motility, number of motile spermatozoa and viability were determined after thawing. Resistance to cryoinjury was compared between groups and also after each refreezing cycle within groups. Results: Motile spermatozoa were recovered after five and two refreeze-thawing cycles in normozoospermic and oligozoospermic specimens, respectively. There were no significant differences in the recovery of motile spermatozoa between thaws within each group of normal and oligozoospermic specimens, but percentage motility and total number of motile spermatozoa were significantly lower in the oligozoospermic one. Specimens from men with cancer were exposed to six refreeze-thawing cycles. Although recovery of motile spermatozoa was significantly impaired after each thawing, there were no significant differences in the recovery of motile sperm between thaws in cancer and non-cancer groups. Conclusions: Human spermatozoa resist repeated cryopreservation using the fast liquid nitrogen vapor method. Normozoospermic specimens withstand refreezing for an average two cycles longer than oligozoospermic ones. Specimens from cancer patients seem to resist repeated cryoinjury similarly to non-cancer counterparts. Resistance to repeated cryoinjury was related to the initial semen quality.

Sperm retrieval techniques for assisted reproduction

“…After processing, epididymal and testicular spermatozoa can be either used for ICSI or cryopreserved. Cryopreserved specimens are thawed and processed using the principles already described (30). Methods for selecting viable sperm for ICSI are currently available in cases immotile spermatozoa only are obtained after processing (27,31).…”

Cryopreservation of Human Spermatozoa by Vitrification vs. Slow Freezing: Canadian Experience

“…It has been reported that motile spermatozoa can be recovered after five refreezing and thawing rounds in normozoospermic samples, but only after two rounds in cases of oligozoospermia (Verza et al, 2009). Spermatozoa of infertile men were also found to be less resistant to damage during cryopreservation compared with spermatozoa from fertile men (Donnelly et al, 2001).…”

Laboratory handling of epididymal and testicular spermatozoa: What can be done to improve sperm injections outcome

“…Epididymal and testicular spermatozoa may be cryopreserved using protocols routinely used for ejaculated sperm. [2122] If sperm is found and frozen, thawing can be done at any time, thus obviating the need to organize two operations (oocyte and sperm retrieval) at the same day. Also, cryopreservation may be an interesting tool to spare left-over specimens that would be discharged after ICSI, especially if the treatment cycle does not result in a pregnancy.…”

“…To date, our cryopreservation technique for surgically retrieval sperm is the standard liquid nitrogen vapor method using TEST-yolk buffer and glycerol as cryoprotectants. [2122] Epididymal specimens are concentrated by washing before freezing, and testicular sperm are freed from the testicular parenchyma, i.e., testicular homogenates are frozen. Cryopreservation of few sperm into an empty zona pellucida and the use of stimulants before freezing may optimize results.…”