Cryoprotectant-Free Cryopreservation of Human Spermatozoa by Vitrification and Freezing in Vapor: Effect on Motility, DNA Integrity, and Fertilization Ability

Abstract

Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 x 10(5) degrees C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (-160 degrees C) with speed in the range 150-250 degrees C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.

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

“…|While several reports indicate no negative effect of freezing on sperm DNA integrity (Duty et al, 2002;V. Isachenko et al, 2004).…”

“…Microdroplets covered by mineral oil in a plastic tissue culture dish placed in liquid nitrogen were also used to cryopreserve individual spermatozoa (Quintans et al, 2000;Sereni et al, 2008). A nylon cryoloop first introduced for embryo freezing was successfully used to cryopreserve small volumes of sperm suspension by both slow freezing and vitrification (Nawroth et al, 2002;Schuster et al, 2003;V. Isachenko et al, 2004;Desai et al, 2004).…”

“…The combination of extremely high rates of cooling/warming and utilization of vitrification media containing proteins and polysaccharides made it feasible to avoid de-vitrification during warming without use of toxic CPAs. The same group compared viability, survival rate and sperm DNA damage between slow freezing and vitrification and found that DNA integrity was independent from the mode of cooling and the presence of cryoprotectants in thawed spermatozoa (V. Isachenko et al, 2004). The acrosome reaction, capacitiation and mitochondrial activity of spermatozoa were compared vis-a-vis slow freezing and vitrification (E. Isachenko et al, 2008).…”

Cryopreservation of a small number of human sperm using enzymatically fabricated, hollow hyaluronan microcapsules handled by conventional ICSI procedures

“…To substitute culture medium into the cryopreservation medium, the HA-ZP or empty ZP-injected sperm were incubated in 10 μL droplets of cryopreservation medium for 5 min at room temperature. The composition of cryopreservation medium was decided based on previous report on sperm freezing by sucrose [19][20][21][22][23]. The cryoprotectant was 0.1 M sucrose (Wako Pure Chemicals, Osaka, Japan) and 20 % synthetic serum substitute (SSS, Irvine Scientific, Santana, USA) in culture medium (sperm washing medium, Irvine).…”

Fertility Cryopreservation

“…Therefore, the amount of high molecular weight components can be 6-16 times higher than in embryos, resulting in enhanced viscosity and glass transition temperature The DNA integrity of vitrified sperm is comparable with that of slowly frozen/thawed spermatozoa. Such findings suggest that a wide range of cooling rates for spermatozoa can be acceptable (Isachenko et al, 2003;2004a;2004b;Nawroth et al, 2005).…”