Clean technique for cryoprotectant-free vitrification of human spermatozoa

Abstract

Human spermatozoa can be successfully cryopreserved without the use of cryoprotectants through vitrification at very high warming rates. This is achieved by plunging a small amount of frozen sperm suspension into a warming medium, or a large amount of sperm suspension into an agitated warming medium. The aim of the present study was to compare the motility of human spermatozoa cryopreserved using four different methodologies of cooling and warming: cryoloops, droplets, open-pulled straws and standard open straws. Evaluation of two parameters, motility and viability rate of spermatozoa, suggests that all four methods are suitable for use in assisted reproductive technology. However, only the use of open-pulled straws as well as standard open straws allows the isolation of spermatozoa from liquid nitrogen with low potential risk of microbial contamination during freezing and storage, and is thereby a clean method of vitrification.

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

“…For vitrification purposes different types of storing strategies have been suggested. These include: cryoloops, electron microscope copper grids, nylon meshes, openpulled straws and standard open straws (V. Isachenko et al, 2005).…”

“…Agarose microspheres were also frozen in 0.25 cc straws by vitrification with better recovery rates (Isaev et al, 2007). Another reported method was to divide the sample into several small aliquots of 15-20-µl and to freeze in 0.2-mm cryopreservation embryo straws cut into smaller sections, sealed on one end (Desai et al, 2004).Conventional and open-pulled straws containing 1 or 5 µl of sperm suspension frozen by vitrification has also been reported (V. Isachenko et al, 2005). However, individual spermatozoa could not be easily sequestered because of possible adherence to the walls of the straws.…”

“…ICSI pipettes were suggested as a container to freeze individual spermatozoa by either the slow method or vitrification (AbdelHafez et al, 2009;Sohn et al, 2003). Cryopreservation of sperm in microdroplets containing 1 or 40 μl of spermatozoa in cryoprotectant placed on a cold surface or directly plunged into liquid nitrogen was also reported (Gil-Salom et al, 2000;Isachenko et al, 2005). 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).…”

“…However, placing drops of semen directly into LN2 raises the issue of the potential risk of microbial or viral cross contamination during freezing and storage (Katkov, 2002). The development of aseptic techniques of vitrification allowing to freeze 5-10 µl of sperm suspension in open-pulled straws (OPS) or 1-2 µl of sample cut standard straw (CSS) placed inside of insemination straw further advanced the methodology of human sperm vitrification (V. Isachenko et al, 2005). The ultra-high freezing rates utilized for vitrification, via direct plunging of specimens into LN 2 , leads to solidification of a solution by an intense increase in viscosity during cooling which avoids water crystallization and damaging ice formation (Katkov et al, 2006).…”

Effect of sperm storage and selection techniques on sperm parameters

“…Sperm are stored during vitrification using various instruments such as cryoloop droplets, open pulled straws, and open straws, and these methods do not differ in terms of motility and viability in post-thaw samples. But open pulled straws were preferred among these four methods cryoloops, droplets, open pulled straw and open straw due to the minimal risk of contamination from LN 2 [Isachenko et al 2005]. Additionally, it is difficult to quantify the amount of sample drawn into an open pulled straw, so to standardize the amount of sample drawn for storage, capillaries of standard diameter may also be used [Isachenko et al 2012].…”