title: Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa.
abstract: Vitrification is a simple and cost-effective method for the storage of human spermatozoa without the use of conventional cryoprotectants, by plunging the sperm suspension directly into liquid nitrogen. As a result, solidification of living cells without the formation of ice crystals is achieved during cooling. This study aimed to compare cryoprotectant-free vitrification to conventional cryopreservation protocols. Semen samples (n = 35) were collected from patients seeking diagnostic assistance at the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. Samples were processed using a discontinuous density-gradient centrifugation method. Washed samples were split into two aliquots and cryopreserved either by means of cryoprotectant-free vitrification (sucrose + 1% albumin) or conventional slow freezing (TEST-yolk buffer). Post-thawing, the sperm motion parameters, mitochondrial membrane potential (Δψm) and DNA fragmentation were compared between the two groups. No significant differences were observed in the sperm motility parameters (P > 0.05). Significantly higher percentages of Δψm (11.99% ± 4.326% versus 6.58% ± 1.026%; P < 0.001) and lower percentages of DNA fragmentation (2.79% ± 1.017% versus 3.86% ± 1.38%; P < 0.01) were observed when comparing cryoprotectant-free vitrification to conventional cryopreservation. Cryoprotectant-free vitrification is a rapid and promising alternative to conventional methods resulting in good-quality spermatozoa post-thaw.
authors.affiliation: Department of Obstetrics and Gynaecology, University of Pretoria, Steve Biko Academic Hospital, Pretoria, South Africa.
authors.forename: M
authors.initials: M
authors.lastname: Slabbert
authors.forename: S S
authors.initials: SS
authors.lastname: du Plessis
authors.forename: C
authors.initials: C
authors.lastname: Huyser
mesh_terms: D000328:Adult; D002499:Centrifugation, Density Gradient; D015925:Cryopreservation / Q000379:methods*; D053938:DNA Fragmentation; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D053078:Membrane Potential, Mitochondrial; D012661:Semen; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility; D013094:Spermatozoa; D058989:Vitrification
keywords: Cryopreservation; DNA integrity; human spermatozoa; mitochondrial membrane potential; vitrification