Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success

Abstract

The use of cryoprotective agents for the conventional cryopreservation of human spermatozoa, oocytes, zygotes, early cleavage stage embryos and blastocysts is an integral part of almost every human IVF programme. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen usually requires high cryoprotectant concentrations with consequent cytotoxic effects. This review covers the history of this problem, and in this light offers an explanation, through physico-chemical concepts, for one of the most recent developments in this area: the recovery of motile and potent spermatozoa after cryoprotectant-free vitrification.

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

“…Expansion of artificial insemination for the dairy industry led to further important research in the field of cryobiology (E. Isachenko, 2003, as sited in Bernstein & Petropavlovski, Polge et al, 1949. Shortly after these practices were initiated with animals, the first pregnancies were reported in humans after insemination with frozen spermatozoa.…”

“…Successful vitrification of human spermatozoa was first reported by the Isaschenkos' group (Nawroth et al, 2002;E. Isachenko et al, 2003).…”

“…To achieve high cooling rates the vitrification specimen volume needs to be kept to a minimum. Specially designed freezing carriers such as cryoloops and electron microscope copper grids have been suggested for vitrification of human spermatozoa (E. Isachenko et al, 2003;Nawroth et al, 2002). 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).…”

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).…”

Cinétique et caractérisation du phénomène “apoptosis-like” induit au cours de la congélation des spermatozoïdes bovins

“…Apres dilution dans le milieu de congelation, on observe une diminution du A~P ; apres ~quilibration, une activation des caspases ; et apres le processus complet de congelation/decongelation, une augmentation de la permeabilite membranaire. Ce resultat sugg~re que ce n'est pas seulement la formation de cristaux de glace qui est & I'origine des differentes modifications cellulaires dues a la congelation/decongelation [6]. Les caracteristiques de I'apoptose dans le spermatozoi'de apparaissent selon la meme cinetique qu'au cours de I'apoptose classique [1,8,14,19].…”