Vitrification of a small number of spermatozoa in normozoospermic and severely oligozoospermic samples.

Despite broad utilization of sperm cryopreservation, little progress has been made to modify freezing protocols or to improve rates of sperm survival. Vitrification is an alternative method for freezing human spermatozoa without toxic permeable cryoprotectants (CPAs). The purpose of our study was to optimize the vitrification and post thaw recovery of a small number of spermatozoa using only nonpermeating CPAs in a closed straw system in normozoospermic and severely oligozoospermic samples. Individual motile spermatozoa (n = 295) were selected from semen samples of 15 normozoospermic and 10 severe oligozoospermia patients. Overall sperm recovery after vitrification was 80% (n = 236) with 80% (n = 189) viability and 41.5% (n = 98) retained post-warming motility. Two different loading techniques were compared to transfer selected spermatozoa into straws in preparation for vitrification: by spontaneous capillary action (CA) and with the aid of a polar body biopsy (PBB) pipette. There was evidence that the PBB loading technique increases the odds of spermatozoa recovery in both subsets (p = 0.01 and p = 0.04) in the normal and abnormal subsamples, respectively.

The three-dimensional image analysis of the chromocenter in motile and immotile human sperm.

Chromosomes in human spermatozoa are arranged non-randomly with the centromeres of non-homologous chromosomes forming a chromocenter. We have compared motile and immotile sperm populations in normozoospermic patients to determine if there is any dissimilarity in the formation of the chromocenter and the nuclear position of chromosome 17. Based on the differences between motile and immotile populations, we propose for the 'optimal' nuclear organization to be defined as containing 1 to 3 chromocenter(s) with central radial and median longitudinal position for the centromere of chromosome 17. By this definition, 42% of motile spermatozoa had 'optima' nuclei, in comparison to 25% of immotile spermatozoa (P < 0.05). Immotile spermatozoa exhibited a greater disruption in the formation of the chromocenter, altered position of the centromere of chromosome 17, and were more prone to chemical decondensation, resulting in higher nuclear and chromocenter volumes. The altered topology of the chromosomes might lead to the disruption of the sequence of events involved in fertilization and early embryonic development.

Vitrification of human laser treated blastocysts within cut standard straws (CSS): novel aseptic packaging and reduced concentrations of cryoprotectants.

Vitrification of laser treated human blastocysts using reduced concentrations of permeable cryoprotectants was carried out by submerging cut standard straws (CSS) into liquid nitrogen. The CSS were made by cutting a standard 0.25 ml straw at an angle of approximately 45 degrees . After laser assisted hatching, 6 day blastocysts (n=250) were loaded into droplets of approximately 0.75 microl in the CSS and were either plunged directly into liquid nitrogen or first encased in a standard 0.5 ml straw (aseptic technique) before being vitrified. Permeable cryoprotectants (ethylene glycol+Me(2)SO) at concentrations of 15% and 20% v:v were tested for their effect on post warming re-expansion and post transfer pregnancy rates. Our results indicate that the use of reduced concentrations of cryoprotectants and aseptic packaging of blastocysts did not have any statistically significant impact on the study outcomes.