Abstract

Human sperm vitrification is a novel method of sperm freezing which achieves cryopreservation due to ultra-rapid cooling rates that prevent ice-crystal formation. However, sperm vitrification protocols are still largely not standardized for routine clinical use and seldom achieve a post warm sperm survival of 25-35%. The study aim was to validate and optimize a simple method of sperm vitrification that yields a high survival rate of spermatozoa for clinical use. Semen samples from 10 normozoospermic patients were subject to a simple swim-up into pre-warmed gamete handling media. Swim-up specimens were mixed in a 1:1 ratio with 0.5 M sucrose. Swim up specimens were then directly dropped in liquid N2. After a week of storage samples where warmed at 42 degree Celsius and sperm motility and viability was estimated. The mean sperm total motility of the fresh sample after the swim up preparation was 94.3 ± 3.06 %. Upon, vitrification followed by warming the mean percentage of total motile sperm fraction recovered was 74.70 ± 5.60 %. The mean sperm progressive motility of vitrified-warmed spermatozoa was 68 ± 8.47 %. The overall mean percentage of motile sperm recovery was 70.05% of the fresh swim up sample in this study. The overall mean sperm viability as assessed using the HOST vitality test was 77.21 ± 7.52%.•This study presents a simple protocol on the 'droplet method' of sperm vitrification.•Sperm cells vitrified using our modified method show a >70% motility and viability rates compared to the routine 25% to 35% of reported survival with the original sperm vitrification/freezing methodologies. This survival is attributed to a crucial change in the warming step.•This method has the advantage of using no toxic cell permeating cryoprotectant or expensive programmable freezing devices.

Highlights

  • This study presents a simple protocol on the 'droplet method' of sperm vitrification

  • A direct extrapolation of these vitrification techniques could not be directlyapplied to the human spermatozoa due to deleterious effects of high concentration of permeating cryoprotectant used in vitrifications protocols which results in 'osmotic shock' [5]

  • The top 0.4 ml containing the motile sperm fraction was gently aspirated out leaving the interface and diluted with the same gamete handling medium to achieve a final concentration of 10 Â 106/ml

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Summary

Background on the Method

Conventional human sperm cryopreservation is considered routine in the medical management of male infertility. Post thaw motility recovery still seldom cross 50% with conventional slow freezing of human sperm [2]. Rate of cooling and warming depend on the concentration and/or type of cryoprotectant utilized during slow freezing. A direct extrapolation of these vitrification techniques could not be directlyapplied to the human spermatozoa due to deleterious effects of high concentration of permeating cryoprotectant used in vitrifications protocols which results in 'osmotic shock' [5]. These methods do not technically require the use of cell permeating cryoprotectant that could result in osmotic shock to the spermatozoa. Isachenko and colleagues have shown that their method of sperm vitrification gave similar rates of sperm motility and DNA integrity when compared with conventional slow freezing[17]. The aim of the study was to optimize the vitirification and post warm recovery of spermatozoa in the absence of permeating cryoprotectant for normozoospermic semen samples

Method
Vitrification Procedure
Notes on the Protocol
Findings
Method Validation & Discussion
Full Text
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