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Suspended animation of human sperm: Ultra-rapid vitrification using a novel microcapillary system
Yahir A. Santiago-Lastra, M.D, Joseph T. McQuaid, MD, Diane Wright, PhD, Cigdem Tanrikut, MD, Thomas Toth, MD.
Massachusetts General Hospital, Boston, MA, USA.

BACKGROUND: Freezing very low concentrations of sperm remains a challenge. Conventional slow-freeze methods require dilution with relatively large volumes and high concentrations of traditional cryoprotectants with potentially lethal osmotic and cytotoxic effects. Development of ultra-rapid vitrification techniques allows for freezing of very low volumes using low concentrations of nontoxic cryoprotectants, such as sugars. Our investigation was undertaken to determine the feasibility of ultra-rapid freezing via vitrification of human sperm samples with 0.5M trehalose using a novel microcapillary system.
METHODS: Fresh ejaculated samples were obtained from healthy men and prepared using the swim-up method in human tubal fluid (HTF) media supplemented with 5% human serum albumin (HSA). All samples had at least 20 million spermatozoa/ml and at least 50% motility prior to swim-up preparation. These samples were then diluted to varying concentrations and divided into equal volumes for vitrification with 0.5M trehalose (a natural sugar) to act as a cryoprotectant, vitrification without cryoprotectant, or no freezing/vitrification (control). Each sample was loaded into a 100µ microcapillary and the ends were heat-sealed to create a closed system. The samples undergoing vitrification were plunged into a liquid nitrogen bath for 20 seconds then warmed in a 37°C solution of phosphate-buffered saline. Each sample was expelled from the capillaries and motility was qualitatively assessed via microscopic visualization. All samples were further assessed by computer-assisted semen analysis or by manual counting by certified andrologists.
RESULTS: Preliminary results demonstrate consistent recovery of motile sperm in samples that were vitrified in 100μ microcapillaries with HTF media plus 5% HSA supplemented by 0.5M trehalose.
CONCLUSIONS: Ultra-rapid vitrification of human sperm samples in microcapillaries is feasible using low volumes of a nontoxic sugar, such as trehalose, as an alternative to traditional toxic cryoprotectants. This is a particularly relevant issue with men who produce very little sperm in the ejaculate and in men who undergo any variety of surgical sperm extraction efforts for either obstructive or nonobstructive azoospermia.


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