title: Rapidly cooled human sperm: no evidence of intracellular ice formation.
abstract: BACKGROUND
  The cellular damage that human spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. Alternatively, the cell damage may be the result of an osmotic imbalance encountered during thawing. This article examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present.
  
  
  METHODS
  In this study, human spermatozoa were cooled at a range of cooling rates from 0.3 to 3000 degrees C/min. The ultrastructure of the samples was examined by cryo scanning electron microscopy and freeze substitution to determine whether intracellular ice formed during rapid cooling and to examine alternative mechanisms of cell injury during rapid cooling.
  
  
  RESULTS
  No intracellular ice formation was detected at any cooling rate. Freeze substitution of cells that had been cooled at 3000 degrees C/min and then slowly warmed showed that the cells had become plasmolysed and had evidence of membrane damage.
  
  
  CONCLUSIONS
  Cell damage to human spermatozoa, at cooling rates of up to 3000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.
authors.affiliation: Asymptote Ltd, St Johns Innovation Centre, Cambridge, UK. jmorris@asymptote.co.uk
authors.forename: G J
authors.initials: GJ
authors.lastname: Morris
mesh_terms: D020285:Cryoelectron Microscopy; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D005614:Freeze Fracturing; D017110:Freeze Substitution; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D007053:Ice / Q000009:adverse effects; D008297:Male; D008855:Microscopy, Electron, Scanning; D009997:Osmotic Pressure; D013094:Spermatozoa / Q000187:drug effects / Q000648:ultrastructure*
chemical_list: D003451:Cryoprotective Agents; D007053:Ice; D005990:Glycerol