Effect of an intermediate hold with vapor freezing on subsequent hypoosmotic swelling in thawed sperm

Cryopreservation and thawing of sperm exerts an adverse effect on functional integrity of the sperm membrane as measured by the hypoosmotic swelling (HOS) test. Supercooling using the liquid nitrogen (LN2) vapor technique may damage membranes by favoring ice crystal formation. Slight slowing of the cooling process may allow escape of intracellular fluid. This study was conducted to evaluate modification of the LN2 vapor technique by employing an intermediate hold in a freezer (to slow down the rate of cooling) on HOS scores on specimens thawed 1 month after freezing. Semen samples were obtained from male partners of infertile couples with a requirement of a baseline HOS score < 70% but > or = 60%. The HOS test was performed on the unprepared semen sample prior to freezing and immediately post-thaw 1 month later on the aliquot frozen with LN2 vapors only vs. the equal fraction subjected to an intermediate hold. The mean initial HOS score was 68.5% and was 47% in thawed specimens that had been cryopreserved with and without an intermediate hold. There were no differences in the percentage of specimens exhibiting a > 50% HOS score following vapor freeze (70%) or vapor freeze with a hold (74%). Thus, these data do not demonstrate any advantage of slowing the vapor freezing process by utilizing an intermediate hold.