Cryopreservation of Human Spermatozoa: An Assessment of Methodology Using Rhodamine 123

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Abstract
Rhodamine 123 fluorescent labeling of the human spermatozoal midpiece was used as a means of monitoring spermatozoal viability. This simple procedure was used in three separate studies to establish differential spermatozoal survival. Twenty ejaculates of reasonable quality were taken from men attending the Cromwell Hospital IVF Clinic. These were split three ways: cultured fresh at 37°C as for IVF, or frozen/thawed and then cultured at 37°C after freezing in either an egg yolk-free glycerol cryoprotectant or an egg yolk citrate medium. An expected overall difference in viability between fresh and frozen/thawed spermatozoa was observed, with no significant difference between the cryoprotective abilities of the two cryoprotectants studied. Four ejaculates were either frozen/thawed in the egg yolk-free cryoprotectant or cultured fresh, and both were subsequently stored at room temperature. Fall-off in frozen/thawed spermatozoal viability was more rapid than for the fresh cultured spermatozoa, although all spermatozoa survived longer at room temperature than at 37°C. Five ejaculates were split to culture their spermatozoa at 37°C in media containing either human or bovine serum albumin, or human fetal cord serum. BSA proved to be the least successful of protein supplements in maintaining spermatozoal viability, with HSA and cord serum giving rise to comparable viability of spermatozoa cultured in each. RH 123 is recommended as an alternative means of assessing human spermatozoal viability, and the results arising from the use of this technique here are discussed with their particular relevance to semen freezing and preparation in IVF centers.