Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men

Cryopreservation is known to impair sperm motility and decrease the fertili zation rate by detrimental effects on acrosomal structure and acrosin activ ity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The prese nt study was designed to determine the effect of the freeze-thawing procedu re on chromatin condensation (aniline blue staining) and the morphology (st rict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured b y the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of m en classified as fertile (n = 20) and subfertile (n = 72), based on their r eproductive history and semen analysis according to WHO guidelines. The mea n percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased si gnificantly (p = 0.0001) after freeze-thawing, in comparison to the value o bserved prior to freezing. By comparing the semen samples between fertile a nd subfertile patients, significantly (p = 0.0009) greater damage was demon strated in the subfertile than in the fertile group. Furthermore, no signif icant difference was observed between the two groups with regard to the mor phological alteration and structural as well as functional damage of the sp erm membrane. In conclusion, the freeze-thawing procedure significantly aff ects chromatin structure and sperm morphology, especially in the head and t he tail regions, and this may explain the lower fertilization rate and IVF/ ICSI outcome when frozen-thawed spermatozoa are used. In addition, this stu dy demonstrates that chromatin condensation is a sensitive parameter for th e evaluation of cryodamage of semen samples from fertile and subfertile pat ients, though subfertile patients with very poor semen characteristics have yet to be studied. It is therefore recommended that chromatin condensation be used as an additional parameter for the assessment of sperm quality aft er freeze-thawing.