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

Abstract

Cryopreservation is known to impair sperm motility and decrease the fertilization rate by detrimental effects on acrosomal structure and acrosin activity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The present study was designed to determine the effect of the freeze-thawing procedure on chromatin condensation (aniline blue staining) and the morphology (strict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured by the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of men classified as fertile (n = 20) and subfertile (n = 72), based on their reproductive history and semen analysis according to WHO guidelines. The mean percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased significantly (p = 0.0001) after freeze-thawing, in comparison to the value observed prior to freezing. By comparing the semen samples between fertile and subfertile patients, significantly (p = 0.0009) greater damage was demonstrated in the subfertile than in the fertile group. Furthermore, no significant difference was observed between the two groups with regard to the morphological alteration and structural as well as functional damage of the sperm membrane. In conclusion, the freeze-thawing procedure significantly affects chromatin structure and sperm morphology, especially in the head and the tail regions, and this may explain the lower fertilization rate and IVF/ICSI outcome when frozen-thawed spermatozoa are used. In addition, this study demonstrates that chromatin condensation is a sensitive parameter for the evaluation of cryodamage of semen samples from fertile and subfertile patients, 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 after freeze-thawing.

Sperm preparation before freezing improves sperm motility and reduces apoptosis in post-freezing-thawing sperm compared with post-thawing sperm preparation

“…During cryopreservation, the dramatic physical and environmental changes of sperm detrimentally affect the sperm membrane, resulting in a large increase in the percentage of poorly motile sperm or sperm with abnormal morphology [2][3][4]. The negative effects relating to rapid decreases in temperature, such as osmotic injury, cellular dehydration, intra-cellular ice crystal formation, and oxidative stress, also lead to damage to sperm DNA, chromatin instability and DNA denaturation [4][5][6][7][8].…”

“…The negative effects relating to rapid decreases in temperature, such as osmotic injury, cellular dehydration, intra-cellular ice crystal formation, and oxidative stress, also lead to damage to sperm DNA, chromatin instability and DNA denaturation [4][5][6][7][8]. Therefore, the reproductive outcome following fertilization with the sperm containing the damaged genetic material may be poor [8][9][10].…”

“…Therefore, the preparation of post-thawed spermatozoa is most commonly performed for selecting the best quality of spermatozoa and discarding the cryoprotectant media before use for IUI or fertilization in IVF. Previous studies demonstrated that sperm preparation after cryopreservation can decrease the percentage of abnormal spermatozoa and increase the good motile spermatozoa [4,5,20].…”

Retention of membrane charge attributes by cryopreserved-thawed sperm and zeta selection

“…In the area of clinical ART, the main concern arising from the use of cryopreserved sperm is the reported freezerelated decrease in strict normal morphology and sperm necrosis leading to lower fertilization and IVF/ICSI pregnancy outcome [18]. The results of the present study demonstrated that improvements in strict normal morphology and reduced sperm necrosis could be achieved after zeta potential processing of the cryopreserved-thawed sperm.…”

“…The mechanism involved in the zeta selection of sperm with strict normal morphology was based on the greater net negative charge of mature sperm due to membrane sialoglycoproteins [21,22], specifically, gp20-CD52 glycopolypeptides, acquired during transit through the epididymis [23]. Researchers have correlated the maturity of sperm membranes with strict normal morphology, an parameter indicative of successful fertilization [7,24,25]. Disruptions in the sperm maturation process, for example, frequent ejaculations, would result in a less negative-charged sperm due to reduced membrane proteins.…”