Analysis of Fullerene’s Action on Human Sperm before and after Cryopreservation

Pavlovich E. V.

CLINICAL AND EXPERIMENTAL MEDICINE

Scentific article

Development of assisted reproductive technologies stipulated the search for methods improving the properties of human sperm. Information about their potential toxicity and effects on human health is necessary for use of nanomaterials in the clinical setting. Application of carbon nanoparticles can increase cryoresistance and recover sperm motility after cryopreservation. Increase the percentage of motility and functional active of human sperm before and after cryopreservation is very important in case of asthenozoospermia. Detailed research before using sperm samples after incubation with fullerenes must be performed considering their motility rate, disorders of membrane integrity and chromatin organization. The effect of carbon nanoparticles on human reproductive system is not completely understood. Usage of carbon nanoparticles for increase of sperm motility before and after cryopreservation can be way to improve the state of human sperm in cryobiological research. The aim of our work was studying of the carbon nanoparticles effect on the morphological and functional characteristics of the human sperm at asthenozoospermia before and after cryopreservation. The proportion of total and progressively motile sperm was estimated. Viability and chromatin state was determined after incubation with fullerenes. As cryoprotective medium 4 % glycerol and 20 % BSA with Hanks’ solution (PAA, Austria) was used. Cryopreservation was performed under cryobank. The processes of of nuclear chromatin decondensation were studied with 7-Amino-Actinomycin (7AAD) (BD) dyes using FACS Calibur Becton-Dickinson flow cytometer. It was evaluated the effect of fullerenes at concentrations of 10, 20, 30, 40, 100, 200 µg/ml on sperm motility after incubation for 1-3 hours. It was established that fullerenes of 10 and 20 µg/ml concentration incubated with human spermatozoa at asthenozoospermia for 1, 2 and 3 hours caused the increase of motility index relative to the control. Carbon nanoparticles at concentrations of 40, 100 and 200 µg/ml had inhibitory effect on the motility of active fraction, and with increasing concentration of the nanoparticles the degree of the spermatozoa motil- ity reduction increased. Cryopreservation of sperm at asthenozoospermia with fullerenes in concentrations of 10 and 20 µg/ml led to the increase in motility relative to the control, while high concentrations of fullerenes used as additives to the cryopreservation medium, reduced sperm motility rate after thawing. The impact of fullerenes on the spermatozoa nuclear chromatin state before and after cryopreservation by flow cytometry was studied. It was shown that fullerenes with concentrations of 10 and 20 µg/ml did not cause any changes in the condensed chromatin state and human sperm membrane integrity at asthenozoospermia before and after freeze-thawing. Cytotoxic effect of fullerenes on human sperm is manifested when concentration growing up to 30-40 µg/ml. Activating effect of low concentrations of fullerenes can be due to amplification of the functional activity of mitochondria, and carbon nanoparticles can inhibit lipid peroxidation process and play a role of cytosorbents. Further increasing the concentration of carbon nanoparticles cause cytotoxic effects on human sperm. Condensed state of chromatin in the final stages of differentiation is very important for the further functioning sperm, because it protects the male genome from harmful influences and is a prerequisite for the return of reorganization in the formation of the male pronucleus.

human sperm, fullerenes, cryopreservation, motility, membrane, chromatin

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