Using Sperm Imaging with Laser Interference Microscopy for Comprehensive Assessment of the Functional State of Cells during Cryopreservation and under the Action of Molecular Hydrogen

Significant advances have been made in sperm cryopreservation but the search for effective sperm cryopreservation technologies is a pressing issue in modern biology and medicine. The most effective cryopreservation leaves 50-60% of viable cells. The paper discusses the use of molecular hydrogen (H2) as a new approach to enhancing sperm protection during freezing and thawing. H2 is a universal antioxidant and limits damage to biomolecules. Visual registration of spermatozoa under the action of H2 was performed using modern microscopy techniques. Laser interference microscopy was used in the work. Laser interference microscopy records the cell surface architectonics depending on the modulation of the optical density of cellular structures. This visualization option provides information on the metabolic level and expands the possibilities for interpreting experimental results. Sample preparation, dyes, and fixatives are not used in interference visualization. The paper presents an analysis of phase images of spermatozoa during cryopreservation and using H2 as a cryoprotector. Verification of the method for analyzing phase characteristics of spermatozoa as an indicator of the metabolic state of cells was performed by analyzing clinical and laboratory parameters of spermatozoa. The phase height of spermatozoa during cryopreservation decreased, the intensity of energy processes decreased, and the oxidative potential of cells increased. A direct correlation was shown between the phase height of spermatozoa and the concentration of ATP, and an inverse correlation was found from the concentration of malondialdehyde (MDA). The use of H2 determined an increase in the phase height of spermatozoa, an increase in energy metabolism, and a decrease in cell oxidation. Changes in the metabolic activity of spermatozoa under the action of H2 were combined with an improvement in sperm fertility. Thus, phase interference microscopy allows for a qualitative and quantitative assessment of the physiological state of spermatozoa. It is an objective method of vital analysis of complex metabolic activity of cells. It can be used for express diagnostics of their functional state.