PROH can readily permeate cell membranes and establish hydrogen bonds with water molecules to prevent ice crystallization. Consequently, PROH causes a slight lowering of the freezing point of the solution. Also, during freezing, the free water solidifies into ice and remaining solution will contain progressively higher concentration of electrolytes, reaching their toxicity level. The cryoprotective agent has the ability to reduce toxic effects of high concentrations of solutes, including salts. The protection may originate from the ability of PROH to replace water molecules around compounds, such as enzymes, hereby protecting them from irreversible conformational changes, which may occur at high salt concentrations (Chang, et al. 2010).

Besides its cryoprotective role, HSA facilitates gamete or embryo manipulation by preventing adsorption to the surface of petri dishes and pipettes through saturation of the potential binding sites.
Also, the increased viscosity of the media, caused by the addition of HSA, promotes the ease of embryo handling and manipulation (Trounson and Gardner 2000).