Novel Approaches to the Cryopreservation of Human Spermatozoa: History and Development of the Spermatozoa Vitrification Technology

Abstract

Cryobiology is very intensively applied in reproductive and veterinary medicine for preservation of gametes, embryos and reproductive tissues. Sub-zero temperatures combined with appropriate cryoprotective agents preserve the physiological and reproductive functions of the cells making long-term storage possible without loss of viability. With the use of cryoprotective agents it has become possible to develop cryopreservation techniques, such as the slow conventional freezing and vitrification that are in use in the present times. In slow controlled-rate conventional freezing extracellular ice crystals are formed whereas in vitrification no ice crystals are formed. Glass formation is compatible with the survival of the cell and the preservation of its intracellular structures provided the type(s) and concentrations of cryoprotectant used are not chemo- or osmotoxic. However, irrespective of the type of cooling method employed the cryosurvival of cells and tissues is influenced by the size and maturity of cells, amounts of intracellular water, quality and quantity of intracellular lipids, type of cells, their function and morphology. The intracellular milieu of cryopreserved cells and tissues remain less understood. The application of nanotechnology may help reveal and help advance our knowledge of the cryobiological principles involved in cryosurvival. At this moment the methods of cryopreservation that merit further investigation are vitrification and lyophilization. Vitrification is cheap if reagents are prepared in-house and the procedure can be performed rapidly. It has been successfully applied for gametes and embryos (of different stages of development), and reproductive cells/tissues, somatic cells and stem cells. However, vitrification is more demanding technically and requires operation and storage at sub-zero temperatures. On the other hand lyophilization deserves further investigation because it is a cheaper form of cryopreservation that may enable cryostorage at less demanding temperatures of 4°C and may even allow transport at ambient temperature. These possibilities are explored in this review.

Human sperm vitrification: the state of the art

“…Reed et al [17] reviewed laboratory and clinical data for all frozen embryo replacement cycles from 2012 to 2015 and concluded that vitrification was more effective, as indicated by higher survival in vitrified embryos compared to slow-cooling cryopreservation. Furthermore, vitrification is also reliable and simple to learn and implement in the laboratory while clinical pregnancy and implantation rates outcomes are similar [17], which is supported by other studies have [18][19][20].…”

“…Indeed, unlike the results noted above, using permeable cryoprotectants in sperm vitrification resulted in compromised or even lower motility than conventional freezing with permeable cryoprotectants [21][22][23]29] (Table 1). Isachenko et al [18,19] used 52 human swim up-prepared ejaculates for vitrification without permeable cryoprotectant. They found that in contrast to conventional freezing with glycerol, vitrified sperm displayed higher motility and integrity rates of cytoplasmic and acrosomal membranes, while no difference in spontaneous cryo-capacitation or acrosome reaction was observed [18].…”

“…It appears that a wide range of cooling rates are applicable. If the cooling speed during vitrification is flexible, aseptic vitrification with double straw containing higher volumes of 0.1 to 0.5 mL would allow the use of these techniques in human ART [18,19]. Interestingly, in human embryo vitrification, embryo survival is higher for large-volume vitrification, which has lower cooling rate comapred to micro-volume vitrification [17].…”

“…Fortunately, efforts to improve the use of closed carriers in sperm vitrification have been made and have shown encouraging results. For example, Isachenko et al [18,19] developed an aseptic technology for human spermatozoa vitrification. They used in 0.5 mL insemination straws for immediate intrauterine insemination and achieved satisfactory outcome with normozoospermic and severely oligozoospermic samples [18].…”

Human sperm vitrification: A scientific report

“…For warming, the 0.5 mL straw is cut at one end, and the inside 0.25 mL straw falls out immediately in 5 mL of warming medium (commercial medium and 0.1% dextran) at a temperature of 37°C. Then, the sample is concentrated by centrifugation at 1800 rpm for 5 minutes, and afterward, the spermatozoa are resuspended in the desired volume according to the purpose of use (ART procedure such as IUI, IVF, or ICSI) 33,34,46 …”