Cryopreservation of human spermatozoa—introduced in the 1960's—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.

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Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

The cryopreservation of spermatozoa was introduced in the 1960s as a route to fertility preservation. Despite the extensive progress that has been made in this field, the biological and biochemical mechanisms involved in cryopreservation have not been thoroughly elucidated to date. Various factors during the freezing process, including sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for poor sperm quality post-thaw. Little is known regarding the new aspects of sperm cryobiology, such as epigenetic and proteomic modulation of sperm and trans-generational effects of sperm freezing. This article reviews recent reports on molecular and cellular modifications of spermatozoa during cryopreservation in order to collate the existing understanding in this field. The aim is to discuss current freezing techniques and novel strategies that have been developed for sperm protection against cryo-damage, as well as evaluating the probable effects of sperm freezing on offspring health.

Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches.

The aim of this review will be to provide a basic understanding of the biophysical processes that accompany the application of cryopreservation in reproductive medicine. The ability to store cells in 'suspended animation' outside the body has become a keystone practice in the development of many modern clinical therapies, and, in fact, the sciences of cryobiology and IVF have developed in parallel over the past 50 years. During this time, some of the underlying principles of the quantitative biophysical aspects of cryobiology have been clarified. Water is the universal biocompatible solvent, but also possesses unique properties for stability of living cells. Whilst low temperatures themselves have defined effects on cell structure and function, it is the phase transition of water to ice that is the most profound challenge for survival. The thermodynamics of dilute aqueous solutions dictate how cells and tissues respond to the freezing process. Current concepts of nucleation, ice crystal growth and solute exclusion from the ice lattice will be discussed to illustrate what cells must negotiate to avoid lethal damage, and the role of cryoprotectants in enhancing recovery. Quantitative formalisms now exist to model and predict how water and solutes move across cell membranes before and during freezing, or how nucleation events will proceed, and these will be outlined. Cryoprotectants have both positive and negative effects on cell function depending on the kinetics of exposure. The concept of tolerable osmotic excursion of cell volume will be discussed, along with the evidence for a 'pseudo-glassy' state for cells during traditional cryopreservation. This will be compared with the recent interest in promoting glassy states in the whole sample using vitrification protocols, outlining the advantages and drawbacks of each approach. Additional methods for controlling ice nucleation have a role to play here, and a brief outline of current technologies will be given. Finally, issues of safety and stability of cryopreserved samples will be set out.

https://www.rbmojournal.com/article/S1472-6483(10)61780-4/pdf

Fundamentals of cryobiology in reproductive medicine

https://www.fertstert.org/article/S0015-0282(08)03942-3/pdf

Effects of cryopreservation on human sperm deoxyribonucleic acid integrity

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 mul suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37 degrees C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5',6,6'-tetrachloro-1-1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1+/-3.2%, P<0.05) when compared with controls (19.4+/-1.9%). The combination of HSA and sucrose (65.2+/-2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6+/-4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.

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Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose.

BACKGROUND: In contrast to the technique of conventional freezing, the vitrification of spermatozoa requires high cooling rates (720 000°K/min), which could be damaging for spermatozoa. The aim of our study was to compare slowly frozen and vitrified spermatozoa in terms of their post-thaw DNA integrity and motility. METHODS: Semen samples were prepared according to the routine swim-up technique and divided into aliquots for comparison of fresh, conventionally frozen and vitrified spermatozoa from the same ejaculate in the presence or absence of cryoprotectants. Spermatozoa motility and DNA integrity were determined. RESULTS: The motility of spermatozoa conventionally (slowly) frozen with a cryoprotectant was similar to that recorded for spermatozoa vitrified in the absence of cryoprotectant (47 versus 52%). The DNA integrity was unaffected by the cryopreservation method or presence of cryoprotectants. CONCLUSION: The vitrification of human spermatozoa in the absence of conventional cryoprotectants is indeed feasible. The DNA integrity of vitrified sperm is comparable with that shown by standard slow-frozen/thawed spermatozoa, yet the method is quick and simple and does not require special cryobiological equipment.

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T. Schöndorf

Papers

DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant‐free vitrification