Sperm cryopreservation: current status and future developments

Keywords: antioxidant, cryopreservation, glycerol, infertility, motility, post-thaw, programmable freezing, reactive oxygen species, spermatozoa, vapour.

Initially discovered by an Italian priest in 1776 (Spallanzani 1776), the cryopreservation of spermatozoa involves cooling these cells to ultra-low temperatures (usually −196°C) followed by storage in liquid nitrogen to be later thawed and inseminated (Mazur 1970; Di Santo et al. 2012). Clinically, this became an increasingly important therapeutic strategy in the mid-to-late 1900s, due to increasing use of artificial insemination (AI) – at a time when there were no treatments for infertile males; and between 1 in 10 and 1 in 4 couples were experiencing conception difficulties (Mazur 1970; Ozmen et al. 2007; Di Santo et al. 2012). Even now, when assisted reproductive technology (ART) in the form of in vitro fertilisation (IVF) or intra-cytoplasmic sperm injection (ICSI) are readily available to treat male infertility, cryopreservation still has an important role to play in the clinical management of infertility. For example, the availability of cryostored spermatozoa may be valuable in instances where the male partner may not be able to produce a fresh semen sample on the day of oocyte retrieval and for banking spermatozoa prior to the initiation of spermatogenesis-compromising medical treatments, such as chemotherapy. This technology may also be beneficial for men who need spermatozoa retrieved via biopsy, to minimise the number of procedures required (Ozmen et al. 2007). Aside from clinical applications, AI with cryopreserved spermatozoa is also central to many animal breeding programs particularly for dairy cattle and Standardbred horses (Bailey et al. 2000).

New freezing methods and cytoprotective agents have been researched since sperm cryopreservation’s clinical implementation, with the most impactful being the accidental discovery of glycerol as a cryoprotectant in 1947 (Polge et al. 1949; Pegg 2002). A protective agent is necessary for freezing sperm, as spermatozoa are likely to experience cryoinjury due to ice formation in the cytoplasm of the cell, changes in membranous lipid composition (cold shock) and osmotic stress from the movement of water and salts across the plasma membrane. Overproduction of, or overexposure to, reactive oxygen species (ROS) can also damage multiple components of these highly specialised cells including the integrity of their nuclear DNA (Aitken et al. 1997; Katkov 2000; Pegg 2002; Ozmen et al. 2007; Amidi et al. 2016; Dutta et al. 2019). The susceptibility of spermatozoa to so many forms of cryodamage and the underlying importance of cryopreservation for the management of fertility in both man and animals, has led significant research to better protect, freeze and thaw spermatozoa for successful application in ART. The purpose of this review is to examine the current status in this area and present possible strategies for the further development of this technology in the future.

During freezing, ice forms in the cellular suspension; this ice is largely made up of water and is unable to dissolve the solutes it previously held, leading to a state of hyperosmolarity. To correct this, cytoplasmic water diffuses out of the spermatozoa to restore the osmotic balance, but in the process dehydrates the cell (Pegg 2002). This dehydration, and the resulting increase in salt concentration within the spermatozoa, can further damage the cells via changes in cellular pH and weakened membrane integrity (Katkov 2000; Pegg 2002; Vadnais and Althouse 2011; Oldenhof et al. 2013). Although cold shock can minimise the fluid leaving/entering the cell by decreasing membrane fluidity; this does not protect the spermatozoa from damage, as the retention of too much fluid can cause membrane rupture when ice forms and expands (Katkov 2000).

Assuming cold shock did not occur, once the solution begins to thaw, the ice melts and extracellular fluid will be brought into the dehydrated spermatozoa to restore osmotic balance. However, as the spermatozoa thaw yet more damage may be induced, not as a consequence of the cryopreservation process per se but as a result of oxidative stress. Such stress may emanate from external sources including damaged leucocytes or spermatozoa in the immediate vicinity, or internally, particularly from the mitochondria (Aitken et al. 1997; Oldenhof et al. 2013; Dutta et al. 2019). Oxidative stress occurs when the exposure of a given cell type to ROS overwhelms the cell’s capacity for antioxidant defence. Typical examples of ROS include superoxide anions (O₂⁻·), peroxyl (·ROO) and hydroxyl (·OH) radicals as well as powerful oxidants such as hydrogen peroxide (H₂O₂), all of which can be created by electron leakage from the mitochondrial electron transport chain (ETC) (Gharagozloo and Aitken 2011; Barati et al. 2020).

Once spermatozoa have been exposed to excessive ROS, the mitochondria become damaged and initiate a self-perpetuating cycle of peroxidative damage. During this process lipid aldehydes such as 4-hydroxynonenal (4-HNE) or acrolein, generated as a result of ROS-induced lipid peroxidation, bind to cysteine rich-proteins involved in the electron transport chain, creating yet more electron leakage and the generation of additional ROS that further drives the peroxidative process (Aitken et al. 2012). Vulnerable mitochondrial proteins in this context are the ATP synthase subunit β (ATP5B), succinate dehydrogenase [ubiquinone] flavoprotein subunit (SDHA) and NADH dehydrogenase [ubiquinone] iron–sulphur protein 2 (NDUFS2) (Aitken et al. 2012; Zhao et al. 2014).

The toxic aldehydes generated as a result of ROS-induced oxidative stress do not only target the mitochondria, but they can also form adducts with several other key proteins that can have a direct impact on sperm function. Thus 4-HNE is known to bind to dynein light chain within the sperm axoneme thereby contributing to the loss of sperm motility observed following cryopreservation (Baker et al. 2015). This toxic lipid aldehyde is also known to bind to cysteine residues within A-kinase anchoring protein 4 (AKAP4) resulting in the disruption of key elements of sperm capacitation pathway mediated by cAMP (Nixon et al. 2019). In addition, 4-HNE has been found to bind to heat shock protein A2 (HSPA2) and, via this mechanism, suppressing the competence of human spermatozoa to bind to the zona pellucida (Bromfield et al. 2015). In addition to lipid peroxidation, the ROS generated as a consequence of cryopreservation can also impair sperm function by oxidising proteins and disrupting the integrity of DNA in the sperm nucleus impairing not just the functional competence of the spermatozoa but also the developmental normality of any embryos created as a result of fertilisation (Aitken et al. 1998a, 2022; Barati et al. 2020).

Spermatozoa do not only suffer from damage during the freezing process, when the combination of ice crystal formation and osmotic stress can disrupt both membrane integrity and intracellular homeostasis. They can also become damaged during the thawing process as a result of a ROS-induced oxidative attack, that can lead to DNA damage and also the disruption of sperm function in terms of motility and the ability of these cells to capacitate in a normal, regulated manner (MacLeod 1943; Aitken et al. 1998a; Oldenhof et al. 2013; Amidi et al. 2016).

Interestingly, in certain species such as the bull, the cellular disruption precipitated by the freezing and thawing process can, besides causing potential lasting damage, also create a type of dysfunctional pseudo-capacitation, or cryocapacitation (Vadnais and Althouse 2011). Cryocapacitation involves a cascade of changes including tyrosine phosphorylation, induction of activated signal transduction pathways triggering hyperactivation, increased membrane fluidity and, ultimately, acrosomal exocytosis (Vadnais and Roberts 2007; Vadnais and Althouse 2011). In part, the induction of this process may reflect the fact that the impact of ROS on sperm function is known to be a two-edged sword. Thus, while high levels of oxidative stress are clearly damaging to these cells for all the reasons given above, low levels of ROS generation are known to promote sperm function, reflecting the fact that capacitation is a redox driven process (Aitken et al. 1998b; Aitken and Drevet 2020). As a result, when human spermatozoa are exposed to increasing levels of oxidative stimulus, the resulting dose response is bell-shaped; low levels of oxidative stress stimulate sperm–oocyte fusion whereas higher levels inhibit this process (Aitken et al. 1998a). This redox drive may be a key factor in the induction of cryocapacitation, since the latter can be prevented in cryopreserved bull spermatozoa by reducing the partial pressure of oxygen in the semen extender (Mustapha et al. 2022).

Similarly, ROS are known to promote cholesterol efflux and a consequential increase in membrane fluidity during normal capacitation via an oxidative mechanism. Thus, ROS generation during capacitation results in the formation of oxysterols in the sperm plasma membrane. The latter are oxidised derivatives of cholesterol that can move more freely out of the membrane to bind acceptor proteins, such as albumin, because they are much more hydrophilic than the parent molecule. As a result, the oxidation of cholesterol by ROS promotes capacitation by facilitating the efflux of this decapacitation factor from the sperm plasma membrane (Aitken 2011). The fact that cryocapacitation can be prevented by exposing the cells to cholesterol in order to compensate for the excessive cholesterol efflux engendered by cryopreservation, again suggests the involvement of ROS in this process (Rajoriya et al. 2020). Finally, an involvement of excess ROS generation in the cryocapacitation phenomenon is strongly suggested by the ability of antioxidants such as reduced glutathione or hypotaurine to counteract this process (Shah et al. 2017; Pons-Rejraji et al. 2021). Cryocapacitation is therefore a disruptive, non-physiological process that reflects the internal disorder generated in spermatozoa as a result of cryopreservation as a consequence of both the physical damage induced during freezing and the initiation of excessive ROS generation as these cells are returned to ambient temperature.

To protect spermatozoa from the multitude of ways in which they can become damaged during cryopreservation, different methods for freezing and varied types of cryoprotective media have been explored.

The main purpose of a cryoprotectant is to reduce cytoplasmic hyperosmolarity during freezing and to prevent intracellular and extracellular ice formation (Karow 1969; Pegg 2002). Glycerol has been widely deployed to protect spermatozoa from cryoinjury following its accidental discovery as a cryoprotectant in the 1940s (Polge et al. 1949; Karow 1969). However, cryoprotective agents (CPAs) had been assessed in the years before this discovery, with the effects of sugars, milk, glycerine and glucose all being studied in the cryoprotection of bacteria in 1913 (Keith 1913; Karow 1969). In light of these studies, there was an expectation that multiple small-molecular mass substances would be discovered with the ability to protect spermatozoa during cryopreservation, not just glycerol. The non-permeable cryoprotective agents evaluated included disaccharides (sucrose, trehalose), and osmotically inactive compounds including polysaccharides (hydroxyethyl starch, maltodextrin), polyvinyl pyrrolidone and proteins (albumin). Membrane permeant CPAs such as dimethyl sulphoxide ethylene glycol, methyl–formamide, or dimethyl–formamide were also assessed and egg yolk base extenders became popular (McGann and Farrant 1976; Oldenhof et al. 2013). However, regardless of the type of cryoprotectant used (penetrating or non-penetrating), the CPA concentration chosen, or whether seminal plasma was present or excluded, no combination could reliably prevent the loss of sperm vitality and motility observed following cryopreservation (Pegg 2002; Best 2015). This lack of progress has prompted new interest in developing additives to supplement the CPAs, that are designed specifically to combat problems spermatozoa experience when undergoing freezing. Prominent within this list of potential additives are antioxidants designed to reduce the ROS-mediated damage highlighted above (Aitken and Krausz 2001; Banihani et al. 2014; Amidi et al. 2016; Dutta et al. 2019).

A wide range of antioxidants have been used in this context, attacking several different stages of the oxidative process. Examples of antioxidants employed for this purpose for the preservation of human semen include curcumin (Riahi et al. 2021), L-proline (Moradi et al. 2022), hypotaurine (Pons-Rejraji et al. 2021), cerium oxide nanoparticles (Hosseinmardi et al. 2022), quercetin (Cheraghi et al. 2021), plant extracts (Ros-Santaella and Pintus 2021), lipoic acid (Shaygannia et al. 2020), L-carnitine and co-enzyme Q10 (Chavoshi Nezhad et al. 2021), the combination of L-α-phosphatidylcholine (PC) and L-acetyl-carnitine (Sicchieri et al. 2021), sulphoraphane (Valipour et al. 2021), vitamin B12 (Hosseinabadi et al. 2020) and polyphenols of various kinds including chlorogenic acid (Noto et al. 2021), green tea extract (Alqawasmeh et al. 2021), resveratrol (Li et al. 2018) and myoinositol (Abdolsamadi et al. 2020). In animal models, additional antioxidants have been assessed in this context but, as far as the authors are aware, have not yet been evaluated for the cryopreservation of human spermatozoa; these reagents include fullerene (Turk et al. 2022), Mito-TEMPO (Esmaeilkhanian et al. 2021) and idebenone (Lone et al. 2019). For a vast majority of these antioxidant trials, the end points assessed have been sperm viability, motility and DNA damage. However, antioxidants can also reduce deprotamination of the sperm nucleus as a result of cryopreservation and this may be one of the ways in which DNA integrity is preserved (Chavoshi Nezhad et al. 2021). Overall, whenever they have been assessed, antioxidants do appear to aid the preservation of sperm function and DNA integrity. However, no systemic comparative trials have yet been conducted to determine which antioxidant, or combination of antioxidants might be optimal for sperm cryopreservation in a given species.

Other additives that can be included alongside CPAs and antioxidants include energy substrates (glucose, fructose, L-carnitine etc.), antifreeze proteins, and other compounds that compensate for osmolarity changes, as well as minerals (higher or lower levels of NaCl and KCl) to manage the ionic composition of the sample (Nash 1962; Katkov 2000; Bilodeau et al. 2001; Ghallab et al. 2017; Gholami et al. 2019; Zandiyeh et al. 2020). However, the concentrations of these additives in cryopreservation media will have to be carefully monitored in futureformulations, as even clinically approved CPAs such as glycerol can become toxic above a critical threshold concentration (Pegg 2002; Best 2015).

For this reason, besides the addition of CPAs, other ways have been explored to increase the survival of cryostored spermatozoa by manipulating the physical process of freezing. Sperm cooling can be conducted via programmed slow or rapid freeze, or even flash-freezing vitrification (Sherman 1963; Stanic et al. 2000; Vutyavanich et al. 2010; World Health Organization 2021). The two most popular choices currently are: (1) slow-freezing, where sperm straws are progressively cooled in a controlled freezer, first slowly at around −1°C/minute until −5°C is reached, then quickly at around −10°C/min until the sample reaches −80°C at which point the straws are plunged into liquid nitrogen (LN); and (2) rapid-freezing, which can involve either the sample straws being incubated at 4°C for a short time before being plunged into LN, or the straw being held above LN vapours for a short time before plunging into LN (Sherman 1963; Vutyavanich et al. 2010). It has been shown that both processes are effective for human spermatozoa, but there is currently no preferred method because of a failure to conduct comparisons under carefully controlled conditions (time, specifics of cooling, CPA or other additives) (Stanic et al. 2000; Vutyavanich et al. 2010). Vitrification is gaining traction as a potential method to freeze spermatozoa, with the cells being frozen so quickly that ice is unable to form, and a glass-like state is achieved (Wowk 2010). Although this process is used in extensively in the freezing of embryos, it is still regarded as an emerging technology as far as human spermatozoa are concerned (Wowk 2010; World Health Organization 2021). Carefully controlled comparative trials are required to determine whether vitrification represents a significant improvement over conventional cryostorage for the long term preservation of human spermatozoa.

Due to the variety of different CPAs, additives, and physical freezing methods; it is difficult to determine the most optimal cryopreservation protocol. Current World Health Organization guidelines simply give instructions for both vapour and mechanical (controlled via machine) based freezing and state that any commercially tested and available CPA may be used (World Health Organization 2021). Therefore, there is currently an urgent need to generate the comparative data to define a standard optimised protocol for each species. In preparation for such analyses, the results gained from a variety of different freezing methods, CPAs and additives have been compiled in the current review. Study dates range between 1982 and 2021, where the cryopreservation medium is clearly defined and the freezing method fully described. On this basis, 32 cryostorage variations have been identified; including 10 publications using human spermatozoa (Table 1) and 47 variations on non-human spermatozoa in a further six publications (Table 2). A comparison of the freezing method and main results for the human and non-human variations are also show in Tables 3, 4 respectively.

Table 1.  The list of constituents given for each CPA and supplement reviewed for human publications

Table 2.  The list of constituents given for each CPA and supplement reviewed for animal publications.

Table 3.  Comparison of human-focused papers demonstrating freezing method, DNA integrity and total motility results. (Mahadevan and Trounson 1983; Stanic et al. 2000; Hammadeh et al. 2001a, 2001b; Banihani et al. 2014; Raad et al. 2018; Gholami et al. 2019; Zandiyeh et al. 2020).

Table 4.  Comparison of animal-focused papers demonstrating freezing method and total motility results. (Purdy and Graham 2004; Moce et al. 2010; Oldenhof et al. 2013; Ghallab et al. 2017; Lee and Kim 2018; Lv et al. 2022).

A detailed analysis of post thaw motilities across mammalian species comparisons was conducted but did not yield any valuable insights due to the considerable variation between studies in the protocols and media employed as well as inter-species variation in the susceptibility of the spermatozoa to cryodamage (Purdy and Graham 2004; Moce et al. 2010; Oldenhof et al. 2013; Ghallab et al. 2017; Lee and Kim 2018; Lv et al. 2022). Although multiple tests were done for each variation, motility was the only result shared by all publications (Table 4). Of this, highest post thaw motility (66%) was recorded in boars using Lactose-Egg-Yolk Glycerol-Orvus-ES-Paste (LEYGO) supplemented with 500 μM of Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) and vapour freezing (Lee and Kim 2018). However, it must be considered that as all mammalian species have varied levels of normal sperm motility, Lee and Kim’s result cannot be accurately compared with the other species listed in this publication.

To avoid this inter-species variation, the majority of publications compared focused on the cryopreservation of human spermatozoa, with 32 detailed projects identified spanning 13 different CPA media, four supplements and three freezing programs. A full list of the CPA employed, supplements included and freezing method, can be found in Table 1 (Peek et al. 1982; Mahadevan and Trounson 1983; Stanic et al. 2000; Hammadeh et al. 2001a, 2001b; Thomson et al. 2009; Raad et al. 2018; Gholami et al. 2019; Zandiyeh et al. 2020).

Similar to the animal models, the most commonly performed post-thaw test for human spermatozoa was the percentage of motility (21/32). Of the 19 experiments, the highest overall motility (52%) was observed using Tris-egg yolk buffer (TYB) CPA, unsupplemented, and a slow program freezing (Stanic et al. 2000). The studies where only the freezing method was changed to a fast program or vapour-phase freezing, achieved 49% and 43% post-thaw motility respectively, indicating that, at least in these cases, the freezing method does impact sperm health (Stanic et al. 2000).

After motility, the second most common post-thaw test was the live sperm count (million/mL). Gholami et al. (2019) obtained the highest post-thaw viable sperm count at 59.28 million/mL, utilising a TEST buffer (type of commercial CPA containing glycerol, DMSO and Tris pH buffer) supplemented with soybean lecithin, using a fast freezing program. The lowest count was only 5 million/mL, obtained using a glycerol egg yolk citrate CPA supplemented with 10 μg/mL of anti-freeze protein III (Zandiyeh et al. 2020).

While studying the behavioural properties of spermatozoa is useful, any results pertaining to the cells’ DNA integrity should also be considered. In this context, Raad et al (2018) observed the smallest increase in DNA fragmentation post-thaw (1.75%), using supplemented Quinn’s Advantage Sperm Freeze; however, Banihani et al (2014) utilising TYB CPA supplemented with L-Carnitine, a dual purpose energy substrate and antioxidant, claimed to actually lower the samples’ DNA fragmentation levels (albeit insignificantly).

Of the 32 treatments using human spermatozoa, there is no one single experiment available to give a concise answer as to which CPA, supplement or freezing method should be implemented from now on (Table 3). The best results gained from each test (vitality, acrosome integrity, motility and DNA fragmentation), covered all three freezing methods, five different CPAs and three types of supplements (Mahadevan and Trounson 1983; Stanic et al. 2000; Hammadeh et al. 2001b; Banihani et al. 2014; Raad et al. 2018; Gholami et al. 2019; Zandiyeh et al. 2020). Of the five CPAs, three contained glycerol nonetheless no other similarities can be discerned. The three supplements were all unrelated, and of the freezing methods vapour freezing was used in most of the highest scoring tests, however, fast-program and slow-program freezing were also conducted in roughly equal measure.

With DNA integrity becoming a vital aspect of sperm health, it is important that future studies include this measure of sperm quality (Banihani et al. 2014; Raad et al. 2018). Most importantly the use of antioxidants to supplement CPAs should be further studied, to determine if the proportion of DNA-fragmented sperm can be reduced, as this may lead to improvements in fertilisation and pregnancy rates and reduce the mutational load carried by the offspring (Thuwanut et al. 2008; Gharagozloo and Aitken 2011; Len et al. 2019; Treulen et al. 2019; Aitken and Bakos 2021; Alyethodi et al. 2021; Wang et al. 2021). Besides this, studies should also consider the effects of thawing time, as many studies and protocols have been created on how to freeze sperm samples, yet there is a significant lack in data on the thawing procedure; with only two relevant papers published on this topic over the past half century (Rowe 1966; Chatterjee and Gagnon 2001).

There have been definite advances in the study of cryopreservation over the last 74 years since the discovery that glycerol is an effective cryopreservation agent for the long-term storage of spermatozoa. Recent research into the effectiveness of antioxidants and the possibility of vitrification are currently major areas of activity and should be continued. However, systematic comparative analyses are needed to determine the optimal antioxidant or combination of antioxidants that should be used, which CPA best protects the spermatozoa from cryoinjury, whether additional supplements such as lipids or anti-freeze proteins are of valuable addition to the cryostorage medium and which freezing method is optimal for a given species. The potential advantages of separating the spermatozoa from seminal plasma prior to cryostorage and the optimal procedure to achieve that separation also remain areas of investigation that are largely unexplored at the present time. The clear differences between species in terms of their cryostorability also indicate that such optimisation procedures will have to be replicated for each species of interest. Furthermore, for all species it will be important to develop entirely defined media that are free of complex biological constituents such as egg yolk or albumin, which are difficult to standardise, may harbour infections and present regulatory hurdles for international transportation.

All tabulated results are available at the end of this review (Tables 1–4).

RJA is Scientific Director of Memphasys Ltd and AH is a PhD student funded by Memphasys Ltd.

A PhD scholarship was provided to AH by Memphasys Ltd.

The study was conceived by RJA and AH. AH generated the drafts and final paper, which RJA and HWB then reviewed and edited.