Human sperm vitrification: the state of the art

Sperm vitrification has been proposed as an alternative after the efforts in past years [7]. Human embryo cryopreservation which has been well established may provide some clues for sperm cryopreservation. 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 [18‐20].

One way to address whether vitrification is superior to conventional freezing is to directly compare the two methods using the same semen samples in the same study (Table 1). With 33 human semen samples, an experiment-controlled study found both slow cryopreservation and vitrification had similar results, but the latter was faster, easier and associated with lower toxicity and cost [28]. Le et al. recently carried out a direct comparison between vitrification and slow cryopreservation [32]. They used 105 human fresh semen samples, exclusive of cryptozoospermia and azoospermia, divided them into washed and unwashed halves, and each group was split into two aliquots: one group cryopreserved by conventional freezing while the other by vitrification. It was demonstrated that conventional freezing resulted in higher motility and viability, while spermatozoa undergoing vitrification were healthier regarding morphology and had fewer defects of sperm head, midpiece and tail [32]. Very recently, Pabon et al. [33] used 47 human sperm samples to compare the efficiency of vitrification and conventional freezing protocols. They found vitrification was optimal for sperm cryopreservation as vitrification protocol resulted in better motility recovery and higher mitochondrial activity [33]. Similar results were reported previously by other researchers [22, 24, 25, 27].

A recent study recruited 20 subfertile men with semen characteristics of severe oligoasthenozoospermia to compare the effects of two approaches and found that the vitrification method using only nonpermeable cryoprotectants was an effective alternative to the conventional slow-freeze technique [31]. Using epididymal and testicular spermatozoa, Epis et al. also demonstrated that vitrification resulted in higher mitochondrial membrane potential and motility than conventional freezing [34].

In order to compare the effectiveness of two methods, it is also legitimate to review and summarize the previously published and related articles. Recently, a systematic review and meta-analysis was undertaken to compare these methods [35]. The authors reviewed a total of 2428 published articles and 13 randomized controlled trials including 486 vitrified and 486 conventional cryopreserved sperm samples. They concluded that vitrification was superior to conventional freezing in post-thaw motility, including both total motility and progressive motility, although the efficacy of vitrification varied by vitrification protocol and sample quality [35].

Collectively, human sperm vitrification has shown increasing potential although the procedure needs further optimization as discussed later.

Different from embryo vitrification, sperm vitrification with highly concentrated, permeable cryoprotectants is not suitable for spermatozoa because mammalian spermatozoa exposed to hypertonic conditions during cryopreservation results in osmotic shock, and causes the sperm tail to coil at the distal end [36]. Most commonly used permeable cryoprotectants include dimethyl sulfoxide, glycerol, glycol, ethylene and methanol, while albumins, dextrans and egg yolk citrate are often used as nonpermeable cryoprotectants. It is known that controlling the osmolarity during cryopreservation is of great importance. Using permeable cryoprotectants in the cryopreservation media increases the osmolarity, which fluctuates between 600 to 1000 mOsm/L. In contrast, sperm vitrification media is isosmolar between 300 to 396 mOsm/L, which can be achieved with the use of nonpermeable cryoprotectants or a combination. Vitrification of spermatozoa, free of permeable cryoprotectants, is important to maintain sperm fertilization potential such as capacitation, acrosome reaction, and the integrity of cytoplasmic and mitochondrial membranes. 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 [21‐23, 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].

Slabbert et al. [20] used 35 human semen samples for permeable cryoprotectant-free vitrification. They found that the cryoprotectant-free technology generated higher mitochondrial membrane potential but lower DNA fragmentation with no difference in post-thaw motility compared to conventional slow freezing [20].

Aizpurua et al. [30] used 18 normozoospermic sperm samples to test a permeable cryoprotectant-free vitrification protocol. They found that it generated better recovery rates of good quality sperm and better maintenance of sperm quality than traditional slow-freezing [30]. In addition, they also demonstrated that permeable cryoprotectant-free vitrification presented a higher percentage of live spermatozoa, better preservation of acrosomes, and lower DNA fragmentation. Furthermore, using α-tubulin immunocytochemistry to show sperm cytoskeleton, they found that vitrified sperm had similar labeling patterns in the tail to fresh sperm, but different from slow freezing [30]. However, Agha-Rahimi et al. [26] vitrified 30 human normospermic samples with and without cryoprotectant, and they found that sperm frozen in permeable cryoprotectant did not show any toxicity or any difference in post-thaw motility, DNA fragmentation, or hyaluronan-binding potential [26].

In terms of nonpermeable cryoprotectants, some might perform better than others at specific concentrations. For instance, Schulz et al. compared two different nonpermeable cryoprotectants in vitrification with healthy volunteer semen samples and found post-warming sperm motility using 0.1 mol/L trehalose (69%) was higher than that of widely used 0.25 mol/L sucrose (58%). Furthermore, similar results were obtained at 6 and 12 h post-thaw. 0.1 mol/L trehalose preserved sperm had improved membrane integrity at 0 h post thaw, although no significant improvements at 6 h or 12 h compared to sucrose [37]. Another study found butylhydroxytoluene (BHT), a synthetic analogue of vitamin E, effectively maintained vitrified sperm function at 1 mmol/L, including higher progressive sperm motility after warming, DNA integrity, and lower reactive oxygen species [38]. Further research is needed to investigate the optimal nonpermeable cryoprotectants, their concentrations, or combinations depending on different human semen parameters or on clinical purposes.

Besides open systems like cryoloop, a number of closed carriers have been developed and applied in vitrification, including straw-in-straw, high security vitrification straw, Cryotip, VitriSafe, Cryopette, cryoLogic, Rapid-i, S3 system, and the S3 μS-VTF device [39‐42], many of which have proven effective for different purposes. More carriers are expected to be developed and optimized to facilitate sperm vitrification in the future. With the rapid progress of 3-D printing technology, it is now possible to design and print a freezing device. This technique could become a flexible, inexpensive and standard approach to produce freezing devices [43]. One of the 3-D printing thermoplastics, polylactic acid, has proved to be well suited to use for cryogenic activities and is routinely used in 3-D printers already. Because engineering grade 3-D printing can be easily standardized and modified, 3-D printing to form a freezing device has shown its potential applications in sperm cryopreservation.

It is believed that, to avoid cryo-damage in vitrification, the loading volume of samples in each carrier must be low enough to achieve high cooling rates [44, 45]. Reducing volume also helps reduce the chance of ice nucleation. By direct plunging into LN₂, sperm samples can reach cooling rates of 2, 000–10, 000 °C/min. However, reducing sample volumes limits efficiency in practice, since too many frozen units with small volumes (≤20 μl) not only make the operation process time-consuming, but also make it hard to collect enough number of motile sperm after warming.

[30] compared the efficiency of slow and ultra-rapid freezing and determined the level of DNA fragmentation after slow freezing-thawing and vitrification-warming. With 18 normal human semen samples, they found that ultra-rapid freezing resulted in higher progressive motility (18% vs 11%), higher mormal morphology rate, vitality, and lower sperm DNA fragmentation (20% vs 27%) compared to slow freezing. They concluded that the sperm ultra-rapid freezing was superior to slow freezing [30]. More recently, Hosseini et al. [46] prepared human samples from normozoospermic ejaculates by swim-up, and low number of human spermatozoa were frozen by directly submerging in LN₂ or its vapor. They found that direct submerging generated higher sperm progressive motility and total motility rates, lower alterations in sperm chromatin indicated by chromomycin-A3 and Aniline Blue staining, but did not affect morphology, acrosome integrity or DNA damage [46].

Using different size of straws for vitrification, a research group vitrified 22 fertile human donor semen samples with few spermatozoa. They used a micro-straw (50–100 μl) as well as traditional straws (0.25 ml and 0.5 ml). Compared to sperm samples frozen in traditional straws, the sperm frozen in micro-straws showed higher sperm motility after freezing-thawing with no difference in morphology, acrosome, or DNA integrity [47]. The authors thought as the micro-straws were thinner and they hold very small volume, the freezing rate was much faster. To freeze a very small number of human spermatozoa, a multi-well mini plate has been newly developed for vitrification using ~ 1 μl droplets. Sperm frozen using this method showed descent recovery rates and post thaw motility [48]. This novel approach was commented by Paffoni and Palini [49] [49].

As noted above, some scientists suggested using fish spermatozoa freezing model to investigate the mechanisms of why cryoprotectant-free vitrification for human ejaculates is better than conventional freezing and vitrification with cryoprotectants [15, 16, 50].

In contrast, Isachenko et al. [51] found the cooling speed didn’t have to be ultra-fast. They compared the quality of human sperm vitrification method (720, 000 °C/min) and relative slow cooling using LN₂ vapor (150–250 °C/min) without any cryoprotectant agents by cryoloop. They found both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa [51]. 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].

Besides the high-speed freezing (2, 000 °C/mL), the warming velocity should also be high so that the water inside spermatozoa passes from glassy state to liquid without ice crystal formation. Using human spermatozoa samples, Sanchez et al. [52] found the temperatures in the devitrification process were essential for preserved morphological membrane integrity and sperm function [52]. Furthermore, Mansilla et al. [53] tried to determine the optimal warming temperature after human spermatozoa vitrification and found the progressive motility in sperm samples warmed at 42 °C (65%) was higher than those at 38 °C (26%) and 40 °C (57%) and plasma membrane function was also better preserved at 42 °C [53]. Ali Mohamed [28] also warmed vitrified human sperm samples in prewarmed medium at 42 °C [28]. Pabon et al. warmed vitrified human spermatozoa micropills (5–10 μl each) in 500 μl medium prewarmed and maintained at 44 °C for 5 s and observed descent post-thaw motility and mitochondrial activity [33]. Another report noted above warmed vitrified human sperm samples (30 μl pellets) in a 37 °C water bath for 5 min resulting in motile and fertile sperm [32], indicating the flexibility of warming procedure.

No freezing method is absolutely safe. Frequent cleaning of used utensils should be the basic measure, including dry shippers, tanks, dewars, canisters, canes, and sample carriers. However, such maintenance may require samples to be removed from storage, which could put the stored specimen at risk [54‐56, 58, 67]. Another basic rule to avoid contamination is to store contaminated samples separately in quarantine to minimize the risk of cross contamination if possible. Commercially produced LN₂ itself could contain cryogenic pathogens and become the source of contamination. However, obtaining a small amount of sterile LN₂ is feasible by sterilizing the air used to create LN₂. For instance, McBurnie and Bardo [68] demonstrated that air filtration with 0.22 μm polytetrafluoroethylene efficiently retained Brevundimonas diminuta with extreme temperatures, high pressures, high flow rates, and high concentration of bacteria before LN₂ manufacturing. Alternatively, filtration of regular but non-sterile LN₂ before the samples are exposed could be even simpler. Indeed, a device called CLAir was developed for use in vitrification of human oocytes and mouse embryos [69]. A 0.22-μm filter equipped inside the canister can produce sterile liquid air at similar temperature to LN₂ so that the samples saved in a sealed canister (esther) are only exposed to sterile liquid air. Liquid air showed the same vitrification outcome with human oocytes and mouse embryos but with no contamination while large amounts of contamination with regular LN₂ were observed. Presumably, such device could be used to prevent contamination in the sperm vitrification process.

Another basic strategy to control contamination is to avoid direct contact with LN₂. It is therefore understandable that closed carriers as used in conventional freezing show lower incidence of contamination in comparison to open ones. Cryoloop, a widely used device in which the specimen is directly submerged into LN₂, generates considerable vitrification effects at the expense of severe sample contamination [55]. As using a closed carrier in the vitrification process is not always feasible, evidence has shown that using liquid nitrogen vapor, instead of LN₂ itself, to store human sperm samples can lower the risk of viral cross-contamination [70]. 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]. Slabbert et al. [20] used 0.5 ml straws to load 300 μl sample to allow sufficient air space inside the straw to prevent rupturing when immersed into LN₂. This method worked successfully with 35 vitrified human semen samples [20]. Diaz-Jimenez et al. [71] cryopreserved six donkey ejaculates, which were vitrified with either 30 μl sperm solution sphere suspensions or 100 μl in 0.25 ml straws with 0.1 M sucrose without glycerol. They found the straw method resulted in higher total and progressive motility while no difference in plasma membrane integrity [71]. It appears that sperm vitrification does not compromise post-thaw motility when using a closed carrier. Straw-in-straw design, or double straw, allows the inner straw contain the specimen and then it is sealed and inserted to outer straw, and then the whole unit is submerged to LN₂ so there is no direct exposure to LN₂. The design showed considerable vitrification results with 82 mouse D2/D3 embryos in 30 μl solution [72] and 113 human oocytes and 93 blastocysts using 1 μl medium [73]. Still, when loaded, the inner straw containing specimen could explode during freezing and warming due to the air pressure fluctuations. Therefore, a thin, narrow-walled capillary was developed to speed up the temperature conduction and reduce the air volume [74‐76]. The contamination incidence of this double straw method should be no greater than that of conventional method.

Intermediate methods exist that are a hybrid of open and closed systems to achieve the benefits of each method. Samples can be vitrified by direct contact to a small amount of purified LN₂, and are then sealed and stored in large quantities regular LN₂. This method has been reported in human embryo vitrification [77], and could be tested for efficacy in sperm vitrification.

Ultraviolet (UV) light could also be a possible solution to reduce the contamination risk to vitrified sperm samples. Treating a small volume of LN₂ with UV light at a suitable dose of radiation has been demonstrated to effectively reduce the number of pathogens, including bacteria, viruses, and fungi [78]. It was reported that 8, 000 μW/cm² of UV light could destroy hepatitis B virus while 330, 000 μW/cm2 destroyed the fungus Aspergillus niger. Most virus become inactivated by UV light at dose of 200, 000 μW/cm² [79] while Zika virus may have higher resistance to UV light [79]. Therefore, UV light could be a viable solution to reducing contamination rates in non-sterile LN₂.

Unfortunately, the application of UV light to LN₂ containing human samples is controversial. The UV light used could also cause severe genetic aberrations to stored spermatozoa, and further to fertilized embryos, although a study with human oocytes has demonstrated no adverse effects [80]. A simple solution is to sterilize LN₂ with UV light before it is used to freeze and store semen samples. Rinsing contaminated samples with sterile LN₂ can significantly reduce the contaminant pathogens. Parmegiani et al. [75] washed human oocytes and embryos purposely contaminated with bacteria (P. aeruginosa, E. coli, and S. maltophilia) and a fungus (A. Niger) three times in LN₂ sterilized by UV light. Washing these samples in sterilized LN₂ eliminated the contamination of both bacteria (0/65) and the fungus (0/25) while the unwashed samples remained highly contaminated with both bacteria (92/117) and fungi (25/25) [75]. Another concern with the use of UV light to sterilize LN₂ is the generation of ozone, which could have detrimental effects on the buffering system in which the cryogenic samples are stored. Luckily, the formation of ozone from UV light is insignificant as ozone is formed by the breakdown of oxygen molecules by the action of UV radiation. When these oxygen atoms separate, they combine with other oxygen molecules to form ozone. However, as LN₂ is virtually free from oxygen, this should not be an issue with UV light sterilization of LN₂ [80].

Generally, there is no easy but cost-effective way to completely eliminate all the potential risks of contamination in sperm vitrification. But based on the improvements in past years, it is possible to control the contamination risk of vitrification to the level of conventional freezing.

Careful optimization of preservation protocols can be tedious, confusing and expensive due to the specific devices and reagents required. Is it possible to develop a universal protocol for all the species and all the samples? It sounds impossible since the cryotolerance of spermatozoa depends on sperm features such as size, shape, and lipid composition, which makes it challenging to generate a single standardized freezing procedure for all species. Even in humans, there is a variety of sperm specimens, such as normospermic, oligospermic, azoospermic samples, testicular sperm aspiration (TESA), and percutaneous epididymal sperm aspiration (PESA) samples with different parameters such as volume, concentration, motility, and seminal plasma features. Furthermore, it is difficult to establish a universal stereotyped model to serve different cryopreservation purposes at human clinic. Rozati et al. [81] reviewed the pitfalls of human sperm cryopreservation, especially the sperm banking for cancer patients [81].

In fact, there was an effort to establish a universal vitrification method for almost all the samples including oocytes, primary cells, stem cells, and genetically modified cells. The method proposed low concentrations of cryoprotectants including 1.5 M propanediol and 0.5 M trehalose in industrial grade microcapillaries made of highly conductive fused silica. It was demonstrated that this universal protocol achieved high recovery and viability rates after vitrification for human mammary epithelial cells, rat hepatocytes, tumor cells from pleural effusions, and multiple cancer cell lines [82]. Unfortunately, due to the different characteristics of spermatozoa from the cell types tested, this method would not likely be superior to the specialized sperm cryopreservation protocols that exist presently.