Human Sperm Vitrification: Review of Recent Progress

Abstract

Sperm vitrification has been used in the field of assisted reproductive technology (ART) for years and has resulted in many healthy live births. Compared to the conventional sperm slow freezing method, vitrification is simpler, quicker, and less expensive, and some vitrification methods are also cryoprotectant free, which has the potential to become an alternative cryopreservation method for human sperm. Human sperm vitrification has been the most commonly used and valuable way to preserve the fertility of males with small numbers of spermatozoa. Recently, new sperm vitrification devices have been developed to help improve volume control. Direct contact during the vitrification process with liquid nitrogen increases the risk of cross-contamination. New strategies have been implemented to minimize the contamination risk. Depending on the variety of semen parameters and patients’ purposes at ART clinics, specific sperm cryopreservation approaches should be personalized to achieve the optimal results for each case.

1. Introduction

Traditional cryopreservation techniques are widely used in assisted reproductive technology (ART) programs all over the world; however, vitrification is a novel technique and has become a quickly growing alternative method for the cryopreservation of human spermatozoa in the past decade. Sperm vitrification usually requires a small loading volume of sample to achieve an extremely high cooling rate. Human sperm vitrification has been the most commonly used way to preserve the fertility of males, including those with severe oligospermia or azoospermia patients, who have undergone a TESE/micro-TESE procedure, which has yielded a small number of spermatozoa. However, in the past few years, many new designs of larger volumes have been developed and have displayed promising results. This review summarizes the recent researches on human sperm vitrification, including comparison studies of conventional cryopreservation methods like slow freezing and vitrification, descriptions of different types of cryoprotectants, methods of human sperm vitrification, as well as the warming, storage of vitrified samples, contamination risk and control during sperm vitrification.

2. Principles of human sperm cryopreservation

There are several different cryopreservation methods, including slow freezing (0.5–0°C/min), rapid freezing (50–400°C/min), ultra-rapid freezing (~2500°C/min), and vitrification (~20,000°C/min), applied accordingly depending on the freezing speed and cryoprotectant concentration and temperature reduction [1]. The principles of cryopreservation are based on the laws of thermodynamics. Sperm has been shown to be sensitive to exposure to high concentrations of cryoprotective agents. Freezing media for human sperm vitrification is significantly different from cryopreservation solutions for oocytes and embryos. Contrary to the slow freezing process, the vitrification process is based on an extremely high cooling rate that prevents intracellular ice crystallization and produces an amorphous, glass-like solid state.

The greatest challenge the cells are facing during the freezing and warming processes is cell damage caused by ice crystals forming a temperature range between −15 and − 60°C. At temperatures between −5 to −15°C, extracellular ice formation occurs and develops an extracellular solid phase. Meanwhile, the inside of the cell remains supercooled, which has a high chemical potential and diffuses out of the cell osmotically. Extracellularly hypertonic results in further removal of water from cells causing almost complete dehydration. The composition of the cryopreservation media plays a vital role in the freezing techniques, fast or slow. Permeable cryoprotectants, including glycerol, ethylene glycol, 1,2 propanediol, and dimethyl sulfoxide (DMSO), are typically used in the cryopreservation of human spermatozoa. Due to their lipophilic properties, they cross the cell membrane, creating an osmotic gradient caused by water outside of the cell. Sperm have been shown to be particularly sensitive to high concentrations of cryoprotectants that are routinely used for oocytes and embryos. Non-permeable cryoprotectants, including sucrose, glucose, and trehalose etc., are of high molecular weight, and consequently are not able to penetrate the cell membrane. They are not directly toxic to sperm, but they nonetheless cause damage due to the osmotic shock encountered during addition and removal. Vitrification does not require an osmotic balance during the freezing period of the cells, as fast dehydration occurs with a super high freezing speed and a hyperosmolar medium. During the process, as the viscosity of the solution increases, the molecules are immobilized and the liquid passed to a solid glass-like phase without the formation of ice crystals in both the intracellular and extracellular environment while displacing the water from the cell. For the vitrification of human sperm, the cryoprotectant-free vitrification method has been developed to reduce cryo-injuries [2].

2.1 Comparison of the studies for human sperm slow freezing versus vitrification

Both slow freezing and vitrification have advantages and disadvantages which can be found in Table 1. One way to investigate the difference between slow freezing and vitrification is directly compared the two methods using the same semen samples in the same study [3]. With 57 human semen samples, Saritha and Bango et al. discovered no substantial motility differences between the two methods [4]. Zhu et al. used 58 washed human semen samples to compare the efficiency of the slow freezing method versus the vitrification process. No differences in motility or DNA stability were found in this study, however, higher progressive motility, plasma membrane, and acrosome integrity were found in the vitrification group with optimal sucrose concentration than in the slow-freezing group [5]. Recently, Pabon et al. compared the efficiency of vitrification versus slow freezing. They found that vitrification resulted in better motility recovery and higher mitochondrial activity compared to slow freezing [6]. Karthikeyan et al. conducted a comparison study using 20 severe oligoasthenozoospermia samples and found higher motility and vitality using vitrification than slow freezing [7]. Spis et al.’s study with one epididymal and one testicular sperm sample demonstrated that vitrification had higher mitochondrial membrane potential and motility in both samples than slow freezing [8]. A systematic meta-analysis review study conducted by Li et al. included a total of 2428 published articles and 13 randomized controlled trials containing 486 vitrified and 486 slow-freezing sperm samples [9]. They concluded that although the efficacy of vitrification varied by vitrification protocols and sample quality, the vitrification method was superior to slow freezing regarding the post-thaw total motility and progressive motility [9]. Taken together, human sperm vitrification has shown higher potential compared to slow freezing, although the vitrification procedures need further to be optimized and standardized for a more definitive conclusion.

	Slow freezing	Vitrification
Cryoprotectants	Glycerol (5–10%), TEST yolk buffer (12% glycerol), IUI -ready cryoprotectant (HEPES-buffered human tubal fluid with 1% human serum album, 4% sucrose, and 6% glycerol)	Glycerol (5–10%), some vitrification methods are free of permeable cryoprotectants
Standardized protocol	Yes	No
Cryopreservation device	Cryovial	Cryoloop, spermVD, cryotip, cryotube, cryogenic Vial 0.25 ml
Food and Drug Administration (FDA) approved	Yes	No
Validated for human sperm cryopreservation and storage	Yes	No
Current clinically applied populations	Male with normal sperm count (>15 million sperm per milliliter)	Male with severe oligospermia or azoospermia who have undergone a TESE/micro-TESE procedure which has yielded a small number of spermatozoa.

Table 1.

Comparison of slow freezing vs. vitrification of human spermatozoa.

2.2 Cryoprotectants and methods of human sperm vitrification/warming

A recent review, including all the previous methods, techniques, and devices for vitrification of human spermatozoa, concluded that the universal method/platform has yet to be developed [3]. Novel solutions specially designed to vitrify a small number of spermatozoa needed to be further explored [10]. Table 2 summarizes the various methods of sperm vitrification and warming which have been developed in the past decades.

Vitrification		Warming	Reference
Device	Cyrotube 0.85 ml	Warmed at room temperature 30–45 min.	[11]
Medium	Glycerol
Procedure	Room temperature 10 min, plunged into liquid nitrogen
Device	Cryogenic Vial 0.25 ml	Warmed in 42°C water bath 1 min, then 37°C water bath till melted.	[5]
Medium	Permeable cryoprotectant free
Procedure	Room temperature 1 min, plunged into liquid nitrogen
Device	1.5 ml straw	Warmed in 42°C medium 20 seconds.	[12]
Medium	Permeable cryoprotectant free
Procedure	Room temperature 10 min, loaded 100 μl in a straw, plunged into liquid nitrogen
Device	Collector-grid 5–10 μl drop	Warmed in 44°C medium 3 min.	[6]
Medium	Permeable cryoprotectant free.
Procedure	Room temperature, plunged into liquid nitrogen
Device	Cryovial.	Quickly submerging the sample in 5 ml G-IVF Plus medium prewarmed to 37°C with gentle agitation. After incubation at 37°C for 5 min, the post-thaw sperm suspension was centrifuged at 300 g for 5 min and resuspended in 100 μl G-IVF Plus medium.	[13]
Medium	Trehalose (0.5 mol l − 1), glycine (100 mmol l − 1) and human serum albumin (1% [w/v])
Procedure	The vitrification medium was slowly added to human semen sample at a 1:1 dilution, and the resultant suspension was incubated at 25°C for 5 min. Approximately 25 μl of aliquots of the sperm suspension were dropped directly into medical-grade liquid nitrogen free from contaminants which resulting in the immediate formation of a 25 μl floating sphere that solidifies and sinks after about 25 seconds. This process was repeated to obtain a sufficient number of spheres (Figure 1). All the spheres were finally packed into a 1.8 ml cryovial and stored in liquid nitrogen.
Device	SpermVD	Warmed at room temperature, spermatozoa were located in the spermVD droplet, transferred to the collection droplet and used for ICSI.	[15]
Medium	Quinn’s Advantage™ Sperm-freezing medium
Procedure	Spermatozoa were transferred from the collection drop to a 0.8-1 μl droplet in the spermVD, and placed the spermVD inside a cryovial, plunged into liquid nitrogen (Figure 2).

Table 2.

Human spermatozoa vitrification and warming procedure.

Vitrification with highly concentrated, permeable cryoprotectants is not suitable for spermatozoa as spermatozoa are osmotically sensitive. Sperm vitrification media is isomolar between 300 and 396 mOsm/L., which can be maintained by non-permeable cryoprotectants or a combination. The most commonly used permeable cryoprotectants include DMSO, glycerol, glycol, and ethylene. Non-permeable cryoprotectants include albumins, dextran, and egg yolk. Isachenko et al. developed cryoprotectant-free vitrification with capillaries method showing higher motility and integrity rates of cytoplasmic and acrosome membranes, and less cryo-injury in human sperm vitrification [2]. They used 52 human swim-up prepared ejaculated samples for vitrification without any permeable cryoprotectants. Some studies reported successful sperm vitrification in straws and cryoloop droplets for small volumes [2]. Azipurua et al. compared a permeable cryoprotectant-free vitrification protocol to the slow freezing method using 18 normal sperm samples and found that improved recovery rates of good quality sperm and better maintenance of sperm quality were generated in the vitrification group compared to the slow-freezing group [16]. Moreover, they demonstrated that vitrification with the cryoprotectant free method produced a higher percentage of spermatozoa, better preservation of acrosomes, and lower DNA fragmentation compared to the slow freezing method [16]. Additionally, they used alpha-tubulin immunochemistry to identify the similar labeling pattern of the sperm skeleton in the tail as fresh sperm, but different from post-thaw sperm from the slow freezing [16]. However, Agha-Rahimi et al. found there was no major difference in post-thaw motility, DNA fragmentation, or hyaluronan-binding potential with or without cryoprotectant for normospermia samples [17].

Regarding nonpermeable cryoprotectants, sucrose (0.25 M final concentration) and serum dextran supplement in a final concentration of 0.1% are generally used for sperm vitrification base medium. Some modifications have been made to the composition of the vitrification medium, for instance, Butylhydroxytoluene (BHT), a synthetic analog of vitamin E, was used as an antioxidant to improve the cryoprotective effects on human spermatozoa [18]. After warming, higher progressive sperm motility, DNA integrity, and lower reactive oxygen species were observed in the vitrification medium supplemented with BHT. The proposed mechanism of BHT is increasing the fluidity of the cell membrane via incorporation into the membrane [18]. Another study found that post-warming sperm motility using 0.1 mol/L trehalose (69%) was higher than that of used sucrose (0.25 M, 58%) with healthy volunteer semen samples [14]. Similar results were found at 6 and 12 h post-thaw. Furthermore, 0.1 mol/L trehalose improved membrane integrity at 0 h post-thaw. No significant improvements were found at 6 and 12 h in terms of membrane integrity [14]. According to these results, the use of trehalose increases tolerance to hypertonic and hypotonic conditions, preventing cell lysis and death during the vitrification and warming process. Additionally, the study demonstrated that post-thaw spermatozoa maintained at room temperature better than maintained at 37°C up to 4 h in terms of viability and mitochondrial membrane potential [14]. Zhou, D et al. developed a modified vitrification method which has a relatively better recovery rate (65.8%) and improved preservation of several sperm quality parameters compared with slow freezing (Figure 1). They used trehalose (0.5 mol/L) glycine (100 mol/L) and human serum albumin (1% w/v) as cryoprotectants to vitrify 28 semen samples from healthy participants [13].

Figure 1.

Illustration of the vitrification process and device by Zhou, D et al. [14]. The vitrification medium was trehalose (0.5 mol/L), glycine (100 mmol/L) and human serum albumin (1% [w/v]). The vitrification medium was added to the semen sample slowly at 1:1 ratio, and was incubated at 25°C for 5 minutes. Then approximately 25 μl of the semen suspension were dropped directly into medical-grade liquid nitrogen-free from contaminants. This process was repeated to obtain a sufficient number of spheres. All the spheres were loaded into a 1.8 ml cryovial and stored in liquid nitrogen for at least 48 h [14].

Collectively, some might perform better than others depending on specific situations/concentrations in terms of cryoprotectants. Further research is needed to investigate the optimal permeable and non-permeable cryoprotectants.

The vitrification process is based on an extremely high cooling rate that prevents ice crystal formation. A semen sample can be processed by swim-up and loading in straws or cryoloops, and then rapidly cooled by direct contact with liquid nitrogen (−196°C). After loading, the straws or cryoloops are put into the precooked aluminum blocks for long storage in liquid nitrogen or vapor phase in a liquid nitrogen tank (−180°C). Zhou, D et al. tested different combinations of carriers, including cryoleaf (Medicult, Jyllinge, Denmark), cryoloop (Hampton Research, Orange, CA, USA), and straw (Cryo Bio System, Paris, France). The highest freeze rate (about 10,000°C per min) is achieved by the cryoleaf and cryoloop system, however, they also exhibit the lowest freezing efficiencies (Figure 1) [13]. Berkovitz et al. tested a novel vitrification device spermVD and found it is an efficient and simple carrier method for freezing a small number of spermatozoa in low-volume droplets that significantly reduces post-thaw search time from hours to minutes, allowing a 96% recovery rate and leading to successful use of sperm for fertilization (Figure 2) [15]. The target populations are patients with a small number of spermatozoa, such as azoospermia patients, who have undergone a TESE/micro TESE procedure or severe oligozoospermia patients.

Figure 2.

Sperm VD: An innovative and efficient medical device for cryopreservation of small numbers of spermatozoa [15]. Vitrification: 1. Place drops of cryoprotectant on the spermVD, then put the spermVD into the culture dish with sperm. 2. Identify sperm and transfer the sperm cells onto the spermVD. 3. Place the spermVD into a cryovial. 4. Plunge the cryovial with the spermVD into liquid nitrogen. Warming: Take the spermVD out of the liquid nitrogen and transfer the cells from the spermVD to sperm washing medium at room temperature [15].

Besides the high-speed freezing, the warming speed should also be high allowing the water inside spermatozoa to pass from a glassy state to liquid without ice crystal formation. Mansilla et al. tested different warming temperatures and found the progressive motility in sperm samples warmed at 42°C (65%) was higher than at 38°C (26%) and 40°C (57%) and plasma membrane function was better preserved at 42°C [19]. Pabon et al. warmed vitrified spermatozoa micro pills (5-10 μl each) in 500 μl prewarmed medium and maintained them at 44°C for 5 seconds and decent post-thaw motility and mitochondrial activity were observed [6]. Zhou D et al. warmed the sample by submerging the spheres in 5 ml G-IVF Plus medium pre-warmed to 37°C accompanied by gentle agitation for 5 min. Post-thaw sperm achieves a statistically significantly higher recovery rate, motility, morphology, and curve line velocity than slow freezing (p < 0.05) [13]. Furthermore, a lower rate of DNA fragmentation and better acrosome protection were observed in the spermatozoa after vitrification than slow freezing (p < 0.05) [13]. Schulz et al. found that 42°C was the optimal temperature to preserve the sperm parameters, including motility and membrane integrity in the warming process [1]. Collectively, the warming process is flexible in terms of temperature.

2.3 Storage of the vitrified sperm samples

Cryopreservation of spermatozoa is the standard of care for fertility preservation in patients who undergo chemotherapy or radiotherapy. There are other reasons why a couple or individual patient needs to cryopreserve the spermatozoa, for instance, before vasectomy or in the case of a traveling husband. The conventional cryopreservation (slow freezing) protocol is standardized and widely used in clinical practice. The spermatozoa being vitrified with non-permeable cryoprotectants reduce the possibility of water inside the cell, allowing storage at lower temperatures. This technique is limited as only small volumes with small numbers of spermatozoa can be cryopreserved. The possibility that vitrified sperm preserve their function at temperature of −80°C could simplify storage, optimizing the space and time as well as the operator’s safety [3]. Lyophilization of spermatozoa is another method that requires additional investigation and validation [20]. Since the lyophilized spermatozoa are immobile, they can only be used in intracytoplasmic sperm injection (ICSI). Therefore, further research needs to be conducted on the optimization, safety, and health of the offspring.

2.4 Contamination risk and control using sperm vitrification

Exposing the semen samples directly to liquid nitrogen increases the risk of contamination. A large variety of bacterial, viral, and fungal species have been found in liquid nitrogen [21]. Unsterilized commercial liquid nitrogen could cause transmission and propagation of diseases. On the other hand, the survival of cryogenic pathogens in liquid nitrogen creates the possibility of cross-contamination between stored contaminated semen samples and liquid nitrogen. Bacteria have a higher tolerance than fungi to freezing. Piasecka–Serafin reported bacteria contamination from infected semen samples to sterile liquid nitrogen and then other sterile semen pellets [22]. Within only 2 h of cryo-storage, about 94% of the sterile samples were contaminated with E. Coli and S. Aureus [22]. Molina et al. compared the contamination risk of bacteria and fungi using open versus closed vitrification devices with human oocytes and embryos [21]. They found the bacteria cross-contamination risk was no greater for open devices than for closed ones in vitrification [21]. But there were Acinetobacter lwoffii, Alcaligenes faecalis ssp. Facecalis, and Sphingomonas Pauli mobilis at the bottom of the storage container. No fungi were observed. The source of these pathogens could be from the cryopreservation environment [21].

The basic procedure to avoid contamination is to store contaminated or infected semen samples separately in quarantine to minimize the risk of cross-contamination. Sterilizing the air used to create a small amount of liquid nitrogen by using the air filter with 0.22 μm polytetrafluoroethylene efficiently retained Brevundimonas diminutive with extreme temperature, high pressures, and high flow rates. Another way is using a 0.22 um filter equipped inside the canister to produce sterile liquid nitrogen at a similar temperature so that the samples sealed in the canister are only exposed to sterile liquid nitrogen air [23].

Another basic rule to control contamination is to avoid direct contact with liquid nitrogen. Using liquid nitrogen vapor, instead of liquid nitrogen itself, to store human semen samples can lower the risk of cross-contamination. Ultraviolet (UV) light could also be a possible way to reduce the contamination risk of vitrified sperm samples [24]. Treating a small volume of liquid nitrogen with 8000 us/cm² UV light could kill the hepatitis B virus, while 330,000 us/cm² destroyed the fungus Aspergillus niger [24]. Most viruses can be deactivated by UV light at a dose of 200,000 uw/cm². But it has been reported that the Zika virus may have higher resistance to UV light [25]. The UV light can also possibly induce genetic aberrations in stored spermatozoa. A simple solution is to sterilize liquid nitrogen with UV light before sperm storage. Another concern is the generation of ozone by UV light, which could cause damage to the buffer system in which the sperm samples are stored. However, the formation of ozone from UV light is insignificant as the liquid quid is free from oxygen.

Although there is no way to completely eliminate all the potential risks of cross-contamination in sperm vitrification, it is possible to control the contamination risk of vitrification to the level of slow freezing.

3. Conclusions

In the past decades, slow freezing of human sperm is still the main method used for sperm cryopreservation. However, vitrification provides a simpler, faster, more cost-effective alternative to conventional methods. Major concerns of the vitrification are the size of the sample and cross contamination with open devices. Optimization of the vitrification medium, sample size, and devices are a promising option. Due to different characteristics of spermatozoa species, including normospermia, oligospermia, azoospermia, testicular sperm aspiration, and testicular sperm extraction samples with different parameters, there is no universal vitrification method to serve different cryopreservation purposes at human clinics. Specific sperm vitrification methods should be individually designed to reach the optimal results depending on the personalized purpose at clinics. Future research in human sperm vitrification should include validation of the vitrification methods and whether the vitrification of sperm can improve clinical ART outcomes.

Acknowledgments

I am grateful to Mr. John Dong’s help with proofreading and grammatical corrections.

Conflict of interest

The authors declare no conflict of interest.

Acronyms and abbreviations

ART

assisted reproductive technology

DMSO

dimethyl sulfoxide

ICSI

intracytoplasmic sperm injection

UV

ultraviolet

BHT

butylhydroxytoluene

FDA

food and drug administration