Authors: María Caballero Sastre & Raquel Pillado González
“Currently, performing any cryopreservation technique results in some degree of damage to the sperm.“
THE HISTORY OF SPERM CRYOPRESERVATION
Human semen cryopreservation has a long history that begins at the end of the nineteenth century. After prior observations on sperm surviving cooling at very low temperatures (-150º C) (2), Mantegazza (1866) first suggested the idea of human sperm banks (3).
Years later, Mantegazza proved it was possible to extend human sperm lifespan up to four days by cooling at moderate sub-zero temperatures (-17º C) (4). Although a few extra days of storability does not make much difference in practical terms, this was the starting point of further research aimed to develop techniques that would allow for longer storage periods.
The next leap forward in this field was the discovery of the cryoprotectant properties of glycerol in 1949. This molecule proved to be an effective cryoprotectant agent (CPA) when combined with bovine sperm, and allowed for the development of new cryopreservation methods through which sperm could preserve their motility and fertilization capabilities even after the freezing-thawing process (5). However, during the following years this new technique was primarily applied to cryopreservation of farm animal semen rather than human semen (6).
In 1953, at the University of Iowa, the first case of live birth from cryopreserved sperm was reported (7). This successful birth, along with reports of high survival percentage (67%) of human sperm after cryopreservation, popularized the utilization of glycerol with human semen. The most common method to use glycerol was the protocol described by Bunge and colleagues, which stated the processing of sperm in a 10%-glycerol solution before freezing it with dry ice (7).
A decade later, the use of liquid nitrogen was introduced for long-term sperm cryopreservation. This new method led to the progressive normalization of sperm freezing as a widespread practice in healthcare (8). With the availability of long-term storage and the extended use of sperm freezing, new methods and variants were developed over time, such as slow freezing, fast freezing, LN2 vapours or lyophilization, which will be later described.
Nowadays, cryopreservation is routinely used in most assisted reproduction centres for numerous reasons:
It is important to possess a clear understanding of the semen cryopreservation process due to its current importance in clinical and research environments. Modern assisted reproduction practices are unthinkable without this tool.
Before being frozen, a sperm sample needs to be appropriately processed in order to separate sperm cells from the seminal plasma. This helps increase the concentration of high quality spermatozoa for a later use. Different techniques for sperm selection have been reviewed in our previous post.
The process of cryopreservation may involve irreversible cellular damage due to a change in the osmotic balance (11). Upon freezing of the extracellular water, the solute concentration increases in this fraction. As a result, the intracellular water is transported out of the cell to restore the osmotic balance, which may lead to cell dehydration and irreversible membrane damage (12, 13). Consequently, CPAs need to be incorporated along the sperm sample following processing. These molecules will protect spermatozoa by reducing intracellular ice formation and by decreasing the freezing point of the medium and the solute concentration present in the extracellular environment (14). There are two kinds of cryoprotective agents: permeable and non-permeable. The permeable ones, such as glycerol, dimethyl sulfoxide or ethylene glycol, protect the intracellular structures and biomolecules. Non-permeable agents, like sucrose, polysaccharides and some proteins, contribute to keeping the osmotic equilibrium, thus preventing cellular dehydration (11).
- Cryopreservation in liquid nitrogen (LN2)
This is the most commonly used method to cryopreserve sperm. It can be subdivided into three methods:
- Cryopreservation in microdroplets
Microdroplets are sometimes used in the clinic to cryopreserve sperm in small volumes. This is preferred in cases such as epididymal sperm aspiration. Around 50-100 μL of the sperm-CPA solution are placed on a dry ice plate up to freezing (Fig. 2); droplets formed in such a way are then kept in vials and plunged into LN2 (18).
This technique consists of cooling the sample at ultrarapid rates, so that the water solidifies (vitrifies) as a glass-like structure rather than forming ice crystals (19). However, even though vitrification should cause minimal damage, this is not always the case in clinical practice. One of the most frequent problems is the requirement for very high CPA concentrations that sperm do not tolerate well (20). Despite this, some studies have demonstrated it is possible to perform vitrification without using CPAs (20-22). Also, the large volume of sperm typically used impedes the cooling of the sample at the appropriate speed, causing ice formation (4). Despite these limitations, a vast proportion of clinics use vitrification as a routine practice due to its practical advantages.
In recent years, a new variant of this technique has been developed. This evolved version is called Solid Surface Vitrification (SSV) (23, 24). For this procedure, the sample or tissue is directly exposed to a metal surface previously precooled at -160º C before the use of LN2 (Fig. 1). This method prevents the apparition of nitrogen bubbles and evaporation that would slow the cooling rate (24). This technique has been previously applied to animal mature oocytes and human gametes and embryos, yielding successful results (23, 24).
OUTCOME COMPARATIVE BETWEEN TECHNIQUES
Because every cryopreservation technique shows different advantages and disadvantages, it is important to bear in mind how sperm will be affected during the thawing process. Parameters such as motility, viability, morphology and DNA integrity are evaluated accordingly.
Regarding LN2 techniques, no studies so far have directly compared post-thaw sperm quality following slow and rapid freezing. The literature shows agreement between studies on the main problem in both processes, which is controlling cooling rates (reviewed in 16). If the cooling rate is too fast, ice crystals may be formed inside the cells. By contrast, if it is too slow, the result could be cell contraction due to osmotic stress (25). This issue can be faced by using an automated programmable freezer, but only when keeping a large number of samples (26).
Even though several reports have compared fast and slow freezing in animal reproduction, conclusions are controversial. Some studies in semen from horse (27) and buffalo (28) have reported better results when using fast thawing, whereas other authors have found no difference between fast and slow thawing rates (29). Nevertheless, it seems clear that the critical point lays on thawing matching the freezing process. Considering rapid freezing, thawing is recommended to be also carried out at a fast rate to avoid formation of intracellular ice crystals. Likewise, for the slow-rate cooling procedure, the sample needs a slow thawing protocol, since cells need more time to rehydrate (30).
On the other hand, all studies on the use of LN2 show similar or slightly better results regarding the aforementioned parameters when using nitrogen vapours (31-33). These results, however, are obtained after short-term storage of samples in nitrogen vapours of up to three months. When stored for longer, sperm quality decreases. Consequently, this method is only recommended for short-term storage (further research would be needed in order to support its application for long-term storage) (34).
Upon comparison between vitrification and LN2 techniques, different results can be highlighted. Certain authors determined that results of sperm parameters such as motility, viability and normal morphology were similar between vitrification and rapid freezing techniques (21). On the contrary, different results were found for DNA fragmentation rates. Whilst some groups found that DNA fragmentation was significantly higher for the rapid freezing technique (12, 21) or for LN2 vapours (24), other groups obtained contradictory or uncertain results (35, 36). For instance, DNA fragmentation has been observed to increase over time when analyzing semen 6h after thawing, compared to recently-thawed samples (35).
Despite the different results obtained, vitrification shows important advantages compared to other available techniques. Some of these advantages are: 1) unnecessary use of CPAs; 2) the technique is simpler and faster compared to conventional slow freezing, due to the fact that once the sample is kept in a proper container (such as cryoloop or straws), it is rapidly plunging into LN2 to be stored; 3) no requirement for programmable freezers; 4) the sample is free of seminal plasma and potential pathogens (vitrification is usually performed after swim-up); 5) no requirement for post-thaw processing (reviewed in 6).
TRENDS IN IMPROVING SEMEN CRYOPRESERVATION
Currently, performing any cryopreservation technique results in some degree of damage to the sperm (4). The severity may differ depending on the initial quality of the sample, being greater in poor quality semen. Luckily, the application of ICSI allows for the successful use of low quality sperm (if necessary) even after having been cryopreserved. Side effects of cryopreservation on sperm include reduced motility, vitality, viability and increased DNA damage. Although motility is the most affected parameter, DNA damage entails greater detrimental effects regarding embryo viability (6).
The majority of the harm produced by cryopreservation occurs during the freezing and thawing phases, the crucial moments being between -15º C and -60º C. It is worth mentioning that a considerable damage is produced by CPAs themselves; these agents cause oxidative stress that derives in the formation of reactive oxygen species (ROS) (8, 10, 37). Furthermore, these components affect the polyunsaturated fatty acids in plasmatic membranes due to lipid peroxidation (10). Consequently, phospholipids reservoirs such as egg yolk are usually added to the freezing media. As a more direct countermeasure, several current research lines look into numerous antioxidant components in order to be added to freezing media, thus avoiding the damage caused by ROS. Examples of these antioxidants are TAT-peroxiredoxin-2 fusion protein, quercetin or melatonin. Preliminary research indicates that their presence results in higher motility and viability rates post-thawing, along with a reduction of intracellular ROS levels (8, 10). Other approaches consider the utilization of protocols that may directly disregard the use of CPAs, such as certain vitrification protocols previously mentioned.
Sperm freezing entails other associated problems, too, such as the loss of chromatin and acrosome integrity that had been observed post-thaw. Recent data have reported differences in the levels of DNA and acrosome integrity after cryopreservation depending on the freezing technique used (24). The difference in DNA integrity levels is suspected to be due to the cold shock faced by the samples (24). This issue could be amended by the development of media able to preserve sperm without freezing, an avenue that is currently being pursued. For instance, Riel and colleagues have reported that the use of an electrolyte-free medium for short-time (1 week) storage of semen yields better levels of DNA integrity in comparison to traditional cryopreservation. If the storage period capacity could be further improved, this might become a rather attractive alternative (38).
Lyophilization or freeze drying is an experimental technique that has been proven less harmful to the DNA (4, 10, 38). In order to perform this method, the sample must be cooled below the triple point of water (Fig. 3). At this temperature solid water (ice) sublimates when the pressure is decreased and exits the cell, leaving it fully dehydrated (4, 10). However, this process irreversibly damages the sperm membrane, thus resulting in non-motile or even non-viable (dead) sperm. Nevertheless, studies on mouse sperm have shown that lyophilized spermatozoa can be used for fertilization with the assistance of ICSI (39). Although the first attempts to use lyophilization on human sperm were in the 50s, today there is still a lacking protocol for this technique that is able to preserve both sperm motility and viability (4).
Figure 3. Water phase diagram showing the relation of the conditions of temperature and pressure for freeze-drying (not in scale). Samples are frozen by reducing the temperature (A to B) and then the pressure is also reduced by aspiration (creating vacuum) so the sample lies below the ‘triple’ point (C) for both temperature and pressure (this is the point where all states co-exist). From here on the sample is subjected to a controlled increase of temperature or to a further decrease of pressure to sublimate ice (for detailed current sperm freeze-drying protocols, see (40) and (41)). Modified from (41).
The main advantages of freeze drying are: the possibility to preserve spermatozoa with high DNA integrity for at least a year and a half (39), the inactivation of viruses that may be present and the fact that liquid nitrogen is not required. Additionally, samples can be stored at 4º C and transported at room temperature (4). To date, this method still remains experimental regarding humans, due to the lack of actual data on the matter (4, 10).
Cryopreservation has gone a long way. Its use in reproductive medicine got to revolutionize the horizon for infertile couples. New doors opened decades ago, and it is fair to reason new ones will open in the near future. Egg donation, social freezing, embryo cryopreservation. Times and timing have changed for patients, and clinics and reproduction centres faced the need for evolution in order to cope with rising approaches.
In spite of the variety of options for semen cryopreservation, all of them present their own limitations. Continuous research allows for the discovery of new ways to correct these flaws; however, there lies a long path ahead, and further studies will be required before any improvement can be incorporated to routine practice.
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Authors: Shuyana Deba, Isabel Sánchez and Sara Sanz
Every procedure carried out in an IVF laboratory, from ovarian puncture and semen capacitation to the embryo transfer, must be performed under specific safety conditions. These standards must be followed to avoid a decrease in gamete/embryo viability (2).
From the in vitro culture, gametes and embryos are exposed to diverse artificial situations that do not take place in nature. In vivo, both fertilization and embryo development in the female reproductive tract occur in the complete absence of light. In this environment, other conditions include oxygen (O2) concentration of 2-8%, pH 7.2-7.4, 37⁰C and gradients of diverse nutrients to which the embryo is exposed (3). Changes in temperature, CO2 and O2 pressure, light exposure or volatile organic compounds may adversely affect embryo quality.
Also, manipulation of embryos by embryologists is as important as air quality and culture conditions. Each human being is covered by about 1012 bacteria (4), which could contaminate embryo cultures if embryologists do not use the necessary clothing, such as laboratory cap, footwear and mask.
Therefore, a daily quality control at different levels should be carried out to obtain good results in IVF cycles. We are going to analyze the effect of some of the elements that can affect germ cells and embryos.
In vivo, mammalian germ cells and embryos are not exposed to light, which might explain why they do not exhibit any protection mechanism against this factor (5,6). In addition, sperm do not have the capacity to repair DNA, unlike oocytes and embryos, which do present some mechanisms for DNA repair (7).
Light variables to be considered are intensity, duration and wavelength. It seems clear that photooxidation increases along with light intensity and duration. However, what type of wavelength would be the most harmful for embryos and germ cells? Energy increases when wavelength is shorter (8). Accordingly, artificial cool white fluorescent light has been demonstrated as the most stressful in mouse embryos. Incandescent light, in turn, seems to be less harmful, and the best outcomes are achieved when warm white fluorescent light is used (7).
How can light affect the quality of these cells?
Indirect effect: Culture and oil photooxidation can affect embryo development (8). In this case, modified components will damage the lipid membranes. Also, if HEPES- or riboflavin-containing media is exposed to light, it results in the formation of hydrogen peroxide, a highly cytotoxic substance (9). Additionally, light can heat up both the plasticware and the oil, resulting in more toxic and damaging components (8,9).
Direct effect: Light can potentially compromise the quality of gametes and embryos, by activating stress-related genes or by ionisation, which may also damage the DNA. This phenomenon would cause DNA fragmentation and mutation, as well as an increase in the apoptotic index and change in the number of mitochondria levels (10).
How can we avoid this effect? (7)
1) Reducing the exposure time.
2) Using warm white fluorescent light in the lab and green filters on microscopes.
3) Adding antioxidants in the media in order to mitigate damages from ROS.
4) Avoiding riboflavin, which is responsible of the phototoxicity in the media.
VOLATILE PARTICLES EFFECT
Since the 1990s, IVF laboratory indoor air quality has taken a high relevance. Thence, focused on creating an optimal environment, laboratories have become clean rooms where filtration of particles is performed by using high-efficiency particle arresting (HEPA) filters, and successful chemical air filtration is achieved by removing volatile organic compounds (VOCs) with solid-phase filtration (e.g., potassium permanganate-impregnated, carbon filters) (11).
Focusing on VOCs (hydrocarbon-based compounds that are emitted by industries, cleaning products, computers, and microscopes among others), several studies have demonstrated their harmful effect on embryos, initially reported by Boone laboratory on mouse embryo development (11). Moreover, VOCs have been shown to increase DNA fragmentation in human sperm, and they can also have detrimental effects on pregnancy rates (12).
Recently, a retrospective study by Munch et al. concluded that, without solid carbon filtration, fertilization, cleavage, and blastocyst conversion rates declined in fresh IVF cycles. Even more, results were found to be even worse in ICSI cycles, probably due to the lack of protective barrier provided by the cumulus cells (13). However, the authors did not observe the same results when embryos had been cryopreserved in an environment with carbon solid filtration but thawed in a laboratory deprived of such systems. The absence of significant changes in cleavage and blastocyst conversion rates, as well as in the proportion of good quality blastocyst developed after thawing suggests that embryos are affected in the peri-fertilization period (13).
Also, products like cosmetics emit VOCs, especially perfumes, colognes, and deodorants. They are highly toxic to embryo development in vitro, primarily due to evaporation of their solvent bases (14,15). After analyzing the results of studies determining the toxicity of VOCs, ideal levels should be below 0.2 ppm but preferably zero (12). Personnel must understand the principles of air quality control, including the function of airflows and airlocks, hygiene, dress code and the use of cleaning agents (16).
pH level depends on bicarbonate concentration of culture media and the CO2 concentration of the incubator. However, other factors like altitude and composition of culture media could affect the pH level, too (17,18). Embryos are able to develop over a range of media pH, considering that they possess an intracellular mechanism to regulate its internal level (17,18). However, it is important to control pH variations because they affect development (17). To control pH level outside the incubator some culture media contain buffers like HEPES or MOPS, but long exposure of embryos to these buffers is not recommended (17). Thawed denuded oocytes and embryos are specially sensitive to pH variations because they do not have an inner system to regulate pH (17). So, an increase in the pH of the medium can affect the physiology and development of oocytes and embryos. Thus, acidification of the medium can even affect the fetal weight and size (18).
As previously mentioned, CO2 is necessary to control pH level of culture media (17,18). The importance of CO2 was demonstrated in 1985, in a study carried out on hamsters (19). The authors cultured hamster embryos in different CO2 concentrations (5% and 10%). They found a higher rate of blastocysts in those cultured at 10% compared to 5%, which demonstrated differences in embryonic development. This results showed that CO2 level is an important factor for embryo culture (19). The capacity of CO2 to get through cell membranes allows for regulation of the inner pH levels in blastomeres. In other studies, it has been shown that the required CO2 concentration to achieve the optimal pH varies in different species. For instance, the required CO2 level in rats is 7.5%, whereas for humans it is 6.5% (19).
Some studies have compared different values of O2 concentration in the incubator and they show that a low level (5-6%) improves results when compared to an ambient level (21%). It has been shown that low O2 levels increase implantation, pregnancy and live birth rates (17,20). It seems that a low O2 level reduces ROS in the culture and the presence of volatile particles in the air, although the exact mechanism of action is still unknown (17).
Standard temperature generally used in IVF laboratories is 37⁰C (17,18). However, optimal temperature is unknown because in the female reproductive tract it could be slightly lower, about 36⁰C. On average, temperature of the Fallopian tube is about 1.5⁰C less, whereas the follicular liquid temperature can reach 2-3⁰C lower than core body temperature (17). It is important to control and prevent temperature variations because it can affect meiotic spindle stability and alter embryonic metabolism. It has been shown that an increase of 2⁰C during 20 minutes potentially alters the integrity of the meiotic spindle, which cannot be completely repaired when temperature is set back to 37⁰C. As a consequence of this increase in temperature, embryos express some stress-response genes that compromise development (18). Interestingly, a small decrease in temperature does not have any effect on oocytes, whereas a large difference can be severely harmful for the meiotic spindle (18).
CULTURE MEDIA EFFECT
Nowadays, there exist two kinds of culture media: one-step media and sequential media (with different compositions for days 0-3 and 3-6) (17,21). All culture media are similar in composition; they contain energy substrates like glucose, pyruvate or lactate, and both organic and inorganic salts, which must be balanced accordingly. Culture media also contain amino acids in different proportions. The exact composition of amino acids in culture media is unknown. One of the most important problems related to the presence of amino acids is the ammonium generated as a product of metabolism. Ammonium has negative effects on embryo and fetal development. To avoid this problem, some culture media contain glutamine, which reduces ammonium production (17,21,22). Also, culture media can be supplemented with macromolecules and other components like HSA, α and β globulins, growth factors, vitamins, lipids, nucleotides, cytokines and hormones (17,22).
What can we conclude?
There are many parameters that should be kept in mind in order to maintain the optimal conditions for both gamete and embryo development in an IVF laboratory. In vitro, cells and embryos are exposed to different stress situations that must be minimized. Therefore, a routine control at different levels needs to be performed, so that the environment in the laboratory is adapted to resemble the reproductive tract and the intrauterine conditions.
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