Fig. 15.1
Mechanism of DNA injury during cryopreservation
Apoptosis markers (caspase activation, DNA damage and phosphatidylserine externalisation) have been linked to male infertility in numerous studies. Spermatozoa positive for active caspase-3 demonstrate phosphatidylserine externalisation and DNA fragmentation more frequently than normal controls. Moreover, low-motility spermatozoa demonstrate a higher level of apoptosis markers as compared to high-motility spermatozoa [68]. Various studies have shown that these apoptosis markers tend to increase in spermatozoa following the freeze/thaw process. It has been proven that normozoospermic semen samples are more resistant to the damage induced by freezing and thawing as compared to oligozoospermic samples. Verza et al. reported that motile sperm could be recovered even after five freeze/thaw cycles in normozoospermic men, while motility could be salvaged after only two freeze/thaw cycles in oligozoospermic samples [69]. The extent of damage is correlated to the degree of oligoasthenoteratozoospermia. Moreover, cryopreserved spermatozoa from cancer patients were also found to be having a higher level of DNA fragmentation as compared to healthy controls.
Though literature suggests that a correlation exists between the presence of activated caspases and sperm DNA fragmentation , there are reports which have found no conclusive evidence for the same, and attribute the sperm DNA damage induced during cryopreservation to oxidative stress rather than apoptosis .
Cryopreservation and Mitochondrial Damage
Transmission electron microscopy (TEM) studies have shown an alteration in the ultrastructure of the mitochondria and plasma membranes, and have confirmed that mitochondrial destruction is secondary to widespread cellular destruction. Changes in mitochondrial membrane potential (M ΔΨ) are assessed using a fluorescent cationic dye, 5,5′, 6,6′ –tetrachloro-1-1′, 3, 3′-tetraethylbenzamidazolocarbocyanin iodide (commonly known as JC-1). Uncoupled mitochondria are suggestive of unhealthy spermatozoa, and hence determination of mitochondrial membrane potential is useful to assess post-thaw sperm survival [70]. In intact mitochondria, M ΔΨ is unaltered and the JC-1 dye aggregates inside the non-damaged mitochondria and fluoresces red. In damaged mitochondria, the M ΔΨ is broken down and the JC-1 dye disperses through the entire cell and fluoresces green.
Cryopreservation and Sperm Motility, Vitality and Morphology
Following freeze/thaw cycles , significant decrease in post-thaw motility and an increase in immotile sperm fraction have been documented. There is a significant decrease in the rapidly progressive fraction (29% pre-freeze vs. 12% post-thaw; p < 0.05) and an increase in immotile fraction (24% pre-freeze vs. 64% post-thaw; p < 0.01). There is a strong correlation between the deterioration in motility post-thaw and vitality (r = −0.848, p < 0.01) [70].
Light microscopic examination has revealed an increase in the percentage of spermatozoa with midpiece detachment and coiled tails (p < 0.05). TEM evaluation following post-thaw showed various ultrastructural abnormalities including decomposition of the plasmalemma, outer acrosomal membrane, acrosomal content, early acrosomal reaction, chromatin condensation anomalies, and diadem defects. There is a significant increase in acrosomal change defect and subacrosomal swelling. Acrosomal change defect is marked by unaltered equatorial segment but an affected apical acrosomal region. Various apical head alterations occurring in acrosomal change defect include lack of continuity, loss of acrosomal content and appearance of vesicles. Subacrosomal swelling, characterised by detachment of inner acrosomal membrane from the nuclear envelope, is another significant observation [70].
Sperm Cryopreservation and Fertility Outcome
Owing to the plethora of injuries incurred to the spermatozoa, the fertilisation potential of sperm is reduced and hence the pregnancy rates following intrauterine insemination and conventional in vitro fertilisation (IVF) are lower with cryopreserved sperm as compared to fresh sperm [71]. Hence, cryopreservation of sperm before intrauterine insemination or conventional IVF is not recommended. However, the results with testicular frozen spermatozoa when used for ICSI are comparable to fresh spermatozoa from the same subject [72]. No differences in fertilisation rate , cleavage rate, embryo quality, clinical pregnancy rate and ongoing pregnancy rates are noted with the use of cryopreserved testicular spermatozoa as compared to fresh sperm [73]. Even with the use of ejaculated spermatozoa, the fertilisation rates are comparable between fresh and frozen groups [74].
Conclusions
Optimisation of freeze/thaw protocols, cryoprotectant concentrations used and semen preparation techniques is warranted to ensure successful application of cryopreservation for semen preservation [75]. Since the spermatozoa are exposed to a variety of ultrastructural cryoinjuries that may not be discernible by light microscopy examination, different and novel cryopreservation methods (e.g. cryoprotectant-free vitrification) and sperm separation techniques (e.g. MACS—magnetic activated cell sorting) should be explored for different patient populations (normal donors and normozoospermic patients, infertile and oligozoospermic men and patients undergoing treatment for malignancy), since post-thaw survival of each specific group is different with the conventional protocols used [76]. Specific measures should be adopted to minimise the perturbations to the spermatozoa during freeze-thawing because of apoptosis and DNA damage.
Appendix 1: Ideal Sperm Freezing Protocol to Avoid Sperm Freezing Injuries
Freezing Steps
- 1.
Pre-warm the Sperm Freezing Medium for a minimum of 2 h at room temperature.
- 2.
After liquefaction, measure the total volume of the ejaculate and carry out semen analysis as required.
- 3.
Ensure that both semen sample and Sperm Freezing Medium are at room temperature and dilute the semen 1:1 (v/v) with the Sperm Freezing Medium. The medium should be added drop by drop onto the semen and the solution carefully mixed after each addition.
- 4.
The mixture is left at room temperature for a minimum of 10 min.
- 5.
Load the diluted semen into straws or cryo-tubes and seal.
- 6.
Suspend the straws horizontally for 30 min, just above the surface of the liquid nitrogen. Cryo-tubes should be attached to a cane and then suspended above the surface of the liquid nitrogen for the same period of time.
- 7.
Finally, transfer the straws or cryo-tubes into the liquid nitrogen and store at −196 °C.
Thawing
- 1.
Warm straws at room temperature for 5 min.
- 2.
Open the straws or cryo-tubes according to the manufacturer’s instructions and remove the thawed semen.
- 3.
Immediately prepare sperm by the density gradient or the swim-up procedure.
[Adapted from ORIGIO Protocol]
Appendix 2: How to Avoid Sperm Freezing Injuries
Serial No | Critical steps to avoid sperm injury | Causes and types of injury | Remarks |
|---|---|---|---|
1. | SFM is egg yolk-free and contains • Glycerol and sucrose as the cryoprotective agents • Glycine which improves post-thaw sperm motility, membrane and acrosome integrity • SSR with insulin shown to be a pro-surviving factor Thermal shock to the sperms may occur if both SFM and semen sample are at different temperatures at the time of procedure Sperms from infertile men have more incidence of disordered chromatin organisation and depict reduced resistance to thermal injury related denaturation as compared with spermatozoa from fertile individuals Freezing unprepared semen in seminal fluid seems to be more resistant to freezing injuries as compared to the frozen prepared sperm sample | Endeavour should be keeping them at room temperature before starting the procedure | |
2. | Using equal volumes of the cryoprotectant media and semen sample (Fig. 15.4) | Osmotic stress may occur to the semen sample during this procedure The phenomenon is marked by the increased coiling of the sperm tail and leading to loss of progressive motility | It is advised to mix them in equal volumes Follow the manufacturer’s guidelines |
3. | Adding the cryoprotectant to the semen sample (Fig. 15.5) | Osmotic stress if the cryoprotectant is added too rapidly to the semen sample. It is therefore important to allow for gradual osmotic adjustment by slowly mixing the Sperm Freezing medium with your sperm sample | Always add the cryoprotectant media drop by drop over a period of 10 minutes to the equal volume of the semen sample |
4. | Osmotic stress may occur if we don’t cool the sample gradually The intracellular damage that spermatozoa may encounter at rapid rates of cooling may be due to the formation of intracellular ice An osmotic imbalance occurs during rapid cooling rates due to a diffusion limited ice crystallisation in the extracellular fluid The amount of ice forming in the extracellular space in the suspension during the cooling is less than expected leading to cellular injuries during thawing | From room temperature move the sample to 4 °C over a period of 20–30 min so that there is no shock to the spermatazoa Then place the mixed sample in the vapour phase for final step of cooling for a minimum 20 min Ice crystals formation breaches the sperm membranes and affects the organelle function. This leads to impaired cell survival Very slow cooling rate regulates the efflux of water from the internal to the external milieu, thus increasing the concentration of solutes and the osmotic pressure inside the cell Such prolonged and gradual cooling leads to cellular volume changes related with the movement of water outside the cell, dehydration, and toxicity damage due to high solute concentration intracellularly | |
5. | Storage in the cryo can | Thermal shock to the semen sample may occur to sperms if the suspension is not stored properly at adequate depth in liquid nitrogen or vapour phase | Ensure that the semen sample is always placed at −196 °C for storage for prolonged periods |
6. | Thawing protocol (Fig. 15.9) | Thermal and osmotic shock Spermatozoa that have been cooled at high rates are subjected to an osmotic shock at low temperatures during thawing, leading to the observed cellular damage The phenomenon of recrystallization of both intracellular and extracellular ice, in frozen thawed semen sample, occurs as formation of small ice crystals with a rate of recrystallization that increases with increasing temperature during thawing or storage Such chilling injuries can alter the structure and integrity of plasma membranes composed of phospholipids and cholesterol. These can also alter mitochondrial membrane fluidity and lead to alteration in mitochondrial membrane potential and release of Reactive Oxygen Species | Always thaw quickly to avoid recrystallization of ice and subjecting sperms to stress and fracture injuries Warm straws/vials at room temperature for 5 minutes and prepare the sample |
7. | Osmotic stress injuries | Gradually add the sperm preparation media to the thawed suspension to avoid sudden osmotic stress injuries | |
8. | Speed of the centrifugation of the semen sample | Sperms are now just recovering from freeze-thaw cycle and are under stress Any unwanted centrifugational stress may lead to membrane injuries | Single wash for less duration at minimal speed |
9. | Timing of use of post-thaw semen sample | Frozen-thawed sperms exhibit different dynamics of DNA fragmentation compared with fresh samples with a more rapid increase in the percentage of DNA damaged spermatozoa | If cryopreservation is required, it is recommended that sperm be used in treatment as soon as possible after thawing |
Fig. 15.2
SFM is egg yolk-free media and contains glycerol and sucrose as the cryoprotective agents
Fig. 15.3
Liquefied semen sample kept at room temperature
Fig. 15.4
Semen sample and sperm freezing media kept at room temperature in equal volumes
Fig. 15.5
Add the cryoprotectant media drop by drop over a period of 10 minutes to the equal volume of the semen sample raw or unprepared
Fig. 15.6
Keeping the vials at room temperature for a period of 10–15 min
