The basic type of cryopreservation is used for cells and tissues in numerous IVF laboratories is vitrification/ultra-rapid freezing. Vitrification combines the use of concentrated cryoprotectant solutions with ultra-rapid cooling in order to avoid the formation of ice. The high osmotic pressure of the concentrated CPAs causes rapid dehydration, and the compressed samples are placed in a vitrification device, which can be immediately immersed in liquid nitrogen.
Samples reach very low temperatures (-196 °C) in a state that has the molecular structure of a viscous liquid without crystals, forming an amorphous glassy solid. The transition from liquid to glass phase is a kinetic phenomenon: increased viscosity delays thermodynamic intermolecular rearrangements and “locks in” a non-equilibrium thermodynamic state. The purpose of vitrification is to completely eliminate the formation of ice in the medium containing the sample at all stages of cooling, storage and heating.
Since rapid cooling is affected via direct contact with liquid nitrogen, programmable freezing machines are not needed. There is an inverse correlation between the cryoprotectant concentrations and cooling rates required, and successful vitrification is based on applying extremely high cooling rates in combination with very high concentrations of cryoprotectant or cryoprotectant mixtures. Cooling is too fast for ice to form or grow noticeably, and the concentration of solute remains constant during cooling.
A balance must be achieved between the lowest level of least hazardous CPA and maximal cooling rate: higher CPA concentrations allow lower cooling rates, and vice versa. Since highly concentrated CPAs can cause toxic and osmotic damage, the preferred strategy is to use the maximum possible cooling and heating rates, and then use the lowest concentration of CPA that will prevent ice formation,
Adequate dehydration and permeation of cells is essential, and therefore exposure time is important,
In order to avoid the formation of both intracellular and extracellular ice, the initial cooling rate must exceed the critical cooling rate (CCR) of the solution,
Warming rates are critical to survival: total development of ice is much more rapid during warming, and warming rates required to avoid significant de-vitrification are far higher than cooling rates required to achieve vitrification.
The cooling rate is affected by several parameters, and different methods have been employed in order to find an effective and practical solution, by varying cryoprotectant solutions, combinations, exposure times and temperature. The addition of sugars reduces the concentration of CPA required. The permeability of mixtures is higher than that of individual components, and different combinations of CPA have also been tried. Vitrification solutions are developed with the lowest possible concentration of CPA compatible with achieving glass formation.
Reducing volume to a minimum reduces potential toxicity and osmotic damage, and methods have been devised to reduce the volume of CPA down to between 0.1 μL and 2 μL. Reducing the drop size or increasing the number of embryos per drop risks diluting the CPA with medium carried over from the culture drop, and this could allow the sample to freeze, with lethal results.
The thawing rate must also be rapid, in order to prevent de-vitrification and ice crystal formation during the transition state. The samples are kept in air (room temperature) for 1–3 seconds. For open systems, the sample is then immersed in dilution medium at 37 °C, and for closed systems, the carrier is immersed in a 37 °C water bath before transferring the sample to the dilution medium. The CPA is diluted in several steps, in order to counterbalance osmotic effects as the CPA leaves the cell.
Although the methodology and protocols that accompany vitrification systems for IVF appear deceptively simple, the principles that ensure viability after warming are a highly complex combination of thermodynamics and cell and molecular biology. Numerous kinetic variables surrounding the physics of aqueous solutions and biological survival can jeopardize success.
Vitrification is now routinely used for human oocytes and embryos. However, questions remain about potential external contamination, as well as the long-term stability of the “glassy state” of the vitrified cells, which are prone to fracture. This may be a hazard under normal working conditions in the IVF laboratory with routine access to storage tanks.
Embryo Cryopreservation Policies
Following fresh embryo transfer in a stimulated IVF cycle, supernumerary embryos are available for cryopreservation in a large number of cycles. In normal IVF practice, more than half of the stimulated IVF cycles can produce excess embryos suitable for cryopreservation. In addition to enhancing the clinical benefits and cumulative conception rate possible for a couple following a single cycle of ovarian stimulation and IVF, a successful cryopreservation program offers other benefits including the possibility of avoiding fresh embryo transfer in stimulated cycles with a potential for ovarian hyperstimulation syndrome, or in which factors that may jeopardize implantation are apparent.
Extended culture to the blastocyst stage is now routine in many IVF laboratories worldwide, and slow-freezing protocols using glycerol as cryoprotectant have largely been replaced by the more successful technique of blastocyst vitrification.
Using strict criteria to select potentially viable blastocysts is crucial to success:
Growth rate: expanded blastocyst stage on Day 5/Day 6,
Overall cell number>60 cells (depending on day of development),
Relative cell allocation to trophectoderm/inner cell mass,
Original quality of early stage embryo: pronucleus formation and orientation, blastomere regularity, mono-nucleation, fragmentation, appropriate cleavage stage for time of development.
Blastocyst vitrification protocols now yield very favorable survival, implantation and clinical pregnancy rates. Commercial kits for blastocyst vitrification are available—as always, the ultimate success of the protocol will be related to the operator’s experience and careful attention to detail. In common with all aspects of human ART, careful research into the consequences of such new therapies continues to be essential. In large expanded blastocysts, collapsing the blastocoelic cavity with an ICSI needle immediately before processing increases survival rates after both slow freezing and vitrification. Poor morphology and delayed expansion (to Day 7) have a negative impact on survival post-vitrification.
Assisted Hatching and Cryopreservation
Freeze-thawing is known to cause hardening of the zona pellucida, and the application of assisted hatching, particularly at the blastocyst stage, has been suggested as beneficial to implantation after freeze-thawing. In some cases, zona pellucida fracture can be a routine result of some cryopreservation protocols. Embryos with existing holes in the zona pellucida following PGD procedures can successfully survive and implant. A recent systematic review confirmed that assisted hatching is consistently of benefit after thawing frozen/vitrified blastocysts.
Clinical Aspects of Frozen Embryo Transfer
Freeze-thawed embryos must be transferred to a uterus that is optimally receptive for implantation, in a post-ovulatory secretory phase. Patients with regular ovulatory cycles and an adequate luteal phase may have their embryos transferred in a natural cycle, monitored by ultrasound and blood or urine luteinizing hormone (LH) levels in order to pinpoint ovulation.
Older patients or those with irregular cycles may have their embryos transferred in an artificial cycle: hormone replacement therapy with exogenous steroids is administered after creating an artificial menopause by downregulation with a gonadotropin-releasing hormone (GnRH) agonist.
In the past, the options for preserving a young woman’s fertility after treatment for malignant disease were very limited: a full IVF treatment cycle with cryopreservation of embryos prior to the initiation of chemotherapy, or oocyte or embryo donation following recovery from the malignant disease. The first option is available only to women with partners to provide a semen sample for fertilization of the harvested oocytes. However, the success of frozen embryo cryopreservation in a competent IVF program is such that these patients maintain a very good chance of achieving a pregnancy after transfer of frozen-thawed embryos following recovery from their disease.
On the other hand, this strategy also raises the risk of creating embryos with a higher than average chance of being orphaned. Many of the legal and ethical problems created by the cryopreservation and storage of embryos can be overcome by preserving oocytes, especially for young women about to undergo treatment for malignant disease that will result in loss of ovarian function.
Oocyte cryopreservation is also indicated in patients with a known family history of premature ovarian failure, and can be advantageous in various clinical scenarios, such as in ovarian hyperstimulation syndrome, unexpected lack of sperm following oocyte retrieval, egg donation programs and in order to extend the duration of natural fertility.
Human oocytes are particularly susceptible to freeze—thaw damage due to their size and complexity. They must not only survive thawing, but also preserve their potential for fertilization and development.
Several intrinsic difficulties are associated with human oocyte freezing, due mainly to their high volume: surface area ratio and low membrane permeability. Intracellular ice formation causes critical damage to the cytoskeleton, which is also sensitive to osmotic stress. Disruption of the meiotic spindle can cause chromosome defects and aneuploidy. Lowering the temperature, or the cryoprotectant agents themselves, may cause an increase in intracellular Ca2+ leading to changes in the intracellular signaling mechanisms and oocyte activation. Finally, since the zona pellucida hardens after freezing, it is necessary to employ ICSI for fertilization of the thawed oocyte.
Freezing can result in parthenogenetic activation, leading to premature release of cortical granules (CGs). It is also important to consider the cytoplasmic maturity of the oocyte at freezing and the potentially toxic effects of cryo-protectants.
Modifications found to improve the effectiveness of oocyte freezing include:
Complete removal of the cumulus and coronal mass, which increases survival rates.
Alteration of sucrose concentrations from 0.1 to 0.2, 0.3 or 0.5 mol/L, which increases oocyte dehydration and survival.
Choline has been used as a substitute for sodium on the basis that cryo-damage to the Na+/K+ pump might lead to high intracellular concentrations of Na+ with a resulting efflux of protons. Choline does not cross the plasma membrane, is less toxic than high sucrose and does not affect osmotic pressure of the cell.
Cryopreserved semen has long been successfully used for artificial insemination (AI), intrauterine insemination (IUI) and IVF. Although freeze—thawing does produce damage to the cells with loss of up to 50% of pre-freeze motility, since large numbers of cells are available, successful fertilization can be achieved even with low cryo-survival rates.
There is, however, a noticeable difference in sperm cryo-survival rates between normal semen and semen with abnormal parameters such as low count and motility. Samples from men who require sperm cryopreservation prior to chemotherapy treatment for malignant disease frequently show very poor cryo-survival rates. The routine introduction of ICSI into IVF practice has surmounted this problem, so that successful fertilization using ICSI is possible even with extremely poor cryo-survival of suboptimal samples.
Sperm survival and pregnancy rates are lower when the frozen samples used are from infertile men compared with samples from fertile donors, and there is evidence to suggest that sperm preparation in order to remove immotile and damaged sperm prior to freezing can help select a sperm population with the best chance of survival. The use of stimulants such as pentoxifylline can also improve survival after thawing.
Cryopreservation of Semen for Cancer Patients
Patients who are to be treated with combined chemotherapy for various types of cancer are frequently young or even adolescent. Recent progress in oncology has given these patients a greatly improved prognosis for successful recovery, and cryopreservation of spermatozoa before initiating treatment can preserve fertility for the majority of patients.
All cancer treatment regimens are toxic to spermatogenesis, and the majority of patients will be azoospermia after 7–8 weeks of treatment. In some cases, spermatogenesis is restored after some years, but in others there is minimal recovery even after a decade.
Currently, there are no pretreatment parameters that can predict a patient’s prognosis for recovery of fertility. The possibility of erectile dysfunction after treatment should also be borne in mind. Patients should be given general advice about the need for contraception when recovery is unpredictable and advised to seek medical help early if fertility is required. Informed consent forms should be signed after discussion and counseling. Ideally, three sperm samples are collected before chemotherapy is initiated.
Sample collection after the start of treatment is preferable to no storage at all. Patients should be informed of the potential risks and receive appropriate counseling in such cases. Spermatogenesis is often already impaired due to the effects of the disease: many demonstrate hypothalamic dysfunction, and in severe cases pituitary gonadotropin secretion is altered. Sperm quality is usually compromised with prior treatment in patients with testicular cancer, leukemia, brain tumors, and sarcoma. The tremendous stress caused by cancer reduces fertility potential by the action of stress hormones in the brain, leading to altered catecholamine secretion and a rise in prolactin and corticotropin-releasing factor, which in turn suppress the release of GnRH. However, in the light of ICSI treatment success rates, semen samples should be frozen regardless of their quality. Prior to sample collection for storage, patients should be screened for hepatitis B and C and HIV.
Patients are naturally concerned that their cancer treatment might cause an increased risk of congenital malformation in a subsequent pregnancy: the results of studies to date are reassuring, although insufficient data have been accumulated for each cancer or treatment regimen. There are now numerous evidences of successful treatment for couples using sperm stored prior to treatment. Type of treatment depends upon the quality of the sample, but pregnancies and live births are reported after AI, IUI, IVF and ICSI.
In the future, autotransplantation of cryopreserved testicular tissue may become an alternative option for young men who are not yet producing sperm or who are unable to produce an ejaculate.