Oocyte Freezing : Advantages and Disadvantages and uses in Today’s Scenario
A woman’s reproductive life span is finite and depends on the number of oocytes with which she is born . Treatment with chemotherapeutic drugs and pelvic radiotherapy for cancer or other serious medical illnesses has the potential to markedly accelerate follicular atresia, placing women who require these treatments at risk of primary ovarian insufficiency. Likewise, genetic conditions such as fragile X premutation and mosaicism for monosomy X also predispose women to primary ovarian insufficiency. Women with these risk factors and others may be candidates for fertility preservation before ovarian failure ensues. However, as stated in the ASRM–SART guideline, “there are not yet sufficient data to recommend oocyte cryopreservation for the sole purpose of circumventing reproductive aging in healthy women because there are no data to support the safety, efficacy, ethics, emotional risks, and cost-effectiveness of oocyte cryopreservation for this indication”
Established fertility-preservation methods for women recognised at this time include fertility-sparing surgery and surgical ovarian transposition, shielding to reduce radiation damage to reproductive organs, and embryo cryopreservation after ovarian stimulation with gonadotropins and in-vitro fertilization (IVF). Currently, all remaining options are still considered experimental.
Cryopreservation techniques play a central role in assisted reproduction. Since initial reports that sperm could survive after being cooled at low temperatures,methods of preserving spermatozoa developed rapidly after accidental discovery of the cryoprotective properties of glycerol on sperm cells. Research on cryopreservation of sperm could advance relatively fast owing to the small size of sperm cells and their high number available for experiments. Since the first reported live birth after insemination with spermatozoa that had been frozen and thawed,clinical treatments have developed fast. Further development of sperm cryopreservation techniques has made the introduction of fertility treatments possible with banked frozen and thawed sperm from donors, and the establishment of sperm banking as a method for preserving male fertility.
With the development of IVF,methods for cryopreservation of embryos were required, as IVF treatment often resulted in surplus embryos. The human embryo has a relatively high permeability to cryoprotectants, and it is notably more resistant to cryodamage than the oocyte. After the first reported human pregnancy after cryopreservation, and thaw of an eight-cell embryo in 1983,methods for cryopreservation of embryos developed successfully. It is estimated that more than half a million babies have been born worldwide after intrauterine replacement of frozen and thawed embryos.
Cryopreservation of oocytes
Freezing of oocytes for fertility preservation is an option for women who do not have a partner and for women who do not wish to preserve embryos because of religious or ethical concerns. Oocyte cryopreservation for fertility preservation may also be an option for adolescent girls in selected cases, as shown in a recent report of a 14-year old girl interested in undergoing hormonal stimulation and oocyte freezing instead of ovarian tissue cryopreservation, which is usually the preferred option for children.
A valid concern in carrying out ovarian stimulation for oocyte freezing in women with breast cancer and other hormone-sensitive tumours is the exposure to supraphysiologial oestradiol levels during gonadotropin stimulation, which has been a main obstacle in offering fertility preservation by oocyte or embryo cryopreservation to those women. Alternative and potentially safer stimulation protocols with tamoxifen, and aromatase inhibitors have been developed. Those stimulation treatments have been shown to be effective,with no detrimental effects on relapse or survival rates in these women.
Freezing unfertilised oocytes, with the aim of later thawing and fertilising them by IVF or intracytoplasmic sperm injection (ICSI) is today a promising option for preserving fertility.. The human oocyte is the largest cell in the body, and its large size and large volume with a high content of water makes it extremely fragile to intracellular ice formation during the freezing and thawing processes. Oocytes, in contrast to embryos and zygotes, also present with lower permeability to cryoprotectants.
Chilling injury and disruption of the meiotic spindle
Concern has also been raised about the disruption of the meiotic spindle observed when oocytes are being cooled to temperatures near 0°C, which has been described as a sign of chilling injury. At low temperatures, tubulin depolymerises, and the support for the structure of the spindle, which maintains the chromosomes aligned on the methaphase plate, disassembles.The meiotic spindle is needed for the correct chromosome segregation during the oocyte maturational processes.
Variations in oocytes’s sensitivity to chilling injury have also been reported among individuals in human oocytes as well as in several animal species, including non-human primates.
Nevertheless, it has been shown that the spindle reappears in the cytoplasm after warming up the oocytes at 37°C, and chromosomal aberrations do not seem to be increased in embryos obtained from frozen thawed oocytes compared with fresh embryos.
Increasing fertilisation rates by intra-cytoplasmic sperm injection
Over many years, the techniques for cryopreservation of oocytes were based on slow freezing protocols originally developed for embryo freezing and success rates of oocyte survival and fertilisation were low. Since the first pregnancy reported in 1986, only a few live births have been achieved. Cryopreservation of oocytes induces hardening of the zona pellucida, which could only be overcome after the development of ICSI.in 1997, microinjection of a sperm in vitro was first used on oocytes that had been frozen and thawed, which resulted in a live birth, and over 100 deliveries were reported in the following decade by using ICSI.
Oocyte freezing by vitrification
The process of vitrification involves the use of high concentrations of cryoprotectants and rapid cooling to achieve a glass-like highly viscous solution without formation of ice crystals. Given the high inter-individual variation in oocyte sensitivity to chilling, it has been suggested that rapid cooling might prevent such injury. In animal studies, cattle oocytes presented with increased survival and fertilisation rates when cooling them at high rates.
The efficiency of vitrification of oocytes has made possible the current establishment of oocyte banking in donor programmes., Survival rates of oocytes thawed after vitrification have been reported to be as high as 96.9%, and the fertilisation rates are currently non-significantly different from those of fresh oocytes (76.3% v 82.2%, respectively). Pregnancy rates have been reported to be as high as 65.2%, and implantation rates 40.8% with vitrified and thawed oocytes. In the largest, propective, randomised-controlled study, including 600 recipients of donated eggs conducted in Spain, no superiority of using fresh oocytes over vitrified egg-banked and thawed oocytes could be demonstrated.
Cryopreservation of immature oocytes
Because immature germinal vesicle stage oocytes have not yet formed spindle, and because they have a higher membrane permeability, it has been suggested that germinal vesicle stage oocytes might be more resistant to chilling injury than mature metaphase II oocytes.Reported survival rates of immature oocytes cryopreserved with slow freezing protocols after thawing vary from 37%to 63%.7 Spindle abnormalities, however, have also been reported after in-vitro maturation, suggesting impaired developmental competence when oocytes are frozen at germinal vesicle stage.
Cryopreservation and transplantation of ovarian tissue
Cryopreservation of ovarian tissue combined with orthotopic transplantation into an irradiated ovary restored fertility in rodents 50 years ago. It took more than 30 years, however, to improve the freezing protocols and transplantation procedures in larger animals and, in particular, in species that could be considered applicable to humans. In fact, the only available cryoprotectant during since the 1930s was glycerol. Although it was successful for freezing sperm, it was highly ineffective for cryopreserving oocytes and ovarian tissue. In the 1970s, more effective cryoprotectants emerged, such as propanediol, ethylene glycol and dimethylsulphoxide (DMSO).
The exhaustive studies of ovarian cryopreservation and transplantation conducted in animals, and particularly those in the lamb model, finally led to significant improvements in cryopreservation and transplantation techniques, and the establishment of a model for applying these investigational procedures to humans. In this model, the ovarian tissue was cryopreserved after improving current slow freezing protocols using DMSO as a cryoprotectant. The ovarian tissue was frozen in slices and the thawed slices were later transplanted orthotopically. In the lamb, recovery of cyclic ovarian activity was demonstrated, and pregnancies and live born lambs achieved, as well as long-term ovarian function. Since then, ovarian tissue has been cryopreserved from several species, with the aim of improving the methods, optimising existing freezing protocols, and developing new techniques, such as vitrification. The first live birth in non-human primates after ovarian tissue transplantation was reported in 2004.
Human ovarian cryopreservation
A method for cryopreservation of human ovarian tissue was first reported in 1996. Cryopreservation of ovarian tissue for fertility preservation is indicated for adult women when ovarian stimulation for oocyte or embryo freezing is not feasible or not desired. For prepubertal girls, the cryopreservation of ovarian tissue is their only option to spare their eggs. As most eggs are within primordial follicles in the ovarian cortex, a significant number of eggs may be preserved by freezing pieces of ovarian cortex, even if they are small.
Ovarian biopsies may be harvested by minimal invasive surgery, such as laparoscopy. Ideally, this procedure should be carried out before starting cytotoxic treatment. Nevertheless, it may still be worth carrying out ovarian cortex harvesting after cytotoxic treatments in girls and young women previously unable to undergo the procedure, because girls and young women normally have high follicle counts in their ovaries. Consensus is lacking about how much ovarian tissue should be harvested for cryopreservation, and some programmes for fertility preservation propose unilateral oophorectomy as standard procedure.
Several cryoprotectants for slow freezing of human ovarian tissue have been investigated, such as glycerol, ethylene glycol, DMSO and propanediol. Follicle survival has been evaluated after thawing pieces of frozen ovarian cortex with those cryoprotectants. The highest follicle survival rates have been obtained with ethylene glycol, whereas glycerol has been associated with the poorest results.
Studies investigating the most favourable cooling rates and dehydration times have also been conducted. After evaluating the tissue by electron microscopic morphology, slow programmed freezing with a relatively long dehydration time became a standard cryopreservation method for human ovarian tissue from the end of the 1990s.
Vitrification of ovarian tissue
A concern of slow-programmed freezing has been the relatively poor survival of the ovarian stroma, which has been demonstrated by transmission-electron microscopy, a method that accurately evaluates cryoinjury of membranes, mitochondria and other organelles not seen in detail with any other methods. The introduction of vitrification techniques for cryopreservation of ovarian tissue has been shown to improve the viability of all compartments of the tissue, with a survival rate of follicles similar to that after slow freezing, much better integrity of ovarian stroma and undamaged morphology of blood vessels.
Vitrification of ovarian tissue used in combination with orthotopic autotransplantation has been successful in rodents and sheep. Human studies comparing slow-freezing protocols with vitrification of ovarian tissue have produced conflicting results, which may be explained by differences in the protocols and the medium used.
As suggested by transmission-electron microscopy, vitrification could be more effective than slow-programmed freezing when cryopreserving ovarian tissue. In one study, thawing of vitrified ovarian tissue resulted in improved oocyte survival compared with slow freezing (89% v 42%, respectively). The methods for vitrification have further developed, achieving a clinical grade closed cGMP-compatible system, in which the ovarian tissue sample will not be in any direct contact with liquid nitrogen during vitrification or storage, thus meeting the requirements of international tissue directives.
Cryopreservation of oocytes and ovarian tissue seem to hold much promise for fertility preservation of women undergoing gonadotoxic treatments. Cryopreservation of oocytes has reached promising standards, and more than 1000 children are born worldwide after fertilisation of frozen and thawed oocytes. Nevertheless, that technique is still considered experimental.
Ovarian-tissue harvesting seems to be safe, but the experience with ovarian transplantation is still limited owing to low utilisation. Autotransplantation of ovarian tissue in women having suffered from systemic haematological malignancies is not recommended, owing to the high risk of retransmission of malignancy. Only women with cancer diagnosis associated with a negligible or no risk of ovarian compromise should be considered for future autotransplantation.