Oocyte Cryopreservation

Oocyte Cryopreservation

Oocyte Cryopreservation

Egg freezing and preservation has enormous potential in reproduction medicine mainly in terms of deferring childbearing until later in life for personal (e.g. pursuing a career) or medical reasons (use of chemotherapeutic drugs and pelvic radiotherapy for cancer) or as part of oocyte donation programs.

Indications for Oocyte Cryopreservation

1. Medical and Genetic Conditions

Chemotherapy and radiation treatment protocols against malignant or benign cancerous tumors subject female patients to major risks for becoming infertile. While the toxic chemical agents and ionizing radiation have not been proven to associate with genetic familial mutations their effect has well been documented to induce premature ovarian failure and therefore infertility. Furthermore, certain patients may require more aggressive interventions such as complete oophorectomy (removal of ovaries) as the most effective line of therapy to prevent metastasis. Adjunct to these, female cancer patients of reproductive age have literally no alternative option than freezing their eggs in a timely fashion to safeguard their fertility. Extrapolating on this, a number of genetic conditions such as mutations in the BRCA gene are contingent to ovarian cancer and while complete salpingo-oophorectomy (removal of both the fallopian tubes and ovaries) is most commonly recommended following childbearing certain candidates may be advised to receive surgery prior to parenting children as means of prophylaxis. Such females and others who are known curriers of gene mutations such as X chromosome deletion or Turner’s syndrome with higher propensity of developing premature ovarian failure can benefit by preserving their eggs using egg freezing technology.

2. Elective Oocyte Cryopreservation to Defer Childbearing

Fertility in the female declines with advancing age and this is a well established fact. As previously mentioned the phenomenon is due to ovarian reserve depletion and increased fragility of the chromosomal constitution in the aging egg cohort. The quantity and quality of eggs deteriorates more rapidly more as from the age of 35 while over 40 the chances for conception become alarmingly limited. In essence, oocyte cryopreservation stops the biological clock and in this respect provides the opportunity to those females whom intention is to delay motherhood, to conceive subsequently. While this notion appears intriguing, marketing egg freezing for the purpose of differing childbearing may raise false hopes in the sense that individual candidates may be encouraged to delay childbearing by virtue of sub conscientious reassurance. Success rates in women who are in their late reproductive years and who may be the most interested in this technology fall at significantly lower values than those corresponding to younger individuals (38 or less). It is therefore advised that prospective candidates receive appropriate counseling pertaining to expected future pregnancy outcomes and associated financial and psychological burdens with the use of this application.

3. Oocyte Cryopreservation Due to Potentially Compromised Embryo Development or for Religious Reasons

IVF patient couples with a history of poor fertilisation rates or compromised embryo development due to male factor may opt to freeze half of the egg harvest during a treatment cycle as means of potentially ‘rescuing’ the given attempt with the use of donor sperm subsequently as the only option. Furthermore oocytes freezing can salvage a IVF treatment cycle following egg retrieval and when the male partner failed to dispose a semen specimen duly.
Certain IVF couples object to freezing embryos on the grounds that these may have to be destroyed if not utilized for a number of years or they may stay as surplus if their families are complete from previously successful attempts. Since donating to other infertile couples or to research is not an option to them, they may opt to utilize a small number of the egg harvest for fertilisation while the rest of the cohort is frozen for potentially subsequent use. Furthermore certain religious groups and ethicists support the notion that the onset of life is the unity of egg and sperm and that destroying a human life can be avoided by freezing eggs. IVF methods have repeatedly raised questions about the moral status of the embryo and its rights. Oocyte cryopreservation abrogates these concerns.

4. Oocyte Cryopreservation for Egg Donation Programs

Egg donation is the most efficient alternative to using own oocytes in IVF treatment for a number of indications. These are analyzed in the egg donation sector of this web page. Fresh egg donations are confronted with a number of difficulties mainly associated with long waiting lists, inefficiencies in coordination and treatment synchronization between donor and recipient, elevated costs and absolute quarantine measures. All these can be circumvented with the incorporation of donor egg cryo-banking. The use of cryopreserved oocytes is beneficial for a collection of reasons most important of which are the ergonomics in the donor selection process, more flexibility in synchronizing treatment protocols in a timely manner, reduced waiting lists, presumed cost effectiveness and overall ease for both donors and recipients.

While the development and introduction of programmable slow freezers for this scientific application reduced the risks of oocyte damage by slowing down the rate of freezing therefore mediating more efficiently cellular dehydration, results had not proven optimal. The modern technique of vitrification which entails an ultra fast freezing method enjoys excellent results consistent with close to 100% survival rates post thawing. Substantial scientific evidence affirms similar fertilisation and term pregnancy rates with vitrified/warmed oocytes when compared with fresh eggs in IVF programs. Research also reported in literature documents that the use of frozen/thawed oocytes does not subject pre-implantation embryos to an increase in chromosomal abnormalities nor a prevalence of birth defects in the resulting offspring when compared to conventional IVF treatments or indeed the general population. Furthermore, prolonged duration of oocyte storage following vitrification does not present a compromising factor for the same parameters.

Similarly with sperm freezing, oocyte cryopreservation using either slow or ultra rapid freezing makes use of special cryoprotectant solutions containing a basal culture medium, cryoprotectant additives. The basal medium consisting mainly of ultra purified water and ingredients such as salts, pH buffers, antibiotics and albumin protein is important for providing cellular support at freezing point. Cryoprotectants can be membrane permeable (i.e. ethylene glycol or DMSO) or membrane impermeable (i.e. sucrose or trehalose). Oocytes are initially suspended in membrane permeable additives which gradually displace their water content while the impermeable counterparts which are subsequently incorporated create a concentration difference between the outside and the inside of the cell (the so called osmotic gradient) supporting the cell dehydration phase. The oocytes tend to shrink initially only to gain normal size and shape after approximately 10-12minutes in suspension while during the second and final step which must be completed within 90 seconds the increased osmotic gradient which is created establishes a glassy or vitreous transition to the egg (hence the term given-vitrification). At this state the eggs are dried on straws prior to being plunged into liquid nitrogen where the freeze spontaneously at -195°C. The straws which are biocompatible are manufactured to withstand the ultra low temperatures and pressures created by expanding liquids and liquid nitrogen. While several oocytes can be vitrified on individual straws, Genesis experience suggests that no more than 4 should be frozen at a time.

Prerequisites to a successful vitrification procedure are the exposure time to both the equilibration and vitrification solutions which must be very rigorous and that the eggs should be literally dried onto the vitrification straws prior to liquid nitrogen immersion. Smaller volumes allow better heat transfer hence facilitating higher cooling rates. Oocytes have an increased susceptibility to cryo-damage when compared to embryos mainly due to their lower volume / surface ratio (therefore being less efficient at taking up cryoprotectant and losing water), the membrane permeability properties and the function of intracellular elements namely organelles necessary for egg survival and viability (prolonged exposure is highly toxic to the egg). Further to this the chromosomal complexes within the eggs themselves are extremely sensitive to both chemical and physical alterations since these are suspended in the cytoplasm (inner part of the egg) rather than being confined in nuclear membranes (each cell of an embryo has a nucleus surrounded by the nuclear membrane).

The thawing (warming) protocol of cryopreserved oocytes is essentially a reversal of the vitrification steps. The straw with the vitrified eggs is rapidly immersed (within less than 1 sec) from liquid nitrogen into a solution containing an increased concentration of sucrose (1M) at 37 °C. This step enables the dissociation of the oocytes from the straw via gradual rehydration and lasts for 60 seconds. The eggs which do not gain normal size and shape within this period are then incubated in a dilution solution at room temperature and for a period of 3 minutes. The suspension which has half the initial concentration of sucrose (0.5M) subjects the eggs to a diluted environment facilitating rehydration whereby removing the cryoprotectant agents and replacing the cellular and intracellular fluid at a precise rate. The use of sucrose which is a non-permeating solute as an osmotic buffer minimizes the risks of an osmotic shock to the eggs while reducing the time duration of the whole process. The eggs are then allowed to settle for further recovery in basal medium for approximately 5 minutes prior to storage at 37 °C under nominal concentrations of carbon dioxide in air and relative humidity which is their physiological environment. Eggs can be subjected to sperm injection for fertilisation approximately 2 hours post the thawing procedure. At the time they should have reached complete recovery.