Cryopreservation of mature and immature oocytes

Although the cryopreservation of human embryos has been used as an important aid in infertility treatment for over fifteen years, resulting in the birth of thousands of normal children through this technology, the same cannot be said for the freezing of human oocytes. Oocyte cryopreservation has potential clinical and psychological benefits for single women of reproductive age who are at risk of losing their ability to produce oocytes due to toxic cancer treatments. While three children were born through oocyte cryopreservation in the 1980s, the technology was not systematically used for clinical purposes until the late 1990s. The vulnerable state of the oocyte and the initially poor success rates were the main reasons for the lack of clinical application. Many of the early concerns about oocyte cryopreservation were based on animal studies, but subsequent evidence from human oocyte research suggested these concerns may have been unfounded and were essentially due to species-specific differences. These human studies, together with recent reports from a series of clinical oocyte cryopreservation programs reporting over 900 live births without any apparent increase in abnormalities, suggest that the technology is a viable option for suitable patients.

Currently, two cryopreservation methods are used to preserve human oocytes: slow-rate freezing and vitrification. Ice formation inside or around a cell is lethal. To avoid ice formation in the slow-rate freezing system, the oocyte is dehydrated in a cryoprotectant solution. To facilitate this process, the temperature is gradually reduced at a controlled rate, during which the solution crystallizes at sub-zero temperatures. Conversely, in vitrification, the high concentration of cryoprotectants and the ultra-rapid cooling prevent solution crystallization both inside and outside the oocyte, instead forming a glassy (vitrified) state.

The difficulty in assessing oocyte cryopreservation is that there are very few controlled comparative data published to confirm improvements, and many existing reports involve a small number of cases. To compare clinical outcomes obtained with these procedures, the estimated efficiency can be calculated by pooling all published results for each method, using both slow-rate freezing and vitrification.

Slow-Rate Freezing

All slow-rate freezing protocols for oocytes are based on the embryo freezing procedure originally developed by Lassalle, which uses a gradual temperature decrease in the presence of propanediol (PROH) and sucrose as cryoprotectants. With a sucrose concentration of 0.1 M, this procedure initially resulted in the survival of about 50% of oocytes, 50% of which were subsequently fertilized after ICSI, with most forming embryos. Since the first reported birth using this procedure, the number of births has gradually increased to nearly 50. Despite this initial success, this method has now been replaced by a series of modified methods.

Recently, modifications to this original method aimed to improve oocyte dehydration efficiency before freezing by using higher levels of sucrose. Although fewer oocytes have been frozen using this modified technique, there is a general trend toward better outcomes. Doubling the sucrose levels resulted in higher survival (71%) and fertilization (80%) rates. A slightly higher proportion of fertilized oocytes formed embryos compared to the low-sucrose protocol, yielding twice as many embryos (53) per 100 oocytes compared to the low-sucrose procedure (23), with more embryos implanting (implantation rate of 17% vs. 10%). This attrition rate would result in 9 fetuses per 100 frozen oocytes. Unfortunately, there are insufficient internal comparative data on these procedures to make meaningful comparisons. Despite promising results, this method has been underutilized compared to subsequent approaches. However, two groups using this method have reported nearly 60 births to date.

Further increases in sucrose levels, up to three times the amount in the initial procedure, have been explored, with higher survival (73%) and fertilization (73%) rates observed again. While a considerable number of embryos (48 per 100 oocytes) are generated, fewer implant, resulting in a final outcome of 3 fetuses per 100 thawed oocytes, similar to the results obtained with the initial procedure. To date, 54 children have been born using the 0.3 M sucrose protocol.

Another approach involved dehydrating the oocyte in a sodium-depleted medium while maintaining the concentration of PROH combined with sucrose at each of the above concentrations: 0.1 M, 0.2 M, and 0.3 M. However, sodium depletion did not yield further improvements in survival, fertilization, or embryonic development rates compared to sucrose levels alone. These results should be interpreted cautiously, as all studies on modified salts are based on a relatively small number of oocytes (~200), and only 16 births have been reported.

Vitrification

Many groups are achieving good results with oocyte vitrification. Vitrification with cryoprotectants similar to those used in slow-rate freezing (propanediol [PROH] and sucrose) combined with another cryoprotectant, ethylene glycol (EG), has resulted in 173 births. Another 77 births were reported following vitrification in a solution where propanediol was replaced with dimethyl sulfoxide (DMSO). All previously reported studies involved a small number of oocytes (<100), which, along with minor differences in methodology that could significantly impact outcomes, makes meaningful comparisons difficult. However, there now appear to be three main methodological protocols, each involving a significant number (>200) of oocytes: (1) EG; (2) EG + DMSO; (3) EG + PROH. Survival and fertilization rates are slightly lower with both EG and EG + PROH protocols (~75% each) compared to the EG + DMSO procedure (93% and 87%, respectively). Furthermore, with the EG + PROH regimen, only half of the fertilized oocytes form embryos, producing only 30 embryos per 100 oocytes and 4 implants per 100 oocytes, similar to some slow-rate freezing results. A confounding factor with this procedure is that some oocytes are immature when collected, which is known to affect further development regardless of cryopreservation.

Larger studies using these vitrification methods are needed to establish whether these improvements can be maintained for all patients and that the pregnancies reported continue to term deliveries.

 

Where results can be attributed to a particular method, these have been reported above. However, many more births have resulted from oocyte cryopreservation. A recent review and survey of groups performing oocyte cryopreservation (2) suggests the true number is in excess of 900; 532 babies from slow freezing and 392 babies from vitrification. The overall neonatal abnormality rate of 1.3% is similar to the rate with natural conception.

Although the data reviewed have mainly been generated from IVF patients undergoing oocyte cryopreservation, many groups have, for a number of years, frozen oocytes from young women with malignant disease. However, due to the delay between cryopreservation and attempts to conceive, only three births have been reported in these circumstances.

 

Controlled ovarian stimulation

Oocyte vitrification has gained ground in recent years, since both the American Society for Reproductive Medicine (ASRM) and European Society of Human Reproduction and Embriology (ESHRE) no longer consider the technique to be experimental (6-7). As a result, it is currently used in different clinical situations in assisted reproduction, including as a fertility preservation strategy prior to gonadotoxic treatment or as elective preservation (8,9). While feasible and effective in the field of oncofertility, oocyte cryopreservation is partly limited by the fact that it requires at least one cycle of controlled ovarian stimulation before oocyte pick-up. For this reason, it can only be offered to postpubertal patients and those who have time to undergo the procedure prior to their gonadotoxic therapy (10). 

Classic ovarian stimulation protocols start in the early follicular phase and proceed to oocyte retrieval when the oocytes are expected to be mature, around 12 days after the beginning of stimulation and according to hormone values and ultrasound features. Since there is no need to achieve synchrony between oocytes and endometrium in cancer patients in the absence of oocyte fertilization and embryo transfer, less time-consuming protocols may be applied. Random-start protocols have allowed us to circumvent some timing limitations in case of cancer treatment, as they can be initiated right after the cancer diagnosis without waiting for the next follicular phase (11,12). These protocols have proved effective and safe in oncofertility settings, with similar outcomes in terms of mature oocyte retrieval (13). 

The standard approach to collect an ideal number of mature oocytes includes stimulation with gonadotropins, associated with a GnRH modulator (agonist or antagonist), to avoid a premature spontaneous LH surge and subsequent spontaneous ovulation or follicle luteinization. As these protocols are associated with a rise in estrogen levels, they are not recommended in patients with hormone-sensitive tumors, like breast cancer expressing estrogen and progesterone receptors. These patients need to be treated with specific protocols with aromatase inhibitors that reduce estradiol levels without compromising oocyte retrieval in terms of number, maturation and further fertilization (14). 

Overall, the ovarian response in cancer patients does not appear to be significantly different from that in women having their oocytes cryopreserved for non-oncological reasons. However, data on this are still contradictory, as some studies show an altered ovarian response in cancer patients while others do not (15-17). Moreover, differences in the ovarian stimulation response may be related to the type of cancer, as reported by some authors who evidenced a difference in outcomes in patients affected by breast cancer (18) and those with BRCA mutations (19,20).

Although demand for oocyte cryopreservation in cancer patients has increased in recent years, there is very little evidence about IVF outcomes in this specific population in the literature. This may be due to only a small proportion of patients returning to use their oocytes, and/or the long time interval between cryopreservation and oocyte use. The largest series ever published to date included 1,073 subjects diagnosed with cancer, 80 of whom returned to use their vitrified oocytes (9). Clinical and ongoing pregnancy rates were 41% and 31% respectively (with 25 healthy babies born). Age at cryopreservation was confirmed to be of utmost importance, with a significant decline in pregnancy outcomes in patients over 35 years of age (21).

 

References: 

1. Gook, D.A. and D.H. Edgar, Human oocyte cryopreservation. Hum Reprod Update, 2007. 13(6): p. 591-605.
2. Porcu, E., et al., Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril, 1997. 68(4): p. 724-6.
3. Yoon, T.K., et al., Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technologies. Fertil Steril, 2007. 88(4): p. 952-6.
4. Kuwayama, M., et al., Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online, 2005. 11(3): p. 300-8.
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