In vitro fertilization and embryo cryopreservation is regarded as the only established method for fertility preservation in female cancer patients. However, a possible delay in treatment of the primary disease due to ovarian stimulation, exposure to supraphysiologic estrogen levels induced by ovarian stimulation, the requirement for a male partner or willingness to use donor sperm for embryo production, legal, ethical, religious issues related to cryopreservation of embryos raise concerns for patients and physicians. Recent improvements achieved with oocyte vitrification have increased the effectiveness of oocyte cryopreservation rendering it a viable option, especially for patients without a male partner. In vitro maturation avoids treatment delay or exposure to increased estradiol levels associated with ovarian stimulation for in vitro fertilization. In vitro maturation combined with embryo or oocyte vitrification provides previously unavailable options for some patients and improves the services provided by a fertility preservation program.
Cancer is a major health problem, and malignant neoplasms constitute the second most common cause of death, second only to cardiac disease. It is estimated that in 2009 approximately 713,220 women in the United States will be diagnosed with cancer. Improved treatment methods have resulted in a steady increase in the survival rates over the last decades. Eventually the number of cancer survivors increase every year. Accordingly, a growing number of women are faced with the risk of infertility resulting from gonadotoxic oncologic treatment. Breast cancer remains the most common cancer in females, representing 27% of all female cancers. Between 2002 and 2006, 12.4% of all breast cancer cases were diagnosed in women younger than 45 years, who are in their reproductive period. Based on this distribution, it can be anticipated that approximately 25,000 reproductive aged women will become candidates for gonadotoxic therapy due to just one type of cancer in only one country in a single year. Moreover, with similar improvements in the treatment of childhood cancers, it is estimated that one in every 250 adults will be a childhood cancer survivor by the year 2010. Patients who are exposed to gonadotoxic agents for the treatment of non-oncologic diseases such as systemic lupus erythematosus, those who are undergoing surgery for endometriosis as well as women with genetic disorders such as Turner syndrome and fragile-X pre-mutation face similar risks, further contributing to the population of women who need fertility preservation procedures.
Given the improvement in survival rates with cancer treatment and developments in the field of reproductive medicine, fertility preservation for female cancer patients has become a very timely and topical issue. With an increased awareness of the options available for fertility preservation, a greater number of women are being offered and are utilizing these technologies.
Today, several methods are available for preserving the reproductive potential of these patients. When the threat to gonads is limited to direct radiotherapy, surgically positioning the ovaries out of the irradiation field is an effective method and can be combined with other methods if deemed necessary. However, in the case of systemic administration of gonadotoxic agents, the gametes at risk should be removed from the body and preserved in some form until they can be reimplanted or used for reproduction upon completion of treatment for the underlying disease. Besides gonadotoxicity, advancing age during treatment is another concern. Fertility declines with age, and in some cases, treatment of the underlying disease may require several years, meaning that the natural decline in fertility may become an issue even in the absence of direct gonadotoxicity.
Administration of gonadotropin releasing hormone analogues or inhibitors of apoptosis, cryopreservation of unfertilized oocytes following in-vitro fertilisation (IVF) or in-vitro maturation (IVM), cryopreservation of embryos following IVF or IVM and ovarian tissue cryopreservation are the current methods available for fertility preservation. At present, embryo cryopreservation following IVF is the only method endorsed by the American Society of Clinical Oncology (ASCO) and the American Society of Reproductive Medicine (ASRM), while the other methods are still considered experimental. However, fertility preservation is a relatively new field in the practice of reproductive medicine and oncology, and it attracts much attention. We are certain that opinions will change shortly in light of rapidly accumulating data from across the world. The method of choice for any particular patient depends on several factors including age, cancer type and stage, pending oncologic treatment, safety of ovarian stimulation, time available before gonadotoxic chemotherapy and the availability of partner sperm or the willingness of single patients to use donor sperm.
In this article, we will review the currently available literature on the cryopreservation of unfertilized oocytes and embryos.
Cryopreservation of cells – controlled slow-freezing versus vitrification
The term cryopreservation refers to the storage of viable cells at low temperature (normally at −196 °C). However, neither human oocytes nor embryos are exposed to such a low temperature under natural conditions. They are prone to damage and subsequent loss of viability and function during cooling to such low temperatures. The three major mechanisms of damage are chilling injury that occurs between +15 °C and −5 °C, ice crystal formation between −5 and −80 °C, and fracture damage; i.e. the mechanical effect of solidified fluid within the cell that occurs between −50 and −150 °C. Once the cell reaches −196 °C, storage at this temperature is the least harmful period of the cryopreservation process. During warming, the cell is subject to the same injuries in reverse order. The occurrence of these events can be prevented to some extent by altering the rate of change in temperature and/or the amount of water in the cell. The size of the sample to be cryopreserved, its surface-to-volume ratio and water content are critical factors determining cryosurvival.
Among the many methods used for cryopreservation of biological samples, the two basic techniques employed in assisted reproduction laboratories are controlled slow-freezing (SF) and, more recently, an ultrarapid cooling method also known as vitrification. Both involve the use of cryoprotectants to minimize ice-crystal formation by drawing water from the cytoplasm through the osmotic effect (non-permeable cryoprotectants); or by preventing ice nucleation and growth either inside the cytoplasm (permeable cryoprotectants) or in the extracellular medium surrounding the cell (extracellular cryoprotectants). Unfortunately, the cryoprotectants currently in use have the potential to harm the cell, either through direct toxicity (permeable cryoprotectants) or through osmotic damage (non-permeable cryoprotectants). Cryoprotectant toxicity is proportional to its concentration and the duration of exposure.
SF is chronologically the earlier technology, and involves cooling the cell at a strictly controlled slow rate that allows water to leave the cell after treatment with relatively low concentrations of cryoprotectants. Eventually, cytoplasmic viscosity is extremely increased without ice formation; i.e., vitrified. SF requires programmable freezing machines, and each round of cooling and thawing takes several hours.
On the other hand, vitrification involves ultrarapid cooling of the sample; i.e., transition from ambient temperature to −196 °C in less than a second. In contrast to SF, the cooling rate is above 10,000 °C/minute. This is achieved by loading the sample onto a carrier and submerging it following treatment with vitrification solution into liquid nitrogen (LN). While rapid cooling prevents chilling injury, this technique requires the use of very high concentrations of cryoprotectants to prevent ice-crystal formation, thus increasing the risk of toxicity. This is counteracted, however, by working with minute amounts. Expensive programmable equipment is not required for the vitrification of human oocytes and embryos. The technique requires minimum set up time but more hands-on time per oocyte/embryo.
One important difference between the two techniques is that while the sample is maintained in a closed container and never comes into direct contact with LN during SF, the solution covering the sample directly contacts LN during vitrification. This has raised concerns about possible disease transmission between open tools used for vitrification. Although the possibility of transmission of viral particles from LN to embryos has been documented under experimental conditions, no actual contamination of cryopreserved oocytes or embryos has ever been reported at the time of writing. However, research is ongoing to develop a closed vitrification system that does not significantly slow heat transfer and decrease the effectiveness of the vitrification process.
Relative merits of the two techniques with regard to oocyte and embryo cryopreservation will be discussed below.
Embryo cryopreservation
The successful cryopreservation of surplus embryos after IVF and resultant pregnancy following frozen-thawed embryo transfer (FET) was first reported in 1983, and the first child after embryo freezing was born in 1984. For more than two decades, embryo cryopreservation (EC) has played an important role in assisted reproduction treatment (ART), providing couples with more than one attempt at embryo transfer after a single ovarian stimulation cycle with IVF, thus improving cumulative pregnancy rates while decreasing exposure to gonadotropins and reducing treatment costs. It is estimated that almost one quarter of the children born after ART are born following cryopreservation of mostly cleavage-stage embryos and, less commonly, blastocysts and oocytes. Most recent data from the Society for Assisted Reproductive Technology and the European IVF Monitoring Program report a pregnancy rate of 34% following FET in women younger than 35 years and an overall pregnancy rate of 19%, respectively. Slow-freezing has been the most widely applied technique for the cryopreservation of embryos, while vitrification has been used more frequently recently. A recent review assessing the medical outcome of ART children born after cryopreservation reported reassuring results. The rate of preterm birth, birth defects and chromosomal abnormalities was not significantly different between children born after transfer of fresh or cryopreserved embryos. Similarly, these children demonstrated similar growth and mental development.
Although the cryopreservation of embryos following a stimulated IVF cycle is considered the only established method of fertility preservation for female cancer patients, several points raise concern about this option. These are: (1) a possible delay of two to five weeks in treatment of the primary disease due to ovarian stimulation depending on the timing of the first consultation with the reproductive endocrinologist in relation to onset of the next menstrual cycle (2) exposure to supraphysiologic estrogen levels induced by ovarian stimulation (3) the requirement for a male partner or willingness to use donor sperm for embryo production (4) legal, ethical, religious issues related to cryopreservation of embryos in general. The method of choice for the cryopreservation of embryos is another relevant issue and these are addressed below.
Does IVF – embryo cryopreservation delay treatment of primary disease?
A retrospective study addressing the above question concluded that having a reproductive medicine consult and subsequent ovarian stimulation followed by oocyte collection did not significantly delay the start of adjuvant chemotherapy in young patients with breast cancer. However, the actual time required for completion of the fertility preservation procedure, which starts with the initial reproductive medicine consultation and technically ends with oocyte collection, depends on the conditions of any particular clinic.
Ovarian stimulation takes between 2 and 5 weeks, depending on the stimulation protocol employed and the timing of the following menstrual cycle of the patient. Studies assessing the effect of the length of time between surgery and the initiation of chemotherapy on the survival of women with breast cancer report no detrimental effect of a delay in treatment if chemotherapy is started within 12 weeks after surgery. However, it must be emphasized that the external validity of these studies is limited to their inclusion criteria, and the potential effect of any delay in oncologic treatment due to fertility preservation procedures must be evaluated on a case-by-case basis together with the treating oncology team. In order to minimize any preventable delay, it is prudent to inform patients about the effects of treatment on fertility and the options for fertility preservation as early as possible in the course of oncologic diagnosis and treatment procedures.
The effect of elevated estrogen levels on underlying disease
The risk of breast cancer is consistently found to be associated with persistently elevated blood estrogen levels. Serum estradiol (E2) levels are increased during ovarian stimulation for IVF and can reach levels twenty times higher than those of a natural cycle. Although the effect of a temporary increase in serum E2 levels on the risk of recurrence of breast cancer is controversial, these facts cause concern among both physicians and patients. Such concerns should not be limited to women with estrogen receptor positive breast cancer, because recent findings also suggest the presence of an indirect mitogenic effect of estrogen on hormone receptor negative breast cancer. Moreover, increased E2 levels can be relevant for patients undergoing fertility preservation treatment due to other oncologic or non-oncologic diseases considered to be estrogen sensitive, such as desmoid tumours, systemic lupus erythematosus or severe endometriosis.
In order to minimize the rise in estradiol levels in breast cancer patients undergoing IVF, Oktay et al. developed an ovarian stimulation protocol involving the concomitant use of an aromatase inhibitor, letrozole, with gonadotropins. Briefly, letrozole was started at a dose of 5 mg/day on the second day of the menstrual cycle and gonadotropins were initiated two days later. A gonadotropin releasing hormone antagonist was used to prevent premature ovulation, and human chorionic gonadotropin (HCG) was administered when at least two follicles reached 19 mm in diameter. Letrozole was reinitiated on the day of oocyte collection in order to prevent a rebound increase in E2 level. The mean peak E2 level was 406 pg/ml (range 58 to 1,166 pg/ml) in 79 women with breast cancer undergoing ovarian stimulation for embryo or oocyte cryopreservation. An average of 10.3 ± 7.75 oocytes were retrieved and 5.97 ± 4.97 embryos/oocytes cryopreserved per patient. Compared to 136 women who opted against ovarian stimulation, the recurrence and relapse-free survival rates were similar after a median follow up of 23.4 months after definitive surgery. However, it is interesting to note that with the same centre 63.3% of breast cancer patients referred for REI consultation declined ovarian stimulation and IVF due to concerns about delay of chemotherapy, effect of ovarian stimulation on cancer or costs associated with treatment.
Ethical and legal issues associated with embryo cryopreservation
When embryos are cryopreserved in a fertility preservation program, the patient/couple should make an advance decision on the fate of these embryos in the event that they are not transferred for any reason including the patient’s failure to survive cancer. It should be documented whether the remaining partner is entitled to use the embryos for his own reproductive end or whether they are to be donated to a third party, used for research or discarded. Considering these issues and making such decisions can be particularly difficult for a patient who has been recently diagnosed with a life-threatening disease and is facing a demanding treatment period. Therefore, patients should be given appropriate counselling using a multidisciplinary approach involving a psychologist and a legal advisor.
Slow freezing or vitrification for embryo cryopreservation
Several trials have been conducted to answer this question in non-cancer patients. A recent systematic review of randomized trials comparing laboratory and clinical outcome with SF or vitrification conclude that pregnancy rates were not statistically significantly different between the two methods (odds ratio (OR): 1.66, 95% confidence interval (CI): 0.98 –2.79, in favour of vitrification). However, vitrification was associated with significantly higher post-thawing survival rates, both for cleavage-stage embryos (OR: 6.35, 95% CI: 1.14–35.26) and for blastocysts (OR: 4.09, 95% CI: 2.45–6.84). Moreover, post-thawing blastocyst development of embryos cryopreserved at the cleavage stage was significantly higher with vitrification than with slow freezing (OR: 1.56, 95% CI: 1.07–2.27). It should be noted that all of the three studies that reported clinical pregnancy rates included in this review reported higher absolute figures in the vitrification arms and, given the significantly better laboratory results together with the favourable trend observed in clinical outcome, we have the impression that lack of statistical significance is more likely to be a matter of sample size. Similar to IVF embryos, we achieved higher survival and pregnancy rates following vitrification of cleavage stage IVM embryos as compared with slow-freezing. In fact, vitrification is the only method of cryopreservation currently employed at the McGill Reproductive Centre. The method of choice should be determined by the ART center based on its own experience and figures.
Embryo cryopreservation
The successful cryopreservation of surplus embryos after IVF and resultant pregnancy following frozen-thawed embryo transfer (FET) was first reported in 1983, and the first child after embryo freezing was born in 1984. For more than two decades, embryo cryopreservation (EC) has played an important role in assisted reproduction treatment (ART), providing couples with more than one attempt at embryo transfer after a single ovarian stimulation cycle with IVF, thus improving cumulative pregnancy rates while decreasing exposure to gonadotropins and reducing treatment costs. It is estimated that almost one quarter of the children born after ART are born following cryopreservation of mostly cleavage-stage embryos and, less commonly, blastocysts and oocytes. Most recent data from the Society for Assisted Reproductive Technology and the European IVF Monitoring Program report a pregnancy rate of 34% following FET in women younger than 35 years and an overall pregnancy rate of 19%, respectively. Slow-freezing has been the most widely applied technique for the cryopreservation of embryos, while vitrification has been used more frequently recently. A recent review assessing the medical outcome of ART children born after cryopreservation reported reassuring results. The rate of preterm birth, birth defects and chromosomal abnormalities was not significantly different between children born after transfer of fresh or cryopreserved embryos. Similarly, these children demonstrated similar growth and mental development.
Although the cryopreservation of embryos following a stimulated IVF cycle is considered the only established method of fertility preservation for female cancer patients, several points raise concern about this option. These are: (1) a possible delay of two to five weeks in treatment of the primary disease due to ovarian stimulation depending on the timing of the first consultation with the reproductive endocrinologist in relation to onset of the next menstrual cycle (2) exposure to supraphysiologic estrogen levels induced by ovarian stimulation (3) the requirement for a male partner or willingness to use donor sperm for embryo production (4) legal, ethical, religious issues related to cryopreservation of embryos in general. The method of choice for the cryopreservation of embryos is another relevant issue and these are addressed below.
Does IVF – embryo cryopreservation delay treatment of primary disease?
A retrospective study addressing the above question concluded that having a reproductive medicine consult and subsequent ovarian stimulation followed by oocyte collection did not significantly delay the start of adjuvant chemotherapy in young patients with breast cancer. However, the actual time required for completion of the fertility preservation procedure, which starts with the initial reproductive medicine consultation and technically ends with oocyte collection, depends on the conditions of any particular clinic.
Ovarian stimulation takes between 2 and 5 weeks, depending on the stimulation protocol employed and the timing of the following menstrual cycle of the patient. Studies assessing the effect of the length of time between surgery and the initiation of chemotherapy on the survival of women with breast cancer report no detrimental effect of a delay in treatment if chemotherapy is started within 12 weeks after surgery. However, it must be emphasized that the external validity of these studies is limited to their inclusion criteria, and the potential effect of any delay in oncologic treatment due to fertility preservation procedures must be evaluated on a case-by-case basis together with the treating oncology team. In order to minimize any preventable delay, it is prudent to inform patients about the effects of treatment on fertility and the options for fertility preservation as early as possible in the course of oncologic diagnosis and treatment procedures.
The effect of elevated estrogen levels on underlying disease
The risk of breast cancer is consistently found to be associated with persistently elevated blood estrogen levels. Serum estradiol (E2) levels are increased during ovarian stimulation for IVF and can reach levels twenty times higher than those of a natural cycle. Although the effect of a temporary increase in serum E2 levels on the risk of recurrence of breast cancer is controversial, these facts cause concern among both physicians and patients. Such concerns should not be limited to women with estrogen receptor positive breast cancer, because recent findings also suggest the presence of an indirect mitogenic effect of estrogen on hormone receptor negative breast cancer. Moreover, increased E2 levels can be relevant for patients undergoing fertility preservation treatment due to other oncologic or non-oncologic diseases considered to be estrogen sensitive, such as desmoid tumours, systemic lupus erythematosus or severe endometriosis.
In order to minimize the rise in estradiol levels in breast cancer patients undergoing IVF, Oktay et al. developed an ovarian stimulation protocol involving the concomitant use of an aromatase inhibitor, letrozole, with gonadotropins. Briefly, letrozole was started at a dose of 5 mg/day on the second day of the menstrual cycle and gonadotropins were initiated two days later. A gonadotropin releasing hormone antagonist was used to prevent premature ovulation, and human chorionic gonadotropin (HCG) was administered when at least two follicles reached 19 mm in diameter. Letrozole was reinitiated on the day of oocyte collection in order to prevent a rebound increase in E2 level. The mean peak E2 level was 406 pg/ml (range 58 to 1,166 pg/ml) in 79 women with breast cancer undergoing ovarian stimulation for embryo or oocyte cryopreservation. An average of 10.3 ± 7.75 oocytes were retrieved and 5.97 ± 4.97 embryos/oocytes cryopreserved per patient. Compared to 136 women who opted against ovarian stimulation, the recurrence and relapse-free survival rates were similar after a median follow up of 23.4 months after definitive surgery. However, it is interesting to note that with the same centre 63.3% of breast cancer patients referred for REI consultation declined ovarian stimulation and IVF due to concerns about delay of chemotherapy, effect of ovarian stimulation on cancer or costs associated with treatment.
Ethical and legal issues associated with embryo cryopreservation
When embryos are cryopreserved in a fertility preservation program, the patient/couple should make an advance decision on the fate of these embryos in the event that they are not transferred for any reason including the patient’s failure to survive cancer. It should be documented whether the remaining partner is entitled to use the embryos for his own reproductive end or whether they are to be donated to a third party, used for research or discarded. Considering these issues and making such decisions can be particularly difficult for a patient who has been recently diagnosed with a life-threatening disease and is facing a demanding treatment period. Therefore, patients should be given appropriate counselling using a multidisciplinary approach involving a psychologist and a legal advisor.
Slow freezing or vitrification for embryo cryopreservation
Several trials have been conducted to answer this question in non-cancer patients. A recent systematic review of randomized trials comparing laboratory and clinical outcome with SF or vitrification conclude that pregnancy rates were not statistically significantly different between the two methods (odds ratio (OR): 1.66, 95% confidence interval (CI): 0.98 –2.79, in favour of vitrification). However, vitrification was associated with significantly higher post-thawing survival rates, both for cleavage-stage embryos (OR: 6.35, 95% CI: 1.14–35.26) and for blastocysts (OR: 4.09, 95% CI: 2.45–6.84). Moreover, post-thawing blastocyst development of embryos cryopreserved at the cleavage stage was significantly higher with vitrification than with slow freezing (OR: 1.56, 95% CI: 1.07–2.27). It should be noted that all of the three studies that reported clinical pregnancy rates included in this review reported higher absolute figures in the vitrification arms and, given the significantly better laboratory results together with the favourable trend observed in clinical outcome, we have the impression that lack of statistical significance is more likely to be a matter of sample size. Similar to IVF embryos, we achieved higher survival and pregnancy rates following vitrification of cleavage stage IVM embryos as compared with slow-freezing. In fact, vitrification is the only method of cryopreservation currently employed at the McGill Reproductive Centre. The method of choice should be determined by the ART center based on its own experience and figures.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
