Abstract
Fertility preservation, which is defined by the National Center for Biotechnology Information as ‘a method of providing future reproductive opportunities before a medical treatment with known risk of loss of fertility’ (1), aims to mitigate the long-term effects of gonadotoxic treatment. Current evidence shows that timely fertility preservation counselling allows patients to better cope with the long-term effects of gonadotoxic treatment, including the potential for infertility (2). This chapter, thus, discusses the present status of fertility preservation in women and explores new developments in the field.
1 Introduction
Fertility preservation, which is defined by the National Center for Biotechnology Information as ‘a method of providing future reproductive opportunities before a medical treatment with known risk of loss of fertility’ (1), aims to mitigate the long-term effects of gonadotoxic treatment. Current evidence shows that timely fertility preservation counselling allows patients to better cope with the long-term effects of gonadotoxic treatment, including the potential for infertility (2). This chapter, thus, discusses the present status of fertility preservation in women and explores new developments in the field.
2 Establishing the Need for Fertility Preservation in Women Exposed to Gonadotoxic Treatment
Gonadotoxic treatments, such as radiotherapy, chemotherapy or surgery to reproductive organs, have been successfully used to treat cancer with considerably improved survival rates for young people diagnosed with cancer in the United Kingdom. As of 2010, there were more than 75,000 cancer survivors of reproductive age in the United Kingdom who were aged between 0 and 24 at the time of diagnosis (3), and the number is expected to further increase. In addition to cancer diagnoses, gonadotoxic treatment has been used for the management of non-oncological systemic diseases, such as autoimmune or haematological diseases (4). Therefore, fertility preservation should also be utilized in non-cancer conditions increasing the number of women who could benefit from it.
Evidence has shown that the reproductive capacity of women exposed to gonadotoxic treatment is reduced (5). Female reproductive capacity is dependent on effective ovarian function for ovulation, patency of fallopian tubes for fertilization and a suitable endometrial environment for implantation and in-utero development to term. Ovarian follicles are sensitive to the effects of agents that cause DNA damage such as radiotherapy and chemotherapy. Radiotherapy and chemotherapy destroy ovarian follicles and, therefore, reduce the ovarian reserve in a dose-dependent manner. Indeed, ovarian primordial follicles are particularly sensitive to radiation, with an estimated lethal dose required to destroy 50% of non-growing follicles (LD50) of about 2 Gy (6). Effective sterilizing dose (ESD) is age dependent and it is estimated to be 20.3 Gy at birth but only 14.3 Gy at 30 years of age (7, 8). As a result, more than 97% of females treated with total body irradiation (20–30 Gy) during childhood will experience premature ovarian insufficiency (9). Moreover, the risk of premature ovarian insufficiency further increases if radiotherapy is combined with chemotherapy. Chemotherapy has two separate effects on ovarian function; an immediate effect induced by the growing follicle loss that is often reversible, and a delayed effect induced by primordial follicle pool depletion. If this reduction in the primordial follicle pool is almost complete, premature ovarian insufficiency may occur immediately after treatment. In addition to radiation-related ovarian damage, the uterus may be damaged by radiation to a field that includes the pelvis. A large retrospective cohort study showed that survivors who received pelvic irradiation are at increased risk of preterm delivery and low birth weight among their children (10). Moreover, pelvic irradiation has been associated with an increased risk of miscarriage and second trimester loss (11).
3 Estimating Fertility Prognosis after Gonadotoxic Treatment
The risk of gonadotoxic treatment to fertility cannot be assessed accurately and, therefore, fertility preservation counselling should be offered to all patients receiving gonadotoxic treatment. Natural progression of a disease means that fertility prognosis could change with the risk of premature ovarian insufficiency being dependent on the planned treatment rather than the disease itself.
Traditionally chronological age and the presence of menses have been used to predict reproductive performance (12). However, this relationship is not absolute (13) and hence there was a need for other markers of fertility potential. Endocrine profiles as well as the use of transvaginal ovarian sonography in the early follicular phase of the menstrual cycle are commonly used as markers of ovarian reserve for cancer survivors (14), with high follicle-stimulating hormone (FSH) levels, low inhibin B concentration, low ovarian volume and low antral follicle count indicating a reduced reproductive potential (15).
Since 2005, measurement of serum levels of anti-mullerian hormone (AMH) has emerged as another useful marker of ovarian reserve, which has been shown to be more sensitive than FSH and inhibin B or ultrasound markers in assessing chemotherapy-induced ovarian follicle loss (16). Moreover, it may also be of value in children where FSH and inhibin B are not useful (17), as AMH is detectable in girls of all ages and rises through childhood. Moreover, prospective studies have demonstrated that pretreatment AMH could predict post-treatment ovarian recovery (18). This could potentially allow for the development of ovarian damage prediction tools that would combine chronological age and AMH concentration (19).
4 Fertility Preservation Strategies for Women
Strategies for mitigating the effects of gonadotoxic treatment on female fertility have been evolving in response to the increasing societal trend to postpone procreation until the end of reproductive years, when the incidence of diseases requiring gonadotoxic treatment is increasing, and the current focus is on quality survival.
Available fertility preservation methods range from well-established techniques, such as embryo and mature oocyte cryopreservation, to experimental techniques, such as ovarian tissue cryopreservation. Currently, the National Institute for Health and Clinical Excellence (NICE) only supports the use of embryo and mature oocyte cryopreservation following ovarian stimulation as the option of choice for fertility preservation (20).
4.1 Embryo Cryopreservation
Embryo cryopreservation is a well-established technique with proven safety and effectiveness. Indeed, embryo cryopreservation is routinely performed in assisted reproductive technique centres worldwide for many different clinical indications such as to store surplus embryos or to prevent ovarian hyperstimulation syndrome (21). Long-term outcome data on the health of children born after frozen embryo transfer have been generally reassuring (22, 23). Embryo cryopreservation can, thus, be safely used to preserve fertility in women who have gone through puberty and have an available partner or are willing to use donor sperm. However, embryo cryopreservation cannot be used for prepubertal girls or women where the timing of initiation of cancer treatment is critical.
Limitations of embryo cryopreservation include the need for controlled ovarian stimulation which requires time, involves risks that could further delay initiation of cancer treatment – such as the risk of ovarian hyperstimulation syndrome – and could potentially negatively affect the prognosis for women with oestrogen-sensitive cancer due to high oestrogen levels (24). Apart from the clinical challenges posed by embryo cryopreservation for cancer patients, complex legal and ethical issues need to be taken into consideration too. These include ownership rights for the frozen embryos and each partner’s rights to their use. In the United Kingdom, cryopreserved embryos are the joint property of the woman and her partner and this could lead to difficult ethical dilemmas and legal decisions regarding their utility or disposition in case of intra-couple disagreement (25) or death of either partner.
4.2 Mature Oocyte Cryopreservation
Mature oocyte cryopreservation could be considered as an alternative to embryo cryopreservation for patients seeking fertility preservation options and who can delay cancer treatment. Indeed, outcomes of cryopreserved-thawed oocytes have significantly improved since the introduction of vitrification and they now appear to be similar to those obtained with fresh oocytes (26). Current evidence has proven that vitrification of mature oocytes yields a high oocyte survival rate when compared with slow freezing and enhances the development of the resultant embryo(s) (27). However, these promising results are coming from mature oocyte vitrification in egg donation programmes and hence caution needs to be applied when using these outcomes to advise cancer patients. Outcome data on oocyte cryopreservation in cancer patients are limited due to the fact that oocyte cryopreservation in this population group is a relatively new approach while treatment and follow-up requirements of cancer patients mandate complete remission of the disease before considering use of cryopreserved oocytes.
Another limiting factor of oocyte cryopreservation is its low final yield. It is thought that around 20 vitrified mature oocytes are required to achieve a live birth with the quoted optimum live birth rate per vitrified oocyte in egg donation programmes being 5.7% (26). The above response to controlled ovarian stimulation protocols may prove challenging for the majority of cancer patients as a recent meta-analysis of retrospective studies showed that cancer patients had lower numbers of both total and mature oocytes in comparison to healthy, age-matched control individuals (28).
4.3 Ovarian Transposition
In ovarian transposition, the ovaries and their vessels are carefully mobilized through an open or laparoscopic approach and transplanted usually to the anterolateral abdominal wall a few centimetres above the umbilicus. Even though ovarian transposition has been shown to preserve ovarian function in the large majority of patients (88.6%–90%) (29), the risk of ovarian failure after pelvic radiotherapy varies from 15% to 40% (30, 31). This risk is further increased if radiotherapy is combined with chemotherapy (6). Moreover, as gynaecologic cancers can coexist, the risk of ovarian involvement in the presence of pelvic cancer should be assessed prior to proceeding to ovarian transposition.
4.4 Ovarian Tissue Cryopreservation
Ovarian tissue cryopreservation with subsequent transplantation has been suggested as a possible fertility preservation option for any female patient undergoing gonadotoxic treatment. Ovarian tissue containing immature follicles has been successfully cryopreserved in several animal and human models and the first successful application of this technology in humans was reported in 2004 (32). During these last fifteen years of clinical progress in ovarian tissue cryopreservation, 30 live births after orthotopic reimplantation of cryopreserved ovarian tissue have been reported (33).
Ovarian tissue cryopreservation is currently the only option available for prepubertal girls and for women who cannot significantly delay cancer treatment (34). However, major limitations still exist that prevent ovarian tissue cryopreservation from being recognized as a proven fertility preservation option. The efficiency of ovarian tissue cryopreservation and reimplantation is difficult to establish because the number of reimplantations performed worldwide remains unknown. Unpublished data, however, from the Donnez group indicate a low success rate (10% reported success rate). Moreover, as the primordial follicle pool and, therefore, the efficacy of ovarian tissue cryopreservation is age dependent, there is the need to define optimal age limits for the use of this fertility preservation technique. There have been several attempts worldwide to set the upper age limit but no recommendations have been made regarding the lower age limit yet as there is no consensus on the age at which reproductive potential is actually reached. The longevity of orthotopic ovarian tissue reimplantation is another issue. Current data supports that this procedure offers a limited window of opportunity to achieve a pregnancy for cancer survivors as the quoted mean duration of ovarian function after transplantation is 4–7 years (35). Moreover, fertility outcomes among women with proven complete restoration of ovarian function after cryopreserved-thawed ovarian tissue reimplantation have been variable. Lastly, the risk of reimplanting malignant cells remains a serious concern related to the use of cryopreserved-thawed ovarian tissue in cancer survivors. A recent review (36) classified malignant diseases into three categories based on the risk of ovarian involvement: the high risk group, which includes malignancies such as leukaemia, neuroblastoma and Burkitt lymphoma, has an estimated risk of ovarian metastasis of 11%; the moderate risk group, which includes malignancies such as non-Hodgkin lymphoma, Ewing sarcoma, has a risk of ovarian metastasis between 0.2% and 11%; and the low risk group, which includes malignancies such as Hodgkin lymphoma, Wilms’ tumour, has a <0.2% risk.
4.5 GnRH Analogues
Gonadotrophin-releasing hormone agonists (GnRHa) have been proposed as adjuvant treatment to minimize the chemotherapy-induced gonadotoxicity and were first tested more than three decades ago (37). The proposed fertility preserving mechanism of action is thought to be based on the hypogonadotrophic state that GnRHa generate. The GnRHa-induced hypogonadotrophic state causes pituitary desensitization and, thus, prevents the increase in FSH, which subsequently interrupts the vicious cycle of chemotherapy-induced follicular demise, supraphysiologic FSH levels and recruitment of further follicles (38). A recent meta-analysis showed that temporary ovarian suppression with GnRHa reduces the risk of premature ovarian insufficiency in breast cancer patients but not in ovarian or lymphoma patients. Moreover, these cancer survivors’ ultimate reproductive ability was unclear (39).
5 Future Fertility Preservation Strategies
A number of well-established and experimental fertility preservation options that are currently available for women exposed to gonadotoxic treatments have been described earlier. However, each one of them has significant limitations and, therefore, research is focused on developing future fertility preservation strategies that would reduce the off-target effects of oncological or non-oncological treatments on the ovaries while minimizing the treatment burden for patients.
For women in whom cryopreserved-thawed ovarian tissue reimplantation is not advisable due to increased risk of reimplanting malignant cells, use of isolated ovarian follicles has been proposed as a safer alternative. In vitro follicle culture aims to develop culture systems that can support complete growth of oocytes from early primordial stages through to maturity. Complete follicle development in vitro from primordial stages has only been achieved in mice (40). However, the success of complete murine in vitro oocyte development has worked as proof of concept and has driven the development of methods to be applied to other species and particularly to humans. Indeed, in recent years a great deal of progress has been made in developing in vitro follicle culture systems for humans, which could have a revolutionary impact on reproductive medicine (41).
Despite advances in in vitro follicle culture, there is still much to do before isolated ovarian follicles can be used successfully as a strategy for obtaining competent oocytes. An alternative approach would, thus, be the development of an artificial ovary by transferring isolated ovarian follicles onto a scaffold that would be able to support the growth and maturation of follicles in vivo. The first step in developing an artificial ovary was accomplished in 2012 with the creation of a biodegradable scaffold consisting of an alginate matrigel matrix onto which isolated preantral follicles were grafted (42). Moreover, an artificial ovary that allowed survival and growth of isolated murine ovarian follicles has recently been achieved using a fibrin scaffold (43).
In the past decade, the isolation of oogonial stem cells in the human ovary (44) raises the possibility of using these stem cells to produce new oocytes to replace the ovarian reserve destroyed by gonadotoxic treatment. Indeed, a number of studies have shown that oogonial stem cells can be isolated, cultured and subsequently developed into oocytes under certain conditions, even though more research is needed to improve the efficacy of oogonial stem cell isolation and development into oocytes. Alternatively, it has been hypothesized but not yet proved that induced pluripotent cell-derived stem cells from embryonic stem cells, the bone marrow or peripheral blood could provide an alternative route for ovarian reserve restoration.
Alternative mitigation strategies for gonadotoxic treatments that have been proposed and assessed in animal models (45) include targeted delivery of chemotherapeutic agents by encapsulating them in nanoparticles with special affinity to cancer cells or the use of agents that inhibit the apoptotic effects of chemotherapy or radiotherapy on primordial follicles.
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