FERTILITY PRESERVATION
Cancer patients today have an increased chance for survival and resumption of a normal life, thanks to dramatic improvements in cancer therapies. When chemotherapy or radiotherapy is required to treat cancer before or during the reproductive years, irreversible loss of procreation capacity remains a major concern. With an increased understanding of human reproduction, several options can be offered in an effort to increase the chances of future fertility. However, an urgent need remains to develop more effective strategies for fertility preservation among cancer survivors. Cancer therapies have two primary adverse effects on fertility potential. Both chemotherapy and pelvic irradiation can decrease ovarian reserve and result in failure (ref). Pelvic irradiation had additional adverse effects on the uterus that increase the subsequent risk of obstetrics complications, including restricted fetal growth and preterm births.1 It is important for both cancer patients and physicians to be aware of the currently available options for preserving fertility when undergoing cancer treatment.
Although multiple fertility preservation strategies have been proposed, most remain experimental and few are practical. Currently available strategies include medical protection during treatment, and pretreatment procedures including ovarian transposition to limit irradiation exposure, assisted reproduction techniques followed by cryopreservation of embryos or oocytes, and ovarian tissue cryopreservation for subsequent transplantation. Of these strategies, only in vitro fertilization followed by embryo cryopreservation is considered to be an established and clinically proven option for women requiring cancer treatment
HOW MANY EGGS DOES ONE HAVE?
The human ovary develops almost completely during the first trimester of gestation. Primordial germ cells migrate from the yolk sac endoderm to reach their final destination in the gonadal ridge by the seventh week of intrauterine life. There these cells develop into oogonia and differentiate by mitosis into primary oocytes. As early as eleven weeks of gestation, some primary oocytes enter their first meiotic division, which becomes arrested in the dictyate stage of prophase I to be resumed around the time of ovulation.
Oogensis, in contrast to spermatogenesis, does not continue after birth in humans (ref). As a result, the oocytes present at birth continue to deplete in number and quality throughout the reproductive lifetime. After a peak of 6 to 7 million primary oocytes is achieved at twenty weeks gestation, the number falls dramatically so that at birth, only 300,000 to 400,000 primary oocytes remain.3,4 By the time of puberty, continued oocyte depletion decreases the number of primary oocytes to approximately 200,000.
The fate of each ovarian follicle is determined by an intricate interplay of a multitude of endocrine, paracrine, autocrine and intracrine factors.5 Independent of gonadotropin stimulation, follicles develop through primordial, primary, secondary and tertiary stages, and acquire an antral cavity. Upon reaching the tertiary antral stage, the vast majority of follicles undergo atresia.
Each month gonadotropin-induced folliculogenesis results in one or two follicles maturing to the preovulatory stage.5 In women of reproductive age, the resultant mature graafian follicle is the main source of ovarian estrogens.6 Folliculogenesis is governed by many genes, proteins, factors, and hormones.7-14 Intercellular communication within each follicle plays a major role in follicular dynamics, which might explain why cells in the neighborhood of those exposed to cancer therapy.
Traditionally, it has been well estabilished that the germline cells (primordial follicles) are non renewable.
This concept was challenged by 2 recent reports15,16 stating that germline cells may be renewed locally15 or from bone marrow stem cells.16 However, this new and exciting findings were not reproduced by other groups and it was challenged itself by the opinion of some other experts in the field.17-19
Chemotherapy-induced POF
It is well known that the use of many anticancer drugs may lead to POF. Chemotherapy-induced gonadotoxicity is almost always irreversible. Histological sections of the ovary after treatment with ovariotoxic drugs show a spectrum of changes ranging from decreased numbers of follicles to absent follicles to fibrosis.20 A multitude of factors contribute towards chemotherapy-induced POF, the most important being patient’s age at the start of therapy, the chemotherapeutic agent used, and its cumulative dose. Since the number of oocytes decreases progressively with age, the risk of POF increases as a woman gets older. Given the multifactorial nature of the insult, the precise incidence of chemotherapy-induced POF is difficult to establish.
Impact of the chemotherapeutic agent on the cell cycle determines to a great extent the magnitude of harm to the ovary; cell-cycle—nonspecific chemotherapeutic agents are more gonadotoxic than cell-cycle—specific ones. Therefore, anticancer chemotherapeutic drugs are not equally ovariotoxic. Out of all cell-cycle—nonspecific anticancer drugs, alkylating agents rank high with regard to gonadotoxicity, and cyclophosphamide, which is commonly used in treating breast cancer, is the most ovariotoxic of its group. Since breast cancer is the most common of all female malignancies, cyclophosphamide is the most frequent chemotherapy-inducing ovarian failure agent.
In one series, sixty percent of women between the ages of thirty and forty years treated with cyclophosphamide suffered from POF and hypergonadotropic amenorrhea, the rate was less than fifty percent in women younger than thirty. Age is not the only determining factor in cyclophosphamide-induced POF; its cumulative dose also strongly influences the POF rate.20 Many other investigators have documented the variable effects of different anticancer regimens on ovarian functions.21,22 Therefore, not all patients receiving multiagent chemotherapy are exposed to the same risk for developing POF
Women receiving high-dose alkylating agent therapy with pelvic irradiation are exposed to the highest risk for POF after cancer treatment. Young women afflicted with Hodgkin’s disease and treated with multiagent chemotherapy and radiation to a field not including the ovaries will mostly remain fertile, but for a shorter period of time, when compared to age-matched controls.23 However, spontaneous pregnancy has been reported in a young woman treated with repeated courses of ifosfamide combined with pelvic irradiation for Ewing’s sarcoma of the pelvis with resultant POF.24
Radiotherapy-induced POF
Radiotherapy is standard treatment for a good number of genital and extragenital cancers afflicting younger reproductive-age women. These include cervical, vaginal, and anorectal cancers, some germ cell tumors, Hodgkin’s disease, and central nervous system (CNS) tumors. Pelvic radiotherapy can damage not only the ovaries but also the uterus. Akin to the impact of chemotherapy, the magnitude and duration of ovarian damage is related to the patient’s age, the total dose of radiation to the ovaries as well as the number of episodes needed to deliver the total dose.
Radiation is more toxic when given in a single dose compared to fractionated doses; fractionation plays an important role in determining the extent of damage to the ovaries.25 The breakpoint for radiation-induced ovarian failure was documented in two studies to be approximately 300 cGy to the ovaries; only 11–13 percent of women experienced ovarian failure below 300 cGy versus 60–63 percent above that threshold value.26 Radiation doses to the ovaries with standard pelvic irradiation will consistently induce ovarian failure. The addition of chemotherapy increases the risk of POF.27,28 Ovarian follicles are particularly susceptible to ionizing radiation-induced DNA damage, resulting in ovarian atrophy and a significant reduction in the ovarian follicle stores.29 Exposing the ovary to radiotherapy results in a dose-dependent reduction in the follicular pool.30 A radiation dose < 2 Gy was reported as enough to destroy 50 percent of the oocyte population (LDL50 < 2 Gy).31 At the cellular level, oocytes exhibit a rapid onset of pyknosis, disruption of the nuclear envelope, chromosome condensation, and cytoplasmic vacuolization. Serum levels of FSH and LH show a progressive rise within four to eight weeks after exposure to radiation, with a decline in serum E2 levels.29,30
Prediction and Diagnosis of Chemotherapy/Radiotherapy-inducedPOF
Important are the diagnostic and prognostic implications of predicting POF in cancer patients. Unfortunately, there is by far, no ideal, sensitive and specific marker that can reliably predict chemotherapy/radiotherapyinduced POF. Although serum FSH levels were elevated in regularly cycling cancer survivors; those of anti-Mullerian hormone (AMH) were lower than age-matched controls. However, antral follicle counts (AFC) were similar in cancer survivors and controls, despite the smaller ovarian volume in cancer patients.32 Moreover, a transient suppression of serum levels of inhibin B was witnessed among prepubertal girls receiving cancer chemotherapy. Subsequently, ovarian reserve in prepubertal girls was assessed by measurement of FSH, coupled with serum inhibin B and was proposed as a potential biomarker of the ovariotoxic effects of cancer chemotherapy in prepubertal girls.33
Basal AFC is another test of ovarian reserve that has been used extensively either alone or with other markers. AMH is produced by the granulosa cells of virtually all types of follicles from primary to the early antral stages. Therefore, it is singled out, among all other hormonal biomarkers that are dependent on the stage of follicular development, by being independent of FSH, LH, and inhibin levels. A stronger correlation of AMH levels was documented with AFC than with other parameters. Growing follicles progressively lose their ability to produce AMH, hence the decreasing peripheral AMH concentrations during ovarian stimulation. There is growing evidence that measurement of serum AMH levels could be a quantitative and possibly a qualitative biomarker of granulosa cell health and activity.34 Where no other markers of ovarian function avail so far for the at-risk prepubertal girls undergoing sterilizing cancer therapy, AMH may have promising prospects.
FERTILITY PRESERVATIION IN WOMEN: WHO MAY CONSIDER IT
In reproductive age women, breast cancer is currently the commonest malignancy that requires immediate fertility intervention; fifteen percent of all breast cancer cases occur in women younger than forty years.35,36 Cervical cancer is the other common malignancy seen in women of reproductive age requiring fertility-preserving intervention. Fifty percent of the 13,000 new cervical cancer cases that were diagnosed in the United States are estimated to be under the age of 35 years.37 Given the peculiar anatomic location, fertility preservation is even more vexing. With an ever expanding list, the indications for fertility preservation currently include gonadotoxic chemotherapy/radiotherapy for malignancies as well as for the treatment of non-malignant disease, including systemic lupus erythematosus, acute glomerulonephritis, and Behcet’s disease. Moreover many other cancer patients with extra-genital cancers are candidates for fertility preservation including those with hematopoietic cancers such as leukemias and lymphomas, musculoskeletal cancers such as Ewing’s sarcoma and osteosarcoma, neuroblastomas, and Wilm’s tumor. Women receiving chemotherapy while undergoing bone marrow transplantation and umbilical cord stem-cell transplantation are potential candidates as well as patients with non-gynecologic cancers including lymphomas, sarcomas and colorectal carcinoma.
Pharmacologically-mediated Protection
Based on the clinically valuable observation that the ovary in premenarchal years appears to be less sensitive to the effect of gonadotoxic agents, pretreatment with a gonadotropin-releasing hormone (GnRH) agonist, as well as a variety of other medications that suppress the hypothalamic-pituitary-ovarian axis, has been attempted.38,39 The rationale behind such treatment was to maintain the prepubertal ovarian quiescence during chemotherapy. Despite being theoretically plausible, conflicting conclusions regarding the effectiveness of this method have been reported by the few observational studies addressing this issue. In an excellent debate that summarizes all the studies on GnRH agonist use, Blumenfeld and colleagues reported a diminished frequency of POF in patients who received a GnRH agonist prior to chemotherapy However, the many limitations of the studies including the retrospective nature of the control group and the shorter follow-up in the GnRH group detract much from their value.40 Whereas similar results were reported by other investigators,41,42 the only prospective controlled study enrolling only eighteen women showed that GnRH analogs were not effective in the prevention of POF.43 The use of GnRH agonist has been criticized simply because if the only mechanism of ovarian protection were FSH suppression, this should not protect the primordial follicle population representing the ovarian reserve. Meanwhile, a direct ovarian effect has also been postulated. Given lack of adequate evidence so far, the controversy on the effectiveness of GnRH agonists will continue and the debate will only come to a consensus after prospective randomized clinical trials with sufficient power. However, patients and oncologists are strong advocates of GnRH use; patients find it simple and oncologists are pleased with an earlier start of medical therapy without undue delays imposed by various ART protocols. Empirical therapy with a variety of suppressive agents such as oral contraceptives or progestins have not shown efficacy in preventing chemotherapy/radiotherapy-induced ovarian damage.
Ovarian Transposition (Oophoropexy)
In patients scheduled to have sterilizing ovariotoxic radiotherapy, it is quite rational to have the ovaries displaced out of the irradiation field in the hope of preserving their function. In patients with low intestinal and genitourinary malignancies or Hodgkin’s disease who receive pelvic irradiation, such a technique helps to bring ovarian radiation exposure to a minimum. Following transposition, radiation dose to the ovaries is reduced to approximately 5-10 percent of that of normally positioned ovaries.44 When the initial dose is 4,500 cGy, the dose to each transposed ovary is 126 cGy for intracavitary radiation, 135–190 cGy for external radiation therapy and 230–310 cGy when para-aortic lymph nodes are also included in irradiation.45 Although medial transposition of the ovaries via suturing the ovaries to the uterus posteriorly and having them shielded during treatment has been tried, it did not provide adequate protection to the ovaries, compared to lateral transposition, which was shown to be more effective.46,
Oocyte Cryopreservation
Freezing mature or immature oocytes may be the only option of practical value in unmarried women and those without a partner. However, given the complex structural nature of the ovary, a multitude of factors come into play to determine the efficacy of oocyte cryopreservation. Compared to preimplantation embryos, subcellular organelles in the oocytes are far more complex, and perhaps more sensitive to thermal injury.54,55 Oocytes can be cyropreserved for years and still retain their reproductive potential. The duration of oocyte storage does not seem to interfere with oocyte survival; a number of pregnancies were reported several years after oocyte cryopreservation in liquid nitrogen.56
The ASRM practice committee in its recent evaluation of the currently available evidence, concluded that despite the limited number of established pregnancies and deliveries resulting from cryopreserved oocytes, no increase in chromosomal abnormalities, birth defects, or developmental deficits have been noted so far among offsprings and recommended that the option of oocyte cryopreservation should be considered as an experimental technique only to be performed under an investigational protocol approved by an internal review board.57 Though the initial pregnancy rates per vitrified–thawed oocyte were unacceptably low, recent technical modifications resulted in improved oocyte survival, fertilization, and pregnancy rates. Interestingly, pregnancy rates are getting better, whether slow-freeze or vitrification methods are used.58 Consequently, oocyte cryopreservation has its place not only as an adjunctive procedure in conventional IVF but also as a viable option for fertile women at risk of losing their fertility. Eagerly awaited are the results of larger prospective controlled clinical trials focusing on the efficacy, consequences, and long-term safety of oocyte cryopreservation. Only when an adequate number of births is achieved and followed-up can oocyte cryopreservation be considered of practical value. Until then, it remains
Embryo Cryopreservation
In 2005, the ASRM stated that embryo cryopreservation was the only established evidence-based and clinically valuable fertility preservation option for women. The remaining options are either too experimental or lacking adequate long-term follow-up to be of virtual applicability at the present time.2 Perhaps, it is the most effective means of fertility preservation, offering women with a partner undergoing ovarian stimulation regimens for IVF, a satisfactory chance of success. Utilizing cryopreserved embryos, delivery rates per embryo transfer have been reported by the Society for Assisted Reproduction Technology (SART) to be 31.8 percent for women under thirty-five years of age.60 Post-thaw embryo survival rates ranged between 35 and 90 percent; implantation rates were reported to be between 8 and 30 percent, and cumulative pregnancy rates over 60 percent.61,62
One of the promising experimental fertility preservation procedures in women at risk of loss of their reproductive potential is ovarian tissue cryopreservation and transplantation. The principle pertains much to autotransplantation rather than transplantation between two genetically distinct humans and hence immunosuppression will not be required. Transplantation of reproductive organs between genetically distinct humans will require long-term use of immunosuppressant agents. Consequently, cryopreservation of ovarian tissue is an integral part of the potential autotransplantation process so that the preserved germ cells can be replaced after completion of the ovariotoxic medical therapy. Better survival is expected to occur with primordial follicles residing at the ovarian cortical strips because of their smaller size and lack of follicular fluid.69 Useful information about transfer methods has been derived from animal models. Gosden and associates established the sheep model for ovarian tissue cryopreservation and transplantation by using sheep ovaries, which resemble those in humans. Follicular survival, endocrine activity, as well as pregnancy and delivery after transplantation of cryopreserved-thawed ovarian cortical strips have been successfully reported.70,71
Primordial follicles obtained from the frozen–thawed ovarian cortical strips can be allowed to mature in vitro; however, such procedure is still under investigation and will only become available in the future. Transplantation studies in SCID mice (severe combined immune deficiency mice) have demonstrated follicular maturation and completion of meiosis I in preparation for ovulation and potential fertilization.94 Technical concerns have been raised as well as concerns about potential viral infection transmission. Ovarian tissue culture with in vitro follicle maturation is valuable to avoid spread of malignant cells.
Given the fact that the primordial cells represent >90 percent of the total follicular pool and by virtue of their ability to withstand cryoinjury, isolated follicle culture from the primordial stage has been tried.95
However, isolated primordial follicles do not mature properly in culture;96 future research is necessary to define the prerequisites of adequate follicular growth and maturation and to decipher the exact role of supporting theca and granulosa cells in this process. In vitro maturation (IVM) of antral follicles, which was originally described for patients with polycystic ovary syndrome (PCOS), may have applications for cancer patients. Unfortunately, its implementation was not successful. IVM of oocytes could be considered a fertility preservation strategy as well; it is a safe and effective treatment offered in some fertility centers for assisted reproduction. Potential merits include avoidance of ovarian stimulation with expensive, and at times dangerous gonadotropins, side effects of the medications, and risks such as ovarian hyperstimulation syndrome. Although primary candidates for IVM of oocytes have been classically PCOS patients with multiple antral follicles, the spectrum of IVM indications is expanding to encompass women with primarily poor-quality embryos in repeated cycles and poor responders to stimulation. Oocyte donation and fertility preservation are two new applications for IVM, especially in women with cancer who are undergoing ovariotoxic therapy. In younger women without partners needing this treatment for fertility preservation, it is combined with oocyte vitrification. Clinical pregnancy rates per cycle in women having IVM are much related to a woman’s age; it is close to 38 percent for infertility treatment up to the age of thirty year, and around 50 percent in recipients of IVM egg donation. Unanswered remains the question of the clinical applicability and practical value of IVM as fertility preservation strategy.97,98 A method using tissue engineering principles for the culture of immature ovarian follicles followed by fertilization of oocytes in vitro has been presented by Xu et al.99 This methodology is a great step forward towards new technology for fertility preservation in female cancer patients.100,1
Cancer patients today have an increased chance for survival and resumption of a normal life, thanks to dramatic improvements in cancer therapies. When chemotherapy or radiotherapy is required to treat cancer before or during the reproductive years, irreversible loss of procreation capacity remains a major concern. With an increased understanding of human reproduction, several options can be offered in an effort to increase the chances of future fertility. However, an urgent need remains to develop more effective strategies for fertility preservation among cancer survivors. Cancer therapies have two primary adverse effects on fertility potential. Both chemotherapy and pelvic irradiation can decrease ovarian reserve and result in failure (ref). Pelvic irradiation had additional adverse effects on the uterus that increase the subsequent risk of obstetrics complications, including restricted fetal growth and preterm births.1 It is important for both cancer patients and physicians to be aware of the currently available options for preserving fertility when undergoing cancer treatment.
Although multiple fertility preservation strategies have been proposed, most remain experimental and few are practical. Currently available strategies include medical protection during treatment, and pretreatment procedures including ovarian transposition to limit irradiation exposure, assisted reproduction techniques followed by cryopreservation of embryos or oocytes, and ovarian tissue cryopreservation for subsequent transplantation. Of these strategies, only in vitro fertilization followed by embryo cryopreservation is considered to be an established and clinically proven option for women requiring cancer treatment
HOW MANY EGGS DOES ONE HAVE?
The human ovary develops almost completely during the first trimester of gestation. Primordial germ cells migrate from the yolk sac endoderm to reach their final destination in the gonadal ridge by the seventh week of intrauterine life. There these cells develop into oogonia and differentiate by mitosis into primary oocytes. As early as eleven weeks of gestation, some primary oocytes enter their first meiotic division, which becomes arrested in the dictyate stage of prophase I to be resumed around the time of ovulation.
Oogensis, in contrast to spermatogenesis, does not continue after birth in humans (ref). As a result, the oocytes present at birth continue to deplete in number and quality throughout the reproductive lifetime. After a peak of 6 to 7 million primary oocytes is achieved at twenty weeks gestation, the number falls dramatically so that at birth, only 300,000 to 400,000 primary oocytes remain.3,4 By the time of puberty, continued oocyte depletion decreases the number of primary oocytes to approximately 200,000.
The fate of each ovarian follicle is determined by an intricate interplay of a multitude of endocrine, paracrine, autocrine and intracrine factors.5 Independent of gonadotropin stimulation, follicles develop through primordial, primary, secondary and tertiary stages, and acquire an antral cavity. Upon reaching the tertiary antral stage, the vast majority of follicles undergo atresia.
Each month gonadotropin-induced folliculogenesis results in one or two follicles maturing to the preovulatory stage.5 In women of reproductive age, the resultant mature graafian follicle is the main source of ovarian estrogens.6 Folliculogenesis is governed by many genes, proteins, factors, and hormones.7-14 Intercellular communication within each follicle plays a major role in follicular dynamics, which might explain why cells in the neighborhood of those exposed to cancer therapy.
Traditionally, it has been well estabilished that the germline cells (primordial follicles) are non renewable.
This concept was challenged by 2 recent reports15,16 stating that germline cells may be renewed locally15 or from bone marrow stem cells.16 However, this new and exciting findings were not reproduced by other groups and it was challenged itself by the opinion of some other experts in the field.17-19
Chemotherapy-induced POF
It is well known that the use of many anticancer drugs may lead to POF. Chemotherapy-induced gonadotoxicity is almost always irreversible. Histological sections of the ovary after treatment with ovariotoxic drugs show a spectrum of changes ranging from decreased numbers of follicles to absent follicles to fibrosis.20 A multitude of factors contribute towards chemotherapy-induced POF, the most important being patient’s age at the start of therapy, the chemotherapeutic agent used, and its cumulative dose. Since the number of oocytes decreases progressively with age, the risk of POF increases as a woman gets older. Given the multifactorial nature of the insult, the precise incidence of chemotherapy-induced POF is difficult to establish.
Impact of the chemotherapeutic agent on the cell cycle determines to a great extent the magnitude of harm to the ovary; cell-cycle—nonspecific chemotherapeutic agents are more gonadotoxic than cell-cycle—specific ones. Therefore, anticancer chemotherapeutic drugs are not equally ovariotoxic. Out of all cell-cycle—nonspecific anticancer drugs, alkylating agents rank high with regard to gonadotoxicity, and cyclophosphamide, which is commonly used in treating breast cancer, is the most ovariotoxic of its group. Since breast cancer is the most common of all female malignancies, cyclophosphamide is the most frequent chemotherapy-inducing ovarian failure agent.
In one series, sixty percent of women between the ages of thirty and forty years treated with cyclophosphamide suffered from POF and hypergonadotropic amenorrhea, the rate was less than fifty percent in women younger than thirty. Age is not the only determining factor in cyclophosphamide-induced POF; its cumulative dose also strongly influences the POF rate.20 Many other investigators have documented the variable effects of different anticancer regimens on ovarian functions.21,22 Therefore, not all patients receiving multiagent chemotherapy are exposed to the same risk for developing POF
Women receiving high-dose alkylating agent therapy with pelvic irradiation are exposed to the highest risk for POF after cancer treatment. Young women afflicted with Hodgkin’s disease and treated with multiagent chemotherapy and radiation to a field not including the ovaries will mostly remain fertile, but for a shorter period of time, when compared to age-matched controls.23 However, spontaneous pregnancy has been reported in a young woman treated with repeated courses of ifosfamide combined with pelvic irradiation for Ewing’s sarcoma of the pelvis with resultant POF.24
Radiotherapy-induced POF
Radiotherapy is standard treatment for a good number of genital and extragenital cancers afflicting younger reproductive-age women. These include cervical, vaginal, and anorectal cancers, some germ cell tumors, Hodgkin’s disease, and central nervous system (CNS) tumors. Pelvic radiotherapy can damage not only the ovaries but also the uterus. Akin to the impact of chemotherapy, the magnitude and duration of ovarian damage is related to the patient’s age, the total dose of radiation to the ovaries as well as the number of episodes needed to deliver the total dose.
Radiation is more toxic when given in a single dose compared to fractionated doses; fractionation plays an important role in determining the extent of damage to the ovaries.25 The breakpoint for radiation-induced ovarian failure was documented in two studies to be approximately 300 cGy to the ovaries; only 11–13 percent of women experienced ovarian failure below 300 cGy versus 60–63 percent above that threshold value.26 Radiation doses to the ovaries with standard pelvic irradiation will consistently induce ovarian failure. The addition of chemotherapy increases the risk of POF.27,28 Ovarian follicles are particularly susceptible to ionizing radiation-induced DNA damage, resulting in ovarian atrophy and a significant reduction in the ovarian follicle stores.29 Exposing the ovary to radiotherapy results in a dose-dependent reduction in the follicular pool.30 A radiation dose < 2 Gy was reported as enough to destroy 50 percent of the oocyte population (LDL50 < 2 Gy).31 At the cellular level, oocytes exhibit a rapid onset of pyknosis, disruption of the nuclear envelope, chromosome condensation, and cytoplasmic vacuolization. Serum levels of FSH and LH show a progressive rise within four to eight weeks after exposure to radiation, with a decline in serum E2 levels.29,30
Prediction and Diagnosis of Chemotherapy/Radiotherapy-inducedPOF
Important are the diagnostic and prognostic implications of predicting POF in cancer patients. Unfortunately, there is by far, no ideal, sensitive and specific marker that can reliably predict chemotherapy/radiotherapyinduced POF. Although serum FSH levels were elevated in regularly cycling cancer survivors; those of anti-Mullerian hormone (AMH) were lower than age-matched controls. However, antral follicle counts (AFC) were similar in cancer survivors and controls, despite the smaller ovarian volume in cancer patients.32 Moreover, a transient suppression of serum levels of inhibin B was witnessed among prepubertal girls receiving cancer chemotherapy. Subsequently, ovarian reserve in prepubertal girls was assessed by measurement of FSH, coupled with serum inhibin B and was proposed as a potential biomarker of the ovariotoxic effects of cancer chemotherapy in prepubertal girls.33
Basal AFC is another test of ovarian reserve that has been used extensively either alone or with other markers. AMH is produced by the granulosa cells of virtually all types of follicles from primary to the early antral stages. Therefore, it is singled out, among all other hormonal biomarkers that are dependent on the stage of follicular development, by being independent of FSH, LH, and inhibin levels. A stronger correlation of AMH levels was documented with AFC than with other parameters. Growing follicles progressively lose their ability to produce AMH, hence the decreasing peripheral AMH concentrations during ovarian stimulation. There is growing evidence that measurement of serum AMH levels could be a quantitative and possibly a qualitative biomarker of granulosa cell health and activity.34 Where no other markers of ovarian function avail so far for the at-risk prepubertal girls undergoing sterilizing cancer therapy, AMH may have promising prospects.
FERTILITY PRESERVATIION IN WOMEN: WHO MAY CONSIDER IT
In reproductive age women, breast cancer is currently the commonest malignancy that requires immediate fertility intervention; fifteen percent of all breast cancer cases occur in women younger than forty years.35,36 Cervical cancer is the other common malignancy seen in women of reproductive age requiring fertility-preserving intervention. Fifty percent of the 13,000 new cervical cancer cases that were diagnosed in the United States are estimated to be under the age of 35 years.37 Given the peculiar anatomic location, fertility preservation is even more vexing. With an ever expanding list, the indications for fertility preservation currently include gonadotoxic chemotherapy/radiotherapy for malignancies as well as for the treatment of non-malignant disease, including systemic lupus erythematosus, acute glomerulonephritis, and Behcet’s disease. Moreover many other cancer patients with extra-genital cancers are candidates for fertility preservation including those with hematopoietic cancers such as leukemias and lymphomas, musculoskeletal cancers such as Ewing’s sarcoma and osteosarcoma, neuroblastomas, and Wilm’s tumor. Women receiving chemotherapy while undergoing bone marrow transplantation and umbilical cord stem-cell transplantation are potential candidates as well as patients with non-gynecologic cancers including lymphomas, sarcomas and colorectal carcinoma.
Pharmacologically-mediated Protection
Based on the clinically valuable observation that the ovary in premenarchal years appears to be less sensitive to the effect of gonadotoxic agents, pretreatment with a gonadotropin-releasing hormone (GnRH) agonist, as well as a variety of other medications that suppress the hypothalamic-pituitary-ovarian axis, has been attempted.38,39 The rationale behind such treatment was to maintain the prepubertal ovarian quiescence during chemotherapy. Despite being theoretically plausible, conflicting conclusions regarding the effectiveness of this method have been reported by the few observational studies addressing this issue. In an excellent debate that summarizes all the studies on GnRH agonist use, Blumenfeld and colleagues reported a diminished frequency of POF in patients who received a GnRH agonist prior to chemotherapy However, the many limitations of the studies including the retrospective nature of the control group and the shorter follow-up in the GnRH group detract much from their value.40 Whereas similar results were reported by other investigators,41,42 the only prospective controlled study enrolling only eighteen women showed that GnRH analogs were not effective in the prevention of POF.43 The use of GnRH agonist has been criticized simply because if the only mechanism of ovarian protection were FSH suppression, this should not protect the primordial follicle population representing the ovarian reserve. Meanwhile, a direct ovarian effect has also been postulated. Given lack of adequate evidence so far, the controversy on the effectiveness of GnRH agonists will continue and the debate will only come to a consensus after prospective randomized clinical trials with sufficient power. However, patients and oncologists are strong advocates of GnRH use; patients find it simple and oncologists are pleased with an earlier start of medical therapy without undue delays imposed by various ART protocols. Empirical therapy with a variety of suppressive agents such as oral contraceptives or progestins have not shown efficacy in preventing chemotherapy/radiotherapy-induced ovarian damage.
Ovarian Transposition (Oophoropexy)
In patients scheduled to have sterilizing ovariotoxic radiotherapy, it is quite rational to have the ovaries displaced out of the irradiation field in the hope of preserving their function. In patients with low intestinal and genitourinary malignancies or Hodgkin’s disease who receive pelvic irradiation, such a technique helps to bring ovarian radiation exposure to a minimum. Following transposition, radiation dose to the ovaries is reduced to approximately 5-10 percent of that of normally positioned ovaries.44 When the initial dose is 4,500 cGy, the dose to each transposed ovary is 126 cGy for intracavitary radiation, 135–190 cGy for external radiation therapy and 230–310 cGy when para-aortic lymph nodes are also included in irradiation.45 Although medial transposition of the ovaries via suturing the ovaries to the uterus posteriorly and having them shielded during treatment has been tried, it did not provide adequate protection to the ovaries, compared to lateral transposition, which was shown to be more effective.46,
Oocyte Cryopreservation
Freezing mature or immature oocytes may be the only option of practical value in unmarried women and those without a partner. However, given the complex structural nature of the ovary, a multitude of factors come into play to determine the efficacy of oocyte cryopreservation. Compared to preimplantation embryos, subcellular organelles in the oocytes are far more complex, and perhaps more sensitive to thermal injury.54,55 Oocytes can be cyropreserved for years and still retain their reproductive potential. The duration of oocyte storage does not seem to interfere with oocyte survival; a number of pregnancies were reported several years after oocyte cryopreservation in liquid nitrogen.56
The ASRM practice committee in its recent evaluation of the currently available evidence, concluded that despite the limited number of established pregnancies and deliveries resulting from cryopreserved oocytes, no increase in chromosomal abnormalities, birth defects, or developmental deficits have been noted so far among offsprings and recommended that the option of oocyte cryopreservation should be considered as an experimental technique only to be performed under an investigational protocol approved by an internal review board.57 Though the initial pregnancy rates per vitrified–thawed oocyte were unacceptably low, recent technical modifications resulted in improved oocyte survival, fertilization, and pregnancy rates. Interestingly, pregnancy rates are getting better, whether slow-freeze or vitrification methods are used.58 Consequently, oocyte cryopreservation has its place not only as an adjunctive procedure in conventional IVF but also as a viable option for fertile women at risk of losing their fertility. Eagerly awaited are the results of larger prospective controlled clinical trials focusing on the efficacy, consequences, and long-term safety of oocyte cryopreservation. Only when an adequate number of births is achieved and followed-up can oocyte cryopreservation be considered of practical value. Until then, it remains
Embryo Cryopreservation
In 2005, the ASRM stated that embryo cryopreservation was the only established evidence-based and clinically valuable fertility preservation option for women. The remaining options are either too experimental or lacking adequate long-term follow-up to be of virtual applicability at the present time.2 Perhaps, it is the most effective means of fertility preservation, offering women with a partner undergoing ovarian stimulation regimens for IVF, a satisfactory chance of success. Utilizing cryopreserved embryos, delivery rates per embryo transfer have been reported by the Society for Assisted Reproduction Technology (SART) to be 31.8 percent for women under thirty-five years of age.60 Post-thaw embryo survival rates ranged between 35 and 90 percent; implantation rates were reported to be between 8 and 30 percent, and cumulative pregnancy rates over 60 percent.61,62
One of the promising experimental fertility preservation procedures in women at risk of loss of their reproductive potential is ovarian tissue cryopreservation and transplantation. The principle pertains much to autotransplantation rather than transplantation between two genetically distinct humans and hence immunosuppression will not be required. Transplantation of reproductive organs between genetically distinct humans will require long-term use of immunosuppressant agents. Consequently, cryopreservation of ovarian tissue is an integral part of the potential autotransplantation process so that the preserved germ cells can be replaced after completion of the ovariotoxic medical therapy. Better survival is expected to occur with primordial follicles residing at the ovarian cortical strips because of their smaller size and lack of follicular fluid.69 Useful information about transfer methods has been derived from animal models. Gosden and associates established the sheep model for ovarian tissue cryopreservation and transplantation by using sheep ovaries, which resemble those in humans. Follicular survival, endocrine activity, as well as pregnancy and delivery after transplantation of cryopreserved-thawed ovarian cortical strips have been successfully reported.70,71
Primordial follicles obtained from the frozen–thawed ovarian cortical strips can be allowed to mature in vitro; however, such procedure is still under investigation and will only become available in the future. Transplantation studies in SCID mice (severe combined immune deficiency mice) have demonstrated follicular maturation and completion of meiosis I in preparation for ovulation and potential fertilization.94 Technical concerns have been raised as well as concerns about potential viral infection transmission. Ovarian tissue culture with in vitro follicle maturation is valuable to avoid spread of malignant cells.
Given the fact that the primordial cells represent >90 percent of the total follicular pool and by virtue of their ability to withstand cryoinjury, isolated follicle culture from the primordial stage has been tried.95
However, isolated primordial follicles do not mature properly in culture;96 future research is necessary to define the prerequisites of adequate follicular growth and maturation and to decipher the exact role of supporting theca and granulosa cells in this process. In vitro maturation (IVM) of antral follicles, which was originally described for patients with polycystic ovary syndrome (PCOS), may have applications for cancer patients. Unfortunately, its implementation was not successful. IVM of oocytes could be considered a fertility preservation strategy as well; it is a safe and effective treatment offered in some fertility centers for assisted reproduction. Potential merits include avoidance of ovarian stimulation with expensive, and at times dangerous gonadotropins, side effects of the medications, and risks such as ovarian hyperstimulation syndrome. Although primary candidates for IVM of oocytes have been classically PCOS patients with multiple antral follicles, the spectrum of IVM indications is expanding to encompass women with primarily poor-quality embryos in repeated cycles and poor responders to stimulation. Oocyte donation and fertility preservation are two new applications for IVM, especially in women with cancer who are undergoing ovariotoxic therapy. In younger women without partners needing this treatment for fertility preservation, it is combined with oocyte vitrification. Clinical pregnancy rates per cycle in women having IVM are much related to a woman’s age; it is close to 38 percent for infertility treatment up to the age of thirty year, and around 50 percent in recipients of IVM egg donation. Unanswered remains the question of the clinical applicability and practical value of IVM as fertility preservation strategy.97,98 A method using tissue engineering principles for the culture of immature ovarian follicles followed by fertilization of oocytes in vitro has been presented by Xu et al.99 This methodology is a great step forward towards new technology for fertility preservation in female cancer patients.100,1