Fertility Preservation: Advances and Controversies Botros RMB Rizk, Mohamed A Bedaiwy
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section 1
1FERTILITY PRESERVATION APPROACHES IN THE FEMALE2

Fertility Preservation in Female Patients: An Overview1

Mohamed A Bedaiwy,
Candice P Holliday,
Botros Rizk

CHAPTER OUTLINE

  • Evolution of Human Oogenesis
  • Anticancer Therapy-Induced Premature Ovarian Failure (POF)
    • POF Induced by Chemotherapy
    • POF Induced by Radiotherapy
  • Chemotherapy/Radiotherapy-Induced POF: Diagnosis and Prediction
  • When is Fertility Preservation Indicated in Women?
  • Strategies for Fertility Preservation
    • Pharmacologically-Based Protection
    • Transposition of the Ovaries (Oophoropexy)
    • Assisted Reproductive Technologies
  • Introduction of Vascular Grafts in Animal Models
  • The Use of Vascularized Grafts in Human Trials
  • Heterologous Ovarian Transplantation
  • In Vitro Maturation of Oocytes
 
INTRODUCTION
Female fertility can be adversely influenced by cancer therapies that affect the ovary and uterus. At the ovarian level, pelvic irradiation and chemotherapy can decrease ovarian reserve and result in ovarian failure. Moreover, pelvic irradiation can adversely affect the uterus, increasing the subsequent risk of obstetrical complications such as intrauterine fetal growth restriction and preterm birth.1 A number of potential options are currently available, however, to help preserve a woman's future fertility, thanks to a better understanding of the intricate mechanisms governing human reproduction. They are: [a] storage of frozen embryos, [b] storage of frozen ovarian tissue or the whole ovary for future transplantation, [c] storage of frozen ovarian tissue or isolated follicles for in vitro growth and maturation, [d] ovarian transposition before radiotherapy, [e] ovarian protection during chemotherapy with either GnRH analogs or antiapoptotic agents (e.g. sphingosine-1-phospate), [f] uterine transplant after successful cancer treatment where a functional uterus no longer exists and [g] in vitro fertilization (IVF) with embryo cryopreservation prior to cancer therapy.
Of those, only the last one—IVF followed by oocyte cryopreservation or embryo cryopreservation—is considered a clinically proven option for women requiring cancer treatment.2 The remaining strategies are considered experimental largely due to unproven efficacy or lack of adequate long-term follow-up.
In this review, the consequences of irradiation and chemotherapy on ovarian function and fertility as well as current and future fertility preservation strategies in female cancer patients will be discussed.
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EVOLUTION OF HUMAN OOGENESIS
By the end of the first trimester of pregnancy, the human ovaries are almost fully formed. By the seventh week of intrauterine life, primordial germ cells reach their final destination in the gonadal ridge after migrating from the yolk sac endoderm. They differentiate into oogonia and eventually into primary oocytes by mitosis. Before the twelfth week, some primary oocytes enter their first meiotic division, which becomes arrested in the dictyotene stage of prophase I. This process resumes with the first ovulation. Contrary to spermatogenesis, oogenesis in humans does not continue after birth. The maximum number of primary oocytes (6 to 7 million) is attained at 20 weeks of intrauterine life. That number falls dramatically to 400,000 at birth and approximately 200,000 at the time of puberty.3,4 Oocyte reserve continues to diminish both in quantity and quality throughout the remaining life cycle.
A multitude of endocrine, paracrine, autocrine and intracrine factors govern the fate of each ovarian follicle.5 During the menstrual cycle, follicles mature in a series of stages (primordial, primary, secondary and tertiary) in a process called folliculogenesis, which is independent of gonadotropin stimulation. The vast majority of follicles undergo atresia after differentiation into tertiary follicles with an antral cavity. During any ovarian cycle, one or two follicles mature to the preovulatory stage as a result of gonadotropin-induced folliculogenesis.5 Throughout reproductive life, the main source of ovarian estrogens remains the mature Graafian follicle.6 Many genes, hormones, proteins and factors are responsible for governing the intricate process of folliculogenesis in humans.7–14 The dynamic nature of folliculogenesis makes it a principal target for the cytotoxic treatment.15 Intrafollicular communication between the cells controls follicular dynamics; exposure to cancer therapy frequently results in the death of many neighboring cells too.6,7,12,13
Traditionally, it has been well-established that the germline cells (primordial follicles) are non-renewable. This concept was challenged by 2 recent reports stating that germline cells may be renewed locally16 or from bone marrow stem cells.17 However, these new and exciting findings were not reproduced by other groups and were challenged by the opinion of some other experts in the field.18–20
 
ANTICANCER THERAPY-INDUCED PREMATURE OVARIAN FAILURE (POF)
 
POF Induced by Chemotherapy
Premature ovarian failure (POF) occurs after exposure to many anticancer drugs. Chemotherapeutic agents can be mainly classified into alkylating agents (e.g. cyclophosphamide, mechlorethamine, etc.), antitumor antibiotics (e.g. bleomycin, adriamycin, etc.), plant alkaloids (e.g. taxanes, vinblastine, etc.), antimetabolites (e.g. 5-fluorouracil, methotrexate, etc.), topoisomerase inhibitors (e.g. etoposide phosphate, etc.) and others (e.g. cisplatin).21 Fifty percent of women over the age of 25 and 25% of women under the age of 25, will develop POF after being treated with mechloroethamine, vincristine, procarbazine and prednisone.22 Unfortunately, chemotherapy-induced ovariotoxicity is almost always an irreversible insult. After treatment with ovariotoxic drugs, the ovary exhibits a spectrum of histological changes ranging from decreased numbers of follicles to absent follicles to fibrosis.23 The variable effects of different anticancer regimens on ovarian functions have been reported by many other investigators.24,25
The incidence of chemotherapy-induced POF cannot be precisely estimated because of the multifactorial nature of the insult. However, a number of factors can increase the risk:
  • The cumulative dose
  • The patient's age at the start of treatment. POF increases with advancing age owing to progressive age-dependent follicular depletion
  • The degree of gonadotoxicity. This varies from one chemotherapeutic drug to another.
Cell cycle specificity of the chemotherapeutic agent is the major determinant for the magnitude of ovarian injury; cell cycle–nonspecific chemotherapeutic agents are more ovariotoxic than cell cycle–specific ones. Alkylating agents are the most gonadotoxic of all cell cycle–nonspecific anticancer drugs. Cyclophosphamide, a common treatment for breast cancer, is the most ovariotoxic of its group. Given the fact that breast cancer is the most common malignancy in women, most cases of chemotherapy-induced ovariotoxicity are induced by cyclophosphamide. Patient age and cumulative dose most strongly influence the POF rate in those taking this drug.23 Less than 50% of women treated with cyclophosphamide, who are younger than 30 years old develop POF. On the other hand, approximately 60% of women between the ages of 30 and 40 years, will develop POF and hypergonadotropic amenorrhea.23
Consequently, the risk of POF is not the same in all patients receiving multiagent gonadotoxic chemotherapy. Women with the highest risk for developing POF are those who receive highdoses of alkylating agents with pelvic irradiation. In contrast, young women with Hodgkin's disease treated with multiagent chemotherapy and radiation to a field that does not include the ovaries will often remain fertile, although usually for a shorter period of time than that of age-matched controls.26 In one case report, a young woman treated with repeated courses of ifosfamide combined with pelvic irradiation for Ewing's sarcoma of the pelvis who developed POF was able to achieve a spontaneous pregnancy.27
 
POF Induced by Radiotherapy
Among young women of reproductive age, radiotherapy is standard treatment for many genital as well as extragenital cancers, including cervical, vaginal and anorectal cancers, some germ cell tumors, Hodgkin's disease and central nervous system (CNS) tumors. Pelvic radiotherapy cannot only damage the ovaries, but also the uterus. The patient's age, total dose of radiation reaching the ovaries and the number of sessions needed to deliver the total dose are the prominent factors that determine both the extent and duration of ovarian damage. This is quite similar to the impact of chemotherapy.
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Fractionating the total dose of radiotherapy significantly reduces the extent of ovarian damage.28 Single-dose radiation is more toxic than fractionated therapy. The approximate threshold for radiation-induced ovarian failure is 300cGy to the ovaries; only 11% to 13% of women developed POF at doses below 300cGy versus 60% to 63% at doses above that threshold.29 Standard pelvic irradiation to the ovaries will consistently result in POF; this is compounded by co-administered chemotherapy.30,31
Ionizing radiation is particularly deleterious to ovarian follicles, resulting in radiation-induced DNA damage, ovarian atrophy and a significant depletion of ovarian follicles.32 Oocytes exhibit a rapid onset of pyknosis, disruption of the nuclear envelope, chromosome condensation and cytoplasmic vacuolization. Within 4 to 8 weeks after exposure to radiation, serum levels of FSH and LH progressively rise while serum E2 levels fall.32,33 Radiotherapy results in a dose-dependent reduction in the follicular pool.33 Fifty percent of the oocyte population is destroyed by a radiation dose < 2 Gy (LDL50< 2 Gy).34
 
CHEMOTHERAPY/RADIOTHERAPY-INDUCED POF: DIAGNOSIS AND PREDICTION
The diagnostic and prognostic implications of predicting POF in cancer patients cannot be overemphasized. Researchers are currently looking for a biomarker that can reliably predict chemotherapy-or radiotherapy-induced POF with satisfactory sensitivity and specificity. Among those potential markers are anti-Mullerian hormone (AMH), serum FSH, inhibin B and basal antral follicle counts (AFC).
AMH is produced by the granulosa cells of virtually all types of follicles, from the primary to early antral stages. Growing follicles progressively lose their ability to produce AMH, which is why peripheral AMH concentrations decrease during ovarian stimulation. Unlike all other hormonal biomarkers, which are dependent on the stage of follicular development, AMH is independent of FSH, LH and inhibin levels.
Serum FSH levels were elevated in cancer survivors with regular menstrual cycles, whereas AMH levels were lower than those of age-matched controls.35 Despite the smaller ovarian volume in cancer patients, AFC were similar between cancer survivors and controls.35 In pre-pubertal girls receiving anticancer chemotherapy, serum levels of inhibin B transiently decreased. Consequently, serum FSH, coupled with serum inhibin B, was proposed as a potential biomarker of the ovariotoxic effects of cancer chemotherapy in pre-pubertal girls.36
Basal AFC have been used extensively alone or with other markers to predict POF. An AFC < 5 is predictive of poor ovarian reserve.37 Measurement of serum AMH levels could be a quantitative and possibly a qualitative biomarker of granulosa cell health and activity.38 For pre-pubertal girls undergoing sterilizing cancer therapy, AMH may have promising prospects.
 
WHEN IS FERTILITY PRESERVATION INDICATED IN WOMEN?
Breast cancer is the most common malignancy seen during the reproductive years that requires an immediate fertility-preserving intervention. One of every 7 breast cancer patients is younger than 40 years old.39, 40 Unfortunately, breast cancer in young women exhibits a high ductal infiltrating effect and most of these women are required to undergo highly gonadotoxic chemotherapy for treatment.41 Another common malignancy seen in younger women that requires fertility-preserving intervention is cervical cancer. Of the 13,000 new cervical cancer cases diagnosed in the United States each year, 50% are estimated to occur in women younger than 35 years of age.42 Moreover, owing to the peculiar anatomic location of the cervix, fertility preservation is even more challenging in those cases. Because it is usually difficult to estimate infertility risk, it is essential to perform conservative surgery when fertility preservation is offered.43
The indications of fertility preservation in women currently include ovariotoxic radiotherapy/chemotherapy for cancer as well as for the treatment of a multitude of nonmalignant conditions such as systemic lupus erythematosus, acute glomerulonephritis, and Behcet's disease. On the other hand, many other patients with extragenital cancers are candidates for fertility-preserving procedures, including those with hematopoietic cancers such as leukemias and lymphomas, musculoskeletal cancers such as Ewing's sarcoma and osteosarcoma, neuroblastomas and Wilms' tumor. Women receiving chemotherapy while undergoing bone marrow transplantation or umbilical cord stem-cell transplantation are potential candidates as well as patients with non-gynecologic cancers including lymphomas, sarcomas and colorectal carcinoma. Some of the salient indications of fertility preservation in women are summarized in Table 1.
 
STRATEGIES FOR FERTILITY PRESERVATION
A spectrum of potentially promising strategies has been assessed for fertility preservation. Although the currently available armamentarium encompasses an ever expanding list, most of those are more experimental than practical. Moreover, none have been tested in a prospective randomized controlled trial (RCT).
 
Pharmacologically-Based Protection
The theoretical rationale behind pharmacologically-based protection came from the intriguing observation that the premenarchal ovary is less sensitive to the effects of ovariotoxic agents. Consequently, researchers have studied pretreatment with a GnRH agonist, as well as many other medications that suppress the hypothalamic pituitary ovarian axis, in an endeavor to maintain the pre-pubertal ovarian quiescence during chemotherapy.44,45 Meanwhile, a direct ovarian effect has also been postulated.44,45
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TABLE 1   Situations necessitating fertility preservation in women
1. Nonmalignant conditions treated with ovariotoxic radiotherapy orchemotherapy:
  1. Multi-system disorders
    • Acute glomerulonephritis
    • Behcet's disease
    • Systemic lupus erythematosus (SLE)
  2. Transplantation procedures:
    • Umbilical cord stem-cell transplantation (SCT)
    • Bone marrow transplantation (BMT)
2. Malignancies treated with ovariotoxic radiotherapy or chemotherapy:
  1. Adult cancers:
    1. Gynecologic cancers:
      • Cancer of the cervix
      • Breast cancer
    2. Non-gynecologic cancers:
      • Sarcomas
      • Lymphomas
      • Colorectal cancer
  2. Childhood malignancies:
    1. Hematopoietic cancers:
      • Lymphomas
      • Leukemias
2. Musculoskeletal cancers:
  • Ewing's sarcoma
  • Osteosarcoma
3. Other cancers:
  • Wilms' tumor
  • Neuroblastomas
3. Bilateral oophorectomy for benign ovarian tumors, endometriosis, or prophylaxis.
However, theory and practice do not always meet. The few observational studies addressing this issue have come to conflicting conclusions. Blumenfeld and colleagues reported a diminished frequency of POF in patients who received a GnRH agonist prior to chemotherapy in their analysis of all studies on GnRH agonist use.46 Most of those studies, however, were limited by short follow-up period in the GnRH group and the retrospective nature of the control groups.46 Other investigators47,48 have come to similar conclusions. When Demeestere et al. (2012) studied triptorelin, a GnRH agonist, for its ability to prevent chemotherapy-induced ovarian failure in patients with lymphoma, 20% of patients in both the control and treatment groups experienced POF during a 1-year follow-up.49 Another recent study of 281 patients suggested that triptorelin-induced temporary ovarian suppression during chemotherapy in premenopausal patients with early-stage breast cancer reduced the occurrence of chemotherapy-induced early menopause.50 On the other hand, a prospective controlled study found that GnRH analogs were not effective in the prevention of POF.51 That particular study, however, was limited by a small sample size.
Patients and oncologists strongly advocate GnRH use; oncologists are satisfied with starting cancer therapy without avoidable delays caused by standard protocols of assisted reproductive technologies (ART) and patients find it simple. Alternatively, counter-arguments downplaying the use of GnRH agonists are centered on the fact that FSH suppression will not protect the primordial follicles constituting the ovarian reserve. Combining a GnRH antagonist with a GnRH agonist, which would add the quick onset of action of a GnRH antagonist to the long-lasting effects of a GnRH agonist, is being considered.52,53 Because prospective randomized studies with statistically accurate numbers of patients and sufficient follow-up are few and showed contradictory results, the evidence of efficacy of GnRH agonists and their protective mechanisms remains lacking and the debate will continue.49,54–57 Empirical use of other suppressive drugs such as combined oral contraceptives (COCs) or progestins has not been successful in preventing chemotherapy/radiotherapy-induced ovarian damage.
 
Transposition of the Ovaries (Oophoropexy)
Another possible fertility preservation option for women scheduled to undergo sterilizing ovariotoxic radiotherapy is surgery, in which the ovaries are moved away from the intended irradiation field. In fact, robotic-assisted ovarian transposition is available.58 Ovarian transposition helps curtail ovarian radiation exposure in patients with low intestinal and genitourinary malignancies and in those with Hodgkin's disease receiving pelvic irradiation. The radiation dose to the ovaries is reduced to approximately 5% to 10% following transposition.59 Given an initial dose of 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 with para-aortic lymph node inclusion in irradiation.60 Lateral transposition of the ovaries was shown to be more effective than medial transposition—with the latter, the ovaries are sutured posteriorly to the uterus and shielded during treatment.61,62
Lateral ovarian transposition can be performed during staging laparotomy for Hodgkin's disease (which is not customary nowadays) and during radical hysterectomy for cervical cancer.63 Laparoscopic ovarian transposition could be considered a valuable option before starting radiotherapy. Moreover, immediate postoperative radiotherapy could be initiated, which could furthermore help prevent POF if the ovaries were to migrate back to the irradiation field.31,64,65 Additionally, the same anesthetic can be used for laparoscopic ovarian transposition as that used to insert a brachytherapy device in cases of cervical or vaginal cancers treated by brachytherapy.66 It can also be performed on an outpatient basis in patients with Hodgkin's disease, leading to a better cosmetic effect and earlier recovery with minimal cost and discomfort.
Following laparoscopic ovarian transposition, almost all women with Hodgkin's disease (stage I and stage II) treated with radiation alone or with minimal chemotherapy retain their ovarian function and fertility.31 Laparoscopic ovarian transposition is not a complication-free procedure, however. POF is still a concern, and symptomatic ovarian cysts may develop as well. There are many potential causes of ovarian failure following ovarian transposition67 and they are summarized in Table 2. Although, COCs can help suppress cyst formation, the underlying etiology remains largely obscure.68
 
Assisted Reproductive Technologies
In patients, who wish to proceed to fertility preservation, ART are probably the most commonly adopted policy, as seen in Table 3.
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TABLE 2   Potential causes of ovarian failure following ovarian transposition before radiotherapy
A. Vascular insult:
  1. Jeopardizing ovarian vessels
  2. Radiation-mediated injury of the vascular pedicle
B. Anatomically-mediated mechanisms:
  1. Transposed ovaries not moved far enough
  2. Ovarian migration back to original position following use of absorbable sutures
 
Oocyte Cryopreservation
In unmarried women and those without a male partner, freezing mature or immature oocytes may be the only practical option. For oocyte cryopreservation, the present rate of pregnancy after thawing, IVF and intrauterine transfer is 57% per embryo.69 A multitude of factors come into play to determine the efficacy of oocyte cryopreservation, given the complex structural nature of the ovary. In contrast to pre-implantation embryos, subcellular organelles in the oocytes are far more complex and perhaps more sensitive to thermal injury.70,71 Oocytes can be cryopreserved for years and still retain their reproductive potential. Moreover, the duration of oocyte cryopreservation does not seem to interfere with oocyte survival; a number of pregnancies have been reported several years after oocyte cryopreservation using liquid nitrogen.72
Based on the evidence available in 2008, the ASRM practice committee concluded that, although no longer considered an experimental, oocyte cryopreservation is associated with a limited number of established pregnancies and deliveries resulting from cryopreserved oocytes.73 However, no increases in chromosomal abnormalities, birth defects, or developmental deficits were noted among offspring at that time. In 2012, the ASRM practice committee released an evaluation of mature oocyte cryopreservation and found that vitrification for oocyte cryopreservation significantly improved oocyte survival and pregnancy rates.74 Further, it stated that there is a decent amount of evidence that the fertilization and pregnancy rates of vitrified/warmed oocytes as a part of IVF/intracytoplasmic sperm injection (ICSI) in young patients was similar to that of IVF/ICSI with fresh oocytes. The practice committee cautioned, however, that the data was not necessarily generalizable because of reporting bias, the use of only healthy, young oocyte donors and the specific cryopreservation protocols used.
Successful human oocyte cryopreservation has recently become a reality. In fact, human GV oocytes also can be successfully cryopreserved after undergoing in vitro maturation (IVM).75 The human oocyte is one of the largest cells in the body measuring approximately 130µm and has a low surface area to volume ratio.76 This makes the oocyte more prone to water retention and damage caused by ice crystals. Avoidance of chilling injury is the most important goal to improve survival rates. Cryoprotectants protect the cells by forming hydrogen bonds with water molecules, hence eliminating ice formation and limiting damage caused by high salt concentration.
From 1986, when the first pregnancy using frozen oocytes was achieved by Chen until 1997 when ICSI was first used to fertilize frozen/thawed oocytes,77 there have only been a few live births from frozen oocytes.76 By 2004, only 100 births were recorded from frozen oocytes.78,79 By 2012, more than 1000 babies have been born following oocyte cryopreservation.76,80
To maintain the long-term viability after storage, living cells must be brought into a state of suspended animation in which they remain for indefinite periods of time.76 The temperature of liquid nitrogen (−196°C) appears to be adequate for these purposes. At these temperatures, water exists in only a solid state and no biologic reactions occur. Human oocytes are viable at 37°C and inactive at −196°C. Therefore, the lapse time of major danger occurs during the temperature decreasing and the rewarming phases.76
TABLE 3   Live births after fertility preservation techniques. Modified from Yasmin E and Davies MC. Pregnancy after fertility preservation. In: Jauniaux E and Rizk B (Eds), pregnancy after assisted reproductive technology. Cambridge University Press; New York. 2012; Chapter 11:pp. 124-36
Method
Evidence
Live Births
Embryo cryopreservation
Wennerholm et al., 2009
>350,000**
Oocyte cryopreservation
Troukoudes et al., 2011
Grynberg et al., 2011
>500
Ovarian tissue cryopreservation and regrafting
Donnez et al., 2004, 2011
Meirow et al., 2005
Anderson et al., 2008
Silber et al., 2008
Sanchez Serrano et al., 2010
15 reported
Ovarian transposition and oophoropexy
Bisharah and Tulandi, 2003
Kuohung et al., 2008
Terenziani et al., 2009
Not quantified
Ovarian suppression with GnRH
Badaiwy et al., 2009
Guiseppe et al., 2007
Nitzschki et al., 2010
Not quantified
**p=0.01
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Conventional slow freezing methods are associated with relatively low success rates, although survival rates are acceptable. Slow freezing methods need low initial cryoprotectant levels, which are associated with lower toxicity. Concentration increases only when the cells decrease their metabolism. At the temperature of −6°C, introducing a seed-ice crystal may be induced. The temperature is then slowly decreased up to −196°C. During thawing, a rapid transitional temperature is preferred to prevent recrystallization of water.
During the last decade, vitrification has been introduced whereby water is prevented from forming ice due to the viscosity of the highly protective cryoprotectant cooled at an extremely rapid rate. The recent advances in vitrification methods have increased oocyte survival to over 80%. Recent studies suggest that vitrification is the most efficient means of oocyte cryopreservation.
Recently, Noyes et al. (2009) published data regarding verified live-born infants after oocyte cryopreservation between 1986 and 2008.80 The authors included 609 live-born babies; 308 from slow freezing, 289 from vitrification and 12 from both methods. An additional 327 live births were documented76 (Table 4, 5). The rate of birth anomalies that resulted was 1.3%, which is comparable to that in spontaneous pregnancies.81
Chian et al. (2008) reported the obstetric and perinatal outcomes of 200 infants born in 165 pregnancies from oocyte vitrification at McGill University in Montreal, Canada.82 The multiple birth rate was 17% (26 twins and 2 triplets). The cesarean section rate was 37% in singleton pregnancies and 96% in multiple pregnancies, whereas the mean birth weight was 2920 +/–37g for singletons and 2231 +/–55g for multiples. The incidence of congenital malformations was 2.5% (two ventricular septal defects, one biliary atresia, one clubfoot and one skin hemangioma).
TABLE 4   Data from oocyte cryopreservation births “series reports”. Modified from Porcu and Bazzocchi, pregnancy outcome after oocyte and embryo cryopreservation. In: Jauniaux E and Rizk B (eds), pregnancy after assisted reproductive technology. Cambridge University Press; New York. 2012; Chapter 10:pp.108-23.
TABLE 5   Obstetric and perinatal outcomes and incidence of congenital malformations in children conceived after oocyte vitrification. Reproduced with permission from: Porcu and Bazzocchi, pregnancy outcome after oocyte and embryo cryopreservation. In: Jauniaux E and Rizk B (Eds), pregnancy after assisted reproductive technology. Cambridge University Press; New York. 2012; Chapter 10:pp.108-23.
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Wennerholm et al. (2009) published data on 148 children born from oocyte slow-freezing and 221 children born after vitrification of oocytes.83
The obstetric and perinatal data on the outcome of children born after oocyte vitrification is of primary importance. In the United States, a national 5 year prospective multi-center observational study (HOPE) has been registered and its reports are awaited with interest.84
 
Embryo Cryopreservation
In 2005, embryo cryopreservation was described by the ASRM as the only established evidence-based and clinically valuable fertility preservation option for women with a male partner. There are two basic techniques employed for cooling of embryos: controlled slow freezing and ultra rapid vitrification.85 Embryo cryopreservation is presumably the most effective means of fertility preservation in women undergoing ovarian stimulation regimens for IVF and it offers them a satisfactory chance of success. The delivery rate per embryo transfer has been reported by the Society for Assisted Reproduction Technology (SART), using cryopreserved embryos, to be 31.8% for women less than 35 years of age.86 Post-thaw embryo survival rates range between 35% and 90% and implantation rates range between 8% and 30%; cumulative pregnancy rates have been reported to be more than 60%.87,88
Consequently, embryo cryopreservation is considered the fertility preservation option with the best outcome. Moreover, long-term follow-up data on the outcome of children born from these procedures are reassuring and confirm that the procedure is safe. A number of limitations do, however, exist. First, it is unsuitable for pre-pubertal or adolescent females and for women without a partner. Second, ovarian stimulation protocols in IVF inevitably lead to extremely high estradiol levels, which may be dangerous for women with estrogen-sensitive tumors such as breast cancer. The typical time interval between breast cancer surgery and initiation of chemotherapy is six weeks. In lieu of the standard protocol, a short flare protocol is adopted, which usually requires less time to achieve follicle recruitment.89 Conventional controlled ovarian hyperstimulation (COH) produces exceptionally high levels of estrogen, potentially affecting the overall prognosis.90 Therefore, some centers offer unstimulated natural-cycle IVF, where a single oocyte is aspirated. Cancellation rates are, however, high and pregnancy rates very low (7.2% per cycle and 15.8% per embryo transfer).91,92
The nonsteroidal antiestrogen tamoxifen92 and the aromatase P450 inhibitor letrozole93 were used as potential alternatives for ovulation induction in the breast cancer patient. Oktay and colleagues reported, in their prospective controlled study comparing tamoxifen to letrozole as ovarian stimulation agents in breast cancer patients, that when combined with low-dose FSH, both drugs had a better outcome than when tamoxifen was used alone.93 Letrozole was preferred to tamoxifen because its use was accompanied with significantly lower peak serum estradiol levels, which is a potential asset for breast cancer patients.93 They further compared the safety and success of ovarian stimulation with letrozole and tamoxifen in breast cancer patients undergoing IVF to cryopreserve their embryos for fertility preservation and reported similar cancer recurrence rates between those who underwent COH and those who did not.94 Because there are various ethical and legal issues associated with embryo cryopreservation, it is encouraged that these patients be cared for using a multidisciplinary approach, including both psychological and legal counseling.95
 
Cryopreservation and Ovarian Tissue Transplantation
Ovarian tissue cryopreservation and transplantation have emerged as promising experimental fertility preservation procedures in women at risk of losing their reproductive capacity. Given the fact that ovarian tissue transplantation is an auto-transplantation rather than transplantation between two genetically distinct humans, immunosuppression is unnecessary. In contrast, long-term immunosuppression is required after transplantation of reproductive organs between genetically distinct humans. Cryopreservation of ovarian tissue is, therefore, an indispensible prerequisite to the potential autotransplantation process, allowing preserved germ cells to be replaced after completion of the ovariotoxic cancer therapy. Owing to their smaller size and lack of follicular fluid, better survival is expected to occur with primordial follicles occupying the ovarian cortical strips.96 Moreover, the advantage of ovarian tissue harvesting is that it can be immediately performed laparoscopically and avoids exposing estrogen sensitive tumorsto the higher estrogen levels associated with ovarian stimulation.97
Animal models have provided invaluable information about transfer methods. Sheep ovaries, which resemble human ovaries, have been used as a model for ovarian tissue cryopreservation and transplantation by Gosden and colleagues.98 Survival and endocrine activity of the follicular apparatus as well as pregnancy and delivery after transplantation of cryopreserved-thawed ovarian cortical strips have been successfully reported.98,99
 
Minimizing Ischemic Damage in Ovarian Tissue Transplantation
In some human ovarian transplants, where non-vascularized grafts were used, an initial ischemic insult was noted, which was postulated to be, at least in part, responsible for the limited lifespan of ovarian function. Therefore, it is vital to reduce the duration of ischemia and provide immediate revascularization of the grafts to prolong their longevity and help preserve their function for as long a duration as possible. Thus, the subject of graft longevity as affected by graft ischemia time or by cryopreservation damage is now the subject of intense study.100
To minimize ischemic damage of the grafts, Almodin and associates described a novel technique in which frozenthawed fragments of one ovary were injected into the cortex of the remaining sterile ovary.101 In that experimental trial, radiotherapy was administered to ewes to induce ovarian failure in one ovary while fragments of the other ovary were kept frozen. Consequently, injection of the thawed fragments of the frozen ovary inside the cortex of the remaining irradiated ovary was carried out adopting a “sowing” procedure, which did require suturing. Rams were used to impregnate the ewes 6 months10 after grafting. By this novel procedure, they documented that prevention of ischemic damage was successful via intracortical grafting of the germinative ovarian tissue.101 Transplantation of ovarian tissue into angiogenic granulation tissue during the process of wound healing was improvised to help prevent the initial ischemic insult to oocytes. The duration of ischemia was reduced by 24 hours, thereby significantly increasing the healthy primordial follicular pool and the reperfused area of the transplanted grafts. Two days after transplantation, the graft became revascularized (functional blood vessels were seen) and functional integrity returned.102
 
INTRODUCTION OF VASCULAR GRAFTS IN ANIMAL MODELS
As a potential way of avoiding ischemic damage to the graft, vascularized grafts have been successfully developed. With these grafts, there is immediate revascularization of the grafted ovarian tissue. The principle of vascularized grafting was challenging in two distinctive perspectives, despite its theoretical plausibility. First, the whole ovary along with the vascular pedicle must be cryopreserved, which is technically challenging. Second, the graft is reattached using microvascular anastomosis, which is a time-consuming and skill-demanding procedure. Reproductive surgeons are, therefore, required to be skillful at microvascular anastomosis; experience can readily be obtained from vascular and plastic surgeons with their vast experience in microsurgery on small vessels.
The ‘ischemia time’ is defined as the duration of time in which the grafted ovarian tissue can withstand ischemia without resultant ischemic tissue damage. Large ovarian cortical strips were reported to withstand an ischemic insult for variable durations103 without inducing significant histological or molecular changes.104 Transplanting an intact fresh ovary together with its vascular pedicle using microvascular reanastomosis has been successfully performed. As with other tissue grafting procedures, if the vascular graft was technically feasible and successful, survival of the ovarian graft was assured.105
Preservation of ovarian function after grafting is all important. Restoration of ovarian function after transplantation of a cryopreserved intact sheep ovary with its vascular pedicle has been documented.106 Consequently, transplantation of fresh as well as cryopreserved-thawed (C-T) intact ovaries to heterotopic sites was established as a relatively simple, easily accessible and technically feasible procedure with a short operative time.106 Moreover, the intricate procedure of orthotopic auto-transplantation of an intact frozen-thawed ovary to the upper genital tract using conventional microvascular principles has also been documented in rats.107
A number of different techniques of auto-transplantation of an intact fresh or frozen-thawed ovary together with its vascular pedicle using microvascular anastomosis in animal models were recently published.108 Special attention was directed towards identifying potential heterotopic locations containing adequate blood vessels that can be used to host and vascularize human ovarian grafts. Moving one step further, contralateral orthotopic auto-transplantation of cryopreserved whole ovaries with microanastomosis of the ovarian vascular pedicle has been successfully performed with very promising results, emphasizing the feasibility, practicality and reproducibility of that principle. Intriguingly, luteal function was demonstrated in 4 sheep and delivery of a healthy lamb less than 2 years after transplantation following spontaneous intercourse has recently been reported. Unfortunately, histological verification of ovarian tissue 18 to 19 months after transplantation has shown that the life span of the graft was short and the follicular survival rate in the grafted ovaries was only 1.7% to 7.6%.109 Despite a functioning vascular anastomosis and fertility, the grafts still had limited survival that could only be maintained for a finite length of time. Apart from decreased graft longevity, the concept of whole organ cryopreservation became established as a promising new fertility preserving option. In the same context, recent models for cryopreservation of an intact uterus110 as well as a whole rabbit ovary have been documented.111
 
THE USE OF VASCULARIZED GRAFTS IN HUMAN TRIALS
A number of human trials using cryopreserved auto-transplanted ovarian cortical strips have been carried out, based on previous successes with animal models using C-T ovarian cortical strips with reported follicular survival, preservation of endocrine function as well as restoration of fertility.98,99 The process of in vitro production of follicles from the cultured ovarian cortical strips, either in isolated fragments of cortex or after tridimensional reconstruction in alginate-based artificial follicles, is still unproven in humans.112,113
Many techniques can be adopted to make use of the cryopreserved ovarian tissue: transplantation back into the host, IVM of primordial follicles and xenografting into a host animal. Considering the first principle, cryopreserved ovarian tissue may be transplanted back into the patient, with the obvious limitation of reintroducing cancer in malignancies known to preferentially involve the ovaries such as leukemias and possibly breast cancer. Ovarian tissue strips are removed from the women and kept frozen in small strips prior to chemotherapy using novel techniques. When pregnancy is desired, the ovarian tissue strips are transplanted back into the patient in an orthotopic or heterotopic site. Given the fact that such ovarian tissue grafts are avascular, any ischemic insult to the transplanted tissue would invariably result in irreversible loss of the whole growing follicle population together, with a significant number of primordial follicles.
Three different surgical techniques of transplanting ovarian cortical strips have been improvised by Oktay and associates.114 Those are orthotopic transplantation into the pelvis and heterotopic transplantation into either the arm or abdominal wall. In one study, the orthotopic transplant stopped functioning nine months after transplantation.114 In the heterotopic transplant, however, function was preserved and resulted in the generation of a four-cell stage embryo that was transferred without pregnancy occurring. Multiple ovarian stimulation cycles were undertaken before this one embryo was obtained.115
11
Worldwide, 12 patients delivered 17 babies in eight centers from frozen ovarian cortex tissue transplants116 (Tables 6 and 7). Donnez et al. (2004) reported the first live birth after orthotopic transplantation of cryopreserved ovarian tissue.117
A 32-year-old woman from Belgium treated for Hodgkin's lymphoma was reportedly the first lady to give birth to a baby after successful orthotopic auto-transplantation of cryopreserved ovarian tissue obtained prior to the start of ovariotoxic chemotherapy.117 Despite developing chemotherapy-induced POF, the re-implantation of her ovarian tissue was successful in resuming ovulatory activity five months later. She became pregnant 11 months after re-transplantation by natural fertilization and gave birth to a healthy baby seven years after ovarian tissue banking.
Another pregnancy was reported in a woman with non-Hodgkin's lymphoma in which a refined IVF stimulation protocol was used following orthotopic auto-transplantation of cryopreserved-thawed ovarian cortical strips.118 Some critics were doubtful as to the exact site of origin of the oocytes that resulted in the two pregnancies in the abovementioned reports. The reason for such skepticism was based on reports of documented spontaneous pregnancies in women with POF after repeated courses of ovariotoxic radiotherapy and/or chemotherapy and the fact that the tissue transplanted to the native ovary does not rule out the chance of resumption of native ovarian activity.27
A recognized complication of ovarian tissue cryopreservation and transplantation is loss of a considerable part of the follicular apparatus during the initial post-transplantation ischemic insult, which can seriously limit its use. After transplantation, almost 70% of follicles are lost. Freezing is not the major cause of such follicular loss, though.98,119,120 Therefore, ovarian tissue freezing has been recommended only for women under the age of 35.114 Proposed sites of ovarian tissue transplantation with and without vascular anastomosis were reported previously by us and others.
 
HETEROLOGOUS OVARIAN TRANSPLANTATION
Silber et al. (2005, 2007) reported on the success of ovarian transplantation from a healthy fertile 24-year-old woman to her monozygotic twin sister, who had suffered from POF at the young age of 14.121,122 Monozygotic twins with discordant ovarian function have provided the basis of this work. Via minilaparotomy, ovarian cortical tissue was transplanted from the fertile sister to her sterile twin sister. Resumption of normal menstrual cyclicity was witnessed 3 months after transplantation, with a consequent drop of serum gonadotropin levels back to normal levels. During the second cycle, she conceived and the pregnancy progressed uneventfully until delivery of a healthy female baby at 38 weeks' gestation.121 Such an outstanding success only helps to emphasize the concept of transplanting large segments of ovarian tissue between monozygotic twins without the need for immunosuppression.
Silber (2012) recently reported on ovarian cortex transplantation in a cohort of nine sets of twins discordant for POF in which ovarian cortical tissue was transplanted from the twin with normal ovarian function to the sister with POF116 (Table 6). Similar to their previous reports, folliculogenesis, hormonal functions and menses were restored in all recipients. Five spontaneous pregnancies were documented in this cohort. 122,123 Donnez and associates124 have recently reported on a successful ovarian allograft between two non-identical twins. Ovarian cortical tissue from the donor sister who hadalready been the donor of bone marrow for a transplant was used. The recipient had received chemotherapy, total body radiation and bone marrow transplantation. The recipient developed spontaneous cycles after receiving the transplant. Moreover, two oocytes and two embryos were obtained.123,124 However, in most patients with an intact immune system, the potential for acute graft rejection and risks of long-term immunosuppressive complications in the mother, such as infection and obstetrical complications may seriously limit its use.123 Following documented ovarian function in a small number of cases following both orthotopic and heterotopic transplantation of thawed ovarian cortical strips, the ASRM practice committee recommended that the procedures of ovarian tissue cryopreservation or transplantation are to be regarded as experimental and performed only under IRB surveillance.73
Del Priore et al. performed uterine extirpation during a multi-organ retrieval from a cadaver and demonstrated the technical feasibility in 8 donors.125 A number of vascular pedicles were utilized including the ovarian, uterine, or internal iliac vessels. Serial histology sections throughout the period of cold ischemia, taken every 15 to 30 minutes, showed no significant change over 12 hours of cold ischemia. They concluded that the human uterus can be obtained from local organ donor networks using existing protocols.125
TABLE 6   Summary table of worldwide frozen ovarian cortex tissue transplant pregnancies. Modified from Silber SJ. Pregnancy after ovarian transplantation. In: Jauniaux E and Rizk B (Eds), pregnancy after assisted reproductive technology. Cambridge University Press; New York. 2012; Chapter 12;pp.137-47.
12
Since a multitude of methodological and technical difficulties are associated with this procedure, the technique of human uterus transplantation is more experimental than practical at this point.
 
IN VITRO MATURATION OF OOCYTES
The rationale behind IVM is the fact that primordial follicles obtained from the frozen-thawed ovarian cortical strips can be allowed to mature in vitro. However, this procedure is still under investigation and will only become available in the future. In severe combined immune deficiency mice (SCID), transplantation studies demonstrated follicular maturation and completion of meiosis I in preparation for ovulation and potential fertilization.126 Concerns have been raised pertaining to technical problems and potential viral infection transmission. To avoid spreading malignant cells, ovarian tissue culture with in vitro follicle maturation can be performed. Isolated follicle culture from the primordial stage has been tried, given the fact that the primordial cells represent >90% of the total follicular pool and by virtue of their ability to withstand cryoinjury.127 However, isolated primordial follicles do not mature properly in culture.128 Originally described for patients with polycystic ovary syndrome (PCOS), IVM of antral follicles may have applications for cancer patients. Unfortunately, its implementation is still being studied. 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 advantages include avoidance of ovarian stimulation with expensive, and at times, dangerous, gonadotropins, side effects of the medications and risks such as ovarian hyperstimulation syndrome (OHSS).
Although primary candidates for IVM of oocytes have classically been PCOS patients with multiple antral follicles, the spectrum of IVM indications is expanding to include women with primarily poor-quality embryos in repeated cycles and poor responders to stimulation. Two new applications for IVM, especially in women with cancer who are undergoing ovariotoxic therapy, are oocyte donation and fertility preservation. It is combined with oocyte vitrification in younger women without partners needing this treatment for fertility preservation. Clinical pregnancy rates per cycle in women choosing IVM are related to their age; approximately 38% for infertility treatment up to the age of 30 and around 50% in recipients of IVM egg donation. Moreover, in a study evaluation the incidence of chromosomal abnormality (CA) in embryos from IVM and IVF cycles, IVM embryos were similar to IVF embryos (58.7% versus 57.4%, respectively).129 For embryos derived from oocytes that matured 48 hours after collection, a higher CA rate was observed as compared with embryos derived from in-vivo matured oocytes and to embryos derived from oocytes that matured in the first 24 hours after collection.
The issue of clinical applicability and practical value of IVM as a fertility preservation strategy is still unsettled.130,131 Xu et al. improvised a method using tissue engineering principles for the culture of immature ovarian follicles followed by fertilization of oocytes in vitro).132 This methodology is a great step forward in the search for new fertility preservation options in female cancer patients.132–134 An additional strategy of fertility preservation, which combines ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue followed by IVM and vitrification, has recently been described.135 Recently, the first healthy baby was delivered following retrieval of immature oocytes in a natural menstrual cycle, followed by IVM and cryopreservation of the oocytes by vitrification.136
TABLE 7   Summary table of the ovarian transplant results with the first nine fresh and three frozen ovary transplants. Modified from Silber SJ. Pregnancy after ovarian transplantation. In: Jauniaux E and Rizk B (Eds), pregnancy after assisted reproductive technology. Cambridge University Press; New York. 2012;Chapter 12;pp.137-47.
13
This provides proof-of-principle evidence that the novel fertility preservation strategy of immature oocyte retrieval, IVM, and vitrification of oocytes can lead to a successful pregnancy and healthy live birth.136
 
CONCLUSION
Most of the currently available strategies to preserve fertility in women are still experimental and do not guarantee subsequent fertility. The only established method in women is IVF with oocyte or embryo cryopreservation prior to cancer therapy. Other proposed strategies to preserve fertility in women with cancer include: [a] storage of frozen embryos, [b] storage of frozen ovarian tissue or the whole ovary for future transplantation, [c] storage of isolated follicles for in vitro growth and maturation and [d] ovarian transposition before radiotherapy. The effectiveness of ovarian protection during chemotherapy with GnRH analogs is yet to be shown.
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