Fertility Preservation in Young Women Undergoing Breast Cancer Therapy. Murat Sonmezer, Kutluk Oktay

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1 This material is protected by U.S. Copyright law. Unauthorized reproduction is prohibited. For reprints contact: Breast Cancer Fertility Preservation in Young Women Undergoing Breast Cancer Therapy Murat Sonmezer, Kutluk Oktay Fertility Preservation Program, Center for Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York, USA Learning Objectives Key Words. Amenorrhea Breast cancer Chemotherapy Fertility preservation After completing this course, the reader will be able to: 1. Explain how chemotherapy for breast cancer impacts ovarian function. 2. Discuss the incidence of ovarian failure after chemotherapy and list the chemotherapeutic agents most likely to cause loss of fertility in breast cancer patients. 3. Describe options for fertility preservation in women undergoing breast cancer therapy. CME Access and take the CME test online and receive 1 AMA PRA category 1 credit at CME.TheOncologist.com Abstract Breast cancer accounts for one third of all neoplasms seen in reproductive-age women and affects tens of thousands of women each year in that age group. The adjuvant chemotherapy regimens used for the treatment commonly affect fertility and cause premature ovarian failure. There have been recent advances in the field of fertility preservation, which can allow many of these breast cancer survivors to have children in the future. The most established option is embryo cryopreservation; oocyte cryopreservation can be considered in single women. Both of these approaches require approximately 2 weeks of ovarian stimulation beginning with the onset of the patient s menstrual cycle. Thus, it is crucial that these patients are referred to appropriate assisted reproduction centers as soon as they are diagnosed with breast cancer. Recently developed ovarian stimulation protocols using tamoxifen and letrozole can be used to increase the margin of safety in these patients. When and if a breast cancer patient does not have time to undergo ovarian stimulation prior to chemotherapy, ovarian cryopreservation for future autotransplantation can be offered as the last resort. The benefit of ovarian protection by gonadotropin-releasing hormone analogues is unproven and unlikely, and thus this treatment should not be offered as the sole method of fertility preservation. The Oncologist 200;11: Downloaded from by guest on September, 2018 Introduction Breast cancer is the most common invasive cancer in women, affecting >215,000 women annually in the U.S. and accounting for 32% of all cancers diagnosed in women Correspondence: Kutluk Oktay, Fertility Preservation Program, Center for Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York, USA. Telephone: ; Fax: ; kuo9001@med.cornell.edu; Web site: Received October 27, 2005; accepted for publication March 24, 200. AlphaMed Press /200/$20.00/0 The Oncologist 200;11: [1]. Approximately 25% of breast cancer cases occur before the age of menopause, and 15% occur under the age of 45 [2 4]. The incidence of breast cancer has increased by 0.5% per year over the past decade, whereas the death rate

2 Sonmezer, Oktay 423 decreased by 1.4% per year during the same period [5]. The decline in mortality is more remarkable in women aged <50 years []. The increase in disease-free survival (DFS) and the decrease in overall mortality are most likely a result of increased awareness of breast diseases, well-established screening programs leading to earlier detection, and better treatment, which includes more liberal use of adjuvant cytotoxic chemotherapy and hormonal therapy [ 9]. Coupled with the increase in the number of women who delay first childbirth beyond the age of 35, the use of adjuvant chemotherapy regimens has resulted in a large proportion of breast cancer patients of reproductive age facing infertility [10 12]. More than 90% of all breast cancers are diagnosed at a local or regional disease stage, with corresponding 5- year survival rates of 97% and 79%, respectively [5]. With improved cure rates from breast cancer, a greater attention has been focused on the long-term adverse effects of cytotoxic treatment that might compromise the quality of life of the survivor. The most commonly used adjuvant chemotherapy regimens use agents with a well-recognized negative impact on fertility [4, 13]. Studies have shown that reproductive concerns play an important role for young women diagnosed with breast cancer [14, 15]. In a study using a Web-based survey, 29% of women stated that infertility concerns influenced their treatment decisions [15]. In young breast cancer patients, the potential benefits of adjuvant cytotoxic treatment should be cautiously weighed against the long-term adverse impact on fertility, especially in very early-stage cancer. Many young breast cancer patients feel that their cancer physicians do not sufficiently inform them about the impact of cancer treatment on their fertility [14 1]. It is vital to provide the most up-to-date and accurate information on the effects of cancer treatment on fertility to these young women who desire greater involvement in their treatment decision making. The Impact of Chemotherapeutic Agents on Ovarian Function The ovaries are endowed with a fixed number of resting primordial follicles at birth, which make up the ovarian reserve. In an adult ovary, primordial follicles constitute approximately 90% of the total follicle pool, each containing an oocyte arrested in prophase of the first meiotic division [17, 18]. Gonadotoxic chemotherapy causes marked follicle loss through, presumably, apoptotic cell death [19 21]. The age of the patient and cumulative dose and type of the cytotoxic agent(s) are the most important factors that determine the likelihood of gonadal failure [4, 13, 22 24] (Table 1). It is not clear if the duration and dose intensity of chemotherapy independently affect the risk for gonadal failure. Alkylating agents are extremely gonadotoxic because they are not cell cycle-specific and can damage resting primordial follicles, whereas cycle-specific agents such as methotrexate and 5-fluorouracil do not have any effect on ovarian reserve. In a mouse study, cyclophosphamide induced follicular damage in a dose-dependent manner through a dose range of mg/kg, whereas destruction of primordial follicles occurred even at the lowest cyclophosphamide dose [24]. With each additional dose of cyclophosphamide administration, an incremental number of primordial follicles are lost and the incidence of ovarian failure increases. Patients who receive cyclophosphamide have a 4- to 9.3-fold greater risk for the development of premature ovarian failure than healthy controls [25, 2]. One of the major weaknesses of preexisting studies assessing the impact of chemotherapy given for breast cancer on fertility is the use of chemotherapy-induced amenorrhea as a surrogate marker for infertility [4, 13, 27 29]. Transient menstrual irregularity or amenorrhea is highly likely to occur in many women during chemotherapy, and a proportion of these patients typically will resume menstruation within months of completion of chemotherapy [4, 30 32]. In a previous report among patients who became amenorrheic after chemotherapy, while <11% aged >40 years resumed menses, 22% 5% of those aged <40 years regained menstruation during follow-up [4]. This is a result of the fact that developing follicles with the main source of steroid production are invariably damaged during the treatment, and it takes 3 Table 1. The degree of gonadal failure associated with chemotherapeutic agents High risk Cyclophosphamide Melphalan Busulfan Nitrogen mustard Cholarambucil Procarbazine Intermediate risk Cisplatin Adriamycin Paclitaxel? a Low or no risk Methotrexate 5-Fluorouracil Vincristine Bleomycin Actinomycin D a Confirmation needed. Downloaded from by guest on September, 2018

3 424 Fertility Preservation in Breast Cancer Patients months to regrow these follicles from the remaining primordial follicles. As amenorrhea is the last event to occur in the scheme of menopausal transition and as many women with diminished ovarian reserve still menstruate regularly, these studies greatly underestimate the extent of the damage. Nevertheless, because no large study with biochemical ovarian reserve assessment is available, we currently base our risk evaluation on the likelihood of amenorrhea after each chemotherapy agent or regimen. On the flip side, resumption of menstruation after chemotherapy does not mean that fertility has been preserved. In a study reported by Schmidt et al. [33], five of the eight women with breast cancer who were treated with cyclophosphamide, methotrexate, and 5-fluorouracil () or cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) had undetectable inhibin B levels (as a moderately sensitive marker of ovarian reserve) despite having regular or irregular menstrual periods. It has been commonly misperceived that young women are spared from the impact of chemotherapy on their ovarian reserve. This stems from the observation that the incidence of immediate amenorrhea is lower in younger women. Because young women have a larger primordial follicle pool, they are less likely to lose all their reserve immediately, but because they have lost a significant proportion of it, they will eventually experience premature ovarian failure [34 38]. On the other hand, permanent gonadal damage can be induced with smaller doses of the implicated chemotherapeutic agents in older women with lower primordial follicle reserve. Thus, even in a women who resumes regular menstruation postchemotherapy, based on pediatric data, the incidence of infertility and premature ovarian failure is extremely high [25, 39]. Ovarian failure is diagnosed by at least two measurements of follicle-stimulating hormone (FSH) above 40 miu/ml, regardless of menstrual bleeding. Continuation of menstruation is not a reliable indication of ovarian function and fertility, as pregnancy rates are extremely low when FSH measurements on the second or third day of the menstrual period exceed 12 miu/ml (20 miu/ml by radioimmunoassay) [40]. Likewise, elevation of estradiol levels above 75 pg/ml on the second or third day of the menstrual period is also associated with compromised fertility [41]. The Incidence of Ovarian Failure Following Adjuvant Chemotherapy for Breast Cancer The reported incidence of chemotherapy-induced amenorrhea with polyagent chemotherapy for breast cancer varies widely, mostly because of nonuniformity of the definition of amenorrhea or menopause as well as variations in age distribution, treatment regimen, and duration of follow-up [4, 13, 42 45]. The incidence of chemotherapy-induced amenorrhea was reported as 8% with classic oral [4, 4]. One cycle of caused loss of ovarian function in 30% of the patients [47]. Bines et al. [4] reported that adjuvant therapy with caused ovarian failure in 40% of women <40 years of age and 7% of those >40 years of age (Table 2). In another study, the risk for menopause was 5% 40% in a 40-year-old woman and 20% 100% in a 50-year-old woman receiving or CEF [13]. In a National Cancer Institute of Canada adjuvant trial, CEF was found to cause amenorrhea in 51% of the patients, whereas caused amenorrhea in 42.% [27]. Overall, the incidence of amenorrhea is in the range of 0% 9% with anthracycline-based regimens [47 49]. It was demonstrated that none of the patients <30 years of age receiving doxorubicin-containing regimens stopped menstruating during the course of chemotherapy, whereas 33% of the women aged years and 9% of those aged years experienced amenorrhea [49]. Even though ovarian toxicity was demonstrated in animal studies, current data are sparse in humans regarding the use of taxanes. In a retrospective analysis, doxorubicin and cyclophosphamide (AC) or AC plus paclitaxel chemotherapy induced amenorrhea in none of the women aged <35 years (n = 11), 14% of those aged years (n = 14), 39% of those aged years (n = 18), and all women aged years (n = 17) [28]. The same authors concluded that the addition of paclitaxel did not substantially increase the risk for amenorrhea. In contrast, results from the Breast Cancer International Research Group (BCIRG) 01 trial showed that the incidence of amenorrhea was higher in the docetaxel, doxorubicin, and cyclophosphamide (TAC) arm compared with the fluorouracil, doxorubicin, and cyclophosphamide (FAC) arm (51.4% vs. 32.8%, respectively) [29]. Moreover, our recent data also revealed a higher incidence of amenorrhea when paclitaxel was added to anthracycline-based regimens [50]. Even though the recent data indicate that paclitaxel is at least moderately gonadotoxic, future studies are required to verify this assumption. Tamoxifen does not cause a loss in primordial follicles in reproductive age women, but its use is associated with a 5-year delay in pregnancy [51]. This delay can then result in ovarian failure, if it has not already been experienced, as the ovarian reserves of these patients have been compromised by adjuvant chemotherapy. Use of Nongonadotoxic Ovarian Suppression Regimens as Adjuvant Treatment Ovarian suppression has been under investigation in place of or in addition to adjuvant chemotherapy for young patients with newly diagnosed hormone-sensitive, early stage breast cancer [52 54]. In a large study comparing The Oncologist Downloaded from by guest on September, 2018

4 Sonmezer, Oktay 425 Table 2. The degree of gonadotoxicity related to the most commonly used chemotherapy regimens in breast cancer; selected trials are summarized Study Hortobagyi et al. [49] Goldhirsch et al. [47] Valagussa et al. [45] Cobleigh et al. [13] Adjuvant regimen Doxorubicin-containing regimen No. of patients Duration of treatment (months) Follow-up to define amenorrhea (months) Rate of amenorrhea % Age 9 24 NA 33/9 3 39/ /34 33/81 <40/ 40 <40/ 40 ± A /50/8 <3/3 40/ AC Bines et al. [4] a 3, /7 <40/ 40 yrs Levine et al. [27] Pagani et al. [137] Goodwin et al. [13] Stone et al. [28] Nabholtz et al. [29] Jonat et al. [55] Castiglione- Gertsch et al. [53] Di Cosimo et al. [43] Vanhuyse et al. [138] Bonadonna et al. [44] Parulekar et al. [139] Martin et al. [140] CEF CEF AC AC + T TAC FAC Goserelin Goserelin + E ± D FEC/EC FEC CEF TAC FAC ± 4 /4 12 NA NA none NA NA a Analysis of 30 studies. The rates of amenorrhea were provided after 12 months of chemotherapy in most of the studies. Abbreviations: A, doxorubicin; AC, doxorubicin and cyclophosphamide; CEF, cyclophosphamide, epirubicin, and fluorouracil;, cyclophosphamide, methotrexate, and fluorouracil; D, docetaxel; E, epirubicin; EC, epirubicin and cyclophosphamide; FAC, fluorouracil, doxorubicin, cyclophosphamide; FEC, fluorouracil, epirubicin, and cyclophosphamide; NA, not available; T, paclitaxel; TAC, paclitaxel, doxorubicin, and cyclophosphamide. Downloaded from by guest on September, 2018 the efficacy and tolerability of monthly goserelin treatment for 2 years (n = 817) with (n = 823), goserelin produced a similar DFS to that seen with in estrogen receptor-positive patients after a median follow-up of years [55]. In contrast, goserelin was found to be inferior to in estrogen receptor-negative patients. Of note, 22.% of the patients receiving goserelin were amenorrheic, as opposed to 77% who received, at 3 years. In another study, nearly half of the patients with early-stage breast cancer considered adjuvant endocrine therapy worthwhile for an absolute gain in survival of 2% from a baseline of either 5% or 85% [52]. It was stated in the National Institutes of Health Consensus Statement of Adjuvant Therapy for Breast Cancer that ovarian ablation with a gonadotropin-releasing hormone agonist (GnRHa) produces results similar to those of some chemotherapy regimens [5]. An International Consensus Panel during the St. Gallen Conference recommended ovarian suppression with or without tamoxifen for hormone receptor-positive, node-negative breast cancer patients having medium-

5 42 Fertility Preservation in Breast Cancer Patients or high-risk disease, and ovarian ablation in conjunction with tamoxifen for patients with node-positive disease [57]. As a result, ovarian ablation appears to be a promising alternative to adjuvant chemotherapy for selected young breast cancer patients who desire to maximize their future chances for childbearing. Is There a Protective Role of GnRH Analogues Against Chemotherapy-Induced Gonadal Damage? It has been highly debated whether cotreatment with a GnRHa can prevent chemotherapy- or radiotherapyinduced gonadal damage. Studies in monkeys showed a protective role of GnRHa cotreatment against chemotherapy-induced follicle loss; however, these studies failed to demonstrate any benefit against radiotherapy-induced gonadal damage [58, 59]. In a recent mouse study, ovarian suppression by either a GnRHa or oral contraceptive did not prove effective against cyclophosphamide-induced ovarian damage [0]. There is a limited number of prospective studies in humans, which are flawed because of short-term follow-up and/or because of a lack of control subjects [1 4]. In the only prospective randomized study, including 30 men and 18 women receiving chemotherapy for Hodgkin s disease, GnRHa treatment was demonstrated to be ineffective in preserving fertility as judged by sperm count and menstrual function [5]. Patients were randomly assigned to receive buserelin (20 men and 8 women) prior to and for the duration of cytotoxic chemotherapy. After 3 years of follow-up, all men in both the study and control groups became oligo/azospermic, while four of the eight women treated with a GnRHa and six of the nine female controls became amenorrheic. In another study, the protective role of goserelin given monthly for 1 year as adjuvant therapy was investigated in 4 premenopausal patients with early breast cancer [3]. Eighteen patients received and 4 patients received an anthracycline-based regimen. Nine patients received highdose chemotherapy and underwent autologous peripheral blood progenitor cell transplantation, and 80% of the patients were irradiated. After a median follow-up of 55 months, 8% of the patients resumed normal menses. There was no control group. Blumenfeld et al. [1] administered a GnRHa concurrently with chemotherapy in 18 women with Hodgkin s or non-hodgkin s lymphomas. The study group was compared with a historical control group of 18 women treated with similar regimens. The percentage of patients resuming spontaneous ovulation and menses was significantly higher in the study group than in the control group (93.7% vs. 37%); however, the mean follow-up was only 1.7 ± 1.0 years in the study group, compared with 7.0 ± 4.9 years in the control group. In a similar study, a GnRHa appeared to be beneficial in both cancer patients (n = 54) and noncancer patients (n = 8) receiving chemotherapy []. The control group (n = 55) was chosen retrospectively from patients who were treated with similar regimens. The percentage of patients who resumed menses and ovulation was significantly higher in the study group than in the control group (~all vs. <50%). However, the length of follow-up was not stated for the treatment group. Based on these findings, one cannot conclude whether the lower incidence of ovarian failure was a result of GnRHa treatment or a shorter follow-up. None of these studies tested whether fertility, not just menstrual function, was preserved. In a recent abstract by Fox et al. [7], whereas GnRHa treatment appeared to reduce the incidence of amenorrhea in a population of relatively older reproductive-age women, reproductive outcome was very poor. Twenty-three of the 24 women resumed menstruation after receiving a GnRHa along with chemotherapy, and went on to attempt to conceive. Six pregnancies occurred in five patients; three resulted in miscarriage, one was terminated because of Down s syndrome, one pregnancy was ongoing, and one delivered [7]. The protective role of GnRH antagonists has also been investigated in rodents. One study reported that the degree of protection achieved by the antagonists is dose dependent, with a lower efficiency when larger doses of cyclophosphamide were used [8]. In contrast, others suggested that GnRH antagonists might deplete ovarian follicles through a direct effect on the ovary in a murine model [9]. In addition to the lack of consistent support from clinical studies, there is currently no biological explanation for how a GnRHa can affect ovarian reserve. Primordial follicles do not express FSH or GnRH-I receptors. A GnRHa, in essence, returns the hormonal milieu to the prepubertal state. A clinical example for why gonadal suppression may not protect ovaries is the fact that prepubertal children receiving high-dose chemotherapy given before hematopoietic stem cell transplantation still suffer from ovarian failure [37]. Nevertheless, because younger patients have a larger ovarian reserve, they might tolerate some loss in the follicular pool, and immediate ovarian function might not be affected in the short term. All patients who receive high-dose gonadotoxic chemotherapy will eventually suffer from premature ovarian failure [70, 71]. In the absence of a prospective randomized study with sufficient power, we do not rely on ovarian suppression as an effective means of fertility preservation. Assisted Reproduction Techniques to Preserve Fertility in Breast Cancer Patients Considering the relative increase in the incidence of breast cancer coupled with the trend to delay childbearing, an The Oncologist Downloaded from by guest on September, 2018

6 Sonmezer, Oktay 427 escalating number of women with breast cancer will be seeking help to prevent and treat chemotherapy-induced infertility using assisted reproductive technologies (ARTs). The current options range from clinically wellestablished, routinely used techniques such as embryo cryopreservation to experimental ones, such as oocyte or ovarian tissue cryopreservation. A typical in vitro fertilization (IVF) cycle includes approximately days of ovarian stimulation with gonadotropins following pituitary downregulation using GnRH analogs in order to achieve multifollicular development. A conventional IVF cycle almost always results in a supraphysiological estradiol level that may be as high as 10- to 15-fold greater than that of a natural cycle. Because conventional ovarian stimulation regimens result in a surge in estradiol levels, they are typically avoided in women with breast cancer [72, 73]. Ovulation induction regimens incorporating tamoxifen and aromatase inhibitors have been put to use [12, 74]. Below, we review the currently available, as well as experimental, techniques to preserve fertility in breast cancer patients and propose a case-based strategy to determine the most appropriate technique for individual patients (Fig. 1). Embryo Cryopreservation Embryo cryopreservation has been used for more than two decades to store excess embryos and to avoid the risk for ovarian hyperstimulation after IVF [75]. The success rate of an IVF cycle using frozen-thawed embryos now approaches those with fresh embryos, and this has become a routine tool that is being used by many ART laboratories throughout the world [7, 77]. Survival rates per thawed embryos are in the range of 35% 90%, implantation rates are in the range of 8% 30%, and cumulative pregnancy rates exceed 0% [23]. If the patient has a partner and sufficient time prior to cytotoxic treatment, embryos generated through IVF can be cryopreserved and stored for future use. Because adjuvant chemotherapy is commonly initiated 4 weeks after surgery, there is usually adequate time to perform ovarian stimulation and IVF. Oocyte retrieval and embryo freezing can also be performed without ovarian stimulation; however, the embryo yield is low and typically no more than a single embryo can be generated [74, 78]. Because the effectiveness of IVF diminishes with every round of chemotherapy and because there is a potential for fertilizing a genetically damaged growing oocyte, it is also not recommended to perform IVF after chemotherapy is initiated [79, 80]. Downloaded from by guest on September, 2018 Figure 1. A proposed algorhythmic approach to decision making for fertility preservation in breast cancer patients. Embryo cryopreservation using letrozole is a novel stimulation protocol in breast cancer patients, and long-term follow-up data are awaited. Abbreviations: Cryo, cryopreservation; FP, fertility preservation; TMX, tamoxifen. Ovarian tissue and oocyte cryopreservation are experimental technologies.

7 428 Fertility Preservation in Breast Cancer Patients Epidemiological and experimental evidence suggests that estrogen plays a significant role in tumorigenesis of breast tissue. Estrogen exposure stimulates mitotic activity in mammary epithelium [72, 73, 81]. Ovarian stimulation increases the number of follicles recruited to grow and increases estrogen production in proportion to the number of follicles recruited. As a result, estrogen can reach levels 10-fold or more higher than those in a natural cycle [82 84] Ovulation Induction with Tamoxifen in Breast Cancer Tamoxifen, a selective estrogen modulator with antiestrogenic actions on breast tissue, is an important part of the adjuvant therapy for early-stage, hormone-sensitive breast cancer. Tamoxifen, a nonsteroidal triphenylethylene antiestrogen, was originally developed as a contraceptive agent in the United Kingdom but was later found to stimulate follicle growth [85 87]. Recently, it has been proven to be effective for chemoprevention of breast cancer [88, 89]. Clomiphene, a related compound of tamoxifen, has been used for ovulation induction for almost more four decades [90]. Tamoxifen was tested as an ovulation induction agent and proved to be as effective as clomiphene for the treatment of patients with anovulatory infertility [91]. Exploiting its dual action as an antineoplastic agent and an ovarian-stimulating drug, albeit data are limited, we recently demonstrated that tamoxifen can be safely used to perform ovarian stimulation and IVF in breast cancer patients [74]. In order to increase embryo yield, 12 women (15 cycles) with breast cancer were stimulated with 40 0 mg of tamoxifen for a mean duration of.9 days, beginning on day 2 3 of the menstrual cycle. Patients underwent IVF and embryo transfer (TamIVF) with either fresh (six cycles) or cryopreserved (nine cycles) embryos, and were compared with a retrospective control group of breast cancer patients who had natural cycle in vitro fertilization (NCIVF). Cycle cancellation was significantly less in patients receiving tamoxifen than in those who underwent NCIVF (1/15 vs. 4/9 patients). In the tamoxifen group, the mean peak estradiol level on the day of human chorionic gonadotropin (hcg) administration was significantly higher than in those who underwent NCIVF (442.4 pg/ml vs. 278 pg/ml). The total number of mature oocytes (1. ± 0.3 vs. 0.7 ± 0.2; p =.03) and total number of embryos (1. ± 0.3 vs. 0. ± 0.2; p =.02) were higher in the tamoxifen group than in the NCIVF group. As a result, TamIVF resulted in the generation of an embryo in every patient (12/12), whereas only three of five patients had an embryo following NCIVF. To prevent patients from spontaneously ovulating prior to egg retrieval, short-acting GnRH antagonists were also administered until the day of hcg administration. Studies with tamoxifen demonstrated that its shortterm use for ovulation induction does not adversely affect oocyte and embryo development [92]. Moreover, no detrimental effect on fetal development was demonstrated [93]. One study showed lower miscarriage rates with tamoxifen than with clomiphene [94]. One can question whether increased estradiol levels as a result of ovarian stimulation with tamoxifen have any detrimental effects on breast cancer. Even though tamoxifen results in an increase in estradiol levels, it also blocks the effects of supraphysiological levels of estrogen on breast tissue and inhibits the growth of breast tumors by competitive antagonism of estrogen at its receptor site. In fact, mean estradiol levels are chronically elevated in breast cancer patients on long-term tamoxifen treatment and can be higher than the levels seen in patients undergoing ovarian stimulation with tamoxifen [95, 9]. Ovulation Induction with Aromatase Inhibitors Aromatase, which is a cytochrome P450 enzyme, catalyses the reaction that converts androgens to estrogens. Aromatase is the rate-limiting step in estrogen synthesis that reduces the amount of estrogen required for estrogen receptor-mediated transcription [97]. Letrozole, which is a third-generation aromatase inhibitor, was developed in the early 1990s. It is a potent and highly selective inhibitor of aromatase that competitively binds the active site of the enzyme [98]. Letrozole significantly suppresses plasma estradiol, estrone, and estrone sulfate levels at doses in the range of mg/day, and it was recently shown to be superior to tamoxifen in the treatment of advanced-stage postmenopausal breast cancer [99, 100]. It was also demonstrated to improve DFS after completion of standard tamoxifen therapy [101]. Letrozole, in contrast to tamoxifen, which acts as a partial estrogen agonist, is not associated with an increased incidence of uterine cancer and venous thromboembolism in long-term use [9, 102]. Aromatase inhibitors have recently been tested as ovulation induction agents. Clinical studies have shown their benefit in ovulation induction alone or in combination with FSH. In poor responders, letrozole was shown to improve ovarian response to gonadotropin stimulation, and to increase the number of preovulatory follicles while decreasing gonadotropin requirement [103, 104]. Aromatase inhibitors can augment the response to ovulation induction, acting both centrally, by blocking negative feedback of estradiol on the pituitary and hypothalamus, and peripherally, by improving follicular sensitivity to gonadotropins. A possible advantage of ovarian stimulation with aromatase inhibitors in breast cancer patients is that the peak estradiol levels are lower than those with conventional stimulation regimens and minimally elevated compared with peak estradiol levels in unstimulated cycles [12, 104]. The Oncologist Downloaded from by guest on September, 2018

8 Sonmezer, Oktay 429 In a prospective controlled study, we demonstrated that the combination of low-dose FSH with letrozole (Letrozole-IVF) or tamoxifen (TamFSH-IVF) produced a higher embryo yield than with tamoxifen alone (Tam-IVF) [12]. Peak estradiol levels were lower with Letrozole-IVF and Tam-IVF than with TamFSH-IVF (380 ± 57, 419 ± 39, and 1,182 ± 271 pg/ml, respectively). The mean estradiol levels in the Letrozole-IVF and Tam-IVF groups were only slightly higher than those seen in natural cycles, which can be as high as pg/ml [74]. The mean durations of follow-up were 09 days, 418 days, 272 days, and 0 days for Tam-IVF, TamFSH-IVF, Letrozole-IVF, and the control groups, respectively, and no difference was observed in recurrence rates between IVF and control patients. When compared with standard IVF cycles performed on noncancer patients, the numbers of oocytes and embryos were lower in the Tam-IVF and TamFSH-IVF groups, whereas they were similar to those with Letrozole-IVF. While the incidence of breast cancer recurrence was approximately 10% in the control and tamoxifen-treated patients, and follow-up was short, no recurrences occurred in the letrozole-treated patients. Because the Letrozole-IVF protocol, in which letrozole and FSH are used together, results in lower estradiol levels with the highest oocyte recovery, and because of its safety profile, we currently prefer this protocol in most breast cancer patients undergoing IVF for embryo or oocyte cryopreservation. Moreover, several live births have already occurred as a result of letrozole-fsh stimulation in patients with breast cancer [105]. It must be emphasized that aromatase inhibitors should not be used during pregnancy because estradiol and its precursors can play a role in fetal development. However, there is no evidence that the exposure of oocytes to letrozole can increase birth defects. When used for in vitro fertilization, embryos are never exposed to systemic letrozole. Even when letrozole is used to assist women to conceive with unfrozen embryos, letrozole has been cleared from the circulation by the time the embryos are transferred. Moreover, a recent study on aromatase-overexpressing mice showed that when these animals were treated with high doses of letrozole for weeks and allowed to conceive 2 weeks later, there was no difference between treated and control animals in terms of litter size, birth weight, and anomalies [10]. Likewise, in vitro exposure of mouse follicles to another aromatase inhibitor, anastrozole, did not increase meiotic spindle defects in oocytes and birth anomalies [107]. Oocyte Cryopreservation When embryo cryopreservation is not feasible, oocytes can be frozen unfertilized. Ovarian stimulation is required, similar to that in IVF cycles for embryo freezing. Unfortunately, oocyte freezing is technically more challenging because of the complex structure of the human oocyte. In a recent meta-analysis, we found that the live birth rate per injected oocytes was approximately 2% for the most commonly used slow-freezing technique [108]. Pregnancy rates were one third to one fourth of the success rates seen with unfrozen oocytes. Thus, we resort to oocyte cryopreservation when embryo freezing cannot be performed, especially in single women who do not wish to use donor sperm for in vitro fertilization. Cryopreservation of Ovarian Tissue In ovarian cryopreservation, the cortex, which contains a rich reserve of primordial follicles, is frozen in thin slices. The success of this procedure is based on the evidence that primordial follicles are less vulnerable to cryodamage because of a high surface volume ratio, low metabolic rate, and the absence of zona pellucida. After the first animal trials of ovarian tissue cryopreservation in the 1950s with glycerol, 40 years have passed to achieve successful birth in animals with the discovery of modern cryoprotectants [ ]. In humans, resumption of endocrine function has been reported after orthotopic [33, 112, 113] and heterotopic [114, 115] transplantation of frozen-thawed ovarian cortical strips. Recently, an embryo was generated from oocytes retrieved from s.c. transplanted ovarian tissue in a breast cancer survivor [11]. Most recently, two live births were reported after orthotopic transplantation of frozen-banked ovarian tissue in lymphoma survivors [117, 118]. In the orthotopic transplantation technique, frozenthawed ovarian cortical pieces can be grafted near the infundibulopelvic ligament or on a postmenopausal ovary. In the heterotopic transplantation method, the tissue is grafted s.c. to the forearm or suprapubic area. The advantage of orthotopic transplantation is that natural conception is possible. However, this technique requires general anesthesia. Heterotopic transplantation does not require general anesthesia or abdominal surgery. It is also easy to monitor follicle development, and to remove the transplanted tissue from the s.c. site when necessary. One of the major concerns in transplanting ovarian tissue from cancer patients is the risk for reseeding cancer cells. In breast cancer, occult ovarian involvement is rare, especially if there is no systemic metastasis and if the pelvic and ultrasound examinations are normal [119]. Previous studies showed that most of the occult metastases belong to the infiltrating lobular histological subtype, which constitutes <15% of all breast cancers and more commonly occurs in postmenopausal women [ ]. Thus, early-stage breast cancer is not a contraindication to perform ovarian Downloaded from by guest on September, 2018

9 430 Fertility Preservation in Breast Cancer Patients tissue cryopreservation and transplantation. However, prior to ovarian transplantation, a thorough histological assessment of a representative piece is required to rule out occult metastasis. In addition, patients with BRCA-1 and BRCA-2 genes are at a higher risk for harboring occult ovarian cancer [125]. Even though ovarian cancer is rare prior to the age of 35, these patients should be counseled about the risk [12]. There have been no reports of cancer recurrence in the limited number of cases published in the medical literature, but ovarian cryopreservation and transplantation remains an investigational protocol. Donor Eggs and Surrogacy IVF with donor eggs is another alternative when a cancer survivor suffers from premature menopause or low ovarian reserve as a result of cancer treatment. The success rates with appropriate egg donors may exceed 0% per embryo transfer. Patients with breast cancer who are considered at high risk for recurrence or who have to be on life-long therapy with tamoxifen or aromatase inhibitors may also resort to gestational surrogacy. However, the laws and regulations regarding this procedure vary substantially among countries and among individual states in the U.S. Future Directions Cryopreservation of the ovary as a whole with its vascular supply might help decrease the rate of follicle loss during the initial ischemia period [127]. Currently, there is no optimum cryopreservation technique available that can effectively preserve both the ovarian tissue and its vascular supply required for reanastomosis. Antiapoptotic treatment with sphingosine-1-phosphate has also proven to be an effective approach in preventing chemotherapyinduced oocyte loss in mice [128]. Xenotransplantation of ovarian tissue to severe combined immunodeficient mice has been suggested when the risk of ovarian involvement with the primary tumor is high [129, 130]. However, aberrant microtubule organization and chromatin patterns, and the theoretical risk for infection with trans-species retroviruses, are among the significant concerns with this experimental procedure [131, 132]. In vitro growth of isolated follicles is another option; however, no success has been achieved in humans thus far [18, 133]. Recent animal studies showed that there may be germ stem cells in adult mice capable of replenishing ovarian reserve and that they may originate from the bone marrow, but the plausibility of this discovery in humans remains to be determined [134, 135]. Conclusions There are a number of options available to preserve fertility in breast cancer patients undergoing chemotherapy. The decisions of whether to resort to fertility preservation and which method to use depends on a number of factors, including the patient s age, the type of adjuvant treatment, the time available before chemotherapy, and the length of delay to childbearing postchemotherapy (Fig. 1). The most recognized option for fertility preservation is embryo cryopreservation with either the partner s or donor sperm. Oocyte cryopreservation is considered in single women who do not wish to use donor sperm. Both approaches require approximately 2 weeks of ovarian stimulation beginning with the onset of the patient s menstrual cycle. Thus, it is crucial that these patients are referred to appropriate ART centers as soon as they are diagnosed with breast cancer. Recently developed ovarian stimulation protocols using tamoxifen and letrozole can be used to potentially increase the margin of safety in these patients. When and if a breast cancer patient does not have sufficient time to undergo ovarian stimulation, ovarian cryopreservation can be offered as the last resort. The benefit of ovarian protection by GnRHa treatment is unproven, and in the opinion of the authors, it should not be offered as a sole method of fertility preservation. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. Downloaded from by guest on September, 2018 References 1 Jemal A, Tiwari RC, Murray T et al. Cancer statistics, CA Cancer J Clin 2004;54: Hankey BF, Miller B, Curtis R et al. Trends in breast cancer in younger women in contrast to older women. J Natl Cancer Inst Monogr 1994;(1): Higgins S, Haffty BG. Pregnancy and lactation after breast-conserving therapy for early stage breast cancer. Cancer 1994;73: Bines J, Oleske DM, Cobleigh MA. Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer. 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