Cancer and fertility preservation in females: where we stand and where we are heading

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It is estimated that, in 2010, one in every 250 adults will be a childhood cancer survivor [1]. In developed or Westernized countries, women are using better methods of contraception and are delaying childbearing for social or financial reasons, so that an increasing number of women are anxious to preserve their fertility when early-stage cancers are discovered [2–5]. According to the most recent cancer statistics, the most com-mon cancers diagnosed in women under the age of 40 years are breast cancer, melanoma, cervical cancer, non-Hodgkin’s lymphoma and leuke-mia [6]. The incidence and 5-year survival rates, respectively, for the various conditions are:

Breast cancer: 0.48% (one in 207 women); •88%

Leukemia: 0.13% (one in 799 women); 46%•

Melanoma of the skin: 0.21% (one in 484 •women); 91%

Non-Hodgkin’s lymphoma: 0.09% (one in •1147 women); 59%

Uterine cervix carcinoma: 0.16% (one in 636 •women); 73%

Uterine corpus carcinoma: 0.06% (one in •1632 women); 84%

In addition, increasing numbers of patients with nonmalignant autoimmune diseases, such as rheumatoid arthritis or systemic lupus

erythematosus, as well as hematological dis-eases [7], are being treated successfully with chemotherapy or radiation therapy.

Cytotoxic therapy often results in premature ovarian failure (POF) [8]. Patients with POF have to face years of menopause and psycho-logical problems, or years of hormone-replace-ment therapy. However, this substitution ther-apy is not capable of replacing the reproductive function of the ovaries.

This article reviews the literature on the topic, discusses the effects of cancer treatment on female fertility and presents the options currently available – thanks to advances in assisted-reproduction technology – for main-taining fertility in women undergoing this type of treatment.

Ovarian anatomy & physiologyThe peak number of oocytes, approximately 6.8 × 106, occurs at 5 months gestation. After this point, there is no further proliferation of germ cells and a progressive atresia occurs. At birth, this number decreases to 1–2 × 106 and at puberty there are only 300,000 left. From these follicles, approximately 300–500 will develop into mature oocytes, while the rest will become atretic. At 51 years of age, the average age of natural menopause in developed countries, there are approximately 1000 cells left [3].

Theodoros Maltaris†, Michael Weigel and Ralf Dittrich†Author for correspondenceDepartment of Obstetrics and Gynecology, Leopoldina Academic Hospital, 97421, Schweinfurt, Germany Tel.: +49 972 1720 6383 Fax: +49 972 1720 [email protected]

It is estimated that, in 2010, one in every 250 adults will be a childhood cancer survivor. This review discusses the impact of current cancer treatment on fertility potential and the assisted-reproduction innovations available today for the most common cancers in young women. As the emerging discipline of fertility preservation is steadily attracting increasing interest, developments in the near future promise to be very exciting. However, in everyday routine work, better interdisciplinary cooperation between gynecological and pediatric oncologists, surgeons, immunologists and endocrinologists is necessary so that individualized options for fertility preservation can be offered in advance of surgical procedures or cancer treatments.

Keywords: cancer • fertility • Hodgkin’s lymphoma

Cancer and fertility preservation in females: where we stand and where we are headingExpert Rev. Endocrinol. Metab. 4(1), 79–89 (2009)

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In healthy women of approximately 37.5 years of age, an acceler-ated atresia of oocytes begins, which is associated with an increase in the level of follicle-stimulating hormone (FSH) [9]. As atresia continues, both the number and quality of oocytes fall below a critical level and the rate of aneuploidy increases. This process leads to a greater risk of spontaneous abortion once pregnancy occurs. This central doctrine of age-dependent follicle depletion has been challenged by recent data that suggest the presence of ovarian stem cells in mice, which could, presumably lead, to a replenishment of follicles [10], a theory that, at least in humans, cannot be supported.

Premature ovarian failure, defined as menopause before the age of 40 years or hypergonadotropic amenorrhea, occurs in up to 0.9% of women in the general population.

The lifecycle of the ovary has four major developments [11]:

The phase of embryogenesis, whereby populations of primordial •germ cells and somatic cells become an integrated ovary mass containing oocytes and granulosa cells located within primor-dial follicles. The first phase of oocyte maturation starts in utero and is gonadotropin independent;

The phase of folliculogenesis, in which oogenesis, granulogen-•esis and thecogenesis occur as a recruited primordial follicle grows and develops to the preovulatory follicle or dies by atresia. This phase begins at puberty and is gonadotropin dependent;

The phase of ovulation, whereby the oocyte, triggered by the •luteinizing hormones, transforms into a mature egg, which is secreted into the oviduct to await fertilization;

The phase of luteogenesis, whereby the follicle luteinizes into •the corpus luteum, which if implantation does not occur, dies by a process termed luteolysis.

Effect of cancer treatment on female fertilityRadiotherapy-induced ovarian damageIonizing radiation has adverse effects on gonadal function at all ages. The degree and persistence of the damage depends on the dose, the irradiation field and the patient’s age, with older women being at greater risk of damage [12]. Cranial irradiation for brain tumors with doses to the hypothalamic–pituitary area in excess of 30 Gy can, in time, cause hypogonadotropic hypogonadism in children [13].

The ovaries are exposed to significant doses of radiation when radiotherapy is used in the treatment of cervical and rectal can-cer, and with craniospinal radiotherapy for CNS malignancies. This can also happen when the pelvic lymph nodes are irradiated for hematological malignancies, such as Hodgkin’s disease, and with total body irradiation before bone marrow transplanta-tion [12]. Gosden et al. demonstrated that there is dose-related depletion of primordial follicles in mouse ovaries after increas-ing radiation doses of 0.1, 0.2 and 0.3 Gy. This explains steri-lization with total depletion of the primordial follicle reserve after exposure to high doses of radiotherapy, and POF at lower doses that cause only partial depletion of the primordial follicle reserve [14].

Various reports have been published on the radiation dosage nec-essary to cause loss of ovarian function. Lushbaugh and Casarett have shown that women under 40 years of age are less sensitive to radiation-induced ovarian damage, with an estimated dose of 20 Gy being required to produce permanent ovarian failure, in comparison with 6 Gy in older women [15]. Chiarelli et al. observed a dose-dependent and distribution-dependent relationship between the risk of POF and the total dose of abdominal pelvic irradia-tion: with doses less than 20 Gy, the relative risk was 1.02; at 20–35 Gy, the relative risk increased to 1.37; and with doses of more than 35 Gy, the relative risk of POF was 3.27. The percent-age of women who suffered infertility correlated with increasing dosages of abdominal pelvic irradiation; treatment with 20–35 Gy caused a 22% rate of infertility and doses greater than 35 Gy led to a 32% rate of infertility [16].

There is also a known radiation effect on the uterus and subse-quent pregnancy outcomes. Uterine radiation is associated with infertility, spontaneous miscarriage and intrauterine growth retar-dation [17]. Direct effects on the uterus after irradiation include irreversible changes in the uterine musculature and blood flow, as well as hormone-resistant endometrial insufficiency. A review by Critchley and Wallace indicated that physiological sex steroid-replacement therapy may improve uterine characteristics in some patients after irradiation at a young age [18].

It is also known that there is a higher rate of obstetric com-plications in patients who have received radiation treatment in comparison with the general population; complications include spontaneous abortions (38 vs 12%), preterm labor (62 vs 9%) and low-birthweight infants (62 vs 6%). However, as long as radiation is not administered during pregnancy, there is no risk of subsequent teratogenicity [19]. These findings confirm studies on women exposed to the radiation following an atomic bomb and on offspring conceived and born to them following exposure, which have shown that the incidence of spontaneous abortion is greater but that the children do not suffer from an increased rate of mutations or major congenital anomalies in comparison with the normal population [20]. Fenig et al. reported an increase in low-birthweight infants and spontaneous abortions, especially if conception occurred less than 1 year after radiation exposure [21]. They advised delaying pregnancy for 1 year after the completion of radiation therapy.

Chemotherapy-induced ovarian damageAll chemotherapeutic drugs act by interrupting vital cell proc-esses and arresting the normal cellular proliferation cycle. Frequently, chemotherapeutic agents are used in combination because of synergistic effects but this also leads to an increase in their adverse effects. In animal experiments, Meirow et al. demonstrated that regular menses and a normal reproduc-tive outcome after chemotherapy are not definite indicators of whether the ovarian follicular reserve has survived the treatment unaffected [22]. The authors also postulated that patients who recover from ovarian failure after high-dose chemotherapy or radiotherapy treatments should not delay childbearing for too many years. These patients should try to conceive after a few

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ReviewFertility preservation for female cancer survivors

years of a disease-free interval but not less than 6–12 months after the treatment, owing to the possible toxic effects of the treatment on growing oocytes [23].

The risk of chemotherapy-related amenorrhea depends on the patient’s age, the specific chemotherapeutic agents used, and the total dose administered.

Effects of ageOlder women have a higher incidence of complete ovarian failure and permanent infertility in comparison with younger women [12,24]. This can be explained by the larger primordial follicle reserve, which declines with age.

Various regimensAlkylating agentsAlkylating agents have a severe effect on human fertility. Known effects are ovarian fibrosis and follicular and oocyte depletion [25–27]. According to Meirow, alkylating agents are associated with the greatest risk among all chemotherapeutic agents for inducing ovar-ian failure (odds ratio [OR]: 3.98 in comparison with unexposed patients) [23]. In a study that examined the development of ovarian failure after cyclophosphamide treatment for lupus erythematosus, the POF incidence was 26%, with the major determining factors being the patient’s age at the start of therapy and the cumula-tive dose [28]. Animal experiments have also shown an increase in abortions and fetal malformations (ten-times higher than in the control group) in pregnancies resulting from oocytes exposed to cyclophosphamide at different stages of oocyte maturation [12]. Meirow also indicated that the effect of cyclophosphamide in mice is not an ‘all-or-nothing’ phenomenon, and that it causes follicular destruction in exponential proportion to increasing doses [23].

Cisplatin & analogsMeirow estimated that cisplatin causes ovarian failure with an OR of 1.77. Studies of cisplatin treatment in female mice have demonstrated that different types of chromosomal damage are induced, with genetic effects in the oocytes resulting in early embryonic mortality and marked aneuploidy [22].

Vinca alkaloidsVinca alkaloids are known aneuploidy inducers. According to Meirow, the OR for ovarian failure was approximately 1 [22]. Many animal experiments have shown high levels of aneuploidy in oocytes exposed to vinblastine [29], which means that these damaged oocytes could produce malformed fetuses.

AntimetabolitesInsufficient data are available on the effects of antimetabolites on female germ cells.

Anthracycline antibioticsAdriamycin and bleomycin are female-specific mutagens and have been shown to induce dominant lethal mutations in maturing/pre-ovulatory oocytes in female mice. Etoposide-induced preferential pericentric lesions and aneuploidy in oocytes [30].

Combination chemotherapyIn everyday practice, women are rarely subjected to just one chemo therapeutic agent; therefore, the results of single-agent administration cannot be determined [24]. Studies that have monitored pregnancies in women exposed to chemotherapy before conception have not reported increased rates of miscarriage or congenital abnormalities in comparison with the general popula-tion [22]. Since these pregnancies occurred long after treatment had ceased, it can be assumed that correction mechanisms exist within the oocyte, or that there are undetected miscarriages at a very early stage due to dominant lethal mutations.

Fertility preservation strategiesTo date, the most effective approach to preserve fertility is embryo cryopreservation. The human embryo is very resistant to damage caused by cryo preservation. The post-thaw survival rate of embryos is in the range of 35–90%, while implantation rates are between 8 and 30%; if multiple embryos are available for cryopreservation, cumulative pregnancy rates can be more than 60% [31]. Delivery rates per embryo transfer using cryopreserved embryos are reported to be in the range of 18–20% [31]. However, this approach requires in vitro fertilization and a participating male partner. If many mature oocytes are retrieved, there is an opportunity to carry out several attempts at embryo transfer from a single cycle. This option may not be acceptable to prepubertal, adolescent girls [32].

Cryopreservation of mature oocytes (following gonadotropin stimulation)Oocyte banking is more problematic than cryopreservation of sperm or embryos. The first obstacle is the sensitivity of oocytes to chilling, probably due to the sensitivity of the spindle apparatus and the higher lipid content of the cells. Cooling and exposure to cryoprotecting agents (CPAs) affect the cytoskeleton and may aggravate the already high incidence of aneuploidy in human oocytes [33]. Exposure to CPAs also causes hardening of the zona pellucida; therefore, all oocyte cryopreservation protocols involve intracytoplasmic sperm injection (ICSI) as a precaution. Fertilization has to be carried out approximately 3–5 h after thawing while the oocyte remains fertile.

Further disadvantages of this method are that cancer patients may not have more than one opportunity for oocyte harvesting before undergoing potentially sterilizing treatment, since a cycle of controlled stimulation requires several weeks and there is nor-mally a delay of a few months before treatment cycles. The suc-cess of the method is also dependent on the total number of eggs harvested (<10 oocytes means a very low chance of pregnancy).

However, with the introduction of ICSI and the publica-tion of reassuring data [34], efforts to cryopreserve oocytes have resumed in recent years, with conventional slow cooling–rapid thawing protocols and later with vitrification. To date, more than 4300 oocytes have been cryopreserved and more than 80 chil-dren have been born, mostly with the conventional slow-cooling method. The overall live-birth rate per cryopreserved oocyte is approximately 2%, which is much lower than that of in vitro fertilization (IVF) using fresh oocytes [35].



These data were confirmed by a recent meta-analysis from Oktay et al., who found that the live-birth rate per injected oocyte was approximately 2% for the most commonly used slow-cooling technique. Pregnancy rates were a third to a quarter of the success rates observed with unfrozen oocytes [36].

Recently, better results regarding the applicability and efficiency of cryopreservation of unfertilzed metaphase II oocytes due to the vitrification method have been published [37].

Cryopreservation of immature oocytes (without gonadotropin stimulation)An alternative strategy that allows a longer recovery time and avoids depolymerization of the spindle is to cryopreserve oocytes at the germinal vesicle stage, when the chromatin is diffuse and the cell is still at prophase I. However, immature oocytes do not appear to cryopreserve better than those at the metaphase II stage. Furthermore, they have to undergo incubation for 24–48 h in culture for meiotic maturation [35].

Cryopreservation of immature oocytes after in vitro maturation (without gonadotropin stimulation)Oocytes are recovered for in vitro maturation (IVM) from fresh tissue or follicular aspirates, before the dominant follicle emerges during the mid-follicular phase of the menstrual cycle (normally 8–10 mm in diameter). Cryopreservation difficulties include the different optimal times of equilibration for the oocyte and its smaller cumulus cells. At present, the reported success of IVM in young women with polycystic ovaries is a pregnancy rate of approximately 25–30% per cycle, with a high miscarriage rate [38].

Oocytes can, thus, be recovered from unstimulated ovaries, as well as from children, and egg harvesting is less expensive and risky and can be repeated frequently. However, this procedure requires further advances in cryotechnology.

Gonadotropin-releasing hormone receptor analogue treatmentMultiple small studies have evaluated the utility of gonado tropin-releasing hormone receptor (GnRH)-α analogue treatment to preserve ovarian function during cytotoxic therapy. Rendering the ovarian follicular development quiescent by suppression of gonadotropins has been proposed to protect women from damage by cytotoxic therapy. This research has suggested that receipt of GnRH-α throughout treatment may increase a woman’s likeli-hood of remaining premenopausal after chemotherapy, although there has been an intensive debate concerning the existence of FSH receptors in primordial follicles and GnRH-α receptors in the human ovary [39,40]. The protection cannot involve induction of quiescence in the already dormant primordial follicle but may involve direct effects of GnRH analogues or indirect effects of gonadotropin suppression on the whole ovary [39,41].

In a prospective randomized study in female Rhesus mon-keys, Ataya et al. demonstrated that GnRH-α may protect the ovary from cyclophosphamide-induced gonadal damage [42]. Meirow, however, was unable to demonstrate a protective effect of GnRH after ablative chemotherapy and radiotherapy in patients

undergoing bone marrow transplantation [43]. Waxman et al. found that buserelin was not effective for fertility preservation in humans. However, it is possible that complete pituitary ovar-ian suppression was not achieved, which might be a necessary prerequisite for these drugs to work [44]. Our own study group showed that GnRH-α did not prevent ovarian follicle loss in a human ovarian xenograft model [45].

Blumenfeld and other research groups were able to demonstrate that the GnRH agonists are well tolerated and may protect long-term ovarian function [46–54], although some studies included only a small number of patients. Blumenfeld has reported on what is probably the largest group of women (55 lymphoma patients) who were started on GnRH analogs 7–10 days before chemotherapy treatment. The rate of POF was 5% in the GnRH analogue/chemotherapy group versus 55% in the group receiving chemotherapy alone [54].

Treatment with GnRH-analogues should begin at least 10 days before the start of chemotherapy due to the initial flare-up effect, which causes an undesirable ovarian stimulation. The application should continue throughout the chemotherapy period in the form of depot injections, so that the downregulating effect remains at least for 2 weeks after the chemotherapy ends. In the case of estrogen-sensitive tumors, a tamoxifen therapy can be initiated after the GnRH-analogue treatment.

However, available studies are limited by small sample size, lack of a randomized control group and lack of definitive information regarding actual fertility outcomes. Randomized controlled trials are currently underway internationally to evaluate this strategy in women with cancer.

The Southwestern Oncology Group is running an ongoing rand-omized evaluation among women with hormone receptor-negative stage I–IIIA breast cancer on whether or not to receive goserelin through treatment. In the UK, the Ovarian Protection Trial in Oestrogen Nonresponsive Premenopausal Breast Cancer Patients Receiving Adjuvant or Neo-adjuvant Chemotherapy trial is simi-lar but is also including women with hormone receptor-positive disease. The potential benefit of ovarian suppression in addition to tamoxifen for women with hormone receptor-positive breast cancer is currently under active investigation in the Suppression of Ovarian Function Trial (SOFT) trial. Other prospective randomized trials, such as the Zoladex Rescue of Ovarian Function study in Germany, the Italian multicenter study for breast cancer patients, the German Hodgkin Lymphoma Group multicenter study, the UK Lymphoma multicenter study, the Spanish Lymphoma multicenter study and the Prevention of Gonadal Toxicity and Preservation of Gonadal Function and Fertility in Young Women with Systemic Lupus Erythematosus Treated by Cyclophosphamide Trial (PREGO) will provide definitive evidence of the role of GnRH-α in ovarian function preservation [40].

In a recent, very interesting review by Octay et al. , the pos-sible hazards of a GnRH-analogue treatment for fertility pres-ervation purposes have been described sufficiently [40]. Not only are GnRH analogues expensive and cause severe menopausal symptoms but, in addition, the direct effects of GnRH agonists on human cancer cells are not understood sufficiently. A variety of human cancers, including those of the breast, ovary and



endometrium, express GnRH receptors. These receptors medi-ate several effects, such as inhibition of proliferation, induction of cell-cycle arrest and inhibition of apoptosis, induced, for example, by cytotoxic drugs [55]. Thus, it cannot be excluded that GnRH-agonist therapy concomitant with cytotoxic chemotherapy might reduce the efficacy of chemotherapy for breast cancer. There are, however, data from randomized stud-ies, as well as the results of the Early Breast Cancer Trialists’ Collaborative Group meta-analysis and the results from the luteinizing hormone-releasing hormone (LHRH) agonists in the Early Breast Cancer Overview group that did not show a different outcome in patients who had received concurrent ovarian suppression with chemotherapy compared with patients treated with chemotherapy alone [56–58]. In a recent meta-anal-ysis, data from 11,906 premenopausal women with early breast cancer randomized in 16 trials were examined. When used as the only systemic adjuvant treatment, LHRH agonists did not significantly reduce recurrence (28.4% relative reduction; 95% confidence interval [CI] consistent with 50.5% reduction to 3.5% increase; p = 0.08) or death after recurrence (17.8; 95% CI: 52.8% reduction to 42.9% increase; p = 0.49) in hor-mone receptor-positive cancers. The addition of LHRH agonists to tamoxifen, chemotherapy, or both, reduced recurrence by 12.7% (95% CI: 2.4–21.9; p = 0.02); and death after recurrence by 15.1% (95% CI: 1.8–26.7; p = 0.03). LHRH agonists showed a similar efficacy to chemotherapy (recurrence: 3.9% increase [95% CI: 7.7% reduction to 17.0% increase]; death after recur-rence: 6.7% reduction [20.7% reduction to 9.6% increase]; both not significant). No trials had assessed an LHRH agonist versus chemotherapy with tamoxifen in both arms. LHRH agonists were ineffective in hormone receptor-negative tumors [56].

On a more practical level, up to 97% of patients suffer from hypoestrogenic symptoms when using a GnRH analogue along with chemotherapy [50]. Furthermore, when used for more than 4 months, patients may experience bone loss, which may not be reversible with longer durations of use [59].

The American Society of Clinical Oncology points out that there is insufficient evidence regarding the safety and effective-ness of GnRH analogs and other means of ovarian suppression on female fertility preservation at this time, and recommends that women interested in ovarian suppression for this purpose are encouraged to participate in clinical trials [60].

Sex steroidsThe oral contraceptive pill has been investigated as an agent to suppress the ovaries during chemotherapy and invoke protection from these cytotoxic agents. Chapman and coll-leagues reported more follicles on ovarian biopsies in three patients receiving combination oral contraceptive pills during chemotherapy than those who did not [61]. On the other hand, Whitehead et al. found no protective effect from combination oral contraceptive pills in patients who received chemotherapy for Hodgkin’s disease [62]. One possible explanation could be that the oral contraceptive pills do not manage to suppress the gonads completely.

Progesterone (P4)In the rat, progesterone was found to have a protective effect when administered 1 week before the start of cyclophosphamide and also during the treatment [63].

Familiari et al. examined the protective effect of medroxypro-gesterone acetate (MPA) on human primordial follicles exposed to cytotoxic drugs. Using electron microscopy, they showed that chemotherapy not only acutely damaged the ovary by reducing the number of follicles, but also chronically damaged the remain-ing follicular quality. MPA was unable to protect the ovary from early follicular atresia [25].

Apoptotic inhibitorsWhen mice oocytes were exposed to doxorubicin in vitro, they underwent a series of changes that produced apoptotic bodies [64]. Apoptosis also plays a significant role in the process of normal germ cell depletion [65], such that the existence of a predetermined genetic pathway that can be aberrantly activated by chemothera-peutic drugs has been suggested [66]. As a logical consequence, the use of apoptosis inhibitors could potentially stop the apoptotic process and protect the patient from POF.

The use of sphingosine-1-phosphate, a known apoptosis inhibitor in mice treated with doxorubicin, was found to protect the oocytes from apoptosis. Furthermore, the oocytes of mice that lacked the enzyme to generate ceramide and acid sphingo myelinase early messengers in the apoptosis sequence, were more resistant to dox-orubicin-induced apoptosis [67]. Sphingosine-1 phosphate also pre-served the fertility of irradiated female mice without any genomic damage to the offspring [68]. These studies show that these agents are promising but are still at a very early experimental stage.

Cryopreservation of ovarian tissue At present, cryopreservation of ovarian tissue appears to be a very promising way of providing the cancer patient with a realistic chance of fertility preservation – a prospect that is also extremely important for psychological reasons [69].

The cryopreservation of ovarian cortical strips has recently emerged as an easy, fast and inexpensive technique, and has already yielded five live births (Table 1) [70–73]. The idea of cryopreserving ovarian tissue is based on the finding that the ovarian cortex har-bors primordial follicles that are more resistant to cryoinjury than mature oocytes, because the oocytes they contain have a relatively inactive metabolism and lack a metaphase spindle, zona pellucida and cortical granules [74]. The clinical indications are almost identi-cal with those for the oocyte, but there are fewer logistical restric-tions and there is a greater fertility potential owing to the far greater number of oocytes preserved. Ovarian tissue cryo preservation may be the only acceptable method for any pre pubertal or premenarchal female patients receiving chemotherapy or pelvic radiotherapy [75]. Follicular viability after cryo preservation and thawing has been demonstrated in several studies [76–78].

The risks of ovarian tissue cryopreservation include reimplanta-tion of the primary tumor and malignant transformation, as well as risks related to the invasiveness of the procedure. Limiting fac-tors of this method are the fact that it is still at the experimental



stage, the availability of the procedure in some selected centers and the limited life of the ovarian grafts. Questions in the field of ovarian tissue cryopreservation that remain unanswered include: the optimal site for retransplantation, the size of the ovarian grafts and the effect of gonadotropin stimulation [69].

Safety & efficacy of in vitro fertilization after chemotherapyAn increas in estradiol during controlled ovarian hyperstimula-tion (COH) may not be safe in women diagnosed with estrogen-sensitive breast cancer who are seeking fertility preservation. It has been clearly shown that estrogen stimulates breast cancer cell growth, even in low concentrations [79,80].

However, the embryo yield with ‘natural cycle’ IVF (without hormone stimulation) is very low [81]. In such cases, alternative stimulation regimens can be used – for example, tamoxifen [82] or aromatase inhibitors [83], although these regimens are less effective without added gonadotropins. Although medications should not be used during pregnancy, studies with tamoxifen and letrozole demonstrated that its short-term use for ovulation induction does not adversely affect oocyte and embryo development. Moreover, no detrimental effect on fetal development was demonstrated. In fact, clomiphene, a related compound of tamoxifen, has been safely used for ovulation induction for almost four decades [84].

In the study by Oktay et al., tamoxifen (40–60 mg) was started on day 2 or 3 of the cycle and was administered daily for 5–12 weeks. The control group consisted of patients with an unstimulated IVF cycle. The tamoxifen group had a significantly higher number of mature oocytes, peak estradiol, and embryos (a mean of 1.6 vs 0.6 embryos) than the natural-cycle group [81].

Third-generation aromatase inhibitors (letrozole, anastrozole, and exmestane) entered practice primarily as first- and second-line treatment agents for the treatment of breast cancer [85,86]. The use of aromatase inhibitors for ovulation induction was first reported in 2001, where letrozole provided superior results to clomiphene

and was associated with 50% lower estra-diol levels [87]. Many groups are currently testing the feasibility of ovarian stimulation with aromatase inhibitors in patients with breast cancer and endo metrial cancer. The patient is stimulated with gonadotropin and an aromatase inhibitor is simultane-ously introduced to reduce serum estradiol levels. Oocyte development is unaffected. A LHRH antagonist is also used to prevent a premature luteinizing hormone surge [88]. Oktay et al. compared the combination of tamoxifen or letrozole with FSH for stimulation in women with breast cancer, with very promising results [81]. Letrozole FSH and tamoxifen-only stimulation were associated with significantly lower peak estradiol levels than tamoxifen FSH stimulation. The combined letrozole FSH protocol resulted in peak estradiol levels close to those seen in unstimulated cycles,

and breast cancer recurrence rates were not increased compared with controls [89].The same group reported the first pregnancy from cryopreserved embryos generated following tamoxifen stimulation [81], and also that breast cancer patients who under-went ovarian stimulation with anastrozole had a significantly higher exposure to estradiol than those who were stimulated with letrozole [90].

Nevertheless, Partridge and Winer have listed a series of questions that remain unanswered [91]:

How does even a brief exposure to high estrogen levels through •the tamoxifen or FSH–letrozole stimulation affect the risk of breast cancer reccurence?

Does a brief exposure to tamoxifen or letrozole before treatment •compromise the chemotherapy effect?

Do these substances have an influence on the quality of oocytes •harvested?

Little is known regarding the efficacy of IVF in female patients who were treated for cancer. Ginsburg et al. examined 15 patients retrospectively in 2001, and discovered they had a poorer response to gonadotropins than women with locally treated cancers, Furthermore, these patients had a significantly diminished ovar-ian response to ovulation induction compared with patients undergoing IVF before cancer treatment [92]. In a retrospec-tive study, Dolmans et al. examined the effect of chemotherapy directly before an IVF treatment in a small number of patients (four patients who underwent IVF in an interval between two chemotherapy regimens vs seven patients who underwent IVF before starting chemotherapy) and reported a dramatic reduc-tion in IVF efficacy even after only one regimen [93]. It was thus recommended that for women whose cancer therapy can be delayed, IVF with embryo cryopreservation should be offered before chemotherapy and not after.

Table 1. Pregnancies and outcome after ovarian tissue cryopreservation and transplantation.

First author

Year Diagnosis Age (years)

Outcome Ref.

Donnez 2004 Hodgkin’s lymphoma 25 Spontaneous pregnancy, live birth

[71]

Meirow 2005 Non-Hodgkin’s lymphoma 28 IVF, live birth [70]

Demeestere 2007 Hodgkin’s lymphoma 29 Spontaneous pregnancy, live birth

[72]

Demeestere 2007 Hodgkin’s lymphoma 31 Spontaneous pregnancy, miscarriage, then live birth

[72]

Andersen 2008 Hodgkin’s lymphoma 25 IVF, miscarriage [73]

Andersen 2008 Hodgkin’s lymphoma 26 IVF, live birth [73]

Andersen 2008 Ewing sarcoma 27 IVF, live birth [73]

IVF: In vitro fertilization.



Pregnancy after cytotoxic therapyIn a recent review of a number of studies in a population of over 15,000 women, with more than 1100 breast cancer patients, it was demonstrated that there were no conclusive data at present that sug-gest any deleterious effects, such as an increased risk for relapse due to subsequent pregnancy in women with a history of breast cancer. A limiting factor of this analysis is that none of these studies is a randomized controlled trial. It is, however, impossible to perform a randomized trial regarding this specific issue, since no woman can be denied the right to get pregnant. Fertility preservation options should, therefore, be discussed with all cancer patients [4].

Regarding the miscarriage rate, studies that have monitored pregnancies in women exposed to chemotherapy before concep-tion could not detect any increased rates of miscarriage or con-genital abnormalities in comparison with the general population. Since these pregnancies occurred long after the cytotoxic treat-ment, it can be assumed that correction mechanisms exist within the oocytes, or that undetected miscarriages occur as a result of dominant lethal mutations at a very early stage [94].

The optimal timing of a subsequent pregnancy after cancer is unclear and depends on the patient’s prognosis, age and personal situation. Meirow and Schiff postulated that patients who recover from ovarian failure after high-dose chemotherapy or radiotherapy treatments should not delay childbearing for too many years. These patients should try to conceive after a disease-free interval of a few years, but not less then 6–12 months after the treatment, owing to the possible toxic effects of the therapy on growing oocytes [94]. A delay of 2–3 years after cancer treatment is conventionally rec-ommended, so that the period associated with the greatest risk of recurrence has passed before a pregnancy. In patients with hor-mone-positive breast cancers, tamoxifen and GnRH analogues do not cause permanent amenorrhea, but this treatment can last up to 5 years, during which a pregnancy is contraindicated [95].

ConclusionYoung female cancer patients are still being counseled poorly with regard to the negative impact of the treatment on their fertility and their options for fertility preservation. The other possibilities of fertility preservation, such as ovarian tissue cryopreservation, IVM or IVF following ovulation induction with aromatase inhib-itors should also be discussed. A pregnancy after cancer treatment does not seem to limit the prognosis.

This review has focused both on the effect of cancer treatments on fertility and on the various assisted-reproduction innovations that are available to provide the cancer patient with the option of future pregnancies. Currently, we are going through a time of uncertainty and revolution concerning the role of ovarian suppres-sion and other fertility preservation measures in the management of early breast cancer, although developments in the near future promise to be very exciting.

Expert commentaryThe field of fertility preservation for female cancer survivors has been growing rapidly, and public as well as scientific inter-est has increased tremendously. This comes as a result of ever

more successful outcomes of cancer treatment and fast-growing reproductive technologies. Although there are some studies that show a positive effect of GnRH-analogues on fertility preserva-tion, there is not sufficient evidence-based data to establish their use as a first-line therapy. There are, at this point, some current prospective randomized studies, but the long-awaited results will probably not be published for another couple of years. In the meantime, the use of GnRH analogues in cancer patients should be offered through a clinical trial following adequate counseling of the patients regarding its potential influence on the effectiveness of chemotherapy.

The cryopreservation of ovarian cortical strips has already yielded the first live births, and is being offered increasingly to patients undergoing cancer treatment. There are important advancements in this reproductive technology that will help to enhance its success and availability for cancer victims.

In everyday routine work, better interdisciplinary coopera-tion between gynecological and pediatric oncologists, surgeons, immunologists and endocrinologists is necessary, so that individu-alized options for fertility preservation can be offered in advance of surgical procedures or cancer treatments.

Five-year viewThe number of female and male cancer survivors will increase further, and the assisted reproduction centers will play a key role in providing the specialized reproductive services requested. ‘Fertility preservation’ specialists will improve the counseling and help to select the best individual method for the ever younger cancer patients, without jeopardizing their cancer survival chances.

National and international fertility preservation networks will coordinate the research and provide better healthcare to cancer patients, with a greater focus on the reproductive outcomes.

Regarding the use of GnRH-analogues over the next few years, we will have the results of the prospective randomized studies currently underway, so that the unclear situation about hormonal suppression will finally be resolved.

Many new technologies, which are now still in a more-or-less experimental state, such as ovarian tissue and oocyte cryopreser-vation, IVM or IVF after ovulation induction with aromatase inhibitors, are expected to develop further and provide reasonable successful outcomes.

The construction of artificial gametes may be made possible by transferring the nucleus of somatic cells into enucleated oocytes and inducing chromosomal halving (haploidization). Ethically, this method is still extremely controversial and is illegal in many coun-tries. However, artificially created gametes could, in the future, provide an ultimate solution for the treatment of infertility [96].

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.



Key issues

As a result of improvements in current treatment therapies, including new surgical techniques, radio- and chemotherapy and • hematopoietic stem cell transplantation, the cure rates for cancer are steadily rising and more young women survive cancer every year to face the long-term complications of the treatment, such as fertility loss.

The risk of chemotherapy-related amenorrhea depends on the patient’s age, the specific chemotherapeutic agents used and the total • dose administered. Regular menses and a normal reproductive outcome after chemotherapy are not certain indicators of whether the ovarian follicular reserve has survived the treatment unaffected.

The primary goal should always be the cancer cure and no compromises should be made regarding the cancer therapy. Fertility • preservation should be an issue when counseling young women, without jeopardizing their survival chances.

The two main fertility preservation tactics include the reduction of reproductive damage by the cancer treatment and assisted • reproduction techniques, such as cryopreservation of embryos, gametes or ovarian tissue.

The new heightened awareness of the effects of various cancer treatments on fertility has coincided with the emergence of new • assisted reproduction and cryopreservation techniques, such as ovarian tissue and oocyte cryopreservation, in vitro maturation or in vitro fertilization after ovulation induction with aromatase inhibitors.

In everyday routine work, better interdisciplinary cooperation between gynecological and pediatric oncologists, surgeons, • immunologists and endocrinologists is necessary so that individualized options for fertility preservation can be offered in advance of surgical procedures or cancer treatments.

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AffiliationsTheodoros Maltaris, MD •Department of Obstetrics and Gynecology, Leopoldina Academic Hospital, 97421, Schweinfurt, Germany Tel.: +49 972 1720 6383 Fax: +49 972 1720 2983 [email protected]

Michael Weigel •Department of Obstetrics and Gynecology, Leopoldina Academic Hospital, Schweinfurt, Germany Tel.: +49 972 1720 6380 Fax: +49 972 1720 2983 [email protected]

Ralf Dittrich •Department of Obstetrics and Gynecology, University-Hospital Erlangen, University of Erlangen-Nuremberg, Erlangen, Germany Tel.: +49 913 1853 3553 Fax: +49 913 1853 3552 [email protected]

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Cancer and fertility preservation in females: where we stand and where we are heading