Ovarian function, reproduction and strategies for fertility preservation after breast cancer

1. Breast cancer and reproductive effects 

Infertility is a dramatic and frequent side effect in men and women of childbearing age who are undergoing chemotherapy for their cancer treatment. This severe and often underestimated complication heavily deteriorates the quality of life of affected patients and risks to reduce the therapeutic efforts and the compliance towards the treatments suggested. Moreover, cancer related infertility is still considered a marginal aspect of the quality of life in cancer patients while “fertility preservation” plays today an emerging role in the culture of industrialized countries and moves extraordinary interests by scientific and economic points of view. Chemotherapy-induced gonadotoxicity could be almost irreversible. Histologic changes in the ovary after the exposure to cytotoxic drugs show different degrees of modifications ranging from decreased numbers of follicles to absent follicles or to irreversible fibrosis. Since more young people are offered adjuvant treatment at earlier stages of cancer, the problem of chemotherapy related gonadic toxicity has considerably increased in the last years. It is also important to consider the new opportunity derived from the current assisted reproductive techniques. By this point of view physicians need to be able to make accurate assessments of risks and benefits of antineoplastic treatments in order to schedule the best approach to preserve the possibility of reproduction in their young patients [1], [2], [3], [4], [5], [6], [7].

Breast cancer is the most common cancer in fertile premenopausal women. Although there are effective curative treatment regimens for early stages of breast cancer, the expanding role of chemotherapy renders many patients infertile irreversibly.

Breast cancer often affects more and more frequently women of reproductive age, with severe consequences from the clinical and psychological point of view [8], [9]. Although the current therapies are effective in a large part of patients, nevertheless cytotoxic chemotherapy causes ovarian reserve depletion; moreover hormonal therapy necessitates a delay in pregnancy, resulting in infertility and gestational side effects.

So patients of reproductive age should be referred to fertility specialists to explore methods of fertility preservation upon diagnosis. Among the different tumors occurring in women, breast cancer constitutes an emerging reproductive problem for three different reasons: the increasing rate of young patients in fertile age, the lack of specific guidelines for gonadic failure prevention after chemotherapy and the conflicting and unresolved debates about the prognosis and risks of pregnancy occurring in women after breast cancer. In addition, we must consider the expanding rate of fertile women with this neoplasia who are definitely cured with an integrated approach and therefore candidates for future reproductive programs.

Breast cancer is the most common invasive cancer in females in Europe. It is estimated that in the year 2000 there were 350,000 new breast cancer cases in Europe, while the number of deaths from breast cancer was estimated to be 130,000. Breast cancer is responsible for 26.5% of all new cancer cases among women in Europe, and 17.5% of cancer deaths [8].

About 25% of breast cancer cases occur before the age of menopause, and 15% occur under the age of 45 [9], [10]. The increase in the number of women who delay first childbirth beyond the age of 35 and the current extensive use of adjuvant chemotherapy regimens has resulted in a large proportion of breast cancer patients of reproductive age facing infertility [11], [12], [13], [14], [15], [16].

Therefore in young breast cancer patients, the potential benefits of adjuvant chemotherapy should be carefully weighed against the long-term adverse impact on fertility, especially in very early stages of cancer and in the case of a clinical indication for endocrine adjuvant therapy. This is more relevant today because of the more extended use and sometimes abuse of chemotherapy in the multidisciplinary treatment of young patients with breast cancer.

There is a strong evidence that many medical oncologists, surgeons and gynaecologists do not sufficiently inform their young patients with breast cancer about the impact of cancer treatment on their fertility [17], [18]. The problem is more relevant in female patients because it appears evident that it is simple and safe to offer a cryopreservation of sperm to male with cancer while it is more complex to give the same opportunity to females or to offer alternative methods of fertility preservation.

Even if there are different possibilities to preserve or restore fertility in women exposed to chemotherapy and other antineoplastic treatments, none of them is considered the standard by a scientific and clinical point of view: in vitro fertilization and embryo cryopreservation, ovarian tissue cryopreservation, follicle and oocyte cryopreservation, and the administration of hormonal agents with a potential protective effect, constitute a very attractive method to avoid or reduce chemotherapy induced ovarian failure.

Usually in the multidisciplinary cancer care programs there is little information about the risk of gonadal failure induced by chemotherapy and the possibility to reduce the incidence and gravity of this side effect in young women as well. There are conflicting hypotheses about the reasons why cancer doctors (oncologists, surgeons, gynaecologists) avoid to explain the risk of infertility while they dedicate more time discussing with their patients about other chemotherapy induced side effects (anemia, neutropenia, alopecia, nausea). By this point of view usually physicians may be induced by the hardness and severity of the disease to discuss with their patients about immediate or potentially life-threatening complications of chemotherapy instead of discussing other late side effects as infertility and ovarian damage.

Cancer doctors are frequently less likely to refer their patients with breast cancer to fertility services in order to offer and discuss integrated and individualized programs useful for the prevention of ovarian failure and for the scheduling of reproductive techniques. So, according to this situation and to the referred data, it appears useful and mandatory to give specific guidelines to better communicate to the young patients with breast cancer an accurate information about the effects of cancer treatment on fertility and to schedule appropriate strategies for reproductive preservation.

2. Chemotherapy and ovarian failure 

There is a strong evidence, according to scientific preclinical and clinical data, that the impact of cancer treatments on fertility is greater than generally perceived and this underestimated effect heavily affects the quality of life of the patients. Ovarian toxicity deriving from chemotherapy was reported almost 60 years ago; chemotherapy has shown to induce both infertility and loss of steroid hormone production in females, but the complex biologic mechanism by which chemotherapy affects the ovary is still unknown. Amenorrhea is the main clinical symptom related to chemotherapy derived gonadal toxicity. Reversible amenorrhea will result when maturing follicles are destroyed by cytotoxic drugs. Permanent amenorrhea or premature ovarian failure will result when all primordial follicles are destroyed. The majority of women who remain amenorrheic 1 year after treatment do not recover ovarian function. Menses recovery after 1 year of amenorrhea occurs in less than 11% of women over 40 years old and in only 12–15% of younger women [12], [13], [14].

The sensitivity of the primordial follicle to cytotoxic drugs is different in different species and probably in different people. Premature ovarian failure is a frequent consequence of exposure of the female gonads to radiotherapy and anticancer drugs; this failure seems related to a rapid depletion of the oocyte reserve, mediated by the apoptotic cell death and ovarian atrophy with disappearance of primordial follicles [19]. Many studies have suggested that premature ovarian failure can improve the breast cancer prognosis for women with receptor positive breast cancer; according to this statement the widespread use of adjuvant endocrine therapy for women with early stage breast cancer has dramatically decreased breast cancer mortality. Unfortunately these women often experiment subsequent infertility and many psychological problems related to this condition.

The risk of chemotherapy-related ovarian failure increases along with aging but the effects of chemotherapy on ovarian function often are unpredictable and it is possible that it could be related to other variables: ovarian follicle reserve, familial ovarian history, kind and number of drugs, dose-intensity and dose-density, type of administration (oral, short infusion, protracted infusion), presence of other diseases, hepatic and renal function, concomitant or sequential radiotherapy and pretreatment fertility of the patient [20], [22]. The age of the patient, cumulative dose and type of the cytotoxic drugs and ovarian reserve at the time of chemotherapy are the most important factors that contribute to the occurrence of chemotherapy induced ovarian failure (Table 1).

Table 1. Risk factors for the occurrence of chemotherapy-induced ovarian failure.

	- Patient age
	- Familial ovarian history
	- Ovarian follicle reserve
	- History of previous ovarian and pelvic surgery
	- Previous chemotherapy
	- Previous pelvic and abdominal radiotherapy
	- Persistent high level of FSH
	- Class of the chemotherapeutic agent (alkylating vs. nonalkylating)
	- Concomitant other diseases
	- Dosing and length of chemotherapy

There is scientific evidence that prepubertal ovaries appear to be more resistant to cytotoxic drugs, probably due to the large number of follicles present at a younger age. In addition genetic and hereditary factors probably play a strategic role in the occurrence of chemotherapy ovarian failure but at the moment the mechanisms involved are unknown. In addition to these different factors (with the leading role of age), the impact of presently available anticancer agents on ovarian function seems related to the type, number and cumulative dose of the drugs used for the treatment of neoplasia. As new drugs are introduced in clinical practice with an expanding use in the adjuvant setting for breast cancer, the long-term effect of these treatments on the human ovary is extremely difficult to evaluate. In this context the future availability of a specific bioassay that can quantify the ovarian impact of anticancer drugs would offer a significant progress in the prevention of chemotherapy induced premature ovarian failure. It is well known that in premenopausal women cytotoxic chemotherapy can induce reversible or permanent ovarian failure [21], [12], [22].

Unfortunately the knowledge on the impact of cancer treatments on gonadal function are based on the assessment of the menstrual cycle, which is not a sensitive marker of fertility. In fact still today the most used indicator of ovarian failure is the occurrence of amenorrhea. The validity of this surrogate sign is questionable and it seems reasonable to investigate with more studies in order to identify new and more specific indicators of ovarian damage. The incidence of chemotherapy induced amenorrhea is directly related to age and varies with the type of chemotherapeutic agent used as well as its dose and schedule [21], [23], [24], [25]. Chemotherapy-related amenorrhea depends considerably on the drug used. We can divide into three different groups the anticancer agents most frequently correlated with ovarian failure in the treatment of breast cancer. In the first group we include agents definitely associated with gonadal toxicity, as cyclophosphamide. In the second group we find drugs that are unlikely to cause gonadal toxicity: methotrexate and 5-fluorouracil. In the third group we include drugs with conflicting data about their gonadal toxicity: doxorubicin, epirubicin, vinca alkaloids (vinorelbine), cisplatin, carboplatin. Alkylants significantly increase the risk of ovarian failure by a factor of 4 [26], and historically these drugs display the most deleterious effect on gonadal function; these agents (for example, cyclophosphamide) show the greatest risk for inducing ovarian failure among all anticancer drugs [23]. In this contex cyclophosphamide has been extensively investigated and is widely known to induce frequently amenorrhea [27]. Scientific data supports the concept that gonadal toxicity seems strongly related to the cumulative dose of cyclophosphamide or other alkylating agents. Alkylants 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 display little toxic effects on ovarian reserve. Other molecules such as antimetabolites (as 5-fluorouracil, methotrexate), antibiotics (adriamycin, epirubicin), platinum derivates, are able to damage the gonadal function with different degrees.

There is few data available concerning the newer agents such as taxanes; moreover data about the toxic effect on fertility of new targeted agents (monoclonal antibodies, small molecules) are also lacking (Table 2).

Table 2. Drugs class and risk of ovarian failure.

	First Group (drugs mostly correlated with ovarian failure)
	•Cyclophosfamide
	•Other alkylantes
	Second Group (drugs that are unlikely to cause gonadal toxicity)
	•Methotrexate
	•5-Fluorouracil
	Third Group (drugs with conflicting results in causing gonadal toxicity)
	•Doxorubicin, epirubicin and other antracyclines
	•Vinca alkaloids (vinorelbine), cisplatin, carboplatin, oxaliplatin
	Fourth Group (drugs without mature data about their gonadal toxicity)
	•Taxanes, topoisomerase inhibitors, new antimetabolites
	•Molecular targeted agents

Due to the few data regarding the risk of male and female infertility associated with cancer chemotherapy and considering the many patient factors that influence fertility, it is difficult for clinicians (surgeons, gynaecologists, oncologists, etc.) to give definite guidance to patients about their risks for their fertility and reproductive results. Given the established role of single agents in the induction of chemotherapy induced ovarian damage, there is strong evidence that the risk of premature ovarian failure seems to increase with the association of more drugs (Table 3).

Table 3. Risk of irreversible and permanent amenorrhea in women treated for breast cancer.

	Risk	Age	Chemotherapy schedule
	High	40 and older	CMF; CEF, CAF×6 cycles
	Intermediate	30–39	CMF; CEF, CAF×6 cycles
		40 and older	AC×4
	Lower	Less than 30	CMF; CEF, CAF×6 cycles
		Less than 40	AC×4
	Very low (<20%)		Vincristine
			Methotrexate
			Fluorouracil
	Unknown		Taxanes
			Oxaliplatin
			Irinotecan
			Monoclonal antibodies
			Tyrosine kinase inhibitors

CMF, CEF, CAF, AC: adjuvant breast cancer therapy with combinations of cyclophosphamide, methothrexate, fluorouracil, doxorubicin, epirubicin.

The classic association of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF schedule) showed a risk of amenorrhea of 61% in patients aged <40 years and 95% in older women [28]. With antracyclines based associations (for example FAC schedule in which a combined infusion of fluorouracil, adriamycin and cyclophosphamide is used), an incidence of menopause was reported more frequently when compared with the CMF schedule. Bines et al. [12] detected amenorrhea in 35% of patients with the AC schedule (adriamycin 60mg/m2, cyclophosphamide 600mg/m2, 4 courses every 3 weeks), Hortobagyi et al. [24] in 59% with the FAC schedule (5-fluorouracil 500mg/m2, doxorubicin 50mg/m2, cyclophosphamide 500mg/m2, 6 courses every 3 weeks), and Levine et al. [29] in 51% with the CEF schedule (oral cyclophosphamide 75 mg/m2 on days 1 through 14, epirubicin 60mg/m2 IV on days 1 and 8 and 5-fluorouracil 500 mg/m2 on days 1 and 8). Some studies have reported a lower cumulative risk of amenorrhea and menopause in antracycline based schedules in comparison with the classic CMF regimen. This effect it is not surprising and it seems related to the higher dosages of cyclophosphamide cumulated in classic CMF schedule (600mg%mq×12 vs. 600mg%mq×6). Unfortunately fewer studies have been conducted on taxane-based regimens and they have shown conflicting results [30], [31], [32].

Moreover, reports using these new drugs were reported in abstract form and have not been confirmed in definitive full papers [33], [34], [35]. A large study showed that the addition of taxane to adriamycin and cyclophosphamide (TAC schedule) increased the risk of chemotherapy-induced amenorrhea, and that the amenorrhea was more likely to be irreversible for women over 40 years old [36]. Other studies have evaluated the impact of the addition of paclitaxel or docetaxel in the occurrence of chemotherapy induced amenorrhea [28], [32], [36], [37]. With some conflicting aspects these reports suggest that the association of taxanes to antracyclines and cyclophosphamide seems to induce little modification in the occurrence of chemotherapy-related amenorrhea. Recently, in a study with an accrual of 191 patients, 4 cycles of AC alone or followed by a taxane were evaluated in order to investigate if the addition of taxane to adriamycin and cyclophosphamide increase the risk of chemotherapy-induced amenorrhea [31]. The study demonstrated that older age and the addition of taxane to AC increase the risk of chemotherapy-induced amenorrhea; moreover the occurrence of amenorrhea was more likely to be irreversible for women >40. In an other study [38] the authors evaluated the incidence and length of amenorrhea induced by two different schedules (6 cycles of FEC100 every 3 weeks and 3 cycles of FEC 100 followed by 3 cycles of docetaxel). The results of the study suggested that sequential treatment induces reversible amenorrhea more frequently than 6FEC courses.

3. Fertility and pregnancy after breast cancer 

Young aged breast cancer survivors very often discuss with the oncologist and gynaecologist about their fertility and the risk of premature menopause that need to be evaluated and appropriately managed. Fertility and reproductive outcome can be transiently or permanently compromised by anticancer treatments as chemotherapy, radiotherapy and endocrine therapy as well, with different mechanisms (decreased number of primordial follicles, gonadal anatomical effects, hormonal regulatory alterations, etc.). Moreover morphological and vascular modifications may also obstacle natural reproduction and successful pregnancy; the long-term effect of these alterations has lead to the use of complex assisted reproductive techniques. According to literature data we know that the incidence of live births after breast cancer is very small. Less than 10% of women previously diagnosed with breast cancer subsequently become pregnant [39], [40]. In women 45 years of age at diagnosis, the rate of full-term pregnancy is only 3% while in younger (<35 years) a rate of 8% was detected [41], [42]. Retrospective case-controlled studies report that full term pregnancy does not adversely effect prognosis [40], [43]. These studies have shown no increase in the incidence of prematurity, stillbirth or congenital malformations in their babies. The follow up and the surveillance of these pregnancies were positive and absolutely reassuring [44], but we have few inconsistent data about the long-term impact of chemotherapy or endocrine therapy on the health of these children. Von Schoultz et al. [42] investigated the prognostic effect of pregnancy 5 years before and 5 years after the detection of breast cancer in a group of 2119 premenopausal women (<50 years); in this experience it was demonstrated that there is no evidence that women with a pregnancy during the 5-year period preceding the detection of breast cancer have a worse prognosis compared with women without pregnancy during the same period. Moreover, in the same study there was no evidence that women with a pregnancy after the diagnosis of breast cancer had a worse prognosis and the Hazard ratio of 0.48 (P=0.14) suggest a possibly reduced risk of distant dissemination in this group of patients. In a danish study the authors examined 173 women with primary breast cancer, who had a full-term pregnancy after breast cancer treatment; in this experience a reduced risk of death (relative risk 0.55) was recognized in comparison with women who did not have a full-term pregnancy [40]. In an other report the overall survival rates at 5 and 10 years, for women with reproductive experience after breast cancer, were reported to be better in an Australian population study [42], compared to the results reported in similar groups of patients [45]. These and other positive results suggest that subsequent pregnancy may give a survival benefit, probably because of the “healthy mother” effect, a bias strongly related to the better prognostic outcome of a self selecting group who is able to conceive after anticancer treatments [46]; nevertheless we have to investigate other conflicting factor potentially involved in this effect. One of the most debated aspects in the clinical care of breast cancer patients is the discussion about the opportunity to schedule a pregnancy after the end of the therapies. Still today the optimal timing to become pregnant, following the diagnosis and treatment, is unknown and it is an important issue for all patients considering both reproductive choices and follow up strategies. In this context patients are strongly recommended to delay pregnancy for at least 2 years after the completion of the therapies for breast cancer; this suggestion is based on the evidence that most recurrences will develop in the first 48 months but it is not agreed by other authors. We are aware that the risk of relapse and the time to recurrence are related to many factors including stage, grading, nodal involvement, endocrine receptor status, HER-2 amplification and other emerging prognostic factors. Some studies [47] stated that patients who achieve complete menstrual activity after high-dose chemotherapy or radiotherapy treatments should not delay childbearing for too many years. A fixed time was not indicated but the authors suggested to start a pregnancy after a disease-free interval of a few years; a shorter time may be dangerous for the possible toxic effects of the therapy on growing oocytes. By this point of view a delay of 2–3 years after the cancer treatment is conventionally recommended, so that the period associated with the greatest risk of recurrence has spent and at least a first step of endocrine treatment has been completed. Therefore, an analysis of a large number of studies in a population of thousands and thousands women with breast cancer, suggests that at present no definitive data confirm negative effects related to subsequent pregnancy in women with a history of breast cancer; moreover, the availability of data showing that women with breast cancer who conceived had a better prognosis when compared with those who did not conceive, support the recommendation to allow pregnancy after the completion of anticancer treatments for breast cancer.

4. Strategies for fertility preservation 

Fertility preservation has become an important issue in the management of breast cancer patients of reproductive age. The more frequent occurrence of breast neoplasia in young women in the last years has expanded this emerging aspect of clinical care in this subset of patients. There is today strong evidence and agreement that all patients of reproductive age with breast cancer should be referred to a fertility specialist to discuss options of fertility preservation. The choice of the most appropriate fertility-preservation procedure depends of different factors and is strongly related to the experience and background of the specialist. Currently there are no treatments which have shown to definitely protect the ovary during the use of chemotherapy for breast cancer. From this point of view the aim to preserve fertility is still far from being reached. For women with breast cancer (particularly in women with hormone-receptor positive disease) fertility preservation is a more complex and debated issue than in other neoplasias, due to the suspect that endocrine manipulations and the occurrence of subsequent pregnancies may increase the risk of cancer recurrence [48]. In evaluating the different options and preferences, critical factors are the evaluation of male fertility and the will to start a pregnancy.

According to the available scientific data, fertility preservation options can be divided into three categories: techniques finalized to reduce the impact of chemotherapy on ovarian function, techniques finalized to remove and preserve ovarian tissue before starting chemotherapy and techniques finalized to produce mature oocytes or fertilized embryos for assisted reproduction. In this context some authors [49] have proposed a model to support the optimal decisions for preserving fertility preservation.

4.2. Other fertilization techniques 

Considering the negative emerging impact of chemotherapy on ovarian tissue in premenopausal fertile women, an escalating number of patients with breast cancer will be included in personalized programs of assisted reproduction in order to maintain their reproductive possibilities. There are different other options available to preserve fertility in breast cancer patients undergoing chemotherapy, in order to select personalized reproductive programs in this subset of patients. The different methods available give many challenges to achieve a pregnancy in this subset of patients and have both advantages and limits (Table 4).

Table 4. Characteristics and limits of methods used for preservation of fertility in females.

	Procedure	Advantage	Disadvantage	Current clinical status
	Hormonal ovarian function suppression with GnRH agonists	Easy to use and reversibility	Safety profile	Investigational
		Delay of chemotherapy by 1–2 weeks
		Relative less invasive	Efficacy not confirmed
	Ovarian tissue cryopreservation and xenotrasplantation	No hormonal stimulation	Low pregnancy ratess	Investigational
		Unlikely to delay chemotherapy
	Embryo cryopreservation	Effective and validated method	Hormonal repeated stimulation	Currently used
	Ovarian stimulation and oocyte cryopreservation	Feasibility	Negative impact in ER+ pts and low pregnancy rates	Currently used

Nevertheless the definitive decision to resume fertility after anticancer treatments for breast cancer depends on a number of well established factors, as the patient's age, the type of adjuvant treatment, the possibility to delay the start of chemotherapy and the possibility to wait for scheduling the pregnancy. Currently, embryo and oocyte cryopreservation (with or without hormonal stimulation), ovarian tissue cryopreservation, ovarian transposition, are different strategies for fertility preservation, although there are other several investigational methods with possible future potential applications in this expanding field.

Embryo cryopreservation is a standard and established fertility preservation method that has been used extensively for storing surplus embryos after in vitro fertilization for infertility. Advantages of this technique are the relatively high pregnancy rates while disadvantages include the need for ovarian stimulation and hyperstimulation, the psychological and ethical problems, the costs, the potentially dangerous delay in starting and completing adjuvant treatment and the risk of chemotherapy interruption and cancellation [60].

With this technique a post-thaw survival rate of embryos ranging from 35% to 90% was reported, while implantations occurred between 8% and 30%; in case of multiple embryos the pregnancy rates occurred in more than 60%; moreover delivery rates were reported in the range of 18–20% [61], [62]. If many mature oocytes are retrieved, it is possible and convenient to carry out several attempts at embryo transfer from a single cycle. In the clinical practice adjuvant chemotherapy commonly starts 4–6 weeks after surgery, so there is an acceptable time to perform ovarian stimulation and IVF before chemotherapy. Oocyte collection and embryo freezing can also be carried out without ovarian stimulation; however in this case the embryo yield is low and typically no more than a single embryo can be produced [63], [64]. Ovarian stimulation with subsequent high estradiol levels can constitute a serious problem in this subset of patients; therefore there is a great interest in the recognition of alternative methods (for example natural cycle IVF). Ovarian stimulation protocols using letrozole, a third generation aromatase inhibitor, given in asociation with FSH, resulted in significantly lower peak estradiol levels than tamoxifen alone or plus FSH. In this case the fertilization rates were similar to the other obtained with conventional ovarian stimulation protocols and 81% of women completed their IVF cycles within 8 weeks of surgery [16], [65].

An alternative method to embryo cryopreservation is oocyte cryopreservation, a method indicated for those patients who do not have a male partner, refuse donor sperm or have critical objections to embryo freezing due to a religious or ethical point of view. At present more than 4300 oocytes have been cryopreserved and more than 80 children have been born, mostly with the conventional slow cooling method. With this technique oocytes are frozen unfertilized and after thawing are fertilized by intracytoplasmic sperm injection. We must also consider that in this procedure, like embryo cryopreservation, there is a need for ovarian stimulation and the success rates are often unsatisfactory: in fact, despite improvements in cryopreservation techniques using slow-freezing protocols, published success rates are three to four times lower than that observed with classic embryo cryopreservation. The overall live birth rate per cryopreserved oocyte is very poor, and it is much lower than that with IVF using fresh oocytes [66]. These data were confirmed by a recent meta-analysis by Oktay and colleagues, who found that the live birth rate per injected oocyte was approximately 2% with the most commonly used slow-freezing technique. Pregnancy rates were one-third to one-quarter of the success rates seen with unfrozen oocytes [67].

Another method, which is still at an experimental stage, is the cryopreservation of immature oocytes (with or without in vitro maturation). This method is currently still associated with a relatively low pregnancy rate, as well as a high rate of miscarriages [68].

The limited success with oocyte thawing is due to the fragility of the meiotic spindle and to intracellular ice crystal formation during the freezing and thawing process [69]. In a meta-analysis [70] comparing the results obtained with the slow-freezing of oocytes and vitrification, the in vitro fertilization success rates with slow-frozen oocytes were significantly lower when compared with the case of IVF with unfrozen oocytes. According to these data the American Society for Reproductive Medicine recommends that oocyte cryopreservation presently should be considered an experimental technique only to be performed under investigational studies.

Cryopreservation of ovarian tissue is another experimental method for fertility preservation. The technique is performed freezing the gonadal tissue (with the primordial follicles) in thin slices. Unfortunately ovarian tissue cryopreservation for future orthotopic or heterotopic reimplantation was associated with poor results in term of successful pregnancies and so still today ovarian cryopreservation and transplantation remains an investigational protocol. There are several reports showing that in humans resumption of endocrine function can occur after orthotopic [71], [72] and heterotopic [73], [74] transplantation of frozen ovarian cortical strips. In literature there have been two reports [75], [76] of full term pregnancies from orthotopic ovarian transplantation in cancer patients (with one spontaneous full term pregnancy and with an other regular delivery after in vitro fertilization).

One criticism against this novel technique is the possibility that reimplanted ovarian tissue may harbour breast cancer micrometastases that may increase recurrence risk, while most of the data is unconclusive concerning this problem. Prior to ovarian transplantation a careful histological assessment of the tissue is mandatory to rule out occult metastasis. Moreover a strict surveillance in patients with BRCA-1 and BRCA-2 mutated genes is important for the risk of occult ovarian cancer [77], [78].

Ovulation induction after breast cancer has been considered for many years a deleterious strategy for the risk that the high estrogen levels may impact the long-term survival negatively. It was extensively demonstrated that estrogens stimulate breast cancer cell growth, even at low concentrations, with potential harmful effect in women treated for invasive breast cancer [79].

Elevated estrogen levels in this subset of patients have been involved in tumorigenesis of breast cells and subsequent risk of breast cancer relapse. Moreover we know that estrogen exposure stimulates mitotic activity in mammary glands; in a conventional in vitro fertilization program the levels of estrogens can reach values 10-fold to 20-fold higher in respect to a natural cycle [80] and this effect generates a great debate about the risk of ovarian stimulation in patient with breast cancer. Therefore, according to the literature data, there is no evidence that the endocrine and immunological effect of pregnancy adversely affect the recurrence rate of breast cancer [81] and therefore it is reasonable to conclude that this risk is low also during the use of common ovarian stimulation techniques. In reproductive medicine, conventional assisted reproductive techniques usually utilize exogenous gonadotropins to recruit multiple follicles for in vitro fertilization programs. In this context it is evident that the best approach is a naturally occurred pregnancy or a natural cycle ART without stimulation; unfortunately the current in vitro fertilization programs are not able to provide successful implantation and full term pregnancy, using a single cryopreserved oocyte or embryo. Fortunately, newer drugs and methods of ovarian stimulation have been developed; these methods are able to obtain multi-follicular recruitment and moreover limit the potential harmful exposure to high levels of estradiol [16], [82], [83], [84].

Paradoxically the same drugs utilized for adjuvant endocrine therapy in breast cancer are today extensively used in IVF programs for ovarian stimulation. Tamoxifen, a selective estrogen modulator with antiestrogenic actions on breast tissue, was originally used as a contraceptive agent but was later investigated as an ovulation stimulator, according to the experience and the results with other SERM (clomiphene) [85], [86]. In the last decades tamoxifen has been the gold standard in the hormonal treatment of early and advanced endocrine responsive breast cancer; although its antagonistic effect on estradiol receptors in breast tissue, endocrine therapy with tamoxifen results in an increase in estradiol levels through inhibiting the negative feedback mechanism of estrogen. In this context it is reasonable that the induction of ovulation with tamoxifen does not adversely affect prognosis if we consider the long time of adjuvant therapy with this hormone and the elevated estradiol levels occurring. The common schedule of treatment with tamoxifen as an agent for ovarian stimulation contemplates 40–60mg of tamoxifen for a mean duration of 7–9 days, beginning on days 2–3 of the menstrual cycle. Oktay and colleagues used tamoxifen in a randomized study to evaluate the fertilization potential in patients with breast cancer. The hormone was given at 40–60mg on day 2 or 3 of the cycle and was administered daily for 5–12 weeks. The control group consisted of patients submitted to unstimulated IVF cycles. In the group titrated with tamoxifen a higher number of mature oocytes, higher peak estradiol, and a higher number of embryos occurred [63].

Other methods of endocrine stimulation of ovarian follicles have been investigated in patients treated for invasive breast cancer. Among these agents the role of aromatase inhibitors has emerged in the last years. These drugs act by blocking the production of estrogens with a peripheral mechanism of action and stimulating the negative feedback of estradiol on the pituitary. Third-generation aromatase inhibitors (letrozole, anastrozole, and exemestane) are extensively used as first-line and second-line therapy for the treatment of advanced breast cancer and for adjuvant treatment of early breast cancer [87], [88]. According to the encouraging results obtained since 2001 in programs of ovulation induction (100), many studies were performed and are currently in progress in order to evaluate the feasibility of ovarian stimulation with aromatase inhibitors in patients with breast cancer [89]. After a preliminary stimulation with gonadotropins, women receive an aromatase inhibitor to reduce serum estradiol levels. The combination of an aromatase inhibitor with gonadotropins was associated with peak estradiol levels similar to those occurring in normal cycles and the risk of breast cancer recurrence was not increased compared with controls [90]. Nevertheless we must consider with precaution these results, considering the limited number of patients investigated.

5. Conclusions 

In the past too many young women with breast cancer were not fully informed about the negative impact of anticancer treatments on their fertility; today, because of the large number of women affected with breast cancer and submitted to adjuvant chemotherapy, many of them are experimenting assisted reproductive strategies to preserve their fertility. Therefore, the number of breast cancer survivors who contact reproductive medicine specialists and gynaecologists to receive clinical indications about their fertility, is dramatically increasing. In this context the questions and the discussions about the reproductive challenges need to be discussed and considered soon after diagnosis and possibly before the start of chemotherapy. Therefore, fertility preservation options should be evaluated and discussed with all young patients desiring future children, considering the potential impact of these techniques on the oncological outcomes. Timing of information is important and crucial for clinical and psychological reasons. The discussion and decisions about the reproductive choices need to be considered immediately after diagnosis and before the start of chemotherapy. A strict link with reproductive specialists and biological banks are mandatory and must be considered as a strategic moment in the integrated approach of the multidisciplinary treatment of breast cancer. Predicting fertility after chemotherapy for breast cancer is still today utopistic and large randomized prospective studies need to be activated in order to better investigate this field.

According to the literature data, pregnancy does not appear to be detrimental after breast cancer, with and without chemotherapy, but tailored and individualized counselling protocols need to be evaluated in terms of prognosis, risk relapse and challenges of reproduction. Since none of the currently available methods for fertility preservation constitutes the gold standard in maintaining future fertility, it is reasonable to encourage the patients to combine different methods of fertility preservation. Moreover patients are usually invited to start a pregnancy at least two years after chemotherapy for breast cancer. Moreover in our opinion the women who regain ovarian functions after cancer therapy should not delay childbearing for too many years.

At moment there are several options available to preserve fertility in breast cancer patients undergoing chemotherapy. In this context the choice of the best strategy depends on a number of different factors, including the patient's age, the type and length of adjuvant treatments, the expertise of the doctor, the need for endocrine therapy, the familial situation, the time available before the start of chemotherapy, the desire for pregnancy.

A positive effect of GnRH analogues on fertility preservation is still debated and investigated; there is insufficient evidence to support the use of GnRH analogues as a first choice in the prevention of chemotherapy induced ovarian failure. According to these conflicting data the routinary use of GnRH analogues in breast cancer patients should be considered in the context of clinical trials after personalized counselling of the women about this therapeutic choice. Other methods of preserving fertility (oocyte and ovarian tissue cryopreservation, in vitro fertilization after ovulation induction, ovarian transposition) should be extensively discussed with the women in order to understand the reproductive results and the potential risk related to the use of these techniques. Unfortunately the most attractive techniques of fertility preservation are not available everywhere, also in industrialized countries, and moreover the high costs limit and slow their clinical application, mainly in developing countries. Nevertheless in our opinion, in the near future, the studies on reproductive and fertilization techniques after breast cancer will dramatically expand, with a positive effect on the quality of life of the young women treated for early (and perhaps advanced) breast cancer.

Reviewers 

Dr. Khalil Zaman, CHUV – Vaud University Hospital, Centre Pluridisciplinaire d’Oncologie (CePO), Rue du Bugnon 46, CH-1011 Lausanne, Switzerland.

Conflict of interest 

The authors disclose no conflicts of interest.