Placenta 29 (2008) S184S190

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Placenta
journal homepage: www.elsevier.com/locate/placenta

Focus on Breast and Ovarian Cancer

A. Borini*, E. Rebellato
Tecnobios Procreazione, Bologna, Italy

a r t i c l e i n f o
Article history: Accepted 5 August 2008 Keywords: Breast cancer Ovarian cancer Ovarian stimulation Oocyte cryopreservation

a b s t r a c t
In reproductive medicine the widespread use of ovarian stimulation has focused interest on the possible relation between induction of ovulation and breast and ovarian cancer. The epidemiological studies published so far are reassuring but not devoid of methodological problems, such as small populations, short follow-ups, and lack of information on confounding factors like oral contraceptive use. In younger patients to preserve fertility before radio- or chemotherapy, the American Society of Clinical Oncology recommended embryo cryopreservation as an established procedure. Moreover, experimental procedure as oocytes cryopreservation, ovarian suppression with GnRH analogues and cortical ovarian tissue freezing probably will be routinely used in the future. A reasonable proposal to protect patients from the risk of estrogen peaks in the case of estrogen receptor positive (ER) breast cancer, ovarian stimulation with tamoxifen or letrozole could be right. Powered studies are necessary to control the safety and effectiveness of the different fertility preservation options. In this review we evaluated the relation between induction of ovulation and breast and ovarian cancer and then the possible options for fertility preservation in patients with these types of tumors. 2008 Elsevier Ltd. All rights reserved.

1. Introduction The possible relation between hormone therapy for induction of ovulation and breast and ovarian cancer is of interest to all specialists in reproductive medicine. In fact, during counselling before an in vitro fertilization (IVF) treatment, the rst question raised very often by patients is if the therapy might increase their health risks, especially of cancer. Therefore, it is necessary to clarify if ovarian stimulation is an independent risk factor for breast and ovarian cancer. Ovulation induction has been used for about 30 years and there are, by now, many studies in the literature trying to provide some answers [15]. 2. Induction of ovulation and ovarian and breast cancer 2.1. Invasive ovarian cancer The aetiology of ovarian cancer is still open to debate. There are few recognized risk factors that could facilitate its early detection. Besides a clear understanding of the earliest recognizable events in ovarian carcinogenesis is difcult just because of an ill-dened premalignant state and the paucity of data from early-stage cancer. In vitro fertilization protocols have become quite widespread, and it is

* Corresponding author. E-mail address: borini@tecnobiosprocreazione.it (A. Borini). 0143-4004/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2008.08.001

necessary to understand if the induction of ovulation might be a possible risk factor for ovarian cancer, especially the epithelial subtypes. There are 2 possible hypotheses for this link. The rst one is the cumulative effect of minor traumas, due to repeated ovulation, that could induce malignant changes in the ovarian epithelium. In fact, conditions as multi-parity, breast feeding and oral contraceptive use, in which ovulation is suppressed, have protective effects on the ovary [6]. The second one is when estrogens could stimulate cell proliferation in the tissues expressing their receptors, such as the granulosa cells [7]. Many studies have tried to clarify if ovarian stimulation could increase the chance of developing ovarian neoplasias as an independent risk factor. Mosgaard et al., for example, reported a casecontrol study including 684 cases of ovarian cancer and 1721 controls, showing no increased risk among treated infertile women, also after separation between nulliparous (Odds Ratio (OR) 0.8) and patients (OR 0.6) [2]. Brinton, on the contrary, presented a retrospective cohort study of 12,193 women evaluated for infertility during the period 1965 1988. The infertile patients had a signicantly elevated ovarian cancer risk compared with the general population (standardized incidence ratio (SIR) 1.98, 95% condence intervals (CI) 1.42.6). When patient characteristics were taken into account, the rate ratios associated with ever usage were 0.82 (95% CI 0.41.5) and 1.09 (95% CI 0.42.8) for clomiphene and for gonadotropins, respectively [3].

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Furthermore, Rossing et al., in a cohort study of 3837 infertile women, found that clomiphene citrate was associated with a relative risk (RR) of 2.3 (95% CI 0.511.4) compared with infertile women not using it. The long-term use of this drug (12 or more cycles) brought relative risk to 11.1 (95% CI 1.582.3) [4]. Finally Shushan et al. reported an adjusted relative risk with clomiphene of 0.9 (95% CI 0.32.3). However, his study was limited by the fact that 36% of the cases had died before further contacts were established, causing a possible selection bias [5]. An interesting review by Mahdavi et al. considered invasive epithelial carcinoma, invasive non-epithelial carcinoma and borderline ovarian tumor. The authors analysed cohort, casecontrol and descriptive studies, nding discordant results. Mahdavi concluded that, at the moment, the ndings on ovarian cancer risk associated with treatment for the induction of ovulation are still reassuring, but well-designed clinical trials are still necessary [1]. The possible limits of these analysis are due to methodological problems: such as small size populations, short follow-up, low prevalence of infertility and fertility drugs use, lack of information on confounding factors, for instance oral contraceptive use, and family history of ovarian neoplasias. 2.2. Borderline ovarian tumor In the literature there are contrasting results also about ` borderline ovarian tumor. In a recent case-control study by Cusido, 42 women with this type of cancer were compared with 257 patients with benign ovarian pathology. No differences were found between the 2 groups, in terms of type of drugs used whether clomiphene citrate (9.5% vs. 6.2%, respectively) or gonadotropins (7.1% vs. 10.1%, respectively) and number of cycles administered (mean number of cycles with clomiphene citrate/gonadotropins was 2.50 1.00 and 3.00 2.64 in the borderline tumor group and 2.44 1.75 and 3.27 2.25 in the control group) [8]. On the contrary, Sanner et al., presenting an historical cohort study of 2768 women treated for infertility, underlined an increased risk of borderline ovarian tumors after clomiphene treatment for ovulatory problems (SIR 7.47; 95% CI 1.5421.83) [9]. Therefore, based on the evidence to date, it is not possible to establish if the induction of ovulation might be a possible risk factor for ovarian cancer. Generally, the results of the studies are reassuring in not conrming a strong link. However, slight, but non-signicant elevation in risk associated with certain subgroups of drugs users, needs a continued monitoring of long-term risks. 2.3. Breast cancer Breast cancer is the most common malignant disease in women of childbearing age, and 15% of all patients are younger than 40 years [1012]. Many investigations have supported the notion that endogenous, as well as, exogenous hormones play an important role in the development of breast cancer [13]. Neither cohort nor case-control studies on the relationship between fertility drugs and breast cancer risk have found any remarkable associations yet. For example, the Australian follow-up studies of IVF patients failed to nd a clear difference in risk between exposed and unexposed subject (the cohort consisted of 29,700 women: exposed group SIR 0.91 [95% CI 0.741.13] and unexposed group SIR 0.95 [95% CI 0.731.23]) [14]. Besides, the study by Gauthier, a cohort of 6602 women treated for infertility, showed no overall signicant association between breast cancer risk and treatment for infertility (RR 0.95, CI 0.821.11), whatever the type and the duration of drugs used [15]. Although Brinton, in a recently published retrospective cohort study of 12,193 infertile women, found that infertile patients had a signicant higher breast cancer risk than the general population

(SIR 1.29, 95% CI 1.11.4). Analyses within the cohort showed adjusted RRs of 1.02 for clomiphene citrate and 1.07 for gonadotrophins, and no substantial relationships to dosage or cycles of use. Slight and non-signicant elevations in risk were seen for both drugs after !20 years of follow-up (RRs 1.39 for clomiphene and 1.54 for gonadotrophins). However, the risk associated with clomiphene for invasive breast cancers was statistically signicant (RR 1.60, 95% CI 1.02.5) [16]. Evidence for the possible importance of the number of treatment cycles as risk factor is provided by Pappo. He described, in women treated with 4 or more IVF cycles, twice the risk to develop breast cancer, although without statistical signicance (SIR 1.9; 95% CI 0.953.81) [17]. All these epidemiological studies, however, are awed: either by inaccurate information on the patterns of drug used, and inaccurate history of well-recognized reproductive risk factors. FSH and LH have no direct effects on breast tissue. The only possible connection between IVF and breast cancer is the elevation of estrogen induced by gonadotropin. Anyway the levels achieved during a stimulation cycle for one or more times do not appear to be sufcient to affect cell proliferation [18]. Ovulation-inducing drugs, however, might promote the growth of pre-existing tumors, as happens with the transient increase in breast cancer incidence after pregnancy [19]. Therefore, to reduce the risk of an early breast cancer immediately after an ovarian stimulation or during pregnancy, it is very important to do a breast ultrasound or a mammography before starting stimulation, to identify pre-existing tumors. Fertility drugs seem to be not dangerous for breast and for ovaries. Clearly additional studies and longer follow-up times are necessary. 3. Fertility preservation: the possible options Another interesting aspect related to patients with breast and/or ovarian cancer is the possibility to preserve their fertility before surgery or radio/chemotherapy. 3.1. Borderline ovarian tumor In the past decades, the incidence of borderline ovarian tumors has increased signicantly with 31% of patients still in their childbearing age (>40). The histology of this type of cancer does not reveal overt stromal invasion, and in younger patients its less aggressive behaviour might lead to opt for conservative surgery, aimed at preserving subsequent fertility. This conservative approach may be a safe solution, with no signicant changes in either recurrence or survival rates compared with radical treatment [20]. Infertility drugs have been lengthy contraindicated in patients already treated for ovarian malignancies. However, recently published reports suggest that IVF might be safe in the patients with previous early-stage borderline tumor [21,22]. From January 1999 to July 2005, Park and his group analysed 10 IVF cycles in 5 patients, previously treated for borderline ovarian tumor. The mean follow-up period after COHIVF initiation was 29.6 (range, 1461) months and no recurrences were observed [21]. Fortin and her group published another multi-centre retrospective study. This time 30 patients treated for early borderline ovarian tumor (stage I) were analysed after IVF [22]. During a median follow-up of 42 months, 4 recurrences were observed and three of them were treated with simple cystectomy. The author suggested that in these tumors infertility drugs might be used safely. The rst approach to preserve fertility in women with borderline ovarian tumor is a conservative surgery. If a condition of

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infertility pre-exists or derives for example from tubal adherences after surgery, IVF seems to be a safe approach. 3.2. Invasive ovarian cancer Fertility-sparing surgery may be safe in early ovarian cancers. They are classied as epithelial and non-epithelial tumors. For the second ones (germ cell tumors and sex cord-stromal tumors) conservative treatment has been considered a good option to preserve fertility in young patients [23,24] In the case of epithelial ovarian cancer the conservative surgery seems safe only in patients with stage IA and grade I (perhaps grade II). Morice and his group [25] analysed 25 patients treated conservatively (19 with stage IA (grade 1 n 9, grade 2 n 10), 1 with stage IC, 2 with stage II and 3 with initial stage unknown). The disease-free survival at 5 years for patients with stage IA, grade 1 was 90%, similar to patients treated radically [26,27]. On the contrary, the survival of grade 2 patients was 68%, compared with 7794% described in literature [26,27]. Four pregnancies in 4 patients were observed, from 4 months to 16 years after surgery. Colombo reviewed the published European articles about the role of conservative surgery in young patients. A total of 152 cases were reported: 88 patients with stage IA, 2 with stage IB, 51 with stage IC, 2 with stage II, 3 with stage IIIA, and 6 with stage IIIC; relapses occurred in 18/152 patients (11.8%). Fifty-three pregnancies were recorded with 38 deliveries, 2 ectopic pregnancies, 6 spontaneous abortions, 4 terminations, and 2 with unknown outcome [28]. Only few reports described the use of assisted reproductive technologies after non-epithelial or epithelial ovarian cancer, reporting single cases and very short follow-ups [2931]. Fortin et al. presented a multicenter retrospective study, in which 40 operated patients, between January 1971 and January 2001, have been included (27 patients had a borderline ovarian tumor, 10 a non-epithelial tumor and 3 an early-stage epithelial invasive carcinoma). With a global follow-up of 372 months, only 3 patients treated for a borderline tumor had a recurrence after induction of ovulation [32]. Actually, ovarian stimulation after epithelial ovarian cancer cannot be considered a safe procedure. There are few studies in the literature and with too small number of patients. Although conservative surgery seems to be a possible option in young patients, after an accurate selection by histologic type (only serous, mucinous and endometrioid) and stage (IA). 3.3. Breast cancer Advances in the treatment of patients with breast cancer, especially when combined with adjuvant chemotherapy, have led to greatly increased survival rates, focusing interest on the effects of long-term treatment. The reproductive function of cancer survivors is frequently impaired, with infertility, premature ovarian failure or osteoporotic fractures. Chemotherapeutic agents would seem to damage primordial follicles by initiating apoptosis, as described by Perez [33,34] in cisplatin-cultured murine oocytes in vitro and by Meirow and Nugent [35] in the granulosa cells of human primordial follicles. Ovarian damage following adjuvant chemotherapy for breast cancer gives variety of symptoms (different modications of menstrual cycle as temporary amenorrhoea and oligomenorrhea), culminating in premature ovarian failure (POF). Chemotherapy-related amenorrhoea (CRA) is still not clearly established by a term describing the occurrence of amenorrhoea following chemotherapy within a year of starting cytotoxic drugs and lasting !6 months. The rate is related to age and type of chemotherapeutic agents [36].

As example, the study presented by Bines underlines the average CRA rate in regimens based on cyclophosphamide, methotrexate and uorouracil (CMF) was 68% (95% condence interval CI 6670%), with a range of 20100% [37]. Summarizing the effects of breast chemotherapy with CMF in women from 30 to 39 years, the risk of amenorrhoea after 6 cycles is estimated between 20% and 80%; more specically: 10% at 35 years, 40% at 40 years, 7080% at 45 years. In women <30 years the risk of amenorrhoea after chemotherapy is very low but the possibility of premature ovarian failure increases [38]. Actually some authors are studying also the possible correlation between CRA and disease-free survival rate or overall survival rate, especially in hormone receptor positive patients. It seems that CRA may have a protective effects in these patients. Vanhuyse studied retrospectively 130 premenopausal patients with localised hormone-sensitive breast cancer, considering disease-free survival rate and overall survival rate [39]. He found a trend towards better disease-free survival when CRA occurred, which did not reach a statistically signicant level, in line with other published data [4042]. Available options to preserve fertility in breast cancer patients are: embryo cryopreservation (only established procedure), reversible ovarian suppression with gonadotropin releasing hormone (GnRH) analogues, oocyte and ovarian tissue cryopreservation. The choice depends on the type and timing of chemotherapy, type of cancer, patients age and the partners status (Table 1). The Ethics Committee of the American Society for Reproductive Medicine considers cryopreservation of the embryo the only well established method of fertility preservation. A partner or semen donor, at least pubertal age and the possibility to undergo ovarian stimulation (sufcient time and no contraindication according to the type of cancer) [43] are the necessary requirements for this choice. Ovarian stimulation requires approximately 2 weeks from the beginning of menses: in breast cancer there is typically a 68 weeks hiatus between surgery and chemotherapy, allowing sufcient time for undergoing ovarian stimulation for fertility preservation purposes. This interval not changes in ER or ER patients [4446]. On the other hand, even if oocyte cryopreservation is still considered experimental, it is a promising technique, requiring no

Table 1 Options for fertility preservation in breast cancer patients Type of technique Embryo cryopreservation Characteristics Unique established method of fertility preservation for Ethics Committee of the American Society for Reproductive Medicine Requires a partner or a semen donor Requires almost 2 weeks for stimulation Safe for ER patients Possible protocols with tamoxifen or letrozole for ER patients Considered experimental Does not require a partner or semen donor No legal or ethical problems Requires almost 2 weeks for stimulation Safe for ER patients Possible protocols with tamoxifen or letrozole for ER patients Considered experimental Needed of laparoscopy Few pregnancies described in the literature Promising for younger patients Part of endocrine therapy Limits ovarian damage Needs further studies

Oocytes cryopreservation

Cortical ovarian tissue cryopreservation

Reversible ovarian suppression

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partner or semen donor. The results are improving day by day especially in the countries where embryo cryopreservation is not legal [47]. The rst protocol used was the same of embryo freezing (slowcooling technique with propane-1,2-diol or dimethyl sulphoxide as intracellular cryoprotectant and 0.1 mol/l sucrose as extracellular cryoprotectant), but many modications have been proposed following the disappointing outcome of the initial attempts [48]. Actually, good results seem to be possible using a modied slow-cooling protocol involving 1.5 mol/l propane-1,2-diol and 0.2 mol/l sucrose during dehydration. Rehydration instead was conducted applying decreasing concentrations of PrOH and 0.3 mol/l sucrose (pregnancy rate per transfer 21.3%, per patient 21.8%, per thawing cycle 18.9%, implantation rate 13.5%, miscarriage rate 11.8%) [49]. To investigate the clinical signicance of oocyte freezing, our group assessed the cumulative pregnancy rate per started cycle derived from the use of fresh and frozen-thawed oocytes [47]. We considered a total of 749 patients treated from 2004 to 2006. The pregnancy rate per embryo transfer in fresh cycles was 38% and in frozen-thawed cycles was 17.2%. Cumulative clinical outcome obtained dividing pregnancies derived from fresh and frozenthawed cycles (355 in total) by the number of patients was 47.4%. It seems that also vitrication technique permitted successful results in oocyte cryopreservation. Kuwayama, vitrifying 64 oocytes, described 91% survival rate, 90% fertilization rate and 43% pregnancy rate per transfer. If conrmed, these success rates would mark a major progress in IVF treatment. However, these dates should be interpreted with caution because of the young patient age (32 6 years) [50]. The same good results were presented by Lucena using the Cryotop vitrication method (87% survival rate, 89% fertilization rate and 56% pregnancy rate) but the majority of pregnancies were obtained in oocyte donation cycles [51]. Recently, Cobo published a comparison between fresh and vitried oocytes coming from the same stimulated cycle in egg donation program. There were no differences in fertilization rate, cleavage rate and embryo quality in the 2 groups. Besides in the vitrication group pregnancy and implantation rate were 65.2% and 40.8%, respectively [52]. All these reports necessitate clinical conrmation through extensive and uniform studies. Cryopreservation of immature germinal vesicle (GV)-stage oocytes has been investigated as an alternative to freezing mature MII stage oocytes [53,54]. In this stage the chromatin is diffuse and surrounded by a nuclear membrane, and the cryopreservation seems to avoid spindle depolymerization and circumvents the risk of polyploidy and aneuploidies [55]. Other major problems associated with GV-stage oocytes freezing were poor maturation and subsequent sub-optimal embryonic development [54,55]. Actually only one human live birth has been reported following cryopreservation of GV-stage oocytes and IVM [56]. 3.4. Ovarian stimulation for embryo and oocyte cryopreservation: protocols with tamoxifen and letrozole For women with ER breast cancer the American Society of Clinical Oncology recommends alternative hormonal stimulation with letrozole or tamoxifen to reduce the risk of estrogen exposure [57]. It is also possible to collect oocytes without ovarian stimulation (natural-cycle IVF), bearing in mind, anyway, that only one oocyte or embryo for every cycle can be obtained by this procedure. The rst drug to be proposed was tamoxifen, a non-steroidal triphenylethylene anti-estrogen rst used in the United Kingdom

as a contraceptive, then in Europe as an ovulation induction agent and nally as drug of choice in breast cancer treatment. In 2003 Oktay treated 12 women with 4060 mg of tamoxifen for about 7 days, starting on day 23 of the menstrual cycle and then compared the results with those obtained in a retrospective control group. Tamoxifen-treated patients obtained almost an embryo, whereas this was not sure in the natural-cycle patients. The very low reserve of embryos obtainable with just one possibility of stimulation is in fact the limit of tamoxifen [44]. Another drug that can probably be used safely in patients with breast carcinoma undergoing IVF is letrozole. This is an aromatase inhibitor that competitively inhibits the activity of aromatase by blocking the conversion of androgenic substances to estrogens in the granulosa cells of ovarian follicles and many other tissues. Letrozole is administrated to women with advanced post-menopausal breast cancer and, recently, also to induce ovulation, alone or in combination with rFSH. It reduces estrogen levels in ovulation induction protocols and stimulates endogenous gonadotrophins production. Oktay did 2 different studies in which he used letrozole for IVF in breast cancer patients [45,46]. The rst compared 47 stage IIIIA ER or ER breast cancer patients stimulated with letrozole and FSH, and a retrospective group of patients submitted to IVF due to tubal factor disease. The letrozole group presented signicantly lower estradiol peak levels (mean SD 483.4 278.9 pg/ml vs. 1464.6 644.9 pg/ml P < 0.001) and total FSH dose (1317.8 578.2 UI vs. 2382.5 1062.8 UI P < 0.001). The other parameters, such as total oocytes, mature oocytes and embryos obtained were similar in the 2 groups. At the time of the publication only 3 cancer survivors have used their embryos, resulted in one live birth (after thawing 3 of the 7 embryos and transfer to a surrogate) and 1 biochemical pregnancy [46]. In his more recent study Oktay compared 3 groups of patients: tamoxifen alone (Tam-IVF), tamoxifen in combination with lowdose follicle stimulating hormone (TamFSH-IVF) or letrozole with FSH (Letrozole IVF) [44]. Both TamFSH-IVF and letrozole IVF patients had greater numbers of follicles, mature oocytes and embryos compared with Tam-IVF cases (follicle >17 mm Tam-IVF 1.2 0.1, TamFSH-IVF 2.6 0.4 and letrozole IVF 3.2 0.4; mature oocytes Tam-IVF 1.5 0.3, TamFSH-IVF 5.1 1.1 and letrozole IVF 8.5 1.6; total embryos Tam-IVF 1.3 0.2, TamFSH-IVF 3.8 0.8 and letrozole IVF 5.3 0.8). On the contrary, peak estradiol levels were lower with letrozole IVF and Tam-IVF (380 57 pg/ml and 419 39 pg/ml vs. 1182 271 pg/ml in TamFSH-IVF group). As to recurrence rates, the patients of the 3 groups were compared with controls patients not submitted to IVF cycles and no differences were found (recurrence rate for the patients who underwent IVF was 3 of 29 patients and for the control group was 3 of 31 patients) [44]. This is a very interesting option for breast cancer patients, but multi-centre prospective randomized trials are necessary to control the safety of the drugs for both women and embryos. 3.5. Reversible ovarian suppression with GnRH analogues There are studies in the literature proposing the administration of a GnRH analogue during chemotherapy to protect the ovaries [5861]. Endocrine therapy is a possible option in the treatment of premenopausal ER breast cancer in association with chemotherapy. In the opinion of several authors it would seem that reversible ovarian suppression by GnRH analogue administered 1 week before chemotherapy might reduce the likelihood of ovarian damage [61,62]. For example in a young womens group with early breast carcinoma, using analogues during chemotherapy (included cyclophosphamide, methotrexate and 5-uorouracil or anthracycline based regimen), Recchia found that, after a median follow-up

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of 75 months, all patients younger than 40 years have resumed their regular menses and 56% of the older ones. The author thought that the effect of the GnRH analogue is not restricted to hormone deprivation: it behaves like a negative growth factor, regulating cancer cell proliferation and inducing apoptosis in chemotherapy survivor cells [61]. As reported by Maltaris [63] in a recent review, the available studies have been criticized owing to the heterogeneity of patients and follow-up protocols, the different regimens and the duration of chemotherapy. International randomized controlled trials are necessary to evaluate this strategy in women with cancer [62,63]. Actually the only one prospective randomized study was a report by Waxman that included 31 men and 18 women receiving chemotherapy for Hodgkins disease. In this study, it seems that GnRH analogues do not preserve fertility [64]. Besides the resumption of menstrual cycle is not a correct indicator of ovarian function and fertility. An interesting work by Anderson described the changes in AMH level during and after chemotherapy. AMH decreased rapidly before FSH increased or irregular menses started. Probably the changes in the levels of this growth factor reected the primary damage of cytotoxic drugs to primordial and primary follicles and it was a good test for residual ovarian reserve, also in presence of regular menstrual cycles [65]. Clearly, further powered studies are necessary to clarify the role of reversible ovarian suppression in early breast cancer patients, not only as an endocrine therapy but also as an option to preserve fertility. 3.6. Ovarian tissue cryopreservation Prepubertal girls and women who cannot delay the start of chemotherapy have cryopreservation of ovarian tissue as an available option. Ovarian tissue can be frozen using 3 different approaches: as fragment of ovarian cortex, as entire ovary with its vascular pedicle or as isolated follicles. Actually the experiences reported in literature use predominantly the rst one. After treatment, when the patient is disease free, the tissue can be reimplanted either into the pelvic cavity (orthotopic site) or in the forearm or abdominal wall (heterotopic site). In early breast cancer the risk of recurrence after reimplantation of cortical ovarian tissue due the presence of malignant cells is extremely low because the possibility of ovarian involvement in early breast cancer is minimal [66,67]. Most of the occult metastases belong to the less common histological types, such as the inltrating lobular cancer that typically occurs in post-menopausal women [66,67]. The real risk is the case of haematologic cancers [68]. Instead Hodgkins disease is nowadays considered just a poor risk [69]. Preoperative imaging and histologic evaluation of the fresh ovarian tissue are essential before storage. Furthermore, postthawing polymerase chain reaction (PCR) with specic markers must be used to evaluate the tissue for minimal residual disease [69]. Because of the risk of transmission of malignant cells, the development of isolated follicle transplantation is an interesting option. Primordial follicles are resistant to cryoinjury because the oocytes contained have an inactive metabolism, lack of meiotic spindle, zona pellucida and cortical granules. Moreover the small size of a primordial follicle facilitates penetration of cryoprotectants. Actually it is possible enzymatically isolate human follicles from their surrounding tissue and, after xenografting in SCID mice, they are able to survive and develop into antral follicles. Probably the formation of a new, well-vascularized stroma-like structure is likely to be a key factor in sustaining follicular growth in this in vivo model [70,71]. Nevertheless further studies are necessary to assess the safety of the procedure and to translate it in humans. The transplantation of whole ovary with vascular anastomosis permits to restore immediate vascularization and to reduce post-

transplantation ischemia, responsible for the reduction of ovarian reserve [72,73]. The laparoscopic ovariectomy must be performed with great care, removing the ovary and a large part (!5 cm) of the infundibulopelvic ligament, allowing dissection of the ovarian vessels, perfusion with a cryoprotective medium, and cryopreservation for subsequent autografting. The period of ischemia between ligation of the ovarian pedicle and ovarian cryopreservation must be as short as possible [72]. Actually in the literature there are some reports about whole human ovary cryopreservation [72,73] but no one transplantation or live birth, that, on the contrary, is described for sheep [74]. This procedure should be encouraged searching for new cryopreservation protocols and surgical techniques. A new strategy of fertility preservation might be, for young women undergoing ovarian cryobanking, retrieval and in vitro maturation of immature germinal vesicles (GV)-stage oocytes followed by vitrication [75,76]. Huang and his group reported an experience with 4 patients who have undergone ovarian cryobanking for Hodgkins lymphoma (n 2), breast cancer (n 1) and rectal cancer (n 1). After the excision of cortical ovarian tissue, the visible antral follicles have been aspirated, in vitro matured and then cryopreserved. The median number of GV-stage oocytes recovered was 3, with a maturation rate varying from 50% to 100% and a median number of vitried oocytes of 3. No patients actually was come back to use the cryopreserved tissue or the vitried oocytes [77]. Clearly, this strategy can be only an adjunct in the protocol of ovarian tissue cryopreservation, for younger patients in which it is possible recovery a sufcient number of GV-stage oocytes. Donnez et al. in 2004 reported the rst pregnancy after orthotopic transplantation of cryopreserved cortical ovarian tissue in a woman with previous stage IV Hodgkins lymphoma. Cortical biopsies had been taken from the left ovary by laparoscopy. The pregnancy started 11 months after transplantation and she delivered a healthy baby. In this case, but there are other reports as well, folliculogenesis took 46 months to start. This time range between implantation and rst estradiol peak is consistent with data obtained from sheep [78]. The other births obtained with cortical ovarian tissue transplantation are described in Table 2. Many reports have been published on heterotopic autotransplantation that describe the restoration of the endocrine
Table 2 Live birth from autotransplantation of cryopreserved human ovarian tissue in disease-free patients References Donnez et al. (2004) [78] Case reports Age of freezing: 25 Orthotopic transplantation Spontaneous pregnancy after 5 weeks Age of freezing: 28 Orthotopic transplantation IVFICSI pregnancy Age of freezing: 24 Orthotopic and heterotopic transplantation Spontaneous pregnancy after 5 months (1) Age of freezing: 26 Orthotopic transplantation IVFICSI pregnancy (2) Age of freezing: 27 Orthotopic transplantation IVFICSI pregnancy Silber et al. (2008) [82] Age of freezing: 25 Orthotopic transplantation Spontaneous pregnancy after 5 months

Meirow et al. (2005) [79]

Demestree et al. (2007) [80]

Andersen et al. (2008) [81]

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function for a few months, but the only pregnancy described by Oktay in 2006, was the outcome of ovulation occurred in a native ovary [43]. The heterotopic autotransplantation was used by the same author also in a breast cancer patient [83]. Silber and his group reported 8 cases of fresh ovarian transplantation in monozygotic twins, discordant for ovarian failure [84,85]. He proposed a minilaparotomy in which a section of approximately one-third of the donor ovarian cortex was laid over the raw medulla of each ovary in the recipient and sutured onto the medulla; in one patient he transplanted the whole ovary. He obtained 6 pregnancies and regular menstrual cycles in the other women for 2 years [82,86]. Clearly this might be another possible option for a restricted group of patients with cancer or for women that would extend their reproductive life span. 4. Conclusions Analysing the available studies in the literature one might conclude that ovarian stimulation is a non-independent risk factor for breast and ovarian cancer. On the contrary, for patients affected by these tumors, the American Society of Clinical Oncology recommended embryo cryopreservation, oocyte or cortical ovarian tissue freezing to preserve fertility, depending on the possibility of delaying chemotherapy and the sensitivity of the tumor. Stimulations with tamoxifen or letrozole are possible options for patients with ER breast cancer. The authors debate the rationale and safety for the use of GnRH analogue ovarian suppression. Finally, ovarian tissue cryopreservation is an interesting option that still needs to be conrmed by well-designed and powerful controlled studies. 5. Conict of interest The authors have no conict of interest. References
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