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Placenta 29 (2008) S184–S190

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Placenta

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

Focus on Breast and Ovarian Cancer

A. Borini*, E. RebellatoTecnobios Procreazione, Bologna, Italy

a r t i c l e i n f o

Article history:Accepted 5 August 2008

Keywords:Breast cancerOvarian cancerOvarian stimulationOocyte cryopreservation

* Corresponding author.E-mail address: borini@tecnobiosprocreazione.it (

0143-4004/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.placenta.2008.08.001

a b s t r a c t

In reproductive medicine the widespread use of ovarian stimulation has focused interest on the possiblerelation between induction of ovulation and breast and ovarian cancer. The epidemiological studiespublished 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 youngerpatients to preserve fertility before radio- or chemotherapy, the American Society of Clinical Oncologyrecommended embryo cryopreservation as an established procedure. Moreover, experimental procedureas oocytes cryopreservation, ovarian suppression with GnRH analogues and cortical ovarian tissuefreezing probably will be routinely used in the future. A reasonable proposal to protect patients from therisk of estrogen peaks in the case of estrogen receptor positive (ERþ) breast cancer, ovarian stimulationwith tamoxifen or letrozole could be right. Powered studies are necessary to control the safety andeffectiveness of the different fertility preservation options.In this review we evaluated the relation between induction of ovulation and breast and ovarian cancerand 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 ofovulation and breast and ovarian cancer is of interest to allspecialists in reproductive medicine. In fact, during counsellingbefore an in vitro fertilization (IVF) treatment, the first questionraised very often by patients is if the therapy might increase theirhealth risks, especially of cancer.

Therefore, it is necessary to clarify if ovarian stimulation is anindependent risk factor for breast and ovarian cancer. Ovulationinduction has been used for about 30 years and there are, bynow, many studies in the literature trying to provide someanswers [1–5].

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 arefew recognized risk factors that could facilitate its early detection.Besides a clear understanding of the earliest recognizable events inovarian carcinogenesis is difficult just because of an ill-defined pre-malignant state and the paucity of data from early-stage cancer. Invitro fertilization protocols have become quite widespread, and it is

A. Borini).

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necessary to understand if the induction of ovulation might bea possible risk factor for ovarian cancer, especially the epithelialsubtypes.

There are 2 possible hypotheses for this link. The first one is thecumulative effect of minor traumas, due to repeated ovulation, thatcould induce malignant changes in the ovarian epithelium. In fact,conditions as multi-parity, breast feeding and oral contraceptiveuse, in which ovulation is suppressed, have protective effects on theovary [6]. The second one is when estrogens could stimulate cellproliferation in the tissues expressing their receptors, such as thegranulosa cells [7].

Many studies have tried to clarify if ovarian stimulation couldincrease the chance of developing ovarian neoplasias as an inde-pendent risk factor.

Mosgaard et al., for example, reported a case–control studyincluding 684 cases of ovarian cancer and 1721 controls, showingno increased risk among treated infertile women, also after sepa-ration between nulliparous (Odds Ratio (OR)¼ 0.8) and patients(OR¼ 0.6) [2].

Brinton, on the contrary, presented a retrospective cohort studyof 12,193 women evaluated for infertility during the period 1965–1988. The infertile patients had a significantly elevated ovariancancer risk compared with the general population (standardizedincidence ratio (SIR) 1.98, 95% confidence intervals (CI) 1.4–2.6).When patient characteristics were taken into account, the rateratios associated with ever usage were 0.82 (95% CI 0.4–1.5) and1.09 (95% CI 0.4–2.8) for clomiphene and for gonadotropins,respectively [3].

A. Borini, E. Rebellato / Placenta 29 (2008) S184–S190 S185

Furthermore, Rossing et al., in a cohort study of 3837 infertilewomen, found that clomiphene citrate was associated with a rela-tive risk (RR) of 2.3 (95% CI 0.5–11.4) compared with infertilewomen not using it. The long-term use of this drug (12 or morecycles) brought relative risk to 11.1 (95% CI 1.5–82.3) [4]. FinallyShushan et al. reported an adjusted relative risk with clomiphene of0.9 (95% CI 0.3–2.3). However, his study was limited by the fact that36% of the cases had died before further contacts were established,causing a possible selection bias [5].

An interesting review by Mahdavi et al. considered invasiveepithelial carcinoma, invasive non-epithelial carcinoma andborderline ovarian tumor. The authors analysed cohort, case-control and descriptive studies, finding discordant results. Mahdaviconcluded that, at the moment, the findings on ovarian cancer riskassociated with treatment for the induction of ovulation are stillreassuring, but well-designed clinical trials are still necessary [1].

The possible limits of these analysis are due to methodologicalproblems: such as small size populations, short follow-up, lowprevalence of infertility and fertility drugs use, lack of informationon confounding factors, for instance oral contraceptive use, andfamily history of ovarian neoplasias.

2.2. Borderline ovarian tumor

In the literature there are contrasting results also aboutborderline ovarian tumor. In a recent case-control study by Cusido,42 women with this type of cancer were compared with 257patients with benign ovarian pathology. No differences were foundbetween the 2 groups, in terms of type of drugs used whetherclomiphene 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/gonadotropinswas 2.50�1.00 and 3.00� 2.64 in the borderline tumor group and2.44�1.75 and 3.27�2.25 in the control group) [8]. On thecontrary, Sanner et al., presenting an historical cohort study of 2768women treated for infertility, underlined an increased risk ofborderline ovarian tumors after clomiphene treatment for ovula-tory problems (SIR¼ 7.47; 95% CI 1.54–21.83) [9].

Therefore, based on the evidence to date, it is not possible toestablish if the induction of ovulation might be a possible risk factorfor ovarian cancer. Generally, the results of the studies are reassuringin not confirming a strong link. However, slight, but non-significantelevation 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 womenof childbearing age, and 15% of all patients are younger than 40years [10–12]. Many investigations have supported the notion thatendogenous, as well as, exogenous hormones play an importantrole in the development of breast cancer [13]. Neither cohort norcase-control studies on the relationship between fertility drugs andbreast cancer risk have found any remarkable associations yet.

For example, the Australian follow-up studies of IVF patientsfailed to find a clear difference in risk between exposed andunexposed subject (the cohort consisted of 29,700 women:exposed group SIR 0.91 [95% CI 0.74–1.13] and unexposed group SIR0.95 [95% CI 0.73–1.23]) [14]. Besides, the study by Gauthier,a cohort of 6602 women treated for infertility, showed no overallsignificant association between breast cancer risk and treatmentfor infertility (RR¼ 0.95, CI 0.82–1.11), whatever the type and theduration of drugs used [15].

Although Brinton, in a recently published retrospective cohortstudy of 12,193 infertile women, found that infertile patients hada significant higher breast cancer risk than the general population

(SIR¼ 1.29, 95% CI 1.1–1.4). Analyses within the cohort showedadjusted RRs of 1.02 for clomiphene citrate and 1.07 for gonado-trophins, and no substantial relationships to dosage or cycles of use.Slight and non-significant elevations in risk were seen for bothdrugs after �20 years of follow-up (RRs¼ 1.39 for clomiphene and1.54 for gonadotrophins). However, the risk associated withclomiphene for invasive breast cancers was statistically significant(RR¼ 1.60, 95% CI 1.0–2.5) [16].

Evidence for the possible importance of the number of treat-ment cycles as risk factor is provided by Pappo. He described, inwomen treated with 4 or more IVF cycles, twice the risk to developbreast cancer, although without statistical significance (SIR¼ 1.9;95% CI 0.95–3.81) [17].

All these epidemiological studies, however, are flawed: either byinaccurate information on the patterns of drug used, and inaccuratehistory of well-recognized reproductive risk factors.

FSH and LH have no direct effects on breast tissue. The onlypossible connection between IVF and breast cancer is the elevationof estrogen induced by gonadotropin. Anyway the levels achievedduring a stimulation cycle for one or more times do not appear tobe sufficient to affect cell proliferation [18]. Ovulation-inducingdrugs, however, might promote the growth of pre-existing tumors,as happens with the transient increase in breast cancer incidenceafter pregnancy [19].

Therefore, to reduce the risk of an early breast cancer immedi-ately after an ovarian stimulation or during pregnancy, it is veryimportant to do a breast ultrasound or a mammography beforestarting stimulation, to identify pre-existing tumors. Fertility drugsseem to be not dangerous for breast and for ovaries. Clearly addi-tional studies and longer follow-up times are necessary.

3. Fertility preservation: the possible options

Another interesting aspect related to patients with breast and/orovarian cancer is the possibility to preserve their fertility beforesurgery or radio/chemotherapy.

3.1. Borderline ovarian tumor

In the past decades, the incidence of borderline ovarian tumorshas increased significantly with 31% of patients still in their child-bearing age (>40).

The histology of this type of cancer does not reveal overt stromalinvasion, and in younger patients its less aggressive behaviourmight lead to opt for conservative surgery, aimed at preservingsubsequent fertility.

This conservative approach may be a safe solution, with nosignificant changes in either recurrence or survival rates comparedwith radical treatment [20].

Infertility drugs have been lengthy contraindicated in patientsalready treated for ovarian malignancies. However, recently pub-lished reports suggest that IVF might be safe in the patients withprevious early-stage borderline tumor [21,22].

From January 1999 to July 2005, Park and his group analysed 10IVF cycles in 5 patients, previously treated for borderline ovariantumor. The mean follow-up period after COH–IVF initiation was29.6 (range, 14–61) months and no recurrences were observed [21].

Fortin and her group published another multi-centre retro-spective study. This time 30 patients treated for early borderlineovarian tumor (stage I) were analysed after IVF [22]. Duringa median follow-up of 42 months, 4 recurrences were observed andthree of them were treated with simple cystectomy. The authorsuggested that in these tumors infertility drugs might be usedsafely.

The first approach to preserve fertility in women with border-line ovarian tumor is a conservative surgery. If a condition of

Table 1Options for fertility preservation in breast cancer patients

Type of technique Characteristics

Embryo cryopreservation Unique established method of fertility preservation forEthics Committee of the American Society forReproductive MedicineRequires a partner or a semen donorRequires almost 2 weeks for stimulationSafe for ER� patientsPossible protocols with tamoxifen or letrozole for ERþpatients

Oocytes cryopreservation Considered experimentalDoes not require a partner or semen donorNo legal or ethical problemsRequires almost 2 weeks for stimulationSafe for ER� patientsPossible protocols with tamoxifen or letrozole for ERþpatients

Cortical ovarian tissuecryopreservation

Considered experimentalNeeded of laparoscopyFew pregnancies described in the literaturePromising for younger patients

Reversible ovariansuppression

Part of endocrine therapyLimits ovarian damageNeeds further studies

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infertility pre-exists or derives for example from tubal adherencesafter 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 classified as epithelial and non-epithelial tumors. For thesecond ones (germ cell tumors and sex cord-stromal tumors)conservative treatment has been considered a good option topreserve fertility in young patients [23,24]

In the case of epithelial ovarian cancer the conservative surgeryseems safe only in patients with stage IA and grade I (perhaps gradeII). Morice and his group [25] analysed 25 patients treatedconservatively (19 with stage IA (grade 1 n¼ 9, grade 2 n¼ 10), 1with stage IC, 2 with stage II and 3 with initial stage unknown). Thedisease-free survival at 5 years for patients with stage IA, grade 1was 90%, similar to patients treated radically [26,27]. On thecontrary, the survival of grade 2 patients was 68%, compared with77–94% described in literature [26,27]. Four pregnancies in 4patients were observed, from 4 months to 16 years after surgery.

Colombo reviewed the published European articles about therole of conservative surgery in young patients. A total of 152 caseswere reported: 88 patients with stage IA, 2 with stage IB, 51 withstage IC, 2 with stage II, 3 with stage IIIA, and 6 with stage IIIC;relapses occurred in 18/152 patients (11.8%). Fifty-three pregnan-cies were recorded with 38 deliveries, 2 ectopic pregnancies, 6spontaneous abortions, 4 terminations, and 2 with unknownoutcome [28].

Only few reports described the use of assisted reproductivetechnologies after non-epithelial or epithelial ovarian cancer,reporting single cases and very short follow-ups [29–31].

Fortin et al. presented a multicenter retrospective study, inwhich 40 operated patients, between January 1971 and January2001, have been included (27 patients had a borderline ovariantumor, 10 a non-epithelial tumor and 3 an early-stage epithelialinvasive carcinoma). With a global follow-up of 372 months, only 3patients treated for a borderline tumor had a recurrence afterinduction of ovulation [32].

Actually, ovarian stimulation after epithelial ovarian cancercannot be considered a safe procedure. There are few studies in theliterature and with too small number of patients. Althoughconservative surgery seems to be a possible option in youngpatients, 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 ledto greatly increased survival rates, focusing interest on the effects oflong-term treatment. The reproductive function of cancer survivorsis frequently impaired, with infertility, premature ovarian failure orosteoporotic fractures. Chemotherapeutic agents would seem todamage primordial follicles by initiating apoptosis, as described byPerez [33,34] in cisplatin-cultured murine oocytes in vitro and byMeirow and Nugent [35] in the granulosa cells of human primordialfollicles.

Ovarian damage following adjuvant chemotherapy for breastcancer gives variety of symptoms (different modifications ofmenstrual cycle as temporary amenorrhoea and oligomenorrhea),culminating in premature ovarian failure (POF).

Chemotherapy-related amenorrhoea (CRA) is still not clearlyestablished by a term describing the occurrence of amenorrhoeafollowing chemotherapy within a year of starting cytotoxic drugsand lasting �6 months. The rate is related to age and type ofchemotherapeutic agents [36].

As example, the study presented by Bines underlines theaverage CRA rate in regimens based on cyclophosphamide, meth-otrexate and fluorouracil (CMF) was 68% (95% confidence interval CI66–70%), with a range of 20–100% [37].

Summarizing the effects of breast chemotherapy with CMF inwomen from 30 to 39 years, the risk of amenorrhoea after 6 cyclesis estimated between 20% and 80%; more specifically: 10% at 35years, 40% at 40 years, 70–80% at 45 years. In women <30 years therisk of amenorrhoea after chemotherapy is very low but thepossibility of premature ovarian failure increases [38].

Actually some authors are studying also the possible correlationbetween 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 patientswith localised hormone-sensitive breast cancer, consideringdisease-free survival rate and overall survival rate [39]. He founda trend towards better disease-free survival when CRA occurred,which did not reach a statistically significant level, in line withother published data [40–42].

Available options to preserve fertility in breast cancer patientsare: embryo cryopreservation (only established procedure),reversible ovarian suppression with gonadotropin releasinghormone (GnRH) analogues, oocyte and ovarian tissue cryopres-ervation. The choice depends on the type and timing ofchemotherapy, type of cancer, patient’s age and the partner’sstatus (Table 1).

The Ethics Committee of the American Society for Reproduc-tive Medicine considers cryopreservation of the embryo the onlywell established method of fertility preservation. A partner orsemen donor, at least pubertal age and the possibility to undergoovarian stimulation (sufficient time and no contraindicationaccording to the type of cancer) [43] are the necessary require-ments for this choice. Ovarian stimulation requires approximately2 weeks from the beginning of menses: in breast cancer there istypically a 6–8 weeks hiatus between surgery and chemotherapy,allowing sufficient time for undergoing ovarian stimulation forfertility preservation purposes. This interval not changes in ERþor ER� patients [44–46].

On the other hand, even if oocyte cryopreservation is stillconsidered experimental, it is a promising technique, requiring no

A. Borini, E. Rebellato / Placenta 29 (2008) S184–S190 S187

partner or semen donor. The results are improving day by dayespecially in the countries where embryo cryopreservation is notlegal [47].

The first protocol used was the same of embryo freezing (slow-cooling technique with propane-1,2-diol or dimethyl sulphoxide asintracellular cryoprotectant and 0.1 mol/l sucrose as extracellularcryoprotectant), but many modifications have been proposedfollowing the disappointing outcome of the initial attempts [48].

Actually, good results seem to be possible using a modifiedslow-cooling protocol involving 1.5 mol/l propane-1,2-diol and0.2 mol/l sucrose during dehydration. Rehydration instead wasconducted applying decreasing concentrations of PrOH and0.3 mol/l sucrose (pregnancy rate per transfer 21.3%, per patient21.8%, per thawing cycle 18.9%, implantation rate 13.5%, miscarriagerate 11.8%) [49].

To investigate the clinical significance of oocyte freezing, ourgroup assessed the cumulative pregnancy rate per started cyclederived from the use of fresh and frozen-thawed oocytes [47]. Weconsidered a total of 749 patients treated from 2004 to 2006. Thepregnancy rate per embryo transfer in fresh cycles was 38% and infrozen-thawed cycles was 17.2%. Cumulative clinical outcomeobtained dividing pregnancies derived from fresh and frozen-thawed cycles (355 in total) by the number of patients was 47.4%.

It seems that also vitrification technique permitted successfulresults in oocyte cryopreservation.

Kuwayama, vitrifying 64 oocytes, described 91% survival rate,90% fertilization rate and 43% pregnancy rate per transfer. Ifconfirmed, these success rates would mark a major progress in IVFtreatment. However, these dates should be interpreted withcaution because of the young patient age (32� 6 years) [50]. Thesame good results were presented by Lucena using the Cryotopvitrification method (87% survival rate, 89% fertilization rate and56% pregnancy rate) but the majority of pregnancies were obtainedin oocyte donation cycles [51].

Recently, Cobo published a comparison between fresh andvitrified oocytes coming from the same stimulated cycle in eggdonation program. There were no differences in fertilization rate,cleavage rate and embryo quality in the 2 groups. Besides in thevitrification group pregnancy and implantation rate were 65.2% and40.8%, respectively [52].

All these reports necessitate clinical confirmation throughextensive and uniform studies.

Cryopreservation of immature germinal vesicle (GV)-stageoocytes has been investigated as an alternative to freezing matureMII stage oocytes [53,54].

In this stage the chromatin is diffuse and surrounded bya nuclear membrane, and the cryopreservation seems to avoidspindle depolymerization and circumvents the risk of polyploidyand aneuploidies [55].

Other major problems associated with GV-stage oocytesfreezing were poor maturation and subsequent sub-optimalembryonic development [54,55].

Actually only one human live birth has been reported followingcryopreservation of GV-stage oocytes and IVM [56].

3.4. Ovarian stimulation for embryo and oocyte cryopreservation:protocols with tamoxifen and letrozole

For women with ERþ breast cancer the American Society ofClinical Oncology recommends alternative hormonal stimulationwith letrozole or tamoxifen to reduce the risk of estrogen exposure[57]. It is also possible to collect oocytes without ovarian stimula-tion (natural-cycle IVF), bearing in mind, anyway, that only oneoocyte or embryo for every cycle can be obtained by this procedure.

The first drug to be proposed was tamoxifen, a non-steroidaltriphenylethylene anti-estrogen first used in the United Kingdom

as a contraceptive, then in Europe as an ovulation induction agentand finally as drug of choice in breast cancer treatment.

In 2003 Oktay treated 12 women with 40–60 mg of tamoxifenfor about 7 days, starting on day 2–3 of the menstrual cycle andthen compared the results with those obtained in a retrospectivecontrol group. Tamoxifen-treated patients obtained almost anembryo, whereas this was not sure in the natural-cycle patients.The very low reserve of embryos obtainable with just one possi-bility of stimulation is in fact the limit of tamoxifen [44].

Another drug that can probably be used safely in patients withbreast carcinoma undergoing IVF is letrozole. This is an aromataseinhibitor that competitively inhibits the activity of aromatase byblocking the conversion of androgenic substances to estrogens inthe granulosa cells of ovarian follicles and many other tissues.Letrozole is administrated to women with advanced post-meno-pausal breast cancer and, recently, also to induce ovulation, alone orin combination with rFSH. It reduces estrogen levels in ovulationinduction protocols and stimulates endogenous gonadotrophinsproduction. Oktay did 2 different studies in which he used letrozolefor IVF in breast cancer patients [45,46]. The first compared 47 stageI–IIIA ERþ or ER� breast cancer patients stimulated with letrozoleand FSH, and a retrospective group of patients submitted to IVF dueto tubal factor disease. The letrozole group presented significantlylower 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� 578.2UI vs. 2382.5�1062.8 UI P< 0.001). The other parameters, such astotal oocytes, mature oocytes and embryos obtained were similar inthe 2 groups. At the time of the publication only 3 cancer survivorshave used their embryos, resulted in one live birth (after thawing 3of the 7 embryos and transfer to a surrogate) and 1 biochemicalpregnancy [46].

In his more recent study Oktay compared 3 groups of patients:tamoxifen alone (Tam-IVF), tamoxifen in combination with low-dose follicle stimulating hormone (TamFSH-IVF) or letrozole withFSH (Letrozole IVF) [44]. Both TamFSH-IVF and letrozole IVFpatients had greater numbers of follicles, mature oocytes andembryos compared with Tam-IVF cases (follicle >17 mm Tam-IVF1.2� 0.1, TamFSH-IVF 2.6� 0.4 and letrozole IVF 3.2� 0.4; matureoocytes Tam-IVF 1.5� 0.3, TamFSH-IVF 5.1�1.1 and letrozole IVF8.5�1.6; total embryos Tam-IVF 1.3� 0.2, TamFSH-IVF 3.8� 0.8and letrozole IVF 5.3� 0.8). On the contrary, peak estradiol levelswere lower with letrozole IVF and Tam-IVF (380� 57 pg/ml and419� 39 pg/ml vs. 1182� 271 pg/ml in TamFSH-IVF group). As torecurrence rates, the patients of the 3 groups were compared withcontrols patients not submitted to IVF cycles and no differenceswere found (recurrence rate for the patients who underwent IVFwas 3 of 29 patients and for the control group was 3 of 31patients) [44].

This is a very interesting option for breast cancer patients, butmulti-centre prospective randomized trials are necessary to controlthe 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 administrationof a GnRH analogue during chemotherapy to protect the ovaries[58–61]. Endocrine therapy is a possible option in the treatment ofpremenopausal ERþ breast cancer in association withchemotherapy.

In the opinion of several authors it would seem that reversibleovarian suppression by GnRH analogue administered 1 week beforechemotherapy might reduce the likelihood of ovarian damage[61,62]. For example in a young women’s group with early breastcarcinoma, using analogues during chemotherapy (includedcyclophosphamide, methotrexate and 5-fluorouracil or anthracy-cline based regimen), Recchia found that, after a median follow-up

Table 2Live birth from autotransplantation of cryopreserved human ovarian tissue indisease-free patients

References Case reports

Donnez et al. (2004) [78] Age of freezing: 25Orthotopic transplantationSpontaneous pregnancy after 5 weeks

Meirow et al. (2005) [79] Age of freezing: 28Orthotopic transplantationIVF–ICSI pregnancy

Demestree et al. (2007) [80] Age of freezing: 24Orthotopic and heterotopic transplantationSpontaneous pregnancy after 5 months

Andersen et al. (2008) [81] (1) Age of freezing: 26Orthotopic transplantationIVF–ICSI pregnancy

(2) Age of freezing: 27Orthotopic transplantationIVF–ICSI pregnancy

Silber et al. (2008) [82] Age of freezing: 25Orthotopic transplantationSpontaneous pregnancy after 5 months

A. Borini, E. Rebellato / Placenta 29 (2008) S184–S190S188

of 75 months, all patients younger than 40 years have resumedtheir regular menses and 56% of the older ones. The author thoughtthat the effect of the GnRH analogue is not restricted to hormonedeprivation: it behaves like a negative growth factor, regulatingcancer cell proliferation and inducing apoptosis in chemotherapysurvivor cells [61].

As reported by Maltaris [63] in a recent review, the availablestudies have been criticized owing to the heterogeneity of patientsand follow-up protocols, the different regimens and the duration ofchemotherapy. International randomized controlled trials arenecessary to evaluate this strategy in women with cancer [62,63].

Actually the only one prospective randomized study wasa report by Waxman that included 31 men and 18 women receivingchemotherapy for Hodgkin’s disease. In this study, it seems thatGnRH analogues do not preserve fertility [64].

Besides the resumption of menstrual cycle is not a correctindicator of ovarian function and fertility. An interesting work byAnderson described the changes in AMH level during and afterchemotherapy. AMH decreased rapidly before FSH increased orirregular menses started. Probably the changes in the levels of thisgrowth factor reflected the primary damage of cytotoxic drugs toprimordial and primary follicles and it was a good test for residualovarian reserve, also in presence of regular menstrual cycles [65].

Clearly, further powered studies are necessary to clarify the roleof reversible ovarian suppression in early breast cancer patients,not only as an endocrine therapy but also as an option to preservefertility.

3.6. Ovarian tissue cryopreservation

Prepubertal girls and women who cannot delay the start ofchemotherapy have cryopreservation of ovarian tissue as an avail-able option. Ovarian tissue can be frozen using 3 differentapproaches: as fragment of ovarian cortex, as entire ovary with itsvascular pedicle or as isolated follicles. Actually the experiencesreported in literature use predominantly the first one. After treat-ment, when the patient is disease free, the tissue can bereimplanted either into the pelvic cavity (orthotopic site) or in theforearm or abdominal wall (heterotopic site).

In early breast cancer the risk of recurrence after reimplantationof cortical ovarian tissue due the presence of malignant cells isextremely low because the possibility of ovarian involvement inearly breast cancer is minimal [66,67]. Most of the occult metastasesbelong to the less common histological types, such as the infiltratinglobular cancer that typically occurs in post-menopausal women[66,67]. The real risk is the case of haematologic cancers [68]. InsteadHodgkin’s disease is nowadays considered just a poor risk [69].

Preoperative imaging and histologic evaluation of the freshovarian tissue are essential before storage. Furthermore, post-thawing polymerase chain reaction (PCR) with specific markersmust be used to evaluate the tissue for minimal residual disease [69].

Because of the risk of transmission of malignant cells, thedevelopment of isolated follicle transplantation is an interestingoption. Primordial follicles are resistant to cryoinjury because theoocytes contained have an inactive metabolism, lack of meioticspindle, zona pellucida and cortical granules. Moreover the smallsize of a primordial follicle facilitates penetration of cryoprotec-tants. Actually it is possible enzymatically isolate human folliclesfrom their surrounding tissue and, after xenografting in SCID mice,they are able to survive and develop into antral follicles. Probablythe formation of a new, well-vascularized stroma-like structure islikely to be a key factor in sustaining follicular growth in this in vivomodel [70,71]. Nevertheless further studies are necessary to assessthe safety of the procedure and to translate it in humans.

The transplantation of whole ovary with vascular anastomosispermits to restore immediate vascularization and to reduce post-

transplantation ischemia, responsible for the reduction of ovarianreserve [72,73]. The laparoscopic ovariectomy must be performedwith great care, removing the ovary and a large part (�5 cm) of theinfundibulopelvic ligament, allowing dissection of the ovarianvessels, perfusion with a cryoprotective medium, and cryopreser-vation for subsequent autografting. The period of ischemia betweenligation of the ovarian pedicle and ovarian cryopreservation mustbe as short as possible [72].

Actually in the literature there are some reports about wholehuman ovary cryopreservation [72,73] but no one transplantationor live birth, that, on the contrary, is described for sheep [74]. Thisprocedure should be encouraged searching for new cryopreserva-tion protocols and surgical techniques.

A new strategy of fertility preservation might be, for youngwomen undergoing ovarian cryobanking, retrieval and in vitromaturation of immature germinal vesicles (GV)-stage oocytes fol-lowed by vitrification [75,76].

Huang and his group reported an experience with 4 patientswho have undergone ovarian cryobanking for Hodgkin’s lymphoma(n¼ 2), breast cancer (n¼ 1) and rectal cancer (n¼ 1). After theexcision of cortical ovarian tissue, the visible antral follicles havebeen aspirated, in vitro matured and then cryopreserved. Themedian number of GV-stage oocytes recovered was 3, witha maturation rate varying from 50% to 100% and a median numberof vitrified oocytes of 3. No patients actually was come back to usethe cryopreserved tissue or the vitrified oocytes [77]. Clearly, thisstrategy can be only an adjunct in the protocol of ovarian tissuecryopreservation, for younger patients in which it is possiblerecovery a sufficient number of GV-stage oocytes.

Donnez et al. in 2004 reported the first pregnancy after ortho-topic transplantation of cryopreserved cortical ovarian tissue ina woman with previous stage IV Hodgkin’s lymphoma. Corticalbiopsies had been taken from the left ovary by laparoscopy. Thepregnancy started 11 months after transplantation and she deliv-ered a healthy baby. In this case, but there are other reports as well,folliculogenesis took 4–6 months to start. This time range betweenimplantation and first estradiol peak is consistent with dataobtained from sheep [78]. The other births obtained with corticalovarian tissue transplantation are described in Table 2.

Many reports have been published on heterotopic auto-transplantation that describe the restoration of the endocrine

A. Borini, E. Rebellato / Placenta 29 (2008) S184–S190 S189

function for a few months, but the only pregnancy described byOktay in 2006, was the outcome of ovulation occurred in a nativeovary [43]. The heterotopic autotransplantation was used by thesame author also in a breast cancer patient [83].

Silber and his group reported 8 cases of fresh ovarian trans-plantation in monozygotic twins, discordant for ovarian failure[84,85]. He proposed a minilaparotomy in which a section ofapproximately one-third of the donor ovarian cortex was laid overthe raw medulla of each ovary in the recipient and sutured onto themedulla; in one patient he transplanted the whole ovary. Heobtained 6 pregnancies and regular menstrual cycles in the otherwomen for 2 years [82,86]. Clearly this might be another possibleoption for a restricted group of patients with cancer or for womenthat would extend their reproductive life span.

4. Conclusions

Analysing the available studies in the literature one mightconclude that ovarian stimulation is a non-independent risk factorfor breast and ovarian cancer. On the contrary, for patients affectedby these tumors, the American Society of Clinical Oncology rec-ommended embryo cryopreservation, oocyte or cortical ovariantissue freezing to preserve fertility, depending on the possibility ofdelaying chemotherapy and the sensitivity of the tumor. Stimula-tions with tamoxifen or letrozole are possible options for patientswith ERþ breast cancer. The authors debate the rationale and safetyfor the use of GnRH analogue ovarian suppression. Finally, ovariantissue cryopreservation is an interesting option that still needs to beconfirmed by well-designed and powerful controlled studies.

5. Conflict of interest

The authors have no conflict of interest.

References

[1] Mahdavi A, Pejovic T, Nezhat F. Induction of ovulation and ovarian cancer:a critical review of the literature. Fertil Steril 2006;85:819–26.

[2] Mosgaard BJ, Lidegaard O, Kjaer SK, Schou G, Anderson AN. Infertility, fertilitydrugs and invasive ovarian cancer: a case control study. Fertil Steril1997;67:1005–12.

[3] Brinton LA, Lamb EJ, Moghissi KS, Scoccia B, Althius MD, Mabie JE, et al.Ovarian cancer risk after the use of ovulation-stimulating drugs. ObstetGynecol 2004;103:1194–203.

[4] Rossing MA, Daling JR, Weiss NS, Moore DE, Self SG. Ovarian tumors ina cohort of infertile women. N Engl J Med 1994;331:771–6.

[5] Shushan A, Paltiel O, Iscovich J, Elchalal U, Peretz Y, Schenker JG. Humanmenopausal gonadotropin and risk of epithelial ovarian cancer. Fertil Steril1996;65:13–8.

[6] Tung KH, Wilkens LR, Wu AH, McDuffie K, Nomura A, Kolonel L, et al. Effect ofanovulation factors on pre and post menopausal ovarian cancer risk: revisitingthe incessant ovulation hypothesis. Am J Epidemiol 2005;161:321–9.

[7] Clinton GM, Hua W. Estrogen action in human ovarian cancer. Crit Rev OncolHematol 1997;25:1–9.

[8] Cusido M, Fabregas R, Pere BS, Escayola C, Barri PN. Ovulation inductiontreatment and risk of borderline ovarian tumors. Gynecol Endocrinol2007;23:373–6.

[9] Sanner K, Conner P, Bergfeldt K, Dickman P, Sundfeldt K, Bergh T, et al. Ovarianepithelial neoplasia after hormonal infertility treatment: long-term follow-upof a historical cohort in Sweden. Fertil Steril 2008 Mar 25 [Epub ahead ofprint].

[10] Weir HK, Thun MJ, Henkey BF, Ries LA, Howe HL, Wingo PA, et al. Annualreport to the nation on the status of cancer, 1975–2000, featuring the uses ofsurveillance data for cancer prevention and control. J Natl Cancer Inst2003;95:1276–99.

[11] Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics.CA Cancer J Clin 2003;53:5–26.

[12] Sonmezer M, Oktay K. Fertility preservation in female patients. Hum ReprodUpdate 2004;10:251–66.

[13] Bernstein L. Epidemiology of endocrine related risk factors for breast cancer.J Mammary Gland Biol Neoplasia 2002;7:3–15.

[14] Venn A, Watson L, Bruinsma F, Giles G, Healy D. Risk of cancer after use offertility drugs with in vitro fertilization. Lancet 1999;354:1586–90.

[15] Gauthier E, Paoletti X, Clavel-Chapelon F, E3N Group. Breast cancer riskassociated with being treated for infertility: results from the French E3Ncohort study. Hum Reprod 2004;19:2216–21.

[16] Brinton LA, Scoccia B, Moghissi KS, Westhoff CL, Althius MD, Mabie JE, et al.Breast cancer risk associated with ovulation-stimulating drugs. Hum Reprod2004;19:2005–13.

[17] Pappo I, Lerner-Geva L, Halevy A, Olmer L, Friedler S, Raziel A, et al. Thepossible association between IVF and breast cancer incidence. Ann Surg Oncol2008;15:1048–55.

[18] Brinton LA, Moghissi KS, Scoccia B, Westhoff CL, Lamb EJ. Ovulation inductionand cancer risk. Fertil Steril 2005;83:261–74.

[19] Lambe M, Hsieh C, Trichopoulos D, Exborn A, Pavia M, Adami HO. Transientincrease in the risk of breast cancer after giving birth. N Engl J Med1994;331:5–9.

[20] Ahyan A, Celik H, Taskiran C, Bozdag G, Asku T. Oncologic and reproductiveoutcome after fertility-saving surgery in ovarian cancer. Eur J Gynaecol Oncol2003;24:223–32.

[21] Park CW, Yang KM, Kim HO, Hong SR, Kim TJ, Lim KT, et al. Outcomes ofcontrolled ovarian hyperstimulation/in vitro fertilization for infertile patientswith borderline ovarian tumor after conservative treatment. J Korean Med Sci2007;22(Suppl.):S134–8.

[22] Fortin A, Morice P, Thoury A, Camatte S, Dhainaut C, Madelenat P. Impact ofinfertility drugs after treatment of borderline ovarian tumor: results ofa retrospective multicentre study. Fertil Steril 2007;87:591–6.

[23] Morice P, Camatte S, Wicart-Poque F, Atallah D, Rouzier R, Pautier P, et al.Results of conservative management of epithelial malignant and borderlineovarian tumours. Hum Reprod Update 2003;9:185–92.

[24] Morice P, Wicart-Poque F, Rey A, Camatte S, Rouzier R, Pautier P, et al.Resultats carcinologiques et fertilite apres traitement conservateur des cancerepitheliaux de l’ovaire. Gynecol Obstet Fertil 2002;30:684–91.

[25] Morice P, Wicart-Poque F, Rey A, El_hassan J, Pautier P, Lhomme C, et al.Conservative treatment of epithelial ovarian cancer. Cancer 2001;92:2412–8.

[26] Villa A, Parazzini F, Acerboni S, Guarnierio P, Bolis G. Survival and prognosticfactors of early ovarian cancer. Br J Cancer 1998;77:123–4.

[27] Dembo AJ, Davy M, Stenwing AE, Berle EJ, Bush RS, Kjorstand K. Prognosticfactors in patients with stage I epithelial ovarian cancer. Obstet Gynecol1990;75:263–73.

[28] Colombo N, Parma G, Lapresa MT, Maggi F, Maggioni A. Role of conservativesurgery in ovarian cancer: the European experience. Int J Gynecol Cancer2005;15:206–11.

[29] Bandera C, Creamer D, Friedman A, Sheets E. Fertility therapy in the setting ofa history of invasive epithelial ovarian cancer. Gynecol Oncol 1995;58:116–9.

[30] Pouly JL, Janny L, Pouly-Vye P, Canis M, Cure A, Dechelotte P. In vitro fertil-ization after stage IC ovarian carcinoma. Hum Reprod 1997;12:1589–90.

[31] Steinkampf MP, Dharia MD, Hammond K. Assisted reproduction in patientswith early-stage ovarian malignancies. Fertil Steril 2003;80:1510–2.

[32] Fortin A, Hazout A, Thoury A, Alves K, Bats AS, Dhainaut C, et al. Assistedreproductive technologies after conservative management of borderline orinvasive ovarian tumors. Gynecol Obstet Fertil 2005;33:488–97.

[33] Perez GI, Knufdon CM, Leykin L, Korsmeyer SJ, Tilly JL. Apoptosis-associatedsignaling pathways are required for chemotherapy-mediated female germ celldestruction. Nat Med 1997;3:1228–32.

[34] Abir R, Ben-Haroush A, Felz C, Okon E, Raanani H, Orvieto R, et al. Selection ofpatients before and after anticancer treatment for ovarian cryopreservation.Hum Reprod 2008;23:869–77.

[35] Meirow D, Nugent D. The effects of radiotherapy and chemotherapy on femalereproduction. Hum Reprod Update 2001;7:534–43.

[36] Berliere M, Dalenc F, Malingret N, Vindevogel A, Piette P, Roche H, et al.Incidence of reversible amenorrhea in women with breast cancer undergoingadjuvant anthracycline-based chemotherapy with or without docetaxel. BMCCancer 2008;8:56.

[37] Bines J, Oleske DM, Cibleigh MA. Ovarian function in premenopausal womentreated with adjuvant chemotherapy for breast cancer. J Clin Oncol1996;14:1718–29.

[38] This P. Breast cancer and fertility: critical review, considerations andperspectives. Bull Cancer 2008;95:17–25.

[39] Vanhuyse M, Fournier C, Bonneterre J. Chemotherapy-induced amenorrhea:influence on disease-free survival and overall survival in receptor-positivepremenopausal early breast cancer patients. Ann Oncol 2005;16:1283–8.

[40] Toma S, Repetto L, Giacchero A, Coialbu T, Costantini M, Addamo GF, et al.Chemotherapy-induced amenorrhea and other clinical and pathological param-eters in the prognosis of breast cancer patients. J Chemother 1992;4:321–5.

[41] Del Mastro L, Venturini M, Sertoli MR, Rosso R. Amenorrhea induced byadjuvant chemotherapy in early breast cancer patients: prognostic role andclinical implications. Breast Cancer Res Treat 1997;43:183–90.

[42] Poikonen P, Saarto T, Elomaa I, Joensuu H, Blomqvist C. Prognostic effects ofamenorrhoea and elevated serum gonadotropin levels induced by adjuvantchemotherapy in premenopausal node-positive breast cancer patients. Eur JCancer 2000;36:43–8.

[43] Ethics Committee of the American Society for Reproductive Medicine.Fertility preservation and reproduction in cancer patients. Fertil Steril2005;83:1622–8.

[44] Oktay K, Buyuk E, Libertella N, Munire A, Rosenwaks Z. Fertility preservationin breast cancer patients: a prospective controlled comparison of ovarianstimulation with tamoxifen and letrozole for embryo cryopreservation. J ClinOncol 2005;22:4347–53.

A. Borini, E. Rebellato / Placenta 29 (2008) S184–S190S190

[45] Oktay K, Buyuk E, Davis O, Yermakova I, Veeck L, Rosenwaks Z. Fertilitypreservation in breast cancer patients: IVF and embryo cryopreservation afterovarian stimulation with tamoxifen. Hum Reprod 2003;18:90–5.

[46] Oktay K, Hourvitz A, Sahin G, Oktem O, Safro B, Cil A, et al. Letrozole reducesestrogen and gonadotropin exposure in women with breast cancer under-going ovarian stimulation before chemotherapy. J Clin Endocrinol Metab2006;91:3885–90.

[47] Borini A, Bianchi V, Bonu MA, Sciajno R, Sereni E, Cattoli M, et al. Evidence-based clinical outcome of oocyte slow cooling. Reprod Biomed Online2007;15:175–81.

[48] Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application ofhuman egg cryopreservation. Hum Reprod 1998;13:3156–9.

[49] Bianchi V, Coticchio G, Distratis V, Di Giusto N, Flamigni C, Borini A. Differ-ential sucrose concentration during dehydration (0.2 mol/l) and rehydration(0.3 mol/l) increases the implantation rate of frozen human oocytes. ReprodBiomed Online 2007;14:64–71.

[50] Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrificationmethod for cryopreservation of human oocytes. Reprod Biomed Online2005;11:300–8.

[51] Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Successful ongoingpregnancies after vitrification of oocytes. Fertil Steril 2006;86:108–11.

[52] Cobo A, Kuwayama M, Perez S, Ruiz A, Pellicer A, Remohı J. Comparison ofconcomitant outcome achieved with fresh and cryopreserved donor oocytesvitrified by the Cryotop method. Fertil Steril 2008;89:1657–64.

[53] Son WY, Park SE, Lee KA, Lee WS, Ko JJ, Yoon TK. Effects of 1,2-propanediol andfreezing on the in vitro development capacity of human immature oocytes.Fertil Steril 1996;66:996–9.

[54] Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S.Cryopreservation in human assisted reproduction is now routine for embryosbut remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl.3):161–77.

[55] Toth TL, Baka SG, Veeck LL, Jones Jr HW, Muhasher S, Lanzendorf SE. Fertil-ization and in vitro development of cryopreserved human prophase I oocytes.Fertil Steril 1994;61:891–4.

[56] Tucker MJ, Wright G, Morton PC, Massey JB. Birth after cryopreservation ofimmature oocytes with subsequent in vitro maturation. Fertil Steril1998;70:578–9.

[57] Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, et al. TheAmerican Society of Clinical Oncology recommendations on fertility preser-vation in cancer patients. J Clin Oncol 2006;24:2917–31.

[58] Blumenfeld Z, Avivi L, Linn S, Epelbaum R, Ben-Shahar M, Haim N. Preventionof irreversible chemotherapy-induced ovarian damage in young women withlymphoma by a gonadotrophin-releasing hormone agonist in parallel tochemotherapy. Hum Reprod 1996;11:1620–6.

[59] Blumenfeld Z. How to preserve fertility in young women exposed tochemotherapy? The role of GnRH agonist cotreatment in addition to cryo-preservation of embryo, oocytes or ovaries. Oncologist 2007;12:1067–9.

[60] Recchia F, Sica G, De Filippis S, Rosselli M, Rea S. Goserelin as ovarianprotection in the adjuvant treatment of premenopausal breast cancer. A phaseII pilot study. Anticancer Drugs 2002;13:417–24.

[61] Recchia F, Saggio G, Amiconi G, Di Blasio A, Cesta A, Candeloro G, et al.Gonadotropin releasing hormone analogues added to adjuvant chemotherapyprotect ovarian function and improve clinical outcomes in young women withearly breast carcinoma. Cancer 2006;106:515–23.

[62] Oktay K, Sonmezer M, Oktem O, Fox H, Emons G, Bang H. Absence ofconclusive evidence for the safety and efficacy of gonadotropin-releasinghormone analogue treatment in protecting against chemotherapy-inducedgonadal injury. Oncologist 2007;12:1055–66.

[63] Maltaris T, Weigel M, Mueller A, Schmidt M, Seufert R, Fischl F, et al. Cancerand fertility preservation: fertility preservation in breast cancer patients.Breast Cancer Res 2008;10:206–16.

[64] Waxman JH, Ahmed R, Smith D. Failure to preserve fertility in patients withHodgkin’s disease. Cancer Chemother Pharmacol 1987;19:159–62.

[65] Anderson RA, Themmen APN, Al-Qahtani A, Groome NP, Cameron DA. Theeffects of chemotherapy and long term gonadotrophin suppression on the

ovarian reserve in premenopausal women with breast cancer. Hum Reprod2006;21:2583–92.

[66] Hann LE, Lui DM, Shi W, Bach AM, Selland DL, Castiel M. Adnexal masses inwomen with breast cancer. US findings with clinical and histopathologiccorrelation. Radiology 2000;216:242–7.

[67] Li CL, Anderson BO, Daling JR, Moe RE. Trends in incidence rate of invasivelobular and ductal breast carcinoma. J Am Med Assoc 2003;289:1421–4.

[68] Sonmezer M, Shamonki MI, Oktay K. Ovarian tissue cryopreservation: benefitsand risks. Cell Tissue Res 2005;322:125–32.

[69] Meirow D, Hardan I, Dor J, Fridman E, Elizur S, Ra’anani H, et al. Searching forevidence of disease and malignant cell contamination in ovarian tissue storedfrom hematologic cancer patients. Hum Reprod 2008 March 15 [Epub ahead ofprint].

[70] Abir R, Fisch B, Nitke S, Okon E, Raz A, Ben Rafael Z. Morphological study offully and partially isolated early human follicles. Fertil Steril 2001;75:141–6.

[71] Dolmans MM, Yuan WY, Camboni A, Torre A, Van Langendonckt A, Martinez-Madrid B, et al. Development of antral follicles after xenografting of isolatedsmall human preantral follicles. Reprod Biomed Online 2008;16:705–11.

[72] Jadoul P, Donnez J, Dolmans MM, Squifflet J, Lengele B, Martinez-Madrid B.Laparoscopy ovariectomy for whole human ovary cryopreservation: technicalaspects. Fertil Steril 2007;87:971–5.

[73] Martinez-Madrid B, Camboni A, Dolmans MM, Nottola S, VanLangendonckt A, Donnez J. Apoptosis and ultrastructural assessment aftercryopreservation of whole human ovaries with their vascular pedicle. FertilSteril 2007;87:1153–65.

[74] Courbiere B, Casquant L, Mazoyer C, Franck M, Lornage J Salle B. Difficultiesimproving ovarian functional recovery by microvascular transplantation andwhole ovary vitrification. Fertil Steril 2008 Apr 26 [Epub ahead of print].

[75] Revel A, Koler M, Simon A, Lewin A, Laufer N, Safran A. Oocyte collectionduring cryopreservation of the ovarian cortex. Fertil Steril 2003;79:1237–9.

[76] Isachenko E, Rahimi G, Isachenko V, Narworth F. In-vitro maturation ofgerminal vesicle oocytes and cryopreservation in metaphase I–II: a possibleadditional option to preserve fertility during ovarian tissue cryopreservation.Reprod Biomed Online 2004;8:553–7.

[77] Huang JYJ, Tulandi T, Holzer H, Lin Tan S, Chian RC. Combining ovarian tissuecryobanking with retrieval of immature oocytes followed by in vitro matura-tion and vitrification: an additional strategy of fertility preservation. FertilSteril 2008;89:567–72.

[78] Donnez J, Martinez-Madrid B, Jadoul P, Van Langendonckt A, Demylle D,Dolmans MM. Ovarian tissue cryopreservation and transplantation: a review.Hum Reprod Update 2006;12:519–35.

[79] Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, et al. Pregnancyafter transplantation of cryopreserved ovarian tissue in a patient with ovarianfailure after chemotherapy. N Engl J Med 2005;353:318–21.

[80] Demeestere I, Simon P, Emiliani S, Del baere A, Englert Y. Fertility preserva-tion: successful transplantation of cryopreserved ovarian tissue in a youngpatient previously treated for Hodgkin’s disease. Oncologist 2007;12:1437–42.

[81] Andersen CY, Rosendahl M, Byskov AG, Loft A, Ottosen C, Dueholm M, et al.Two successful pregnancies following autotransplantation of frozen/thawedovarian tissue. Hum Reprod 2008 Jul 3 [Epub ahead of print].

[82] Silber SJ, De Rosa M, Pineda J, Lenahan K, Grenia D, Gorman K, et al. A series ofmonozygotic twins discordant for ovarian failure: ovary transplantation(cortical versus microvascular) and cryopreservation. Hum Reprod2008;23:1531–7.

[83] Oktay K. Spontaneous conception and live birth after heterotopic ovariantransplantation: is there a germ line stem cell connection? Hum Reprod2006;21:1345–8.

[84] Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, et al. Embryodevelopment after heterotopic transplantation of cryopreserved ovariantissue. Lancet 2004;363:837–40.

[85] Silber SJ, Lenahan KM, Levine DJ, Pineda JA, Gorman KS, Friez MJ, et al. Ovariantransplantation between monozygotic twins discordant for premature ovarianfailure. N Engl J Med 2005;353:58–63.

[86] Silber SJ, Gosden RG. Ovarian transplantation in a series of monozygotic twinsdiscordant for ovarian failure. N Engl J Med 2007;356:1382–4.