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Breast cancer treatment and ovarian function

Published:September 20, 2022DOI:https://doi.org/10.1016/j.rbmo.2022.09.014

      Abstract

      The aim of this study was to provide an update on ovarian function and the mechanisms of gonadal damage after exposure to chemotherapy in breast cancer survivors. The alkylating agents are toxic to both primordial and growing follicles. However, anti-metabolite drugs are more likely to destroy preantral and antral follicles. Younger patients are more likely to have a higher ovarian reserve, and therefore, more likely to retain some residual ovarian function after exposure to gonadotoxic regimens. However, there can be significant variability in ovarian reserve among patients of the same age. Furthermore, patients with critically diminished ovarian reserve may continue to menstruate regularly. Therefore age and menstrual status are not reliable indicators of good ovarian reserve and might give a false sense of security and result in an adverse outcome if the patient is consulted without considering more reliable quantitative markers of ovarian reserve (antral follicle count and anti-Müllerian hormone) and fertility preservation is not pursued. In contrast to well-documented ovarian toxicity of older chemotherapy regimens, data for newer taxane-containing protocols have only accumulated in the last decade and data are still very limited regarding the impact of targeted therapies on ovarian function.

      Keywords

      Introduction

      Breast cancer is the most frequent neoplasm among females and accounts for 30% of all new cancer diagnoses (
      • Siegel R.L.
      • Miller K.D.
      • Jemal A.
      Cancer statistics, 2019.
      ). Breast cancer is also frequently seen in childhood cancer survivors among patients with recurrent cancer, especially those with Hodgkin's lymphoma treated with chest radiation (
      • Dracham C.B.
      • Shankar A.
      • Madan R.
      Radiation induced secondary malignancies: a review article.
      ). The 5-year relative survival rate has reached 99% among women with localized breast cancer (
      • Siegel R.L.
      • Miller K.D.
      • Jemal A.
      Cancer statistics, 2019.
      ). Increased life expectancy has raised concern about fertility among young survivors of breast cancer, many of whom suffer from amenorrhoea, infertility or premature ovarian failure (POF) after exposure to gonadotoxic adjuvant chemotherapy regimens. Moreover, postponement of pregnancy for years will further increase the risk of infertility in breast cancer patients due to ageing-related decline in ovarian reserve. It is essential that at the initial visit to counsel and inform patients about the risk of infertility, personalized risk assessment and treatment options are offered to preserve their fertility. Although there is an abundance of data on the incidence of amenorrhoea in breast cancer patients treated with older classical chemotherapy regimens such as cyclophosphamide, methotrexate and 5-fluorouracil (CMF) and cyclophosphamide, epirubicin and 5-fluorouracil (CEF), amenorrhoea rates associated with newer chemotherapy protocols have begun to emerge recently. Therefore, the aim of this study was to provide a comprehensive update on the risk of POF in breast cancer survivors in different chemotherapy protocols in the light of available markers to assess pre- and post-chemotherapy ovarian reserve and function. Finally, current fertility preservation strategies under the guidance of the most recent recommendations are reviewed.

      Materials and methods

      A total of 58 peer-reviewed articles and abstracts were identified between 1985 and 2021 after searching PubMed as well as the proceedings of professional society meetings relevant to cancer and reproductive medicine (American Society of Clinical Oncology [ASCO], San Antonio Breast Cancer Symposium, European Cancer Summit, European Society for Medical Oncology, American Society for Reproductive Medicine [ASRM] and European Society of Human Reproduction and Embryology [ESHRE]).

      The mechanisms of chemotherapy-induced ovarian damage

      Most of the follicles are found at dormant primordial follicle stage in the adult human ovary. The number of primordial follicles determines ovarian reserve while growing follicle fractions, particularly those small antral follicles that produce anti-Müllerian hormone (AMH), constitute functional ovarian reserve (
      • Moolhuijsen L.M.E.
      • Visser J.A.
      Anti-Mullerian hormone and ovarian reserve: update on assessing ovarian function.
      ;
      • Oktem O.
      • Urman B.
      Understanding follicle growth in vivo.
      ) (Figure 1). Among the components of the female reproductive system, the ovary is the most sensitive organ to the cytotoxic effects of chemotherapy regimens. Chemotherapy drugs of the alkylating category are the most toxic to the ovaries, causing extensive organ damage and massive follicle destruction characterized by apoptotic death of oocyte and somatic cells (Figure 2) (
      • Oktem O.
      • Oktay K.
      A novel ovarian xenografting model to characterize the impact of chemotherapy agents on human primordial follicle reserve.
      ;
      • Plowchalk D.R.
      • Mattison D.R.
      Phosphoramide mustard is responsible for the ovarian toxicity of cyclophosphamide.
      ). Accumulating evidence indicates that, in addition to the direct toxic effects of chemotherapy agents on the follicular apparatus, there are some additional mechanisms that play a role in the development of chemotherapy-induced ovarian damage/failure. Ovarian vascular damage is one of those proposed mechanisms (Figure 2). Previous studies have demonstrated that chemotherapy drugs injure vascular structures in the cortical and medullary portions of the human ovary, resulting in obliteration, fibrosis and decreases in vascular endothelial growth factor (VEGF) and microvascular intensity (
      • Bildik G.
      • Akin N.
      • Senbabaoglu F.
      • Sahin G.N.
      • Karahuseyinoglu S.
      • Ince U.
      • Taskiran C.
      • Selek U.
      • Yakin K.
      • Guzel Y.
      • Ayhan C.
      • Alper E.
      • Cetiner M.
      • Balaban B.
      • Mandel N.M.
      • Esen T.
      • Iwase A.
      • Urman B.
      • Oktem O.
      GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro.
      ;
      • Meirow D.
      • Dor J.
      • Kaufman B.
      • Shrim A.
      • Rabinovici J.
      • Schiff E.
      • Raanani H.
      • Levron J.
      • Fridman E.
      Cortical fibrosis and blood-vessels damage in human ovaries exposed to chemotherapy. Potential mechanisms of ovarian injury.
      ). The decrease in ovarian vascularization may impede blood supply, aggravating follicle loss and accelerating ovarian ageing. The second proposed mechanism is the so-called burn-out phenomenon. It was shown that exposure to cyclophosphamide activates the phosphatidylinositol 3-kinase signalling pathway, which causes premature activation of primordial follicles and hence ‘burn-out’ or early depletion of the follicle pool of the ovary (Figure 2) (
      • Kalich-Philosoph L.
      • Roness H.
      • Carmely A.
      • Fishel-Bartal M.
      • Ligumsky H.
      • Paglin S.
      • Wolf I.
      • Kanety H.
      • Sredni B.
      • Meirow D.
      Cyclophosphamide triggers follicle activation and ‘burnout’; AS101 prevents follicle loss and preserves fertility.
      ). Based on the findings above, the investigators also observed that primordial follicles were lost after chemotherapy exposure. Only a few were positive after staining with the apoptosis marker, TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling, which suggests that the reduction in primordial follicle numbers is caused by accelerated growth initiation rather than a direct toxic effect of the chemotherapy (
      • Morgan S.
      • Lopes F.
      • Gourley C.
      • Anderson R.A.
      • Spears N.
      Cisplatin and doxorubicin induce distinct mechanisms of ovarian follicle loss; imatinib provides selective protection only against cisplatin.
      ). Because AMH has an inhibitory effect on the activation of primordial follicles, declining AMH concentrations as a result of destruction of AMH producing small antral follicles after exposure to chemotherapy drugs might cause premature activation and therefore, subsequent atresia of primordial follicles (
      • Spears N.
      • Lopes F.
      • Stefansdottir A.
      • Rossi V.
      • De Felici M.
      • Anderson R.A.
      • Klinger F.G.
      Ovarian damage from chemotherapy and current approaches to its protection.
      ). The third theory, conducted with mice ovaries, explains the different mechanisms of follicle loss. For example, while cisplatin exposure causes oocyte-specific damage, doxorubicin induces more selective damage to the mitotic granulosa cells of secondary follicles (
      • Morgan S.
      • Lopes F.
      • Gourley C.
      • Anderson R.A.
      • Spears N.
      Cisplatin and doxorubicin induce distinct mechanisms of ovarian follicle loss; imatinib provides selective protection only against cisplatin.
      ). Taken together, it appears that there are multiple pathogenetic mechanisms underlying chemotherapy-induced ovarian damage and follicle loss in the ovary.
      Figure 1
      Figure 1Adult human ovary. Dormant primordial follicles constitute almost 90% of the follicle stockpile, whereas the growing follicle fraction is made up of the follicles from the primary stage onwards. Scale bar: 100 µm.
      Figure 2
      Figure 2The molecular mechanisms of chemotherapy-induced ovarian damage and follicle loss. 1: Direct damage and apoptotic death of the oocytes and somatic cells in the ovary (TUNEL staining before and after cyclophosphamide exposure). 2: Vascular damage within the ovarian stroma post-exposure to chemotherapy (PCAM-1/CD31, endothelial cell marker). 3: Premature activation and subsequent atresia of primordial follicles (burn-out theory).
      Although chemotherapy-induced gonadal toxicity is a significant concern for premenopausal women with breast cancer, the fundamental role of chemotherapy in improving survival is undeniable. As a striking example, the 15-year survival rate in breast cancer patients under the age of 40 dramatically increased to 70.2% in patients who received chemotherapy compared with 60.1% in those who did not (
      • Clarke M.
      • Collins R.
      • Darby S.
      • Davies C.
      • Elphinstone P.
      • Evans V.
      • Godwin J.
      • Gray R.
      • Hicks C.
      • James S.
      • MacKinnon E.
      • McGale P.
      • McHugh T.
      • Peto R.
      • Taylor C.
      • Wang Y.
      Early Breast Cancer Trialists’ Collaborative Group
      Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials.
      ). Different chemotherapy regimens are used in the treatment of patients with breast cancer, some of which include cyclophosphamide-based alkylating agents. Cyclophosphamide is in fact a pro-drug, which undergoes a 4-hydroxylation step in the liver and forms 4-hydroxycyclophosphamide. After a spontaneous ring opening, 4-hydroxycyclophosphamide gives rise to the generation of the final products phosphoramide mustard and acrolein. Phosphoramide mustard is responsible for the gonadotoxic effects while acrolein causes urinary toxicity of the drug. The formation of guanosine adducts that prevent DNA replication and damage to mitochondria and other organelles within cells are the primary mechanisms of the cyclophosphamide-induced cellular cytotoxicity (
      • Plowchalk D.R.
      • Mattison D.R.
      Phosphoramide mustard is responsible for the ovarian toxicity of cyclophosphamide.
      ;
      • Spears N.
      • Lopes F.
      • Stefansdottir A.
      • Rossi V.
      • De Felici M.
      • Anderson R.A.
      • Klinger F.G.
      Ovarian damage from chemotherapy and current approaches to its protection.
      ). A human ovarian xenograft model provided in-vivo evidence that administration of cyclophosphamide triggers apoptotic death of not only the oocytes and somatic cells of primordial follicles but also the surrounding stroma in the grafts, starting as early as 24 h after administration (
      • Oktem O.
      • Oktay K.
      A novel ovarian xenografting model to characterize the impact of chemotherapy agents on human primordial follicle reserve.
      ).
      Chemotherapeutics used in breast cancer patients vary based on cell cycle specificity (Figure 3). The alkylating agents and anthracyclines (not cell cycle specific agents) exert cytotoxic effects at every stage of the cell cycle, causing more widespread damage in the ovary. However, cell cycle specific agents (e.g. 5-fluorouracil and methotrexate) are more likely to impact growing preantral and antral follicles than primordial follicles due to their higher metabolic demand. Patients older than 40 are likely to have a lower ovarian reserve and so chemotherapeutics may result in a more significant reduction of residual ovarian functions. By contrast, younger patients (below the age of 40) are more likely to retain menstrual function (22–56% versus 11%) (
      • Bines J.
      • Oleske D.M.
      • Cobleigh M.A.
      Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer.
      ). It should also be remembered that age is not always a reliable indicator of ovarian reserve as there could be a mismatch between chronological age and ovarian reserve status in some women, underscoring the importance of using more reliable and specific markers of ovarian reserve, such as antral follicle count on ultrasound examination and serum AMH concentrations.
      Figure 3
      Figure 3Cell cycle stages targeted by chemotherapy drugs. Those drugs that exert their anti-proliferative impact at a specific cell cycle stage, such as mitosis-specific taxanes and S-phase-specific anti-metabolite drugs, are less detrimental than alkylating agents, which act in a cell cycle non-specific manner and cause more widespread destruction.
      Acute ovarian failure (AOF) is the cessation of menstrual function during or shortly after the completion of cancer treatment and is caused by the destruction of the growing follicles. AOF may be reversible if the primordial follicle pool is not completely depleted and follicle development from primordial to antral follicles resumes later on. However, it should be kept in mind that the return of menses does not guarantee a normal reproductive lifespan, because a subset of these women will ultimately experience POF (premature menopause) before the age of 40 due to a diminished primordial follicle pool. If the magnitude of ovarian damage is severe enough to destroy all primordial follicles, as in cases exposed to very high dose alkylating agents and/or ionizing radiation, permanent ovarian failure may develop without resumption of menses. Studies have assessed the risk of amenorrhoea after various chemotherapeutic exposures, and these regimes are summarized in Table 1 and shown in detail in Supplementary Table 1.
      Table 1Summary of the studies assessing the risk of amenorrhea following exposure to various chemotherapy regimens for breast cancer
      Chemotherapy protocol (study references)No. of included patientsDuration of chemotherapy (months)Chemotherapy-induced amenorrhea rate range (%)
      Age <40Age >40
      CMF only

      • Bonadonna G.
      • Rossi A.
      • Valagussa P.
      Adjuvant CMF chemotherapy in operable breast cancer: ten years later.
      ;
      Ludwig Breast Cancer Group
      A randomized trial of adjuvant combination chemotherapy with or without prednisone in premenopausal breast cancer patients with metastases in one to three axillary lymph nodes.
      ;
      • Goldhirsch A.
      • Gelber R.D.
      • Castiglione M.
      The magnitude of endocrine effects of adjuvant chemotherapy for premenopausal breast cancer patients. The International Breast Cancer Study Group.
      ;
      • Richards M.A.
      • O'Reilly S.M.
      • Howell A.
      • George W.D.
      • Fentiman I.S.
      • Chaudary M.A.
      • Crowther D.
      • Rubens R.D.
      Adjuvant cyclophosphamide, methotrexate, and fluorouracil in patients with axillary node-positive breast cancer: an update of the Guy's/Manchester trial.
      ;
      • Bianco A.R.
      • Del Mastro L.
      • Gallo C.
      • Perrone F.
      • Matano E.
      • Pagliarulo C.
      • De Placido S.
      Prognostic role of amenorrhea induced by adjuvant chemotherapy in premenopausal patients with early breast cancer.
      ;
      • Campora E.
      • Pronzato P.
      • Amoroso D.
      • Bertelli G.F.
      • Venturini M.
      • Baldini E.
      • Brunetti I.
      • Sertoli M.R.
      • Conte P.
      • Rosso R.
      Prognostic factors in node positive primary breast cancer patients treated with adjuvant CMF.
      ;
      • Valagussa P.
      • Moliterni A.
      • Zambetti M.
      • Bonadonna G.
      Long-term sequelae from adjuvant chemotherapy.
      ;
      • Cobleigh M.A.
      • Bines J.
      • Harris D.
      • LaFollette S.
      • Lincoln S.T.
      • Wolter J.M.
      Amenorrhea following adjuvant chemotherapy for breast cancer.
      ;
      • Bines J.
      • Oleske D.M.
      • Cobleigh M.A.
      Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer.
      ;
      • Levine M.N.
      • Bramwell V.H.
      • Pritchard K.I.
      • Norris B.D.
      • Shepherd L.E.
      • Abu-Zahra H.
      • Findlay B.
      • Warr D.
      • Bowman D.
      • Myles J.
      • Arnold A.
      • Vandenberg T.
      • MacKenzie R.
      • Robert J.
      • Ottaway J.
      • Burnell M.
      • Williams C.K.
      • Tu D.
      Randomized trial of intensive cyclophosphamide, epirubicin, and fluorouracil chemotherapy compared with cyclophosphamide, methotrexate, and fluorouracil in premenopausal women with node-positive breast cancer. National Cancer Institute of Canada Clinical Trials Group.
      ;
      • Pagani O.
      • O'Neill A.
      • Castiglione M.
      • Gelber R.D.
      • Goldhirsch A.
      • Rudenstam C.M.
      • Lindtner J.
      • Collins J.
      • Crivellari D.
      • Coates A.
      • Cavalli F.
      • Thurlimann B.
      • Simoncini E.
      • Fey M.
      • Price K.
      • Senn H.J.
      Prognostic impact of amenorrhoea after adjuvant chemotherapy in premenopausal breast cancer patients with axillary node involvement: results of the International Breast Cancer Study Group (IBCSG) Trial VI.
      ;
      • Goodwin P.J.
      • Ennis M.
      • Pritchard K.I.
      • Trudeau M.
      • Hood N.
      Risk of menopause during the first year after breast cancer diagnosis.
      ;
      • Jonat W.
      • Kaufmann M.
      • Sauerbrei W.
      • Blamey R.
      • Cuzick J.
      • Namer M.
      • Fogelman I.
      • de Haes J.C.
      • de Matteis A.
      • Stewart A.
      • Eiermann W.
      • Szakolczai I.
      • Palmer M.
      • Schumacher M.
      • Geberth M.
      • Lisboa B.
      Goserelin versus cyclophosphamide, methotrexate, and fluorouracil as adjuvant therapy in premenopausal patients with node-positive breast cancer: The Zoladex Early Breast Cancer Research Association Study.
      ;
      • Castiglione-Gertsch M.
      • O'Neill A.
      • Price K.N.
      • Goldhirsch A.
      • Coates A.S.
      • Colleoni M.
      • Nasi M.L.
      • Bonetti M.
      • Gelber R.D.
      Adjuvant chemotherapy followed by goserelin versus either modality alone for premenopausal lymph node-negative breast cancer: a randomized trial.
      ;
      • Di Cosimo S.
      • Alimonti A.
      • Ferretti G.
      • Sperduti I.
      • Carlini P.
      • Papaldo P.
      • Fabi A.
      • Gelibter A.
      • Ciccarese M.
      • Giannarelli D.
      • Mandala M.
      • Milella M.
      • Ruggeri E.M.
      • Cognetti F.
      Incidence of chemotherapy-induced amenorrhea depending on the timing of treatment by menstrual cycle phase in women with early breast cancer.
      ;
      • Bonadonna G.
      • Moliterni A.
      • Zambetti M.
      • Daidone M.G.
      • Pilotti S.
      • Gianni L.
      • Valagussa P.
      30 years’ follow up of randomised studies of adjuvant CMF in operable breast cancer: cohort study.
      ;
      • Parulekar W.R.
      • Day A.G.
      • Ottaway J.A.
      • Shepherd L.E.
      • Trudeau M.E.
      • Bramwell V.
      • Levine M.
      • Pritchard K.I.
      Incidence and prognostic impact of amenorrhea during adjuvant therapy in high-risk premenopausal breast cancer: analysis of a National Cancer Institute of Canada Clinical Trials Group Study – NCIC CTG MA.5.
      ;
      • Vanhuyse M.
      • Fournier C.
      • Bonneterre J.
      Chemotherapy-induced amenorrhea: influence on disease-free survival and overall survival in receptor-positive premenopausal early breast cancer patients.
      ;
      • Kil W.J.
      • Ahn S.D.
      • Shin S.S.
      • Lee S.W.
      • Choi E.K.
      • Kim J.H.
      • Son B.H.
      • Ahn S.H.
      • Kim W.K.
      • Kim S.B.
      Treatment-induced menstrual changes in very young (<35 years old) breast cancer patients.
      ;
      • Petrek J.A.
      • Naughton M.J.
      • Case L.D.
      • Paskett E.D.
      • Naftalis E.Z.
      • Singletary S.E.
      • Sukumvanich P.
      Incidence, time course, and determinants of menstrual bleeding after breast cancer treatment: a prospective study.
      ;
      • Lee S.
      • Kil W.J.
      • Chun M.
      • Jung Y.S.
      • Kang S.Y.
      • Kang S.H.
      • Oh Y.T.
      Chemotherapy-related amenorrhea in premenopausal women with breast cancer.
      ;
      • Jung M.
      • Shin H.J.
      • Rha S.Y.
      • Jeung H.C.
      • Hong S.
      • Moon Y.W.
      • Kim H.S.
      • Oh K.J.
      • Yang W.I.
      • Roh J.K.
      • Chung H.C.
      The clinical outcome of chemotherapy-induced amenorrhea in premenopausal young patients with breast cancer with long-term follow-up.
      ;
      • Sukumvanich P.
      • Case L.D.
      • Van Zee K.
      • Singletary S.E.
      • Paskett E.D.
      • Petrek J.A.
      • Naftalis E.
      • Naughton M.J.
      Incidence and time course of bleeding after long-term amenorrhea after breast cancer treatment: a prospective study.
      ;
      • Najafi S.
      • Djavid G.E.
      • Mehrdad N.
      • Rajaii E.
      • Alavi N.
      • Olfatbakhsh A.
      • Najafi M.
      • Bahrami A.
      • Heidari K.
      Taxane-based regimens as a risk factor for chemotherapy-induced amenorrhea.
      98741–124–7434–97
      CEF or FEC

      • Levine M.N.
      • Bramwell V.H.
      • Pritchard K.I.
      • Norris B.D.
      • Shepherd L.E.
      • Abu-Zahra H.
      • Findlay B.
      • Warr D.
      • Bowman D.
      • Myles J.
      • Arnold A.
      • Vandenberg T.
      • MacKenzie R.
      • Robert J.
      • Ottaway J.
      • Burnell M.
      • Williams C.K.
      • Tu D.
      Randomized trial of intensive cyclophosphamide, epirubicin, and fluorouracil chemotherapy compared with cyclophosphamide, methotrexate, and fluorouracil in premenopausal women with node-positive breast cancer. National Cancer Institute of Canada Clinical Trials Group.
      ;
      • Luporsi E.
      • Weber B.
      The effects of breast cancer chemotherapy on menstrual function.
      ;
      • Goodwin P.J.
      • Ennis M.
      • Pritchard K.I.
      • Trudeau M.
      • Hood N.
      Risk of menopause during the first year after breast cancer diagnosis.
      ;
      • Di Cosimo S.
      • Alimonti A.
      • Ferretti G.
      • Sperduti I.
      • Carlini P.
      • Papaldo P.
      • Fabi A.
      • Gelibter A.
      • Ciccarese M.
      • Giannarelli D.
      • Mandala M.
      • Milella M.
      • Ruggeri E.M.
      • Cognetti F.
      Incidence of chemotherapy-induced amenorrhea depending on the timing of treatment by menstrual cycle phase in women with early breast cancer.
      ;
      • Parulekar W.R.
      • Day A.G.
      • Ottaway J.A.
      • Shepherd L.E.
      • Trudeau M.E.
      • Bramwell V.
      • Levine M.
      • Pritchard K.I.
      Incidence and prognostic impact of amenorrhea during adjuvant therapy in high-risk premenopausal breast cancer: analysis of a National Cancer Institute of Canada Clinical Trials Group Study – NCIC CTG MA.5.
      ;
      • Samuelkutty S
      • G O.
      • Mohrmann S
      • Hille S
      • Zwiefel K
      • Schuett G
      • Nitz U.
      Chemotherapy-induced amenorrhea (CIA) in patients treated with adjuvant CEF/CMF or EC/docetaxel: analysis from a phase III randomized EC/Doc Trial.
      ;
      • Vanhuyse M.
      • Fournier C.
      • Bonneterre J.
      Chemotherapy-induced amenorrhea: influence on disease-free survival and overall survival in receptor-positive premenopausal early breast cancer patients.
      ;
      • Venturini M.
      • Del Mastro L.
      • Aitini E.
      • Baldini E.
      • Caroti C.
      • Contu A.
      • Testore F.
      • Brema F.
      • Pronzato P.
      • Cavazzini G.
      • Sertoli M.R.
      • Canavese G.
      • Rosso R.
      • Bruzzi P.
      Dose-dense adjuvant chemotherapy in early breast cancer patients: results from a randomized trial.
      ;
      • Roche H.
      • Fumoleau P.
      • Spielmann M.
      • Canon J.L.
      • Delozier T.
      • Serin D.
      • Symann M.
      • Kerbrat P.
      • Soulie P.
      • Eichler F.
      • Viens P.
      • Monnier A.
      • Vindevoghel A.
      • Campone M.
      • Goudier M.J.
      • Bonneterre J.
      • Ferrero J.M.
      • Martin A.L.
      • Geneve J.
      • Asselain B.
      Sequential adjuvant epirubicin-based and docetaxel chemotherapy for node-positive breast cancer patients: the FNCLCC PACS 01 Trial.
      ;
      • Berliere M.
      • Dalenc F.
      • Malingret N.
      • Vindevogel A.
      • Piette P.
      • Roche H.
      • Donnez J.
      • Symann M.
      • Kerger J.
      • Machiels J.P.
      Incidence of reversible amenorrhea in women with breast cancer undergoing adjuvant anthracycline-based chemotherapy with or without docetaxel.
      ;
      • Zhou W.B.
      • Yin H.
      • Liu X.A.
      • Zha X.M.
      • Chen L.
      • Dai J.C.
      • Tao A.D.
      • Ma J.J.
      • Ling L.J.
      • Wang S.
      Incidence of chemotherapy-induced amenorrhea associated with epirubicin, docetaxel and navelbine in younger breast cancer patients.
      ;
      • Sakurai K.
      • Matsuo S.
      • Enomoto K.
      • Amano S.
      • Shiono M.
      Menstruation recovery after chemotherapy and luteinizing hormone-releasing hormone agonist plus tamoxifen therapy for premenopausal patients with breast cancer.
      ;
      • Zhou W.
      • Ding Q.
      • Liang X.
      • He Z.
      • Zha X.
      • Liu X.
      • Wang S.
      The risk of amenorrhea is related to chemotherapy-induced leucopenia in breast cancer patients receiving epirubicin and taxane based chemotherapy.
      ;
      • Meng K.X.
      • Tian W.
      • Zhou M.Q.
      • Chen H.L.
      • Deng Y.C.
      Impact of chemotherapy-induced amenorrhea in breast cancer patients: the evaluation of ovarian function by menstrual history and hormonal levels.
      ;
      • Tiong V.
      • Rozita A.M.
      • Taib N.A.
      • Yip C.H.
      • Ng C.H.
      Incidence of chemotherapy-induced ovarian failure in premenopausal women undergoing chemotherapy for breast cancer.
      292560–5049–92
      AC-containing regimen

      • Hortobagyi G.N.
      • Buzdar A.U.
      • Marcus C.E.
      • Smith T.L.
      Immediate and long-term toxicity of adjuvant chemotherapy regimens containing doxorubicin in trials at M.D. Anderson Hospital and Tumor Institute.
      ;
      • Lower E.E.
      • Blau R.
      • Gazder P.
      • Tummala R.
      The risk of premature menopause induced by chemotherapy for early breast cancer.
      ;
      • Stone E.R.
      • Slack R.S.
      • Novielli A.
      Rate of chemotherapy related amenorrhea (CRA) associated with adjuvant adriamycin and cytoxan (AC) and adriamycin and cytoxan followed by taxol (AC+T) in early stage breast cancer.
      ;
      • Ball J.
      • Fox K.R.
      Age-associated incidence of chemotherapy-related amenorrhea (CRA) following adjuvant doxorubicin, cyclophosphamide, and paclitaxel (AC/TAXOL) in early-stage breast cancer.
      ;
      • Nabholtz J.M.
      Phase III trial comparing TAC (docetaxel, doxorubicin, cyclophosphamide) with FAC (5-fluorouracil, doxorubicin, cyclophosphamide) in the adjuvant treatment of node positive breast cancer (BC) patients: interim analysis of the BCIRG 001 study.
      ;
      • Borde F.
      • Chapelle-Marcillac I.
      • Fumoleau P.
      • Hery M.
      • Bonneterre J.
      • Kerbrat P.
      • Namer M.
      • Fargeot P.
      • Roche H.
      Role of chemo-induced amenorrhea in premenopausal, node-positive, operable breast cancer patients: 9-year follow-up results of French Adjuvant Study Group (FASG) data base.
      ;
      • Fornier M.N.
      • Modi S.
      • Panageas K.S.
      • Norton L.
      • Hudis C.
      Incidence of chemotherapy-induced, long-term amenorrhea in patients with breast carcinoma age 40 years and younger after adjuvant anthracycline and taxane.
      ;
      • Kramer R.
      • Tham Y.L.
      • Sexton K.
      • Friedman L.
      • Weiss H.
      Chemotherapy-induced amenorrhea is increased in patients treated with adjuvant doxorubicin and cyclophosphamide (AC) followed by a taxane (T).
      ;
      • Martin M.
      • Pienkowski T.
      • Mackey J.
      • Pawlicki M.
      • Guastalla J.P.
      • Weaver C.
      • Tomiak E.
      • Al-Tweigeri T.
      • Chap L.
      • Juhos E.
      • Guevin R.
      • Howell A.
      • Fornander T.
      • Hainsworth J.
      • Coleman R.
      • Vinholes J.
      • Modiano M.
      • Pinter T.
      • Tang S.C.
      • Colwell B.
      • Prady C.
      • Provencher L.
      • Walde D.
      • Rodriguez-Lescure A.
      • Hugh J.
      • Loret C.
      • Rupin M.
      • Blitz S.
      • Jacobs P.
      • Murawsky M.
      • Riva A.
      • Vogel C.
      Adjuvant docetaxel for node-positive breast cancer.
      ;
      • Basser R.L.
      • O'Neill A.
      • Martinelli G.
      • Green M.D.
      • Peccatori F.
      • Cinieri S.
      • Coates A.S.
      • Gelber R.D.
      • Aebi S.
      • Castiglione-Gertsch M.
      • Viale G.
      • Price K.N.
      • Goldhirsch A.
      Multicycle dose-intensive chemotherapy for women with high-risk primary breast cancer: results of International Breast Cancer Study Group Trial 15-95.
      ;
      • Colleoni M.
      • Gelber S.
      • Goldhirsch A.
      • Aebi S.
      • Castiglione-Gertsch M.
      • Price K.N.
      • Coates A.S.
      • Gelber R.D.
      Tamoxifen after adjuvant chemotherapy for premenopausal women with lymph node-positive breast cancer: International Breast Cancer Study Group Trial 13-93.
      ;
      • Kil W.J.
      • Ahn S.D.
      • Shin S.S.
      • Lee S.W.
      • Choi E.K.
      • Kim J.H.
      • Son B.H.
      • Ahn S.H.
      • Kim W.K.
      • Kim S.B.
      Treatment-induced menstrual changes in very young (<35 years old) breast cancer patients.
      ;
      • Petrek J.A.
      • Naughton M.J.
      • Case L.D.
      • Paskett E.D.
      • Naftalis E.Z.
      • Singletary S.E.
      • Sukumvanich P.
      Incidence, time course, and determinants of menstrual bleeding after breast cancer treatment: a prospective study.
      ;
      • Tham Y.L.
      • Sexton K.
      • Weiss H.
      • Elledge R.
      • Friedman L.C.
      • Kramer R.
      The rates of chemotherapy-induced amenorrhea in patients treated with adjuvant doxorubicin and cyclophosphamide followed by a taxane.
      ;
      • Reh A.
      • Oktem O.
      • Oktay K.
      Impact of breast cancer chemotherapy on ovarian reserve: a prospective observational analysis by menstrual history and ovarian reserve markers.
      ;
      • Zekri J.M.
      • El-Helw L.M.
      • Purohit O.P.
      • Hatton M.Q.
      • Coleman R.E.
      Epirubicin/vinorelbine adjuvant chemotherapy in young women with breast cancer is associated with preservation of menstrual function.
      ;
      • Han H.S.
      • Ro J.
      • Lee K.S.
      • Nam B.H.
      • Seo J.A.
      • Lee D.H.
      • Lee H.
      • Lee E.S.
      • Kang H.S.
      • Kim S.W.
      Analysis of chemotherapy-induced amenorrhea rates by three different anthracycline and taxane containing regimens for early breast cancer.
      ;
      • Lee S.
      • Kil W.J.
      • Chun M.
      • Jung Y.S.
      • Kang S.Y.
      • Kang S.H.
      • Oh Y.T.
      Chemotherapy-related amenorrhea in premenopausal women with breast cancer.
      ;
      • Swain S.M.
      • Land S.R.
      • Ritter M.W.
      • Costantino J.P.
      • Cecchini R.S.
      • Mamounas E.P.
      • Wolmark N.
      • Ganz P.A.
      Amenorrhea in premenopausal women on the doxorubicin-and-cyclophosphamide-followed-by-docetaxel arm of NSABP B-30 trial.
      ;
      • Abusief M.E.
      • Missmer S.A.
      • Ginsburg E.S.
      • Weeks J.C.
      • Partridge A.H.
      The effects of paclitaxel, dose density, and trastuzumab on treatment-related amenorrhea in premenopausal women with breast cancer.
      ;
      • Perez-Fidalgo J.A.
      • Rosello S.
      • Garcia-Garre E.
      • Jorda E.
      • Martin-Martorell P.
      • Bermejo B.
      • Chirivella I.
      • Guzman C.
      • Lluch A.
      Incidence of chemotherapy-induced amenorrhea in hormone-sensitive breast cancer patients: the impact of addition of taxanes to anthracycline-based regimens.
      ;
      • Sukumvanich P.
      • Case L.D.
      • Van Zee K.
      • Singletary S.E.
      • Paskett E.D.
      • Petrek J.A.
      • Naftalis E.
      • Naughton M.J.
      Incidence and time course of bleeding after long-term amenorrhea after breast cancer treatment: a prospective study.
      ;
      • Ganz P.A.
      • Land S.R.
      • Geyer Jr., C.E.
      • Cecchini R.S.
      • Costantino J.P.
      • Pajon E.R.
      • Fehrenbacher L.
      • Atkins J.N.
      • Polikoff J.A.
      • Vogel V.G.
      • Erban J.K.
      • Livingston R.B.
      • Perez E.A.
      • Mamounas E.P.
      • Wolmark N.
      • Swain S.M.
      Menstrual history and quality-of-life outcomes in women with node-positive breast cancer treated with adjuvant therapy on the NSABP B-30 trial.
      ;
      • Najafi S.
      • Djavid G.E.
      • Mehrdad N.
      • Rajaii E.
      • Alavi N.
      • Olfatbakhsh A.
      • Najafi M.
      • Bahrami A.
      • Heidari K.
      Taxane-based regimens as a risk factor for chemotherapy-induced amenorrhea.
      ;
      • Okanami Y.
      • Ito Y.
      • Watanabe C.
      • Iijima K.
      • Iwase T.
      • Tokudome N.
      • Takahashi S.
      • Hatake K.
      Incidence of chemotherapy-induced amenorrhea in premenopausal patients with breast cancer following adjuvant anthracycline and taxane.
      ;
      • Koga C.
      • Akiyoshi S.
      • Ishida M.
      • Nakamura Y.
      • Ohno S.
      • Tokunaga E.
      Chemotherapy-induced amenorrhea and the resumption of menstruation in premenopausal women with hormone receptor-positive early breast cancer.
      ;
      • Vriens I.J.
      • De Bie A.J.
      • Aarts M.J.
      • de Boer M.
      • van Hellemond I.E.
      • Roijen J.H.
      • van Golde R.J.
      • Voogd A.C.
      • Tjan-Heijnen V.C.
      The correlation of age with chemotherapy-induced ovarian function failure in breast cancer patients.
      11,7734–65.3–9844–96
      Taxane-containing regimen

      • Stone E.R.
      • Slack R.S.
      • Novielli A.
      Rate of chemotherapy related amenorrhea (CRA) associated with adjuvant adriamycin and cytoxan (AC) and adriamycin and cytoxan followed by taxol (AC+T) in early stage breast cancer.
      ;
      • Ball J.
      • Fox K.R.
      Age-associated incidence of chemotherapy-related amenorrhea (CRA) following adjuvant doxorubicin, cyclophosphamide, and paclitaxel (AC/TAXOL) in early-stage breast cancer.
      ;
      • Nabholtz J.M.
      Phase III trial comparing TAC (docetaxel, doxorubicin, cyclophosphamide) with FAC (5-fluorouracil, doxorubicin, cyclophosphamide) in the adjuvant treatment of node positive breast cancer (BC) patients: interim analysis of the BCIRG 001 study.
      ;
      • Fornier M.N.
      • Modi S.
      • Panageas K.S.
      • Norton L.
      • Hudis C.
      Incidence of chemotherapy-induced, long-term amenorrhea in patients with breast carcinoma age 40 years and younger after adjuvant anthracycline and taxane.
      ;
      • Kramer R.
      • Tham Y.L.
      • Sexton K.
      • Friedman L.
      • Weiss H.
      Chemotherapy-induced amenorrhea is increased in patients treated with adjuvant doxorubicin and cyclophosphamide (AC) followed by a taxane (T).
      ;
      • Martin M.
      • Pienkowski T.
      • Mackey J.
      • Pawlicki M.
      • Guastalla J.P.
      • Weaver C.
      • Tomiak E.
      • Al-Tweigeri T.
      • Chap L.
      • Juhos E.
      • Guevin R.
      • Howell A.
      • Fornander T.
      • Hainsworth J.
      • Coleman R.
      • Vinholes J.
      • Modiano M.
      • Pinter T.
      • Tang S.C.
      • Colwell B.
      • Prady C.
      • Provencher L.
      • Walde D.
      • Rodriguez-Lescure A.
      • Hugh J.
      • Loret C.
      • Rupin M.
      • Blitz S.
      • Jacobs P.
      • Murawsky M.
      • Riva A.
      • Vogel C.
      Adjuvant docetaxel for node-positive breast cancer.
      ;
      • Samuelkutty S
      • G O.
      • Mohrmann S
      • Hille S
      • Zwiefel K
      • Schuett G
      • Nitz U.
      Chemotherapy-induced amenorrhea (CIA) in patients treated with adjuvant CEF/CMF or EC/docetaxel: analysis from a phase III randomized EC/Doc Trial.
      ;
      • Petrek J.A.
      • Naughton M.J.
      • Case L.D.
      • Paskett E.D.
      • Naftalis E.Z.
      • Singletary S.E.
      • Sukumvanich P.
      Incidence, time course, and determinants of menstrual bleeding after breast cancer treatment: a prospective study.
      ;
      • Tham Y.L.
      • Sexton K.
      • Weiss H.
      • Elledge R.
      • Friedman L.C.
      • Kramer R.
      The rates of chemotherapy-induced amenorrhea in patients treated with adjuvant doxorubicin and cyclophosphamide followed by a taxane.
      ;
      • Reh A.
      • Oktem O.
      • Oktay K.
      Impact of breast cancer chemotherapy on ovarian reserve: a prospective observational analysis by menstrual history and ovarian reserve markers.
      ;
      • Han H.S.
      • Ro J.
      • Lee K.S.
      • Nam B.H.
      • Seo J.A.
      • Lee D.H.
      • Lee H.
      • Lee E.S.
      • Kang H.S.
      • Kim S.W.
      Analysis of chemotherapy-induced amenorrhea rates by three different anthracycline and taxane containing regimens for early breast cancer.
      ;
      • Lee S.
      • Kil W.J.
      • Chun M.
      • Jung Y.S.
      • Kang S.Y.
      • Kang S.H.
      • Oh Y.T.
      Chemotherapy-related amenorrhea in premenopausal women with breast cancer.
      ;
      • Swain S.M.
      • Land S.R.
      • Ritter M.W.
      • Costantino J.P.
      • Cecchini R.S.
      • Mamounas E.P.
      • Wolmark N.
      • Ganz P.A.
      Amenorrhea in premenopausal women on the doxorubicin-and-cyclophosphamide-followed-by-docetaxel arm of NSABP B-30 trial.
      ;
      • Abusief M.E.
      • Missmer S.A.
      • Ginsburg E.S.
      • Weeks J.C.
      • Partridge A.H.
      The effects of paclitaxel, dose density, and trastuzumab on treatment-related amenorrhea in premenopausal women with breast cancer.
      ;
      • Perez-Fidalgo J.A.
      • Rosello S.
      • Garcia-Garre E.
      • Jorda E.
      • Martin-Martorell P.
      • Bermejo B.
      • Chirivella I.
      • Guzman C.
      • Lluch A.
      Incidence of chemotherapy-induced amenorrhea in hormone-sensitive breast cancer patients: the impact of addition of taxanes to anthracycline-based regimens.
      ;
      • Su H.I.
      • Sammel M.D.
      • Green J.
      • Velders L.
      • Stankiewicz C.
      • Matro J.
      • Freeman E.W.
      • Gracia C.R.
      • DeMichele A.
      Antimullerian hormone and inhibin B are hormone measures of ovarian function in late reproductive-aged breast cancer survivors.
      ;
      • Sukumvanich P.
      • Case L.D.
      • Van Zee K.
      • Singletary S.E.
      • Paskett E.D.
      • Petrek J.A.
      • Naftalis E.
      • Naughton M.J.
      Incidence and time course of bleeding after long-term amenorrhea after breast cancer treatment: a prospective study.
      ;
      • Ganz P.A.
      • Land S.R.
      • Geyer Jr., C.E.
      • Cecchini R.S.
      • Costantino J.P.
      • Pajon E.R.
      • Fehrenbacher L.
      • Atkins J.N.
      • Polikoff J.A.
      • Vogel V.G.
      • Erban J.K.
      • Livingston R.B.
      • Perez E.A.
      • Mamounas E.P.
      • Wolmark N.
      • Swain S.M.
      Menstrual history and quality-of-life outcomes in women with node-positive breast cancer treated with adjuvant therapy on the NSABP B-30 trial.
      ;
      • Najafi S.
      • Djavid G.E.
      • Mehrdad N.
      • Rajaii E.
      • Alavi N.
      • Olfatbakhsh A.
      • Najafi M.
      • Bahrami A.
      • Heidari K.
      Taxane-based regimens as a risk factor for chemotherapy-induced amenorrhea.
      ;
      • Okanami Y.
      • Ito Y.
      • Watanabe C.
      • Iijima K.
      • Iwase T.
      • Tokudome N.
      • Takahashi S.
      • Hatake K.
      Incidence of chemotherapy-induced amenorrhea in premenopausal patients with breast cancer following adjuvant anthracycline and taxane.
      ;
      • Zhou W.
      • Ding Q.
      • Liang X.
      • He Z.
      • Zha X.
      • Liu X.
      • Wang S.
      The risk of amenorrhea is related to chemotherapy-induced leucopenia in breast cancer patients receiving epirubicin and taxane based chemotherapy.
      ;
      • Meng K.X.
      • Tian W.
      • Zhou M.Q.
      • Chen H.L.
      • Deng Y.C.
      Impact of chemotherapy-induced amenorrhea in breast cancer patients: the evaluation of ovarian function by menstrual history and hormonal levels.
      ;
      • Tiong V.
      • Rozita A.M.
      • Taib N.A.
      • Yip C.H.
      • Ng C.H.
      Incidence of chemotherapy-induced ovarian failure in premenopausal women undergoing chemotherapy for breast cancer.
      ;
      • Ruddy K.J.
      • Guo H.
      • Barry W.
      • Dang C.T.
      • Yardley D.A.
      • Moy B.
      • Marcom P.K.
      • Albain K.S.
      • Rugo H.S.
      • Ellis M.J.
      • Shapira I.
      • Wolff A.C.
      • Carey L.A.
      • Overmoyer B.A.
      • Hudis C.
      • Krop I.E.
      • Burstein H.J.
      • Winer E.P.
      • Partridge A.H.
      • Tolaney S.M.
      Chemotherapy-related amenorrhea after adjuvant paclitaxel-trastuzumab (APT Trial).
      ;
      • Koga C.
      • Akiyoshi S.
      • Ishida M.
      • Nakamura Y.
      • Ohno S.
      • Tokunaga E.
      Chemotherapy-induced amenorrhea and the resumption of menstruation in premenopausal women with hormone receptor-positive early breast cancer.
      ;
      • Vriens I.J.
      • De Bie A.J.
      • Aarts M.J.
      • de Boer M.
      • van Hellemond I.E.
      • Roijen J.H.
      • van Golde R.J.
      • Voogd A.C.
      • Tjan-Heijnen V.C.
      The correlation of age with chemotherapy-induced ovarian function failure in breast cancer patients.
      41814–86–10017–100
      AC = anthracycline; CEF or FEC = cyclophosphamide, epirubicin and 5-fluorouracil; CMF = cyclophosphamide, methotrexate and 5-fluorouracil.

      Alkylating regimens

      CMF

      A typical chemotherapy regime for adjuvant treatment of breast cancer is CMF (cyclophosphamide, methotrexate and 5-fluorouracil). According to 24 studies in the literature, the incidence of amenorrhoea after the CMF regime ranges from 0% (
      • Bianco A.R.
      • Del Mastro L.
      • Gallo C.
      • Perrone F.
      • Matano E.
      • Pagliarulo C.
      • De Placido S.
      Prognostic role of amenorrhea induced by adjuvant chemotherapy in premenopausal patients with early breast cancer.
      ) to 97% (
      • Richards M.A.
      • O'Reilly S.M.
      • Howell A.
      • George W.D.
      • Fentiman I.S.
      • Chaudary M.A.
      • Crowther D.
      • Rubens R.D.
      Adjuvant cyclophosphamide, methotrexate, and fluorouracil in patients with axillary node-positive breast cancer: an update of the Guy's/Manchester trial.
      ). The differences in the age of the patients enrolled, duration of therapy and length of the follow-up period are probably responsible for the observed variations in the reported amenorrhoea rates among the studies.
      • Bines J.
      • Oleske D.M.
      • Cobleigh M.A.
      Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer.
      reported that ovarian failure incidence after the CMF regime is lower in patients younger than 40 compared with those aged 40 and over (40% versus 76%, respectively). The amenorrhoea risk is higher in older patients because of age-related decline in their ovarian reserve. As a striking example, none of the 25 patients under the age of 35 developed amenorrhoea after six courses of CMF, whereas 33.3% and 80% of the patients aged 36–40 and 41–45 years, respectively, developed amenorrhoea after receiving the same regimen (
      • Bianco A.R.
      • Del Mastro L.
      • Gallo C.
      • Perrone F.
      • Matano E.
      • Pagliarulo C.
      • De Placido S.
      Prognostic role of amenorrhea induced by adjuvant chemotherapy in premenopausal patients with early breast cancer.
      ). It was reported in another study that completion of six courses of the CMF chemotherapy regimen was associated with 0, 19–38 and 76–96% risk of amenorrhoea in breast cancer patients at ages <30, 30–40 and >40, respectively (
      • Partridge A.
      • Gelber S.
      • Gelber R.D.
      • Castiglione-Gertsch M.
      • Goldhirsch A.
      • Winer E.
      Age of menopause among women who remain premenopausal following treatment for early breast cancer: long-term results from International Breast Cancer Study Group Trials V and VI.
      ).

      CEF or FEC

      Cyclophosphamide combined with epirubicine and 5-fluorouracil (CEF or FEC) is another regimen used in the treatment of breast cancer.
      • Parulekar W.R.
      • Day A.G.
      • Ottaway J.A.
      • Shepherd L.E.
      • Trudeau M.E.
      • Bramwell V.
      • Levine M.
      • Pritchard K.I.
      Incidence and prognostic impact of amenorrhea during adjuvant therapy in high-risk premenopausal breast cancer: analysis of a National Cancer Institute of Canada Clinical Trials Group Study – NCIC CTG MA.5.
      compared CEF with CMF in premenopausal patients with breast cancer for the risk of chemotherapy-induced amenorrhoea (CIA) and found that the risk appears to be higher in patients treated with the CEF regime (76% versus 64%, respectively; relative risk 1.2; 95% confidence interval [CI] 1.0–1.3). Furthermore, although the cumulative dose of cyclophosphamide was higher in the CMF treatment arm at 6 months post-random assignment, there was no difference at 12 months. In oestrogen receptor positive (ER+) subgroup analysis, relapse-free survival and overall survival were significantly associated with amenorrhoea at 12 months (respectively, hazard ratio 0.51, 95% CI 0.32–0.82, P = 0.005; hazard ratio 0.40, 95% CI 0.22–0.72, P = 0.002) (
      • Parulekar W.R.
      • Day A.G.
      • Ottaway J.A.
      • Shepherd L.E.
      • Trudeau M.E.
      • Bramwell V.
      • Levine M.
      • Pritchard K.I.
      Incidence and prognostic impact of amenorrhea during adjuvant therapy in high-risk premenopausal breast cancer: analysis of a National Cancer Institute of Canada Clinical Trials Group Study – NCIC CTG MA.5.
      ). Other studies also found comparable rates of amenorrhoea after CMF and CEF regimens (
      • Di Cosimo S.
      • Alimonti A.
      • Ferretti G.
      • Sperduti I.
      • Carlini P.
      • Papaldo P.
      • Fabi A.
      • Gelibter A.
      • Ciccarese M.
      • Giannarelli D.
      • Mandala M.
      • Milella M.
      • Ruggeri E.M.
      • Cognetti F.
      Incidence of chemotherapy-induced amenorrhea depending on the timing of treatment by menstrual cycle phase in women with early breast cancer.
      ;
      • Goodwin P.J.
      • Ennis M.
      • Pritchard K.I.
      • Trudeau M.
      • Hood N.
      Risk of menopause during the first year after breast cancer diagnosis.
      ;
      • Levine M.N.
      • Bramwell V.H.
      • Pritchard K.I.
      • Norris B.D.
      • Shepherd L.E.
      • Abu-Zahra H.
      • Findlay B.
      • Warr D.
      • Bowman D.
      • Myles J.
      • Arnold A.
      • Vandenberg T.
      • MacKenzie R.
      • Robert J.
      • Ottaway J.
      • Burnell M.
      • Williams C.K.
      • Tu D.
      Randomized trial of intensive cyclophosphamide, epirubicin, and fluorouracil chemotherapy compared with cyclophosphamide, methotrexate, and fluorouracil in premenopausal women with node-positive breast cancer. National Cancer Institute of Canada Clinical Trials Group.
      ;
      • Vanhuyse M.
      • Fournier C.
      • Bonneterre J.
      Chemotherapy-induced amenorrhea: influence on disease-free survival and overall survival in receptor-positive premenopausal early breast cancer patients.
      ). If the amenorrhoea rate is calculated based on the eight studies including 1190 breast cancer patients for the occurrence of CEF-related amenorrhoea, the risk appears to be around 51% after six courses at 12 months of follow-up (
      • Berliere M.
      • Dalenc F.
      • Malingret N.
      • Vindevogel A.
      • Piette P.
      • Roche H.
      • Donnez J.
      • Symann M.
      • Kerger J.
      • Machiels J.P.
      Incidence of reversible amenorrhea in women with breast cancer undergoing adjuvant anthracycline-based chemotherapy with or without docetaxel.
      ;
      • Di Cosimo S.
      • Alimonti A.
      • Ferretti G.
      • Sperduti I.
      • Carlini P.
      • Papaldo P.
      • Fabi A.
      • Gelibter A.
      • Ciccarese M.
      • Giannarelli D.
      • Mandala M.
      • Milella M.
      • Ruggeri E.M.
      • Cognetti F.
      Incidence of chemotherapy-induced amenorrhea depending on the timing of treatment by menstrual cycle phase in women with early breast cancer.
      ;
      • Goodwin P.J.
      • Ennis M.
      • Pritchard K.I.
      • Trudeau M.
      • Hood N.
      Risk of menopause during the first year after breast cancer diagnosis.
      ;
      • Levine M.N.
      • Bramwell V.H.
      • Pritchard K.I.
      • Norris B.D.
      • Shepherd L.E.
      • Abu-Zahra H.
      • Findlay B.
      • Warr D.
      • Bowman D.
      • Myles J.
      • Arnold A.
      • Vandenberg T.
      • MacKenzie R.
      • Robert J.
      • Ottaway J.
      • Burnell M.
      • Williams C.K.
      • Tu D.
      Randomized trial of intensive cyclophosphamide, epirubicin, and fluorouracil chemotherapy compared with cyclophosphamide, methotrexate, and fluorouracil in premenopausal women with node-positive breast cancer. National Cancer Institute of Canada Clinical Trials Group.
      ;
      • Parulekar W.R.
      • Day A.G.
      • Ottaway J.A.
      • Shepherd L.E.
      • Trudeau M.E.
      • Bramwell V.
      • Levine M.
      • Pritchard K.I.
      Incidence and prognostic impact of amenorrhea during adjuvant therapy in high-risk premenopausal breast cancer: analysis of a National Cancer Institute of Canada Clinical Trials Group Study – NCIC CTG MA.5.
      ;
      • Vanhuyse M.
      • Fournier C.
      • Bonneterre J.
      Chemotherapy-induced amenorrhea: influence on disease-free survival and overall survival in receptor-positive premenopausal early breast cancer patients.
      ;
      • Venturini M.
      • Del Mastro L.
      • Aitini E.
      • Baldini E.
      • Caroti C.
      • Contu A.
      • Testore F.
      • Brema F.
      • Pronzato P.
      • Cavazzini G.
      • Sertoli M.R.
      • Canavese G.
      • Rosso R.
      • Bruzzi P.
      Dose-dense adjuvant chemotherapy in early breast cancer patients: results from a randomized trial.
      ;
      • Zhou W.B.
      • Yin H.
      • Liu X.A.
      • Zha X.M.
      • Chen L.
      • Dai J.C.
      • Tao A.D.
      • Ma J.J.
      • Ling L.J.
      • Wang S.
      Incidence of chemotherapy-induced amenorrhea associated with epirubicin, docetaxel and navelbine in younger breast cancer patients.
      ). In the multicentre PACS 01 trial,
      • Berliere M.
      • Dalenc F.
      • Malingret N.
      • Vindevogel A.
      • Piette P.
      • Roche H.
      • Donnez J.
      • Symann M.
      • Kerger J.
      • Machiels J.P.
      Incidence of reversible amenorrhea in women with breast cancer undergoing adjuvant anthracycline-based chemotherapy with or without docetaxel.
      indicated comparable amenorrhoea rates between patients treated with six cycles of FEC (6FEC) versus three cycles of FEC followed by three cycles of docetaxel (3FEC/3D) at the end of chemotherapy (93% versus 92.8%). On the other hand, at 1 year after completion of chemotherapy, returning hormone concentrations to premenopausal levels and resumption of menses were more frequently observed in the 3FEC/3D arm than in the 6FEC arm (respectively, 43% versus 29%, 35.5% versus 23.7%) (
      • Berliere M.
      • Dalenc F.
      • Malingret N.
      • Vindevogel A.
      • Piette P.
      • Roche H.
      • Donnez J.
      • Symann M.
      • Kerger J.
      • Machiels J.P.
      Incidence of reversible amenorrhea in women with breast cancer undergoing adjuvant anthracycline-based chemotherapy with or without docetaxel.
      ).
      • Zhou W.B.
      • Yin H.
      • Liu X.A.
      • Zha X.M.
      • Chen L.
      • Dai J.C.
      • Tao A.D.
      • Ma J.J.
      • Ling L.J.
      • Wang S.
      Incidence of chemotherapy-induced amenorrhea associated with epirubicin, docetaxel and navelbine in younger breast cancer patients.
      showed different amenorrhoea rates between various chemotherapy protocols; 44.87% for FEC and 30.30% for TE (docetaxel–epirubicin), and 23.08% for NE (navelbine–epirubicin). Furthermore, although significant differences were not found between the FEC and TE groups, the amenorrhoea rate was significantly higher in the FEC compared with the NE regimen (
      • Zhou W.B.
      • Yin H.
      • Liu X.A.
      • Zha X.M.
      • Chen L.
      • Dai J.C.
      • Tao A.D.
      • Ma J.J.
      • Ling L.J.
      • Wang S.
      Incidence of chemotherapy-induced amenorrhea associated with epirubicin, docetaxel and navelbine in younger breast cancer patients.
      ).

      Anthracyclines

      Anthracyclines act as inhibitors of the nuclear topoisomerase II enzyme, promoting chromosome disentanglement. They therefore prevent topoisomerase II DNA complex formation, leading to the accumulation of DNA fragments and eventual death of the cell (
      • Spears N.
      • Lopes F.
      • Stefansdottir A.
      • Rossi V.
      • De Felici M.
      • Anderson R.A.
      • Klinger F.G.
      Ovarian damage from chemotherapy and current approaches to its protection.
      ). Cell viability decreases with mitochondrial damage and free radical formation. Clinically used anthracyclines are doxorubicin, daunorubicin, epirubicin and idarubicin. The gonadal effects of doxorubicin have been more thoroughly investigated than other members of the family, using several animal models (
      • Spears N.
      • Lopes F.
      • Stefansdottir A.
      • Rossi V.
      • De Felici M.
      • Anderson R.A.
      • Klinger F.G.
      Ovarian damage from chemotherapy and current approaches to its protection.
      ) and two human studies (
      • Li F.
      • Turan V.
      • Lierman S.
      • Cuvelier C.
      • De Sutter P.
      • Oktay K.
      Sphingosine-1-phosphate prevents chemotherapy-induced human primordial follicle death.
      ;
      • Soleimani R.
      • Heytens E.
      • Darzynkiewicz Z.
      • Oktay K.
      Mechanisms of chemotherapy-induced human ovarian aging: double strand DNA breaks and microvascular compromise.
      ). These studies show that doxorubicin reduces ovulation rate, blastocyst formation and reproductive lifespan and specifically causes granulosa cell apoptosis.
      Anthracycline-based regimens are less harmful than alkylating regimens due to the lower total cyclophosphamide dosages. Therefore, amenorrhoea rates are lower than the classic CMF regimen. For example, 74% of patients treated with CMF developed amenorrhoea in contrast to 42% receiving the AC regimen (adriamycin [doxorubicin] and cyclophosphamide) (
      • Anderko L.
      • Roffenbender J.S.
      • Goetzel R.Z.
      • Howard J.
      • Millard F.
      • Wildenhaus K.
      • Desantis C.
      • Novelli W.
      Promoting prevention through the Affordable Care Act: workplace wellness.
      ). Similarly,
      • Petrek J.A.
      • Naughton M.J.
      • Case L.D.
      • Paskett E.D.
      • Naftalis E.Z.
      • Singletary S.E.
      • Sukumvanich P.
      Incidence, time course, and determinants of menstrual bleeding after breast cancer treatment: a prospective study.
      found that amenorrhoea rates in patients treated with AC and CMF regimens were 53% and 83%, respectively. On the other hand,
      • Hortobagyi G.N.
      • Buzdar A.U.
      • Marcus C.E.
      • Smith T.L.
      Immediate and long-term toxicity of adjuvant chemotherapy regimens containing doxorubicin in trials at M.D. Anderson Hospital and Tumor Institute.
      reported that amenorrhoea risk in breast cancer patients treated with anthracycline-based regimens was 80%. In addition, no patients under 30 years of age experienced menstrual abnormalities, whereas 96% of those aged 40–49 developed amenorrhoea, which was permanent for most women over 40 but was reversible for 50% of patients under 40 years of age. Subsequent studies confirmed that the AC regimen did not cause amenorrhoea in patients younger than 35 during 2 years of follow-up (
      • Sukumvanich P.
      • Case L.D.
      • Van Zee K.
      • Singletary S.E.
      • Paskett E.D.
      • Petrek J.A.
      • Naftalis E.
      • Naughton M.J.
      Incidence and time course of bleeding after long-term amenorrhea after breast cancer treatment: a prospective study.
      ) or caused amenorrhoea in only 13% of cases under the age of 40 (
      • Abusief M.E.
      • Missmer S.A.
      • Ginsburg E.S.
      • Weeks J.C.
      • Partridge A.H.
      The effects of paclitaxel, dose density, and trastuzumab on treatment-related amenorrhea in premenopausal women with breast cancer.
      ).
      • Kil W.J.
      • Ahn S.D.
      • Shin S.S.
      • Lee S.W.
      • Choi E.K.
      • Kim J.H.
      • Son B.H.
      • Ahn S.H.
      • Kim W.K.
      • Kim S.B.
      Treatment-induced menstrual changes in very young (<35 years old) breast cancer patients.
      studied menstrual changes in breast cancer patients under 35 years of age in a retrospective study. They found similar amenorrhoea occurrences (25 [31.3%] in those treated with CMF, and 34 [42.5%] in anthracycline-based regimens [adriamycin, AD]; P = 0.142) and menstruation resumed in 83.1% of patients (80% of those treated with CMF and 85.3% with AD). The study indicates that CMF and AD protocols have similar amenorrhoea or recovery rates within 54 months of the follow-up period (
      • Kil W.J.
      • Ahn S.D.
      • Shin S.S.
      • Lee S.W.
      • Choi E.K.
      • Kim J.H.
      • Son B.H.
      • Ahn S.H.
      • Kim W.K.
      • Kim S.B.
      Treatment-induced menstrual changes in very young (<35 years old) breast cancer patients.
      ).

      Taxanes

      Taxanes (paclitaxel and docetaxel) stabilize mitotic spindles by attaching to tubulin within existing microtubules (
      • Kim Y.Y.
      • Kim W.O.
      • Liu H.C.
      • Rosenwaks Z.
      • Kim J.W.
      • Ku S.Y.
      Effects of paclitaxel and cisplatin on in vitro ovarian follicle development.
      ;
      • Trudeau M.E.
      Docetaxel: a review of its pharmacology and clinical activity.
      ). As a result, the normal microtubule breakdown during cell division is impaired. In addition, it leads to activation of the mitotic checkpoint, which induces reversion to the G-phase of the cell cycle and apoptosis (mitotic arrest). Taxanes can also harm the oocyte as they prevent depolymerization of the meiotic spindles during meiosis I and II (
      • Mailhes J.B.
      • Carabatsos M.J.
      • Young D.
      • London S.N.
      • Bell M.
      • Albertini D.F.
      Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes.
      ).

      Paclitaxel

      According to the existing data, the adverse effects of paclitaxel on ovaries are still controversial. For instance, one study comparing the CIA incidences between ACT (doxorubicin and cyclophosphamide followed by a taxane) and AC (anthracycline) regimens demonstrated that the CIA incidence was higher in the ACT than the AC alone regimen (64% versus 55%). Furthermore, the study also found amenorrhoea rates were higher (82% versus 55%) and more likely to be irreversible in older women (over the age of 40) (
      • Tham Y.L.
      • Sexton K.
      • Weiss H.
      • Elledge R.
      • Friedman L.C.
      • Kramer R.
      The rates of chemotherapy-induced amenorrhea in patients treated with adjuvant doxorubicin and cyclophosphamide followed by a taxane.
      ). In a prospective observational study,
      • Reh A.
      • Oktem O.
      • Oktay K.
      Impact of breast cancer chemotherapy on ovarian reserve: a prospective observational analysis by menstrual history and ovarian reserve markers.
      found no significant difference in amenorrhoea rates within 6 months of follow-up after chemotherapy (ACT group, 29%; AC group, 41.7%). However, within the second follow-up period (28 months after chemotherapy; mean), they found higher amenorrhoea rates in the ACT patients and suggested that gonadotoxicity of paclitaxel may manifest itself at more extended follow-up periods (35.7% in the ACT group versus 9.1% in the AC group) (
      • Reh A.
      • Oktem O.
      • Oktay K.
      Impact of breast cancer chemotherapy on ovarian reserve: a prospective observational analysis by menstrual history and ovarian reserve markers.
      ). On the other hand, another study with a similar design obtained the opposite findings by showing that while taxane use was related to a higher amenorrhoea risk in the first year of follow-up, after 2 years, AC and ACT regimens yielded similar amenorrhoea rates (66.7% versus 73.3%, respectively) (
      • Han H.S.
      • Ro J.
      • Lee K.S.
      • Nam B.H.
      • Seo J.A.
      • Lee D.H.
      • Lee H.
      • Lee E.S.
      • Kang H.S.
      • Kim S.W.
      Analysis of chemotherapy-induced amenorrhea rates by three different anthracycline and taxane containing regimens for early breast cancer.
      ). Regarding the long-term effects of taxane on the ovary,
      • Okanami Y.
      • Ito Y.
      • Watanabe C.
      • Iijima K.
      • Iwase T.
      • Tokudome N.
      • Takahashi S.
      • Hatake K.
      Incidence of chemotherapy-induced amenorrhea in premenopausal patients with breast cancer following adjuvant anthracycline and taxane.
      showed that even though the amenorrhoea rates during chemotherapy are significantly higher in the anthracycline + taxane arm than in the anthracycline (93.9% versus 73.6%, P < 0.05), persistent amenorrhoea rates were similar; 24.5% versus 11.8% (P > 0.05) after 3 years of follow-up. The authors concluded that patient age at diagnosis (≤35 versus ≥36) was significantly and independently associated with chemotherapy-related amenorrhoea within long-term follow-up. In addition, time to resume menstruation was longer in patients in the ACT than in the AC group (342 versus 92 days). Similar findings were also obtained in another study in 466 premenopausal breast cancer patients who were treated with AC, ACT and CMF regimens; the likelihood of resumption of menstrual bleeding after 6 months of amenorrhoea exhibited significant variation according to the chemotherapy protocols (P = 0.002; 68% with AC, 57% with ACT and 23% with CMF). Nevertheless, no significant difference was reported between the chemotherapy protocols for recovery after 12 months of amenorrhoea. Among the patients in the CMF group, 10% resumed menstrual bleeding after an initial 2-year period of amenorrhoea, but none had regular menses. The majority of patients treated with CMF continued to have amenorrhoea (84%), followed by ACT (68%) and AC (58%) (
      • Sukumvanich P.
      • Case L.D.
      • Van Zee K.
      • Singletary S.E.
      • Paskett E.D.
      • Petrek J.A.
      • Naftalis E.
      • Naughton M.J.
      Incidence and time course of bleeding after long-term amenorrhea after breast cancer treatment: a prospective study.
      ). The findings of other studies did not indicate an increased risk of amenorrhoea after sequential addition of a taxane to standard adjuvant anthracycline-based chemotherapy compared with historic controls (
      • Fornier M.N.
      • Modi S.
      • Panageas K.S.
      • Norton L.
      • Hudis C.
      Incidence of chemotherapy-induced, long-term amenorrhea in patients with breast carcinoma age 40 years and younger after adjuvant anthracycline and taxane.
      ); or showed that the likelihood of remaining amenorrheic was not statistically different in patients who received ACT versus AC at 33 months of follow-up (OR 1.59; 95% CI 0.8–3.2) (
      • Abusief M.E.
      • Missmer S.A.
      • Ginsburg E.S.
      • Weeks J.C.
      • Partridge A.H.
      The effects of paclitaxel, dose density, and trastuzumab on treatment-related amenorrhea in premenopausal women with breast cancer.
      ). Interestingly, one study even reported that taxane-based chemotherapy was associated with an increased probability of recovery of menses after CIA (
      • Minisini A.M.
      • Menis J.
      • Valent F.
      • Andreetta C.
      • Alessi B.
      • Pascoletti G.
      • Piga A.
      • Fasola G.
      • Puglisi F.
      Determinants of recovery from amenorrhea in premenopausal breast cancer patients receiving adjuvant chemotherapy in the taxane era.
      ).

      Docetaxel

      Docetaxel is a semi-synthetic analogue of paclitaxel. Several clinical trials have provided information about the gonadotoxic potential of docetaxel. One of these trials, mentioned earlier in this review, is PACS 01. The results of the PACS 01 trial showed that although amenorrhoea rates were comparable in the FEC and 3FEC/3D arms (93% versus 92.8%), more patients in the 3FEC/3D arm recovered menses (35.5% versus 23.7%, P < 0.05) at 1 year after completion of chemotherapy. Furthermore, the study revealed that patients under 40 years of age treated with taxane-containing regimens had an increased incidence of reversible amenorrhoea (20.5% versus 10.5%, P = 0.025). By contrast, the incidence of amenorrhoea was high in both groups for women >40 years of age (
      • Berliere M.
      • Dalenc F.
      • Malingret N.
      • Vindevogel A.
      • Piette P.
      • Roche H.
      • Donnez J.
      • Symann M.
      • Kerger J.
      • Machiels J.P.
      Incidence of reversible amenorrhea in women with breast cancer undergoing adjuvant anthracycline-based chemotherapy with or without docetaxel.
      ). The BCIRG 01 trial demonstrated that the CIA rates were lower in the FAC (fluorouracil, doxorubicin and cyclophosphamide) arm than the TAC (docetaxel, doxorubicin and cyclophosphamide) regimen (32.8% versus 51.4%, respectively) (
      • Nabholtz J.M.
      Phase III trial comparing TAC (docetaxel, doxorubicin, cyclophosphamide) with FAC (5-fluorouracil, doxorubicin, cyclophosphamide) in the adjuvant treatment of node positive breast cancer (BC) patients: interim analysis of the BCIRG 001 study.
      ). The NSABP B-30 trial showed that the estimated rate of resumption of menses at 24 months from day 1 of cycle 4 or 9 weeks after start of chemotherapy was 45.3% for women <40 years, 10.9% for women 40–50 and 3.2% for women >50 years treated with doxorubicin and cyclophosphamide followed by docetaxel (
      • Swain S.M.
      • Land S.R.
      • Ritter M.W.
      • Costantino J.P.
      • Cecchini R.S.
      • Mamounas E.P.
      • Wolmark N.
      • Ganz P.A.
      Amenorrhea in premenopausal women on the doxorubicin-and-cyclophosphamide-followed-by-docetaxel arm of NSABP B-30 trial.
      ).
      Also, a subgroup analysis of this trial compared three treatment groups containing sequential doxorubicin (A) and cyclophosphamide (C) followed by docetaxel (T) [AC-T], concurrent TAC or AT, among 2445 patients. Amenorrhoea 12 months after a random assignment was significantly different between groups: 69.8% for AC-T, 57.7% for TAC and 37.9% for AT (P < 0.001). The lowest rate of amenorrhoea was in the AT group without tamoxifen. This can provide useful information for patients who want to preserve their fertility (
      • Ganz P.A.
      • Land S.R.
      • Geyer Jr., C.E.
      • Cecchini R.S.
      • Costantino J.P.
      • Pajon E.R.
      • Fehrenbacher L.
      • Atkins J.N.
      • Polikoff J.A.
      • Vogel V.G.
      • Erban J.K.
      • Livingston R.B.
      • Perez E.A.
      • Mamounas E.P.
      • Wolmark N.
      • Swain S.M.
      Menstrual history and quality-of-life outcomes in women with node-positive breast cancer treated with adjuvant therapy on the NSABP B-30 trial.
      ).
      Another study has shown that the CIA rate was 90.2% with docetaxel/capecitabine (TX) and doxorubicin/cyclophosphamide (AC), 73.5% with AC followed by paclitaxel, and 72.1% with FAC at 1 year, and 66.7%, 73.3% and 58.9%, respectively, at 3 years after completion of chemotherapy. At 1 year, age (P < 0.001) and taxane use (P = 0.002), and after 2 years, age and tamoxifen use were significant factors for CIA in a multivariate analysis (
      • Han H.S.
      • Ro J.
      • Lee K.S.
      • Nam B.H.
      • Seo J.A.
      • Lee D.H.
      • Lee H.
      • Lee E.S.
      • Kang H.S.
      • Kim S.W.
      Analysis of chemotherapy-induced amenorrhea rates by three different anthracycline and taxane containing regimens for early breast cancer.
      ).
      In a follow-up study
      • Najafi S.
      • Djavid G.E.
      • Mehrdad N.
      • Rajaii E.
      • Alavi N.
      • Olfatbakhsh A.
      • Najafi M.
      • Bahrami A.
      • Heidari K.
      Taxane-based regimens as a risk factor for chemotherapy-induced amenorrhea.
      analysed 226 breast cancer patients treated with conventional CMF, anthracycline-based or anthracycline–taxane-based chemotherapy protocols. The CIA rates were 52.5%, 66.7% and 78.7% for CMF, anthracycline and anthracycline–taxane regimens, respectively. Thus, the frequency of CIA was significantly higher in the taxane-based chemotherapy group (P = 0.015), and the two most significant factors for the development of CIA were anthracycline–taxane-based protocols (OR 4.059; 95% CI 1.6–9.8) and age >40 (OR 3.5; 95% CI 1.9–6.6) (
      • Najafi S.
      • Djavid G.E.
      • Mehrdad N.
      • Rajaii E.
      • Alavi N.
      • Olfatbakhsh A.
      • Najafi M.
      • Bahrami A.
      • Heidari K.
      Taxane-based regimens as a risk factor for chemotherapy-induced amenorrhea.
      ). On the contrary, another study including 165 patients treated with FEC (5-fluorouracil, epirubicin and cyclophosphamide), sequential ECT (epirubicin and cyclophosphamide followed by docetaxel), FEC-T (FEC followed by docetaxel) and concurrent ECT regimens found that regimens including taxane did not increase the rate of CIA compared with FEC regimens (P < 0.05 for all) (
      • Zhou W.
      • Ding Q.
      • Liang X.
      • He Z.
      • Zha X.
      • Liu X.
      • Wang S.
      The risk of amenorrhea is related to chemotherapy-induced leucopenia in breast cancer patients receiving epirubicin and taxane based chemotherapy.
      ). One study comparing CAF (tegafur + pirarubicin + ifosfamide) and DTC (docetaxel + pirarubicin + ifosfamide) in 164 women found that DTC carries a significantly higher risk of POF than the CAF regimen (
      • Long J.P.
      • Wan F.
      • Zhang F.
      • Zhou J.
      • Don L.F.
      DTC chemotherapy regimen is associated with higher incidence of premature ovarian failure in women of reproductive age with breast cancer.
      ). A meta-analysis of eight studies with 2124 patients found that the adjusted OR for CIA in patients receiving taxanes versus no taxanes combined with anthracyclines is 1.45 (0.94–2.23), suggesting a weak association between taxanes and the occurrence of amenorrhoea (
      • Zavos A.
      • Valachis A.
      Risk of chemotherapy-induced amenorrhea in patients with breast cancer: a systematic review and meta-analysis.
      ).

      Lapatinib and trastuzumab

      Trastuzumab is an anti-HER2 monoclonal antibody, and lapatinib is a tyrosine kinase inhibitor. Both drugs have been approved for human epidermal growth factor receptor 2 (HER2/neu receptor) positive breast cancer therapy (
      • Lambertini M.
      • Campbell C.
      • Bines J.
      • Korde L.A.
      • Izquierdo M.
      • Fumagalli D.
      • Del Mastro L.
      • Ignatiadis M.
      • Pritchard K.
      • Wolff A.C.
      • Jackisch C.
      • Lang I.
      • Untch M.
      • Smith I.
      • Boyle F.
      • Xu B.
      • Barrios C.H.
      • Baselga J.
      • Moreno-Aspitia A.
      • Piccart M.
      • Gelber R.D.
      • de Azambuja E.
      Adjuvant anti-HER2 therapy, treatment-related amenorrhea, and survival in premenopausal HER2-positive early breast cancer patients.
      ). Almost 25% of breast cancers overexpress HER2/neu receptor (
      • Morris S.R.
      • Carey L.A.
      Trastuzumab and beyond: new possibilities for the treatment of HER2-positive breast cancer.
      ). Trastuzumab improves progression-free survival and overall survival in both metastatic and early-stage HER2/neu positive breast cancers (
      • Piccart-Gebhart M.J.
      • Procter M.
      • Leyland-Jones B.
      • Goldhirsch A.
      • Untch M.
      • Smith I.
      • Gianni L.
      • Baselga J.
      • Bell R.
      • Jackisch C.
      • Cameron D.
      • Dowsett M.
      • Barrios C.H.
      • Steger G.
      • Huang C.S.
      • Andersson M.
      • Inbar M.
      • Lichinitser M.
      • Lang I.
      • Nitz U.
      • Iwata H.
      • Thomssen C.
      • Lohrisch C.
      • Suter T.M.
      • Ruschoff J.
      • Suto T.
      • Greatorex V.
      • Ward C.
      • Straehle C.
      • McFadden E.
      • Dolci M.S.
      • Gelber R.D.
      Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.
      ). In a cross-sectional study evaluating ovarian reserve by measuring AMH concentrations in 25 premenopausal breast cancer survivors, trastuzumab was found to be associated with serum AMH concentration in the survivors with normal cycles, suggesting that this drug does not adversely affect ovarian reserve (
      • Morarji K.
      • McArdle O.
      • Hui K.
      • Gingras-Hill G.
      • Ahmed S.
      • Greenblatt E.M.
      • Warner E.
      • Sridhar S.
      • Ali A.M.F.
      • Azad A.
      • Hodgson D.C.
      Ovarian function after chemotherapy in young breast cancer survivors.
      ). In line with these findings, another study observed no increase in the likelihood of CIA for patients receiving trastuzumab on short-term (12 weeks) or long-term (52 weeks) regimens. In that study of 431 patients after a median follow-up of 33 months (range 6–114 months) the likelihood of remaining amenorrheic was not statistically different in patients who received AC-T versus AC (OR 1.59; 95% CI 0.8–3.2), dose-dense treatment versus treatment every 3 weeks (OR 0.56; 95% CI 0.25–1.3) or AC-T + trastuzumab (OR 0.6;95% CI 0.22–1.61). Amenorrhoea was significantly associated with tamoxifen use and age at diagnosis (
      • Abusief M.E.
      • Missmer S.A.
      • Ginsburg E.S.
      • Weeks J.C.
      • Partridge A.H.
      The effects of paclitaxel, dose density, and trastuzumab on treatment-related amenorrhea in premenopausal women with breast cancer.
      ).
      The APT Trial (Adjuvant Paclitaxel and Trastuzumab for node-negative HER-2positive breast cancer) was a single-arm phase 2 adjuvant study analysing 64 premenopausal patients who had received 12 weeks of paclitaxel and trastuzumab followed by 9 months of trastuzumab monotherapy. The study found an amenorrhoea rate of 28% (95% CI 18–41%) at long term (median 4 years after chemotherapy initiation), which was lower than would be expected with standard adjuvant chemotherapy regimens (
      • Ruddy K.J.
      • Guo H.
      • Barry W.
      • Dang C.T.
      • Yardley D.A.
      • Moy B.
      • Marcom P.K.
      • Albain K.S.
      • Rugo H.S.
      • Ellis M.J.
      • Shapira I.
      • Wolff A.C.
      • Carey L.A.
      • Overmoyer B.A.
      • Hudis C.
      • Krop I.E.
      • Burstein H.J.
      • Winer E.P.
      • Partridge A.H.
      • Tolaney S.M.
      Chemotherapy-related amenorrhea after adjuvant paclitaxel-trastuzumab (APT Trial).
      ).
      In the multicentre, open-label, phase 3 ALTTO (Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation; BIG 2-06) Trial, 2862 HER2 positive premenopausal breast cancer patients were randomized to four groups (lapatinib alone, trastuzumab alone, their sequence or their combination). The rates of treatment-induced amenorrhoea did not show a difference in the four arms, being 72.6%, 74.0%, 72.1% and 74.8%, respectively. However, because the study group did not include patients without anti-HER2 therapies, they could not address the potential gonadotoxic effect of these therapies beyond the chemotherapy (
      • Geyer C.E.
      • Forster J.
      • Lindquist D.
      • Chan S.
      • Romieu C.G.
      • Pienkowski T.
      • Jagiello-Gruszfeld A.
      • Crown J.
      • Chan A.
      • Kaufman B.
      • Skarlos D.
      • Campone M.
      • Davidson N.
      • Berger M.
      • Oliva C.
      • Rubin S.D.
      • Stein S.
      • Cameron D.
      Lapatinib plus capecitabine for HER2-positive advanced breast cancer.
      ;
      • Xu Z.Q.
      • Zhang Y.
      • Li N.
      • Liu P.J.
      • Gao L.
      • Gao X.
      • Tie X.J.
      Efficacy and safety of lapatinib and trastuzumab for HER2-positive breast cancer: a systematic review and meta-analysis of randomised controlled trials.
      ). Although the results of these studies are encouraging, more data are required to reach a definitive conclusion on the gonadal effect of anti-HER2 therapies.

      Tamoxifen

      Tamoxifen selectively modulates oestrogen receptors and demonstrates its anti-oestrogenic actions by competing with oestrogen at its receptor site, and it has been used as a first-line treatment of oestrogen receptor positive breast cancer for almost four decades (
      • Rodriguez-Wallberg K.A.
      • Oktay K.
      Fertility preservation in women with breast cancer.
      ). Despite its long and widespread use, the association between tamoxifen use and the development of amenorrhoea is vague. In the current literature, amenorrhoea rates after tamoxifen treatment vary, and several studies found no significant differences between patients who were and were not treated with tamoxifen (
      • Di Cosimo S.
      • Alimonti A.
      • Ferretti G.
      • Sperduti I.
      • Carlini P.
      • Papaldo P.
      • Fabi A.
      • Gelibter A.
      • Ciccarese M.
      • Giannarelli D.
      • Mandala M.
      • Milella M.
      • Ruggeri E.M.
      • Cognetti F.
      Incidence of chemotherapy-induced amenorrhea depending on the timing of treatment by menstrual cycle phase in women with early breast cancer.
      ;
      • Fornier M.N.
      • Modi S.
      • Panageas K.S.
      • Norton L.
      • Hudis C.
      Incidence of chemotherapy-induced, long-term amenorrhea in patients with breast carcinoma age 40 years and younger after adjuvant anthracycline and taxane.
      ;
      • Oktem O.
      • Urman B.
      Options of fertility preservation in female cancer patients.
      ). Furthermore, prospective analysis of the IBCSG (International Breast Cancer Study Group) Trial 13-93 showed comparable amenorrhoea rates in women who did and did not receive tamoxifen after chemotherapy (88% versus 84%) (
      • Colleoni M.
      • Gelber S.
      • Goldhirsch A.
      • Aebi S.
      • Castiglione-Gertsch M.
      • Price K.N.
      • Coates A.S.
      • Gelber R.D.
      Tamoxifen after adjuvant chemotherapy for premenopausal women with lymph node-positive breast cancer: International Breast Cancer Study Group Trial 13-93.
      ). On the other hand, other studies revealed higher amenorrhoea rates with tamoxifen treatment. For instance, in a study of 466 premenopausal breast cancer patients who were administered AC, ACT and CMF protocols with and without tamoxifen and followed up at 6-month intervals, the percentage of patients who resumed menstrual bleeding at 6, 12 and 24 months after completion of chemotherapy was 68, 42 and 26% for AC; 57, 32 and 14% for ACT; and 23, 16 and 0% for CMF. The odds of experiencing an initial 6- and 24-month period of amenorrhoea were 2.96 (95% CI 1.68–5.24) and 2.54 (95% CI 1.32–4.88) times greater, respectively, for women treated with tamoxifen compared with those who were not (
      • Sukumvanich P.
      • Case L.D.
      • Van Zee K.
      • Singletary S.E.
      • Paskett E.D.
      • Petrek J.A.
      • Naftalis E.
      • Naughton M.J.
      Incidence and time course of bleeding after long-term amenorrhea after breast cancer treatment: a prospective study.
      ). A retrospective study demonstrated that patients aged over 40 had twice the risk of staying amenorrheic (OR 2.51; 95% CI 1.20–5.20), but such an increased risk was not observed in patients under the age of 40 at diagnosis (
      • Abusief M.E.
      • Missmer S.A.
      • Ginsburg E.S.
      • Weeks J.C.
      • Partridge A.H.
      The effects of paclitaxel, dose density, and trastuzumab on treatment-related amenorrhea in premenopausal women with breast cancer.
      ).
      In a retrospective cohort study, a total of 250 women who were premenopausal at diagnosis were divided into two groups: those who received tamoxifen and those who did not. Neither group of patients received chemotherapy. Menstrual pattern changes were more frequently observed in the tamoxifen group (amenorrhoea: 22% versus 3%, P < 0.001). The mean age of menopause onset was 51.0 for both groups and did not seem to be associated with the duration of tamoxifen use or the age. They concluded that tamoxifen use without chemotherapy is not associated with an earlier age onset of menopause and is unlikely to significantly accelerate ovarian ageing (
      • Chien A.J.
      • Duralde E.
      • Hwang R.
      • Tsung K.
      • Kao C.N.
      • Rugo H.S.
      • Melisko M.E.
      • Esserman L.J.
      • Munster P.N.
      • Cedars M.
      • Kerlikowske K.
      • McCulloch C.E.
      • Rosen M.P.
      Association of tamoxifen use and ovarian function in patients with invasive or pre-invasive breast cancer.
      ). A meta-analysis has evaluated that tamoxifen therapy significantly increased CIA incidence in premenopausal breast cancer patients, with an OR of 1.48 (95% CI 1.28–1.70, P = 0.00001) (
      • Zhao J.
      • Liu J.
      • Chen K.
      • Li S.
      • Wang Y.
      • Yang Y.
      • Deng H.
      • Jia W.
      • Rao N.
      • Liu Q.
      • Su F.
      What lies behind chemotherapy-induced amenorrhea for breast cancer patients: a meta-analysis.
      ). Similar results were obtained in another meta-analysis (
      • Zavos A.
      • Valachis A.
      Risk of chemotherapy-induced amenorrhea in patients with breast cancer: a systematic review and meta-analysis.
      ). The mechanism underlying amenorrhoea after tamoxifen use still remains obscure. A follow-up study has indicated no difference in AMH concentrations between patients receiving anti-oestrogenic versus non-anti-oestrogenic therapy, suggesting that tamoxifen may be responsible for amenorrhoea but not for ovarian failure (
      • Rosendahl M.
      • Andersen C.Y.
      • Ernst E.
      • Westergaard L.G.
      • Rasmussen P.E.
      • Loft A.
      • Andersen A.N.
      Ovarian function after removal of an entire ovary for cryopreservation of pieces of cortex prior to gonadotoxic treatment: a follow-up study.
      ). A recent study demonstrated that ovarian reserve significantly decreased in the AC and CMF protocols compared with the tamoxifen-only treatment (P < 0.0001 for both AC versus tamoxifen and CMF versus tamoxifen). Furthermore, substantially diminished AMH concentrations were only seen in patients receiving AC and CMF regimens (P = 0.53). However, patients treated with the tamoxifen-only protocol showed minor changes in the age-adjusted AMH concentrations, and adjuvant tamoxifen treatment after the AC protocol did not impact AMH recovery (
      • Goldfarb S.B.
      • Turan V.
      • Bedoschi G.
      • Taylan E.
      • Abdo N.
      • Cigler T.
      • Bang H.
      • Patil S.
      • Dickler M.N.
      • Oktay K.H.
      Impact of adjuvant chemotherapy or tamoxifen-alone on the ovarian reserve of young women with breast cancer.
      ). Tamoxifen may be used for ovulation induction as clomiphene citrate is also used for patients with anovulatory infertility. In addition, it can be used in breast cancer patients undergoing IVF treatment based on its dual action (
      • Oktay K.
      • Buyuk E.
      • Davis O.
      • Yermakova I.
      • Veeck L.
      • Rosenwaks Z.
      Fertility preservation in breast cancer patients: IVF and embryo cryopreservation after ovarian stimulation with tamoxifen.
      ,
      • Oktay K.
      • Buyuk E.
      • Libertella N.
      • Akar M.
      • Rosenwaks Z.
      Fertility preservation in breast cancer patients: a prospective controlled comparison of ovarian stimulation with tamoxifen and letrozole for embryo cryopreservation.
      ).

      Assessment of chemotherapy-induced loss in ovarian function

      Accurate assessment and prediction of loss of ovarian function after chemotherapy for premenopausal young women with breast cancer are essential for future fertility and clinical decisions regarding the need for subsequent adjuvant ovarian suppression. Most of the previously published clinical studies have used amenorrhoea as a surrogate marker of ovarian toxicity (
      • Oktem O.
      • Oktay K.
      Quantitative assessment of the impact of chemotherapy on ovarian follicle reserve and stromal function.
      ). However, menstrual status may not be a reliable indicator of the impact of chemotherapy (
      • Bines J.
      • Oleske D.M.
      • Cobleigh M.A.
      Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer.
      ). A proportion of these patients resumed menses in the following months, depending on the age of the patients and the type and dose of chemotherapy regimens. Patients younger than 40 are more likely to retain or regain menstrual function than those aged over 40 (22–56% versus 11%) (
      • Bines J.
      • Oleske D.M.
      • Cobleigh M.A.
      Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer.
      ). Furthermore, patients with a critically diminished ovarian reserve and elevated FSH values may continue to menstruate (
      • Reh A.
      • Oktem O.
      • Oktay K.
      Impact of breast cancer chemotherapy on ovarian reserve: a prospective observational analysis by menstrual history and ovarian reserve markers.
      ).
      There is solid evidence that AMH and antral follicle count (AFC) are the most reliable quantitative markers of ovarian reserve. Although basal FSH concentration on cycle day 2 or 3 is still used in screening tests assessing ovarian reserve by some studies, it shows significant inter-cycle variability (
      • Kwee J.
      • Schats R.
      • McDonnell J.
      • Lambalk C.B.
      • Schoemaker J.
      Intercycle variability of ovarian reserve tests: results of a prospective randomized study.
      ). Therefore, measuring FSH concentrations practically has no meaning in the assessment of ovarian reserve. AMH is produced by the granulosa cells (
      • van Rooij I.A.
      • Broekmans F.J.
      • te Velde E.R.
      • Fauser B.C.
      • Bancsi L.F.
      • de Jong F.H.
      • Themmen A.P.
      Serum anti-Mullerian hormone levels: a novel measure of ovarian reserve.
      ). Serum AMH concentrations correlate well with the number of antral follicles in the ovary (
      • Gruijters M.J.
      • Visser J.A.
      • Durlinger A.L.
      • Themmen A.P.
      Anti-Mullerian hormone and its role in ovarian function.
      ) and the number of oocytes retrieved in the IVF cycles (
      • Ebner T.
      • Sommergruber M.
      • Moser M.
      • Shebl O.
      • Schreier-Lechner E.
      • Tews G.
      Basal level of anti-Mullerian hormone is associated with oocyte quality in stimulated cycles.
      ). Serum AMH concentrations have declined in parallel with diminishing ovarian reserve after chemotherapy administration (
      • Oktay K.
      • Hourvitz A.
      • Sahin G.
      • Oktem O.
      • Safro B.
      • Cil A.
      • Bang H.
      Letrozole reduces estrogen and gonadotropin exposure in women with breast cancer undergoing ovarian stimulation before chemotherapy.
      ;
      • Partridge A.H.
      • Ruddy K.J.
      • Gelber S.
      • Schapira L.
      • Abusief M.
      • Meyer M.
      • Ginsburg E.
      Ovarian reserve in women who remain premenopausal after chemotherapy for early stage breast cancer.
      ). Similarly, inhibin B has also been used as an ovarian reserve marker (
      • Anders C.
      • Marcom P.K.
      • Peterson B.
      • Gu L.
      • Unruhe S.
      • Welch R.
      • Lyons P.
      • Behera M.
      • Copland S.
      • Kimmick G.
      • Shaw H.
      • Snyder S.
      • Antenos M.
      • Woodruff T.
      • Blackwell K.
      A pilot study of predictive markers of chemotherapy-related amenorrhea among premenopausal women with early stage breast cancer.
      ;
      • Su H.I.
      • Sammel M.D.
      • Green J.
      • Velders L.
      • Stankiewicz C.
      • Matro J.
      • Freeman E.W.
      • Gracia C.R.
      • DeMichele A.
      Antimullerian hormone and inhibin B are hormone measures of ovarian function in late reproductive-aged breast cancer survivors.
      ). A study investigating AMH, inhibin B and FSH as ovarian reserve markers in breast cancer survivors at reproductive age found that compared with age-matched controls, cancer subjects had significantly lower AMH and inhibin B and higher FSH. Also, participants with a pretreatment AMH concentration >2 ng/ml recovered at a rate of 11.9%, whereas participants with pretreatment AMH concentrations ≤2 ng/ml recovered at a rate of 2.6% per month after therapy. AFC on the third day of the menstrual cycle also appears to be a reliable marker of the ovarian follicle pool (
      • Lutchman Singh K.
      • Muttukrishna S.
      • Stein R.C.
      • McGarrigle H.H.
      • Patel A.
      • Parikh B.
      • Groome N.P.
      • Davies M.C.
      • Chatterjee R.
      Predictors of ovarian reserve in young women with breast cancer.
      ). Of these markers, AMH seems to be the earliest and most reliable indicator of ovarian ageing and reduction in post-chemotherapy ovarian reserve and in predicting the return of menses after chemotherapy for breast cancer (
      • Anderson R.A.
      • Themmen A.P.
      • Al-Qahtani A.
      • Groome N.P.
      • Cameron D.A.
      The effects of chemotherapy and long-term gonadotrophin suppression on the ovarian reserve in premenopausal women with breast cancer.
      ,
      • Anderson R.A.
      • Kelsey T.W.
      • Perdrix A.
      • Olympios N.
      • Duhamel O.
      • Lambertini M.
      • Clatot F.
      Diagnostic and predictive accuracy of anti-mullerian hormone for ovarian function after chemotherapy in premenopausal women with early breast cancer.
      ;
      • Chai J.
      • Howie A.F.
      • Cameron D.A.
      • Anderson R.A.
      A highly-sensitive anti-Mullerian hormone assay improves analysis of ovarian function following chemotherapy for early breast cancer.
      ).

      How to preserve fertility in patients with breast cancer

      The use of gonadotoxic chemotherapy regimens in adjuvant/neoadjuvant settings, together with the need to delay pregnancy while undergoing hormonal treatment (tamoxifen with or without gonadotrophin-releasing hormone [GnRH] agonists) for up to 10 years in oestrogen receptor positive disease will cause a substantial reduction in ovarian reserve in breast cancer patients. Therefore, counselling for fertility preservation is required in these patients if they have not completed childbearing at the time of diagnosis. In addition, the clinical guidelines by ASCO and the practice committee opinion of the ISFP (International Society for Fertility Preservation) emphasize that all cancer patients interested in future fertility should be referred for consideration of fertility preservation (
      • Kim S.S.
      • Donnez J.
      • Barri P.
      • Pellicer A.
      • Patrizio P.
      • Rosenwaks Z.
      • Nagy P.
      • Falcone T.
      • Andersen C.
      • Hovatta O.
      • Wallace H.
      • Meirow D.
      • Gook D.
      • Kim S.H.
      • Tzeng C.R.
      • Suzuki S.
      • Ishizuka B.
      • Dolmans M.M.
      Recommendations for fertility preservation in patients with lymphoma, leukemia, and breast cancer.
      ;
      • Lee S.J.
      • Schover L.R.
      • Partridge A.H.
      • Patrizio P.
      • Wallace W.H.
      • Hagerty K.
      • Beck L.N.
      • Brennan L.V.
      • Oktay K.
      American Society of Clinical Oncology recommendations on fertility preservation in cancer patients.
      ). In guidelines from the ASRM (American Society for Reproductive Medicine) and the SART (Society for Assisted Reproductive Technology), the designated fertility preservation methods are embryo/oocyte freezing and, very recently, ovarian tissue cryopreservation. Additional approaches are GnRH agonist adjunct to the chemotherapy and IVM (in-vitro maturation).

      Embryo freezing

      Embryo cryopreservation is one of the most well-known fertility preservation techniques for patients with adequate time before cancer therapy begins. The clinical pregnancy rate per frozen–thawed embryo transfer is well documented based on the SART guidelines and the European IVF Monitoring Programme. Actual rates exceeded 40% in women younger than 35 (
      • De Geyter C.
      • Wyns C.
      • Calhaz-Jorge C.
      • de Mouzon J.
      • Ferraretti A.P.
      • Kupka M.
      • Nyboe Andersen A.
      • Nygren K.G.
      • Goossens V.
      20 years of the European IVF-monitoring Consortium registry: what have we learned? A comparison with registries from two other regions.
      ). However, conventional ovarian stimulation protocols are characterized by multi-follicular growth and elevated (10–20 times) blood concentrations of endogenous oestrogens over the natural cycle. Therefore, tamoxifen (selective oestrogen receptor modulator) and letrozole (aromatase inhibitor) were added to ovarian stimulation protocols to minimize the possible risks of rising oestrogen concentrations during stimulation (
      • Azim A.A.
      • Costantini-Ferrando M.
      • Oktay K.
      Safety of fertility preservation by ovarian stimulation with letrozole and gonadotropins in patients with breast cancer: a prospective controlled study.
      ;
      • Oktay K.
      • Buyuk E.
      • Libertella N.
      • Akar M.
      • Rosenwaks Z.
      Fertility preservation in breast cancer patients: a prospective controlled comparison of ovarian stimulation with tamoxifen and letrozole for embryo cryopreservation.
      ,
      • Oktay K.
      • Oktem O.
      • Reh A.
      • Vahdat L.
      Measuring the impact of chemotherapy on fertility in women with breast cancer.
      ). A very recent study demonstrated that endogenous serum progesterone concentrations are reduced as well, in addition to oestrogen in breast cancer patients undergoing ovarian stimulation with aromatase inhibitor in comparison to those stimulated without aromatase (
      • Lalami I.
      • Labrosse J.
      • Cedrin-Durnerin I.
      • Comtet M.
      • Vinolas C.
      • Krief F.
      • Sifer C.
      • Peigne M.
      • Grynberg M.
      Is letrozole during ovarian stimulation useful in breast cancer patients undergoing fertility preservation to reduce early luteal progesterone levels following GnRH-agonist trigger?.
      ). Although these studies show promising results, there is a need for more extensive prospective studies with long-term follow-up periods in patients with breast cancer to demonstrate the safety of these approaches.
      Another important issue is the time interval between ovarian stimulation and initiation of chemotherapy in breast cancer patients. There is generally a 4–6-week interval between administration of chemotherapy protocols and surgical treatment in breast cancer patients, whereas chemotherapy is initiated before surgery as neo-adjuvant therapy in patients with advanced stage disease. Ovarian stimulation to collect oocytes for oocyte and/or embryo cryopreservation typically lasts 10–14 days. The studies that assessed the impact of the length of time between surgery and the administration of the chemotherapy regimens on the success of treatment provided reassuring findings by showing that there was no harmful effect of a delay if the therapy is used within 12 weeks after the operation (
      • Cold S.
      • During M.
      • Ewertz M.
      • Knoop A.
      • Moller S.
      Does timing of adjuvant chemotherapy influence the prognosis after early breast cancer? Results of the Danish Breast Cancer Co-operative Group (DBCG).
      ;
      • Jara Sanchez C.
      • Ruiz A.
      • Martin M.
      • Anton A.
      • Munarriz B.
      • Plazaola A.
      • Schneider J.
      • Martinez del Prado P.
      • Alba E.
      • Fernandez-Aramburo A.
      Influence of timing of initiation of adjuvant chemotherapy over survival in breast cancer: a negative outcome study by the Spanish Breast Cancer Research Group (GEICAM).
      ;
      • Lohrisch C.
      • Paltiel C.
      • Gelmon K.
      • Speers C.
      • Taylor S.
      • Barnett J.
      • Olivotto I.A.
      Impact on survival of time from definitive surgery to initiation of adjuvant chemotherapy for early-stage breast cancer.
      ). Unfortunately, some breast cancer patients might not have sufficient time to undergo conventional ovarian stimulation protocols for oocyte or embryo freezing because some of them have to start chemotherapy without any significant delay, necessitating an urgent ovarian stimulation for oocyte or embryo freezing prior to cancer therapy (
      • Cakmak H.
      • Rosen M.P.
      Random-start ovarian stimulation in patients with cancer.
      ). Several ovarian stimulation protocols have been developed to overcome this problem, such as initiating ovarian stimulation randomly (so-called random-start ovarian stimulation), either at the late follicular or luteal phases; and starting gonadotrophins concomitantly with GnRH antagonist. Current data indicate that unconventional ovarian stimulation protocols can shorten the interval between ovarian stimulation and oocyte retrieval, and the number of oocytes retrieved in these cycles and their competency for euploid embryo development appear to be comparable to conventional stimulation protocols (
      • Cakmak H.
      • Rosen M.P.
      Random-start ovarian stimulation in patients with cancer.
      ;
      • Cimadomo D.
      • Vaiarelli A.
      • Colamaria S.
      • Trabucco E.
      • Alviggi C.
      • Venturella R.
      • Alviggi E.
      • Carmelo R.
      • Rienzi L.
      • Ubaldi F.M.
      Luteal phase anovulatory follicles result in the production of competent oocytes: intra-patient paired case-control study comparing follicular versus luteal phase stimulations in the same ovarian cycle.
      ;
      • Danis R.B.
      • Pereira N.
      • Elias R.T.
      Random start ovarian stimulation for oocyte or embryo cryopreservation in women desiring fertility preservation prior to gonadotoxic cancer therapy.
      ;
      • Llacer J.
      • Moliner B.
      • Luque L.
      • Bernabeu A.
      • Lledo B.
      • Castillo J.C.
      • Guerrero J.
      • Ten J.
      • Bernabeu R.
      Luteal phase stimulation versus follicular phase stimulation in poor ovarian responders: results of a randomized controlled trial.
      ;
      • Muteshi C.
      • Child T.
      • Ohuma E.
      • Fatum M.
      Ovarian response and follow-up outcomes in women diagnosed with cancer having fertility preservation: comparison of random start and early follicular phase stimulation – cohort study.
      ;
      • Sonmezer M.
      • Turkcuoglu I.
      • Coskun U.
      • Oktay K.
      Random-start controlled ovarian hyperstimulation for emergency fertility preservation in letrozole cycles.
      ;
      • Ubaldi F.M.
      • Capalbo A.
      • Vaiarelli A.
      • Cimadomo D.
      • Colamaria S.
      • Alviggi C.
      • Trabucco E.
      • Venturella R.
      • Vajta G.
      • Rienzi L.
      Follicular versus luteal phase ovarian stimulation during the same menstrual cycle (DuoStim) in a reduced ovarian reserve population results in a similar euploid blastocyst formation rate: new insight in ovarian reserve exploitation.
      ;
      • Vaiarelli A.
      • Cimadomo D.
      • Alviggi E.
      • Sansone A.
      • Trabucco E.
      • Dusi L.
      • Buffo L.
      • Barnocchi N.
      • Fiorini F.
      • Colamaria S.
      • Giuliani M.
      • Argento C.
      • Rienzi L.
      • Ubaldi F.M.
      The euploid blastocysts obtained after luteal phase stimulation show the same clinical, obstetric and perinatal outcomes as follicular phase stimulation-derived ones: a multicenter study.
      ,
      • Vaiarelli A.
      • Cimadomo D.
      • Gennarelli G.
      • Guido M.
      • Alviggi C.
      • Conforti A.
      • Livi C.
      • Revelli A.
      • Colamaria S.
      • Argento C.
      • Giuliani M.
      • De Angelis C.
      • Matteo M.
      • Canosa S.
      • D'Alfonso A.
      • Cimadomo V.
      • Rienzi L.
      • Ubaldi F.M.
      Second stimulation in the same ovarian cycle: an option to fully-personalize the treatment in poor prognosis patients undergoing PGT-A.
      ).

      Oocyte freezing

      Oocyte freezing is an established method of fertility preservation for young women who do not have a partner and do not wish to use donor spermatozoa. Its experimental label was removed and oocyte vitrification was accepted as an established method of fertility preservation in 2012 by the ASRM (
      Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology
      Mature oocyte cryopreservation: a guideline.
      ). Oocyte freezing should also be considered in women who still wish to have their oocytes frozen without insemination, even in the presence of a husband or partner's spermatozoa, for other important social reasons such as partner abandonment, a phenomenon that is more frequently observed in women diagnosed with cancer (
      • Glantz M.J.
      • Chamberlain M.C.
      • Liu Q.
      • Hsieh C.C.
      • Edwards K.R.
      • Van Horn A.
      • Recht L.
      Gender disparity in the rate of partner abandonment in patients with serious medical illness.
      ). Vitrification is the standard method for oocyte cryopreservation as the post-warming survival rates of the oocytes and the IVF success rates of resulting embryos are superior to the conventional slow freezing technique. The ongoing pregnancy, top quality embryo, embryo cleavage and fertilization rates are comparable between the vitrified and the fresh oocyte groups (
      • Cobo A.
      • Diaz C.
      Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials.
      ;
      • Cobo A.
      • Garcia-Velasco J.A.
      • Remohi J.
      • Pellicer A.
      Oocyte vitrification for fertility preservation for both medical and nonmedical reasons.
      ). Available data indicate that cancer diagnosis itself might have a negative impact on the post-warming oocyte survival rates and cumulative live birth rates (CLBR), although the underlying molecular mechanisms are not clearly understood. When a comparison was made between the oncology patients and those undergoing elective fertility preservation under the age of 35 it appears that there are significant differences in the oocyte survival rate (91.4% versus 81.2%), clinical pregnancy rate (65.9% versus 42.8%) and CLBR (68.8% versus 42.1%) (
      • Cobo A.
      • Garcia-Velasco J.
      • Domingo J.
      • Pellicer A.
      • Remohi J.
      Elective and onco-fertility preservation: factors related to IVF outcomes.
      ). Another important issue is the question of how many oocytes should be vitrified to obtain a reasonable chance of pregnancy or live birth. As mentioned above, cancer patients are more likely to have lower CLBR compared to those undergoing elective oocyte freezing. Age of the patient is one of the most deterministic factors because CLBR gradually decline with advancing age (
      • Cobo A.
      • Garcia-Velasco J.A.
      • Remohi J.
      • Pellicer A.
      Oocyte vitrification for fertility preservation for both medical and nonmedical reasons.
      ). Embryo biopsy for preimplantation genetic testing is another potential confounder that might affect the success of IVF in patients with inherited BRCA positive breast cancer. Overall, it can be stated that live birth rate per warmed oocyte is 4–6% for patients under age 35. As an example, CLBR with 95% CI for five oocytes used for patients under age 35 are 9.1 (0.7–19) and 15.8 (8.4–23.1) for cancer patients versus elective cases, respectively (
      • Cobo A.
      • Garcia-Velasco J.A.
      • Remohi J.
      • Pellicer A.
      Oocyte vitrification for fertility preservation for both medical and nonmedical reasons.
      ). In a longitudinal cohort multicentric study,
      • Rienzi L.
      • Cobo A.
      • Paffoni A.
      • Scarduelli C.
      • Capalbo A.
      • Vajta G.
      • Remohi J.
      • Ragni G.
      • Ubaldi F.M.
      Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study.
      revealed that more than eight oocytes are required to enhance live birth rates (22.6 versus 46.4%). Conversely, live birth rates are dramatically reduced when fewer oocytes (≤8) are available in women older than 38 years (12.6 versus 27.5%). These figures are beneficial in providing actual information on the success rate of oocyte freezing when counselling breast cancer patients who desire to have their oocytes frozen with the aim of achieving a successful pregnancy in the future. Another critical issue concerning gamete freezing is the risk of congenital anomalies in the descendants. Fortunately, no noticeable increase in congenital anomaly rates has been documented compared with natural conceptions (
      • Noyes N.
      • Porcu E.
      • Borini A.
      Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies.
      ). Furthermore, no adverse effect on the grade of the oocytes and embryos has been detected with tamoxifen or letrozole used during ovarian stimulation (
      • Meirow D.
      • Raanani H.
      • Maman E.
      • Paluch-Shimon S.
      • Shapira M.
      • Cohen Y.
      • Kuchuk I.
      • Hourvitz A.
      • Levron J.
      • Mozer-Mendel M.
      • Brengauz M.
      • Biderman H.
      • Manela D.
      • Catane R.
      • Dor J.
      • Orvieto R.
      • Kaufman B.
      Tamoxifen co-administration during controlled ovarian hyperstimulation for in vitro fertilization in breast cancer patients increases the safety of fertility-preservation treatment strategies.
      ;
      • Revelli A.
      • Porcu E.
      • Levi Setti P.E.
      • Delle Piane L.
      • Merlo D.F.
      • Anserini P.
      Is letrozole needed for controlled ovarian stimulation in patients with estrogen receptor-positive breast cancer?.
      ).

      Do breast cancer patients already have reduced ovarian reserve or impaired response to ovarian stimulation?

      The results of several recent studies raised the question of whether malignancy itself impacts ovarian reserve or response to ovarian stimulation for gamete cryopreservation before any therapeutic intervention. In a study with 108 recently diagnosed breast cancer patients and 99 healthy women in a control group,
      • Su H.I.
      • Flatt S.W.
      • Natarajan L.
      • DeMichele A.
      • Steiner A.Z.
      Impact of breast cancer on anti-mullerian hormone levels in young women.
      compared AMH, inhibin B and FSH concentrations. Although the AMH concentrations did not differ significantly in younger patients with breast cancer, in a subgroup of older patients (above 37 years of age), AMH concentrations showed a downward trend (
      • Su H.I.
      • Flatt S.W.
      • Natarajan L.
      • DeMichele A.
      • Steiner A.Z.
      Impact of breast cancer on anti-mullerian hormone levels in young women.
      ). The other two studies with a similar design analysed ovarian response in cancer patients undergoing ovarian stimulation for fertility preservation compared with healthy controls (
      • Domingo J.
      • Guillen V.
      • Ayllon Y.
      • Martinez M.
      • Munoz E.
      • Pellicer A.
      • Garcia-Velasco J.A.
      Ovarian response to controlled ovarian hyperstimulation in cancer patients is diminished even before oncological treatment.
      ;
      • Friedler S.
      • Koc O.
      • Gidoni Y.
      • Raziel A.
      • Ron-El R.
      Ovarian response to stimulation for fertility preservation in women with malignant disease: a systematic review and meta-analysis.
      ). Their results showed that hormone-dependent cancers and malignancy are associated with a weaker response to ovarian stimulation and a lower number of oocytes retrieved before oncological treatment. However, the pooled results of a recent meta-analysis with 10 case–control retrospective studies (a total of 722 cycles of women with cancer and 1835 cycles of healthy women) showed a negligible effect of a cancer diagnosis on fertilization rates (95% CI –0.29 to 0.183, P = 0.273) and the mean number of total oocytes (95% CI –0.23 to 0.12, P = 0.517), mature oocytes (95% CI –0.23 to 0.01, P = 0.104) and two-pronuclear embryos (95% CI –0.32 to 0.04, P = 0.136) (
      • Turan V.
      • Quinn M.M.
      • Dayioglu N.
      • Rosen M.P.
      • Oktay K.
      The impact of malignancy on response to ovarian stimulation for fertility preservation: a meta-analysis.
      ).
      Women with germline BRCA mutations (gBRCA) were also reported to have lower AMH concentrations (Johnson et al., 2017). Recently a meta-analysis including five datasets on 828 patients has shown that women with gBRCA1/2 had lower serum AMH concentrations compared with women without gBRCA1/2 mutation after adjustments (23%; 95% CI 4–38; P = 0.02). This significant difference persisted in adjusted analysis when only women with breast cancer were included (25% lower; 95% CI 9–38; P = 0.003). However, only the women with gBRCA1 mutation had lower AMH concentrations (33%; 95% CI 12–49; P = 0.004). In those with gBRCA2 mutation the difference was not significant (7% lower; 95% CI 31% lower to 26% higher; P = 0.64). The findings could be explained by DNA repair deficiency in women with gBRCA pathogenic variants. Therefore, it would be plausible to counsel patients with gBRCA about the possibility of decreased ovarian reserve (
      • Turan V.
      • Lambertini M.
      • Lee D.Y.
      • Wang E.
      • Clatot F.
      • Karlan B.Y.
      • Demeestere I.
      • Bang H.
      • Oktay K.
      Association of germline BRCA pathogenic variants with diminished ovarian reserve: a meta-analysis of individual patient-level data.
      ).

      Safety of ovarian stimulation

      Exposure to higher oestradiol concentrations may be detrimental to tumour proliferation and dissemination in women with hormone-dependent cancer. It is therefore crucial to investigate the safety of ovarian stimulation in these women. There is a paucity of data regarding the safety of IVF in women with breast cancer. The first study to determine the effect of ovarian stimulation in breast cancer evaluated 79 women and compared recurrence rates with 136 women who did not choose any fertility-preserving procedure. After a median follow-up of 23.4 months (7.5–63.6 months) post-chemotherapy, the hazard ratio for recurrence after IVF was 0.56 (95% CI 0.17 to 1.90), and the survival was not compromised in women who underwent ovarian stimulation compared with controls (
      • Azim A.A.
      • Costantini-Ferrando M.
      • Oktay K.
      Safety of fertility preservation by ovarian stimulation with letrozole and gonadotropins in patients with breast cancer: a prospective controlled study.
      ). The same group published another follow-up study in 2016. The mean follow-up after diagnosis was 5.0 years in women with breast cancer who underwent ovarian stimulation and 6.9 years in the control group. The recurrence and survival rates were similar between the two groups (P = 0.61) (
      • Kim J.
      • Turan V.
      • Oktay K.
      Long-term safety of letrozole and gonadotropin stimulation for fertility preservation in women with breast cancer.
      ). Recently,
      • Condorelli M.
      • Bruzzone M.
      • Ceppi M.
      • Ferrari A.
      • Grinshpun A.
      • Hamy A.S.
      • de Azambuja E.
      • Carrasco E.
      • Peccatori F.A.
      • Di Meglio A.
      • Paluch-Shimon S.
      • Poorvu P.D.
      • Venturelli M.
      • Rousset-Jablonski C.
      • Senechal C.
      • Livraghi L.
      • Ponzone R.
      • De Marchis L.
      • Pogoda K.
      • Sonnenblick A.
      • Villarreal-Garza C.
      • Cordoba O.
      • Teixeira L.
      • Clatot F.
      • Punie K.
      • Graffeo R.
      • Dieci M.V.
      • Perez-Fidalgo J.A.
      • Duhoux F.P.
      • Puglisi F.
      • Ferreira A.R.
      • Blondeaux E.
      • Peretz-Yablonski T.
      • Caron O.
      • Saule C.
      • Ameye L.
      • Balmana J.
      • Partridge A.H.
      • Azim H.A.
      • Demeestere I.
      • Lambertini M.
      Safety of assisted reproductive techniques in young women harboring germline pathogenic variants in BRCA1/2 with a pregnancy after prior history of breast cancer.
      conducted a retrospective multicentric study including breast cancer survivors who underwent ovarian stimulation. The median follow-up time was 4.6 years (2.4–12.5) in women undergoing ovarian stimulation, and the adjusted follow-up time for the non-exposed group was 6.9 years. Breast cancer relapsed in 7.7% of women who underwent ovarian stimulation (20.5% in the control group) and no significant difference was found compared with the control group (P = 0.23) (
      • Condorelli M.
      • Bruzzone M.
      • Ceppi M.
      • Ferrari A.
      • Grinshpun A.
      • Hamy A.S.
      • de Azambuja E.
      • Carrasco E.
      • Peccatori F.A.
      • Di Meglio A.
      • Paluch-Shimon S.
      • Poorvu P.D.
      • Venturelli M.
      • Rousset-Jablonski C.
      • Senechal C.
      • Livraghi L.
      • Ponzone R.
      • De Marchis L.
      • Pogoda K.
      • Sonnenblick A.
      • Villarreal-Garza C.
      • Cordoba O.
      • Teixeira L.
      • Clatot F.
      • Punie K.
      • Graffeo R.
      • Dieci M.V.
      • Perez-Fidalgo J.A.
      • Duhoux F.P.
      • Puglisi F.
      • Ferreira A.R.
      • Blondeaux E.
      • Peretz-Yablonski T.
      • Caron O.
      • Saule C.
      • Ameye L.
      • Balmana J.
      • Partridge A.H.
      • Azim H.A.
      • Demeestere I.
      • Lambertini M.
      Safety of assisted reproductive techniques in young women harboring germline pathogenic variants in BRCA1/2 with a pregnancy after prior history of breast cancer.
      ). Consistent with this report, two recent retrospective analyses of Swedish registry data found no increased risk of recurrence after pregnancies conceived naturally compared with through assisted reproduction in women with breast cancer (
      • Fredriksson A.
      • Rosenberg E.
      • Einbeigi Z.
      • Bergh C.
      • Strandell A.
      Gonadotrophin stimulation and risk of relapse in breast cancer.
      ;
      • Rosenberg E.
      • Fredriksson A.
      • Einbeigi Z.
      • Bergh C.
      • Strandell A.
      No increased risk of relapse of breast cancer for women who give birth after assisted conception.
      ). Moreover, another Swedish nationwide cohort study compared the long-term reproductive outcomes of women who did and did not undergo fertility preservation after a breast cancer diagnosis. The study demonstrated a significantly higher live birth rate after breast cancer in women who underwent fertility preservation than those who did not (adjusted hazard ratio 2.3; 95% CI 1.6–3.3). The 5-year and 10-year cumulative incidence of post-breast cancer live births was 19.4% and 40.7% among fertility preservation-exposed women versus 8.6% and 15.8% among comparators, respectively. Assisted reproduction was much more common in the fertility preservation group (adjusted hazard ratio 4.8; 95% CI 2.2–10.7). The study also showed that the all-cause mortality rate was lower in women exposed to fertility preservation (adjusted hazard ratio 0.4; 95% CI 0.3–0.7), with a 5-year cumulative incidence of death of 5.3% (95% CI 3.1–9.0%) versus 11.1% (95% CI 8.7–14.1%) for women with versus without fertility preservation (
      • Marklund A.
      • Lundberg F.E.
      • Eloranta S.
      • Hedayati E.
      • Pettersson K.
      • Rodriguez-Wallberg K.A.
      Reproductive outcomes after breast cancer in women with vs without fertility preservation.
      ). Taken collectively, ovarian stimulation does not appear to have a negative impact on disease-free survival in breast cancer survivors. However, randomized controlled studies with long-term follow-up are needed to obtain a more conclusive result.

      Ovarian tissue cryopreservation

      Ovarian tissue cryopreservation is the only available option to preserve fertility of breast cancer patients in whom cancer therapy cannot be delayed because of a rapidly growing or advanced stage tumour, or where ovarian stimulation for embryo or oocyte freezing is contraindicated. This technique does not need ovarian stimulation or preparation, and ovarian tissue can be gathered laparoscopically. The tissue is processed into thin cortical pieces and cryopreserved via slow freezing or vitrification (
      • Oktay K.
      • Oktem O.
      Ovarian cryopreservation and transplantation for fertility preservation for medical indications: report of an ongoing experience.
      ;
      • Yding Andersen C.
      • Mamsen L.S.
      • Kristensen S.G.
      Fertility preservation: freezing of ovarian tissue and clinical opportunities.
      ). In the current literature, more than 120 pregnancies have been demonstrated after auto-transplantation of frozen–thawed ovarian cortical pieces (
      • Sheshpari S.
      • Shahnazi M.
      • Mobarak H.
      • Ahmadian S.
      • Bedate A.M.
      • Nariman-Saleh-Fam Z.
      • Nouri M.
      • Rahbarghazi R.
      • Mahdipour M.
      Ovarian function and reproductive outcome after ovarian tissue transplantation: a systematic review.
      ). The ASRM removed the experimental label from this procedure and accepted it as an established strategy to preserve fertility in designated patients (
      Practice Committee of the American Society for Reproductive Medicine
      Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion.
      ). A very recent meta-analysis of 85 studies in 735 women showed that the pooled rates were 37% (95% CI 32–43%) for pregnancy, 28% (95% CI 24–34%) for live birth and 37% (95% CI 30–46%) for miscarriage following cryopreserved ovarian tissue transplantation (
      • Khattak H.
      • Malhas R.
      • Craciunas L.
      • Afifi Y.
      • Amorim C.A.
      • Fishel S.
      • Silber S.
      • Gook D.
      • Demeestere I.
      • Bystrova O.
      • Lisyanskaya A.
      • Manikhas G.
      • Lotz L.
      • Dittrich R.
      • Colmorn L.B.
      • Macklon K.T.
      • Hjorth I.M.D.
      • Kristensen S.G.
      • Gallos I.
      • Coomarasamy A.
      Fresh and cryopreserved ovarian tissue transplantation for preserving reproductive and endocrine function: a systematic review and individual patient data meta-analysis.
      ).
      The ovarian cortex possesses primordial follicles with oocytes arrested in the diplotene prophase of the first meiotic division. The potential mechanisms of resistance to cryo-injury by primordial follicles are the moderately high surface/volume ratio, low metabolic rate and the lack of zona pellucida. These features make successful ovarian tissue cryopreservation feasible (
      • Oktem O.
      • Urman B.
      Understanding follicle growth in vivo.
      ). Most ovarian tissue has been cryopreserved via slow freezing, which has been successful, but has not yet been fully optimized. Moreover, ovarian tissue vitrification has yielded encouraging results (
      • Amorim C.A.
      • Dolmans M.M.
      • David A.
      • Jaeger J.
      • Vanacker J.
      • Camboni A.
      • Donnez J.
      • Van Langendonckt A.
      Vitrification and xenografting of human ovarian tissue.
      ;
      • Sheikhi M.
      • Hultenby K.
      • Niklasson B.
      • Lundqvist M.
      • Hovatta O.
      Clinical grade vitrification of human ovarian tissue: an ultrastructural analysis of follicles and stroma in vitrified tissue.
      ). Nevertheless, it may be too premature to replace slow freezing with vitrification for ovarian tissue. It has previously been demonstrated that vitrified human ovaries contain fewer primordial follicles and produce less AMH in vitro compared with those cryopreserved by slow freezing (
      • Oktem O.
      • Alper E.
      • Balaban B.
      • Palaoglu E.
      • Peker K.
      • Karakaya C.
      • Urman B.
      Vitrified human ovaries have fewer primordial follicles and produce less antimullerian hormone than slow-frozen ovaries.
      ). In another study that investigated tissue damage at the molecular level after vitrification using various markers of DNA damage and repair, apoptosis and autophagy, increased DNA damage and apoptosis were observed in bovine and human ovarian tissue after vitrification and warming (
      • Kim S.S.
      • Limback D.
      • He L.
      • Albertini D.
      Assessment of autophage, apoptosis and dna damage/repair process in cryopreserved ovarian cortex.
      ).
      Patient age is a vital factor to consider, because the chances of restoring ovarian function and fertility is closely associated with the number of follicles in the ovarian graft; 10–15% of the follicles will be lost during the freezing and thawing. The greatest loss is observed after transplantation, as nearly half of the ovarian reserve (primordial stockpile) will be lost during the ischaemic period until revascularization is established in the grafts (
      • Baird D.T.
      • Webb R.
      • Campbell B.K.
      • Harkness L.M.
      • Gosden R.G.
      Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196 C.
      ). This fact explains why a good ovarian reserve in the candidate is a prerequisite for the success of the procedure. Current experience with human ovarian transplantation implies that women over 38 years might not be good candidates for ovarian tissue cryopreservation, as the chance of pregnancy with ovarian transplantation is particularly low (
      • Kim S.S.
      • Donnez J.
      • Barri P.
      • Pellicer A.
      • Patrizio P.
      • Rosenwaks Z.
      • Nagy P.
      • Falcone T.
      • Andersen C.
      • Hovatta O.
      • Wallace H.
      • Meirow D.
      • Gook D.
      • Kim S.H.
      • Tzeng C.R.
      • Suzuki S.
      • Ishizuka B.
      • Dolmans M.M.
      Recommendations for fertility preservation in patients with lymphoma, leukemia, and breast cancer.
      ). Measuring AFC and serum AMH concentrations is advised before ovarian tissue cryopreservation for a more reliable assessment of ovarian reserve. The site of choice for transplant could be either orthotopic or heterotopic. In orthotopic transplantation, frozen–thawed ovarian cortical pieces are engrafted near the infundibulopelvic ligament (if both ovaries were previously removed) or on the current non-functional ovary. The heterotopic transplantation technique generally uses the ovarian tissue pelvic side wall. The benefit of orthotopic transplantation is the opportunity for natural conception. However, this method is a surgical procedure with general anaesthesia, whereas heterotopic transplantation can be performed with local anaesthesia. It is easy to monitor follicle development and remove the grafts if needed. Orthotopic auto-transplantation of frozen–thawed ovarian tissue has resulted in more than 130 live births worldwide (
      • Oktay K.
      • Oktem O.
      Ovarian cryopreservation and transplantation for fertility preservation for medical indications: report of an ongoing experience.
      ;
      • Sheshpari S.
      • Shahnazi M.
      • Mobarak H.
      • Ahmadian S.
      • Bedate A.M.
      • Nariman-Saleh-Fam Z.
      • Nouri M.
      • Rahbarghazi R.
      • Mahdipour M.
      Ovarian function and reproductive outcome after ovarian tissue transplantation: a systematic review.
      ). However, only one live birth has been reported after heterotopic transplantation (
      • Tammiste T.
      • Kask K.
      • Padrik P.
      • Idla K.
      • Rosenstein K.
      • Jatsenko T.
      • Veerus P.
      • Salumets A.
      A case report and follow-up of the first live birth after heterotopic transplantation of cryopreserved ovarian tissue in Eastern Europe.
      ). The reasons for reduced success rates after heterotopic transplantation can be multi-factorial. For example, the suboptimal conditions of the heterotopic site could impact the development and quality of oocytes; the environmental characteristics of the pelvis at the heterotopic site are not identical to those at the orthotopic site; the setting of transplantation sites can affect the survival of grafted ovarian tissue; cryo-injury can damage the follicles during freezing and thawing, leading to an ischaemic condition after transplantation. Studies showed increased empty follicles, abnormal oocytes and low average fertilization rates after replacing frozen–thawed ovarian tissue, even at the orthotopic sites (
      • Donnez J.
      • Jadoul P.
      • Pirard C.
      • Hutchings G.
      • Demylle D.
      • Squifflet J.
      • Smitz J.
      • Dolmans M.M.
      Live birth after transplantation of frozen-thawed ovarian tissue after bilateral oophorectomy for benign disease.
      ;
      • Schmidt K.T.
      • Rosendahl M.
      • Ernst E.
      • Loft A.
      • Andersen A.N.
      • Dueholm M.
      • Ottosen C.
      • Andersen C.Y.
      Autotransplantation of cryopreserved ovarian tissue in 12 women with chemotherapy-induced premature ovarian failure: the Danish experience.
      ).
      There is a risk of re-seeding of cancer cells within the grafts after transplantation, although this risk appears to be minimal. While occult cancer cells are frequently detected in the ovarian grafts, particularly in Hodgkin's lymphoma (
      • Bittinger S.E.
      • Nazaretian S.P.
      • Gook D.A.
      • Parmar C.
      • Harrup R.A.
      • Stern C.J.
      Detection of Hodgkin lymphoma within ovarian tissue.
      ) and leukaemia (
      • Dolmans M.M.
      • Marinescu C.
      • Saussoy P.
      • Van Langendonckt A.
      • Amorim C.
      • Donnez J.
      Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe.
      ), reintroduction of cancer cells and recurrence of the breast and other cancers have not been reported after ovarian tissue transplantation. However, there are certain conditions in which ovarian transplantation should not be performed. BRCA positive cancer survivors is such an example. These patients may already carry mutations in the ovarian cortical grafts, which may give rise to malignant tumour formations after transplantation. In addition, other breast cancer patients without BRCA mutations or other identified mutations with a strong family history of breast/ovarian/pancreatic cancer, and patients with stage 4 breast cancer, should be discouraged from having their ovarian tissue transplanted. It is imperative that an accurate analysis of the ovarian tissue is performed for cancer relapse/reoccurrence and re-seeding and an individual multidisciplinary evaluation of the risk for each fertility restoration is required for each candidate patient for ovarian tissue transplantation.

      GnRH analogues

      GnRH analogues suppress the secretion of LH and FSH from the anterior pituitary gland, creating a hypoestrogenic hypogonadotrophic state. So far several RCT have been conducted to assess the protective effects of GnRH analogues against ovarian failure induced by chemotherapy drugs. Unfortunately, the results of these RCT and meta-analysis of these studies yielded conflicting results (
      • Elgindy E.A.
      • El-Haieg D.O.
      • Khorshid O.M.
      • Ismail E.I.
      • Abdelgawad M.
      • Sallam H.N.
      • Abou-Setta A.M.
      Gonadatrophin suppression to prevent chemotherapy-induced ovarian damage: a randomized controlled trial.
      ;
      • Lambertini M.
      • Moore H.C.F.
      • Leonard R.C.F.
      • Loibl S.
      • Munster P.
      • Bruzzone M.
      • Boni L.
      • Unger J.M.
      • Anderson R.A.
      • Mehta K.
      • Minton S.
      • Poggio F.
      • Albain K.S.
      • Adamson D.J.A.
      • Gerber B.
      • Cripps A.
      • Bertelli G.
      • Seiler S.
      • Ceppi M.
      • Partridge A.H.
      • Del Mastro L.
      Gonadotropin-releasing hormone agonists during chemotherapy for preservation of ovarian function and fertility in premenopausal patients with early breast cancer: a systematic review and meta-analysis of individual patient-level data.
      ;
      • Li Z.Y.
      • Dong Y.L.
      • Cao X.Z.
      • Ren S.S.
      • Zhang Z.
      Gonadotropin-releasing hormone agonists for ovarian protection during breast cancer chemotherapy: a systematic review and meta-analysis.
      ;
      • Munhoz R.R.
      • Pereira A.A.
      • Sasse A.D.
      • Hoff P.M.
      • Traina T.A.
      • Hudis C.A.
      • Marques R.J.
      Gonadotropin-releasing hormone agonists for ovarian function preservation in premenopausal women undergoing chemotherapy for early-stage breast cancer: a systematic review and meta-analysis.
      ;
      • Munster P.N.
      • Moore A.P.
      • Ismail-Khan R.
      • Cox C.E.
      • Lacevic M.
      • Gross-King M.
      • Xu P.
      • Carter W.B.
      • Minton S.E.
      Randomized trial using gonadotropin-releasing hormone agonist triptorelin for the preservation of ovarian function during (neo)adjuvant chemotherapy for breast cancer.
      ;
      • Yang B.
      • Shi W.
      • Yang J.
      • Liu H.
      • Zhao H.
      • Li X.
      • Jiao S.
      Concurrent treatment with gonadotropin-releasing hormone agonists for chemotherapy-induced ovarian damage in premenopausal women with breast cancer: a meta-analysis of randomized controlled trials.
      ). In fact, the studies assessing the protective role of GnRH analogues in preventing chemotherapy-related ovarian failure are not limited to patients with breast cancer (
      • Hickman L.C.
      • Llarena N.C.
      • Valentine L.N.
      • Liu X.
      • Falcone T.
      Preservation of gonadal function in women undergoing chemotherapy: a systematic review and meta-analysis of the potential role for gonadotropin-releasing hormone agonists.
      ;
      • Senra J.C.
      • Roque M.
      • Talim M.C.T.
      • Reis F.M.
      • Tavares R.L.C.
      Gonadotropin-releasing hormone agonists for ovarian protection during cancer chemotherapy: systematic review and meta-analysis.
      ). If GnRH has the potential to protect the ovary from CIA, what is the mechanism of its action? Although the mechanism is unknown (or does not exist), there are some proposed mechanisms, such as decreased delivery of chemotherapeutics into ovarian tissue due to reduced ovarian blood flow, pseudomenopausal state created by GnRH administration, or other actions of GnRH in the ovary (
      • Lambertini M.
      • Horicks F.
      • Del Mastro L.
      • Partridge A.H.
      • Demeestere I.
      Ovarian protection with gonadotropin-releasing hormone agonists during chemotherapy in cancer patients: from biological evidence to clinical application.
      ). Follicle development from the primordial to the antral stage is a continuous process in the ovary. It is not controlled by gonadotrophin hormones, which are mainly responsible for the growth of a cohort of antral follicles and the selection of dominant follicles for the ovulation (
      • Oktem O.
      • Urman B.
      Understanding follicle growth in vivo.
      ). Furthermore, neither GnRH ligands (GnRH-I, GnRH-II) nor their receptor (GnRHR) are expressed in the follicles from the primordial to the early antral stage in the human ovary. In preovulatory follicles, both the receptor and ligands of GnRH are localized predominantly to the granulosa cell layer. In the corpus luteum, significant concentrations of the ligands and GnRH receptors have been observed in granulosa luteal cells. GnRH isoforms and the type I GnRHR are also localized to the ovarian surface epithelium (
      • Choi J.H.
      • Gilks C.B.
      • Auersperg N.
      • Leung P.C.
      Immunolocalization of gonadotropin-releasing hormone (GnRH)-I, GnRH-II, and type I GnRH receptor during follicular development in the human ovary.
      ). Therefore it is doubtful that GnRH exerts any protective effect against chemotherapy by preserving ovarian reserve after binding to its receptor expressed on the ovarian follicles.
      While the evidence from clinical studies supports the administration of concurrent GnRH administration with chemotherapy to protect ovarian function, the data are still limited regarding post-treatment reproductive function and ovarian reserve. Due to insufficient data, concurrent administration of GnRH agonists with chemotherapy regimens should not be the first-line choice for fertility preservation for breast cancer patients. In malignancies other than breast cancer, GnRH agonists should not be routinely offered as an option for ovarian function protection and fertility preservation without discussing the uncertainty about its benefit (
      • Anderson R.A.
      • Amant F.
      • Braat D.
      • D'Angelo A.
      • Chuva de Sousa Lopes S.M.
      • Demeestere I.
      • Dwek S.
      • Frith L.
      • Lambertini M.
      • Maslin C.
      • Moura-Ramos M.
      • Nogueira D.
      • Rodriguez-Wallberg K.
      • Vermeulen N.
      The ESHRE Guideline Group on Female Fertility Preservation
      ESHRE guideline: female fertility preservation.
      ).

      IVM of oocytes

      Vitrification of mature oocytes harvested after ovarian stimulation has recently become an established method to preserve fertility of cancer patients. But when there is insufficient time or a contraindication for ovarian stimulation, cryopreservation of immature oocytes at the germinal vesicle (GV) stage can be an attractive alternative to cryopreservation of mature oocytes. However, a study investigating mouse GV oocytes after vitrification showed they are vulnerable to cryo-injury and their capacity for normal maturation and fertilization are easily compromised (
      • Van Blerkom J.
      Maturation at high frequency of germinal-vesicle-stage mouse oocytes after cryopreservation: alterations in cytoplasmic, nuclear, nucleolar and chromosomal structure and organization associated with vitrification.
      ). They have to be matured to metaphase II stage before cryopreservation because their maturational capacity after warming is lower than fresh oocytes (
      • Lee J.A.
      • Barritt J.
      • Moschini R.M.
      • Slifkin R.E.
      • Copperman A.B.
      Optimizing human oocyte cryopreservation for fertility preservation patients: should we mature then freeze or freeze then mature?.
      ). Several perturbations have been described such as premature chromosomal condensation, externalization of chromatin fragments into the cytoplasm, perturbations in the organization and distribution of microtubules and mitochondria, and alteration in protein synthesis activity in the cytoplasm during maturation of mouse GV stage oocytes (
      • Van Blerkom J.
      Maturation at high frequency of germinal-vesicle-stage mouse oocytes after cryopreservation: alterations in cytoplasmic, nuclear, nucleolar and chromosomal structure and organization associated with vitrification.
      ).
      Immature oocytes can be retrieved without ovarian stimulation in both follicular and luteal phases before exposure to gonadotoxic chemotherapy regimens, which avoids supraphysiological oestrogen concentrations. Immature oocytes can also be retrieved from antral follicles, and both techniques can be combined (
      • Fasano G.
      • Moffa F.
      • Dechene J.
      • Englert Y.
      • Demeestere I.
      Vitrification of in vitro matured oocytes collected from antral follicles at the time of ovarian tissue cryopreservation.
      ).
      Using immature oocytes for fertility preservation was assessed in a retrospective cohort analysis of 66 breast cancer patients. Patients were divided into two groups: immature oocytes collected and matured in vitro (Group 1, n = 35) and vitrified or fertilized and preserved as vitrified embryos (Group 2, n = 31). The IVM preservation results were (Group 1 versus Group 2, respectively): 11.4 ± 8.8 versus 9.7 ± 6.4 for the average number of retrieved oocytes; 64.2% versus 53.2% for the maturation rates; 7.9 ± 6.6 versus 5.8 ± 2.7 for average numbers of mature oocytes for vitrification or fertilization and lastly 3.8% versus 8.1% for the pregnancy rates per vitrified oocyte and embryo (
      • Shalom-Paz E.
      • Almog B.
      • Shehata F.
      • Huang J.
      • Holzer H.
      • Chian R.C.
      • Son W.Y.
      • Tan S.L.
      Fertility preservation for breast-cancer patients using IVM followed by oocyte or embryo vitrification.
      ). In conclusion, IVM combined with oocyte or embryo freezing can be a feasible option for some breast cancer patients because it bypasses the delay of cancer therapy and potential supraphysiological oestrogen concentration exposure. However, the process requires more expertise than the typical IVF strategy.

      Pregnancy in breast cancer

      An increasing number of women are achieving pregnancy during or after breast cancer. A better understanding of the impact of pregnancy on survival of breast cancer patients is of paramount importance. Breast cancers diagnosed during pregnancy and up to 1 year post-partum are generally more aggressive tumours, characterized by unfavourable clinicopathological features such as high tumour grade, low level of expression of oestrogen and progesterone receptors, in comparison to those breast cancers diagnosed in non-gravid women of the same age (
      • Basaran D.
      • Turgal M.
      • Beksac K.
      • Ozyuncu O.
      • Aran O.
      • Beksac M.S.
      Pregnancy-associated breast cancer: clinicopathological characteristics of 20 cases with a focus on identifiable causes of diagnostic delay.
      ;
      • Iqbal J.
      • Amir E.
      • Rochon P.A.
      • Giannakeas V.
      • Sun P.
      • Narod S.A.
      Association of the timing of pregnancy with survival in women with breast cancer.
      ;
      • Tretli S.
      • Kvalheim G.
      • Thoresen S.
      • Host H.
      Survival of breast cancer patients diagnosed during pregnancy or lactation.
      ). But the impact of pregnancy on survival remains a matter of debate (
      • Amant F.
      • von Minckwitz G.
      • Han S.N.
      • Bontenbal M.
      • Ring A.E.
      • Giermek J.
      • Wildiers H.
      • Fehm T.
      • Linn S.C.
      • Schlehe B.
      • Neven P.
      • Westenend P.J.
      • Muller V.
      • Van Calsteren K.
      • Rack B.
      • Nekljudova V.
      • Harbeck N.
      • Untch M.
      • Witteveen P.O.
      • Schwedler K.
      • Thomssen C.
      • Van Calster B.
      • Loibl S.
      Prognosis of women with primary breast cancer diagnosed during pregnancy: results from an international collaborative study.
      ;
      • Azim Jr., H.A.
      • Santoro L.
      • Russell-Edu W.
      • Pentheroudakis G.
      • Pavlidis N.
      • Peccatori F.A.
      Prognosis of pregnancy-associated breast cancer: a meta-analysis of 30 studies.
      ;
      • Hartman E.K.
      • Eslick G.D.
      The prognosis of women diagnosed with breast cancer before, during and after pregnancy: a meta-analysis.
      ;
      • Iqbal J.
      • Amir E.
      • Rochon P.A.
      • Giannakeas V.
      • Sun P.
      • Narod S.A.
      Association of the timing of pregnancy with survival in women with breast cancer.
      ;
      • Jerzak K.J.
      • Lipton N.
      • Nofech-Mozes S.
      • Boles D.
      • Slodkowska E.
      • Pond G.R.
      • Warner E.
      Clinical outcomes and prognostic biomarkers among pregnant, post-partum and nulliparous women with breast cancer: a prospective cohort study.
      ;
      • Park S.
      • Lee J.S.
      • Yoon J.S.
      • Kim N.H.
      • Park S.
      • Youn H.J.
      • Lee J.W.
      • Lee J.E.
      • Lee J.
      • Hur H.
      • Jeong J.
      • Kim K.C.
      • Bae S.Y.
      The risk factors, incidence and prognosis of postpartum breast cancer: a nationwide study by the SMARTSHIP Group.
      ). Surgery for breast cancer is not harmful for pregnancy, but chemotherapy is not recommended during the first trimester of pregnancy. Hormonal and targeted therapies and radiation are contraindicated throughout pregnancy. The choice of treatment can therefore be complicated, as there is conflict between the ideal treatment for the mother and the well-being of the fetus. If a woman is diagnosed with breast cancer early in pregnancy and needs urgent chemotherapy, termination of pregnancy should be considered. In short, women diagnosed with breast cancer during or shortly after pregnancy should be informed thoroughly about the risks during counselling.

      Other considerations in fertility preservation

      Aside from the above-mentioned medical concerns, the psychological aspects of infertility treatments in breast cancer patients are relatively understudied. In reviewing existing literature from 1996 to 2008,
      • Tschudin S.
      • Bitzer J.
      Psychological aspects of fertility preservation in men and women affected by cancer and other life-threatening diseases.
      revealed that infertility among breast cancer patients represents issues of self-esteem, identity, sexuality and self-image rather than yearning to have a child. Another moderately understudied topic is the practice manners of physicians in guiding patients with cancers to fertility preservation centres. Numerous patients discern that their primary physicians for cancer therapy do not adequately advise them about the consequences of cancer therapy on fertility or the possibilities of preserving it. However, some oncologists are less concerned about the impact of a treatment on fertility and rarely guide them to reproductive endocrinologists. For example, 30% rarely consider a woman's desire for fertility when scheduling treatment. On the other hand, gynaecological oncologists are more likely to consider fertility than other oncologists (93% versus 60%). They are also more likely to administer a less gonadotoxic regimen to support preserving fertility (61% versus 37%). Most oncologists (86%) would be ready to sacrifice less than a 5% reduction in disease-free survival if a regimen offered better fertility outcomes; 36% of patients would be willing to sacrifice >5% (
      • Forman E.J.
      • Anders C.K.
      • Behera M.A.
      Pilot survey of oncologists regarding treatment-related infertility and fertility preservation in female cancer patients.
      ).
      A survey based on the American Medical Association Physician Masterfile database revealed that although the ASCO guidelines recommend that “all patients of childbearing age should be informed about fertility preservation”, less than half of US physicians are following these guidelines. However, female physicians are more likely to refer such patients to a reproductive endocrinologist (
      • Quinn G.P.
      • Vadaparampil S.T.
      • Lee J.H.
      • Jacobsen P.B.
      • Bepler G.
      • Lancaster J.
      • Keefe D.L.
      • Albrecht T.L.
      Physician referral for fertility preservation in oncology patients: a national study of practice behaviors.
      ). These findings reveal that fertility preservation is still inadequately understood among physicians. Furthermore, it is crucial to underline the vital position of oncologists at tertiary centres in advising and guiding cancer patients with worries about their future fertility.
      Although there seems to be no increasing trend in overall divorce rates in patients with cancer, there is a gender discrepancy in these rates. An intriguing investigation examining the role gender played in so-called ‘partner abandonment’ demonstrated that a woman is six times more likely to be divorced soon after a cancer diagnosis than if a man in the relationship is the patient (20.8% versus 2.9%) (
      • Glantz M.J.
      • Chamberlain M.C.
      • Liu Q.
      • Hsieh C.C.
      • Edwards K.R.
      • Van Horn A.
      • Recht L.
      Gender disparity in the rate of partner abandonment in patients with serious medical illness.
      ), emphasizing the importance of unfertilized gamete storage in female cancer patients with a higher risk of divorce.

      Conclusion

      Breast cancer continues to be the most prevalent neoplasm in women. Many issues need to be addressed in these patients, from diagnosis to therapy-related adverse health conditions. Contemporary breast cancer management needs a multidisciplinary approach. The current review has mainly focused on the reproductive issues and fertility preservation methods in patients with breast cancer. Several options are viable for preserving fertility in patients planning to start chemotherapy for breast cancer. The most appropriate choice should be chosen considering several characteristics, including the patient's age or desire for the length of delay to childbearing post-chemotherapy, the type or the time before starting adjuvant treatment. Oocyte and embryo freezing are currently the most effective strategies to preserve fertility in breast cancer patients. Addition of aromatase inhibitors to ovarian stimulation appears to be a safe approach to reduce endogenous oestrogen concentration. Ovarian tissue cryopreservation should be offered to women without sufficient time for ovarian stimulation. The protective effect of GnRH agonists during chemotherapy on the ovaries against chemotherapy-induced damage and follicle loss continues to be a matter of heated debate. GnRH agonists during chemotherapy can be considered as an option for ovarian function protection in premenopausal breast cancer patients receiving chemotherapy; however, limited evidence exists on their protective effect on the ovarian reserve and the potential for future pregnancies. Therefore, GnRH agonists should not be considered an equivalent or alternative option for fertility preservation but can be offered after cryopreservation techniques or when they are not possible, according to the most recent guidelines of fertility preservation. Individualized detailed genomic analyses of patients and breast tumours in future could help select the most effective chemotherapy regimen while minimizing their gonadotoxic effects on the ovary. It should be remembered that infertility and premature menopause should not only be considered as adverse reproductive outcomes in breast cancer survivors, they can also significantly increase the psychological burden on these patients. Therefore these patients should always be counselled and treated for psychological well-being.

      Appendix. Supplementary materials

      References

        • Abusief M.E.
        • Missmer S.A.
        • Ginsburg E.S.
        • Weeks J.C.
        • Partridge A.H.
        The effects of paclitaxel, dose density, and trastuzumab on treatment-related amenorrhea in premenopausal women with breast cancer.
        Cancer. 2010; 116: 791-798
        • Amant F.
        • von Minckwitz G.
        • Han S.N.
        • Bontenbal M.
        • Ring A.E.
        • Giermek J.
        • Wildiers H.
        • Fehm T.
        • Linn S.C.
        • Schlehe B.
        • Neven P.
        • Westenend P.J.
        • Muller V.
        • Van Calsteren K.
        • Rack B.
        • Nekljudova V.
        • Harbeck N.
        • Untch M.
        • Witteveen P.O.
        • Schwedler K.
        • Thomssen C.
        • Van Calster B.
        • Loibl S.
        Prognosis of women with primary breast cancer diagnosed during pregnancy: results from an international collaborative study.
        J. Clin. Oncol. 2013; 31: 2532-2539