Fertility in cancer patients after cryopreservation of one ovary

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1 Reproductive BioMedicine Online (2013) 26, ARTICLE Fertility in cancer patients after cryopreservation of one ovary KT Schmidt a,b, *, A Nyboe Andersen a, T Greve b, E Ernst c, A Loft a, C Yding Andersen b a The Fertility Clinic, Copenhagen University Hospital, Rigshospitalet 9, DK-2100 Copenhagen, Denmark; b The Laboratory of Reproductive Biology, Copenhagen University Hospital, Rigshospitalet, DK-2100 Copenhagen, Denmark; c The Fertility Clinic, University Hospital of Aarhus, 8200 Skejby, Aarhus, Denmark * Corresponding author. address: kirsten.tryde.schmidt@rh.regionh.dk (KT Schmidt). Kirsten Tryde Schmidt is a consultant at the Fertility Clinic at Copenhagen University Hospital. In 2005 she defended her PhD thesis on cryopreservation of ovarian tissue in young cancer patients. Since then, her main interest has been fertility preservation, and she now leads the clinical programme on fertility preservation in cancer patients in Copenhagen. Since January 2012, she has also worked part time at Complete Fertility Centre in Southampton, England to head the fertility preservation programme there. Abstract This questionnaire study describes the fertility and ovarian function in 143 adult female cancer survivors with only one ovary due to cryopreservation of the other. The women were asked about their ovarian function (as defined by the presence of a spontaneous menstrual cycle), pregnancies and their outcome. The mean follow-up time was 58 months after cryopreservation (range months). The risk of premature ovarian failure was high in the group of patients with leukaemia (13/15; 87%) but low in the breast cancer group (5/54; 9%). Fifty-seven women had actively tried to become pregnant after end of treatment; of these, 41 women obtained a total of 68 pregnancies resulting in 45 live births and five ongoing pregnancies, 15 spontaneous abortions, one ectopic pregnancy and two elective abortions. In the remaining 86 women without a pregnancy wish, there had been five elective abortions. Ninety-three per cent of the pregnancies were after natural conception and only four cases were a result of fertility treatment. The overall risk of premature ovarian failure was low (22%). Patients who retain their ovarian function after treatment of a malignant disease have a good chance of becoming pregnant. RBMOnline ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cancer, chemotherapy, cryopreservation, fertility, ovarian failure, ovary Introduction A side effect to cancer treatment with chemotherapy and/or radiation therapy in girls and premenopausal women is premature ovarian failure (POF). The risk is dependent on age of the patient, dose and type of drugs used, with an increased risk with increasing age, treatment with alkylating agents, abdominal irradiation or bone marrow transplantation (BMT) (Larsen et al., 2003; Meirow, 2000; Wallace et al., 2005a,b). To circumvent this unwanted and, for many women, serious side effect, cryopreservation of ovarian tissue has been developed as a means of preserving fertility /$ - see front matter ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

2 Fertility after cancer treatment 273 (Tryde Schmidt et al., 2004; von Wolff et al., 2009). Thawing and transplantation of pieces of the cryopreserved ovarian tissue to the woman from whom it originated has lead to restoration of ovarian function, as confirmed by return of menses and endogenous hormone production in many women (Bedaiwy et al., 2008) and to the birth of 19 children worldwide (Andersen et al., 2008, 2012; Demeestere et al., 2007, 2010; Dittrich et al., 2012; Donnez et al., 2004, 2011; Ernst et al., 2010; Meirow et al., 2005; Revel et al., 2011; Roux et al., 2010; Sanchéz-Serrano et al., 2010; Silber, 2012; Silber and Gosden, 2007; Silber et al., 2005, 2008a,b, 2010). However, this procedure should ideally only be offered to those patients who actually become menopausal as a result of their treatment and it remains uncertain who will develop POF (Anderson and Wallace, 2008). Most clinics offering ovarian tissue cryopreservation for fertility preservation agree that there should be a certain risk of POF, at least around 30 50%, before this procedure should be offered to cancer patients (Anderson et al., 2008; Moffa and Biacchiardi, 2007; von Wolff et al., 2009). However, treatment protocols vary and have developed over time and often consist of several types of drugs in combination. Therefore, it is difficult to predict the individual risk of ovarian failure. Secondly, the removal of one ovary will halve the overall ovarian reserve of the woman and could by itself negatively influence fertility. The purpose of this study was to assess both ovarian function and fertility in patients, who have had cryopreservation of one ovary performed before gonadotoxic treatment. This is of great importance to the oncologist, fertility specialist or general practitioner who is counselling the patient before or after cancer treatment. This study centre has offered ovarian tissue cryopreservation since 1999 and have thus accumulated a large cohort over time, offering a unique possibility to perform this study. Further, easy accessibility of each individual patient via her own personal identification number is possible in Denmark, which makes loss to follow up negligible. Materials and methods As shown in the flow chart in Figure 1, the study population was selected among a cohort of 410 women who had ovarian cryopreservation from 1999 to Of these 410 patients, 57 later died from their disease. Included women were above 18 years of age who had undergone unilateral oophorectomy over 24 months prior to the study (between 1999 and 2008) for fertility preservation due to a planned treatment with chemotherapy and/or radiation therapy. Excluded women were those who had received transplantation with frozen thawed ovarian tissue (n = 17), girls younger than 18 at follow up and women with ovarian tissue cryopreservation for less than 24 months. A questionnaire was posted to all participants. It included data on menstrual cycle, pregnancies after treatment and their outcome, use of hormones and birth control. Additionally, the women were asked about the treatment they had received and whether they intended to use their cryopreserved tissue in the future if necessary. Background cohort n=410 Eligible for study n=191 Returned the questionnaire n=149 Study population n=143 Not meeting inclusion criteria n=202 died n=57 <18 years n=49 < 24 months since cryopreservation Autotransplantation n=17 Excluded n=42 did not return questionnaire n=33 Untracable adress n=6 Emigrated n=3 Participation rate 78% Excluded due to bilateral oophorectomy n=6 Figure 1 Flow chart showing the cohort of girls and women with cryopreserved ovarian tissue from 1999 to June 2010 at Rigshospitalet, Copenhagen, Denmark and the study population. In total, 191 women fulfilled the inclusion criteria and their addresses were found in a Danish central register via each woman s personal identification number. The address was untraceable in six cases, and three women had emigrated. In total, 33 women did not return the questionnaire or did not want to participate, but 149 women filled out and returned the questionnaire (participation rate 78%). Six were excluded due to bilateral oophorectomy and thus a total of 143 women participated in the study. The mean follow-up time of these women was 58 months (range months). As shown in Table 1, the diagnoses of the 143 women were as follows: 54 with breast cancer (38%), 40 with lymphoma (29 Hodgkin, eight non-hodgkin, three unknown histology; 28%), 15 with leukaemia (10%), nine with sarcoma (6%) and 15 with other malignant diagnoses (three brain tumours, four ovarian cancers, two molas, two cervical cancers, two colon cancers, one kidney tumour and one pharynx cancer) (10%). Additionally the study included 10 women who had benign non-malignant diagnoses, but all of whom were exposed to a potentially gonadotoxic treatment: seven with an autoimmune disease (5%) and three with aplastic anaemia (2%).

3 274 KT Schmidt et al. Table 1 Patient characteristics of 143 women with cryopreserved ovarian tissue. n Age (years, mean, range) Treatment (n) At cryopreservation At follow up Chemotherapy only Irradiation below the diaphragm + chemotherapy Preconditioning protocol before BMT *** Breast cancer (22 38) 35.5 ( ) 54 Lymphoma (16 34) 29.1 ( ) 36 4 Sarcoma (13 27) 24.4 ( ) 8 1 Leukaemia (13 31) 27.3 ( ) 3 12 Other malignant (15 34) 28.1 ( ) 11 4 Aplastic anaemia 3 25 (23 26) 29.8 ( ) 3 Autoimmune diseases (16 28) 28.4 ( ) 7 Total BMT *** = bone marrow transplantation. All women had undergone a laparoscopic unilateral oophorectomy prior to treatment for fertility preservation as described previously. The cortex was isolated 1 2 mm in thickness, cut into fragments of 5 5 mm and cryopreserved using the slow protocol. Ethylene glycol was used as cryoprotectant (Schmidt et al., 2003a). In Denmark cryopreservation of ovarian tissue is centralized to one centre, so patients living in other parts of the country were operated on locally, and the ovary was transported cooled on ice for up to 4 h to Copenhagen, where the actual cryopreservation took place (Schmidt et al., 2003b). The women were selected for oophorectomy on the basis of a one-to-one counselling from a fertility specialist with special interest in fertility preservation based on the following criteria: 30 50% assumed risk of ovarian failure, <35 years of age, no disseminated disease and no contraindications against the operative procedure or anaesthesia. Details of ethical approval The study was approved by the Danish Data Protection Agency ( ). According to Danish legislation, questionnaire studies do not need ethical approval. Cryopreservation of ovarian tissue was originally approved by the Minister of Health and by the ethical committee of Copenhagen and Frederiksberg (J/KF/01/170/99). Results Treatment Table 1 includes data on treatment with chemotherapy, irradiation below the diaphragm and BMT. It is seen that, among the 143 women, 119 received chemotherapy alone for their disease, four received chemotherapy and radiotherapy below the diaphragm in combination and 20 had a BMT. Fertility before treatment Among the 143 women, 50 (35%) had been pregnant at least once before cryopreservation of their ovaries, and these pregnancies had resulted in the delivery of a total of 38 children to 31 women. Thus 31/143 (22%) were parous. Ovarian function after treatment Table 2 gives the number of women with self-reported POF as well as the number with intact ovarian function after treatment, according to diagnosis. Intact ovarian function was defined according to whether or not the woman still had a menstrual cycle without the use of hormonal contraception. Apart from the small group of women with autoimmune diseases (n = 7) in which none reported amenorrhoea, the highest chance of intact ovarian function despite gonadotoxic treatment was found in the breast cancer group, where 46 (85%) maintained a menstrual cycle. In three breast cancer patients (6%) it was uncertain whether the ovarian function was intact or not as they were on oral contraception or had an intrauterine system containing a progestogen. Only five breast cancer patients (9%) were amenorrhoeic; two of these had had a recurrence of their disease requiring treatment with additional chemotherapy. The women with the highest risk of POF were found in the group with leukaemia, where 13/15 (87%) were amenorrhoeic; all but one had received BMT. In the remaining groups, the chance of an intact ovarian function was between 56 73%. Table 3 gives the history of the women reporting to have POF. Fertility after treatment Among the 57 women who had tried to become pregnant after their treatment, 41 (72%) had succeeded. The mean follow-up period of these women was 66 months (range months). Of the whole sample population, 84 women had not yet wished to conceive, but five had experienced an unwanted pregnancy. Thus, a total of 46 women (32%) had been pregnant after their treatment. The mean follow-up period of those without a pregnancy wish was 53 months (range ). In the 86 women without a pregnancy wish, eight (9%) had been advised against a pregnancy by their oncologist and 11 (13%) were still on anti-oestrogenic treatment.

4 Fertility after cancer treatment 275 Table 2 POF diagnosis Number of women with/or without premature ovarian failure according to diagnosis. Breast cancer (n = 54) Lymphoma (n = 40) Leukaemia (n = 15) Sarcoma (n=9) Autoimmune disease (n =7) Aplastic anaemia (n =3) Others (n = 15) Positive 5 (9) 7 (18) 13 (87) 2 (22) 0 1 (33) 3 (20) Negative 46 (85) 27 (68) 0 5 (56) 5 (71) 2 (67) 11 (73) Uncertain 3 (6) 6 (15) 2 (13) 2 (22) 2 (29) 0 1 (7) Values are n (%). Table 3 Patient Medical history in 31 women with premature ovarian failure after treatment. Diagnosis Treatment Age at diagnosis Age at follow up Recurrence of disease 1 ALL BMT ** Yes 2 CML BMT No 3 Sarcoma BMT Yes 4 ALL BMT No 5 CML BMT Yes 6 CML BMT Yes 7 ALL BMT No 8 ALL BMT No 9 Ovarian cancer Chemotherapy alone No 10 AML BMT No 11 Hodgkin Chemotherapy alone Yes 12 AML BMT No 13 Hodgkin Chemotherapy alone No 14 Hodgkin BMT Yes 15 Hodgkin BMT Yes 16 AML BMT No 17 Hodgkin Chemotherapy alone No 18 Hodgkin Chemotherapy alone Yes 19 AML BMT No 20 ALL Chemotherapy alone No 21 Sarcoma Chemotherapy alone No 22 Breast cancer Chemotherapy alone No 23 AML BMT No 24 Brain tumour Chemotherapy alone Yes 25 Cervical cancer Chemotherapy and No radiation 26 Non-Hodgkin BMT No 27 Breast cancer Chemotherapy alone Yes 28 Breast cancer Chemotherapy alone Yes 29 Breast cancer Chemotherapy and No radiation 30 Aplastic BMT No anaemia 31 Breast cancer Chemotherapy alone No ALL = acute lymphoblastic leukaemia; AML = acute myeloid leukaemia; BMT ** = bone marrow transplantation; CML = chronic myeloid leukaemia. Of the 46 women who had been pregnant after treatment 28 had been pregnant only once, 13 twice, three patients three times and two patients had been pregnant five times. Thus, the 46 women experienced a total of 73 pregnancies. The course of the 73 pregnancies was as follows: 45 ended in a live birth, five are currently ongoing, 15 ended in a spontaneous abortion, seven pregnancies were unwanted and terminated, and one pregnancy was ectopic. Forty-four of the 45 pregnancies that were carried to term were singleton pregnancies and one was a twin pregnancy. Of the 46 children born, 29 were boys and 17 were girls. The mean gestational age at delivery was 38.5 weeks

5 276 KT Schmidt et al. Table 4 Number of women with a pregnancy wish who have obtained a pregnancy and delivered in each of the seven diagnostic groups. Diagnosis n Women with a pregnancy wish Women obtaining a pregnancy Breast cancer (73) 14 (64) Lymphoma (74) 13 (68) Leukaemia Sarcoma (100) 1 (33) Autoimmune disease Aplastic anaemia Others (89) 6 (67) Total (72) 34 (60) Values are n or n (% of women with a pregnancy wish). Women delivered or with an ongoing pregnancy (range weeks) and the mean birthweight was 3442 g (range g). Sixty-eight of the 73 pregnancies were spontaneous conceptions (93%), three were a result of intrauterine insemination, one pregnancy was after IVF and in one pregnancy, the origin was not stated. Of the 41 women who had become pregnant spontaneously, 20 stated that they became pregnant within the first month of trying, seven had become pregnant after 1 3 months, eight after 3 6 months, four after 12 months and in two cases the time to pregnancy was not stated. In Table 4, the number of women with an active pregnancy wish, number of pregnancies and deliveries can be seen for each of the seven diagnostic groups. Attitudes towards future motherhood One hundred (70%) of the 143 women returning the questionnaire stated that they would like to become pregnant in the future, 19 (13%) did not, 17 (12%) were uncertain and seven patients (5%) did not answer that question. Of the 19 patients who did not want to become pregnant in the future, 12 already had children before their malignancy or after their treatment. When asked whether they thought they would like to make use of their cryopreserved tissue in the future if it became necessary, 115 (80%) women said yes, 21 (15%) said no, three did not know and four patients did not answer the question. Discussion When offering cryopreservation of ovarian tissue for fertility preservation, the clinician has to optimize the balance between the expected advantages and risks of this procedure. The risk of ovarian failure due to the planned treatment has to be outweighed against the risk of any complications occurring during the collection of the ovarian tissue, which could potentially harm the patient and possibly postpone chemotherapy or radiation therapy. Secondly, removing half of the woman s stock of primordial follicles may by itself shorten her reproductive life span. Finally, by removing one ovary, the normal function of the contralateral oviduct is critical in order to conceive. POF was defined according to whether the woman had a menstrual cycle or not. This may seem a crude assessment of ovarian function, but this study centre has previously shown in a follow up of 92 women with cryopreserved ovarian tissue that regular menstruation was a good indicator of ovarian function (Rosendahl et al., 2008). The present study found that the risk of POF after treatment was not as high as initially anticipated. Only 31 of the 143 participating women (21.7%) became amenorrhoeic after treatment, and of these more than half (n = 17) had received BMT. Indeed, BMT following a preconditioning protocol consisting of high-dose alkylating agents and/or total body irradiation has been associated with a high risk of POF (Schmidt et al., 2010). This constitutes a paradox, since the patients who most need ovarian tissue cryopreservation are also those in whom the study centre is most reluctant to autotransplant the tissue after the patient has been cured due to the possible risk of reintroducing the disease in the case of leukaemia. For early stage breast cancer patients and lymphoma patients, on the other hand, this risk is small and several studies now demonstrate that the risk of malignant cell contamination in the ovaries of these patients is low (Meirow et al., 2008; Rosendahl et al., 2011; Seshadri et al., 2006; Sánchez-Serrano et al., 2009). The reason why this study centre continues to offer ovarian tissue cryopreservation to leukaemia patients, however, is that many of them are very young at the time of diagnosis and thus with time may benefit from the probable technological progress in the years to come, such as in-vitro maturation of primordial follicles or enzymic isolation of follicles from the thawed tissue. Very encouragingly, this study centre recently published a study on 25 leukaemia patients who had some of their cryopreserved thawed ovarian tissue transplanted for 20 weeks to 25 nude mice and found that none of the mice revealed any signs of disease (Greve et al., 2012). It appears thus, that the ovaries from leukaemia patients in complete remission do not seem to contain viable malignant cells, which may make it safe to transplant tissue from these patients. But of course, further studies are needed before offering this to leukaemia patients. Among the women with a malignant disease, the group of women with the best chance of maintaining menstrual cycles after treatment was women with breast cancer, where at least 85% had an intact ovarian function after treatment with chemotherapy. In Denmark the standard

6 Fertility after cancer treatment 277 protocol to premenopausal women with early stage operable breast cancer is three series of cyclophosphamide and epirubicine followed by three series of taxotere. Each series of cyclophosphamide consists of 600 mg/m 2 body surface, and thus three series in a normalweight patient corresponds to a total of 3000 mg of cyclophosphamide. It is well known that alkylating agents such as cyclophosphamide are gonadotoxic, but it is also known that the risk of POF is dependent on the cumulative dose given and the age of the patient, with a higher risk the higher the dose and the older the patient (Abir et al., 2008; Boumpas et al., 1993; Meirow, 2000; Meirow et al., 1999). The incidence of POF in women treated for breast cancer varies in the literature. In a large study of 595 women with breast cancer it was found that the long-term incidence of amenorrhoea (3 years after diagnosis) in women <35 years was similar to that of women who did not receive chemotherapy (10%), but it increased to 50% in women aged years and to 85% in women >40 years (Petrek et al., 2006). A recent review concluded that young women <35 years are less likely to experience POF and that anthracycline-based protocols appear to have a lower incidence of POF than the older protocols (Hickey et al., 2009). Based on the findings in the present study, it appears that women following the study centre s protocol for the treatment of breast cancer have only a minor risk of becoming infertile and may not require ovarian tissue cryopreservation, at least not in the younger women. Interestingly, the patients with autoimmune diseases (n = 7) all retained an intact ovarian function despite treatment with chemotherapy. This is contrary to other studies which have found a risk of POF of up to 60% in women <40 years of age treated with cyclophosphamide for systemic lupus erythematosus (Laskari et al., 2010; Manger et al., 2006). Fifty-seven of the 143 women (40%) have so far tried to obtain a pregnancy and 72% (n = 41) have succeeded. Thus, a reassuringly high percentage of the women with a pregnancy wish actually do conceive, and some have more than one child and consider their families complete. With 45 live births and five ongoing pregnancies there seems to be a sound fertility potential in this group of women without making use of the cryopreserved ovarian tissue. It appears thus, that removing half the stock of the woman s primordial follicles does not seem to harm her fertility, since 72% of the women with a pregnancy wish have so far succeeded in obtaining a pregnancy, which is in accordance with a previous study on women with just one ovary, who have fertility as equally good as those with two ovaries (Lass, 1999). Overall, most pregnancies were found in the group with breast cancer, but also women having suffered from lymphoma, sarcoma or other malignancies had a good prognosis. Not surprisingly, no pregnancies were found in the leukaemia group. Of all the 46 women who had been pregnant after treatment, 42 had achieved their pregnancy spontaneously, and 20 women (48%) had become pregnant within the first month of trying and another nine women (21%) within the first 3 months of trying. Thus, two-thirds of the women obtained a pregnancy within 3 months, which is comparable to time to pregnancy in a normal population (Bonde et al., 1998; Zinaman et al., 1996). With a mean gestational age of 38.5 weeks and a mean birthweight of 3442 g, there does not seem to be any adverse effects of a previous cancer diagnosis in terms of the woman s ability to carry a pregnancy to term and to provide a healthy environment for the baby in utero. As a curious finding, two-thirds of the children born were boys. There is no ready explanation to this apparently skewed sex ratio, but the numbers are of course small. The majority of the women participating in this study have not actively tried to become pregnant yet (n = 84). It will obviously be interesting to continue to follow the fertility in this group of women, to observe whether it differs from the women, who had already actively tried to become pregnant. A proportion of the women (n = 33) had not returned the questionnaire. It could be that the women who choose not to participate did so because of an unfulfilled pregnancy wish and would have found it too painful to be confronted with their unwanted infertility. This study cannot provide this information, but future long-term follow-up studies will hopefully give a more realistic picture of the fertility in female cancer survivors with only one ovary due to cryopreservation of the other. Another point to be considered when looking at ovarian function in female cancer survivors is the long-term risk of POF. Although most of the women in this study seem to have an intact ovarian function more than 2 years after cancer treatment, previous studies have shown that these women may have a partial loss of ovarian function despite a regular menstrual cycle (Bath et al., 2003; Larsen et al., 2003). Larger and longer follow-up studies are obviously needed to assess the long-term risk of POF in female cancer survivors. It is reassuring, however, that women with only one ovary do not seem to have a reduced fertility potential to conceive, either naturally or via IVF (Lass, 1999), although it still remains uncertain at what age they will enter menopause. The follicular depletion that invariably occurs in connection with cancer treatment, whether or not one ovary has been removed, makes it difficult to predict the age of menopause in these women. But 80% of the women in this study stated that they intended to use the cryopreserved tissue if it became necessary. Thus, the option remains for the women to use their tissue to maintain menstrual cycles, fertility and endogenous hormone production. Finally, experience from this study group and others show that, for those women who do become menopausal as a result of their cancer treatment, autotransplantation of the cryopreserved ovarian tissue may ensure long-term duration of ovarian function, thus offering them a chance of conceiving and of avoiding the side effects of POF (Andersen et al., 2012; Grudzinskas, 2012; Lieberman, 2012). In conclusion, this study found a fairly low overall risk of POF of 22% in a cohort of women with cryopreserved ovarian tissue. The risk was highest in the group with leukaemia or other malignancies requiring treatment with BMT and lowest in the women with breast cancer. It is recommended that women suffering from a disease requiring treatment with BMT should be offered fertility preservation and that women suffering from cancers not requiring treatment with BMT should be offered fertility preservation after assessment of the planned protocol and individual counselling. Younger women suffering from breast cancer may not need cryopreservation of ovarian tissue with the actual protocol

7 278 KT Schmidt et al. employed. For female cancer survivors who have received a potentially gonadotoxic treatment and have had one of their ovaries removed for cryopreservation, it is reassuring to know that most of them will not lose their ovarian function, at least not during the first years after treatment, and that it will allow many of them to be able to obtain a pregnancy during that period. Acknowledgements The financial support from the Danish Agency for Science and Innovation (grant no ) is greatly appreciated. References Abir, R., Ben-Haroush, A., Felz, C., Okon, E., Raanani, H., Orvieto, R., Nitke, S., Fisch, B., Selection of patients before and after anticancer treatment for ovarian cryopreservation. Hum. Reprod. 23, Andersen, C.Y., Rosendahl, M., Byskov, A.G., Loft, A., Ottosen, C., Dueholm, M., Schmidt, K.L.T., Andersen, A.N., Ernst, E., Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue. Hum. 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