Anti-Müllerian hormone follow-up in young women treated by chemotherapy for lymphoma: preliminary results

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1 Reproductive BioMedicine Online (2009) 20, ARTICLE Anti-Müllerian hormone follow-up in young women treated by chemotherapy for lymphoma: preliminary results Christine Decanter a,b,, Franck Morschhauser b,c, Pascal Pigny b,d, Catherine Lefebvre a,b,cécile Gallo a,b, Didier Dewailly a, a Department of Endocrine Gynaecology and Reproductive Medicine, Hôpital Jeanne de Flandre, CHRU, Lille, France; b Faculty of Medicine, Université de Lille II, Lille, France; c Department of Haematology, Hôpital Huriez, CHRU, Lille Cedex, France; d Laboratory of Endocrinology, Parc Eurasanté, CHRU, Lille, France Corresponding authors. addresses: christine.decanter@chru-lille.fr (C Decanter), ddewailly@chru-lille.fr (D Dewailly). Christine Decanter is a medical doctor in endocrinology and reproductive medicine working in the IVF Centre of the Lille University Hospital, France. She is responsible for fertility preservation at the Centre and her special interests are in PCOS, hormonal markers during ovarian stimulation and ovarian preservation. Abstract Susceptibility of the ovarian reserve to chemotherapy is highly variable from one patient to another and poorly documented. To better characterize the evolution of follicular depletion in patients treated for lymphoma, serum anti-müllerian hormone (AMH) assay was used. A total of 30 young women (mean age 24 years) were prospectively recruited before initiation of chemotherapy for lymphoma. They were assigned either to an adriamycin, bleomycin, vinblastine and dacarbazine protocol (ABVD group) or to a protocol that included cyclophosphamide (non-abvd group). AMH assays were performed before and during chemotherapy, and then every 3 months after the end of treatment for a period of 1 year. In all patients, AMH concentrations fell drastically just after the start of chemotherapy and were close to the detection limit at the end of the treatment. In the ABVD group, AMH concentrations increased from the third month after the end of chemotherapy and returned to pretreatment values 12 months after the end of chemotherapy. Conversely, no significant change was observed in the non-abvd group throughout the follow-up period. In conclusion, longitudinal analysis of AMH during chemotherapy highlights differences between protocols that could contribute to an understanding of ovarian toxicity and, ultimately, be useful in fertility preservation counselling. RBMOnline ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: AMH, chemotherapy, follicular depletion, follow-up, lymphoma /$ - see front matter ª 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 AMH follow-up after chemotherapy 281 Introduction With the continuous improvement of cure rate over the last decades, reproductive capacity after treatment of lymphoma has become an important quality-of-life issue. Chemotherapy protocols for Hodgkin s lymphoma or non- Hodgkin s lymphoma are notoriously ovariotoxic by damaging primordial, pre-antral and antral follicles (Familiari et al., 1993; Lutchman Singh et al., 2005; Marcello et al., 1990; Meirow and Nugent, 2001; Meirow et al., 1999). This occurs mainly through an apoptotic process (Meirow, 2000; Meirow and Nugent, 2001; Oktem and Oktay, 2007a; Perez et al., 1997) but also by inducing cortical fibrosis and blood vessel injury (Meirow et al., 2007). Most of these protocols abruptly arrest ovarian function during the treatment period. In some women, particularly in those who received alkylating agents, damage is permanent, leading to premature ovarian failure and infertility (Blumenfeld et al., 2002; Lutchman Singh et al., 2005; Meirow and Nugent, 2001). In other cases, recovery may occur, but the ovarian recovery pattern after discontinuing chemotherapy is highly variable. Patient age, type of drug and dose-dependent effects account partly for this heterogeneity (Familiari et al., 1993; Meirow et al., 1999). However, other pathophysiological mechanisms may underlie these differences. Lastly, while the follicular toxicity of alkylating agents such as cyclophosphamide is well documented (Blumenfeld et al., 2002; Lutchman Singh et al., 2005; Meirow and Nugent, 2001), that of multi-agent regimens remains poorly documented. Most of the studies that have addressed the question of fertility after lymphoma treatment have focused on markers such as cycle length, pregnancy occurrence and/or basal serum FSH concentrations (Byrne et al., 1992; Mackie et al., 1996). Nevertheless, recent studies have shown that follicular depletion may occur despite maintenance of regular menstrual cycles (Bath et al., 2003; Larsen et al., 2003). Therefore, more accurate indicators are needed in order to properly inform the women about their reproductive capacity after treatment. They may also help in determining adequate fertility preservation strategies. Recent studies have suggested that anti-müllerian hormone (AMH) could be a valuable indicator of follicular depletion in women treated for breast cancer (Anderson et al., 2006; Lutchman Singh et al., 2007; Rosendahl et al., 2009) or for lymphoma (Bath et al., 2003; Lie Fong et al., 2008; Rosendahl et al., 2009; van Beek et al., 2007). AMH is secreted by granulosa cells (GC) of preantral and antral follicles and appears to be the best marker of growing follicles (Durlinger et al., 2002; Seifer and Maclaughlin, 2007). Indeed, AMH concentrations correlate closely to the antral follicle count at ultrasonography (Fanchin et al., 2003a; van Rooij et al., 2002). Although this hormone is not secreted by primordial follicles, its serum concentration is considered to accurately reflect the primordial follicle pool since the number of growing follicles is tightly related to the number of primordial follicles (Gougeon et al., 1994). Moreover, serum AMH concentration does not change significantly with the menstrual cycle (Hehenkamp et al., 2006; Streuli et al., 2008; Tsepelidis et al., 2007) and seems to be poorly influenced by gonadotrophin suppressing treatments (Mohamed et al., 2006; Somunkiran et al., 2007; Streuli et al., 2008), except in case of gonadotrophin-releasing hormone (GnRH) agonist suppression longer than 6 consecutive months (Anderson et al., 2006). Lastly, AMH assays are easier to perform and more reproducible than the antral follicle count (Fanchin et al., 2005). A prospective clinical and hormonal follow-up of all of the lymphoma patients was designed before they started their treatment. This study took advantage of AMH as a convenient and non-invasive marker to assess the acute and long-term effects of different chemotherapy regimens on the ovarian follicles. Serial and repeated serum AMH measurements were performed prior to, during and after treatment in young women with either Hodgkin s lymphoma or non-hodgkin s lymphoma. The results presented here were obtained in the first 30 patients who completed at least 1 year of this follow-up. Materials and methods Subjects A total of 30 young women (mean age 24 ± 4.7 years; years) with a diagnosis of Hodgkin s lymphoma (n = 22) or non-hodgkin s lymphoma (n = 8) according to WHO classification (Harris et al., 1999) were recruited before initiation of chemotherapy from 2006 to Of the 30 women, 17 were assigned to an adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) protocol (ABVD group; four cycles in nine cases and eight cycles in eight cases) and 13 to a protocol that included potentially more ovariotoxic drugs such as cyclophosphamide, ifosfamide or melphalan (R-CHOP, ACVBP, BEAM, BEACOPP and MINE) (non-abvd group). Chemotherapy protocols are detailed in Table 1. The choice of these protocols was based on severity and extent of the haematologic disease. Each of them was randomized. All of the patients received a once-monthly GnRH agonist injection (leuprorelin acetate, Enantone 3.75 mg; Takeda, France) during the whole treatment period, according to Table 1 Protocol Details of the drugs and doses used in chemotherapy protocols. Drug and dose (mg/m 2 ) ABVD Adriamycin 25, bleomycin 10, vinblastine, dacarbazine 375 R-CHOP Adriamycin 50, cyclophosphamide 750, vincristine 1.4, prednisone 40 ACVBP Adriamycin 75, cyclophosphamide 1200, bleomycin 10, vindesin 2 BEACOPP Adriamycin 25, cyclophosphamide 650, etoposide, procarbazine 100, vincristine 1.4 BEAM BCNU 300, etoposide 100, cytarabine 200, melphalan 140, procarbazine 100, prednisone 40 MINE Mitoguazon 500, etoposide 150, ifosfamide 1500, vinorelbine 15

3 282 C Decanter et al. the study centre s policy. The advantage of this treatment is that it avoids bleeding during chemotherapy and it has a gonadotrophin-suppressing effect. Each of the 30 patients was followed for at least 1 year according to the protocol described below. The study excluded those patients who had an ovary removed for cryopreservation. Study design Serum samples for AMH assays were obtained and frozen for further assays before initiation of chemotherapy (AMH0), 15 days after the first cycle of chemotherapy (AMH1), 15 days before the last cycle of chemotherapy (AMH2) and every 3 months after the end of chemotherapy (AMH+3, +6, +9 and +12) for each patient. This prospective longitudinal study has been approved by the institutional review board of the Lille University Hospital and patients gave their informed consent. AMH assay This study used a new second-generation enzyme immunoassay for AMH (Beckman-Coulter-Immunotech, Villepinte, France), with greater sensitivity (detection limit of 0.7 pmol/l and a functional sensitivity of 2.5 pmol/l) to measure AMH concentrations. Intra- and inter-assay coefficients of variation were <5.5% and <9.0%, respectively. Statistical analysis Significant differences between frequencies or mean values of the two groups were calculated by using the chi-squared test or the non-parametric Mann Whitney U-test, respectively. ANOVA with post-hoc analysis using the Bonferroni correction was used to detect any significant variation of the tested parameters between the different times of sampling. All statistic procedures were run on Statistical Package for Social Sciences version 11.5 (SPSS Inc., Chicago, IL, USA). Results Clinical follow-up The characteristics of the patients before the initiation of the treatment and duration of chemotherapy are summarized in Table 2. The mean serum AMH concentrations before chemotherapy (AMH0) were not different between the two groups. The range of individual values of serum AMH concentration in the whole population ( pmol/l) was similar to the one that previously reported in normal ovulatory women aged years, i.e., pmol/l (Pigny et al., 2006). Not surprisingly, all patients were amenorrhoeic during chemotherapy and co-treatment with the GnRH agonist. At the end of treatment, seven of 30 patients (23%) asked to be immediately switched to an oral contraceptive pill (OCP) without waiting for the occurrence of a first spontaneous cycle. Among the 23 patients who did not take OCP, nine out of 11 in the ABVD group (82%) and three out of nine in the non- ABVD group (33%) had spontaneous menses during the first 3 months (P < 0.03), while three other patients remained amenorrhoeic. Four patients more from the non-abvd group recovered menses at month 6 despite undetectable AMH concentrations for three of them. At months 6, 9 and 12, 11 of 30 patients (37%) were taking OCP. All of the patients who did not take OCP (63%) had spontaneous menses. Hormonal follow-up Whole population The serum AMH variations before, during and after treatment in the whole population are shown in Figure 1. Mean Table 2 groups. Pretreatment data and duration of chemotherapy in the whole group and in the ABVD and non-abvd Total group (n = 30) ABVD (n = 17) Non-ABVD (n = 13) P-value between groups Mean age ± SD (years) 24.0 ± ± ± 4.6 NS Mean body mass index ± SD (kg/m 2 ) 22.7 ± ± ± 6.2 NS Hodgkin s disease NA Non-Hodgkin s disease NA Regular cycle NA Oligomenorrhoea NA Taking OCP (%) 19 (63) 15 (88) 6 (46) Mean serum AMH ± SD (pmol/l) 21.7 ± ± ± 13.7 NS Stem cell transplantation NA R-CHOP protocol NA ACVBP protocol NA BEAM protocol NA MINE protocol NA BEACOPP protocol NA Mean duration of chemotherapy; range (months) 5.15; ; ; 3 6 NS Values are numbers unless otherwise stated; ABVD = adriamycin, bleomycin, vinblastine and dacarbazine; AMH = anti- Müllerian hormone; NA = not applicable; NS = not statistically significant; OCP = oral contraceptive pill.

4 AMH follow-up after chemotherapy AMH (pmol/l) CHEMOTHERAPY // p<0.001 AMH (pmol/l) CHEMOTHERAPY // // ABVD non ABVD AMH 0 AMH 1 AMH 2 AMH +3 AMH +6 AMH +9 AMH +12 AMH 0 AMH 1 AMH 2 AMH +3 AMH +6 AMH +9 AMH +12 Figure 1 Longitudinal evolution of mean (± SD) serum AMH concentration in the whole group of patients (n = 30). The P- value indicates that this parameter varied significantly as a function of time by ANOVA. AMH0 = before initiation of chemotherapy, AMH1 = 15 days after the first cycle of chemotherapy, AMH2 = 15 days before the last cycle of chemotherapy and AMH+3, +6, +9, +12 = every 3 months after the end of chemotherapy. \ = significantly (P < 0.05) different from the mean AMH0 value by ANOVA with post-hoc analysis. // indicates that the time lag between AMH1 and AMH2 was not the same for every patient. AMH concentrations fell significantly (P < 0.001) as soon as 15 days after initiation of chemotherapy and were close to the detection limit of the assay at the end of the treatment period. Post-treatment AMH concentrations increased by the third month of follow-up. By ANOVA test with posthoc analysis, the values for AMH1, AMH2, AMH+3, AMH+6 and AMH+9, but not AMH+12, were all significantly (P < 0.05) lower than the mean AMH0 value. None of the AMH+3, AMH+6, AMH+9 and AMH+12 values were significantly different from the AMH2 value (Figure 1). ABVD versus non-abvd protocols The population was divided into two groups according to whether they received potentially more ovariotoxic drugs such as cyclophosphamide, ifosfamide or melphalan or not (non-abvd or ABVD group, respectively). As shown by Figure 2, mean AMH concentrations fell drastically and rapidly after the start of chemotherapy in a similar fashion between both groups. However, in the ABVD group, the drop in AMH concentrations became significant only at the end of treatment (AMH0 versus AMH2: P < 0.05) while in the non-abvd group, mean AMH1 concentration was already different from AMH0 (P < ). After the end of chemotherapy, the rise of AMH concentrations was much more pronounced in the ABVD group than in the non-abvd group (Figure 2), with significant (P < 0.05) differences between the two groups for AMH+3, +6, +9 and +12. In the ABVD group, only AMH+12 concentrations were significantly different from AMH2 (P < 0.02) and returned to pretreatment values (Figure 2). There were however large individual variations among patients, with a significant trend to higher values in the nine patients having received Figure 2 Longitudinal evolution of mean (±SD) serum AMH concentration in the ABVD group (circles, n = 17) and non-abvd group (squares, n = 13). AMH0 = before initiation of chemotherapy, AMH1 = 15 days after the first cycle of chemotherapy, AMH2 = 15 days before the last cycle of chemotherapy and AMH+3, +6, +9, +12 = every 3 months after the end of chemotherapy. \ = significantly (P < 0.05) different from the non- ABVD mean value by ANOVA with post-hoc analysis. // indicates that the time lag between AMH1 and AMH2 was not the same for every patient. four treatment cycles than in the eight patients exposed to eight cycles (P < by ANOVA). In the non-abvd group, mean AMH+3, +6, +9 and +12 concentrations remained significantly lower than AMH0 values (P < ) and not significantly different from AMH2 concentrations, even after excluding patients who have received the strongest protocol for bone mineral transplantation. The mean duration of chemotherapy (months) in each group was identical (5 months) (Table 2). Discussion So far, few studies have focused on AMH concentration, chemotherapy and reproductive function (Anderson et al., 2006; Bath et al., 2003; Lie Fong et al., 2008; Rosendahl et al., 2009). Bath et al. and van Beek et al. have shown that AMH concentrations in young adult women who received cancer treatment during childhood were significantly lower in comparison to healthy controls. Lie Fong et al. (2008) and Lutchman Singh et al. (2007) have shown the same results, respectively, in 25 young women treated for haematological malignancies and in 22 patients treated for breast cancer. The data of Lutchman Singh et al. (2007) were mainly cross-sectional and longitudinal study of AMH concentrations could be performed in only three patients. By using repeated measurements, an abrupt and prompt drop in AMH concentrations has been shown just after initiation of chemotherapy, with both regimens, despite a good ovarian reserve before treatment in all of the young patients. This suggests that almost all of the in-growth follicles were rapidly damaged and/or impaired in their AMH secretion, even with the softer regimen used in lymphoma (ABVD). This study s results are in agreement with those of Anderson et al. (2006) and Rosendahl et al. (2009) who

5 284 C Decanter et al. also performed longitudinal evaluation of AMH concentrations during chemotherapy. However, the population of Anderson et al. (2006) consisted of 50 patients who were treated by different regimens of adjuvant chemotherapy for breast cancer, all including cyclophosphamide. In addition, patients were much older than in the present study (mean age 41 years, range years, with only seven patients younger than 35). The age issue is important, since serum AMH concentrations decreases after the age of about 30 years in normal fertile women, as recently demonstrated by van Disseldorp et al. (2008). The influence of age was minimal in this study since all of the patients were less than 33 years of age, with a mean age of 24 years. Rosendahl et al. (2009) have performed a longitudinal study of AMH concentrations in a small and heterogeneous population (16 patients) mixing patients with breast cancer or lymphoma. They showed a marked fall in AMH concentrations as soon as 1 week after the initiation of treatment. Nevertheless, contrasting with this study s data, almost all of the patients had one ovary removed for cryopreservation before or just after initiation of chemotherapy, which may partly skew the results. The main finding of the present study is that different ovarian recovery patterns were observed according to chemotherapy protocols after the end of treatment. While Anderson et al. (2006) did not observe such a phenomenon in their patients who all received cyclophosphamide, Rosendahl et al. (2009) also reported significantly higher AMH values in patients who did not receive alkylating agents, but only in the first month after treatment. The results in the ABVD group suggest that the primordial follicle pool has been less damaged than in the non-abvd protocol, thus allowing the rapid emergence of new growing and AMHsecreting follicles. Accordingly, most of the non-hormonally treated patients from the ABVD group (82%) recovered spontaneous cycles as soon as 4 6 weeks after the end of chemotherapy, while this was the case in only 33% patients from the non-abvd. It remains to be determined whether this follicular resurgence will be sustained with time and whether different patterns of AMH concentration profile during the ovarian recovery phase have any clinical relevance and prognosis value concerning fertility capacity. The data in the non-abvd group are in keeping with experimental studies in rats and rhesus monkeys treated with cyclophosphamide, showing that the primordial follicle depletion was rapid and drastic (Ataya and Ramahi-Ataya, 1993). Moreover, in a novel ovarian xenografting model studying the in-vivo impact of chemotherapeutic agents on human primordial follicles, Oktem and Oktay (2007b) have shown a rapid and significant reduction in primordial follicle density with a 53% and 93% follicle loss 24 and 48 h after the first injection of cyclophosphamide, respectively. Apoptosis indicators were detectable in significant concentrations by TdT (terminal deoxynucleotidyl transferase)-mediated dudp nick-end labelling as soon as 12 h after the first injection of cyclophosphamide. The fact that alkylating agents are not cell-cycle specific and, thus, do not require cell proliferation for their cytotoxic action can explain why both resting and in-growth follicles are damaged. In addition to the type of chemotherapy, exposure time and cumulative doses may also influence the recovery of ovarian function, as suggested by the preliminary data in patients exposed either to four or eight cycles of ABVD. Nevertheless, in the whole population of the present study there was no difference in the mean duration of chemotherapy between ABVD and non-abvd group (5.2 months versus 5.1 months, respectively). Exposure time to GnRH agonist co-treatment might also be suspected to have interfered with the results. If so, it is nevertheless unlikely that it could have been responsible for such a rapid and dramatic effect on AMH concentrations. Moreover, it has been previously suggested that, although studies are few and not always specifically designed to address this issue, serum AMH concentrations were not significantly decreased by GnRH agonists (Fanchin et al., 2003b; Jayaprakasan et al., 2008; Mohamed et al., 2006) except if treatment lasts more than 6 months (Anderson et al., 2006). In the present study, only eight women received GnRH agonist co-treatment for more than 5 months. Likewise, it has been shown that OCP have no significant effect on AMH concentrations (Somunkiran et al., 2007; Streuli et al., 2008). It is also unlikely that taking OCP, that concerned 23% of patients, could have introduced any significant bias during the follow-up period. Incidentally, the number of patients taking OCP in the ABVD group before starting chemotherapy was significantly higher than in the non-abvd group; none of these patients had continued taking OCP during chemotherapy. Thus, even if a potential protective effect of this anti-gonadotrophic treatment cannot be excluded, it has to be further studied in a much larger population. At this point, it is not yet possible to define a patient and/or an ovarian profile that could be more susceptible to the gonadotoxicity of chemotherapy. In the same way, the clinical relevance of low AMH concentrations after chemotherapy relating to chances of pregnancy, which is the main question, has to be further studied. Only a long-term and systematic follow-up in a larger population will allow this issue to be addressed. The present study is therefore still prospectively continuing in order to confirm and to develop these preliminary results. In conclusion, these longitudinal data confirm that serum AMH allows the detection of differences in ovarian toxicity between chemotherapy regimens. However, it remains to be determined as to whether these different patterns of serum AMH profile during the ovarian recovery phase have any clinical relevance and prognostic value concerning fertility capacity. This should help ultimately in fertility preservation strategies, fertility counselling and future family planning in women exposed to ovariotoxic drugs. Acknowledgements The authors thank Mrs F Becquin and all the staff of the Laboratoire de Biochimie et Hormonologie, Parc Eurasanté, Centre Hospitalier Régional Universitaire de Lille, for their excellent technical help. 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