Mathilde Chabut
email: mathilde.chabut@chu-rouen.fr

Pascale Schneider

Blandine Courbiere

Paul Saultier

Yves Bertrand

Marie-Dominique Tabone

Cécile Pochon

Stéphane Ducassou

Catherine Paillard

Virginie Gandemer

Justyna Kanold

Jean-Hugues Dalle

Maryline Poiree

Geneviève Plat

Sandrine Thouvenin

Dominique Plantaz

Nicolas Sirvent

Sara Weinhard

Julie Berbis

André Baruchel

Guy Leverger

Zeinab Hamidou

Pascal Auquier

Gérard Michel

Ovarian Function and Spontaneous Pregnancy After Hematopoietic Stem Cell Transplantation for Leukemia Before Puberty: An L.E.A. Cohort Study

Keywords: Leukemia, Premature ovarian insufficiency, Hematopoietic stem cell transplantation

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Leukemia is the most common childhood cancer, accounting for one third of cases. Over the last decades, innovation in diagnosis and treatment have increased the overall survival rate to over 80 %. [START_REF] Bertuccio | Childhood cancer mortality trends in Europe, 1990 2017, with focus on geographic differences[END_REF] The remarkable improvement in the prognosis of children with acute leukemia (AL) has been mainly achieved through the administration of risk-adapted therapy, including intensive chemotherapy and hematopoietic stem cell transplantation (HSCT). As a consequence, there is a growing population of adult survivors that will face the long term side effects of cancer treatments such as growth failure, hypothyroidism, gonadal or uterine dysfunction or alteration of quality of life. [START_REF] Berbis | A French Cohort of Childhood Leukemia Survivors: Impact of Hematopoietic Stem Cell Transplantation on Health Status and Quality of Life[END_REF][START_REF] Courbiere | Uterine volume is dramatically decreased after Hematopoietic Stem Cell Transplantation during childhood regardless of the conditioning regimen[END_REF] Indeed, Berbis and al in 2013 described that more than 70% of childhood leukemia survivors from the French cohort L.E.A. (Leucémie de l'Enfant et de l'Adolescent) were found to have at least one late side effect.

The human ovary contains a fixed number of non-growing follicles (NGFs) established before birth that declines with age until the menopause occurs, physiologically around the age of 50. Ovarian reserve damage causes hypergonadotropic hypogonadism with a high risk of premature ovarian insufficiency (POI), defined by ESHRE (European Society of Human Reproduction and Embryology) as oligo/amenorrhea for at least 4 months, and an elevated FSH level > 25 IU/l on two occasions > 4 weeks apart before the age of 40. [START_REF] Webber | ESHRE Guideline: management of women with premature ovarian insufficiency[END_REF] The ovarian reserve is particularly sensitive to the toxicity of cancer treatment, especially conditioning regimens preparing for HSCT, which cause disorders of both endocrine and reproductive functions. Indeed, POI and infertility are among the most frequent late effects after HSCT. [START_REF] Berbis | A French Cohort of Childhood Leukemia Survivors: Impact of Hematopoietic Stem Cell Transplantation on Health Status and Quality of Life[END_REF][START_REF] Borgmann-Staudt | Fertility after allogeneic haematopoietic stem cell transplantation in childhood and adolescence[END_REF] POI includes pubertal development impairment; amenorrhea can be primary but also secondary depending on the residual post-HSCT ovarian reserve. According to various published data, the incidence of POI ranges from 44 to 100% patients who received HSCT in childhood for either malignant or non-malignant hematological disorders. [START_REF] Diesch-Furlanetto | Late Effects After Haematopoietic Stem Cell Transplantation in ALL, Long-Term Follow-Up and Transition: A Step Into Adult Life[END_REF][START_REF] Sarafoglou | Gonadal function after bone marrow transplantation for acute leukemia during childhood[END_REF][START_REF] Elchuri | Longitudinal Description of Gonadal Function in Sickle-cell Patients Treated With Hematopoietic Stem Cell Transplant Using Alkylator-based Conditioning Regimens[END_REF] Risk factors for POI after HSCT have been described : younger age at HSCT predicted spontaneous menarche whereas myeloablative conditioning (MAC) regimen was shown as the main risk factor for ovarian failure. [START_REF] Vatanen | Ovarian function after allogeneic hematopoietic stem cell transplantation in childhood and adolescence[END_REF] Indeed, the age at HSCT is known to be critical for ovarian function : HSCT in prepubertal girls means better chances of recovery and achievement of puberty. [START_REF] Borgmann-Staudt | Fertility after allogeneic haematopoietic stem cell transplantation in childhood and adolescence[END_REF][START_REF] Sanders | Ovarian function following marrow transplantation for aplastic anemia or leukemia[END_REF][START_REF] Spinelli | Ovarian recovery after total body irradiation and allogeneic bone marrow transplantation: long-term follow up of 79 females[END_REF] Fertility impairment in women transplanted during childhood and adolescence is high, ranging around 83%. [START_REF] Borgmann-Staudt | Fertility after allogeneic haematopoietic stem cell transplantation in childhood and adolescence[END_REF] Pregnancy rate after HSCT is described in the litterature as quite low, below 1%. [START_REF] Salooja | Pregnancy outcomes after peripheral blood or bone marrow transplantation: a retrospective survey[END_REF][START_REF] Diesch-Furlanetto | Pregnancy and pregnancy outcomes after hematopoietic stem cell transplantation in childhood: a cross-sectional survey of the EBMT Pediatric Diseases Working Party[END_REF] In this context, fertility preservation before HSCT remains a major challenge for childhood cancer survivors.

In AL, ovarian stimulation and oocyte vitrification is possible only after puberty, often constrained by cancer treatment emergency and not recommended immediately after the first-line of chemotherapy cycles due to a mutagenic risk. In AL and for prepubertal girls, the only fertility preservation strategy available before HSCT is ovarian tissue cryopreservation (OTC).

The aim of our study was to evaluate puberty development, long term ovarian function, and occurrence of spontaneous pregnancy on a population of prepubertal HSCT survivors who underwent HSCT for acute leukemia in childhood.

MATERIALS AND METHODS

Characteristics of childhood cancer survivor women

Women described here were all included in the L.E.A. program. L.E.A. is the long-term follow-up program involving childhood acute leukemia survivors in France. Patients were included in L.E.A. if they met the following criteria: treated for acute leukemia after 1980 in one of the participating centers, younger than 18 years of age at the time of diagnosis and agreed (or their parents/legal guardians) to participate in the study. Participants are summoned to the follow-up clinic at predefined dates, starting one year after completion of anti-leukemic therapy. [START_REF] Berbis | Cohort Profile: The French Childhood Cancer Survivor Study For Leukaemia (LEA Cohort)[END_REF] These visits are repeated every two years until the age of 20 and at least 10 years after complete remission, and every four years thereafter. The program began in 2004 and relies on the constitution of a multicenter historical and prospective cohort, which includes both incident cases (diagnosed after the starting date of the participation of the center in the L.E.A. program) and prevalent cases (diagnosed between 01/01/1980 and the starting date of the participation of the center in the L.E.A. program).

Patients were eligible for the present study if they had been included in the L.E.A. program between 2004 and 2020, were female, were treated with HSCT after a MAC regimen, without menarche at the time of transplant and more than 18 years old at the time of last evaluation.

Patients were excluded from the study if they underwent more than 2 HSCT. Among 186 women who fulfilled these inclusion/exclusion criteria, clinical and biological data were obtained in 178 and all these 178 women were included in the study group.

All patients (or their parents) provided written informed consent. This study was approved by the Sud Mediterranee V ethics committee (opinion n° 2012-A00984-39), in compliance with the General Data Protection Regulation.

Evaluation of the ovarian function after hematopoietic stem cell transplantation

In the L.E.A. program, post-HSCT ovarian function evaluation was systematically proposed to all adolescents and women who had previously received HSCT. Typically, during the medical L.E.A. visits, hormone levels were tested and various clinical data were collected including spontaneous menarche/induction of puberty, puberty progress, sex steroid hormone replacement therapy (HRT), primary or secondary amenorrhea, pregnancy occurrence and outcome.

POI was defined by ESHRE as the association of an oligo/amenorrhea for at least 4 months, and an elevated FSH level > 25 IU/l on two occasions at 4 weeks apart before the age of 40. [START_REF] Webber | ESHRE Guideline: management of women with premature ovarian insufficiency[END_REF] Spontaneous pregnancy was defined as a pregnancy obtained without any medically assisted reproductive procedure.

Depending on the level of damage to the ovarian reserve, three groups of ovarian hormonal status were distinguished. Girls who didn't have spontaneous menarche and needed HRT to induce puberty were classified as having immediate POI (IPOI). Girls who had spontaneous puberty and then presented a POI after a period of residual ovarian function were classified as having secondary POI (SPOI). Finally, women with neither clinical nor biological evidence of POI and without HRT at time of their last evaluation constituted the group without POI (noPOI).

Quality of Life (QoL) was evaluated with the SF-36 self-questionnaire (the Medical Outcome Study Short Form 36 Health Survey). [START_REF] Leplège | The French SF-36 Health Survey[END_REF] Statistical methods are described in the supplemental data.

RESULTS

Childhood hematopoietic stem cell transplantation survivors' characteristics

We included 178 women from 15 L.E.A. centers. Their characteristics are summarized in Table 1.

The median age at HSCT was 7. In the studied cohort, 169 patients were treated with one HSCT and 9 were treated with two HSCT. Out of the 169 patients treated with one HSCT, 135 received an allogeneic and 34 an autologous HSCT. Among the 9 patients with two HSCT, 2 had autologous followed by allogeneic HSCT and 7 had 2 allogeneic HSCT.

Thirteen patients from the studied population had an ovarian tissue cryopreservation (OTC) before HSCT.

Conditioning regimen before hematopoietic stem cell transplantation

Conditioning regimen depended on protocols in use at the time of HSCT, underlying diseases and their status, and history of previous CNS irradiation. All 178 women had received a MAC regimen, among which 116 had total-body irradiation (TBI) and 62 a Busulfan-based conditioning protocol (Table 1).

Most women prepared with TBI were treated with the commonly used French pre-transplant radiation regimen : they (107/116, 92%) received a total dose of 12 Gy divided in 6 fractions (i.e. 2 Gy twice daily during 3 days) with lung shielding at 8 Gy. Only 8 other women received a fractionated 10 to 11 Gy total dose and one a single 8 Gy dose. The cytostatic drugs used in association with TBI were: cyclophosphamide only in 53 patients, cyclophosphamide and fludarabine in 2, melphalan only in 3, cytarabine and melphalan in 50 and etoposide in 8 patients. Mean calculated cumulative alkylating dose per patient, using the cyclophosphamide equivalent dose (CED) was 4661 mg/m 2 (range: 0 to 15435). [START_REF] Green | The cyclophosphamide equivalent dose as an approach for quantifying alkylating agent exposure: A report from the childhood cancer survivor study: Cyclophosphamide Equivalent Dose[END_REF] All 62 women who did not receive TBI were given busulfan (BU) at a myeloablative dose.

BU was given per os or IV in 16 divided doses (4 doses per day for 4 days) at a total dose of 16 mg/kg or 480 mg/m 2 when given per os, and according to a previously published weightbased protocol when given IV. In addition to BU, patients also received cyclophosphamide (49 patients), melphalan (6 patients), aracytine and melphalan (5 patients) or fludarabine and thiotepa (2 patients). Mean cumulative CED dose was 9285 mg/m 2 (range: 5600 to 19 235).

Ovarian function after hematopoietic stem cell transplantation in leukemia survivors

Out of the 178 included women, 106 (59.6%) needed an induction of puberty with HRT whereas 72 (40.4%) had spontaneous menarche. Among the 72 patients with spontaneous menarche, 33 developed POI secondarily and 39 women still had a residual ovarian function (Figure 1). Table 2 resumes the mean FSH levels before introduction of HRT in the IPOI and SPOI groups and at last evaluation for the noPOI group (Table 2).

In the IPOI group, the median age for the introduction of HRT to induce puberty was 13.7 years [12.7-14.9] and the median age at last evaluation was 25.5 years [21.3-30].

In the SPOI group, the median age for spontaneous menarche was 13 years [11.9-14.1] , the median age for the introduction of HRT was 15.1 years [14.2-16.4] and the median age at last evaluation was 24.3 years [20.3-29.6].

In the noPOI group, the median age for spontaneous menarche was 12.7 years [12-13.5] and they still had spontaneous menstruations at 24.8 years [21.3-29.8].

Cumulative incidence of POI in women who had spontaneous menarche is shown in Figure 2.

During the follow-up, among the 72 women of the cohort who had spontaneous menarche, 33 (45.8%) progressed to a POI. The cumulative incidence of POI occurrence increased rapidly affecting 40% of women within 5 years of having spontaneous menarche and more slowly thereafter, reaching 45% at 10 years (Figure 2). 3. There is a continuous effect of age at time of HSCT on ovarian status: using the median age and the quartile distribution of the cohort as a cut-off, older children at time of HSCT had a higher risk of developing POI. Indeed, more than 80% of the patients who underwent HSCT after the age of 10.9 years were in the IPOI group. In contrast, more than 65% of the patients who underwent HSCT before the age of 4.79 years had spontaneous menarche and almost 50% of them were in the noPOI group at last evaluation (Figure 3) Thirteen patients from the study cohort had fertility preservation with OTC . Among them, 12 (92.3%) were in the IPOI group, one in the SPOI but none belonged to the noPOI group.

Risk factors for premature ovarian insufficiency in hematopoietic stem cell transplantation survivors

Potential risk factors influencing ovarian function are analyzed in Table

OTC is a significant risk factor for POI. However, there was a statistically significant association between older age at first HSCT and history of OTC (p<0.001), more than 90% of women with history of OTC being older than 7.70 years old at HSCT (median age of the study cohort)(Table 3).

Spontaneous pregnancy and outcomes in hematopoietic stem cell transplantation survivors

Among the 178 women included in our cohort, 22 had at least one spontaneous pregnancy (12 %), with a total number of 37 spontaneous pregnancies leading to 17 live births, 14 miscarriages, 4 legal abortions and 2 therapeutic abortions.

Potential factors influencing the occurrence of spontaneous pregnancy in our study cohort are shown in Table 4. We observed a continuous effect of age at last evaluation on the occurrence of spontaneous pregnancies, older patients at last evaluation having higher probabilities of spontaneous pregnancy. Age at HSCT was not a prognostic factor, perhaps due to a statistically significant correlation between age at HSCT and age at last evaluation (Figure 4). Women from the noPOI group had a significantly better chance of spontaneous pregnancy than women in the IPOI and SPOI groups. Interestingly, none of those who had OTC enjoyed spontaneous pregnancy, although the difference did not reach the level of statistical significance (Table 4).

Premature ovarian insufficiency and spontaneous pregnancies consequences on quality of life

We compared the QoL profile in our study population with that of the French general population. [START_REF] Leplège | Le questionnaire MOS SF-36: Manuel de l'utilisateur et guide d'interprétation des scores[END_REF] There was a highly statistically significant decline in the mental component score of the study population, as well as for almost all subscales of SF-36 (additional Table 1A). However, we didn't find any statistically significant effect of the women's ovarian status or the occurrence of spontaneous pregnancy on their quality of life (additional Table 1B and1C).

DISCUSSION

We report the evolution of residual ovarian function and spontaneous pregnancies in 178 adult females treated before puberty for AL by HSCT prepared with a MAC regimen.

Ovarian function after HSCT has been described in other studies but the survivors cohorts were often heterogeneous with various indications such as leukemia, Hodgkin lymphoma or severe aplastic anemia. [START_REF] Sarafoglou | Gonadal function after bone marrow transplantation for acute leukemia during childhood[END_REF][START_REF] Jadoul | How does bone marrow transplantation affect ovarian function and fertility?[END_REF] A strength of our study is to have included a large and homogeneous population of women, all treated before puberty for a similar pathology with similar treatments.

Temporary recovery of ovarian function has been previously described on prepubertal and midpubertal girls. [START_REF] Spinelli | Ovarian recovery after total body irradiation and allogeneic bone marrow transplantation: long-term follow up of 79 females[END_REF][START_REF] Diesch-Furlanetto | Pregnancy and pregnancy outcomes after hematopoietic stem cell transplantation in childhood: a cross-sectional survey of the EBMT Pediatric Diseases Working Party[END_REF] Based on these data, we chose to focus on girls who were prepubertal at the time of HSCT and had attained adulthood at last evaluation. Another significant strength of our study is its extended longitudinal follow up duration (median duration of 18 years after HSCT).

Several other studies which evaluated gonadal function after cancer treatment did not respond with a clear and uniform definition of ovarian impairment with, for example, heterogeneous thresholds of gonadotropin plasma levels. [START_REF] Sanders | Ovarian function following marrow transplantation for aplastic anemia or leukemia[END_REF][START_REF] Chemaitilly | Premature Ovarian Insufficiency in Childhood Cancer Survivors: A Report From the St. Jude Lifetime Cohort[END_REF] Our study was based on the European Society of Human Reproduction and Embryology (ESHRE 2016), definition of POI. [START_REF] Webber | ESHRE Guideline: management of women with premature ovarian insufficiency[END_REF] Moreover, some previous studies have reported pregnancies in cancer survivors after HSCT without specifying whether these were spontaneous or required assisted reproductive techniques such as oocytes donation. [START_REF] Borgmann-Staudt | Fertility after allogeneic haematopoietic stem cell transplantation in childhood and adolescence[END_REF][START_REF] Vatanen | Ovarian function after allogeneic hematopoietic stem cell transplantation in childhood and adolescence[END_REF] In this study, we only report spontaneous pregnancies with a view to assess as accurately as possible residual ovarian function.

A number of risk factors have already been described regarding ovarian toxicity after MAC regimen for bone marrow transplantation. The age at HSCT is critical: POI is more frequent after HSCT in adult women, whereas in prepubertal girls, there is a higher probability of spontaneous menarche. [START_REF] Borgmann-Staudt | Fertility after allogeneic haematopoietic stem cell transplantation in childhood and adolescence[END_REF][START_REF] Sanders | Ovarian function following marrow transplantation for aplastic anemia or leukemia[END_REF][START_REF] Spinelli | Ovarian recovery after total body irradiation and allogeneic bone marrow transplantation: long-term follow up of 79 females[END_REF] Our data show that age at HSCT strongly influences residual ovarian function: more than 65% of patients who underwent HSCT before the age of 4.8 years had spontaneous menarche and almost 50% didn't have POI at last evaluation whereas more than 85% of patients who received HSCT after the age of 10.9 years didn't have spontaneous menarche and needed induction of puberty with HRT. Jadoul et al [START_REF] Jadoul | Clinical and biologic evaluation of ovarian function in women treated by bone marrow transplantation for various indications during childhood or adolescence[END_REF] observed that patients under the age of 10 at the time of HSCT for malignant and nonmalignant diseases showed significant and independently-associated higher rates of clinically proven persistent ovarian function. The protective effect of young age might be related to the higher number of NGFs in the ovarian reserve of younger girls. During the reproductive life, the decline in NGFs occurs in an exponential way. [START_REF] Wallace | Human Ovarian Reserve from Conception to the Menopause[END_REF] We were surprised to observe a significantly increased chance of keeping functional ovaries when girls were treated before the age of 5 (48.8 %); Similarly, we report a gradual increase in the risk for POI with age at HSCT beginning much earlier than the previously described threshold of 10 years.

The identification of women endowed with residual ovarian function after HSCT and the timing until the definitive POI occurs remains a major challenge for counseling a long-term fertility preservation strategy such as oocyte cryopreservation after puberty. According to our study, 40% of transplanted girls have spontaneous menarche but among them, 40% will progress to POI within 5 years of menarche, which is quite a short critical therapeutic window to consider fertility procedure. [START_REF] Das | Ovarian reserve and response to IVF and in vitro maturation treatment following chemotherapy[END_REF] Indeed, it could be discussed to perform an oocyte banking before definitive POI in these young adolescents as already described in case of risk of POI in women with a history of benign ovarian tumor and high risk of ovarian depletion. [START_REF] Legrand | Oocyte accumulation for fertility preservation in women with benign ovarian tumours with a history of previous surgery, multiple or large cysts[END_REF] Infertility is a very frequent late effect after HSCT. [START_REF] Mosher | Quality of life concerns and depression among hematopoietic stem cell transplant survivors[END_REF] Pregnancy in transplanted women has been documented by Salooja et al. with a pregnancy rate of 0,6% (232/37362). This study included girls and adult women transplanted at different ages ranging from 6 to 36 with a median age at HSCT of 19 years. [START_REF] Salooja | Pregnancy outcomes after peripheral blood or bone marrow transplantation: a retrospective survey[END_REF] The study of Diesch-Furlanetto et al. focused on pregnancies (naturally or by artificial insemination) both in childhood disease survivor transplanted women and partner pregnancies of childhood disease survivor transplanted males with a pregnancy rate of 0.7% (406/61047). The studied population included less than 30% of leukemia as HSCT indication. [START_REF] Diesch-Furlanetto | Pregnancy and pregnancy outcomes after hematopoietic stem cell transplantation in childhood: a cross-sectional survey of the EBMT Pediatric Diseases Working Party[END_REF] Indeed, pregnancies after HSCT have been documented in several studies but with heterogeneous inclusion criteria : underlying diseases including malignant and non-malignant ones [START_REF] Chiodi | Menstrual patterns, fertility and main pregnancy outcomes after allogeneic haematopoietic stem cell transplantation[END_REF][START_REF] Loren | Pregnancy after Hematopoietic Cell Transplantation: A Report from the Late Effects Working Committee of the Center for International Blood and Marrow Transplant Research (CIBMTR)[END_REF] ; including patients transplanted at any age [START_REF] Salooja | Pregnancy outcomes after peripheral blood or bone marrow transplantation: a retrospective survey[END_REF] ; including men and women indifferently or without reporting whether the conception method was natural or involved medically assisted reproduction. [START_REF] Borgmann-Staudt | Fertility after allogeneic haematopoietic stem cell transplantation in childhood and adolescence[END_REF][START_REF] Diesch-Furlanetto | Pregnancy and pregnancy outcomes after hematopoietic stem cell transplantation in childhood: a cross-sectional survey of the EBMT Pediatric Diseases Working Party[END_REF] To our knowledge, our study is the first one to assess spontaneous pregnancy in women who received HSCT at a prepubertal age. In the other cohort studies, the follow-up was too short to include pregnancies at ages for which family planning is considered. However, compared with the mean age of healthy women at their first child's birth (29 years old), our studied women delivered 5 years earlier (mean 24,3 years old). [START_REF] Diesch-Furlanetto | Pregnancy and pregnancy outcomes after hematopoietic stem cell transplantation in childhood: a cross-sectional survey of the EBMT Pediatric Diseases Working Party[END_REF] With a median age at last evaluation of 24.9 years old, we probably underestimate the total number of pregnancies after HSCT.

We describe that more than 12% of women had spontaneous pregnancies. Surprisingly, we report more than 10% of legal abortions in these pregnancies with half of them in the IPOI group.

One of the main messages to highlight from this result is the absolute necessity to replace the HRT with a contraceptive method for the young adults to avoid non desired pregnancies, especially in girls with a history of induction of puberty.

Fertility impairment being such an important late effect after cancer treatment, fertility preservation procedures before treatment is one of the most important issue for survivor patients and for their quality of life. [START_REF] Brodigan | Safety of Surgical Fertility Preservation Procedures in Children Prior to Hematopoietic Stem Cell Transplant[END_REF] OTC is the only option for fertility preservation in prepubertal girls. On the other hand, we observed an impairment of residual ovarian function in girls who have benefited from an OTC. In our study, only around 7% of the women had undergone an OTC before HSCT. OTC appears as a statistically significant risk factor for POI with more than 90% of these women being in the group IPOI. However, there is a potential age bias, because women who underwent OTC were older at time of HSCT and we previously described age at HSCT as an independent risk factor for POI. In addition, we did not describe any spontaneous pregnancy in women with a history of OTC. Consequently, we suggest that OTC before HSCT for AL might influence future ovarian function and fertility but this result must be confirmed in other cohorts because of the relatively small number of women and the potential age bias.

More than 200 live births after OTC and autotransplantation [START_REF] Dolmans | Importance of patient selection to analyze in vitro fertilization outcome with transplanted cryopreserved ovarian tissue[END_REF][START_REF] Dolmans | Transplantation of cryopreserved ovarian tissue in a series of 285 women: a review of five leading European centers[END_REF] have been reported worldwide, which gives OTC a rightful place in fertility preservation to patients of different ages [START_REF] Chambon | Cryopreservation of ovarian tissue in pediatric patients undergoing sterilizing chemotherapy[END_REF][START_REF] Meirow | Transplantations of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria[END_REF] or treated for various diseases, especially non malignant ones. [START_REF] Hovatta | Ovarian function and in vitro fertilization (IVF) in Turner syndrome[END_REF] In AL however, there is a concern about possible contamination of ovarian tissue by residual leukemic disease, which can potentially induce disease recurrence after ovarian autograft. [START_REF] Courbiere | Tumor cell contamination in ovarian tissue cryopreserved before gonadotoxic treatment: should we systematically exclude ovarian autograft in a cancer survivor?[END_REF] AL remains a discussed indication for OTC. [START_REF] Imbert | Safety and usefulness of cryopreservation of ovarian tissue to preserve fertility: a 12-year retrospective analysis[END_REF][START_REF] Shapira | Fertility Preservation in Young Females with Hematological Malignancies[END_REF] One animal model study concluded that reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe. [START_REF] Dolmans | Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe[END_REF] Consequently, only a few women around the world have been transplanted after leukemia and this was done with maximal safety procedures to ensure that there was no residual disease in the graft. [START_REF] Rodriguez-Wallberg | Successful pregnancies after transplantation of ovarian tissue retrieved and cryopreserved at time of childhood acute lymphoblastic leukemia -A case report[END_REF][START_REF] Shapira | First delivery in a leukemia survivor after transplantation of cryopreserved ovarian tissue, evaluated for leukemia cells contamination[END_REF][START_REF] Silber | Cryopreservation and transplantation of ovarian tissue: results from one center in the USA[END_REF][START_REF] Sonmezer | First pregnancy and live birth in Turkey following frozen-thawed ovarian tissue transplantation in a patient with acute lymphoblastic leukemia who underwent cord blood transplantation[END_REF] Even for centers that do not consider autotransplantation of OTC in AL, OTC could be discussed in view of the emerging technology of in vitro folliculogenesis in human. To date, the ''artificial ovary'' technology has been successful in nonhuman animals, but not yet in humans. [START_REF] Pampanini | Fertility Preservation for Prepubertal Patients at Risk of Infertility: Present Status and Future Perspectives[END_REF][START_REF] Mclaughlin | Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system[END_REF] Finally, the usefulness of OTC in young girls suffering from AL is questionable if ovarian tissue autograft couldn't be proposed. In the end, other studies assessing the potential impact of OTC on future ovarian function and fertility are needed to answer this important question.

Teinturier et al [START_REF] Thomas-Teinturier | Age at menopause and its influencing factors in a cohort of survivors of childhood cancer: earlier but rarely premature[END_REF] , showed that unilateral oophorectomy in cancer survivors was associated with a 7-year earlier age at onset of menopause.

Several previous studies have documented an impairment of quality of life in childhood leukemia survivors. [START_REF] Shapira | Fertility Preservation in Young Females with Hematological Malignancies[END_REF][START_REF] Chiou | Health-related quality of life and cognitive outcomes among child and adolescent survivors of leukemia[END_REF][START_REF] Maunsell | Quality of Life Among Long-Term Adolescent and Adult Survivors of Childhood Cancer[END_REF][START_REF] Speechley | Health-Related Quality of Life Among Child and Adolescent Survivors of Childhood Cancer[END_REF] Studies focusing on HSCT survivors also showed a decrease in their perceived QoL. [START_REF] Berbis | Quality of life in minor siblings of childhood leukemia survivors, long-term after diagnosis: A LEA study (for Leucemies de l'Enfant et de l'Adolescent-childhood and adolescent leukemia): Quality of life in minor siblings of childhood leukemia survivors[END_REF][START_REF] Clarke | Clinical outcomes and health-related quality of life (HRQOL) following haemopoietic stem cell transplantation (HSCT) for paediatric leukaemia: Quality of life after paediatric bone marrow transplant[END_REF] Our study showed significantly lower scores of QoL in HSCT survivors compared to the French general population especially on the mental component score. However, we were unable to detect an impact of the ovarian function status on QoL, perhaps because the impairment of QoL in these cases is multifactorial and might require a larger cohort to become visible.

Our study reported that spontaneous puberty and spontaneous pregnancies are possible and not so rare after HSCT. We evidenced a more resilient residual ovarian function especially when HSCT was performed before the age of 5. Age at HSCT remains a major risk factor for ovarian dysfunction with a continuous effect on ovarian function. It is important to identify the therapeutic window of fertility preservation for those who had spontaneous menarche. As spontaneous pregnancies may occur, it is essential to propose contraceptive methods. More studies with a larger cohort of survivors are necessary to identify the potential impact of OTC on ovarian function in leukemia survivors who received HSCT during childhood. ACCEPTED MANUSCRIPT / CLEAN COPY

TABLES

  7 [IQR25-75: 4.79-10.90] years old and the median age at last evaluation was 24.9 [21.08-30.1] years old. The median follow up duration since HSCT was 18 years [14.2-23.3].

Figure 1 :

 1 Figure 1: Ovarian function evolution of the survivors cohort after hematopoietic stem cell

Figure 2 :

 2 Figure 2: Cumulative incidence of occurrence of premature ovarian insufficiency in women

Figure 3 :

 3 Figure 3: Ovarian status depending on age at hematopoietic stem cell transplantation in the

Figure 4 :Figure 1 ACCEPTED

 41 Figure 4: Correlation between age at hematopoietic stem cell transplantation and age at last

Figure 2 ACCEPTED

 2 Figure 2

Figure 3 ACCEPTED

 3 Figure 3

Figure 4

 4 Figure 4

Table 1 :

 1 Hematopoietic stem cell transplantations and survivors study population characteristics

	Study patients n = 178	%

Table 2 :

 2 Mean FSH levels before introduction of HRT in the IPOI and SPOI group and at last evaluation for the noPOI group

	FSH levels are shown in UI/L with the mean± standard error
	IPOI (Immediate Premature Ovarian Insufficiency); SPOI (Secondary Premature Ovarian Insufficiency); noPOI
	(without Premature Ovarian Insufficiency at last evaluation)

Table 3 :

 3 Evaluation of potential risk factors impacting ovarian status after hematopoietic stem cell transplantation for leukemia in childhood

	ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; HSCT, hematopoietic stem cell
	transplantation; TBI, total body irradiation; GvHD, graft vs host disease; BU, Busulfan; OTC, ovarian tissue
	cryopreservation
	IPOI (Immediate Premature Ovarian Insufficiency); SPOI (Secondary Premature Ovarian Insufficiency); noPOI
	(without Premature Ovarian Insufficiency at last evaluation)
	Ages are expressed in quartiles.

Table 4 :

 4 Evaluation of potential factors influencing spontaneous pregnancy occurrence after hematopoietic stem cell transplantation for leukemia in childhood

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; HSCT, hematopoietic stem cell transplantation; TBI, total body irradiation; GvHD, graft vs host disease; BU, Busulfan; OTC, ovarian tissue cryopreservation; IPOI (Immediate Premature Ovarian Insufficiency); SPOI (Secondary Premature Ovarian Insufficiency); noPOI (without Premature Ovarian Insufficiency at last evaluation). Ages are expressed in quartiles.

  In the studied cohort, of a total of 178 eligible survivors, 106 patients (59.6%) are in the group IPOI, 33 patients (18.5%) are in the SPOI group and 39 patients (21.9%) are in the group noPOI. IPOI (Immediate Premature Ovarian Insufficiency); SPOI (Secondary Premature Ovarian Insufficiency); noPOI (without Premature Ovarian Insufficiency at last evaluation)

SUPPLEMENTARY DATA

Quality of life (QoL) focusing on health was evaluated with the SF-36 self-questionnaire (the Medical Outcome Study Short Form 36 Health Survey), a widely used QoL measure that provides a non-disease-specific measure of adult functioning and well-being, allowing for comparison with a broad range of same-age norm groups. [START_REF] Berbis | A French Cohort of Childhood Leukemia Survivors: Impact of Hematopoietic Stem Cell Transplantation on Health Status and Quality of Life[END_REF][START_REF] Speechley | Health-Related Quality of Life Among Child and Adolescent Survivors of Childhood Cancer[END_REF][START_REF] Berbis | Quality of life in minor siblings of childhood leukemia survivors, long-term after diagnosis: A LEA study (for Leucemies de l'Enfant et de l'Adolescent-childhood and adolescent leukemia): Quality of life in minor siblings of childhood leukemia survivors[END_REF] SF36 includes 36 items describing eight dimensions. Two summary scores are also calculated from the subscales, a physical component score and a mental component score. All scores range between 0 and 100, with higher scores indicating better QoL.

Statistical analysis

Statistical analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). The SF 36 scores were compared according to ovarian function and spontaneous pregnancies in our study population. Quality of life scores were also compared with age-and sexmatched controls from the French population.

Continuous age variables

Cumulative incidence of occurrence of POI in women who had spontaneous menarche over time were estimated using the Kaplan-Meier method. The proportional hazards assumption was assessed via examination of the log-minus-log survival plot. For the analysis of cumulative incidence rates of occurrence of POI, patients were censored when there was no POI identified.

ADDITIONAL TABLE