# Use of tamoxifene-controlled ovarian hyperstimulation for fertility preservation before breast cancer treatment: A prospective cohort study with a 5-year follow-up

A. Dezellus, S. Mirallie, F. Leperlier, B. Sauterey, P.-E. Bouet, A. Dessaint, S. Duros, A.S. Gremeau, M.-A. Mouret-Reynier, L.M. Durand, et al.

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## To cite this version:

A. Dezellus, S. Mirallie, F. Leperlier, B. Sauterey, P.-E. Bouet, et al.. Use of tamoxifene-controlled ovarian hyperstimulation for fertility preservation before breast cancer treatment: A prospective cohort study with a 5-year follow-up. The Breast, 2024, 77, pp.103776. 10.1016/j.breast.2024.103776. hal-05084637

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# Use of tamoxifene-controlled ovarian hyperstimulation for fertility preservation before breast cancer treatment: A prospective cohort study with a 5-year follow-up

A. Dezellus<sup>a, *</sup>, S. Mirallie<sup>b</sup>, F. Leperlier<sup>b</sup>, B. Sauterey<sup>a</sup>, P.-E. Bouet<sup>c</sup>, A. Dessaint<sup>d</sup>, S. Duros<sup>e</sup>, A.S. Gremeau<sup>f</sup>, M.-A. Mouret-Reynier<sup>g</sup>, L.M. Durand<sup>h</sup>, L. Venat<sup>i</sup>, P. De Blay<sup>j</sup>, M. Robert<sup>a</sup>, T. Freour<sup>b</sup>, M. Campone<sup>a, k</sup>, A. Blanc-Lapierre<sup>a</sup>, V. Bordes<sup>a, 1</sup>

## ARTICLE INFO

**Keywords:**

Breast cancer

Fertility preservation

Tamoxifen

Ovarian stimulation

Neoadjuvant chemotherapy

## ABSTRACT

**Purpose:** Fertility issues are of great concern for young women undergoing treatment for breast cancer (BC). Fertility preservation (FP) protocols using controlled ovarian stimulation (COS) with letrozole have been widely used with overall good results. However, letrozole cannot be used in every country in this context. This study aimed to assess the efficacy of tamoxifen for COS in women with early BC undergoing FP.

**Methods:** This multicentric prospective study included patients aged 18–40, diagnosed with stage I, II and III invasive BC, undergoing tamoxifen-COS before adjuvant or neoadjuvant chemotherapy (NAC). The primary endpoint was the efficacy of tamoxifen-COS protocol evaluated by the number of oocytes collected and vitrified. Secondary endpoints included the time interval before chemotherapy, breast cancer (BC) recurrence rates, and reproductive outcomes.

**Results:** Ninety-five patients were included between 2014 and 2017, aged 31.5 ± 4 years on average. 37.9 % received NAC and 62.1 % received adjuvant chemotherapy. FP procedure was successful in 89.5 % of the cycles. The mean number of collected and vitrified oocytes was 12.8 ± 7.9 and 9.8 ± 6.2, respectively. The mean duration of COS was 10.4 ± 1.9 days. Median time before chemotherapy initiation was 3.6 weeks (IQR 3.1; 4.1) for women receiving NAC. Five-year relapse-free and overall survival rates were in-line with those expected in this population. Twenty-one women had spontaneous full-term pregnancies, while 5 underwent IVF cycles with frozen-thawed oocytes, without pregnancy.

**Conclusion:** Tamoxifen-COS protocols appear to be feasible before adjuvant or NAC treatment in young BC patients and efficient in terms of oocyte yield.

<footnote><sup>a</sup> Institut de Cancérologie de l'Ouest, Saint Herblain et Angers, France</footnote>

<footnote><sup>b</sup> Service de Médecine et Biologie du Développement et de la Reproduction, CHU de Nantes, France</footnote>

<footnote><sup>c</sup> Service de Médecine et Biologie du Développement et de la Reproduction, CHU Angers, France</footnote>

<footnote><sup>d</sup> Centre Eugène Marquis, Rennes, France</footnote>

<footnote><sup>e</sup> Service de Médecine et Biologie du Développement et de la Reproduction, CHU de Rennes, France</footnote>

<footnote><sup>f</sup> Service de Médecine et Biologie du Développement et de la Reproduction, CHU de Clermont-Ferrand, France</footnote>

<footnote><sup>g</sup> Centre Jean Perrin, Clermont-Ferrand, France</footnote>

<footnote><sup>h</sup> Service de Médecine et Biologie du Développement et de la Reproduction, CHU de Limoges, France</footnote>

<footnote><sup>i</sup> Service d'oncologie, CHU de Limoges, France</footnote>

<footnote><sup>j</sup> Service de Gynécologie-Obstétrique CH de Vendée, La Roche-sur-Yon, France</footnote>

<footnote><sup>k</sup> Université Nantes/Angers, Inserm, CNRS, CRCI2NA, France</footnote>

<footnote>* Corresponding author. Institut de Cancérologie de l'Ouest – Service de Prévention et de Promotion de la santé, Boulevard Jacques Monod, 44805, Saint Herblain, France.</footnote>

<footnote>E-mail address: aliette.dezellus@ico.unicancer.fr (A. Dezellus).</footnote>

<footnote n="1">¹ This work could not have been realized without the engagement of Dr. Bordes, who sadly passed away before the study was completed.</footnote>
# 1. Introduction

The worldwide incidence of Breast Cancer (BC) among young women has increased throughout the last 20 years [1,2], making it the most frequently diagnosed form of cancer among women of reproductive age, i.e. <40 years [3]. Fortunately, hormonal, cytotoxic and targeted therapies have improved survival over the last decade, reaching an average 5-year survival rate over 80 % in the United States of America and in Europe [4]. Young women with early BC can be treated with chemotherapy including gonadotoxic molecules, such as anthracyclins and cyclophosphamide. Age and initial ovarian reserve are the main factors determining how resilient the ovaries will be to chemotherapy treatment, as previously shown [5]. Given that many young women with BC have not completed family planning, fertility preservation management is crucial [6]. At present, various Fertility Preservation (FP) methods exist: embryo, oocyte or ovarian tissue cryopreservation. Gonadotropin Releasing Hormone (GnRH) agonists can also be used during chemotherapy to diminish ovarian damage but their efficacy in improving pregnancy rates post-BC treatment remains controversial [7–10]. For patients with a moderate risk of premature ovarian failure, oocytes preservation, after controlled ovarian stimulation (COS), seems to be the most suitable and efficient strategy [11,12].

In EBC patients, particularly those with hormone receptor-positive disease, international guidelines advocate for controlled ovarian stimulation (COS) with an aromatase inhibitor or a selective estrogen receptor modulator like tamoxifen [6,8]. While the successful use of letrozole in combination with COS for FP in BC patients has been reported in several studies [13–15], its use is not permitted for this indication in some countries such as France. Conversely, tamoxifen can be used in premenopausal women in France, but its efficacy for COS has only been evaluated in a few studies with small sample sizes [16–20]. Besides this pending question of which aromatase inhibitor or estrogen receptor modulator should be used, the issue of COS and FP before NAC also remains debated [21,22]. Currently, there is concern that FP might delay the onset of NAC, and that COS could increase tumor growth and metastatic risk. Recent meta-analyses have nevertheless provided more robust evidence on the safety of controlled COS, primarily with letrozole-COS protocol [21–23]. Further prospective studies with long-term follow-up are, however warranted to strengthen the evidence and determine the role of tamoxifen in COS for FP in young women with early BC.

The primary objective of this prospective study with extended follow-up was to assess the efficacy of Tamoxifen-COS before adjuvant or NAC in young patients with early BC.

# 2. Materials and methods

## (1) Inclusion criteria

Six cancer centers participated in the study. The inclusion period spanned from February 2014 to July 2017. Inclusion criteria were: patients aged 18–40, a stage I, II, and III BC undergoing chemotherapy in an adjuvant or neoadjuvant setting. Women with low ovarian reserve, defined as an anti-Müllerian Hormone (AMH) < 1 ng/mL and/or antral follicular count <5, were excluded. Women with a previous history of BC or other malignancies in the past five years, pregnancy at the time of inclusion, and a thrombo-embolic event in the past 6 months were also excluded.

## (2) Study protocol and procedures

Following BC diagnosis, patients were referred by their oncologist to the Reproductive Medicine unit of the local university hospital for FP counselling. Oocyte rather than embryo cryopreservation was proposed, except upon specific request from the patients. Ovarian stimulation could be started at a random cycle date, as already described [24,25]: in

Initial Follicular Phase (IFP) (between D1 and D3), in Late Follicular Phase (LFP) (D4-D14) or in Luteal Phase (LP) (D15-D28). COS was performed with recombinant Follicle Stimulating Hormone (FSH), with a daily dose of 150–450 International Unit (IU) according to the patient's age, BMI and ovarian reserve. Hormonal and ultrasonography monitoring was performed according to standard practice, and ovulation was triggered with either human recombinant Chorionic Gonadotropin hormone or Gonadotropin Releasing Hormone agonist. Transvaginal ultrasound guided oocyte retrieval was scheduled 36-h after ovulation. Mature oocyte vitrification was performed with RapidVit Oocyte kit (Vitrolife, Sweden) according to the manufacturer's instructions. Concomitantly with COS, 60 mg of tamoxifen was given daily to all patients until the day of triggering as previously done in pilot studies [16,17,20]. This protocol has been approved by the French National Agency for Medicines and Health Products Safety (ANSM) (ref 130764A-22).

## (3) Outcomes

The main objective was FP feasibility, evaluated by the number of patients who had at least one oocyte cryopreserved and the mean number of oocytes vitrified per patient. Secondary objectives were the assessment of time before initiation of chemotherapy, number of oocytes collected according to the type of stimulation (i.e. IFP, LFP or LP), BC recurrence defined as the detection of local relapse, distant metastases, or contralateral invasive BC, disease-free survival (DFS, i.e. time from diagnosis until evidence of disease recurrence or death from any cause), overall survival (OS, i.e. time from diagnosis to death from any cause) and number of pregnancies, either natural or following assisted reproductive technologies (ART). The study was designed for a five-year follow-up with 6-month reports by oncologists in accordance with clinical guidelines.

## (4) Statistical analysis

Comparison between groups were performed using Chi-square or Fisher exact tests for qualitative parameters, and Student's t-test, Anova, Mann-Whitney U test or Kruskal-Wallis tests for quantitative parameters. Patients without death or BC recurrence, who were lost during follow-up were censored on the last visit. The Kaplan-Meier method was applied to estimate 5-year DFS and OS. The median follow-up was calculated using the reverse Kaplan-Meier estimator. We did not impute missing data, except that the value 15 was imputed when the day was missing for the end of chemotherapy or histological diagnosis dates. All p values were based on two-sided tests and were considered significant if less than 0.05. We used SAS version 9.4 for the analyses.

This multicentric prospective study (ClinicalTrials.gov identifier NCT02890082) received approval from the Committee for the protection of persons (CPP OUEST IV 25/13). All participants signed a written consent form, and the study was conducted in accordance with the Declaration of Helsinki. Data were collected via an eCRF (Ennov Clinical).

# 3. Results

## (1) Patients' characteristics

A total of 101 patients were screened and 95 were analyzed (Fig. 1). Patients and treatment characteristics are presented in Table 1. Most patients had grade II-III carcinoma (94.6 %) and Estrogen Receptor (ER) positive-cancer (67 %). Sixty-nine patients (72.6 %) received the standard polychemotherapy sequential “FEC→Taxane” regimen administered in 3 consecutive “FEC” cycles (5-Fluorouracil 500mg/m2, Epirubicin 100mg/m2, Cyclophosphamide 500mg/m2), followed by 3 cycles of taxanes (docetaxel 100Mg/m2, repeated every 21 days) or paclitaxel every week during 12-weeks. Thirty-six patients (37.9 %)
<figure><img src="image_5.png" /><figcaption>Fig. 1. Flow chart.</figcaption></figure>

Abbreviations: IFP: Initial Follicular Phase; LFP: Late Follicular Phase; LP: Luteal Phase.

<caption>**Table 1**</caption>

Demographic and histological characteristics. Results are presented as number of patients (%) or means ± standard deviation when appropriate.

<table><thead><tr><th></th><th>All patients<br>n = 95</th><th>Neoadjuvant<br>chemotherapy n = 36<br>(37.9 %)</th><th>Adjuvant<br>therapy n = 59<br>(62.1 %)</th></tr></thead><tbody><tr><td><strong>Demographic characteristics</strong></td><td></td><td></td><td></td></tr><tr><td>Age at diagnosis<br>(years)</td><td>31.5 ± 4</td><td>30.8 ± 3.8</td><td>31.9 ± 4.1</td></tr><tr><td>No pregnancy history,<br>N(%)</td><td>52 (54.7)</td><td>23 (63.9)</td><td>29 (49.2)</td></tr><tr><td>Single at the diagnosis,<br>N(%)</td><td>23 (24.2)</td><td>7 (19.4)</td><td>16 (27.1)</td></tr><tr><td>BMI (kg/m²)</td><td>23.1 ± 4.8</td><td>23.5 ± 4.8</td><td>22.9 ± 4.8</td></tr><tr><td>BRCA 1/2 mutation<br>carriers (69 women<br>tested), N(%)</td><td>16 (23.2)</td><td>6 (21.4)</td><td>10 (24.4)</td></tr><tr><td><strong>Histological characteristics</strong></td><td></td><td></td><td></td></tr><tr><td>Invasive ductal<br>carcinoma, N(%)</td><td>89 (93.7)</td><td>35 (97.2)</td><td>54 (91.5)</td></tr><tr><td>Invasive lobular<br>carcinoma, N(%)</td><td>2 (2.1)</td><td>0</td><td>2 (3.4)</td></tr><tr><td>Other histological<br>category, N(%)</td><td>4 (4.2)</td><td>1 (2.8)</td><td>3 (5.1)</td></tr><tr><td>Grade II-III, N(%)</td><td>89 (94.6)</td><td>35 (97.2)</td><td>54 (91.5)</td></tr><tr><td>ER positive, N(%)</td><td>63 (67)</td><td>19 (52.8)</td><td>44 (74.6)</td></tr><tr><td>HER2 positive, N(%)</td><td>29 (31.2)</td><td>14 (38.9)</td><td>15 (25.4)</td></tr><tr><td>triple negative, N(%)</td><td>24 (25.3)</td><td>15 (41.7)</td><td>9 (15.3)</td></tr><tr><td>Node-positive, N(%)</td><td>29 (30.5)</td><td>10 (27.8)</td><td>19 (32.2)</td></tr></tbody></table>

Abbreviations: BMI, Body Mass Index; ER, estrogen receptor; HER2: human epidermal growth factor receptor 2.

received NAC.

## (2) Outcome of FP cycles

FP procedure was successful and led to ovum pickup in 85 patients (89.5 %). Ten patients (10.5 %) had COS cancelled because of poor response to stimulation (n = 8), high risk of ovarian hyperstimulation syndrome (n = 1) or personal reasons (n = 1).

The mean number of oocytes collected was 12.8 ± 7.9, and the mean number of mature oocytes vitrified was 9.8 ± 6.2. Ten patients opted to preserve both oocytes and embryos, with a mean of 9.5 ± 3.9 vitrified oocytes and a mean of 3.6 ± 3.7 cryopreserved embryos. On average, the duration of COS was 10.4 ± 1.9 days (range 7–17). Forty-three women (45.3 %) started COS in the IFP, 17 (17.9 %) in the LFP and 35 (36.8 %) in the LP. No significant differences were found in the number of oocytes collected and vitrified, dose of FSH and ovarian stimulation length according to the phase of the cycle in which COS was started (Table 2). The mean number of oocytes vitrified was not significantly different when COS was performed before NAC compared to

<caption>**Table 2**</caption>

Comparison of patient's characteristics and outcomes according to the type of stimulation. Data are presented as mean ± standard deviation (range), median (interquartile range) and number (%).

<table><thead><tr><th>Cycle outcomes</th><th>Initial<br>follicular<br>phase</th><th>Late<br>follicular<br>phase</th><th>Luteal phase</th><th>p-value</th></tr></thead><tbody><tr><td>Number of patients</td><td>43</td><td>17</td><td>35</td><td>0.81</td></tr><tr><td>Age (y)</td><td>31.3 ± 4</td><td>32.0 ± 4.5</td><td>31.4 ± 3.9</td><td>0.83</td></tr><tr><td>BMI (kg/m²)</td><td>23.5 ± 4.1</td><td>22.5 ± 5.3</td><td>22.9 ± 5.2</td><td>0.47</td></tr><tr><td>AMH (ng/ml)</td><td>4.4 ± 3.7<br>(0.4–15.8)</td><td>4.8 ± 6.1<br>(0.5–27)</td><td>4.4 ± 4<br>(0.1–22.7)</td><td>0.98</td></tr><tr><td>AFC</td><td>22.5 ± 15<br>(8–86)</td><td>18.2 ± 10.1<br>[9–41]</td><td>22.3 ± 9.8<br>(5–50)</td><td>0.24</td></tr><tr><td>Duration of ovarian<br>stimulation (days)</td><td>10 ± 1.8<br>[7–14]</td><td>11.1 ± 2.4<br>[7–17]</td><td>10.6 ± 1.7<br>[7–14]</td><td>0.08</td></tr><tr><td>Total dose of<br>gonadotropins (IU)</td><td>2268 ± 714</td><td>2568 ± 714</td><td>2408 ± 753</td><td>0.47</td></tr><tr><td>Total number of<br>oocytes retrieved</td><td>12.9 ± 8.8<br>(1–43)</td><td>9.7 ± 5.7<br>[1–21]</td><td>14.2 ± 7.2<br>[1–30]</td><td>0.16</td></tr><tr><td>Mature Oocytes<br>cryopreserved</td><td>10.0 ± 7.3<br>[1–35]</td><td>7.7 ± 4.0<br>[1–13]</td><td>10.4 ± 5.3<br>[1–22]</td><td>0.38</td></tr><tr><td>Cycle cancellation</td><td>2 (4.7 %)</td><td>3 (17.6 %)</td><td>5 (14.3 %)</td><td>0.20</td></tr></tbody></table>

Abbreviations: BMI, Body Mass Index; AMH, anti-Müllerian Hormon; AFC: Antral Follicle Count, IU: International Unit.

before adjuvant chemotherapy (11.1 ± 7.6 vs 9 ± 5.2, p = 0.27).

The mean number of mature oocytes vitrified was not statistically different between BRCA mutation carriers (n = 16) and other patients (n = 53) (9.1 ± 4.6 vs 10.2 ± 6.6, p = 0.77).

## (3) Time before chemotherapy start

In the adjuvant group, the median time from surgery to the initiation of chemotherapy was 6.0 weeks (q1: 5, q3: 7). In the neoadjuvant group, the median time from the first oncological visit to the initiation of chemotherapy was 3.6 weeks (q1 = 3.1; q3 = 4.1). Time to initiation of chemotherapy was not significantly different when COS was started in IFP, LFP or LP (6.4, 6.0 and 5.6 vs 3.7, 3.7 and 3.3 weeks in the adjuvant and in the neoadjuvant group, respectively (p = 0.5)).

## (4) Pregnancy follow-up

During the 5-year follow-up, five patients (5.3 %) returned to the local reproductive medicine unit to use their gametes, with a mean time interval of 4.1 years after FP. Seven embryo transfers with frozen-thawed oocytes were performed among these patients, but none resulted in pregnancy. The patients were 36 years-old at the time of diagnosis (q1-q3: 35–37 year). Meanwhile, 21 women (22.1 %) delivered after natural pregnancies with a mean time interval of 2.8 years after
diagnosis. Women who achieved natural pregnancies were younger and had better ovarian reserves at diagnosis compared to those who required ART after completing anticancer therapy (median age 30 [q1-q3: 27–32] vs 35.4 [q1-q3: 29–40] years); median AMH 3.2 ng/mL; [q1-q3: 2.2–5.8] vs 2.08 [q1-q3: 0.06–3.12]; *p* < 0.05 respectively).

### (5) Oncological follow-up

The median follow-up was 5.5 years (95 % confidence interval [CI] 5.4–5.7).

After a 5-year oncological follow-up, 19 patients experienced BC recurrence, of which 9 died (5-year DFS of 82 % [95%CI: 72%–88 %] and a 5-year OS rate of 90 % [95%CI: 81%–95 %], see Kaplan Meier curves on Figs. 2 and 3). Among relapses, 3 were local, 1 was regional and 14 were distant metastases. Two patients were lost to follow-up.

## 4. Discussion

The study provides further evidence supporting the efficacy of tamoxifen-COS protocol for FP in young women with early BC. The overall efficiency of the FP procedure was 89.5 %, with a mean number of vitrified oocytes per patient of 9.8 ± 6.2. These results are consistent with previous pilot studies evaluating tamoxifen-COS protocols [16,17], as well as reports based on letrozole-COS protocols (Table 3), although these studies were heterogeneous [21]. Two recent randomized controlled trials have further confirmed that alternative ovarian stimulation protocols involving tamoxifen or letrozole do not significantly affect the number of mature oocytes obtained [18,26]. In our study, 8 cycles were cancelled because of poor ovarian response to stimulation (8.4 %) and one because of a high risk of Ovarian Hyperstimulation Syndrome. This proportion is in overall agreement with the existing literature (Table 3), but very little information is given about cycle cancellations in the other studies, preventing a precise comparison. Of note, only ten women chose to preserve embryos, mainly because embryos cannot be reused if the couple separates.

Tamoxifen is the only antiestrogen therapy authorized for ovarian stimulation in France. Unlike in some countries, where letrozole is more widely used for FP, its use in ovarian stimulation is currently not permitted in France. One of the main advantages of COS with letrozole compared to tamoxifen is significantly decreased peak estradiol levels. The mechanism of using tamoxifen during COS is to modulate the ER and prevent the extra growth of estrogen responsive tumors during COS due to increased serum estrogen levels. A previous study shows that using 60 mg of tamoxifen daily during COS is sufficient to reach endoxifen levels that are considered therapeutically effective to inhibit

<figure><img src="image_6.png" /><figcaption>Fig. 2. Disease-free survival Kaplan-Meier curve.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 3. Overall survival Kaplan-Meier curve.</figcaption></figure>

BC growth [27]. The study reported no undesirable effects associated with tamoxifen-COS, including no thromboembolic events. Although tamoxifen has been widely used since the 1970's, at first for ovarian stimulation in polycystic ovarian syndrom [28], French authorities issued a warning on tamoxifen in 2021, based on old toxicology studies on rats, suggesting a potential risk of genotoxicity with aneugen and clastogenic mechanisms [29,30]. However, the ANSM ultimately provided reassurance regarding tamoxifen-COS safety (ref MEDMSANAT-2022-04-0090_2013-01981-40). This was based on the understanding that oocytes are not exposed to tamoxifen before the preantral follicle stage. Indeed, oocytes' DNA is quiescent until the end of prophase I and tamoxifen does not interact with this type of DNA, unlike some alkylating agents.

Considering the time interval before initiation of chemotherapy, the adjuvant and the neoadjuvant treatments should be considered separately. In the cohort studied here, 60 % of the patients underwent FP between surgery and adjuvant chemotherapy with a median time before chemotherapy of 6.0 weeks, while 40 % underwent FP before NAC with a median time before chemotherapy of 3.6 weeks. A recent meta-analysis by Arecco reported a 6-day delay in the time to chemotherapy start in patients who underwent COS for FP, with no detrimental prognostic effect observed [22]. In the current study, the initiation of adjuvant chemotherapy never exceeded 60-days, which is considered a reasonable cutoff, even for patients with more aggressive tumors [31]. In neoadjuvant cases, there is more urgency to start chemotherapy. However, due to a good cooperation between oncologists and ART units in all participating centers, the time interval did not exceed 4-weeks, which was deemed optimal [32].

Besides the fear of delaying chemotherapy mentioned above, some oncologists may have concerns that FP procedures could lead to a higher rate of metastasis in patients who have not yet undergone surgery (NAC setting). However, the meta-analysis conducted by Arecco also provided reassuring data regarding this concern. The analysis observed fewer recurrences and deaths in women who underwent COS before starting chemotherapy compared to patients who were not exposed to FP strategies, with no difference in DFS between the two groups [22]. In the meta-analysis, only 2 studies [33,34] reported data specifically on BC recurrences in patients who received COS before NAC compared to controls. Encouragingly, these studies indicated a reduced risk of recurrence among patients who underwent COS before starting NAC (RR0.22; *p* = 0.021).

In our study, the 5-year DFS was 82 %. Considering the composition of our cohort, which consists of young patients, many of whom are women with grade II-III disease, node positivity, triple-negative tumors, or a combination of these, this finding is consistent with existing
<caption>**Table 3**</caption>

Review of studies using tamoxifen and/or letrozole-COS stimulation for FP before BC treatment. Results are presented as means (standard deviation) and number of patients (%) when appropriate.

<table><thead><tr><th>First author, year (ref)</th><th>Study design</th><th>Tamoxifen/Letrozole</th><th>Number of patients</th><th>Number of oocytes collected</th><th>Number of embryos cryo-preserved</th><th>Number of oocytes cryo-preserved</th><th>Duration of ovarian stimulation (days)</th><th>Total use of gonadotropins (IU)</th><th>Cycle cancellation (number, rate)</th></tr></thead><tbody><tr><td>Oktay, 2005 [20]</td><td>Prospective randomized controlled study</td><td>Tamoxifen</td><td>9</td><td>6.9 (1.1)</td><td>3.8 (0.8)</td><td>/</td><td>8.9 (0.8)</td><td>/</td><td>/</td></tr><tr><td>Quintero, 2010 [17]</td><td>Retrospective cohort study</td><td>Letrozole</td><td>11</td><td>12.3 (2.5)</td><td>5.3 (0.8)</td><td>/</td><td>9.1 (0.5)</td><td>/</td><td>/</td></tr><tr><td>Meirow, 2014 [16]</td><td>Prospective non-randomized controlled study</td><td>Tamoxifen</td><td>28</td><td>8.8 (7.1)</td><td>/</td><td>/</td><td>10.9 (2.7)</td><td>4217(1363)</td><td>/</td></tr><tr><td>Kim, 2016 [13]</td><td>Prospective non-randomized controlled study</td><td>Letrozole</td><td>116</td><td>13.3 (8.4)</td><td>6.1 (4.7)</td><td>12.6 (3.5)</td><td>/</td><td>2053 (1243)</td><td>4 (3.3 %)</td></tr><tr><td>Quinn, 2017 [14]</td><td>Retrospective non-randomized controlled study</td><td>Letrozole</td><td>144</td><td>20.1 [1]</td><td>/</td><td>14.1 (0.8)</td><td>10.3 (0.2)</td><td>2370 (60)</td><td>7 (4.7 %)</td></tr><tr><td>Alvarez, 2018 [15]</td><td>Retrospective non-randomized controlled study</td><td>Letrozole</td><td>140</td><td>12.9 (8.7)</td><td>7.1 (4.4)</td><td>9.6 (6.3)</td><td>9.1 (5.1)</td><td>2633 [09,12]</td><td>5 (3.45 %)</td></tr><tr><td>Letourneau, 2021 [26]</td><td>Prospective randomized controlled study</td><td>Tamoxifen</td><td>44 50</td><td>16.9 (10.9)</td><td>/</td><td>12 (8.6) 11.6 (7.5)</td><td>9.8 (1.5) 10.2 (1.3)</td><td>2234 (858) 2283 (689)</td><td>1 (1.1 %)</td></tr><tr><td>Balkenende, 2022 [18]</td><td>Prospective randomized controlled study</td><td>Tamoxifen</td><td>54</td><td>12.5 (10.4)</td><td>5.6 (4.5)</td><td>10.2 (7.9)</td><td>10.6 (2.4)</td><td>2371 (537)</td><td>1 (1.9)</td></tr><tr><td></td><td></td><td>Letrozole</td><td>53</td><td>14.2 (9.4)</td><td>5.2 (3.9)</td><td>10.2 (8.1)</td><td>10.2 (3.2)</td><td>2225 (716)</td><td>2 (3.8)</td></tr></tbody></table>

literature [35–37]. All patients included in our study received chemotherapy, which differs from most FP studies. The POSITIVE trial demonstrated that the temporary interruption of endocrine therapy in women with ER-positive BC who attempted pregnancy, did not increase the short-term risk of recurrence. In this trial, only 62.0 % of the patients had received chemotherapy [38]. Embryo/oocyte cryopreservation at BC diagnosis, followed by embryo transfer after endocrine therapy interruption, yielded higher pregnancy rates and was not associated with worse prognosis [39]. Of the 518 enrolled patients in the POSITIVE trial, 179 patients (36 %) had undergone ovarian stimulation for embryo/oocyte cryopreservation at diagnosis; 83 (46 %), 36 (20 %) and 65 (37 %) of the patients reported using gonadotrophins alone, gonadotropins with tamoxifen, or gonadotropins with letrozole for ovarian stimulation, respectively.

In our study, all pregnancy plans were discussed with the patient's oncological advisor, and a staging scan, mammography, and possibly breast magnetic resonance imaging were conducted before given the green light. A time interval of two to three years after the end of treatment was recommended, at the discretion of each oncologist. Only a small number of women sought ART with frozen-thawed oocytes during the 5-year follow-up, which is not surprising according to the literature. Indeed, Oktay reported in a 14-year follow-up study that time to return for frozen embryo transfer after FP was 5.25 years on average [40]. However, it should be noted that 18 women (55 %) required a gestational carrier in this study, which may not be permitted in all countries. Due to the potential time interval between FP and utilization of ART, the study's follow-up period was extended to 10 years with patients' consent. This decision aligns with the findings from a Swedish cohort study, which demonstrated a cumulative incidence of post-BC live births of 19.4 % at 5 years and 40.7 % at 10 years [41]. Actually, at least three live births occurred after ART cycles since then. The number of spontaneous pregnancies in our cohort confirms the moderate gonadotoxicity of BC treatment but it should be highlighted that our cohort was very young with a median age of 31.5 ± 4 years old. Results could help oncologists to better inform very young patients about their chances of conceiving naturally post-treatment. In our study, women with low ovarian reserve at BC diagnosis, defined as an AMH<1 ng/mL, were excluded because multi-follicular ovarian stimulation is not efficient in

these patients. However, low AMH is not predictive of infertility, especially in post-cancer patients, with a non-negligible number of spontaneous pregnancies observed in BC survivors with undetectable AMH [42,43]. Additionally, in a cohort of young BRCA carriers with a median age at diagnosis of 35 years, Lambertini reported a 22 % cumulative incidence of pregnancy at 10 years. Of these pregnancies, 20.8 % occurred with the use of ART [44]. However, limited data are available on the gonadotoxicity of newer treatments such as CDK4/6 inhibitors [45], making it challenging to provide clear information about infertility risk for very young patients.

The study's main strength is its prospective design with a 5-year follow-up period. However, limitations of the study should be considered. First, it is difficult to assess the efficacy of tamoxifen for COS as an alternative to letrozole without a comparison group of patients undergoing letrozole-COS. Second, although a 5-year follow-up is longer than many previous studies on FP, it may not be long enough to evaluate the efficacy of tamoxifen-COS protocols in terms of pregnancy outcomes. Third, the safety of using tamoxifen-COS protocols could be better assessed by comparing it with patients who did not undergo ovarian stimulation. Much larger cohorts are needed to document the oncological safety of tamoxifen-COS.

## 5. Conclusion

In conclusion, our study, conducted on a long-term prospective cohort, reaffirms the efficacy of tamoxifen as an alternative to letrozole for COS in terms of oocyte yield for young women with early BC. Through effective collaboration between oncologists and ART centers, COS did not significantly delay the initiation of chemotherapy, even in the neoadjuvant setting. However, longer-term follow-up is necessary to fully understand the clinical implications and outcomes associated with the use of vitrified-warmed oocytes. As BC treatments are evolving towards a wider use of NAC and extended novel therapies, it is of utmost importance to propose efficient and safe FP protocols.

## Funding

This study was supported by a grant from the French Ministry of
Health (Reference PHRC 2009 20-17).

### CRediT authorship contribution statement

**A. Dezellus:** Writing – original draft, Validation, Resources, Investigation, Formal analysis. **S. Mirallie:** Writing – review & editing, Conceptualization. **F. Leperlier:** Writing – review & editing, Resources. **B. Sauterey:** Resources. **P.-E. Bouet:** Resources. **A. Dessaint:** Resources. **S. Duros:** Resources. **A.S. Gremeau:** Resources. **M.-A. Mouret-Reynier:** Resources. **L.M. Durand:** Resources. **L. Venat:** Resources. **P. De Blay:** Resources. **M. Robert:** Writing – review & editing. **T. Freour:** Writing – review & editing. **M. Campone:** Supervision, Conceptualization. **A. Blanc-Lapierre:** Methodology. **V. Bordes:** Resources, Investigation, Formal analysis, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: None of the authors have any conflicts of interest related to this study.

Florence Leperlier reports honoraria for lectures and presentations from Ferring Pharmaceuticals, Gedeon Richter, and IBSA, unrelated to this work.

Pierre-Emmanuel Bouet reports travel and meeting support from Merck, Gedeon-Richter, Theramex, Ferring, Organon and IBSA. P-E.B. have undertaken consultancy work for Merck and Gedeon-Richter and have received research grants from Theramex, Ferring, MSD, and Gen-evrier, not related to the present work.

Thomas Freour has undertaken consultancy work for Vitrolife France and Gedeon-Richter. T.F. reports travel and meeting support from Gedeon-Richter, Theramex, Ferring and IBSA. T.F. reports honoraria for lectures and presentations from Ferring Pharmaceuticals, Theramex, Gedeon Richter and Merck Serono, unrelated to this work.

Marie Robert reports travel fees and congress fees from AstraZeneca, Gilead and Creafirst, and has undertaken consultancy work for AstraZeneca and Lilly.

Mario Campone is on the advisory board of AstraZeneca, Novartis, Sanofi, Lilly, Pfizer, Seagen, Gilead and Daiichi-Sankyo. M.C. has undertaken consultancy work for AstraZeneca, Novartis, Daiichi-Sankyo, PET-Therapy, Menarini and DIACCURATE, and he is a speaker for Novartis, Lilly and Amgen. He reports travel support from Pfizer, Novartis, Roche, AstraZeneca and Daiichi-Sankyo.

### Acknowledgments

The authors have a special thought to the principal investigator of the PRESAGE study, Virginie Bordes, who prematurely died after a long fight against breast cancer. They also wish to thank the Institut de Cancerologie de l'Ouest DRCI staff for their help in setting-up, coordinating and monitoring the study, as well as the research staff of each investigating center.

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