# AMH as a marker for resumption of ovarian function after chemotherapy: an IPD meta-analysis and systematic review

Charissa van Zwol-Janssens, Mandy van M. Rosmalen, Joop S.E. Laven, Kazem Nasserinejad, Jenny Visser, Richard Anderson, Irit Ben-Aharon, Thomas Freour, Kathryn Ruddy, H. Irene Su, et al.

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## To cite this version:

Charissa van Zwol-Janssens, Mandy van M. Rosmalen, Joop S.E. Laven, Kazem Nasserinejad, Jenny Visser, et al. AMH as a marker for resumption of ovarian function after chemotherapy: an IPD meta-analysis and systematic review. Cancer Treatment Reviews, 2026, 142, pp.103068. 10.1016/j.ctrv.2025.103068. inserm-05474255

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# AMH as a marker for resumption of ovarian function after chemotherapy: an IPD meta-analysis and systematic review

Charissa van Zwol – Janssens<sup>a,*</sup>, Mandy van M. Rosmalen<sup>b</sup>, Joop S.E. Laven<sup>a</sup>, Kazem Nasserinejad<sup>c</sup>, Jenny A. Visser<sup>d</sup>, Richard A. Anderson<sup>e</sup>, Irit Ben-Aharon<sup>f</sup>, Thomas Freour<sup>g</sup>, Kathryn J. Ruddy<sup>h</sup>, H. Irene Su<sup>i</sup>, Yvonne V. Louwers<sup>a,1</sup>, Agnes Jager<sup>b,1</sup>

## ARTICLE INFO

### Keywords:

- Breast cancer

- Premenopausal

- Ovarian function

- Chemotherapy induced amenorrhea

## ABSTRACT

**Background:** In premenopausal women with breast cancer, chemotherapy often leads to amenorrhea that could be temporary or permanent. Anti-Müllerian hormone (AMH) is a potential biomarker predicting resumption of ovarian function, an outcome that aids in the decision making for endocrine therapy. This study aimed to determine the predictive value of pre-chemotherapy AMH levels for resumption of ovarian function.

**Methods:** We conducted a systematic review and individual patient data (IPD) meta-analysis. Online databases were searched using terms including: AMH, prediction, menses, menses recovery, amenorrhea, chemotherapy-related amenorrhea, anovulation, menopause, infertility, ovarian reserve, premenopausal, breast cancer, chemotherapy. The study protocol was registered with PROSPERO (CRD42021233966).

**Results:** The systematic review included 31 studies, with 26 contributing to the meta-analysis. Eleven studies provided IPD from 1,029 women. Ovarian function resumption rates varied from 24 % to 61 % depending on follow-up time. Pre-chemotherapy AMH was significantly higher in women whose ovarian function resumed, standardized mean difference 0.94 (95 % CI 0.65–1.22) and showed good predictive ability for resumption of ovarian function (AUC 0.79–0.83). However, the identified cut-off values for pre-chemotherapy AMH gave high false negative rates and differed a lot between studies which was much determined by follow-up time, age and used AMH assay.

**Conclusion:** Pre-chemotherapy AMH shows association with ovarian function resumption, but a clinically reliable cut-off across assays could not be established using individual patient data. Therefore, due to high inter-assay variability, AMH is not suitable for optimizing endocrine therapy in premenopausal breast cancer patients at this time. Standardizing AMH assays can help in further research into a cut-off value.

## Introduction

Breast cancer is the most common cancer among women, with 12–20 % of women in reproductive age [1]. These women have a 5-year

survival rate of 72–80 % after treatment [2]. Premenopausal women with primary breast cancer are mostly treated with (neo)adjuvant chemotherapy, which improves survival rate and reduces the risk of recurrence, but it is also gonadotoxic. This toxicity can cause ovarian

<footnote><sup>a</sup> Division Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Erasmus MC, University Medical Center Rotterdam, the Netherlands</footnote>

<footnote><sup>b</sup> Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University MC, Rotterdam, the Netherlands</footnote>

<footnote><sup>c</sup> Division of Statistics, Department of Hematology, Erasmus MC Cancer Institute, Erasmus University MC, Rotterdam, the Netherlands</footnote>

<footnote><sup>d</sup> Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands</footnote>

<footnote><sup>e</sup> Centre for Reproductive Health, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh EH16 4UU, United Kingdom</footnote>

<footnote><sup>f</sup> Division of Oncology, Rambam Health Care Campus, Haifa, Israel</footnote>

<footnote><sup>g</sup> CHU Nantes, Service de Médecine et Biologie de la Reproduction & Nantes Université, Inserm, CR2TI Nantes, France</footnote>

<footnote><sup>h</sup> Mayo Clinic, Rochester, MN, USA</footnote>

<footnote><sup>i</sup> Department of Obstetrics, Gynecology and Reproductive Sciences and Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA</footnote>

<footnote>* Corresponding author at: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynaecology, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD, Rotterdam, the Netherlands.</footnote>

<footnote>E-mail address: c.janssens@erasmusmc.nl (C. van Zwol – Janssens).</footnote>

<footnote n="1">¹ Shared last authorship.</footnote>
injury, potentially causing chemotherapy-induced amenorrhea (CIA). This may be temporary or if the damage is permanent, it's known as premature ovarian insufficiency (POI). Women with hormone-sensitive breast cancer who develop POI after chemotherapy often have improved survival outcomes [3,4]. Moreover, recognizing menopausal status following treatment can aid in tailoring therapy.

Anti-Müllerian hormone (AMH) is a reliable indicator of ovarian reserve, indirectly reflecting the number of primordial follicles [5]. Studies have shown that pre-chemotherapy AMH levels can predict the likelihood of resumption of ovarian function after chemotherapy [6]. Resumption of ovarian function involves the ovaries resuming their natural activity after chemotherapy, both menstruation and the production of estrogens and other hormones are surrogates for resumption of ovarian function. This is especially important in women with hormone-sensitive breast cancer. Endocrine therapy in these women can vary depending on whether they experience resumption of ovarian function after chemotherapy. Women who resume ovarian function benefit from ovarian suppression using a gonadotropin-releasing hormone (GnRH) agonist. This medication can help prevent the ovaries from producing estrogen which can stimulate the growth of breast cancer cells [7]. On the other hand, postmenopausal women, including those with CIA, may be treated with aromatase inhibitors (AIs). AIs inhibit the conversion of androgens into estrogens providing an effective way to manage hormone receptor-positive breast cancer. It's important to note that treating women with resumption of ovarian function with AIs alone can be ineffective and potentially detrimental [8]. These women need a combination of ovarian suppression and AI therapy to achieve optimal outcomes.

Understanding the factors that influence whether a woman will become postmenopausal or experience ovarian function recovery after chemotherapy is crucial for optimizing treatment decisions. This knowledge allows healthcare providers to tailor endocrine therapy to maximize its effectiveness and minimize potential risks. To address this we conducted a systematic review and meta-analysis of existing studies. In addition, by analyzing the pooled individual patient data (IPD) from these studies, we aimed to establish a cut-off value for pre-chemotherapy AMH that could accurately predict resumption of ovarian function in premenopausal breast cancer patients.

## Methods

### Systematic review

This study was conducted according to ‘A guide to systematic review and meta-analysis of prognostic factor studies’ (from Cochrane) [9] and reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [10]. We registered this meta-analysis with PROSPERO (CRD42021233966). To answer our main research question, we divided it into three sub-questions: the incidence of resumption of ovarian function, the difference in pre-chemotherapy AMH levels in women with and without resumption of ovarian function, and the predictive value of pre-chemotherapy AMH levels for resumption of ovarian function.

MEDLINE, EMBASE, Web of Science Core Collection, Google Scholar and Cochrane Library were searched for articles published up to July 31st 2023. The key words used in search were “AMH”, “prediction”, “menses”, “menses recovery”, “amenorrhea”, “chemotherapy-related amenorrhea”, “anovulation”, “menopause”, “infertility”, “ovarian reserve”, “premenopausal”, “breast cancer”, “chemotherapy”.

Duplicates were removed and articles were screened by two independent reviewers (CvZ, MvR) in two steps, firstly all articles were screened based on title and abstract, secondly only the selected articles were screened full text. The following inclusion criteria were used: premenopausal women diagnosed with primary non-metastatic breast cancer, measurements of AMH levels at least before chemotherapy was started (pre-chemotherapy), and information regarding ovarian

function after chemotherapy should be available. Exclusion criteria were duplicate datasets/ cohorts, non-comparable population (e.g. when women with amenorrhea were excluded or AMH assays were compared), non-English papers and review articles. Disagreements were resolved by discussion with the other reviewer, and judgment was based on consensus.

Data were extracted using the checklist for critical appraisal and data extraction for systematic reviews of prediction modeling studies for prognostic factors (CHARMS-PF) [9] by one of the reviewers (CvZ).

All included articles underwent the quality in prognostic factor studies (QUIPS) [9] tool to examine each study's risk of bias by two independent reviewers (CvZ, MvR). Disagreements were resolved by discussion with the other reviewer, and judgment was based on consensus.

Extracted data for the systematic review included description of study design, cohort size, inclusion criteria, mean/median pre-chemotherapy age, chemotherapy regimen, GnRH agonist co-treatment, AMH sampling timepoints, AMH assay used and unit in what it was expressed, cohort mean/ median pre-chemotherapy AMH, predictive value of pre-chemotherapy (pre-CT) AMH, number of women with resumption of ovarian function and main conclusion on AMH.

### Individual patient data (IPD) meta-analysis

The IPD was requested from the corresponding authors of all included studies through 4 separate emails over a period of 6 months, these involved pre-chemotherapy AMH levels and any other AMH levels during or after chemotherapy, pre-chemotherapy age, menstrual cycle data (before and after chemotherapy), chemotherapy regimens, GnRH agonist or other hormone therapy as co-treatment, tumor stage and (hormone) receptor status, follow-up time, intoxications (smoking, alcohol use), ethnic origin and BMI and/ or m². Women who received GnRH agonists concurrently with chemotherapy (n = 123) or whose post-chemotherapy GnRH agonist start date was unknown (n = 34) were fully excluded. Conversely, women who started post-chemotherapy GnRH agonists with a known start date (n = 17) or underwent bilateral oophorectomy (n = 33) were censored at the time of the intervention and included in the analysis only for the follow-up durations prior to the procedure.

### Statistical analysis of the IPD meta-analysis

To identify pre-chemotherapy AMH value as a possible predictor for resumption of ovarian function we performed an IPD meta-analysis. Data from studies that reported mean pre-chemotherapy AMH values in the article of women with resumption of ovarian function were used to answer the following two sub-questions: 1. the incidence of resumption of ovarian function and 2. the difference in pre-chemotherapy AMH levels in women with and without resumption of ovarian function. Resumption of ovarian function is defined as any activity of the ovaries and is defined differently across studies (e.g. resumption of regular menstrual cycle, estradiol production, etc.), see *Supplemental Table S1*. Standardized mean difference of pre-chemotherapy AMH values were calculated between women with resumption of ovarian function and chemotherapy induced amenorrhea at one year after chemotherapy using the random effects model to account for heterogeneity. The meta-analysis package of Review Manager 5.4.1 was used to perform this analysis.

To explore the predictive value of pre-chemotherapy AMH (sub-question 3) we have pooled the available data per follow-up time. Due to differences in follow-up time, we pooled the data in studies that reported 6 months, 12 months and 24 months of follow-up. We have calculated the pooled sensitivity and specificity and made a summary ROC curve using a random effects model in the meta-analysis package of R studio version 4.3.2.

In a subgroup analysis, we have used ROC analysis to identify the
optimal cut-off point for pre-chemotherapy AMH, age and AMH combined with age to predict resumption of ovarian function after chemotherapy in the pooled IPD data. In addition, we also did a subgroup analysis for all the different AMH assays, because the specific assay is an important factor for heterogeneity between the studies.

## Results

### Study characteristics

Fig. 1 shows the number of studies assessed and the reason for exclusion in the PRISMA flowchart. The search identified 1291 records, and after removal of duplicates and screening for quality and relevance to the study question based on title and abstract and full-text, 31 studies were included for the systematic review. The corresponding authors of these 31 studies were contacted to share their study IPD. In total, data from 2034 women (of 26 studies) were included in the meta-analysis, of whom individual patient data were available for 1029 women (of 11 studies). These included women had a mean age of 38.3 years and 64.2 % of the women had a hormone receptor positive tumor. In *Supplemental Table S2* we show a comparison of the baseline characteristics of the studies that sent the IPD and those that did not, showing some studies significantly differed in age, BMI and AMH, although the range was similar.

### Systematic review of resumption of ovarian function

In total, 27 studies reported information concerning menstrual cyclicity after chemotherapy [11–37] (*Supplemental Table S1*). Studies

reporting on 6 months of follow-up showed that 18–53 % of the patients had resumption of ovarian function [16–20]. Studies reporting on 12 months of follow-up showed that 18–90 % of the patients had resumption of ovarian function [11,15,17,18,20–22,24,28,33,36–38]. Studies reporting on 24 months of follow-up showed that 23–92 % of the patients had resumption of ovarian function [13,14,19,25,34,35]. Studies reporting other follow-up durations can be found in *Supplemental Table S1*.

### IPD meta-analysis of resumption of ovarian function

In *Supplemental Table S3* we show the numbers and percentages of women with resumption of ovarian function at different follow-up times of the studies included in the IPD meta-analysis. When we combined all studies reporting on 6 months follow-up after chemotherapy, 223 (23.8 %) of the 937 women had resumption of ovarian function. With all studies reporting on 12 months of follow-up, 731 (48.3 %) of the 1,513 women had resumption of ovarian function. And with all studies reporting on 24 months of follow-up, 505 (61.2 %) of the 825 women had resumption of ovarian function. Analysis of studies reporting resumption percentages below the average revealed that these studies often had a higher mean age. Conversely, studies reporting higher resumption percentages tended to have a lower mean age (see *Supplemental Table S1* and S3).

### Systematic review of pre-chemotherapy AMH levels

Studies reporting pre-chemotherapy AMH levels (n = 12) in both groups of women who had resumption of ovarian function after

<figure><img src="image_5.png" /><figcaption>Fig. 1. PRISMA flowchart.</figcaption></figure>
chemotherapy and those that did not had resumption of ovarian function, mainly showed higher AMH levels in those that had resumption of ovarian function [11,12,14,16,18,22,26,28,35,39,40] at different follow-up durations (Supplemental Table S1). Only one study showed similar AMH level in both groups with one year follow-up [36]. Furthermore, the mean or median age of women who had resumption of ovarian function was lower than in women who had chemotherapy induced amenorrhea [11,12,14,16,18,22,26,28,35].

## IPD meta-analysis of pre-chemotherapy AMH levels

In Fig. 2 we show the results of the IPD meta-analysis of the eight studies reporting mean AMH values combined with the IPD, we calculated a standardized mean difference of 0.94 (95 % CI 0.65 – 1.22), with higher levels observed in women experiencing resumption of ovarian function compared to those with CIA at 2-year follow-up. There was a moderate heterogeneity between the studies (I² = 52 %). In Supplemental Figure S1 we show the results of the meta-analysis of 11 studies with a follow-up of 12 months, we calculated a standardized mean difference of 0.77 (95 % CI 0.54–1.00).

## Systematic review of predictive value of pre-chemotherapy AMH

In total 16 studies reported the predictive value of pre-chemotherapy AMH for resumption of ovarian function [11–14,16,21–25,28,29,31,35,37,41] (Supplemental Table S1). Six studies reported an AUC for chemotherapy induced amenorrhea, which was between 0.77 and 0.91 [12–14,16,22,35]. Five studies reported odds ratios. Henry et al. reported that detectable pre-chemotherapy AMH (≥0.16 ng/ml) was associated with recovery of ovarian function (OR: 108; 95 % CI 5.8- >2000; p < 0.002) [21]. Leonard et al. and Li et al. reported an OR of pre-chemotherapy AMH of 0.43 and 0.31, respectively, for post-CT chemotherapy induced amenorrhea [23,24]. Ruddy et al. 2014 reported for every one ng/mL increase in pre-chemotherapy AMH, there was a 59 % decrease in the odds of chemotherapy induced amenorrhea at 18 months, OR 0.41 [28]. In their follow up study in 2020, Ruddy et al. reported the odds of having post chemotherapy menses increased with higher pre-chemotherapy AMH in a different population (OR 1.24) [29]. Anders et al. reported that AMH levels below the median (0.7 ng/ml) gave a relative risk (RR) of 1.83 for developing chemotherapy induced amenorrhea [11]. Su et al. found that higher AMH levels were associated with resumption of ovarian function, unadjusted hazard ratio (HR) of 1.53 [31]. Mailliez et al. reported that the risk of longer amenorrhea duration decreased with standardized pre-chemotherapy AMH, HR/unit: 0.91 [25]. Dillon et al. reported that pre-chemotherapy AMH levels >2.0 ng/ml gave a significantly higher chance of resumption of ovarian function (p = 0.003) [41]. Similarly, Zhong et al. found that detectable AMH levels (above 1.1 ng/ml) gave a

significantly higher chance of resumption of ovarian function (p = 0.013) [37]. Three studies reported that there was no significant predictive value of pre-chemotherapy AMH for resumption of ovarian function [32,36,38].

## IPD meta-analysis of predictive value of pre-chemotherapy AMH

In Fig. 3 we show the results of the meta-analysis of all studies reporting sensitivity and specificity of pre-chemotherapy AMH for predicting resumption of ovarian function and studies which provided IPD at different timepoints. At 6 months (Fig. 3a), the pooled sensitivity was 83 % (95 % CI 77–88 %) and pooled specificity was 64 % (95 % CI 51–76 %). The used or optimal cut-off for pre-chemotherapy AMH to predict resumption of ovarian function at 6 months ranged from 0.77 to 3.32 ng/ml, with a mean of 1.68 ng/ml and median of 1.31 ng/ml. With these different AMH cut-off values 17 % of women were false negatives (i.e., they had an AMH below the cut-off value, but their ovarian function did resume). The AUC for prediction of resumption of ovarian function at 6 months in the summary ROC curve analysis was 0.83. At 12 months (Fig. 3b), the pooled sensitivity was 80 % (95 % CI 73–85 %) and the pooled specificity was 75 % (95 % CI 59–85 %). The used, or optimal, cut-off for pre-chemotherapy AMH to predict resumption of ovarian function at 12 months ranged from 0.16 to 2.49 ng/ml, with a mean of 0.99 ng/ml and median of 0.70 ng/ml. With these different AMH cut-off values, 20 % of women were false negatives. The AUC for prediction of resumption of ovarian function at 12 months was 0.79. At 24 months (Fig. 3c) the pooled sensitivity was 76 % (95 % CI 71–79 %) and the pooled specificity was 78 % (95 % CI 66–86 %). The used, or optimal, cut-off for pre-chemotherapy AMH to predict resumption of ovarian function at 24 months ranged from 0.41 to 1.55 ng/ml, with a mean of 0.84 ng/ml and median of 0.74 ng/ml. With these different AMH cut-off values 24 % of women were false negatives. The AUC for prediction of resumption of ovarian function at 24 months was 0.80.

In the subgroup analysis, we have pooled all individual data in one dataset without accounting for random effect to identify a cut-off value for pre-chemotherapy AMH at the different follow-up durations. For resumption of ovarian function at 6 months the optimal cut-off value calculated with ROC-analysis was 1.33 ng/ml with a sensitivity of 82 % and a specificity of 61 % (Fig. 4a). For resumption of ovarian function at 12 months the optimal cut-off value was 0.91 ng/ml with a sensitivity of 75 % and a specificity of 59 % (Fig. 4b). For resumption of ovarian function at 24 months the optimal cut-off value was 1.12 ng/ml with a sensitivity of 63 % and a specificity of 78 % (Fig. 4c). When focusing on continuation of endocrine therapy with AIs, we need a pre-chemotherapy AMH cut-off value that can predict CIA with a high certainty. Therefore, in addition to calculating the optimal AMH cut-off value using ROC analysis, we also examined a range of highly sensitive cut-off values. At the 6-month follow-up, a cut-off of 0.10 ng/ml

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Results of the IPD meta-analysis: Standardized mean difference pre-chemotherapy AMH for resumption of ovarian function and CIA at 24 months Resumption = resumption of ovarian function; CIA = chemotherapy-induced amenorrhea.</figcaption></figure>
a. pre-chemotherapy AMH for resumption of ovarian function at 6 months

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

b. pre-chemotherapy AMH for resumption of ovarian function at 12 months

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

**Fig. 3.** Results of meta-analysis: Forrest plot and summary ROC curve of the studies reporting predicting resumption of ovarian function with pre-chemotherapy AMH Forrest plot and SROC curves for prediction of resumption of ovarian function: a = pre-chemotherapy AMH for resumption of ovarian function at 6 months (based on 6 studies, including 174 women with resumption of ovarian function and 574 without resumption of ovarian function); b = pre-chemotherapy AMH for resumption of ovarian function at 12 months (based on 9 studies, including 398 women with resumption of ovarian function and 509 without resumption of ovarian function); c = pre-chemotherapy AMH for resumption of ovarian function at 24 months (based on 8 studies, including 389 women with resumption of ovarian function and 271 without resumption of ovarian function). Note, in the study of Zhong et al women who received GnRH-agonist during chemotherapy were included (21 of 28 women with resumption of ovarian function received GnRH agonist during chemotherapy).
c. pre-chemotherapy AMH for resumption of ovarian function at 24 months

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>Fig. 3. (continued).</figcaption></figure>

c. pre-chemotherapy AMH for resumption of ovarian function at 24 months

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>Fig. 3. (continued).</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Fig. 4.** Results of the IPD meta-analysis: ROC curves for prediction of resumption of ovarian function with pre-chemotherapy AMH and age, ROC curves for prediction of resumption of ovarian function, based on 11 studies included in the IPD analysis: a = pre-chemotherapy (pre-CT) AMH for resumption of ovarian function at 6 months (131 women with resumption of ovarian function and 692 without resumption of ovarian function); b = pre-chemotherapy AMH for resumption of ovarian function at 12 months (381 women with resumption of ovarian function and 543 without resumption of ovarian function); c = pre-chemotherapy AMH for resumption of ovarian function at 24 months (481 women with resumption of ovarian function and 212 without resumption of ovarian function); d = pre-chemotherapy age for resumption of ovarian function at 6 months (131 women with resumption of ovarian function and 692 without resumption of ovarian function); e = pre-chemotherapy age for resumption of ovarian function at 12 months (381 women with resumption of ovarian function and 543 without resumption of ovarian function); f = pre-chemotherapy age for resumption of ovarian function at 24 months (481 women with resumption of ovarian function and 212 without resumption of ovarian function); g = pre-chemotherapy AMH combined with age for resumption of ovarian function at 6 months (131 women with resumption of ovarian function and 692 without resumption of ovarian function); h = pre-chemotherapy AMH combined with age for resumption of ovarian function at 12 months (381 women with resumption of ovarian function and 543 without resumption of ovarian function); i = pre-chemotherapy AMH combined with age for resumption of ovarian function at 24 months (481 women with resumption of ovarian function and 212 without resumption of ovarian function). AUC = area under the curve; sens = sensitivity; spec = specificity.</figcaption></figure>
demonstrated a high sensitivity of 99.2 % and a negative predictive value of 99.1 %. However, its specificity was very low at 16 % (Supplemental Table S4). While longer follow-up periods led to a decrease in specificity and negative predictive values, it is known that a 24-month follow-up provided greater confidence that individuals who didn't experience a resumption of ovarian function would continue to do so. Finally, we found that pre-chemotherapy AMH levels alone had similar AUC values compared to pre-chemotherapy AMH combined with age, or age on its own (Fig. 4d-i).

Given that pre-chemotherapy AMH and age have similar AUCs, we investigated whether pre-chemotherapy AMH has additional predictive value for resumption of ovarian function. We used a backward logistic regression model with both age and pre-chemotherapy AMH and found that age (p < 0.001) and pre-chemotherapy AMH (p = 0.002) were both independently associated with resumption of ovarian function at 6 months, but age had a slightly larger beta (age -0.163; AMH 0.090). For resumption of ovarian function at 12 months, both age (p < 0.001) and pre-chemotherapy AMH (p = 0.005) were independently associated, but age had a slightly larger beta (age -0.180 and AMH 0.092). Similarly, for resumption of ovarian function at 24 months, both younger age and higher pre-chemotherapy AMH were independent significantly associated (both p < 0.001), but pre-chemotherapy AMH had a bigger beta (age -0.202 and AMH 0.410). We investigated the association of chemotherapy regimens, smoking, alcohol use, ethnic origin, and BMI with the resumption of ovarian function. Among the 436 women with known smoking status (42.4 % of the cohort), ever smoking was negatively associated with the resumption of ovarian function at 24 months (p = 0.035). No other covariates showed a significant association.

Next to age we found that the AMH assay that was used impacted the results. In *Supplemental Figure S2* we show the distribution of AMH values at different ages per assay. This shows that four of the six assays (Gen I Beckman coulter, Gen II Beckman coulter, DSL and Roche elecys) measured lower values over all ages, where the other two measured higher values. It is important to note that each blue dot represents a unique woman, not the same woman measured with different assays. Therefore, other covariates might explain this difference. In *Supplemental Figure S3* we show the different cut-off values for each assay at the different follow-up durations. We found a wide variation of optimal cut-off values ranging from 0.18 to 2.27 ng/ml depending on the AMH assay and follow-up period.

## Quality assessment

Selection bias was classified as low in 14 studies, moderate in 7 studies and high in 10 studies (Supplemental Table S5). Mainly, study attrition was poorly documented, the study population and the method for measuring the outcome was not described.

## Discussion

Our IPD meta-analysis including more than 1,000 premenopausal breast cancer patients aimed to calculate a cut-off value of pre-chemotherapy AMH levels for predicting resumption of ovarian function after chemotherapy. We found that ovarian function resumption rates varied from 25 % to 67 % depending on follow-up duration. Notably, women with higher pre-chemotherapy AMH levels were significantly more likely to experience resumption of ovarian function compared to those with CIA. Although our meta-analysis demonstrates good predictive values for pre-chemotherapy AMH in predicting resumption of ovarian function, a clinically applicable cut-off value cannot be definitively established. This is complicated by the use of various AMH assays without an international standard, as well as the importance of being able to state with certainty that an individual will not experience resumption of ovarian function before initiating the optimal adjuvant endocrine treatment.

Interestingly, we found that pre-chemotherapy AMH and age

exhibited similar AUCs. While the finding that AMH is independent predictor confirms its unique biological relevance distinct from chromological aging, the similar AUCs suggest the incremental predictive value of adding AMH to age is modest. However, other studies have suggested that pre-chemotherapy AMH levels may offer additional prognostic value [16,31]. This could possibly be due to the high heterogeneity of the study populations, as well as the use of different assays in the studies. Given that AMH as a predictor did not demonstrate superiority over age, and considering that age remains a simple, cost-free, and clinically validated predictor while AMH is constrained by the current lack of international assay standardization, these findings suggest that age serves as a good substitute predictor. When comparing the results of the meta-analysis with those of the subgroup analysis of the combined IPD data, we observed slightly higher AUC in the meta-analysis. The AUC in the meta-analysis represents the results in the summary ROC curve, which is based on various study-specific cut-off points. This suggests that the heterogeneity of the populations and of assays used across studies influenced the lower AUC for pre-chemotherapy AMH observed in the subgroup analysis, where all different assays were pooled without accounting for random effects. Consistent with our findings on the distribution of AMH levels across different ages per assay, previous research confirms that AMH assays are not interchangeable and cannot be directly converted or compared [42]. For this reason, we conducted subgroup analyses for each individual assay. These subgroup analyses revealed substantial variations in optimal cut-off values, ranging from 0.22 ng/ml to 2.27 ng/ml. This tenfold difference in cut-off magnitude highlights the substantial impact of inter-assay variability on the clinical interpretation of AMH levels, effectively precluding the definition of a universal predictive threshold. Such discrepancies in cut-off values across different assays hinder the implementation of AMH as a clinical predictor. While AMH can be used as a clinical predictor, a single cut-off value across all assays cannot be applied. Furthermore, it is difficult to calculate robust cut-off values using this data due to the very small subgroup sizes per different assay. Moving forward, addressing this variability is paramount. Current international efforts, such as the establishment of the World Health Organization (WHO) International Standard for AMH [43], are crucial steps towards the necessary standardization of assays, which may enable the derivation of a reliable, universally applicable cut-off value in future research.

Although we analyzed multiple follow-up durations in our meta-analysis, our results suggest that the 24-month follow-up is most clinically relevant, as it better reflects long-term ovarian function than earlier time points and women with permanent CIA may be switched from tamoxifen to an AI after two years. We initially hypothesized that by using pre-chemotherapy AMH levels as a predictor of resumption of ovarian function, we no longer needed this two-year wait period. However, the false negative rate is too high to safely use our cut-off of pre-chemotherapy AMH for this purpose. On the other hand, pre-chemotherapy AMH can be used to identify women with likely resumption of ovarian function who need to be treated with a GnRH agonist, preferably before their ovarian function resumes. So, although we do not have a cut-off value of pre-chemotherapy AMH for this purpose, a high AMH level may still be informative in the treatment of premenopausal women with breast cancer.

In this meta-analysis, our objective was to inform decision-making for endocrine therapy by analyzing individual patient data, we additionally examined the impact of chemotherapy regimens, smoking, alcohol use, ethnic origin, and BMI on the resumption of ovarian function. We observed minimal variation in chemotherapy regimens, with over 95 % utilizing cyclophosphamide and over 80 % including taxane, which precluded the detection of a statistical difference for this factor. However, a significant association was found with smoking status, where women who had ever smoked or were current smokers had a lower chance of resumption of ovarian function. Despite these insights, the high volume of missing data for several factors unfortunately limited
our ability to incorporate them into a robust prediction model for
ovarian function resumption.

It is important to note that this study is subject to certain limitations. While a random-effects model was employed to mitigate the impact of heterogeneity in study designs, patient populations, AMH assays, and chemotherapy regimens, we found a moderate statistical heterogeneity (I² = 52 %), suggesting that caution is required when interpreting the pooled estimate regarding residual confounding and the external validity of our results. Especially, for the results of the subgroup analysis that were analyzed without taking this variability into account. Despite all effort to retrieve all data, the incomplete retrieval of IPD restricted generalizability and introduced selection bias, which was highlighted by observed differences in baseline characteristics (age and pretreatment AMH) between contributing and non-contributing studies. This incomplete retrieval of IPD also resulted in small numbers in the subgroup analysis stratified by assay. Furthermore, the necessary exclusion and censoring of women who underwent GnRH agonist treatment or oophorectomy introduced a specific selection bias. However, this strict approach was crucial to isolate the effect of chemotherapy and ensure the internal validity of the natural ovarian function endpoint. Although we studied the endpoint 'resumption of ovarian function', considerable variation existed in its definition across the included studies, ranging from only menstrual recovery to hormonal criteria. This inconsistency in defining the outcome introduces heterogeneity that may limit the direct comparability of results and could potentially impact the precision of the pooled estimates. Finally, the varying follow-up durations among studies may have impacted the accuracy of long-term predictions.

In conclusion, our IPD meta-analysis of over 1,000 patients demonstrated that pre-chemotherapy AMH levels are significantly associated with resumption of ovarian function following chemotherapy in premenopausal breast cancer patients. However, a single cut-off value that predicts post-chemotherapy ovarian function across all AMH assays could not be definitively established due to substantial inter-assay variability. Therefore, development and implementation of standardized AMH assays to facilitate robust inter-hospital collaborations and ensure consistent and comparable results across diverse clinical settings are urgently needed. Despite the use of IPD, representing the highest level of study quality, our findings show that the use of pre-chemotherapy AMH level in predicting the absence of resumption is limited by an unacceptably high false negative rate. However, pre-chemotherapy AMH can be informative in identifying women likely to experience ovarian function resumption and thus guide GnRH agonist treatment.

CRediT authorship contribution statement

**Charissa van Zwol – Janssens:** Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft. **Mandy van M. Rosmalen:** Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. **Joop S.E. Laven:** Conceptualization, Methodology, Supervision. **Kazem Nasserinejad:** Formal analysis, Investigation, Methodology, Writing – review & editing. **Jenny A. Visser:** Writing – review & editing. **Richard A. Anderson:** Resources, Writing – review & editing. **Irit Ben-Aharon:** Resources, Writing – review & editing. **Thomas Freour:** Resources, Writing – review & editing. **Kathryn J. Ruddy:** Resources, Writing – review & editing. **H. Irene Su:** Resources, Writing – review & editing. **Yvonne V. Louwers:** Conceptualization, Methodology, Supervision, Writing – review & editing, Writing – review & editing. **Agnes Jager:** Conceptualization, Methodology, Supervision, Writing – review & editing.

Funding

This research received no specific grant from any funding agency in
the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
M.M. van Rosmalen reports a relationship with Astrazeneca that in-
cludes: consulting or advisory. Y.V. Louwers reports a relationship with
Erasmus MC that includes: funding grants. Y.V. Louwers reports a
relationship with Ferring that includes: speaking and lecture fees. Y.V.
Louwers reports a relationship with Merck & Co Inc that includes:
speaking and lecture fees. J.A. Visser reports a relationship with Eras-
mus MC that includes: non-financial support. J.S.E. Laven reports a
relationship with Ansh Labs LLC that includes: funding grants. J.S.E.
Laven reports a relationship with Ferring that includes: funding grants.
J.S.E. Laven reports a relationship with Merck & Co Inc that includes:
funding grants. J.S.E. Laven reports a relationship with National In-
stitutes of Health that includes: funding grants. J.S.E. Laven reports a
relationship with Ansh Labs LLC that includes: consulting or advisory. J.
S.E. Laven reports a relationship with Ferring that includes: consulting
or advisory. J.S.E. Laven reports a relationship with Gedeon Richter Plc
that includes: consulting or advisory. J.S.E. Laven reports a relationship
with Roche Diagnostics that includes: consulting or advisory. J.S.E.
Laven reports a relationship with Titus Health care that includes:
consulting or advisory. R.A. Anderson reports a relationship with Roche
Diagnostics that includes: funding grants. R.A. Anderson reports a
relationship with Beckman Coulter Inc that includes: funding grants. R.
A. Anderson reports a relationship with Ferring that includes: funding
grants. R.A. Anderson reports a relationship with IBSA that includes:
consulting or advisory. R.A. Anderson reports a relationship with Merck
that includes: consulting or advisory. T. Freour reports a relationship
with Gedeon Richter Plc that includes: consulting or advisory. T. Freour
reports a relationship with IBSA that includes: consulting or advisory. T.
Freour reports a relationship with Vitrolife that includes: consulting or
advisory. H.I. Su reports a relationship with National cancer institute
that includes: funding grants. H.I. Su reports a relationship with Robert
Wood Johnson Foundation that includes: funding grants. H.I. Su reports
a relationship with Defense for the California Breast Cancer Research
Program that includes: funding grants. If there are other authors, they
declare that they have no known competing financial interests or per-
sonal relationships that could have appeared to influence the work re-
ported in this paper.

Acknowledgments

We extend our sincere thanks to the medical library staff of Erasmus
MC for their invaluable assistance with the systematic literature search.
We are grateful to Dr. Cristina Silva and Prof. dr. Hui-Chun I. Su for
kindly providing the individual participant data. Finally, we would like
to thank the NCTN Data Archive of the National Cancer Institute's
(NCI's) National Clinical Trials Network (NCTN). This manuscript was
prepared using data from Datasets NCT00433511-D6 from the NCTN
Data Archive of the NCI's NCTN. Data were originally collected from
clinical trial NCT number NCT00433511-D6; 'Biomarker prediction of
chemotherapy-related amenorrhea in premenopausal women with
breast cancer participating in E5103'. All analyses and conclusions in
this manuscript are the sole responsibility of the authors and do not
necessarily reflect the opinions or views of the clinical trial in-
vestigators, the NCTN, or the NCI.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ctrv.2025.103068.

Data availability

The data underlying this article were provided by third parties. No
new data were generated for this IPD meta-analysis.

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