The percentage of women delaying childbearing continues to increase, and demand for an accurate assessment of ovarian reserve has grown. Reproductive aging varies markedly among women. Anti-Müllerian hormone (AMH) is recognized as a reliable predictor of ovarian response in assisted reproduction and is a good indicator of ovarian reserve in other clinical settings (1). To demonstrate the value of AMH as a biomarker, a reliable and commercially available assay is essential (2). The original second generation (Gen II) AMH assay was developed by modification of the Immunotec (IOT) and Diagnostic Systems Laboratory (DSL) assay. The Gen II assay uses the same antibody from the DSL system, but adopted calibration against the standard from the IOT system (3). Recent publications reported a discrepancy in AMH values obtained using the original Gen II assay compared to those with the IOT assay (4), as well as a significant decrease in values compared to those with the DSL assay (567). Beckman-Coulter released a field safety notice stating that all kit lots (≤ 326,119) were affected by complement interference, which caused a time-dependent 70% reduction in the AMH value, and they revised their assay methodology (i.e., premixing the sample with a buffer before plating) (8). This technical issue is of critical importance, and it may have hampered the clinical use of the AMH.

Reference values do not have a meaning per se unless they are referred to in a particular context, which is usually a physiological one; thus, those values represent a specific population (9). Reference values and centile charts for AMH could serve as a tool for clinicians and patients when assessing ovarian reserve at a given age, which would assist in decision-making and selecting treatment options. The samples for reference value studies must be collected under conditions representative of those used in clinical practice (10). We previously reported on age-specific AMH values measured using the IOT assay in a cohort of 16,972 infertility women, and we confirmed that the quadratic model is the most appropriate for describing the AMH-age relationship (11). Since the revised Gen II assay is a substitution for the IOT and original Gen II assay, new age-specific AMH values are urgently needed.

The aim of this study was to establish age-specific reference values and centile chart for the revised Gen II assay. To achieve this objective, we studied mass anonymized AMH data, which were obtained in the same laboratory during routine clinical use.

Table 1 shows the baseline demographic characteristics of the study cohort, which was randomly divided into a training or validation set. The P value from the Wilcoxon-Mann-Whitney test showed no significant differences in age and the AMH values between the training and validation set (P = 0.332 and 0.421, respectively). In the training set, 5 regression models were fitted, and the parameters for each model were estimated (Table 2). Table 3 presents summary statistics for the sum of the absolute residuals, R², and AIC in the validation set. The linear model had the largest sum of absolute residuals and the lowest R². Despite a small difference in the goodness of fit among the nonlinear models, the quadratic, cubic, and power models had the lower sum of absolute residuals and AIC. To determine which model would best fit the data, additional statistical analyses were performed. According to the partial F test (Table 4), a quadratic and cubic model provided a significantly better fit than a linear model. There was no significant difference between the quadratic and cubic models. When 2 models fit the data equally well, a simpler model was preferred because of the ease of interpretation. Thus, we chose the quadratic model over the cubic model. Finally, the quadratic and power models appeared to provide the best fit to our data. For the sake of general usability and accessibility, we chose the quadratic model as the most appropriate clinical model for describing the association between AMH and age.

Fig. 1 shows centile charts of the predicted AMH values against the age acquired by the IOT assay and by the revised Gen II assay; both were based on the most suitable quadratic models. There is a little difference between the shapes of 2 centile charts; the centile charts of the revised Gen II assay had a wider separation in the younger age group and a narrower separation in the older age group. The observed, not predicted, age-specific AMH values measured using the revised Gen II assay in 15,801 infertility patients and corresponding percentiles are summarized in Table 5.

Discrepancy in AMH values measured using different kits is a major concern that hampers their application in the reproductive field. Our study aimed to establish age-specific reference values for the revised Gen II assay with population-based data.

With the introduction of the revised Gen II assay, it is necessary to establish new cut-off values and age-specific reference values of AMH. The manufacturer alleged that the existing reference established with the IOT assay can be applied when interpreting the AMH values with the original Gen II without any problems theoretically. However, adapting clinical cut-off values from the IOT assay to the original Gen II without verification was not recommended because of a different antibody pair. This is actually correct, considering the aforementioned AMH discrepancy. We derived reference values for AMH in a large population-based infertility cohort aged 25–45 years. There are 2 approaches for establishing age-specific AMH reference values. One way is to construct percentile values from observed population data (Table 5), and the other way is using predicted AMH values based on the best model fitted to the population data (Fig. 1). Raw centiles can be calculated from appropriate data, but the perturbations will unlikely reflect the underlying AMH decrease with age at the population level. Therefore, it is reasonable to use statistical methods to derive a series of smoothed curves showing how the centiles of AMH change when plotted against age (13). We previously reported that the quadratic model optimally described AMH-age relation in an analysis of 16,972 women with AMH measurements obtained using the IOT assay (11). The current study also demonstrated that the quadratic model was best fit for describing the AMH-age relation irrespective of the types of AMH assay.

A limitation of this study was that the centile charts were constructed to provide reference values of AMH for infertile women, not for a normal population. Because of our large databases, reference values for the infertile population could be extrapolated to the general population with some assumptions. However, it is necessary to confirm and validate the reference values obtained by using a more scientifically sound a priori selection of the general population (9).

Although a low ovarian reserve does not mean decreased fertility potential, it is clear that a low AMH value is associated with a worse outcome during assisted conception. Consequently, many clinicians will advise patients with a low AMH value not to delay commencing family planning or to consider a rapid in vitro fertilization conversion as an infertility treatment.

To the best of our knowledge, this is the largest population-based study to establish age-specific reference values of AMH using the revised Gen II assay. Further studies of larger general population are consistently required to verify the reference AMH values.