Performance of the two new fully automated anti-müllerian hormone immunoassays compared with the clinical standard assay

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1 Human Reproduction, Vol.30, No.8 pp , 2015 Advanced Access publication on June 20, 2015 doi: /humrep/dev127 ORIGINAL ARTICLE Reproductive endocrinology Performance of the two new fully automated anti-müllerian hormone immunoassays compared with the clinical standard assay Josef van Helden 1, * and Ralf Weiskirchen 2 1 Laboratory Diagnostic Center University Hospital RWTH Aachen, MVZ Dr. Stein und Kollegen, Wallstrasse 10, D Moenchengladbach, Germany 2 Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH University Hospital Aachen, Aachen, Germany *Correspondence address. jvhelden@labor-stein.de Submitted on January 22, 2015; resubmitted on May 4, 2015; accepted on May 14, 2015 study question: How do the two new fully automated anti-müllerian hormone (AMH) assays released in September 2014 by two different diagnostic companies perform compared with the clinical standard assay, namely the AMH Gen II enzyme-linked immunosorbent assay (ELISA)? summary answer: Both fully automated AMH assays perform in a nearly identical fashion compared with the AMH Gen II assay, with a higher analytical sensitivity. what is known already: Owing to the lack of standardization, the results of AMH ELISA assays are sometimes difficult to compare. The BCI AMH Gen II assay became the clinical reference assay over the last few years. Two newly developed fully automated, highly sensitive AMH immunoassays, based on the AMH Gen II antibody composition have become available since September study design, size, duration: Previously characterized serum samples from 155 women were used to measure AMH with the three immunoassays, focusing on the aspect of predicting ovarian reserve. participants/materials, setting, methods: Samples from 94 women with an unfilled desire for a child diagnosed as infertile/subfertile, 29 samples women with polycystic ovary syndrome and 32 women approaching menopause were included to the study. The precision and the linearity in dilutions of the two new AMH assays were determined and the assay results were compared with the clinical reference (the modified version of the BCI AMH Gen II assay) and to the antral follicle counts of the study participants. Cutoff values for the discrimination between each of two predefined groups were calculated using receiver operating characteristic analysis. main results and the role of chance: The performance evaluation of the fully automated AMH assays resulted in a within-run and intermediate precision of % and % with the one and % or % with the other immunoassay, respectively. Pearson s coefficient of correlation was for the method comparison between both assays with a bias of ng/ml and a slope of The discrimination of the new immunoassays between subfertile women and women approaching menopause was significantly better compared with the BCI Gen II assay (87.5 versus 68.8%, P, 0.05). limitations, reasons for caution: Owing to the low number of study subjects in each group, the results have to be confirmed in further studies. wider implications of the findings: The findings of the study are in good agreement with studies that used the Ultra Sensitivite AMH and the pico AMH ELISA assays. The application of AMH measurement onto an automated immunoassay platform is a major step forward, allowing health care providers rapid access to the AMH result and facilitating the adoption of AMH measurement into daily clinical practice. study funding/competing interest(s): We declare no financial relationships or competing interests. Key words: endocrinology / fertilization / follicle development / growth factors / inhibins and activins & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 Three anti-müllerian hormone assays compared 1919 Introduction Anti-Müllerian hormone (AMH) is expressed in granulosa cells of small pre-antral ovarian follicles, where it is important for ovarian folliculogenesis in which it represses follicle recruitment and maturation (Durlinger et al., 2002) by inhibiting the influence of FSH on growing follicles (Teixeira et al., 2001). After an initial increase until the third decade of life, AMH levels slowly decrease with increasing age. Using the most widely used enzyme-linked immunosorbent assays (ELISAs), AMH becomes undetectable some 3 5 years before complete exhaustion of the stock of primordial follicles and final menopause (Sowers et al., 2008). The AMH levels may vary significantly in women of the same chronological age due to individual variability in the velocity of follicle pool depletion and the initial size of the follicle pool which isthe reason for the broad range of age at the time of menopause (Dewailly et al., 2014). AMH is the best currently available parameter to measure the ovarian reserve under different clinical situations (van Rooijet al., 2002). InIVF therapy, theamhlevel significantly correlates with the number of retrievable oocytes after stimulation with gonadotrophins (Hazout et al., 2004; Nakhuda et al., 2007; Elgindy et al., 2008) and has prognostic value concerning the outcome of a pregnancy (Leader et al. 2012). The AMH result enables the dose of gonadotrophin to be adjusted for each individual. In case of high AMH levels, hyperstimulations can be avoided by reducing the dosage and in case of low levels and a reduced ovarian reserve the standard dosage can be elevated or an alternative method of stimulation can be used. AMH is a prognostic factor for the probability of pregnancies as well as live birth after IVF (Nelson et al., 2007; Elgindy et al., 2008), independent of the age of the pregnant woman. Additionally, AMH measurement can be applied for the forecasting of ovarian lifespan, detecting ovarian dysfunction, such as polycystic ovary syndrome (PCOS), and quantifying the loss of ovarian reserve after chemotherapy in patients with cancer (Decanter et al., 2010; Dewailly et al., 2014). Measurement of serum AMH was first reported in the 1990s, with the development of three AMH ELISAs with a sensitivity of 0.5 ng/ml. The assay sensitivity was improved in the following years (Broer et al., 2014) and in year 2000 an ultrasensitive assay with an analytical sensitivity of 0.1 ng/ ml was introduced (Long et al., 2000). This assay, known as the IOT assay, became commercially available through Immunotech a Beckman Coulter company. Another assay was developed by Diagnostic System Lab (DSL) that was expected to measure total AMH by using a pair of highly specific monoclonal antibodies, which recognize epitopes in both the pro-region (F2B/7A) and mature regions (F2B/12H), prepared by Groome et al. (2011). With the acquisition of DSL by Beckman Coulter, the two existing assays were replaced by one new ELISA, the BCI AMH Gen II assay that was calibrated to the IOT AMH ELISA, yielding a sensitivity of 0.08 ng/ml (Kumar et al., 2010). The same pair of antibodies used in the BCI Gen II assay were recently used by Roche and BCI in the Elecsys and Access assays, respectively (Gassner and Jung, 2014). This study presents the first comparison of the Roche Elecsys and Beckman Coulter Access assays to determine whether either or both of these assays meet the requirements as a robust clinical tool. Materials and Methods Clinical samples Preselected defined clinical leftover samples of fresh serum from three reproductive centers in North Rhine Westphalia were collected. The blood samples wereobtained from 94 women with an unfilled desire for achild diagnosed as infertile/subfertile. They were taken between Days 2 and 4 of the cycle. Simultaneously, the antral follicle count (AFC) was determined by transvaginalsonography by the patients gynecologists and reported on the laboratory request form. The samples were divided into 3 groups, 32 with an AFC between 0 and 7, 31 with an AFC between 8 and 15 and another 31 with an AFC.15. The groups were defined according to two cutoffs published for AFC (Ferraretti et al., 2011; van Tilborg et al., 2012). Additionally, 29 samples from women with PCOS according to the Rotterdam criteria (Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group, 2004) and 32 samples from women approaching menopause according to the clinical information on the request form, aged (mean + SD) years were collected. Unused aliquots were stored frozen at 2308C until analysis. The frozen serum specimens that were used were thawed only once for precision studies. AMH immunoassays The modified version of AMH Gen II ELISA (cat. no. A79765, lot no ) is an enzymatically amplified two-site immunoassay. Serum samples were prediluted and pipetted manually into the microtitre plate. Wash steps were performed with a hydro FLEX automated plate washer (TecanCrailsheim, Germany). The absorbance measured with a Genios plate reader (TecanCrailsheim, Germany) is directly proportional to the concentration of AMH in the samples. In this assay, a set of seven AMH calibrators is used to generate a calibration curve for the calculation of the AMH concentrations in the samples. In the Elecsys w AMH assay (cat. no , lot no ), the patient sample is mixed with a biotinylated AMH-specific mouse monoclonal capture antibody (F2B/12H) and a second sulfo-ruthenium-labeled AMH mouse monoclonal antibody (F2B/7A). Results are determined via a lot-specific calibration curve which is generated specifically for the instrument by two-point recalibration with AMH CalSet and a master curve provided via the reagent barcode. The Access AMH assay (cat. no. B13127, lot no ) is a simultaneous one-step sandwich chemiluminescence immunoassay. The capture antibody (F2B/12H) is already bound on paramagnetic particles and the second antibody (F2B/7A) is alkaline phosphatase labeled. The light production is directly proportional to the concentration of AMH in the sample. The amount of analyte in the sample is determined from a stored, six-point calibration curve. The assays were performed according to the instructions of the manufacturers. Both automated assays have been claimed to be standardized according to the Beckman Coulter AMH Gen II ELISA. Precision The precision study for the Elecsys and Access assays was carried out using the kit controls of each assay, and five self-prepared pool sera at different AMH concentrations (range ng/ml). Within-run precision was determined by 21 replicate measurements in a single run. Mean, SD and coefficients of variation (CV) were calculated. Intermediate precision was calculated according to a modified protocol of the Clinical and Laboratory Standards Institute (CLSI)/NCCLS guideline EP5-A2 (NCCLS, 2004) using aliquots of control material and the five self-prepared pooled sera at different concentrations which were thawed immediately before the analysis as

3 1920 van Helden and Weiskirchen recommended by the manufacturer and were analyzed in three determinations per run and two runs per day on 10 days (with n ¼ 60 measurements). Linearity in dilution To get an impression of the linearity in dilution of the new automated AMH assays, two different serum samples, one with a high concentration close to the upper end of the measuring range and one with a low concentration, of 2 ng/ml, were used to study the linearity in range between 0.2 and more than 20 ng/ml, that nearly represents the whole measuring range of the assays. The samples were diluted using an analyte-free sample diluent (Access Sample Diluent A, cat. no , lot no G). Each dilution series contained nine linear dilution steps (9+1, 8 + 2, 7 + 3, 6 + 4, etc.). In each run, measurements of the dilutions were performed in triplicate, the undiluted sera and the analyte-free sample were analyzed in six replicate measurements. The method was considered to be linear if the measured AMH values differed by,10% from the expected AMH concentrations. Method comparison studies A technical method comparison (Passing-Bablok regression) of the two fully automated AMH assays and the clinical standard assay BCI AMH Gen II was carried out with 89 routine samples distributed over nearly the whole measuring range. In a clinical method comparison, 155 characterized serum samples from women with an unfulfilled desire for a child, with PCOS and women approaching menopause were tested with the three different AMH assays: the Beckman Coulter AMH Gen II ELISA and the fully automated AMH assays on the Roche Modular E170 w analyzer and on the Beckman Coulter Access2 w analyzer in single determinations spread over at least five runs. If previously frozen serum samples were used, the AMH values were simultaneously determined with both methods. Evaluation of decision levels for the assessment of ovarian reserve To calculate the ability of the assays to predict the ovarian reserve and to distinguish between the preselected and defined groups of clinical samples, receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cutoff value for the discrimination between each of two predefined groups. Sensitivities, specificities, areas under the curve (AUC) and the percentage of misclassified samples were calculated and compared between the different assays. Statistical analysis The statistical calculations and the t-test were performed with the BiAS program for Windows, version (epsilon-verlaggbrhochheim, Darmstadt, Germany). The analysis of regression was performed using Bland Altman s method comparison (Bland and Altman, 1986) and with the method of Passing and Bablok (1983). The standard t-test was performed for determination of differences between the analytical methods. Results The clinical samples were divided into five groups; women of late reproductive age approaching menopause (n ¼ 32), women with an unfulfilled desire for a child and an AFC of 0 7 (AFC 0 7, n ¼ 32), with an AFC between 8 and 15 (AFC 8 15, n ¼ 31), with an AFC more than 15 (AFC.15, n ¼ 31) and women with PCOS (n ¼ 29). The age and the results of the AMH determinations with the three different AMH methods, namely the AMH Gen II ELISA, the Access AMH immunoassay and the Roche Elecsys AMH immunoassay, are displayed in Table I. All AMH results were in good agreement among the three methods except in the group of women approaching menopause, where AMH was significantly higher when measured with the AMH Gen II ELISA. The lowest AMH levels were found in the group of women of late reproductive age, approaching menopause (mean ¼ ng/ml) However, with increasing AFC, an increase in AMH levels could be detected (AFC 0 7, mean ¼ ng/ml; AFC 8 15, mean ¼ 1.63 ng/ml; AFC.15, mean ¼ 4.46 ng/ml). The highest AMH levels were detected in the group of women with PCOS (mean ¼ 11.3 ng/ml). Precision The performance evaluation of the two fully automated AMH immunoassays for AMH serum pools between 0.1 and 18 ng/ml resulted in a within-run and an intermediate precision CV of between % and % with the new Roche Elecsys immunoassay and % and % with the new Access immunoassay, respectively. The results of the precision study are displayed in Table II. Both automated AMH immunoassays performed with a high degree of precision and the imprecision was, as expected, even lower when compared with the data reported for the manual AMH Gen II ELISA. Linearity in dilution Both automated methods exhibited high levels of linearity. The coefficient of correlation between the expected theoretical values was for the Elecsys AMH immunoassay and for the Access AMH immunoassay. The percentage of recovery varied between 95.5 and 105.3% (mean 100.4%) with the Elecsys AMH immunoassay and between 94.3 and 101.4% (mean 97.8%) with the Access method. Both methods performed nearly identically regarding linearity and recovery. The complete results of the linearity study are displayed in Table III. Table I Distribution of age and AMH levels in the test population, as assessed using three AMH assays. Group ID Age, years AMH Gen II, ng/ml AMH Access, ng/ml AMH Elecsys, ng/ml... Women approaching menopause (n ¼ 32) AFC 0 7 (n ¼ 32) AFC 8 15 (n ¼ 31) AFC.15 (n ¼ 31) PCO (n ¼ 29) AFC, antral follicle count; PCO, women with polycystic ovary syndrome; values are mean + SD.

4 Three anti-müllerian hormone assays compared 1921 Table II Within-run precision and intermediate precision of Elecsys and Access AMH assays. Sample Concentration... Within-run precision... Intermediate Precision... Elecsys AMH Access AMH Elecsys AMH... Access AMH... Elecsys AMH... Access AMH... Mean, ng/ml Mean, ng/ml SD, ng/ml CV, % SD, ng/ml CV, % SD, ng/ml CV, % SD, ng/ml CV, %... Human serum pool Human serum pool Human serum pool Human serum pool Human serum pool Preci Control AMH Preci Control AMH Access Control Access Control Access Control Within-run precision in a single run (n ¼ 21) and intermediate precision of Elecsys and Access AMH immunoassays in repeated measurements of different human serum pools and assay control samples according to a modified protocol (EP-5A2) of the CLSI (10 days, two runs per day in triplicate (n ¼ 60). CV, coefficient of variation. Table III Linearity in dilution of Elecsys and Access AMH immunoassays. AMH Elecsys AMH Access Expected Measured SD, ng/ml CV, % Deviation, % Expected Measured SD, ng/ml CV, % Deviation, % concentration, mean, concentration, mean, ng/ml ng/ml ng/ml ng/ml... Serum Serum Linearity in dilution of Elecsys and Access AMH immunoassays in triplicate measurements of different dilutions of two human sera samples. Criterion of linearity:,10% deviation from expected concentration. Method comparison A comparison of 89 routine samples analyzed with the Access AMH immunoassay and the AMH Gen II ELISA led to the following correlation: Passing/Balblok Access AMH ¼ 0.91 (AMH Gen II) 0.033, r ¼ The comparison between the Elecsys AMH immunoassay and the AMH Gen II ELISA resulted in: Elecsys AMH ¼ 0.88 (AMH Gen II) 0.039, r ¼ The sample concentration was determined between the assay specific limit of detection (LoD) and 17.0 ng/ml. Both assays showed a good correlation to the established ELISA test regarding the measuring range above 1 ng/ml. Samples below 1 ng/ml had showed poor correlation because of the higher imprecision and the lower sensitivity of the ELISA test especially in this concentration range

5 1922 van Helden and Weiskirchen (Fig. 1A and B) depending on the higher imprecision of the Gen II ELISA close to its LoD. The comparison between the two novel fully automated immunoassays led to a correlation of Elecsys AMH ¼ 0.97 (Access AMH) , r ¼ Both assays yield nearly identical values, even in the lower concentration range (Fig. 1C). The box plot diagram in Fig. 2 demonstrates AMH values in women close to menopause (PM), women grouped according to AFC and patients with PCOS. The lowest AMH values were detected in the group of women approaching menopause. An increase of the AMH level with increasing AFC could be detected with all tested AMH assays. The highest levels of AMH were found in women with POCS. To calculate the ability of the assays to predict the ovarian reserve and to distinguish between the preselected and defined groups of clinical samples, ROC analysis were performed to determine the optimal cutoff value for the discrimination between each of two predefined groups. Figure 3 is an example for the ability of the assays to differentiate Figure 1 Passing-Bablok regression analysis. The AMH concentrations (ng/ml) obtained with Beckman AMH Gen II ELISAversus Beckman Access AMH immunoassay (A) and Elecsys AMH immunoassay (B) and Beckman Access and Elecsys AMH immunoassay (C) for 89 samples were compared. Passing- Bablok regression analysis results: Access AMH ¼ 0.91 (AMH Gen II) (r ¼ 0.996), Elecsys AMH ¼ 0.88 (AMH Gen II) (r ¼ 0.998) and Elecsys AMH ¼ 0.97 (Access AMH) (r ¼ 0.991). Identical samples were tested (n ¼ 89). Dashed line: line of unity. Insets A, B and C: range up to 1 ng/ml each magnified. Note undetectable values in AMH Gen II ELISA, whereas measurable low AMH values were detected with the Elecsys and Access AMH assays. A sample of a 33-year old African women displayed different results: 3.7 ng/ml with the Elecsys assay, 7.0 ng/ml with the Access assay and 8.6 with the AMH Gen II ELISA and these results were confirmed by a second determination.

6 Three anti-müllerian hormone assays compared 1923 Figure 2 Distribution of AMH according to AFC. The distributions of AMH were analyzed in women approaching menopause (n ¼ 32), subfertile women with low AFC of 1 7 (1 7, n ¼ 32), women with AFC of 8 15 (8 15, n ¼ 31), women with AFC.15 without PCOS (.15, n ¼ 31) and women with PCOS (n ¼ 29). Access AMH concentrations (ng/ml) are presented as box plots with mean and quartiles Q1 Q3 and mean + SD. The Box plot of the Elecsys AMH level distribution is not shown. between women at late reproductive age approaching menopause and women that still have a menstrual cycle and a desire for a child but a low AFC. The complete results of the ROC analysis are displayed in Table IV. A significant difference between the calculated cutoffs sensitivities of the fully automated assays and the manual ELISA could be demonstrated. Because of the better sensitivity and the higher precision, the differentiation between the two groups of women approaching menopause and women with an AFC between one and seven with low AMH levels is better with the new fully automated AMH immunoassays of Roche and Beckman Coulter. A significant difference between these two assays could not be detected. Also there was no difference between all three assays in the discrimination between the other groups with higher AMH concentrations. Table V presents an overview of the key performance parameters of the newly developed fully automated AMH assays. Although the Roche assay has apparently better precision and the Beckman assay 2 3-fold lower limits of quantification (LoQ) and detection these did not impact on the clinical interpretation of the test results reported here. Additional differences exist concerning the total assay duration, the sample volume, the stability of the reagents after loading onto the system and the calibration. Discussion The comparison and evaluation of two fully automated immunoassays for the determination of serum AMH is described. Both assays, the Elecsys AMH and the Access AMH, exhibited excellent analytical performance that was superior to the current clinical standard assay, the BCI AMH Gen II ELISA, which make them suitable for routine use in the laboratory. The similar performance of both assays was not unexpected because of the use of the same pair of monoclonal antibodies in both tests that are also part of the wildly used AMH Gen II ELISA. Nevertheless, astonishing was the high degree of accordance with both assays. A method comparison showed only a difference of 3% between the AMH values measured with both assays with a coefficient of correlation near to 1.0 and no bias, although the difference between the Elecsys assay and the AMH Gen II ELISA was 12% in this study and 19% in a recent study performed by others (Gassner and Jung, 2014). In our study, we found a difference of 9% between the Access assay and the AMH Gen II ELISA, possibly because of the high inter-laboratory variability of the manual AMH assay that is documented in AMH external ring trial schemes (Zuvela et al., 2013; Clarket al., 2014). Therefore, these results have to be confirmed by other users. Nevertheless, an international standardization of the AMH determination is required because of comparability to other AMH assays, e.g. the high sensitive Ansh Labs AMH assays. In the respective National Institute for Health and Clinical Excellence (2013) guidelines clear cutoffs are suggested for the measurement of AMH to give an indication for ovarian reserve and for treatment decisions. However, these cutoffs only make sense when the measurement of AMH over the time and between laboratories shows a high degree of comparability. Fully automated assays with a high lot-to-lot precision and recovery are an important step to fulfill these criteria. The limits of blank, LoD and LoQ takenfrom the instructions for use of the immunoassays were 0.007, 0.01 and 0.03 ng/ml for the Elecsys AMH immunoassay and , and 0.01 ng/ml in the Access AMH immunoassay, respectively. The LoD were nearly identical in both methods, while the LoQ of the Access AMH immunoassay showed an even higher sensitivity than the AMH Elecsys method. Compared with the current clinical standard AMH Gen II ELISA assay, both new methods are superior concerning their analytical sensitivity. The sensitivity of the new fully automated AMH assays is superior, or at least comparable with, two new AMH immunoassays developed by Ansh Labs, the Ultra-Sensitive AMH and the pico AMH ELISA. The Ultra-Sensitive AMH ELISA with an LoD of 0.05 ng/ml was reported to perform comparably with the AMH Gen II assay but with AMH results 40% higher than measured with the Gen II assay. The pico AMH ELISA was reported to have a higher analytical sensitivity with an LoD of,0.005 ng/ml (Welsh et al., 2014). Highly sensitive assays are useful for epidemiologic studies, for example, investigating the reproductive life span in a population, or clinical situations where expected values of AMH are very low. The new fully automated AMH assays fulfill this requirement. The AMH concentrations determined by both assays were in high correlation with the number of antral follicles, confirming the study of Anderson et al. (2015) who performed a prospective study into the value of the Elecsys AMH assay for the assessment of the growing follicle pool. Welsh et al. (2014) postulated that reliable AMH measurement at the very low end of the measuring range will offer new diagnostic and clinical possibilities. The quantum leap forward was the improvement in sensitivity and precision of the current clinical standard assay AMH Gen II ELISA method that could not discriminate between infertile and subfertile women. In one study, the cutoff values were calculated for the discrimination between infertile women approaching menopause and three groups of women with different AFC representing the different

7 1924 van Helden and Weiskirchen Figure 3 ROC curve analysis including the datafor women approaching menopause and subfertile women with a low AFC (0 7). (A) AMH Gen II (Var2) compared with Access AMH (Var1), (B) AMH Gen II compared with Elecsys AMH (Var3), (C) Elecsys AMH compared with Access AMH. AMH Gen II: cutoff 0.16, AUC 87.6%, sensitivity 68.8%, specificity, 93.9%, error rate 18.7%; AMH Access: cutoff 0.056, AUC 90.7%, sensitivity 87.5%, specificity, 87.9%, error rate 12.4%; AMH Elecsys: cutoff 0.065, AUC 91.2%, sensitivity 87.5%, specificity, 90.9%, error rate 10.8%. probability for a pregnancy and a fifth group of women with a confirmed PCOS (La Marca and Sunkara, 2014). The calculated cutoffs between the three groups of women with different AFC were in accordance with formerly reported cutoff values (Wallace et al., 2011) even if these cutoff values were generated with the unmodified version of the AMH Gen II assay which was sensitive to complement interference. In the present study, additional cutoff values were calculated to discriminate between infertile women approaching menopause and

8 Three anti-müllerian hormone assays compared 1925 Table IV ROC analysis for the discrimination between each of two predefined groups. AMH Gen II AMH Access AMH Elecsys Sens, % Spec, % AUC, % Error rate, % Optimal cutoff, mg/l Sens, % Spec, % AUC, % Error rate, % Optimal cutoff, mg/l Sens, % Spec, % AUC, % Error rate, % Optimal cutoff, mg/l... PM/ * * / / / PCO Comparison of different AMH assays. PM, women approaching menopause (n ¼ 32); 0 7, subfertile women with an AFC between 0 and 7, (n ¼ 32); 8 15, women with an AFC between 8 and 15, (n ¼ 31);.15, women with an AFC higher than 15, (n ¼ 31); Sens, sensitivity; Spec, specificity; AUC, area under the curve; error rate, percentage of misclassified samples. * Significant difference compared with the AMH Gen II ELISA (P, 0.05). Table V Overview of key performance indicators of automated AMH immunoassays. AMH Elecsys AMH Access... Total duration 18 min 40 min Assay principle Sandwich immunoassay Sandwich immunoassay Sample volume 50 ml 20ml LoD 0.01 ng/ml mg/l LoQ 0.03 ng/ml 0.01 ng/ml Measuring range* ng/ml ng/ml Within laboratory,6.5%,10.7% precision (CV) # Sample material* Serum/lithium heparinate Serum/lithium heparinate On board stability* 8 weeks 31 days Calibration Two points Six points Calibration stability* At least one per lot, 28 days 31 days recommended Dilution Possible Possible High dose hook effect.1400 ng/ml.1000 ng/ml * According to assay-specific instructions of use. # Concentration range tested ( ng/ml). subfertile women with a low AFC with a sensitivity and specificity of 90%. The cutoffs were calculated with and ng/ml below the LoD of the AMH Gen II Elisa. Last of all, cutoffs for the differentiation between women with high AFC and women with high AFC and a confirmed POCS were calculated by ROC analysis. AMH levels above 5.42 determined with the Access assay and AMH levels above 5.21 ng/ml determined with the Elecsys assay predict the occurrence of PCOS with a sensitivity and specificity of 90%, which fully confirms the data of a previous report (Iliodromiti et al., 2013). Nevertheless, because of the relatively small sample size of each group, these preliminary results have to be confirmed by others with higher numbers of women in each group. The main differences between the automated assays lie in different key performance parameters such as the assay duration. While the determination of an AMH value on Access systems needs 40 min, the total assaytime on the Roche analyzers is only 18 min. This could be of importance when the patient has to wait for the result for an optimization of the dosage of the hormone stimulation therapy. The difference in the sample volumes (30 ml) needed mayonly very rarely be of importance in cases of difficult blood taking. The number of calibrators and the calibration interval can be of relevance concerning the test costs. In conclusion, the application of AMH measurement onto an automated immunoassay platform was the major step forward to allow rapid access of an AMH result to the health care providers and adopting its measurement into daily clinical practice. The new automated assays perform in a similar manner and the decision to use one assay can be made depending on the technical configuration and individual requirements of a particular institution. Nevertheless, evaluationof reproducibility of the automated AMH assays in external ring trial schemes involving multiple laboratories should be awaited.

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