Short communicationMeasurement of serum Anti-Müllerian Hormone by Beckman Coulter ELISA and DSL ELISA: Comparison and relevance in Assisted Reproduction Technology (ART)
Abstract
Background
Numerous studies have evocated the clinical usefulness of serum AMH levels as a predictor of ovarian response and pregnancy in assisted reproductive technology cycles. Nevertheless, the analysis of the literature shows a great dispersion in serum AMH concentrations obtained with different methods from almost comparable populations.
Methods
We compared two commercial immunoassays (AMH Beckman Coulter ELISA and AMH DSL ELISA) and we evaluated the AMH levels in serum as a prognosis value for ovarian response and pregnancy in assisted reproductive technology cycles.
Results
We found a close linear relationship between the two methods but AMH levels were almost 4.6-fold lower with the DSL kit than with the Beckman Coulter kit. We found a significant and positive correlation between the number of mature ovocytes inseminated and AMH levels obtained with the two methods. Whatever the ELISA used, we found no significant difference between AMH level of pregnancy and non pregnancy groups. Indeed, using the Beckman Coulter method, all pregnant patients had serum AMH levels over 1.4 μg/L. Conversely, no cut-off value can be found for the DSL kit.
Conclusion
Our results show clearly for the first time that AMH results are method dependent even if the correlation obtained between the two methods remained excellent. The Beckman Coulter AMH ELISA should produce clinical agreement when used for prognosis purposes on patients undergoing assisted reproduction.
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Cited by (114)
Towards international standardization of immunoassays for Müllerian inhibiting substance/anti-Müllerian hormone
2018, Reproductive BioMedicine OnlineIs formulated and lyophilized, recombinant human Müllerian inhibiting substance, also known as anti-Müllerian hormone (AMH), suitable for the preparation of a WHO international standard to calibrate AMH immunoassays?
The AMH content of a trial preparation, coded SS-581, was determined by five laboratories using seven immunoassay methods. Participants were requested to report the content of the preparation in terms of their method calibrators through the measurement of a minimum of five concentrations in the linear part of the dose-response curve. Participants were also asked to measure, concomitantly, a panel of six serum samples containing AMH at concentrations of 0.1–13.0 ng/ml.
Across all assays, including two automated assays in development, the geometric mean content was 361.76 ng/ampoule with a geometric coefficient of variation (GCV%) of 39.95%. When measured by immunoassays that were commercially available at the time of the study, the mean content was 423.08 ng/ampoule, with a GCV% of 26.67%. The inter-method geometric means of five serum samples with an AMH concentration >0.3 ng/ml and measured concomitantly with dilutions of SS-581 varied with a range of GCV% of 14.90–22.35%, which may reflect the use of serum sample value transfer to calibrate current immunoassays, some of which use non-human AMH calibrators. The AMH in trial preparation SS-581 was shown to be biologically active in the Müllerian duct regression assay.
A reference material prepared using human recombinant AMH is a promising candidate for the preparation of an international standard for AMH for immunoassays calibrated to recombinant human AMH.
Multicenter evaluation of the Access AMH antimüllerian hormone assay for the prediction of antral follicle count and poor ovarian response to controlled ovarian stimulation
2018, Fertility and SterilityTo evaluate a new fully automated antimüllerian hormone (AMH) assay for prediction of poor ovarian response (POR) to ovarian stimulation defined as four or fewer oocytes retrieved.
Prospective cohort study.
Thirteen private and academic fertility centers in the United States.
A total of 178 women undergoing their first in vitro fertilization (IVF) cycle eligible for the study were consented and enrolled, with data available from 160 women for prediction of POR and 164 women for AMH correlation with antral follicle count (AFC).
None.
Cutoff point for AMH that predicts POR. Correlation of AMH with AFC, and cutoff point for AMH that correlates with antral follicle count >15.
The mean AMH among the poor responders was 0.74 ng/mL, compared with 3.20 ng/mL for normal to high responders. The AMH cutoff at 90% specificity for predicting POR with the use of the receiver operating characteristic (ROC) curve was 0.93 ng/mL, with an associated sensitivity of 74.1%. For prediction of POR, ROC analysis showed that AMH (area under the ROC curve [AUC] = 0.929) was significantly better than FSH (AUC = 0.615; P<.0001). AMH was positively correlated with AFC (Spearman rho = 0.756). The AMH at 90% sensitivity for AFC >15 was 1.75, with specificity of 59.1%.
A fully automated AMH assay can be a useful biomarker for predicting POR in IVF cycles. Because AMH cutoff points vary depending on the assay used, future studies should continue to calibrate test results to clinically important outcomes.
Estudio multicéntrico del test Access de la hormona antimülleriana (AMH) para la predicción del recuento de folículos antrales y de la baja respuesta a la estimulación ovárica controlada
Evaluar un nuevo test completamente automatizado de medición de la hormona antimülleriana (AMH) para la predicción de la pobre respuesta a la estimulación ovárica (PRO), definida como la obtención de cuatro o menos ovocitos.
Estudio prospectivo de cohorte.
Trece centros de fertilidad académicos y privados en Estados Unidos.
Un total de 178 mujeres que realizaron su primer ciclo de Fertilización in vitro (FIV) dieron su consentimiento y fueron incluidas, con datos disponibles de 160 mujeres para predicción de PRO, y datos de 164 mujeres para correlación de AMH con el recuento de folículos antrales (CFA).
Ninguna.
Punto de corte de AMH predictivo para la PRO. Correlación de AMH con el recuento de folículos antrales >15.
LA media de AMH entre las pobres respondedoras fue de 0.74 ng/mL, en comparación con 3.20 ng/mL para normo a hiperrespondedoras.
El punto de corte de AMH al 90% de especificidad para predecir PRO mediante el uso de la curva característica operativa del receptor (ROC) fue de 0,93 ng/ml, con una sensibilidad asociada de 74.1%. Para la predicción de PRO, el análisis de ROC mostró que la AMH (área bajo la curva ROC [AUC]=0,929) fue significativamente mejor que la FSH (AUC=0.615; P <.0001). La AMH se correlacionó positivamente con el recuento de foliculos antrales (RFA) (Spearman rho=0.756). La AMH a un 90% de sensibilidad para el RFA > 15 fue de 1.75, con una especificidad de 59.1%.
El análisis de la AMH totalmente automatizado puede ser un biomarcador útil para predecir la PRO en ciclos de FIV. Debido a que los puntos de corte de la AMH varían según el ensayo utilizado, son necesarios más estudios para calibrar los resultados del test según los resultados clinicos.
How to deal with the different serum AMH kits in France in 2017?
2017, Gynecologie Obstetrique Fertilite et SenologieLe dosage d’hormone anti-mullérienne (AMH) est devenu un incontournable en pratique clinique quotidienne, en particulier avant une prise en charge en assistance médicale à la procréation (AMP). Cependant, de nombreuses trousses de dosage existent qui ne rendent pas des résultats équivalents, pouvant induire une confusion dans l’interprétation des valeurs d’AMH par le clinicien. Jusqu’à récemment, seuls existaient des dosages Elisa manuels (principalement Gen II Beckman, EIA/AMH Immunotech et les deux dosages d’Anshlab), dont les résultats n’étaient pas interchangeables. Les valeurs seuils hautes et basses, utiles en particulier pour l’adaptation thérapeutique en AMP, étaient variables entre les trousses. L’arrivée sur le marché fin 2014 de deux dosages automatisés (Access Dxi Beckman et AMH Elecsys Roche) semble améliorer la sensibilité et la reproductibilité des mesures de l’AMH ce qui pourrait simplifier l’interprétation du dosage et ainsi la prise en charge de nos patients. Cette revue synthétise les comparatifs entre les différents dosages d’AMH disponibles en 2017 pour aider le clinicien dans sa pratique quotidienne.
Serum AMH measurement became a key element in clinical practice, especially before using Assisted reproductive techniques (ART). However, many AMH kits exist giving different AMH results, leading to a confusion in their interpretation. Until recently, only manual ELISA kits existed (mainly Gen II Beckman, EIA/AMH Immunotech and two Anshlab kits) reporting non-interchangeable results. High and low AMH cut-off values, mainly useful to adapt therapeutics in ART, were different between kits. Since the end of 2014, the arrival of two automatic assays (Access Dxi Beckman and AMH Elecsys Roche) seems to improve the sensitivity and the reproducibility of AMH measurement. It could simplify the interpretation of AMH values and improve our clinical choices. This review synthetizes the main comparisons between the different AMH kits available in 2017 to help clinicians in their daily clinical practice.
Comparability of the effect of storage time and temperature on serum anti-Müllerian hormone measurement between original and modified enzyme-linked immunosorbent assay
2017, Clinica Chimica ActaTo explore the effect of modified enzyme-linked immunosorbent assay on the AMH results is increased or decreased, and to investigate the effect of storage time and temperature on AMH measurements with and without sample premixing assay buffer using the Kangrun ELISA method.
Serum AMH concentration were measured by ELISA, consistency between two kits, and comparability between original and the modified assay under different stored conditions were analyzed by Passing-Bablok regression analysis and Bland-Altman bias evaluation.
There was a strong consistency between AMH concentrations measured in Kangrun ELISA and Ansh Labs ultra-sensitive AMH ELISA. Pre-mixing serum specimens with assay buffer gave consistent results compared with original assay. Modified protocol can reduce the amplitude of increase affected by sample aged and give the most consistent results regardless of storage conditions.
Pre-mixing protocol did not influence the results of fresh serum or frozen serum incubation < 3 days at 4 °C and − 80 °C, but when specimens detected after collection and stored in other storage conditions, should be pre-mixed with assay buffer to insure its accuracy.
Anti-Müllerian hormone as a marker of ovarian function
2016, Revista Argentina de Endocrinologia y MetabolismoPor décadas, el significado clínico de la hormona antimülleriana (HAM) ha estado limitado a su papel crítico en el desarrollo sexual fetal. Sin embargo, en los últimos 20 años esta ha surgido también como marcador de función ovárica.
La HAM tiene funciones específicas como regulador del crecimiento folicular, desempeñando su papel como señal de retroalimentación negativa. Jugaría un papel importante tanto en la regulación del número de folículos en crecimiento (inhibiendo el reclutamiento), como en su selección para ser ovulados (inhibiendo a FSH).
La HAM es sintetizada como una pre-prohormona. En el citoplasma cada monómero es clivado generando un fragmento N-terminal: 110 KDa (región pro) y otro C-terminal: 25 KDa (región madura o nativa), unidos en forma no covalente por 2 puentes disulfuro. El dominio C-terminal es el bioactivo, uniéndose al receptor, pero necesita del fragmento N-terminal para desencadenar respuesta biológica. En circulación podemos encontrar una mezcla de la forma pro-HAM y del complejo C-terminal/N-terminal, que serían medidos por los ensayos disponibles.
Distintos autores han demostrado que la HAM es un marcador precoz de la disminución y agotamiento de la reserva ovárica. Muestra una estrecha correlación con la reserva folicular y la capacidad reproductiva, más que la FSH y el estradiol.
La revisión realizada no deja lugar a dudas sobre la utilidad de la HAM en la etapa fértil. Ha mostrado ser una excelente herramienta para caracterizar pobres respondedoras en los procedimientos de fertilización asistida, alertar precozmente en mujeres jóvenes sobre reserva ovárica baja, en relación con su edad cronológica y expresar un número de folículos en crecimiento elevado, como en síndrome de ovario poliquístico, para evitar una hiperestimulación ovárica. El creciente número de pacientes que decidieron retrasar su maternidad y su papel en la fisiología ovárica han posibilitado que la HAM integre hoy la evaluación de mujeres con alteraciones de la fertilidad.
For many years, the clinical significance of the anti-Müllerian hormone (AMH) was limited to its critical role in foetal sexual development. However, in the last 20 years it has also emerged as a marker of ovarian function.
AMH has specific functions as a regulator of follicular growth, playing its role as negative feedback signal. It may also play an important role in the regulation of the number of growing follicles (inhibiting the recruitment) as well as in their selection to be ovulated (inhibiting FSH).
AMH is synthesised as a pre-pro-hormone. In the cytoplasm each monomer is cleaved, generating one N-terminal fragment: 110 KDa (pro region) and another C-terminal fragment: 25 KDa, non-covalently bound by two disulphide bridges. The C-terminal domain is bioactive, binding to the receptor, but requires the N-terminal fragment to trigger a biological response. A mixture of pro-AMH complex and C-terminal/N-terminal complex can be found in the bloodstream, which can be measured by the assays available.
Several authors have shown that AMH is an early marker of the decrease and depletion of ovarian reserve. It shows a close correlation with follicular reserve and reproductive capacity more than FSH and oestradiol.
This review leaves no doubt about the usefulness of AMH in the fertile phase. It has proven to be an excellent tool in characterising poor responders in assisted reproduction procedures, as an early alert in young women of a low ovarian reserve in relation to their chronological age, as well as in expressing a number of follicles in high growth, as in polycystic ovary syndrome, to avoid ovarian hyperstimulation. The growing number of patients who have decided to delay motherhood and the role of AMH in ovarian physiology has led it to an integral part of the assessment of women with impaired fertility.
The impact of excision of benign nonendometriotic ovarian cysts on ovarian reserve: a systematic review
2016, American Journal of Obstetrics and GynecologyBenign nonendometriotic ovarian cysts are very common and often require surgical excision. However, there has been a growing concern over the possible damaging effect of this surgery on ovarian reserve.
The aim of this metaanalysis was to investigate the impact of excision of benign nonendometriotic ovarian cysts on ovarian reserve as determined by serum anti-Müllerian hormone level.
MEDLINE, Scopus, ScienceDirect, and Embase were searched electronically.
All prospective and retrospective cohort studies as well as randomized trials that analyzed changes of serum anti-Müllerian hormone concentrations after excision of benign nonendometriotic cysts were eligible. Twenty-five studies were identified, of which 10 were included in this analysis.
Two reviewers performed the data extraction independently.
A pooled analysis of 367 patients showed a statistically significant decline in serum anti-Müllerian hormone concentration after ovarian cystectomy (weighted mean difference, –1.14 ng/mL; 95% confidence interval, –1.36 to –0.92; I2 = 43%). Subgroup analysis including studies with a 3-month follow-up, studies using Gen II anti-Müllerian hormone assay and studies using IOT anti-Müllerian hormone assay improved heterogeneity and still showed significant postoperative decline of circulating anti-Müllerian hormone (weighted mean difference, –1.44 [95% confidence interval, –1.71 to –1.1; I2 = 0%], –0.88 [95% confidence interval, –1.71 to –0.04; I2 = 0%], and –1.56 [95% confidence interval, –2.44 to –0.69; I2 = 22%], respectively). Sensitivity analysis including studies with low risk of bias and excluding studies with possible confounding factors still showed a significant decline in circulating anti-Müllerian hormone.
Excision of benign nonendometriotic ovarian cyst(s) seems to result in a marked reduction of circulating anti-Müllerian hormone. It remains to be established whether this reflects a real compromise to ovarian reserve.