anti-Müllerian Hormone Assay Research Articles

Anti Mullerian Hormone: A Potential Marker for Recruited Non Growing Follicle of Ovarian Pool in Women with Polycystic Ovarian Syndrome

Polycystic ovarian disease is one of the most common causes of infertility in women of reproductive age. Anti- mullerian hormone (AMH), a member of transforming growth factor (TGF) family which is secreted by granulosa cells of growing follicle, is found to be increased to three to four fold in Poly Cystic Ovarian Syndrome (PCOS) patients as evidenced by previous studies. But the level of AMH in relation to the infertile status of PCOS was not studied yet. The present study was focused to determine the discriminative power of AMH in infertility subjects with regular cycles and infertility subjects associated with PCOS. The subjects under study were one hundred and twenty infertile women of age group ranging from 27-35 years. Subjects, were further divided into sixty infertile with regular cycles as controls (Group1) and sixty infertile subjects with PCOS as cases (Group 2). Hormones like FSH, E2 and AMH were assayed for all the subjects. Mean and student t- test for all hormones were compared between controls and cases. The diagnostic power of AMH pertaining to sensitivity and specificity was evaluated by Receiver operating characteristic (ROC) curve. Serum AMH level were two fold higher in PCOS patients than in controls. The mean value of AMH also shows a test of significance between the two groups. The area under the receiver operating characteristic curve for the AMH assay was 0.95 in infertile group when 3.34ng/ml was used as cut off point indicating its better discriminative power and good diagnostic potency. Setting the AMH value at 3.34ng/ml sensitivity, specificity,Positive Predictive Value(PPV) and Negative Predictive Value(NPV) were observed 98% ,93%, 93% and 98% respectively. The diagnostic potency of Area Under Curve (AUC) for AMH in infertile subjects reflects that AMH is a potential marker for recruited non growing follicles rather than a simple marker for ovarian reserve as it is predominantly produced by small follicles rather than a simple marker for ovarian reserve.

Anti–müllerian hormone and ovarian reserve in systemic lupus erythematosus

To study the level of anti-müllerian hormone (AMH) and its relationship to age and previous exposure to cyclophosphamide (CYC) in patients with systemic lupus erythematosus (SLE). Consecutive female patients ages 18-52 years who had menses at least once during the preceding 12 months and fulfilled ≥4 American College of Rheumatology criteria for SLE were recruited. AMH was determined using an enzyme-linked immunosorbent assay (ELISA) kit. Serum AMH levels were compared in patients with and without previous use of immunosuppressive agents. The relationship of the AMH level to the patient's age and CYC exposure was studied by linear regression and receiver operating characteristic (ROC) curve analysis. A total of 216 patients were studied (mean±SD age 35.1±10.1 years, mean±SD SLE duration 7.6±5.9 years). The mean±SD AMH level was significantly lower in patients previously exposed to CYC therapy than in those who had not been exposed after adjustment for age (1.58±2.92 versus 1.73±2.11 ng/ml; P=0.04). The median time interval between the AMH assay and the last dose of CYC administered was 6.7 years (interquartile range 3.4-8.5). AMH levels in users versus nonusers of other immunosuppressive agents, including mycophenolate mofetil, azathioprine, and the calcineurin inhibitors, were not statistically different. Linear regression revealed increasing age (beta -0.32, P=0.02) and each 5 gm of CYC exposure (beta -0.28, P=0.047) were independently associated with a lower AMH level. In patients ages 30 years and younger, a cumulative CYC dose cutoff of 5.9 gm yielded a sensitivity of 0.75 and a specificity of 0.80 for the prediction of undetectable AMH level on ROC curve analysis. AMH is a sensitive marker for ovarian damage due to previous CYC exposure in women with SLE.

Correlation between three assay systems for anti-Müllerian hormone (AMH) determination

PurposeAnalysis of anti-Müllerian hormone (AMH) is becoming of recognized importance in reproductive medicine, but assays are not standardized. We have evaluated the correlation between the new Gen II ELISA kit (Beckman-Coutler) and the older ELISA kits by Immunotech (IOT) and Diagnostic Systems Laboratories (DSL).MethodsA total of 56 archived serum samples from patients with subfertility or reproductive endocrine disorders were retrieved and assayed in duplicate using the three AMH ELISA kits . The samples covered a wide range of AMH concentrations (1.9 to 142.5 pmol/L).ResultsWe observed good correlations between the new (AMH Gen II) and old AMH assay kits by IOT and DSL (R2 = 0.971 and 0.930 respectively). The regression equations were AMH (Gen II) = 1.353 × AMH (IOT) + 0.051 and AMH (Gen II) = 1.223 × AMH (DSL) – 1.270 respectively.ConclusionsAMH concentrations using the Gen II kit are slightly higher than those from the IOT and DSL kits. Standardization of assay results worldwide is urgently required but this analysis facilitates the interpretation of values obtained historically and in future studies using any of the 3 assays available. Meanwhile, adapting clinical cut-offs from previously published work by direct conversion is not recommended.

Presence of anti‐müllerian hormone in the squirrel monkey (Saimiri boliviensis): gender and seasonal differences

Anti-mullerian hormone (AMH) is a glycoprotein of the transforming growth factor-β superfamily secreted by male Sertoli cells and female ovarian granulosa cells. This study determined squirrel monkey AMH levels as influenced by gender and seasonality. Squirrel monkey sera AMH were measured by an enzymatically amplified two-site immunoassay. A significant difference (P<0.001) was found in AMH levels between male (mean=3.46 ng/ml) and female squirrel monkeys (mean=22.12 ng/ml). A significant difference (P<0.05) was found in male AMH levels between breeding (mean=4.21 ng/ml) and non-breeding seasons (mean=2.78 ng/ml). No significant differences were found between female groups. Anti-mullerian hormone levels in female squirrel monkeys are the highest in any primate species reported, whereas in the male, levels are within reported ranges. The AMH assay may allow us soon to assess the squirrel monkey fertility potential as a function of various factors.

Anti-Müllerian Hormone Serum Values and Ovarian Reserve: Can It Predict a Decrease in Fertility after Ovarian Stimulation by ART Cycles?

BackgroundA variety of indicators of potentially successful ovarian stimulation cycles are available, including biomarkers such as anti-Mullerian hormone. The aim of our study was to confirm the usefulness of serum anti-Mullerian hormone assay in predicting ovarian response and reproductive outcome in women eligible for ART cycles.MaterialsForty-six women undergoing ART cycles at the Centre for Reproductive Medicine in Parma were recruited from March-to-June 2010. Inclusion criteria: age<42 years; body-mass-index = 20–25; regular menstrual cycles; basal serum FSH concentration <12 IU/L and basal serum estradiol concentration <70 pg/mL. The couples included in our study reported a variety of primary infertility causes. All women underwent FSH stimulation and pituitary suppression (GnRH-agonist/GnRH-antagonist protocols). Women were considered poor-responders if thay had ≤3 oocytes; normal-responders 4–9 oocytes and high-responders ≥10 oocytes. Serum samples for the AMH assays were obtained on the first and last days of stimulation. A P value ≤0.05 was considered statistically significant.ResultFSH levels increased significantly when AMH levels decreased. The total dose of r-FSH administered to induce ovulation was not correlated to AMH. The number of follicles on the hCG, serum estradiol levels on the hCG-day, and the number of retrieved oocytes were significantly correlated to AMH. The number of fertilized oocytes was significantly correlated to the AMH levels. No significant correlation was found between obtained embryos or transferred embryos and AMH. Basal serum AMH levels were significantly higher than those measured on the hCG-day, which appeared significantly reduced. There was a significant correlation between AMH in normal responders and AMH in both high and poor responders.ConclusionsOur data confirm the clinical usefulness of AMH in ART-cycles to customize treatment protocols and suggest the necessity of verifying an eventual permanent decrease in AMH levels after IVF.

Anti-Mullerian hormone: poor assay reproducibility in a large cohort of subjects suggests sample instability

What is the variability of anti-Müllerian hormone (AMH) concentration in repeat samples from the same individual when using the Gen II assay and how do values compare to Gen I [Diagnostic Systems Ltd (DSL)] assay results? The Gen II AMH assay displayed appreciable variability, which can be explained by sample instability. AMH is the primary predictor of ovarian performance and is used to tailor gonadatrophin dosage in cycles of IVF/ICSI and in other routine clinical settings. Thus, a robust, reproducible and sensitive method for AMH analysis is of paramount importance. The Beckman Coulter Gen II ELISA for AMH was introduced to replace earlier DSL and Immunotech assays. The performance of the Gen II assay has not previously been studied in a clinical setting. We studied an unselected group of 5007 women referred for fertility problems between 1 September 2008 and 25 October 2011; AMH was measured initially using the DSL AMH ELISA and subsequently using the Gen II assay. AMH values in the two assays were compared using a regression model in log(AMH) with a quadratic adjustment for age. Additionally, women (n = 330) in whom AMH had been determined in different samples using both the DSL and Gen II assays (paired samples) identified and the difference in AMH levels between the DSL and Gen II assays was estimated using the age-adjusted regression analysis. A subset of 313 women had repeated AMH determinations (n = 646 samples) using the DSL assay and 87 women had repeated AMH determinations using the Gen II assay (n = 177 samples) were identified. A mixed effects model in log(AMH) was utilized to estimate the sample-to-sample (within-subject) coefficients of variation of AMH, adjusting for age. Laboratory experiments including sample stability at room temperature, linearity of dilution and storage conditions used anonymized samples. In clinical practice, Gen II AMH values were ∼20% lower than those generated using the DSL assay instead of the 40% increase predicted by the kit manufacturer. Both assays displayed high within-subject variability (Gen II assay CV = 59%, DSL assay CV = 32%). In the laboratory, AMH levels in serum from 48 subjects incubated at RT for up to 7 days increased progressively in the majority of samples (58% increase overall). Pre-dilution of serum prior to assay, gave AMH levels up to twice that found in the corresponding neat sample. Pre-mixing of serum with assay buffer prior to addition to the microtitre plate gave higher readings (72% overall) compared with sequential addition. Storage at -20°C for 5 days increased AMH levels by 23% compared with fresh samples. The statistical significance of results was assessed where appropriate. The analysis of AMH levels is a retrospective study and therefore we cannot entirely rule out the existence of differences in referral practices or changes in the two populations. Our data suggests that AMH may not be stable under some storage or assay conditions and this may be more pronounced with the Gen II assay. The published conversion factors between the Gen II and DSL assays appear to be inappropriate for routine clinical practice. Further studies are urgently required to confirm our observations and to determine the cause of the apparent instability. In the meantime, caution should be exercised in the interpretation of AMH levels in the clinical setting. S. Roberts is supported by the NIHR Manchester Biomedical Research Centre.

Anti-Mullerian hormone as a predictor of follicular reserve in ovarian insufficiency: special emphasis on FSH-resistant ovaries

Anti-Müllerian hormone (AMH) is secreted by ovarian granulosa cells and its serum levels reflect ovarian follicle reserve. The main objective of this study was to test the use of AMH assay in identifying women with primary amenorrhea (PA) and existing follicles and to study follicle phase dependent AMH secretion. Serum levels of AMH were measured in subjects with FSH-resistant ovaries (FSHRO, n= 12), primary ovarian insufficiency (POI) with PA (n= 11) or secondary amenorrhea (SA n= 20) of unknown etiology, and controls (n= 23), and in Turner syndrome (TS) [45,X (n= 18), mosaicism (n= 7), structural X chromosome abnormalities (SCA, n= 10)], and healthy controls (n= 34). Serum levels of AMH in women with FSHRO were comparable with those in control women (2.76 ± 2.37 versus 3.77 ± 2.36 ng/ml) and significantly higher than in women with PA (0.05 ± 0.04 ng/ml; P < 0.001) or SA of unknown origin (0.12 ± 0.20 ng/ml; P < 0.001). TS girls/women with 45,X or SCA had low serum AMH levels (0.13 ± 0.09 and 0.27 ± 0.19 ng/ml) compared with their controls (3.34 ± 2.23 ng/ml) or subjects with mosaicism (2.33 ± 2.81 ng/ml). AMH expression was detected in granulosa cells of women with FSHRO but not in any of the 45,X fetal ovarian specimens. A serum AMH assay could be used to identify patients with decreasing ovarian reserves and POI. Moreover, our results support the notion that AMH is secreted mainly by small non-selected follicles, since follicular granulosa cells were AMH-positive and serum AMH levels were normal/low normal in women with FSHRO, who lack follicle development beyond the small antral stage.

Reproductive late effects in female survivors of childhood cancer.

Childhood cancer treatments can cause female reproductive late effects. Radiation to the hypothalamic-pituitary-ovarian axis is associated with altered menarche, miscarriage, and implantation failure. Patients who receive chemotherapy and/or ovarian radiation are at risk of premature ovarian failure; the risk increases with increasing radiation dose, alkylating agent score, combination therapy, and older age at treatment. Ovarian reserve may be assessed using antimullerian hormone assay and ultrasound measurements of ovarian volume and antral follicle count; however, their efficacy is poorly established in this cohort. Fertility preservation options including cryopreservation, oophoropexy, and gonadotropin-releasing hormone analogues may be initiated prior to treatment, although most are still considered experimental. Uterine radiation has been linked to pregnancy complications including miscarriage, premature delivery, stillbirth, low-birth-weight and small-for-gestational-age infants. This paper summarises the literature on female reproductive late effects. The information should facilitate counseling and management of female survivors throughout their reproductive lives.

L’hormone antimüllérienne dans le syndrome des ovaires polykystiques

Anti-Müllerian hormone (AMH) is known to be secreted in women by granulosa cells of growing follicles from the primary to the large antral follicle stage. Serum and intrafollicular AMH levels are elevated in patients with polycystic ovary syndrome (PCOS) due to increased number of small follicles and an increased secretion within each of these small follicles. This excess of AMH is strongly suspected to play a role in the follicular arrest of PCOS by a negative action on aromatase expression and on action of FSH. The interest of serum AMH assay is also clinically important because AMH level is more sensitive, more specific and reproducible from one center to another as the follicle count. Rates above 5 ng/ml or 35 pmol/l may be considered as a diagnostic criterion for PCOS.

Antimüllerian hormone as a predictor of controlled ovarian hyperstimulation outcome: comparison of two commercial immunoassay kits

This study was performed to compare antimüllerian hormone (AMH) levels measured by two commercially available AMH measuring kits currently available, and to evaluate the AMH levels as predictor of controlled ovarian hyperstimulation (COH) outcome using the two different kits. The two assays for AMH measurement provide similar results, and serum AMH levels measured by the two kits both could be used as COH outcome predictors with similar reference values.

The reproducibility of serum anti-Müllerian hormone in subfertile women: within and between patient variability

Serum anti-Müllerian hormone concentrations vary significantly over time and this should be taken into account when tailoring treatment protocols for patients undergoing controlled ovarian hyperstimulation (COH). Compared with FSH, serum anti-Müllerian hormone may have greater discriminatory power because of its modest intrapatient variation and the larger interpatient variation.

Development of a second generation anti-Müllerian hormone (AMH) ELISA

AMH is a glycoprotein dimer composed of two 72kDa monomers linked by disulfide bridges. It belongs to the transforming growth factor-β family. AMH performs various physiological functions. In males, AMH is secreted by the Sertoli cells of the testis. During embryonic development, AMH is responsible for Müllerian duct regression. AMH continues to be produced by the testicles until puberty and then decreases slowly to residual post-puberty values. In females, AMH is produced in small amounts by ovarian granulosa cells after birth, until menopause, and then becomes undetectable. A two-step, sandwich-type enzymatic microplate assay has been developed to measure AMH levels in 20μL of sample in less than 3h. AMH calibrators range from 0.2 to 28ng/mL. The antibodies used in the assay bind to the mature region of AMH, which is more stable to proteolysis compared to prohormone region. The AMH Gen II assay (Beckman Coulter, Inc., Webster, Texas) was standardized to the Immunotech (IOT, Beckman Coulter, Inc., Marseilles, France) AMH assay. AMH Gen II, when compared to IOT using 120 serum samples in the range of 0–20.4ng/mL yielded a correlation coefficient of 0.98 and a slope of 1.0. Total imprecision, calculated on four samples over 40 runs, four replicates per run, between two lots using CLSI EP5-A guidelines, was 5.7% at 3.8ng/mL, 7.7% at 4.4ng/mL, 5.8% at 14ng/mL and 5.3% at 16.4ng/mL. The average analytical sensitivity calculated by the interpolation of the mean plus two standard deviations of 16 replicates of the zero calibrator on two independent lots was 0.08ng/mL. Dilution and spiking studies showed an average recovery of 91–110%. Lot-to-lot comparison of two independent lots testing 38 serum samples (1.5–33ng/mL range) yielded a slope of 1.01, intercept of −0.08ng/mL and r of 0.99. When potential interferents (hemoglobin, triglycerides, and bilirubin) were added at two times the physiological concentrations, AMH concentrations were within ±10% of the control. A highly specific and reproducible microplate AMH Gen II assay has been developed to standardize the measurement of AMH between methods. The performance of the AMH Gen II assay is ideal for investigation into the physiologic roles of AMH in men and women.

Maternal serum anti‐Mullerian hormone level is not superior to chronological age in predicting Down syndrome pregnancies

To compare the difference in maternal serum anti-Mullerian hormone (AMH) level between Down syndrome pregnancies and unaffected pregnancies, and to evaluate its performance as a screening marker for Down syndrome pregnancy. A total of 145 pregnancies affected by foetal Down syndrome and 290 unaffected controls matched with maternal age and gestational age were selected, and their archived first or second trimester serum retrieved for AMH assay. There was no significant difference in maternal serum AMH level between pregnancies affected and unaffected by foetal Down syndrome. Our first trimester serum samples had higher AMH concentration compared to second trimester samples. Maternal serum AMH level, as a marker of ovarian age, is not superior to chronological age in predicting Down syndrome pregnancies. Despite the cross-sectional nature of our study, the variation of maternal serum AMH concentration with gestational age warrants further investigation.

Anti-Müllerian hormone follow-up in young women treated by chemotherapy for lymphoma: preliminary results

Susceptibility of the ovarian reserve to chemotherapy is highly variable from one patient to another and poorly documented. To better characterize the evolution of follicular depletion in patients treated for lymphoma, serum anti-Müllerian hormone (AMH) assay was used. A total of 30 young women (mean age 24years) were prospectively recruited before initiation of chemotherapy for lymphoma. They were assigned either to an adriamycin, bleomycin, vinblastine and dacarbazine protocol (ABVD group) or to a protocol that included cyclophosphamide (non-ABVD group). AMH assays were performed before and during chemotherapy, and then every 3months after the end of treatment for a period of 1year. In all patients, AMH concentrations fell drastically just after the start of chemotherapy and were close to the detection limit at the end of the treatment. In the ABVD group, AMH concentrations increased from the third month after the end of chemotherapy and returned to pretreatment values 12months after the end of chemotherapy. Conversely, no significant change was observed in the non-ABVD group throughout the follow-up period. In conclusion, longitudinal analysis of AMH during chemotherapy highlights differences between protocols that could contribute to an understanding of ovarian toxicity and, ultimately, be useful in fertility preservation counselling.

Effect of cell cycle specificity of chemotherapeutic agents on the gonadal functions of a pediatric cancer population: role of anti-mullerian hormone (AMH)

OBJECTIVE: The objectives of the study are to 1) determine whether AMH levels could be used to quantify gonadal damage inflicted by gonadotoxic chemotherapy and 2) to examine the effect of cell cycle specificity of the chemotherapeutic agents on the gonadal reserve as evidenced by the post-treatment AMH assay.DESIGN: Cohort study.MATERIALS AND METHODS: Baseline blood samples were obtained from 34 cancer patients (group I: 27 males and 7 females) and 10 normal controls (group II: 7 males and 3 females). Group I was composed of patients treated with chemotherapy for hematological, musculoskeletal and soft tissue cancers in 18,6 and 10 patients, respectively. Patients were followed until the completion of chemotherapy (range1-3 years). A second blood sample was collected at the completion of chemotherapy. AMH assay was completed using a commercially available kit.RESULTS: Using Spearman correlation, baseline AMH levels were significantly correlated with age of the study population (R=0.41, P=0.007). Patients and controls were comparable regarding their age at the initiation of chemotherapy. Baseline AMH levels were comparable between patients and controls irrespective of gender, age, pubertal status and type of malignancy (Boys7.83±4.75ng/ml vs 17.24±7.77 ng/ml; P=0.056) and (Girls 1.53±1.39 ng/ml vs 1.86±0.55 ng/ml; P=0.12)}. Post-chemotherapy AMH levels were not significantly different from baseline in boys (9.86±7.30 ng/ml vs 7.83±4.75 ng/ml, P=0.28). On the other side, post-chemotherapy AMH levels were significantly lower from baseline in girls (0.81±1.11 ng/ml vs 1.53±1.39 ng/ml, P=0.02). In boys and girls there was no significant reduction of post-chemotherapy AMH levels in subjects who received cell cycle specific chemotherapy. Similarly, there was no significant reduction of post-chemotherapy AMH levels in male subjects who received cell cycle non specific chemotherapy, P=0.06. On the contrary, girls who received cell cycle non specific chemotherapy showed significantly lower AMH levels compared to baseline (0.22 0.35 ng/ml vs 1.28 ±1.43 ng/ml; P=0.01).CONCLUSIONS: AMH assay is a sensitive marker for ovarian functions before and after puberty in patients at risk of gonadal damage secondary to chemotherapy. Ovarian reserve appears to be more sensitive to the insult of cell cycle non specific chemotherapeutic agents than the testicular reserve. AMH assay could be added to the initial work up of all patients undergoing chemotherapy to assess the damage to their reproductive potential. OBJECTIVE: The objectives of the study are to 1) determine whether AMH levels could be used to quantify gonadal damage inflicted by gonadotoxic chemotherapy and 2) to examine the effect of cell cycle specificity of the chemotherapeutic agents on the gonadal reserve as evidenced by the post-treatment AMH assay. DESIGN: Cohort study. MATERIALS AND METHODS: Baseline blood samples were obtained from 34 cancer patients (group I: 27 males and 7 females) and 10 normal controls (group II: 7 males and 3 females). Group I was composed of patients treated with chemotherapy for hematological, musculoskeletal and soft tissue cancers in 18,6 and 10 patients, respectively. Patients were followed until the completion of chemotherapy (range1-3 years). A second blood sample was collected at the completion of chemotherapy. AMH assay was completed using a commercially available kit. RESULTS: Using Spearman correlation, baseline AMH levels were significantly correlated with age of the study population (R=0.41, P=0.007). Patients and controls were comparable regarding their age at the initiation of chemotherapy. Baseline AMH levels were comparable between patients and controls irrespective of gender, age, pubertal status and type of malignancy (Boys7.83±4.75ng/ml vs 17.24±7.77 ng/ml; P=0.056) and (Girls 1.53±1.39 ng/ml vs 1.86±0.55 ng/ml; P=0.12)}. Post-chemotherapy AMH levels were not significantly different from baseline in boys (9.86±7.30 ng/ml vs 7.83±4.75 ng/ml, P=0.28). On the other side, post-chemotherapy AMH levels were significantly lower from baseline in girls (0.81±1.11 ng/ml vs 1.53±1.39 ng/ml, P=0.02). In boys and girls there was no significant reduction of post-chemotherapy AMH levels in subjects who received cell cycle specific chemotherapy. Similarly, there was no significant reduction of post-chemotherapy AMH levels in male subjects who received cell cycle non specific chemotherapy, P=0.06. On the contrary, girls who received cell cycle non specific chemotherapy showed significantly lower AMH levels compared to baseline (0.22 0.35 ng/ml vs 1.28 ±1.43 ng/ml; P=0.01). CONCLUSIONS: AMH assay is a sensitive marker for ovarian functions before and after puberty in patients at risk of gonadal damage secondary to chemotherapy. Ovarian reserve appears to be more sensitive to the insult of cell cycle non specific chemotherapeutic agents than the testicular reserve. AMH assay could be added to the initial work up of all patients undergoing chemotherapy to assess the damage to their reproductive potential.

Clinical uses of anti-Müllerian hormone assays: pitfalls and promises

To investigate whether the controversy about fluctuations of anti-Müllerian hormone (AMH) levels during the menstrual cycle results from differences between the immunoassays currently available: the Beckman Coulter Immunotech kit (Fullerton, CA) and the Diagnostic Systems Laboratories kit (Webster, TX). Prospective trial. Fertility clinics of two tertiary university hospitals. One hundred sixty-eight blood samples from three different populations. Serial samples at set intervals from the LH surge were taken in a fourth population of 10 volunteers. We remeasured AMH levels by using the Diagnostic Systems Laboratories kit in 168 blood samples in which AMH initially had been measured by using the Beckman Coulter assay. We also conducted serial AMH measurements (n = 7) during the menstrual cycle of 10 women. Linear regression of AMH levels determined by using 2 different assays and analysis of variance of serial measurements in the menstrual cycle. We found a linear relationship between the 2 methods, with a correlation coefficient of 0.88. When repeated individual AMH measures were longitudinally analyzed in relation to the LH surge, a slight but significant decrease was observed after ovulation. Differences in AMH fluctuations during the menstrual cycle reported in recent publications do not result from the use of different AMH assays. The changes in AMH levels after ovulation are slight, yet statistically significant. However, the fluctuations observed are smaller than intercycle variability and therefore are not clinically relevant as far as AMH measurements for clinical purposes are concerned. In daily practice, AMH therefore can be measured anytime during the menstrual cycle.

Serum anti-müllerian hormone (AMH) measurement: inter-assay agreement and temperature stability

Two different immunoassay systems are currently in use to measure serum AMH, one produced by Diagnostic Systems Laboratories (DSL) and one produced by Immunotech (both Beckman Coulter, Inc companies). Previously, the Immunotech system was more widely used in publications and is currently in use outside the United States. However, the DSL system is currently the only commercially available system in the United States. Limited published data is available which compares the two systems. There were two objectives to this study: 1) better characterize the relationship between the Immunotech and DSL systems; 2) test the stability of serum specimens over 48 hours at different temperatures. ELISA analysis of serum samples. Objective 1) AMH measurement was performed on samples from serum from 38 different donors using both the Immunotech and DSL AMH assays. Objective 2) Multiple AMH measurements were conducted using the DSL assay and serum from seven different donors. Specimens were separated into different aliquots, the first immediately frozen at −20°C and the subsequent samples frozen after 4, 8, 24, and 48 hours at 4°C and room temperature (22°C). Data from the 38 serum samples comparing the two AMH assays revealed a relationship defined by the line Immunotech = (1.5) DSL + 0.7 (ng/ml) with good agreement (R2 = 0.98). The mean AMH value ratio of samples incubated for 48 hours at 4°C to samples immediately frozen, was 1.01 with a 95% CI of (0.94–1.08). After 48 hours at 22°C, the mean AMH value ratio to immediately frozen samples was 1.09 with a 95% CI of (0.98–1.20). Values obtained using the DSL AMH immunoassay can be linearly converted to Immunotech AMH values with some degree of variation. DSL AMH measurement is stable for at least 48 hours at 4°C, with values likely to differ by less than eight percent of values from immediately frozen samples. With samples at room temperature for 48 hours, AMH values were likely to differ by less than twenty percent of values from immediately frozen samples.

O-24: Diphasic pattern of anti-mullerian hormone (AMH) in ovulatory cycles as evidenced by means of an ultra-sensitive assay: New insights into ovarian function

AMH levels have been recognized for a decade as a reliable index of ovarian reserve. However the assay initially developed for testis investigation by Josso et al (JCEM 1990) was designed for the determination of high AMH levels in diluted serum, but was not sensitive enough for the measurement of low AMH levels in diluted samples. On the other hand, a significant matrix effect was observed with neat serum, resulting in underestimation of low AMH levels. As a consequence, the first generation AMH assay was unable to find any significant change in AMH levels during normal menstrual cycles. The aim of the study was to reassess ovarian secretion of AMH by means of a new ultrasensitive assay (DSL, Houston, Texas), suitable for precise and free of matrix effect measurements on diluted female serum samples. AMH as well as other ovarian hormone and gonadotropin levels were measured in women enrolled in a multicenter international trial. They received daily for 3 months either a placebo or the progesterone receptor antagonist VA 2914 (HRA Pharma, Paris, France). The trial was approved by the local Ethics Committee and conducted in compliance with the Declaration of Helsinki The subjects were sampled every 3 days (D) throughout the 3rd cycle of treatment. FSH and LH were measured by means of AutoDelfia reagents (PerkinElmer France), estradiol and progesterone by RIA (DiaSorin-France and CIS biointernational, France, respectively,) and inhibin A by Elisa (Oxford Bioinovation-DSL-France) In 12 placebo treated ovulatory cycles, AMH levels (pmol / l, mean ± sem) exhibited a diphasic pattern, with a peak on D -7 relative to LH peak : 26 ± 3.2, a nadir on D0 : 19.1 ± 3.5 , and a second peak in luteal phase on D10 after LH peak : 25.4 ± 4.3. These variations were significant (p<0.05). There were no correlations between AMH levels and FSH, LH, inhibin A, or progesterone levels. Interestingly AMH levels were negatively correlated with estradiol levels : r = 0.27, p < 0 .02. As expected since both hormones are indexes of luteinization, inhibin A and progesterone levels were strongly correlated : r = 0.62, p <0.001. In 21 anovulatory cycles observed in women treated with the progesterone receptor antagonist, AMH levels exhibited a quite different pattern : no significant changes from D3 to D14 of cycle, significantly higher levels in the second phase of the cycle (D18-D28) : 28.2 ± 2.2 relative to the first phase (D3-D14) : 24.2 ± 1.9, p<0.05. Again no correlations were found between AMH and gonadotropin or ovarian hormone levels. In addition AMH and estradiol levels were not correlated in these anovulatory cycles. It should be noted however that estradiol levels in the second phase of the anovulatory cycles were lower than in luteal phase Thanks to this new AMH assay, we report for the first time a diphasic pattern of AMH levels throughout the menstrual cycle, contrasting with previous reports of unsignificant changes during the cycle. In addition the inverse relationship of estradiol and AMH in ovulatory cycles suggest a possible role of estradiol in the regulation of AMH secretion by the ovary.

Serum Anti-Müllerian Hormone as a Surrogate for Antral Follicle Count for Definition of the Polycystic Ovary Syndrome

Despite its frequency, the polycystic ovary syndrome (PCOS) is still a difficult diagnosis in endocrinology, gynecology, and reproductive medicine. To help solve this issue, the Rotterdam consensus conference proposed to include the ultrasonographic follicle count as a new diagnostic criterion, in addition to hyperandrogenism and oligo-anovulation. Unfortunately, its assessment does not offer sufficient reliability worldwide. The aim of our study was to check whether anti-Müllerian hormone (AMH) measurement in the serum could be a surrogate for antral follicle count in the diagnostic criteria of PCOS. Serum AMH was measured with a second-generation immunoassay in a cohort of 73 PCOS patients and 96 controls, and its diagnostic power was evaluated by receiver operating characteristic curves. PCOS was diagnosed according to the Rotterdam definition. Serum AMH levels were 3-fold higher in PCOS patients than in controls (81.6 vs. 33.5 pmol/liter; P < 0.001) and were significantly related to the follicle number in the two groups. The area under the receiver operating characteristic curve for the AMH assay was 0.851, indicating a good diagnostic potency. Setting the threshold at 60 pmol/liter offered the best compromise between specificity (92%) and sensitivity (67%). The serum AMH level is an accurate marker of the ovarian early antral follicle number and offers a good diagnostic potency. In situations where accurate ultrasonographic data are not available, AMH could thus be used instead of the follicle count as a diagnostic criterion and incorporated as such in the Rotterdam definition of PCOS.

99 Anti Mullerian Hormone (AMH/MIS) Elisa: A Diagnostic Tool in Neona-Tology And Paediatry

Background: Dimorphic expression of the Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance (MIS), in ovary and testis is crucial for normal differentiation of reproductive structures. Its absence in the female embryo allows Müllerian ducts to form the uterus, oviducts and upper vagina. AMH assay in girls is restricted to granulosa cancer and evaluation of ovarian follicular status. In males, AMH is produced by the testes reflecting Sertoli cell maturation. AMH induces Müllerian duct regression and is involved in testicular differentiation and function. Clinical applications of AMH assay include external abnormal genitalia, precocious puberty and hypogonadotropic hypogonadism. Howether, AMH measurement belongs to specialised laboratories which performed home made assays. Recently, Immunotech® has developed an assay available in all clinical laboratories.We established usual AMH values for this assay and we improved its interest. Methods: AMH measurement required only 25 μl of plasma and 3 hours (detection range: 0–200 ng/ml; sensitivity: 0.3 ng/ml; within-assay and between-assaies coefficients of variation: below 6% and 9% respectively). Usual values were established on blood from, 48 eutrophic fetuses (18 to 37 weeks of gestation), 101 full-term healthy newborns, and 425 healthy infants aged from one day to 10 years. 88 children with external gonadal abnormalities (14 clitoral hypertrophy; 50 microphallus; 43 hypospadias; 25 cryptorchidism; 9 non palpable testis) were evaluated. Clinical overlap (ambiguous genitalia) was observed in 42 cases. Results: In female, AMH was undetectable before birth and then weakly produced (< 6 ng/ml). In males, AMH increases from fetal life, pickes up between 1 and 12 months of life (99.7 ng/ml) with wide interindividual variations and then decrase with no overlap with female values. In clitoral hypertorphia, isolated microphallus and isolated hypospadias, AMH concentrations were within the usual range in 90% of cases. AMH was decreased in 73% of isolated cryptorchidism cases and was undetectable in anorchia. In ambiguous genitalia, AMH concentrations were increased, decreased or normal. Conclusions: At birth, AMH levels seems useful when investigating ambiguous genitalia antenatally suspected. In children with isolated microphallus or hypospadias, normal AMH values exclude testis dysfunction. A single AMH measurement distinguishes between anorchia and cryptorchidism when testis palpation is abnormal. In ambiguous genitalia, AMH measurement must be combined with other investigations.