External replication was carried out in an independent cohort. A total of 321 women from the Chicago PCOS cohort were included. The Chicago PCOS cohort was approved by the Northwestern IRB (#STU00008096). Patients were included when they met the 1990 National Institutes of Health (NIH) criteria for PCOS, requiring the presence of both clinical and/or biochemical HA and OD. Cases fulfilling these criteria also meet the Rotterdam criteria for PCOS. Serum AMH levels were measured with the picoAMH assay [Ansh Labs®, Webster, TX, USA].
Picoamh assay
Manufactured by Ansh Labs
Sourced in United States
The PicoAMH assay is a laboratory test used to measure the levels of Anti-Müllerian Hormone (AMH) in biological samples. AMH is a protein produced by the ovaries and testes, and its measurement provides information about reproductive function. The PicoAMH assay is a sensitive and precise analytical tool designed to quantify AMH concentrations in a variety of sample types.
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10 protocols using picoamh assay
1
Serum AMH Levels in PCOS Women
2
Quantifying Disease Activity and Serum AMH Levels
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Disease activity was measured using a tender and swollen joint count for 28 joints (disease activity score in 28 joints [DAS28]) combined with serum C‐reactive protein (CRP) levels (DAS28‐CRP) 23 . Details on the measurement of RF and ACPAs can be found in the original report on the PARA study 22 .
Serum samples were stored at −80°C. Serum AMH levels (μg/l or ng/ml) were measured using picoAMH assay developed by Ansh Labs24 . The limit of detection (LoD) was 0.0012 μg/l.
Serum samples were stored at −80°C. Serum AMH levels (μg/l or ng/ml) were measured using picoAMH assay developed by Ansh Labs
3
Measuring Disease Activity and AMH Levels
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Disease activity was measured during the PARA study visits using the Disease Activity Score assessing 28 joints (DAS28) for tenderness and swollenness, combined with serum C reactive protein levels.23 (link)Serum samples were stored at −80°C. Serum AMH levels were measured in the samples from all preconception and 6 month postpartum PARA study assessments, as well as in the newly acquired samples from the follow-up visit.
AMH values were measured using the picoAMH assay, provided by Ansh labs (Houston, Texas, USA).24 25 (link) Interassay and intra-assay coefficients of variation were both <5%.
AMH values were measured using the picoAMH assay, provided by Ansh labs (Houston, Texas, USA).24 25 (link) Interassay and intra-assay coefficients of variation were both <5%.
4
AMH Measurement Protocols Across Epidemiological Studies
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Included studies measured AMH in either serum or plasma using different manual AMH ELISA assays. The picoAMH assay (Ansh Labs, Webster, TX) was used to measure circulating AMH levels in the Generations Study, both Nurses’ Health Studies, the Doetinchem Cohort Study and SWAN. For the Sister Study, the ultrasensitive AMH ELISA (Ansh Labs, Webster, TX, USA) was used, and for ALSPAC, AMH was measured using the Gen II AMH ELISA (Beckman Coulter UK Ltd, High Wycombe, UK). Antibodies differ between the Ansh Labs and Beckman Coulter assays (Moolhuijsen and Visser, 2020 ), but detailed information about these differences in antibodies is not available. The methodology for handling AMH measurements below the assay limit of detection (LOD) also differed across studies. A detailed overview of these study-specific details has been included in Supplementary Table SI . Across studies, the percentage of measurements under the assay-specific LODs ranged from 0% to 24.2%.
5
Verifying AMH Assay Discrepancies
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A recent case report suggested that the amino acid substitution corresponding to rs10417628 (AMH locus) reduces AMH detection by the picoAMH assay from Ansh Labs without influencing AMH bioactivity (Hoyos et al., 2020 (link)). We sought to verify this finding in a subsample of the Doetinchem Cohort Study, for which AMH was measured using both the picoAMH assay and the less sensitive Gen II assay from Beckman Coulter. In addition, we requested median AMH levels across rs10417628 genotype categories from both ALSPAC mothers and daughters, because ALSPAC also used the AMH Gen II assay from Beckman Coulter.
6
Detailed PCOS Diagnosis and Measurement Protocol
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In total, 655 women of reproductive age and European ancestry from the previously described Rotterdam PCOS cohort were included in this study (Day et al., 2018 (link)). The Rotterdam PCOS cohort, the CyclusOLigoAmennorroe (COLA) study, was approved by the institutional review board (Medical Ethics Committee) of the Erasmus Medical Center (04-263). PCOS was diagnosed based on the Rotterdam criteria requiring at least two out of the following three diagnostic criteria: (i) oligo- or amenorrhea, defined as chronic menstrual cycle interval >35 days or >188 days, respectively; (ii) clinical HA, defined as modified Ferriman Gallwey (FG) score above 5 and/or biochemical HA, defined as elevated (>4.5) free androgen index [testosterone × 100/SHBG] (Bui et al., 2015 (link)); and (iii) polycystic ovaries, defined as 12 or more follicles of 2–9 mm in each ovary and/or an ovarian volume >10 ml (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004 ). Serum AMH levels were measured with the picoAMH assay [Ansh Labs®, Webster, TX, USA]. Serum testosterone was measured using radioimmunoassays (Diagnostic Products Corporation). Serum FSH and LH were measured using the Siemens Immulite 2000XPi, and total follicle count (TFC) and ovarian volume were assessed as described earlier (van Santbrink et al., 1997 (link); Kevenaar et al., 2008 (link)).
7
AMH Measurement in Plasma Samples
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The plasma samples from round 1 were stored in EDTA aliquots at –30 °C. The samples derived from rounds 2–5 were stored in EDTA aliquots at –80 °C. Prior to the current study, the samples were thawed once for additional measurements and immediately refrozen. For the current study, stored plasma samples of rounds 1–5 were utilized. In March 2015, all the available samples of each participant had been retrieved from storage and were shipped on dry ice to AnshLabs (Webster, Texas, USA), where they were temporarily stored at –20 °C until the analyses were performed. AMH levels were measured with the picoAMH assay (AnshLabs), because of its low limit of detection and the small aliquot size necessary, which is crucial for cohort studies with a limited pool of biological samples. The plasma samples of each individual were measured in a single assay run, by a single laboratory operator. In total, two laboratory operators performed all measurements. At a mean level of 91.2 pg/mL, the coefficient of variation was 4.0 %. At 290.3 pg/mL, the coefficient of variation was 4.8 %. The limit of quantification was 3.0 pg/mL and the limit of detection 1.8 pg/mL. There were no indications of plate drift, with all coefficients of variation within plate columns and rows under 5 %.
8
Replicating PCOS Biochemical Criteria
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Internal replication was carried out in a follow-up sample of the discovery PCOS cohort. A total of 458 women of European ancestry, diagnosed based on the Rotterdam PCOS criteria, were included. The diagnostic criteria for biochemical HA was adjusted to a cutoff value of 2.9 for patients entering the dataset after 20 August 2012, due to the use of UPLC-MS/MS instead of radioimmunoassay for the measurement of serum testosterone (Bui et al., 2015 (link)). Serum AMH levels were measured with the picoAMH assay [Ansh Labs®, Webster, TX, USA].
9
PCOS Cohort Genotyping Analysis
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All genotyping specifications for the PCOS cohorts have been described earlier in a large genome-wide association study (GWAS) meta-analysis in PCOS patients (Day et al., 2018 (link)). Samples used in this study were selected from the Rotterdam PCOS cohort based on the presence of AMH levels measured by the picoAMH assay [Ansh Labs®, Webster, TX, USA] (Day et al., 2018 (link)). All two-allelic SNPs in the region Chr19:2 245 353–2 250 827 bp (Build 37, hg19) were selected. This region covers 3969 bp of the human AMH promoter and 1505 bp downstream of the protein coding region of AMH, covering the first three exons of the AMH protein. Only SNPs with a minor allele frequency (MAF) >1% and imputation quality (r2) >0.75 were included for the analysis. The quality control check analysis was described previously (Day et al., 2018 (link)). Linkage disequilibrium (LD) between SNPs was calculated using the Ensembl library 1000GENOMES:phase_3:CEU (Zerbino et al., 2018 (link)).
10
Serum AMH Quantification and Batch Correction
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