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.
Amh gen 2 assay
Manufactured by Beckman Coulter
Sourced in United States
The AMH Gen II assay is a diagnostic laboratory test used to measure the levels of anti-Müllerian hormone (AMH) in the blood. AMH is a protein produced by the granulosa cells of the ovary and is an important marker of ovarian reserve. The AMH Gen II assay provides a quantitative measurement of AMH levels, which can be used to assess ovarian function and fertility status.
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11 protocols using amh gen 2 assay
1
Verifying AMH Assay Discrepancies
2
Hormonal Biomarkers in Breast Cancer
3
Serum Hormone Profiling in Women
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Blood samples were collected from all participants in tubes without anticoagulants on the same day of ultrasonography in the early follicular phase. Sera were obtained by centrifugation for the determination of serum hormone levels. Serum hormone levels, including AMH, FSH, LH, and total and free testosterone were measured using enzyme immunoassays. The serum AMH level was measured using the AMH Gen II assay (Beckman Coulter Inc., Brea, CA, USA). The serum FSH level was measured using a Roche Elecsys FSH assay (Roche, Indianapolis, IN, USA), and the serum LH level was measured using a Roche Elecsys LH assay (Roche, USA). The last menstrual period was either spontaneous or induced by the administration of medroxyprogesterone acetate (Provera, 10 mg/day for 7 days).
4
Comprehensive Hormonal Assessment in IVF
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Blood tests were performed on cycle day 2 and the hCG trigger day. The concentrations of serum AMH in all subjects were measured before the start of IVF treatment. The concentrations of FSH, LH, E2, and progesterone were quantitatively determined by chemiluminescence assay (Abbott Biologicals B.V., Olst, Overijssel, The Netherlands) as described in our previous work [32 (link)]. We defined the detection limit as follows: FSH = 0.06 mIU/mL, LH = 0.09 mIU/mL, E2 = 10 pg/mL, and P = 0.1 ng/mL. For AMH measurement, all samples were immediately assayed with a commercial ELISA kit (AMH Gen II assay, Beckman Coulter, Brea, CA) according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation did not exceed 6% and 16%, respectively.
5
AMH Levels in IVF/ICSI Patients
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Blood and FF were taken from all patients undergoing
IVF or ICSI and ET on the day of follicular
puncture, processed by being centrifuged for
10 minutes at 350×g and 5˚C, shock-frozen and
kept at −80˚C. After pick-up of the oocytes, the
FF samples underwent the same procedures as the
blood. AMH levels in serum and FF were measured
in duplicate by a solid-phase enzyme-linked
immunosorbent assay (ELISA) using an AMH kit
(AMH Gen II Assay, Immunotech, Beckman Coulter
Company, Kiel, Germany). This assay uses the
quantitative sandwich enzyme immunoassay technique.
The AMH precision from manufacture was
CV=3.2-12.3% for intra-assay and CV=5.8-14.2%
for inter-assay. Only those cases in which both FF
and serum could be collected simultaneously on
the day of oocyte retrieval were included in this
study.
IVF or ICSI and ET on the day of follicular
puncture, processed by being centrifuged for
10 minutes at 350×g and 5˚C, shock-frozen and
kept at −80˚C. After pick-up of the oocytes, the
FF samples underwent the same procedures as the
blood. AMH levels in serum and FF were measured
in duplicate by a solid-phase enzyme-linked
immunosorbent assay (ELISA) using an AMH kit
(AMH Gen II Assay, Immunotech, Beckman Coulter
Company, Kiel, Germany). This assay uses the
quantitative sandwich enzyme immunoassay technique.
The AMH precision from manufacture was
CV=3.2-12.3% for intra-assay and CV=5.8-14.2%
for inter-assay. Only those cases in which both FF
and serum could be collected simultaneously on
the day of oocyte retrieval were included in this
study.
6
Modified Beckman Coulter AMH Gen II Assay
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The modified Beckman Coulter AMH Gen II assay was used for all AMH analyses in this study. The AMH Gen II assay is a two-step sandwich-type enzymatic microplated assay. Problems with the robustness of the Gen II assay were solved with the use of a modified version of the AMH Gen II assay kit (reference A79765), including an additional assay step before calibrators were added (premixing). This additional step eliminates the complement and thereby overcomes the not optimal assay reproducibility of the original AMH Gen II assay. The standards cover a range of 0-22 ng/mL. The sensitivity is reported to be 0.1-0.21 ng/mL. Reported intra-and interassay CVs were <2% and <12%, respectively.
7
Serum AMH Level Decline after Surgery
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Blood samples were collected before surgery and on the third day after surgery to determine the serum AMH levels from all study participants following the guidelines of the Declaration of Helsinki. The serum AMH level was measured using a commercially available enzyme-linked immunosorbent assay kit (AMH Gen II assay; Beckman Coulter, Inc.), and the lowest detection limit of the kit was 0.08 ng/mL with both the intra- and interassay coefficients of variation below 8.0% [8 (link)16 (link)].
The rate of decline in AMH levels was calculated using the following formula [8 (link)16 (link)]:
(preoperative AMH level – postoperative Day 3 AMH level)/(preoperative AMH levels) × 100 (%).
The rate of decline in AMH levels was calculated using the following formula [8 (link)16 (link)]:
(preoperative AMH level – postoperative Day 3 AMH level)/(preoperative AMH levels) × 100 (%).
8
Granulosa Cells Metabolic Profiling
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Glucose uptake was evaluated by using the Glucose Uptake-Glo™ Assay (Promega, Charbonnieres, France). Lactate concentrations in the culture medium and pyruvate and cholesterol contents in granulosa cells were evaluated by using the following spectrophotometric assays, respectively (Sigma, l'Isle d'Abeau Chesnes, France; Clinisciences, Nanterre, France; Biolab, Maizy, France). Analysis was performed on at least three different cultures in duplicate for each condition. The results of each assay were normalized per 10 6 cells.
Steroid (pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, 17αhydroxyprogesterone, Δ4 androstenedione, testosterone, and dihydrotestosterone) levels were determined by LC-MS/MS (mmol/L) as described by Meunier et al. (Meunier et al., 2015) .
The serum AMH level was evaluated on five control and three transgenic mice by using an enzyme-linked immunoassay (AMH Gen II assay®) and read with a Beckman Coulter® (France) as already described in mice (Valeri et al., 2020) . The values are expressed in pmol/l.
Steroid (pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, 17αhydroxyprogesterone, Δ4 androstenedione, testosterone, and dihydrotestosterone) levels were determined by LC-MS/MS (mmol/L) as described by Meunier et al. (Meunier et al., 2015) .
The serum AMH level was evaluated on five control and three transgenic mice by using an enzyme-linked immunoassay (AMH Gen II assay®) and read with a Beckman Coulter® (France) as already described in mice (Valeri et al., 2020) . The values are expressed in pmol/l.
9
Serum Hormone Levels in IVF Patients
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Patient serum samples were collected at three time points during IVF: on day 2 or 3 of the menstrual cycle, i.e. before gonadotropin administration; on the day of hCG/GnRHa administration, and 35–37 h after hCG/GnRHa administration. In contrast to the other sex hormones, the serum level of AMH (AMH Gen II assay, Beckman Coulter, Brea, CA) was measured by ELISA before the IVF cycle. All serum samples were collected into tubes and centrifuged at 1300 × g at 4°C for 10 min and stored at −80°C for further analyses. Serum estradiol (E2), luteinizing hormone (LH), progesterone (P4), and/or FSH levels were measured by chemiluminescence assay (Abbott Biologicals B.V., The Netherlands).
10
Reproductive Hormone Assays Protocol
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