Inter-assay variation and reproducibility of progesterone measurements during ovarian stimulation for IVF

In recent years there is increasing evidence that elevated progesterone levels during ovarian stimulation for IVF / ICSI have a negative impact on the ART-outcome. However, different progesterone assays were used in the previous studies and different assays might produce varying results. This retrospective study evaluated the reproducibility and reliability of different progesterone assays with a special focus on progesterone levels below 1.5 ng/ml, as this range is crucial for early detection of progesterone rise during ovarian stimulation for IVF. A total of 413 blood samples were categorized in different progesterone ranges and whether they were retrieved on the day of final oocyte maturation and the results were compared regarding their reproducibility and reliability. To compare the reproducibility between the different progesterone assays, the Intraclass Correlation Coefficient (ICC) was calculated and interpretation of the ICC results was done according to Cicchetti, ranging from poor to excellent. The correlation of the assays was excellent when all samples were compared including samples retrieved on day of final oocyte maturation, however in the ranges of progesterone levels 1.0 ng/ml to < 1.5 ng/ml, 0.8 ng/ml to < 1.0 ng/ml and < 0.8 ng/ml, the ICC varied between poor and excellent. The assays “gen III” and “Architect” showed an excellent reproducibility of progesterone results throughout all ranges of progesterone levels. This analysis demonstrates, that different progesterone assays have a limited reproducibility and that the results depend on the assay used and the range of progesterone level. This fact leads to two important conclusions. Firstly the limited reproducibility might lead to substantially different treatment decisions in ovarian stimulation treatment for IVF and secondly critical interpretation of thresholds, provided by meta-analysis, is crucial despite the risk that the so far gained clinical experience might become irrelevant and has to be adjusted to the results, obtained by each assay.


Precautions and warnings
For in vitro diagnostic use. Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines. Safety data sheet available for professional user on request. Avoid foam formation in all reagents and sample types (specimens, calibrators and controls).

Reagent handling
The reagents in the kit have been assembled into a ready-for-use unit that cannot be separated. All information required for correct operation is available via the cobas link.

Storage and stability
Store at 2-8 °C. Do not freeze. Store the cobas e pack upright in order to ensure complete availability of the microparticles during automatic mixing prior to use.

Specimen collection and preparation
Only the specimens listed below were tested and found acceptable. Serum collected using standard sampling tubes or tubes containing separating gel. Li-heparin, K 2 -EDTA and K 3 -EDTA plasma. Criterion: Slope 0.9-1. For optimum performance of the assay follow the directions given in this document for the analyzer concerned. Refer to the appropriate operator's manual for analyzer-specific assay instructions. Resuspension of the microparticles takes place automatically prior to use. Place the cooled (stored at 2-8 °C) cobas e pack on the reagent manager. Avoid foam formation. The system automatically regulates the temperature of the reagents and the opening/closing of the cobas e pack.

Calibration
Traceability: The Elecsys Progesterone III assay is traceable via ID-GC/MS (isotope dilution gas chromatography/mass spectrometry) to highly purified progesterone by weight analogously to BCR-348R and ERM-DA347. 6 The predefined master curve is adapted to the analyzer using the relevant CalSet. Calibration frequency: Calibration must be performed once per reagent lot using fresh reagent (i.e. not more than 24 hours since the cobas e pack was registered on the analyzer). Calibration interval may be extended based on acceptable verification of calibration by the laboratory. Renewed calibration is recommended as follows: ▪ after 8 weeks when using the same reagent lot ▪ after 28 days when using the same cobas e pack on the analyzer ▪ as required: e.g. quality control findings outside the defined limits Quality control For quality control, use PreciControl Universal. In addition, other suitable control material can be used. Controls for the various concentration ranges should be run individually at least once every 24 hours when the test is in use, once per cobas e pack, and following each calibration. The control intervals and limits should be adapted to each laboratory's individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits. If necessary, repeat the measurement of the samples concerned. Follow the applicable government regulations and local guidelines for quality control.

Calculation
The analyzer automatically calculates the analyte concentration of each sample (either in nmol/L, ng/mL or in μg/L).

Limitations -interference
The effect of the following endogenous substances and pharmaceutical compounds on assay performance was tested. Interferences were tested up to the listed concentrations and no impact on results was observed. In addition, the following special drug was tested. No interference with the assay was found.

Clomiphene citrate 100
In rare cases, interference due to extremely high titers of antibodies to analyte-specific antibodies, streptavidin or ruthenium can occur. These effects are minimized by suitable test design. For diagnostic purposes, the results should always be assessed in conjunction with the patient's medical history, clinical examination and other findings. Depending on the biological variance of the diluted patient sample and the human serum matrix used for production of Diluent Estradiol/Progesterone, lower recovery of diluted samples may be observed.

Expected values
The expected ranges were determined by testing specimens drawn from 147 apparently healthy males, 142 apparently healthy, post-menopausal women over the age of 50, and from 416 apparently healthy pregnant women between the ages of 17 and 45 (137 in the first trimester, 140 in the second trimester, and 139 in the third trimester). The expected range for healthy women was determined by weekly blood drawing over a period of 3 months from 26 apparently healthy women between the ages of 18 and 45 that were not taking any hormonal contraceptives. Based on a central 90 % interval, the following ranges were obtained: Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.

Specific performance data
Representative performance data on the analyzer is given below. Results obtained in individual laboratories may differ.

Precision
Precision was determined using Elecsys reagents, pooled human sera and controls in a protocol ( The sample concentrations were between 0.050 and 59.0 ng/mL.

Analytical specificity
For the Elecsys Progesterone III assay, the following cross-reactivities were found at the respective additive concentration, tested with progesterone concentrations of approximately 0.3 ng/mL and 5 ng/mL: