LAUSR

Serum measurements of 17β‐estradiol and progesterone are widely used to verify menstrual cycle status and confirm contraceptive use, often through commercially available immunoassay kits. However, no studies have investigated whether blood collection tube chemistries influence hormone concentrations in young females, despite assays permitting the use of different biofluids with similar reference ranges. In this study, venous blood was sampled from physically active females (n = 25) using Ethylenediaminetetraacetic acid (EDTA) and serum vacutainers, and 17β‐estradiol and progesterone concentrations were measured using competitive immunoenzymatic assays. Median plasma concentrations of 17β‐estradiol and progesterone were 44.2% (plasma 40.75 vs. serum 28.25 pg/ml) and 78.9% (plasma 1.70 vs. serum 0.95 ng/ml) higher than serum concentrations, respectively (P < 0.001 for both). Strong positive correlations were observed between plasma and serum concentrations for 17β‐estradiol (r = 0.72; P < 0.001) and progesterone (r = 0.89; P < 0.001). The mean bias and limits of agreement for plasma versus serum were 12.5 pg/ml (−20.6 to 45.5 pg/ml) for 17β‐estradiol and 1.01 ng/ml (−5.6 to 7.6 ng/ml) for progesterone. Ovarian hormone levels were consistently higher in EDTA plasma compared with serum, with these matrices not yielding statistically equivalent results. Despite these differences, the strong correlations and good agreement suggest that both matrices are suitable for biomarker analysis. Researchers using EDTA plasma should account for the higher hormone concentrations when applying inclusion or exclusion criteria, because adjustments might be necessary to ensure appropriate participant classification.