Quantification of anti-Müllerian hormone (AMH) in dried blood spots: validation of a minimally invasive method for assessing ovarian reserve

Abstract

Background: Biological markers of ovarian reserve have the potential to advance research on fecundability, infertility and reproductive aging. Anti-Müllerian hormone (AMH) has emerged as a clinically useful measure of ovarian reserve, but the requirement for venous blood is an obstacle to application in non-clinical settings. This paper validates a new method for quantifying AMH in dried blood spot (DBS) samples--drops of whole blood collected on filter paper following a simple finger stick.

Methods: Matched serum and DBS samples were obtained from n=101 women of reproductive age, and AMH values were compared using regression analyses and scatter plots. The precision, reliability, linearity, recovery and lower detection limit of the DBS assay were evaluated, as well as the stability of AMH in DBS across a range of storage conditions.

Results: There was a strong agreement between AMH concentrations measured in DBS and serum samples across the entire assay range. Analysis of within-assay (percent coefficient of variation, 4.7-6.5%) and between-assay (3.5-7.2%) variability indicated a high level of assay precision and reliability, respectively. The minimum detectable dose of AMH was 0.065 ng/ml. Concentrations of AMH remained stable in DBS samples stored for 2 weeks at room temperature, and for 4 weeks when refrigerated.

Conclusions: The DBS assay performs at a level that is comparable to serum-based methods, with the advantage of lower burdens and costs associated with blood collection that may be advantageous for research in clinical as well as non-clinical settings on the causes and consequences of variation in ovarian reserve.

Figures

Figure 1

Scatterplot and Passing and Bablok regression analysis of the association between AMH concentrations obtained from matched serum and DBS samples for n = 101 reproductive age women. Regression results were as follows: y = 1.000(x); r = 0.97, P < 0.0001.

Figure 2

Scatterplot of the difference in AMH concentrations between DBS and serum samples as a function of the average AMH concentration across the two sample types (n = 101). Mean difference = −0.016; limits of agreement (Bland and Altman, 1999) upper 95% = 0.858: lower 95% = −0.890.

Figure 3

Association between age and mean AMH concentration in DBS samples from n = 77 reproductive age women without a diagnosis of infertility (open bars), and for n = 22 women with a diagnosis of infertility (black bars). The number of observations for each group is indicated in italics.