We have developed and validated a minimally invasive method for quantifying AMH in DBS samples in order to facilitate research on ovarian reserve. Analysis of assay performance indicates that the DBS method returns AMH results that are accurate, precise, reliable and in strong agreement with the current gold standard serum-based assay method. In addition, since our protocol uses commercially available supplies and commonly available enzyme immunoassay equipment, barriers to implementation are relatively low.
The age distribution of DBS AMH concentrations in women in our validation sample is generally consistent with previously published studies using serum samples (Lee et al., 1996
; Seifer et al.
, 2011). Our sample was recruited for the purposes of assay validation, and the small, non-representative nature of the sample may account for the lower AMH concentrations among 18–20 year-olds, although this pattern is in agreement with a recent study in which AMH concentrations were shown to peak at age 25 and subsequently decline (Kelsey et al., 2011
). The pattern of association between AMH and age in our sample is identical whether we use serum- or DBS-based results; any deviations from prior research therefore likely derive from sample composition rather than assay methodology.
Previously, validated methods for quantifying gonadotrophins and steroid hormones in DBS have promoted comparative, community-based research on human reproductive function for almost two decades (Worthman and Stallings, 1994
). Our AMH method adds an important biomarker to this methodological toolkit, and takes advantage of the low costs and simplified logistics associated with collecting DBS samples. Recent applications have yielded high rates of participant compliance in multiple community- and population-based studies (Borders et al., 2007
; Williams and McDade, 2009
; McDade, 2011
). For example, in a large, nationally representative study of young adults, 94% of participants consented to provide a DBS sample (Harris, 2010
Requirements for storage and transportation are simplified by the fact that DBS samples can be stacked and stored in air-tight containers and kept at ambient temperatures. Results of our stability analysis indicate that samples can be stored for 2 weeks at normal room temperature without loss of AMH, and that this period can be extended to 4 weeks or longer with refrigeration. However, the AMH in DBS is sensitive to elevated temperatures, with more rapid degradation with exposure to temperatures common in tropical regions, and during summer months in other areas. Efforts should therefore be made to protect samples from prolonged exposure to high temperatures during storage and shipping.
While there are many advantages to DBS sampling, disadvantages associated with quantifying AMH in DBS should also be considered. First, proper placement of whole blood on the filter paper is essential, since the dispersion of analytes within the sample will be inconsistent if blood is blotted or smeared onto the paper, or if a drop of blood is placed on the top of a previously collected drop. In addition, the AMH assay requires a relatively large quantity of whole blood: six 3.2 mm discs are required for duplicate analyses, which is the volume that can be obtained from one large drop of blood (~50 μl). The filter papers we used include pre-printed circles as guides for blood placement, and by collecting at least one large drop of blood that fills the border of this circle one can be assured of having enough sample. While the process of collecting DBS samples is relatively straightforward, implementing procedures that ensure sufficient sample volume and that avoid blotting and smearing are important for successful quantification of AMH.
Lastly, measuring AMH in DBS complicates efforts to compare results with studies using serum-based methods. Current AMH reference values apply to plasma and serum (Almog et al., 2011
), and DBS values will differ substantially due to the presence of lysed erythrocytes and associated matrix effects. This issue is not of particular concern for relative comparisons within studies using DBS samples, but it poses challenges for comparisons with clinical or epidemiological data sets assaying AMH in serum, and for the application of clinically relevant cut-off values. This limitation can be overcome by comparing serum and DBS AMH concentrations for a large set of matched samples to derive a conversion formula that produces serum equivalent values based on DBS results. Our serum-DBS comparison indicates a high level of correlation in AMH values, but a much larger, and more diverse sample is required to establish a reliable and generalizable conversion factor. Alternative approaches include the development of AMH reference values specific to the DBS assay, or for investigators to develop study-specific conversion factors based on matched serum and DBS samples collected from a subset of participants.
AMH is a promising measure of ovarian reserve, with potential utility for clinical practice related to reproductive function, aging and the reproductive capacity of ovaries compromised by iatrogenic treatments. Methodological tools that facilitate research on AMH in non-clinical settings, with larger, more diverse and representative samples, provide opportunities for clarifying the physiological significance of AMH, and for broadening our understanding of the determinants and consequences of variation in female reproductive function, aging and disease.