Stability of chimpanzee (Pan troglodytes) urinary reproductive hormones during long-term preservation on filter paper

Urine contains multiple water-soluble hormones, which are valuable non-invasive biomarkers for the monitoring of reproductive status and health. An effective method for drying urine on filter paper was previously developed to preserve wildlife urine samples where electrical equipment was not available for this; however, the stability of samples preserved in this way remains to be verified. Here, we developed and validated a method to elute multiple water-soluble reproductive hormones from filter paper that had been stored for an extended period of time. Aliquots of urine from chimpanzees were adsorbed on filter papers, air dried and stored for 1 year at room temperature. Estrone-3-conjugate (E₁C), pregnanediol-3-glucuronide (PdG), estriol-3-glucuronide (E₃G), and chorionic gonadotropin (CG) were eluted into deionized water from the filter papers and measured using enzyme immunoassays (EIAs). The mean recoveries of E₁C, PdG, and creatinine from filter papers stored for 1 year were 69.5%, 128.7%, and 83.8%, respectively. The profiles of E₁C and PdG from preserved filter papers significantly correlated with those derived from a direct analysis of the frozen urine of menstruating chimpanzees. We detected E₃G and CG from 1-year-old filter papers for urine collected during early pregnancy, but the recovery of E₃G was low and CG profiles did not correlate with those of the original frozen urine samples. The method proposed here for the elution and measurement of reproductive hormones in urine preserved for a long period of time on filter paper provides a practical and simple way to monitor the reproductive status of chimpanzees. We propose that this method can also be utilized in field studies of other wild nonhuman primates.