Anti-Mullerian Hormone concentrations in whole blood, plasma, and serum under different incubation periods and temperatures

Daniela Saade
Undergraduate (Animal Sciences – Health)
Dr. Alvaro Garcia Guerra
Animal Sciences

Anti-Mullerian Hormone (AMH) is produced by ovarian follicles and is utilized as a predictor for embryo production in assisted reproductive technologies (ART). However, AMH is prone to preanalytical instability. Thus, the objective of the present study was to investigate the effect of sample type (plasma, serum or whole blood), transport time (6 to 48 h) and temperature (4ºC or 20ºC) on circulating AMH concentration. Blood samples were collected from 20 Angus cows at a random stage of the estrous cycle. Blood was drawn into evacuated tubes providing three sample types: plasma, serum and whole blood. Whole blood samples were directly stored at -20ºC, while one plasma and serum sample were centrifuged and stored at -20ºC (time 0). Additional samples (plasma and serum) from each cow were incubated at 4ºC or 20ºC for either 6, 12, 24, or 48 h. After incubation, samples were centrifuged and stored at -20ºC. Samples were assayed for AMH using a commercially available ELISA (AnshLabs® Bovine AMH). Data were analyzed using linear mixed models (SAS 9.4) and are presented as mean ± SEM. There was a positive correlation (P<0.05) between AMH concentrations determined in the different sample types at time 0. Correlation coefficients were 0.65 (plasma vs serum); 0.78 (serum vs whole blood); and 0.85 (plasma vs whole blood). Mean circulating AMH concentration, however, was greater (P<0.001) in plasma (793 ± 94 pg/ml) than whole blood (587 ± 68 pg/ml) and serum (542 ± 73 pg/ml). During incubation at either 4ºC or 20ºC AMH concentration was affected by sample type (P<0.001) and incubation period (P<0.01). In addition, during incubation at 4ºC there was a sample type by incubation period interaction (P=0.002). Serum AMH concentration increased (P<0.0001) from 0 to 48 hours when incubated at either 4ºC or 20ºC, while plasma AMH concentration remained unchanged (P>0.10) during incubation at either 4ºC or 20ºC. In conclusion, plasma samples present the greatest initial circulating AMH concentration, and will remain relatively stable for up to 48 h. Thus, when assessing AMH for selection of cattle for ART it is recommended to use plasma samples