Aconitate 2 (ACO2) and pyruvate kinase M2 (PKM2) are good predictors of human sperm freezability

The aim of this study was to find the useful and reliable biomarkers to predict human sperm freezability. According to progressive motility of the thawed sperm, ejaculate samples were classified as good freezability ejaculates (GFE) and poor freezability ejaculates (PFE). Before starting cryopreservation protocols, we chose ACO2 and PKM2 as probable markers of sperm freezing capacity to compare their expressions between GFE and PFE group using quantitative real time PCR (qRT PCR), western blot and immunofluorescence. Moreover, Pearson's linear correlation was used to assess the association between ACO2 and PKM2 expressions before cryopreservation and the sperm motility before cryopreservation. The expressions of ACO2 and PKM2 were significantly higher in GFE group compared to PFE group in both qRT-PCR and western blot analysis. Besides, the immunofluorescent staining confirmed these results and showed that ACO2 localized in the sperm midpiece, and PKM2 was located in the body and tail of spermatozoa. Pearson's linear correlation demonstrated a significant positive correlation between sperm freezability and normalized band volume of ACO2 and PKM2 before cryopreservation. In this study, the expressions of ACO2 and PKM2 before cryopreservation were higher in GFE group than PFE group, which suggested that these two proteins could be used as predictive markers of human sperm freezability.