Application of the Methylated Markers (Spectrin Beta and DEAD-Box Protein) for Definitive Differentiation Between Fresh and Aged Semen by evaluating Their Role in Identifying Semen From Mixed Body Fluids

Background: Semen identification is assumed a crucial proof of sexual assault. Moreover, body fluids at the crime scene of a human being, such as blood, semen, and saliva, are often mixed. Methods: Hence, in our study, we aimed to use methylation analysis targeting DNA epigenetic markers Spectrin beta chain (B_SPTB_03) and DEAD-box protein (DDX4) to differentiate between fresh semen (less than 4 hours) and aged semen (after 24 hours) as well as to differentiate between semen alone and semen mixed with other body fluids (blood and saliva) in the fresh and dried state. Results: Our findings showed statistically significant differences in the methylation patterns of the SPTB and DDX4 loci to distinguish semen from mixed body fluids in fresh and old samples. We were able to obtain two novel cutoff values to differentiate between fresh and aged semen, which are (52.25) with the SPTB marker and (70.75) with the DDX4 marker. Conclusion: It is concluded that the methylation approach based on the epigenetic markers of Spectrin beta chain and DEAD-box protein (B_SPTB_03 and DDX4) successfully identified fresh from aged semen and semen-derived alleles from mixed stains, hence it is recommended to be employed in forensic practice.