Pitfalls in body fluid identification – age independent DNA methylation markers for vaginal secretions and menstrual blood in sexual assaults

In recent years, determination of cellular origin of DNA has been acknowledged as an important tool in forensic genetics. It complements identification of the DNA donor by STR analysis, since it could provide further information about the origin of DNA found at a crime scene. A workflow (body fluid identification (BFI) workflow) developed in 2023 for determination of seven different body fluids in forensic genetic casework by DNA methylation analysis has been very helpful in context with sexual assaults, especially rapes. The verification of vaginal secretions and/or menstrual blood, for which no (or no reliable) preliminary tests or rapid tests are available, can be decisive in these cases. During case work, it was hypothesized that the methylation pattern of vaginal secretions cannot be assumed to be universal but rather might depend on the female’s hormonal status or age. Validation studies have confirmed this assumption. Both, vaginal secretions from young girls before sexual maturity and from women who have already reached menopause showed a deviation from the established and validated DNA methylation percentages of the BFI workflow published in 2023. For this reason, additional vaginal marker V1(cg09765089) was established and validated in this study, so that in combination with marker V2 already published Konrad et al., (Int J Legal Med 137(6):1683–1692, 1) 99% of vaginal secretion samples could be identified, regardless of woman’s age or hormone status. Additionally, the new blood marker B6 (cg17518965) was integrated in the eBFI workflow (extended body fluid identification workflow), as the previous blood marker could not provide unambiguous identification of the target secretion in children in the context of sexual assaults. Overall, we have analyzed 453 samples in this study using bisulfite conversion and pyrosequencing. In summary, our revised eBFI workflow is a simple and useful tool for forensic analysis to identify vaginal secretions and/or menstrual blood in the context of sexual assaults, regardless of female’s hormonal status or age.