Delayed reporting of sexual assaults is very common. This could be due to instances of victim incapacitation, fear to come forward or, in cases of assault of young children. The ability to obtain an autosomal STR (aSTR) profile of the semen donor from the living victim diminishes rapidly as the post-coital interval is extended. It is normally not possible to obtain the male aSTR profile when the post-coital interval exceeds 48-72 hours. In many cases, this is not due to the absence of sperm cells, but rather the failure to detect the genetic signature of the male donor. Often Y-STR analysis is employed in place of, or subsequent to, aSTR analysis in these cases. While Y-STRs are extremely useful, a full Y-STR profile does not hold the same statistical significance as a full aSTR profile. We hypothesize that a viable approach to overcoming the limitations described, and to be able to obtain aSTR profiles from extended interval post coital samples, would be to carry out direct aSTR typing on selectively enriched, purified, and pooled rare individual sperm cells obtained by direct physical recovery from the sample using simplified micromanipulation.
In this study, we applied our previously established micromanipulation method to sperm. The simplified micromanipulation approach utilities a water-soluble adhesive to collect individual sperm cells and transferring them directly into a lysis solution. After lysis, STR amplification reagents are added to the same reaction tube eliminating any need for sample transfer. Both Y STR and aSTR increased cycle number amplifications were performed on 1 - 5, 10 and 25 sperm cell samples collected from five different semen donors. Each cell number was evaluated using five replicates, with a consensus profile generated from the replicates.
For aSTRs, average allele recovery ranged from ~20 – 73% for 1-5 sperm cells (with increasing percentages observed with increasing cell number). For 10 and 25 sperm cells, 86.7%, and 99.0% profile recovery was observed, respectively. A similar trend was obtained for Y-STRs with average percent recoveries of ~13-65% (1-5 sperm cells), 78% (10 sperm cells) and 94% (25 sperm cells). More variability was observed with profile recovery for Y-STR analysis as expected, as only 50% of individual sperm cells collected will contain a Y chromosome. We demonstrate the ability to improve profile recovery by employing consensus profiling, with full or near full profiles obtained with five to twenty replicates. We also demonstrate the ability to apply this method to simulated sperm-epithelial cell (buccal and vaginal) admixtures with single source aSTR and Y STR profiles obtained from collected sperm cells.