Abstract
Determining the presence of sperm cells on an item or swab is often a crucial component of sexual offence investigation. However, traditional histological staining techniques used for the morphological identification of spermatozoa lack both specificity and sensitivity, making analysis a complex and time-consuming process. New methods for the detection of sperm cells based on aptamer recognition may be able to overcome these issues. In this work, we present the selection of ssDNA aptamers against human sperm cells using Cell-SELEX and massively parallel sequencing technologies. A total of 14 rounds of selection were performed following a modified Cell-SELEX protocol, which included additional steps for the isolation of spermatozoa from seminal fluid. Massively parallel sequencing using the Illumina Miseq platform was conducted on enriched aptamer pools to elucidate the structure of potential binders. A custom bioinformatics pipeline was also developed using Galaxy for the automated processing of sequencing datasets. This data revealed several promising aptamer candidates, which were shown to selectively bind sperm cells through both microscale thermophoresis and enzyme-linked oligonucleotide assays. These aptamers have the potential to increase the efficiency of sexual offence casework by facilitating sperm detection.Graphical abstract
Highlights
Despite their increasing frequency, conviction rates for sexual offences remain much lower than other violent crimes
As human sperm cells used for in vitro studies are typically obtained from whole seminal fluid, rather than via traditional culture techniques, pre-selection cell growth and preparation methods were substituted with a density gradient centrifugation–based sperm cell isolation process [20]
A randomized N40 ssDNA library containing fixed flanking regions was incubated with a defined number of sperm cells at the start of each round of selection
Summary
Conviction rates for sexual offences remain much lower than other violent crimes. Only 1.5% of rapes reported to the police result in the conviction of a perpetrator [1] Half of these reported cases fail to progress past the investigative stage of the criminal justice process due to ‘evidence issues’ [2]. Methods used by forensic laboratories to confirm the presence of semen are currently based on the microscopic examination of sperm cells extracted from suspected fluid samples [4]. This examination is often facilitated by the use of histological staining techniques designed to enhance sperm cell visualization, allowing for easier assessment of cell size, number, and morphology [5]. Despite routine use in forensic laboratories for over 40 years, stains such as haematoxylin-eosin, Christmas tree, and alkaline fuchsin are known to lack both specificity and sensitivity, making the identification of sperm samples extremely challenging (especially if they are degraded, mixed, or contaminated) [6]
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