An intimate solution

Kimberley Bexon takes us through a new approach to tackle sexual assaults through microRNA analysis

When dealing with sexual assault cases it is important that the body fluids present in the samples found are identified. The majority of these cases will have a mixture of intimate body fluids and therefore distinguishing them can provide support to the prosecution or the defence side of the story.

This is however a difficult task and current techniques are not sufficiently robust. Tests for blood such as leucomalachite green (LMG) and Kastle–Meyer (KM) are presumptive and can give false positives, such as with grass, rust, and any other oxidative substances. The acid phosphatase (AP) test for semen, which targets the acid phosphatase enzyme secreted from the prostate, cannot be used to distinguish from vaginal material as it is also present, albeit at lower concentrations. These tests are currently not robust enough and cannot be used reliably in mixed body fluid samples.

There are several problems that face forensic scientists that new research aims to solve – presently there are no techniques for distinguishing between saliva and vaginal material. The inability to make this distinction means that any DNA profiles obtained cannot reliably support either the prosecution’s or the defendant’s version of the alleged event.

Another problem that forensic scientists face without a suitable body fluid identification tool is that vasectomised males do not produce spermatozoa, which contains the nuclear DNA required for DNA profiles. In addition, a confirmatory histological test relies upon the presence of spermatozoa. Consequently, in cases where the male does not express spermatozoa (through vasectomies or infertility), the identification of the male or the body fluid becomes problematic. For example, if a male DNA profile is obtained, it could come from urethral epithelial cells or from another body fluid such as saliva. The NHS state that approximately one in five young men suffer from oligozoospermia (low sperm count)¹. This would suggest that there is a high likelihood that semen samples found from a sexual assault crime scene could contain little or no sperm, thus demonstrating the need to be able to identify seminal fluid without the presence of spermatozoa.

The extraction of semen is also difficult; the proteinaceous coating surrounding the spermatozoa head must be degraded by dithiothreitol (DTT) in order to release the genetic material. However, the DTT can also damage any other genetic material that may be present in the sample, such as vaginal material. When dealing with unknown samples from a sexual assault this process cannot be performed as semen might not be present and it can damage the other body fluids present, it is therefore vital that a technique is established that can show significant expression of semen without the need of the additional lysis step of DTT.

The final challenge is that when a violent sexual assault occurs, it is likely that trauma blood may be present. However, it is not currently possible to differentiate between venous blood from trauma and menstrual blood using conventional techniques. Therefore, a suspect may provide a defence that sexual intercourse occurred consensually while the female was menstruating, thus negating the DNA profile obtained from penile swabs or condoms.

The use of a robust body fluid identification test would address these problems and help progress the case. One such method for distinguishing body fluids is the characterisation of miRNA markers. Such miRNA molecules are endogenous, non-coding RNAs that are approximately 22 nucleotides in length and are vital in the regulation of genes². They cause the translational repression or cleavage of mRNA and thus are responsible for gene silencing. Their key role in targeting specific genes means that some are body fluid specific and thus useful, robust tools for body fluid identification purposes. On average there are approximately 50,000 strands of distinct miRNA present in each cell³. In addition, their short size and protein protection gives them higher stability than the longer mRNA molecules. These factors; their specificity, abundance, and stability, makes them ideal markers for use in forensic casework.

[caption id="attachment_42354" align="aligncenter" width="480"] Table 1: Body fluid samples collected for screening study.[/caption]

A full body fluid panel screening study was then designed with body fluids being collected from multiple volunteers; the number of samples can be seen in Table 1. Current literature suggests that various factors could affect the expression of miRNAs therefore it was important to try and include as many of these factors in the sample groups. Samples were obtained from individuals of varying ages, ethnicities, contraception, sterilisations, diseases, pregnancy histories, stages of menopause, medications and various smoking and drinking habits. Detailed questionnaires were obtained that provided answers to these factors and allowed for a more comprehensive analysis of screening data.

These samples then underwent DNA extraction followed by stem-loop reverse transcription and qPCR. Each sample was analysed using >20 markers that were suggested as being body fluid specific. All markers were tested on each sample to determine if it was present or absent in certain body fluids and therefore show which could be a viable identification tool. Markers must significantly show that they are robust and specific to a particular body fluid. Menstrual blood will contain markers from venous blood and vaginal material therefore expression of these body fluid markers will be expected. It is important that the marker for menstrual blood must be absent in venous blood and vaginal material to be able to distinguish it from other body fluids. In short, menstrual blood markers must be more than just a mixture of vaginal material and venous blood markers.

MiRNA markers have been analysed by many research groups for body fluid specificity, however an area with very little research is that once markers are identified as showing statistically significant expression in a singular body fluid, how might the expression change over certain durations of time. The most dynamic being the menstrual cycle, it is not only very varied between individuals due to factors such as ethnicity and lifestyle habits⁴, but can also be affected by many external factors. For example, contraception such as the hormonal injection or implant causes menses to cease, as does breast feeding an infant. The cycle can also be affected by illnesses such as cancers or anorexia and also treatments such as radiotherapy. The markers need to be robust enough to still show significant enough expression in varied individuals that many suffer from or none of these factors.

[caption id="attachment_42355" align="aligncenter" width="400"] Figure 1: Mechanisms of miRNA. Taken from 5.[/caption]

When studying the markers throughout the menstrual cycle the markers for menstrual blood should be completely absent throughout the rest of the cycle, if they are expressed when menses is absent it cannot be used as a marker. In contrast markers that are specific to other body fluids such as vaginal material must stay at a constant level of expression throughout the cycle; if processes such as the ovum being released causes a decrease in expression of the vaginal material marker to such a point that it is below the threshold of other markers it the body fluid cannot be distinguished. This would mean that if a sexual assault occurred at this point in a female’s cycle it may infer vaginal penetration did not occur.

A scholarship was awarded from the Chartered Society of Forensic Sciences to fund this research and therefore enable a study to be designed that could assess the expression of a full body fluid panel over a 31 day period. The length of 31 days was chosen to ensure all females went through a full menstrual cycle and to provide overlap for further comparison of expression. Five volunteers provided two low vaginal swabs at the same time each day for the duration of the study. The swabs were then extracted within two hours of sampling; one swab underwent DNA extraction using a QIAamp DNA Mini Kit with the other swab being extracted using a total RNA RNeasy Mini Kit. These two methods were utilised to not only assess expression levels but to determine which method of extraction was more efficient for extracting miRNA. The samples were then analysed using stem-loop reverse transcription and qPCR using the miRNA markers that had previously been determined specific for each body fluid.

Future work will consist of obtaining a substantial vasectomised sample group and analysing them against regular semen samples to determine if miRNA from the spermatozoa can be detected and which miRNA are specific to seminal fluid and will therefore be able to determine vasectomised or low sperm count samples.

References

1. NHS (2012) “Low sperm count (oligozoospermia)”, http://www.nhs.uk/Conditions/low-sperm-count/Pages/Introduction.aspx
2. Wang, Z., J. Zhang, et al. (2013). "Screening and confirmation of microRNA markers for forensic body fluid identification." Forensic Science International: Genetics 7(1): 116-123.
3. Bartel, D. P. (2004). "MicroRNAs: Genomics, Biogenesis, Mechanism, and Function." Cell 116(2): 281-297.(Bartel 2004)
4. Liu, Y., E. B. Gold, et al. (2004). "Factors Affecting Menstrual Cycle Characteristics." American Journal of Epidemiology 160(2): 131-140.
5. Kirton, L. (2010). “Analysis of miRNA and mRNA associated with Epithelial Mesenchymal Transition” The Art of the PFUG. S. Cox.

The author 

Kimberley Bexon is a Ph.D. student at the University of Huddersfield. Her research is focused on using microRNA to identify body fluids associated with sexual assaults. She is an Associate member of the Chartered Society of Forensic Sciences and was successful in gaining their research scholarship to fund her research.

Acknowledgements

The authors would like to acknowledge the support from the Chartered Society of Forensic Sciences.