Forensic Lab-on-a-Disc for enhanced sperm cell purification in differential extraction workflows.

Background: Sexual assault remains one of the most underreported and complex medico-legal challenges worldwide. The accurate detection and interpretation of forensic evidence play a pivotal role in substantiating allegations, guiding legal proceedings, and delivering justice. However, the effectiveness of forensic evidence can be influenced by several factors including the timing of examination, the condition of the victim, the availability of trained professionals, and procedural limitations. Objective: To evaluate the accuracy and limitations of forensic evidence in medico-legal sexual assault cases and assess the relationship between evidence recovery, injury documentation, and time to examination. Methods: This descriptive cross-sectional study was conducted at the Department of Forensic Medicine, Bacha Khan Medical College Mardan, and Allama Iqbal Medical College Lahore, Pakistan, from January 2022 to March 2023. A total of 70 medico-legal cases of alleged sexual assault were reviewed. Data were extracted on demographic details, time of presentation, types of forensic evidence collected, presence of physical injuries, and DNA test outcomes. Descriptive statistics were used to analyze the data. Results: The majority of victims were females (75.7%), with adolescents (13–18 years) representing the most affected group. A significant portion of cases (54.3%) presented within 24 hours of assault. Semen was detected in 44.3% of cases, spermatozoa in 32.9%, and a DNA match with the alleged perpetrator was found in 37.1% of cases. Genital injuries were present in 41.4% of victims. However, 55.7% of cases showed no biological evidence, often due to delayed reporting, hygiene measures, or procedural errors. Conclusion: While forensic evidence can significantly aid in the investigation of sexual assault cases, it has inherent limitations. Early examination, proper evidence collection, and multidisciplinary collaboration are critical for maximizing the utility of forensic findings. A victim-centered, evidence-informed, and context-sensitive approach is essential for ensuring justice in sexual assault investigations. Keywords: Sexual assault, forensic evidence, medico-legal, DNA analysis, injury documentation, limitations.

Forensic DNA analysis of semen-vaginal fluid mixed stains is essential and necessary in sexual assault cases. Here, we used a magnetic bead conjugated acrosin binding protein (ACRBP) antibody to separate and enrich sperm cells from mixed stains. Previously, western blotting indicated that ACRBP was specifically expressed in sperm cells, but not in female blood and epithelial cells, while immunofluorescence data showed ACRBP was localized to the acrosome in sperm cells. In our study, sperm were separated from mixed samples at three sperm cell/female buccal epithelial cell ratios (103:103; 103:104; and 103:105) using a magnetic bead conjugated ACRBP antibody. Subsequently, 23 autosomal short tandem repeat (STR) loci were amplified using the Huaxia™ Platinum PCR Amplification System and genotyped using capillary electrophoresis. The genotyping success rate for STR loci was 90% when the sperm to female buccal epithelial cell ratio was > 1:100 in mixed samples. Our results suggest that the magnetic bead conjugated ACRBP antibody is effective for isolating sperm cells in sexual assault cases.

Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility (MHC) class I, CD45 and cytokeratin expression. Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified. We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape.

The conditions under which contact DNA analysis yields useful recovery of materials is not well known. Using simulated models of grabbing and struggling, the recovery of male contact DNA transferred from male “assailants” to female “victims” was investigated. The upper and lower arm areas of female subjects (sites for the modeled assaults) were swabbed for male DNA. Extracted DNA was quantified using the Quantifiler® Duo DNA Quantification kit and amplified using the AmpFlSTR® Identifiler® and the AmpFlSTR® YfilerTM PCR Amplification kits. The qPCR quantification results detected male “assailant” DNA from the female “victims”. However, no viable male profiles were obtained when the DNA was subjected to short tandem repeat (STR) analysis with the Identifiler® and Yfiler™ kits. The grab and struggle scenarios yielded up to 20 pg/μL and 40 pg/μL of male contact DNA from female subjects, respectively, DNA amounts which are below the detection threshold of most commercial human STR identification kits. Although contact DNA levels are highly variable, this study showed that the likelihood of DNA deposition on skin from assailant to victim was substantially greater when a struggle ensues. The results of the present study are limited by small sample size, but imply that further research for improving recovery and amplification techniques of contact DNA should be sought to increase the probative value of this evidence in sexual assault investigations.

Enhancing the forensic sexual assault investigations with LAMP-based male DNA detection

The persistence of moisturizer products on human skin in relation to sexual assault investigations

Preliminary classification scheme of silicone based lubricants using DART-TOFMS

During double fertilization of angiosperms, the two sperm cells are transported conjointly to the female gametophyte, where one sperm cell fuses with the egg cell and the other sperm cell with the central cell. These fusions have to take place in a controlled manner to avoid undesired gamete fusion events and prevent polyspermy. Only little is known about gamete recognition and coordination of gamete fusion in plants. The aim of this work was to characterize the function of the Arabidopsis egg cell-expressed EGG CELL 1 (EC1) gene family during gamete recognition and to identify putative interaction partners. The EC1 gene family comprises five members that encode cysteine-rich proteins, which are secreted from the egg cell during fertilization. Triple knockout mutants were additionally transformed with an RNAi construct targeting the remaining two genes. In these plants (ec1+/-) the fusion of the sperm cells with the female gametes was impaired resulting in a reduced seed set. Detailed analyses of ec1+/- plants showed that in 45% of the ovules of ec1+/- plants sperm cells did not fuse with the female gametes and that non-fused sperm cells were always observed as pairs, which indicated that EC1 might function in sperm cell separation. This hypothesis was supported by the observation that single sperm cells of mutant pollen seemed to be able to fuse in ec1 ovules. With the aim to identify interaction partners of EC1, a pollen cDNA library was screened using a yeast-two-hybrid approach. Two putative interactors were found: (i) a protein containing two ubiqutin-like (UBL) domains (UbDKγ3), which is probably involved in substrate delivery to the 26S proteasome and (ii) a regulatory subunit of the Phosphatase 2A (PP2A B’θ). The putative interaction with a PP2A subunit and predicted phosphorylation sites at the C-terminus of EC1 indicated that phosphorylation might play a role in EC1. The transient expression of a phospho-mimicking variant of EC1 fused to eGFP in N. benthamiana leaves was more stable, i.e. showed fluorescence, compared to the wild type form of EC1. Moreover, a proteasome inhibitor experiment with plants expressing EC1.1 fused to eGFP under control of the 35S promoter suggested that misexpressed EC1 is rapidly degraded via the ubiquitin-proteasome pathway. Based on these findings, it was hypothesized that the pollen tube delivers the regulatory subunit of PP2A, which triggers dephosphorylation of the secreted EC1 and thereby marks it for degradation. This was supported by the observation that misexpressed PP2A B’θ in the synergid cell partially phenocopied the ec1 phenotype. This work shows that EC1 is essential during double fertilization probably for gamete recognition or sperm cell separation. After fertilization and in all other cells, EC1 is unstable, its degradation is highly regulated and any protein accumulation is avoided.

Objective To observe sperm changes before and after the preparation of the density gradient centrifugation and swim-up,to confirm the role of the preferred technology in improvement the quality of the sperm,and to explore internal relations between them.Methods Semen samples were prepared by sperm prepared technology (the density gradient centrifugation and swim-up).Then early apoptosis,mitochondrial membrane potential,reactive oxygen specials were evaluated by flow cytometry analysis.Sperm parameters were examined by computer assisted sperm analysis.The nDNA integrity was evaluated by the single cell gel electrophoresis.Results Sperm motility and the percentage morphologically normal spermatozoa improved significantly after the density gradient centrifugation and swim-up compared with raw semen.MMP were significantly correlated with the percentage a sperm and VSL (r=0.641,0.708,P ＜ 0.05).ROS had the negative correlation with the percentage morphologically normal spermatozoa in non-leucocytospermia patients (r=-0.629,-0.685,P ＜ 0.05).Conclusion The method of density gradient centrifugation and swim-up was safe and effective; MMP reflect sperm motility; morphologically unnormal spermatozoa sperm is an important source of ROS in non-leucocytospermia patients. Key words: Spermatozoa; Apoptosis; Mitochondrial membrane potential; Reactive oxygen specials; Single cell gel electrophoresis

In forensic field, during investigation of sexual assault, there are many problems facing the criminal investigators to get success in obtaining significant evidence and performing DNA profile. Prostatic specific antigen detection is a good rapid test to detect seminal stains before DNA typing even in azospermic men. Aim of the Study: The aim of this work case was to highlight the importance of the prostate-specific antigen (PSA) by rapid test in the seminal stain examination before STR analysis to solve a rape case. Methods: Vaginal swabs, clothes, urine samples from a rape victim were examined by PSA strip test. DNA from all of blood samples of victim and suspects were extracted using QIAamp® DNA Mini Kit then qualification for all samples using Quantifier Duo DNA Quantification kit. PCR amplification (DNA Typing) of 15 autosomal STR markers along with amelogen was done using AmpFlSTR Identifiler™ kit. Results: Prostatic specific antigen was positive in clothes and vaginal swabs of the victim and negative in the urine samples. After getting complete DNA profiles from vaginal swabs, it completely matched with one of the two suspects. Conclusion: Prostatic specific antigen detection is rapid, easy, cheap and effective method in seminal stain identification.

A retrospective study on the transfer, persistence and recovery of sperm and epithelial cells in samples collected in sexual assault casework

Simple SummaryFor the breeding of cattle using artificial insemination, the cryopreservation of sperm cells is an essential tool. To predict the male fertility potential, it is of utmost importance to control the sperm quality after freezing–thawing, before sending the semen samples to be used at farms for inseminations. Sperm motility is a crucial parameter to keep track of and can be analyzed using the Computer-Assisted Sperm Analyzer, which can accurately and objectively address the question of sperm function with a user-friendly method at the AI station. The sperm cells can be divided into different subpopulations according to swimming pattern and velocity, like rapid progressive, rapid non-progressive and slow-motility spermatozoa. In this study, Norwegian Red bull sperm motility was studied and compared between fresh and frozen–thawed sperm cells after more in vivo-like conditions, using sperm selection via a swim-up method and incubation at body temperature over time. The difference between the bulls was mainly due to the subpopulation with a rapid progressive swimming pattern, which was also the only population significantly correlating with fresh and frozen–thawed spermatozoa from the same bull. These results indicate that rapid, progressive sperm cells are the possible indicators of bull spermatozoa functionality and freezability.Discrete subpopulations of motile sperm cells have been found for several species and are implicated to be important for sperm functionality. The aim of this present study was to examine the motile subpopulations in swim-up-selected bull spermatozoa and the relationship between subpopulations in fresh and frozen–thawed sperm cells. In experiment 1, swim-up (SWUP)-selected and non-selected (control) sperm cells were analyzed using a Computer-Assisted Sperm Analyzer (CASA). In experiment 2, the semen from nine bulls was cryopreserved and analyzed using CASA both before and after freezing and after incubation at physiological temperatures. The SWUP population had a higher proportion of total motility, progressivity, and velocity compared to the control (p < 0.05). Likewise, both incubation over time and cryopreservation affected motility and motility parameters (p < 0.05). The population of rapid progressive (RapidP) sperm cells dominated the SWUP fraction and was higher than in the control samples (p < 0.05). Furthermore, RapidP was also the main part of fresh semen, but decreased significantly over time during incubation and due to cryopreservation. In conclusion, RapidP was the main population in SWUP-selected spermatozoa and seems to be an important subpopulation contributing to the differences between treatments and in response to the freezing of sperm cells.

The Effect of Platelet Activating Factor on Motility Properties of Human Sperm Cells In Vitro

From crime scene to courtroom: A review of the current bioanalytical evidence workflows used in rape and sexual assault investigations in the United Kingdom

Poor quality ejaculates appear associated with reduced zona penetration and poor fertilization in vitro in some felid species or populations. Even morphologically normal sperm from these teratospermic ejaculates are compromised in ability to penetrate the zona pellucida and fertilize oocytes. A domestic cat model and zona piercing were used to study the function of normal and malformed sperm. Male cats naturally producing different proportions of morphologically normal sperm (group I, > 60%; group II, 45-55%; group III, < 40%) were identified. Electroejaculates were subjected to sperm washing (WS) and half of these to swim-up (SU) processing following WS, and all samples were assessed for a sperm motility index (SMI) over time. Zonae of salt-stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Control (nonmanipulated) SS oocytes (n = 430) and ZnPd-SS oocytes (n = 430) were coincubated with sperm in vitro. The proportion of morphologically normal sperm/ejaculate differed (P < 0.05) among groups averaging 68.8% for group I, 53.5% for group II, and 34.7% for group III. After SU processing, the percent normal sperm among groups was similar (P > 0.05; range, 70.8-80.2%). Average SMI for all groups was high (> 70) at 0 hour and decreased over time, but the descent of slope was greater (P < 0.05) for group III than groups I and II. The proportion of oocytes penetrated was not influenced (P > 0.05) by sperm-processing treatment. For all groups, inner zona penetration was greater (P < 0.05) in ZnPd-SS than SS oocytes (group I, 52.3% vs. 34.9%; group II, 37.9% vs. 20.3%; group III, 32.0% vs. 17.2%).(ABSTRACT TRUNCATED AT 250 WORDS)