The significance of prostate-specific antigen in forensic examinations of sexual violence: A case report

Introduction

In cases of sexual assault where the suspects are believed to be male, it is important to forensically identify sperm as a source on the victim's body to confirm male sexual contact. ¹ Semen is the ejaculate produced by the male reproductive system, containing spermatozoa (sperm cells) and seminal fluid. It is a complex mixture that facilitates reproduction by transporting and protecting sperm. Seminal fluid constitutes over 95% of human semen, while testicular secretions, including spermatozoa, compose about 5%. Semen also includes other cellular components, such as immature germ cells and immune cells. Seminal fluid specifically refers to the liquid portion of semen. It is produced by accessory reproductive glands: the seminal vesicles contribute 65–75% of the volume, rich in fructose, prostaglandins, and fibrinogen; the prostate gland provides 20–30%, containing enzymes, prostate-specific antigens (PSA), and minerals; and the bulbourethral glands contribute a small amount (∼1%), mainly a mucus-like secretion.^2–6 The most recognized and reliable method of semen identification is the microscopic detection of spermatozoa. However, microscopic examinations are time-consuming. PSA tests are used in forensic science to perform rapid semen screening on vaginal swab samples from suspected victims of sexual assault. PSA is one of the constituent proteins of prostatic fluid, and its function is to liquefy semen. ¹ According to various studies, PSA is reliably detected in vaginal samples up to 48 h after unprotected sex, but the sensitivity of the test decreases significantly after 24 h.^7–9 PSA can be useful in examining cases where the perpetrator is oligospermic, azoospermic, or has undergone a vasectomy.^10,11 In some sexual assault cases, clothing or bedding is key evidence. Semen stains on the clothes or bedding of sexual assault victims can remain as evidence even after being washed. The PSA test is also an effective method for screening for sperm stains on fabric, even after washing.^12,13

Methods and materials

Impersonalized criminal case materials obtained from the publicly accessible Lithuanian court information system (LITEKO) and materials from the forensic medical examination conducted by the State Forensic Medicine Service (SFMS). All procedures were performed in compliance with the relevant laws and institutional guidelines, and the Vilnius Regional Biomedical Research Ethics Committee approved them (approval number 2024/2-1566-1029). A literature search was conducted using the PubMed and Google Scholar databases. After excluding unsuitable sources, 38 relevant scientific articles were selected and analyzed.

Case report

A pre-trial investigation was launched following a complaint by an 85-year-old woman, who alleged that she had been raped by her 63-year-old son-in-law with whom she lives. A possible piece of physical evidence—the woman's panties—was collected at the scene of the incident. A gynecological examination of the victim was conducted approximately 8.5 h after the alleged incident. During the examination, it was determined: the vaginal mucosa is pink, without lesions, and there is no vaginal discharge. Swabs were taken from the affected woman's vagina. The gynecological history was not collected because the victim was inadequate, and contact with her was difficult. Subsequently, a specialist report from the serology and DNA laboratory was submitted: traces of seminal fluid were found in the vaginal swab (following a PSA test), and traces of seminal fluid, along with the victim's DNA and a partial DNA profile of the suspect, were found on the victim's underwear. No spermatozoa were detected in the samples. The suspect was not examined, and no penile swabs were taken.

The first-instance court (district court) found the man guilty and sentenced him to 3 years and 6 months of imprisonment for rape. The district court based its verdict on the findings of the forensic medical expert: the victim may have been sexually assaulted, as traces of seminal fluid were found in her vagina, and the defendant's partial DNA was found on her underwear.

The convicted person has submitted an appeal to the second-instance court (appeal instance, regional court). During the appellate court proceedings, repeated and additional explanations of the experts’ and specialists’ conclusions were heard. In the conclusions of another forensic medical expert, as well as a specialist oncologist, it was indicated that the PSA test does not confirm the presence of seminal fluid, as PSA can also be produced by a woman's body. The victim's medical history indicates that her body may have produced PSA due to her suffering from hirsutism and having cysts in her breasts, which markedly increase PSA production. The conclusions of the specialist geneticist and the specialist immunologist indicate that PSA is only a protein and that it is not possible to determine DNA from it. The immunology specialist explained in the second-instance court that PSA and seminal fluid are not synonyms, that DNA is obtained from a cell with a nucleus, and that PSA is only a protein that is completely identical in both men and women. Additionally, a genetics specialist stated that the DNA of a cohabiting person can transfer onto another person's body or clothing and can remain even after washing. No spermatozoa were found during the forensic examinations. It was unclear from which biological material the partial DNA profile of the accused was identified on the victim's underwear. DNA cannot be determined from PSA, so this could have been biological contamination from living together—such as washing clothes together, touching, sneezing, etc. There were no changes in the victim's genital organs. From the victim's medical history, it is known that the woman suffers from Alzheimer's disease and senile dementia. According to testimonies from witnesses who knew the woman, she had previously expressed sexual fantasies about engaging in sexual relations. Additionally, they noted that she was not mentally healthy.

The regional court annulled the verdict of the district court, arguing as follows: the mental state of the victim indicates a clear unreliability of her testimony, and there are no other objective and indisputable pieces of evidence, as the alleged traces of seminal fluid could, in fact, have been a PSA produced by the victim. The version that the partial DNA of the accused was found on the victim's underwear while they were living together and leading a shared household cannot be dismissed. In the appellate court, the specialist who provided conclusions to the first-instance court was questioned again, and she believed that the PSA test only reacts to male antigens. Additionally, the specialist had not encountered cases where a woman's body would produce PSA. The victim died during the pre-trial investigation, so there were no objective possibilities to verify whether her body actually produced PSA. All this data allowed the court to draw a strong conclusion that the charges were unproven, and the conviction that was made was unfounded, therefore it was annulled.

Discussion

Although PSA is produced in the prostate and secreted into seminal fluid, it is not exclusive to the prostate.^1,14 The periurethral glands in women (Skene's glands) are referred to as the “female prostate.” ¹⁴ Studies have shown that in 45–80% of cases, PSA testing of female periurethral secretions was positive.^1,14 PSA can be detected in women's bodies in various pathological conditions (breast cancer, ovarian cancer, hirsutism, polycystic ovary syndrome, breast cysts, fibroadenomas).^1,15 When a woman is exposed to androgenic steroids (endogenous or exogenous), androgens stimulate androgen receptor-positive cells to produce and secrete PSA.^15,16 Female breast tissue can also produce PSA and release it into the systemic circulation. ¹⁵ Measurable amounts of PSA may be excreted in breast milk in women taking progestin oral contraceptives. ¹ Elevated PSA levels are also observed in cases of breast cancer and benign breast diseases (breast cysts, fibroadenomas). In cases of breast cysts and fibroadenomas, serum PSA levels can increase by 1000 times and may reach or even exceed the PSA concentration level of healthy men.^15,17 In our case, the victim's medical history included hirsutism and breast cysts, which may have contributed to the elevated PSA levels in the woman's body. The victim had died during the pre-trial investigation, so there was no opportunity to verify whether her body could actually produce PSA. The autopsy was not performed. In Lithuania, an autopsy is performed based on a decision by competent authorities or institutions, or at the request of the deceased person's relatives. In this case, no such decision was made, and no request from the relatives was received.

Older women are more likely to have a positive PSA test. ¹ The false-positive rate for PSA was 94.4% in vaginal swabs from women over 60 years of age. The proportion of false-positive results increased with age. False-positive specimens are defined as those with positive PSA test results but negative microscopy and police examination results. This occurs because after menopause the ratio of estrogen to androgens decreases, which stimulates PSA production. These results indicate that PSA tests are relatively unreliable and misleading, especially when obtained from vaginal swab samples of older women. ¹ In our case, the victim was 85 years old, which may also have been an important factor contributing to the woman's increased PSA production. Detectable PSA has been reported in women who claimed no sexual intercourse or only condom-protected intercourse, with detection rates ranging from 10% to 48% depending on the study population. ⁸ Notably, PSA is also produced by women's periurethral glands, particularly in those using contraceptives, making it possible to test PSA-positive even without recent intercourse. ¹⁸ Furthermore, studies have shown that lubricated condoms can produce false-positive PSA results due to interactions between latex and substances used in the lubrication. ¹⁹ This highlights the need for caution when interpreting PSA results in isolation, especially in the absence of supporting evidence such as the presence of sperm.

The most reliable and widely used method for detecting semen is microscopic identification of sperm cells. A fluorescence microscopy technique using human sperm-specific antibodies (e.g. SPERM HY-LITER™) with laser capture microdissection is much more specific to human sperm than traditional semen staining methods. Perhaps the most useful feature of this test is its ability to stain sperm cells that are completely obscured by other non-target cells.^18,20–22 The DNA analysis of various body fluid stains at crime scenes facilitates the identification of individuals but does not allow for determining the biological material from which the DNA was obtained. Meanwhile, conventional serological or immunological testing methods used to identify body fluids, which are based on detecting protein markers, cannot identify individuals. Even if forensic stain test is positive for a specific body fluid, the DNA profile obtained may come from another body fluid or tissue source. The method of epigenetic DNA methylation analysis reveals that chromosomal regions known as tDMRs (tissue-specific differentially methylated regions) exhibit different DNA methylation profiles across various tissues and body fluids. This method can improve forensic investigations by enabling not only the identification of individuals but also the determination of the biological source of the DNA. This analysis is particularly useful for distinguishing semen from other body fluids, as well as for analyzing body fluid mixtures (e.g. semen and vaginal fluid).^23–25

Textiles are key evidence in some sexual assault cases. In some cases, clothing or bedding may have been washed before the investigations. Sperm stains that have been washed more than once can still be positive for PSA. After one wash, the PSA test was positive in 92% of cases, and after four washes, the PSA remained positive in 45% of sperm spots. ¹³ The best PSA test results were obtained with cotton fabrics. The proportion of positive PSA results decreased with increasing washing temperature for all fabrics, except cotton. ²⁶ PSA and DNA can remain on textiles after washing.^13,27,28 However, it should be noted that DNA from a regular user of a washing machine may also be transferred to unsoiled clothes. ²⁸ In a study conducted by other scientists, 98% of once-washed stains yielded informative DNA profiles corresponding to semen donors. After performing several washing procedures on the same fabrics, the DNA quantities decreased, but full DNA profiles could still be obtained. ²⁹ In another study, it was found that during laundering, the transfer of DNA from both spermatozoa and vaginal secretions onto unstained clothing can be sufficient to produce interpretable genetic profiles. Furthermore, in families where sexual abuse had never occurred, DNA from relatives living in the same household was detected on children's underwear. In all 144 swabs collected using tampons, no DNA meeting the amplification threshold was detected, whereas most fabric cuttings yielded interpretable DNA results. This strongly indicates that swabbing is ineffective at collecting spermatozoa and epithelial DNA transferred during washing. Instead, spermatozoa transferred during laundering were likely absorbed or trapped deeper into the fabrics of unstained clothing rather than settling on their surface. Swab samples yielding high quantities of DNA in the sperm fraction would suggest that sperm was directly deposited onto the child's underwear. Additionally, spermatozoa DNA was found on the inner drum of the washing machine. ³⁰ In our case, both the DNA profile of the male and the DNA profile of the female were detected in the examined area of the panties. The victim shared a household with the suspect, so the woman's DNA could have been present on the panties while wearing them, just as the man's DNA could have been present due to their cohabitation (using the same washing machine, drying, touching clothes, etc.). A partial DNA profile is established when an allele is missing in one of the loci. In cases where samples contain very low amounts of DNA, are exposed to extreme environmental conditions, or are improperly stored, it can be difficult to obtain a complete DNA profile; however, partial profiles are still useful in determining whether an individual may be associated with the investigated circumstances. ³¹

Nonmotile spermatozoa can be found in vaginal discharge stains 12 to 48 h after ejaculation. Dried discharge on clothing remains quite stable, allowing sperm to be detected for more than 1 year. ¹⁰ Our victim was examined by a gynecologist, and vaginal swabs were taken about 8.5 h after the suspected incident. However, no spermatozoa were found in either the vaginal swabs or on the victim's underwear. It is known that the PSA test can be useful in examining cases where the suspect is oligospermic, azoospermic, or has undergone a vasectomy.^11,12 In this case, azoospermia was not confirmed in the suspect. However, the detection of human DNA and the identification of a person require eukaryotic cells (spermatozoa, epithelial cells, leukocytes), while PSA is merely a protein that is not suitable for personal identification.^31–33 Therefore, in our case, the mere finding of PSA cannot serve as proof that traces of the suspect's seminal fluid were found.

In vaginal swabs, it is often difficult to detect spermatozoa due to improper sampling, the suspect having undergone a vasectomy, or the use of a condom. In sexual assault cases, tests of penile washings or swabs are useful for solving the crime. In some cases, traces of lubricant from condoms can serve as important evidence when they can be linked to similar findings on the suspect's and the victim's vaginal and penile swabs. Foreign bodies, blood, saliva, hair, and other biological materials found on a man's penis can indicate recent sexual intercourse. ³⁴ In this case, the victim's vaginal swab did not contain the suspect's DNA. However, tests for the suspect's penile washings or penile swabs were not conducted. These tests are important in sexual offense cases, as they allow for the collection of a sufficient amount of the victim's DNA within 24 h after sexual intercourse. ³⁴

Sexual violence against elderly women is an underrecognized issue, with older survivors reporting lower victimization rates compared to younger women. Contributing factors include a lack of data on violence against older women, particularly in low- and middle-income countries, and survey tools designed for younger women that fail to capture the experiences of older victims. The stereotype that older adults are “asexual” further limits adequate responses to their sexual health needs.^35–37 Sexual abuse of older people typically occurs in the victim's home or in an institutional setting where the victim lives, with the perpetrator being someone known to the victim, such as a spouse or other family member. Persons with physical and/or cognitive disabilities, in particular dementia, are at higher risk of victimization. Despite the long-term physical and psychological impacts of sexual violence, these effects on older adults are often overlooked.^36–38 In our case, despite the woman being elderly and having mental health problems, she personally approached the police.

Conclusions

The PSA test is important for the detection of seminal fluid, but it is not suitable for personal identification. When determining criminal liability, it is necessary to follow the principle of in dubio pro reo, according to which all doubts and uncertainties that cannot be eliminated must be interpreted in favor of the person being charged. In cases of sexual violence, the proper interpretation of laboratory tests is essential, taking into account all the circumstances of the incident as well as the victim's medical history, particularly in countries where the range of assays conducted is more limited. It is important to evaluate all tests performed in conjunction to ensure an accurate interpretation. A mistake could unjustly lead to the conviction of an individual.

We followed the CARE reporting guidelines in preparing the structure of this case report. ³⁹

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