The study was prompted by a real forensic case that illustrates the problem vividly. An eight-year-old male victim reported that anal penetration had occurred, and intimate swabs were sent for forensic testing. Short Tandem Repeat analysis, the standard technique for matching DNA to individuals, revealed a mixture containing an unknown major component that matched the suspect’s profile. Yet tests for semen, saliva and blood all returned negative results. Skin contact was considered an unlikely explanation for the high amount of DNA detected, and the investigators suggested pre-ejaculate as a potential source. Without a validated method to confirm the presence of this fluid, however, the source of the DNA could not be determined, and the DNA report alone could not prove that sexual activity had occurred. The detection of pre-ejaculate, the authors note, could have substantially contributed to reconstructing the course of events.

Pre-ejaculate, colloquially known as pre-cum, is produced by the bulbourethral glands, also called Cowper’s glands, which sit below the prostate within the male pelvic floor. The fluid is released during sexual arousal and serves as a natural lubricant, while also preparing the urethra for ejaculation by creating a sperm-friendly pH environment. Its composition is known to be heterogeneous, containing glucose, leukocytes, occasional epithelial cells, enzymes and glycoproteins. Importantly, the volume of secretion varies enormously between individuals, with some men producing little to none and others producing comparatively large amounts. The fluid is not only secreted before ejaculation but also during the ejaculatory phase itself, which means that pre-ejaculate and semen can become mixed in a single sample, complicating any attempt to attribute findings to one fluid or the other.

One of the most contested questions in the literature concerns whether pre-ejaculate contains spermatozoa. Previous studies have reached conflicting conclusions: some have reported the presence of sperm in collected samples, while others have found none. The German team addressed this uncertainty directly by screening every sample microscopically before analysis. Ninety-four male donors aged 20 to 59 collected samples at home using Copan FLOQSwabs, which were transported in thermal envelopes and frozen at minus 20 degrees Celsius. Using the Sperm HY-LITER kit, a human-specific monoclonal antibody-based fluorescence staining method that visualises sperm heads, the researchers examined each sample under a Zeiss Axioscope 5 fluorescence microscope. Spermatozoa were detected in 35 of the 94 samples, which were excluded from further analysis, even when only a single sperm cell was observed. The remaining 58 samples, genuinely sperm-free, formed the basis of the study.

The exclusion of nearly half the samples is itself revealing. The authors attribute the presence of sperm in so many collected specimens to contamination, most likely residual semen left in the urethra from a prior ejaculation that was washed out by the pre-ejaculate. This finding is consistent with earlier studies that reported divergent results on sperm content, and it underscores the practical difficulties of collecting pre-ejaculate without admixture. The researchers also acknowledge a residual limitation: ejaculate from azoospermic donors, who produce no detectable sperm, could theoretically have been misclassified as pre-ejaculate. This risk was reduced, though not eliminated, by recruiting donors of reproductive age and providing written instructions to collect the fluid early enough to avoid mixing with ejaculate.

With the sperm-free samples secured, the team tested how three standard forensic assays for seminal fluid reacted to pre-ejaculate, and the results were strikingly inconsistent. The RSID-Semen test, which detects semenogelin, a protein specific to seminal fluid, returned negative in all 58 samples. The Seratec PSA-Semiquant test, which detects prostate-specific antigen, was positive in 84.5 percent of samples. The Phosphatesmo KM test for acid phosphatase produced a substantial proportion of ambiguous results, with 31.0 percent of samples that could not be clearly classified; of the remainder, 29.3 percent were positive and 39.7 percent negative. The pattern suggests that low quantities of PSA and acid phosphatase may be present in pre-ejaculate, producing positive results in some samples but not others, while semenogelin, being specific to seminal fluid, is absent altogether.

These divergent test results carry serious implications for forensic practice. Because PSA and acid phosphatase assays are commonly used as presumptive tests for semen, a positive result on such a test could in principle originate from pre-ejaculate even in the complete absence of semen or seminal fluid. Conversely, a negative result, for example on a semenogelin assay, might lead investigators to exclude a sample from further DNA analysis, with the risk of missing crucial case information. The authors emphasise that this inconsistency reflects the poorly understood molecular composition of pre-ejaculate and complicates forensic interpretation in casework. Their findings align with a 2021 review by Kelly and colleagues, which first highlighted the forensic relevance of pre-ejaculate, and with prior reports that semenogelin assays yield negative results for pre-ejaculate while PSA and acid phosphatase may be present.

The DNA analysis delivered the study’s headline finding. Yields reached up to 436 nanograms, an amount far exceeding what is typically needed for genetic profiling. From 49 of the 58 samples, full STR profiles could be generated; these samples contained a mean total DNA amount of 23.616 nanograms and a median of 2.768 nanograms. Two further samples produced partial profiles, with ten fully typed STR markers and possible allelic dropouts in the remaining six systems, from DNA quantities of just 0.068 and 0.044 nanograms; despite the dropouts, both profiles would meet the criteria for submission to the national DNA database. No profile could be generated from seven samples, which had a mean DNA yield of only 0.027 nanograms. The DNA quantities observed in the study would comfortably explain the trace amounts found in the rape case that inspired the investigation, where stains in the anal canal and perianal area contained 1.272 and 6.712 nanograms respectively.

The authors are candid about the limitations of their methodology. Because donors collected samples unsupervised at home, neither the volume of fluid nor the swab handling could be standardised, and this variability likely contributed to the wide spread in DNA concentrations. A single swab was used across all analyses to rule out sperm contamination as far as possible, and DNA was extracted from the sample pad of the immunochromatographic test cassette rather than directly from the swabs, meaning only a fraction of the available material was utilised. Direct smearing of swabs onto microscope slides and filter papers, done to minimise sample loss, raises the possibility that insufficient transfer in individual samples produced false-negative results. Positive semen and negative water controls were run with each batch to monitor detection reliability, but they cannot confirm adequate material transfer in every sample.

Perhaps the deepest open question concerns where the DNA in pre-ejaculate actually comes from. Microscopic examination found isolated cells or cell-like structures in 23 of the sperm-free samples, while in the remaining 35 the findings were inconclusive. Some samples showed no detectable cells at all despite containing enough DNA for full STR typing, leaving the cellular origin of the genetic material unresolved. It remains unclear whether the DNA derives from urethral epithelial cells or from cell-free DNA, and the cellular composition of the fluid appears highly variable and poorly characterised. No validated method currently exists for the specific detection of pre-ejaculate as a distinct body fluid. The authors argue that further research is essential to characterise it reliably, both to confirm its presence at crime scenes and to differentiate it from semen. As they conclude, pre-ejaculate appears to be a key DNA source in sexual offences, and its current invisibility to routine forensic testing means that evidence central to reconstructing events may be going unnoticed.

Subject of Research: Forensic characterization of pre-ejaculate as a DNA source in sexual assault investigations

Article Title: Pre-ejaculate – an overlooked body fluid with high amounts of DNA

Article References: Lienesch, M., Sibbing, U., Schürenkamp, M., Vennemann, M., & Bauer, H. (2026). Pre-ejaculate – an overlooked body fluid with high amounts of DNA. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-04003-9

Image Credits: AI Generated

DOI: 10.1007/s00414-026-04003-9

Keywords: pre-ejaculate, forensic science, DNA, STR profiling, bulbourethral glands, semen detection, sexual assault, body fluid identification, prostate-specific antigen, acid phosphatase, semenogelin, International Journal of Legal Medicine