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

The findings arrive at a moment when the scientific community is increasingly aware that polycystic ovary syndrome, or PCOS, is not a single disease but a spectrum of overlapping phenotypes. Affecting an estimated five to ten percent of women of reproductive age, the syndrome is defined by the 2003 Rotterdam ESHRE/ASRM criteria, which require two of three features: oligo-ovulation or anovulation, clinical or biochemical hyperandrogenism, and polycystic ovarian morphology on ultrasound. That broad definition captures women whose experiences of the disorder can differ dramatically. Some struggle primarily with irregular cycles and fertility; others are marked by the visible stigmata of androgen excess, including coarse terminal hair growth on the face and body, a condition known as hirsutism, along with acne and androgenic alopecia. Understanding which biological signals distinguish these phenotypes has become a central goal of reproductive endocrinology.

The rationale for studying PSA in this context rests on a surprising body of evidence accumulated over nearly three decades. Although PSA, encoded by the KLK3 gene, is produced abundantly by prostatic epithelial cells, immunoreactive PSA has also been detected in female tissues, including the breast, the salivary glands, and the endometrium, and measurable amounts circulate in the blood of healthy women at concentrations far below those seen in men. Crucially, early work by Melegos, Diamandis and colleagues in 1997 demonstrated that female serum PSA rises in conditions of androgen excess, and subsequent studies showed that antiandrogen drugs lower PSA levels in hirsute women, providing pharmacological evidence that the protein functions as a downstream readout of androgen action in female tissues. In other words, PSA in women is not a prostate marker at all; it is a marker of what androgens are actually doing to target tissues.

This biological logic made PSA an attractive candidate for the Turkish research team, led by Seyma Donmez Sahip and Batuhan Turgay of the University of Health Sciences at Ankara Bilkent City Hospital, with Turgay also affiliated with Ankara University’s Reproductive Health center. Their hypothesis was straightforward: if PSA reflects androgen activity, and if hirsute women with PCOS experience more intense androgenic stimulation of hair follicles and sebaceous glands than their non-hirsute counterparts, then hirsute patients should display higher serum concentrations of both total and free PSA. To test this, the investigators enrolled ninety women diagnosed with PCOS by Rotterdam criteria between May 2022 and January 2023, stratifying them into hirsute and non-hirsute groups using the modified Ferriman-Gallwey score, the standard clinical instrument for visually grading terminal hair growth across nine androgen-sensitive body areas.

The methodology reflects careful attention to the confounders that plague hormonal research. Venous blood samples were drawn on cycle days two or three, when baseline reproductive hormones are most reliably interpreted, and the panel included total and free PSA alongside total and free testosterone, DHEA-S, luteinizing hormone, and metabolic parameters. Insulin resistance was quantified using the homeostatic model assessment, HOMA-IR, a calculation derived from fasting glucose and insulin that estimates how hard the pancreas must work to maintain normal blood sugar. This detail matters because insulin resistance is not merely a metabolic side effect of PCOS; it is widely regarded as a central driver of the syndrome, amplifying ovarian and adrenal androgen production while suppressing hepatic synthesis of sex hormone-binding globulin, the carrier protein that keeps circulating testosterone biologically inert.

When the two phenotypic groups were compared directly, the results partially confirmed expectations. Hirsute women had significantly higher body mass index, with a p-value of 0.044, higher acne prevalence at p equal to 0.032, and higher free testosterone at p equal to 0.014, painting a coherent picture of a phenotype in which androgenic and metabolic disturbances cluster together. Interestingly, menstrual disturbance ran in the opposite direction: amenorrhea and oligomenorrhea were more frequent among non-hirsute women, a reminder that the severity of ovulatory dysfunction and the intensity of cutaneous androgen effects do not march in lockstep. Ovarian morphology and alopecia showed no significant differences between the groups, and, contrary to the primary hypothesis, neither total nor free PSA concentrations differed significantly between hirsute and non-hirsute patients, with all such comparisons yielding p-values above 0.05.

But the story changed when the researchers abandoned group comparisons in favor of correlation analysis across the whole cohort. Using Spearman rank correlation, a non-parametric method appropriate for biological variables that do not follow normal distributions, the team uncovered strikingly strong positive associations. PSA correlated with the Ferriman-Gallwey hirsutism score at a rank correlation coefficient of 0.896, with total testosterone at 0.779, with HOMA-IR at 0.838, with DHEA-S at 0.807, and with luteinizing hormone at 0.804, all with p-values below 0.001. In clinical research, correlation coefficients of this magnitude are rare; values approaching 0.9 indicate that two variables rise and fall together almost in lockstep across the study population. The implication is that PSA behaves as a continuous integrator of androgenic and metabolic load, even if the binary split into hirsute and non-hirsute categories is too coarse to reveal differences in group means.

This distinction between categorical and continuous analysis carries real methodological weight. Phenotypic classification by Ferriman-Gallwey score necessarily dichotomizes a graded biological variable, and thresholds for hirsutism vary by ethnicity and remain imperfectly standardized. A molecule whose expression is driven by tissue-level androgen action may respond smoothly to the full range of androgen exposure, so that averaging PSA values across a threshold-defined group obscures the very dose-response relationship that correlation analysis exposes. The strong PSA-HOMA-IR correlation adds a further layer, suggesting that PSA may capture the intertwined androgen-insulin axis that many investigators consider the engine of PCOS pathophysiology, in which hyperinsulinemia potentiates luteinizing hormone-driven ovarian theca cell androgen synthesis and reduces binding-protein-mediated sequestration of free testosterone.

The new findings also sit within a contested literature. Prior studies of PSA in PCOS have produced mixed results: some reported elevated PSA in affected women compared with controls, a 2019 systematic review and meta-analysis in Hormone and Metabolic Research found overall differences supporting PSA as a potential androgen-excess marker, and work in adolescent girls explored its diagnostic value, while other investigations failed to replicate clear group-level differences. The Ankara study’s design differs from much of this literature in that it compared PSA across PCOS phenotypes rather than between PCOS and healthy controls, asking a subtler question about within-syndrome heterogeneity. The answer, that PSA does not separate hirsute from non-hirsute patients but correlates powerfully with hirsutism severity, androgen concentrations, and insulin resistance, suggests that PSA’s utility may lie not as a diagnostic classifier but as a quantitative index of androgenic-metabolic activity.

For patients and clinicians, the practical implications remain preliminary. PSA is not proposed as a replacement for testosterone measurement or for careful clinical assessment of hirsutism, acne, and cycle regularity, and the study’s authors emphasize that their results underscore the heterogeneity of PCOS and the need for comprehensive phenotypic evaluation rather than a single biomarker shortcut. Yet the research adds to a growing recognition that molecules once considered sexually dimorphic in absolute terms may carry meaningful physiological information in both sexes, and that the androgen signaling pathway, so central to PCOS, leaves measurable fingerprints throughout the body. If future longitudinal studies confirm that serum PSA tracks with long-term metabolic risk in women with PCOS, a cheap and widely available immunoassay, already deployed in laboratories worldwide, could find an unexpected second career in women’s health, transforming a prostate cancer biomarker into a lens on the hormonal and metabolic complexity of one of medicine’s most heterogeneous syndromes.

Subject of Research: Serum prostate-specific antigen as a marker of androgenic and metabolic activity in hirsute and non-hirsute phenotypes of polycystic ovary syndrome

Article Title: Serum Prostate-Specific Antigen Concentrations in Polycystic Ovary Syndrome: A Comparative Analysis of Hirsute and Non-Hirsute Phenotypes

Article References: Sahıp, S. D., Eyol, M. M., Ersoy, E., Erden Deveci, E., Bahadır, I. B., & Turgay, B. (2026). Serum Prostate-Specific Antigen Concentrations in Polycystic Ovary Syndrome: A Comparative Analysis of Hirsute and Non-Hirsute Phenotypes. Reproductive Sciences. https://doi.org/10.1007/s43032-026-02187-4

Image Credits: AI Generated

DOI: 10.1007/s43032-026-02187-4

Keywords: polycystic ovary syndrome, prostate-specific antigen, hirsutism, hyperandrogenism, PCOS phenotypes, insulin resistance, free testosterone, biomarkers, reproductive endocrinology, Ferriman-Gallwey score, HOMA-IR, Rotterdam criteria

The study, published in Holistic Integrative Oncology, drew on five cycles of the National Health and Nutrition Examination Survey, or NHANES, the rolling federal program that collects standardized laboratory measurements from a cross-section of the civilian, noninstitutionalized US population. The researchers assembled a final analytic population of 7,174 men with concurrent measurements of serum cotinine, total PSA, and free PSA. The free-to-total PSA ratio, a value clinicians use to sharpen the interpretation of borderline PSA results, was calculated directly from the two measured fractions. Cotinine, measured with isotope dilution high-performance liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry, served as the objective window onto recent nicotine exposure — capturing not only active smoking but also secondhand smoke and other nicotine sources that self-reported questionnaires routinely miss or misclassify.

What the analysis found was a story of two biomarkers going their separate ways. In fully adjusted multivariable linear regression models that accounted for age, race and ethnicity, body mass index, education, marital status, estimated kidney function, alcohol use, family income, and survey cycle, serum cotinine showed essentially no independent association with total PSA — the regression coefficient hovered at effectively zero, with a P value of 0.801. Yet the same exposure variable was consistently and significantly associated with a lower free-to-total PSA ratio: each additional nanogram per milliliter of cotinine corresponded to a decrease of roughly 0.005 percentage points in the ratio (95% confidence interval -0.00705 to -0.00283; P = 0.000005). The signal survived every tier of statistical adjustment, growing rather than shrinking as confounders were layered in, and the direction remained consistent when cotinine was log-transformed to temper the influence of heavy smokers.

The discordance between the two endpoints is what makes the result scientifically interesting. Total PSA reflects the overall circulating concentration of a kallikrein-related serine protease secreted by the androgen-responsive prostate gland, a concentration that climbs with benign prostatic hyperplasia, prostatitis, and prostate cancer alike. The free-to-total ratio, by contrast, depends not on how much PSA is in the blood but on how it is distributed between molecular forms — free PSA floating unbound versus PSA complexed with proteins such as alpha-1-antichymotrypsin. Because the two endpoints track different aspects of PSA chemistry, a compound that shifts the composition of circulating PSA without changing its total concentration would produce exactly the pattern observed: a null association with total PSA and a robust inverse association with the ratio.

Recognizing that a single cotinine measurement captures only a narrow biological window — days, not decades — the team added a second exposure dimension. From self-reported cigarette histories, they calculated pack-years, the classic epidemiological measure of cumulative smoking burden, for 6,132 of the participants, 3,433 of whom were current or former smokers. Correlation analyses confirmed that cotinine and pack-years point in the same direction but are far from interchangeable: the Pearson correlation between raw values was a modest 0.209, the Spearman rank correlation 0.270, and only after log-transforming both variables did the relationship strengthen to 0.337. A heavy lifetime smoker who quit years ago can carry low cotinine today; a recent relapser can carry high cotinine on a thin lifetime foundation. The two metrics measure genuinely different things.

That difference mattered in the sensitivity models. When pack-years replaced cotinine as the exposure variable in fully adjusted regressions, the association with PSA ratio vanished entirely (β = -0.00750, 95% CI -0.02010 to 0.00509; P = 0.243), and the log-transformed version of pack-years fared no better. The cotinine signal, in other words, cannot be explained away as a shadow of lifetime cigarette burden. One provocative secondary pattern emerged for total PSA: log-transformed pack-years showed a statistically significant inverse association in sensitivity analyses, hinting that decades of cumulative exposure may relate to total PSA through chronic tissue, vascular, or endocrine remodeling in ways that an acute biomarker cannot capture. The authors are careful to stress that nothing in these data should be read as evidence that smoking protects the prostate — tobacco exposure has established harmful effects across organ systems and is associated with worse outcomes after prostate cancer diagnosis, including higher mortality and progression risk.

What might cotinine actually be doing to PSA chemistry? The mechanistic clues point toward nicotinic acetylcholine receptor signaling and androgen biology. Cotinine, which persists in serum far longer than nicotine, binds cell-surface cholinergic alpha5 nicotinic receptors, which experimental studies have shown are upregulated in prostate cancer and drive tumor cell proliferation and invasion. Experimental work also suggests cotinine can interact with the androgen receptor and suppress its expression in animal prostate tissue. Because PSA secretion is androgen-regulated, cotinine-related modulation of androgen receptor signaling could preferentially reduce the epithelial secretion of free PSA, shifting a greater share of immunoreactive PSA toward the alpha-1-antichymotrypsin-complexed fraction — lowering the ratio while leaving total PSA nearly untouched. Tobacco-driven systemic inflammation could contribute as well by altering the acute-phase protein milieu in which PSA complexes circulate. These mechanisms remain inferential in a cross-sectional dataset, but they explain the observed geometry of the results with unusual precision.

The interpretation demands caution at every turn. NHANES participants were not enrolled because of suspected prostate disease, and no biopsy adjudication exists for this cohort, so the findings speak to biomarker-level variation in a general population, not to cancer incidence, diagnostic thresholds, or clinical decision-making. Serum cotinine is also shaped by CYP2A6-mediated metabolism, which varies across racial and genetic backgrounds, meaning identical cotinine values may reflect different actual nicotine intake. The authors noted that the cotinine-PSA ratio association appeared more pronounced in certain subgroups, including non-Hispanic White and non-Hispanic Black participants, men aged 40 to 49 and 70 to 79, and those with a body mass index above 18.5, but stratified patterns in cross-sectional data carry their own fragility. Smokers may also differ systematically in screening behavior, comorbidity, and healthcare access, and despite the standardized survey protocol, differential participation and missing laboratory data cannot be excluded as sources of selection.

Within those constraints, the study stakes out a genuinely novel position: it is, according to the authors, the first population-based analysis to link cotinine to prostate-related endpoints in a nationally representative sample. Its strength lies in the objective laboratory measurement of exposure, the large sample, and the statistical persistence of the PSA ratio association after extensive adjustment. Its limitation is time — a single cotinine measurement, a single PSA snapshot, and no way to order cause and effect. The authors call for longitudinal studies with repeated cotinine measurements, validated cumulative smoking metrics, nicotine metabolite profiling that includes trans-3′-hydroxycotinine, and adjudicated prostate disease outcomes to determine whether recent exposure status, cotinine metabolism, or something else entirely drives the association.

For clinicians and epidemiologists alike, the practical message is narrower than the biological one. Nothing in this analysis justifies changing PSA screening practice or interpreting an individual patient’s PSA ratio through the lens of a cotinine blood level. But the results do suggest that tobacco-related biological status is a real and quantifiable source of variation in one of the most widely used prostate biomarkers in medicine — a reminder that the numbers generated by screening assays are not static properties of a gland but dynamic readouts that absorb the chemical history of the person they come from. As molecular epidemiology continues to separate the recent exposure a biomarker captures from the cumulative burden a questionnaire recalls, studies like this one map the fault lines where those two measures diverge, and where the biology of tobacco meets the biochemistry of cancer screening.

Subject of Research: The association between serum cotinine, a biomarker of tobacco exposure, and prostate-related clinical endpoints including PSA measures

Article Title: Association between serum cotinine levels and prostate-related clinical endpoints

Article References: Wang, Z., Ge, Q., Anwaier, A., Xu, W., & Ye, D. (2026). Association between serum cotinine levels and prostate-related clinical endpoints. Holistic Integrative Oncology, 5(1), Article 76. https://doi.org/10.1007/s44178-026-00292-7

Image Credits: AI Generated

DOI: 10.1007/s44178-026-00292-7

Keywords: serum cotinine, prostate-specific antigen, free-to-total PSA ratio, tobacco exposure, NHANES, prostate cancer, pack-years, nicotine metabolism, prostate biomarkers, epidemiology, urology, cancer screening