Semen Research Articles

Forensic analysis of mixed semen and vaginal fluids via ATR-FTIR spectroscopy for identification purposes in sexual aggression cases

BackgroundForensic science is an interdisciplinary field that applies principles and techniques from the natural and physical sciences to matters of law and criminal justice. As an academic discipline, forensic science encompasses a broad range of scientific methods used to analyze physical evidence from crime scenes to aid in investigations, legal proceedings, and the enforcement of justice. Within this field, forensic genetics plays a pivotal role by employing genetic material obtained from biological samples at crime scenes to establish associations between suspects and criminal acts, particularly in cases involving sexual assault. Furthermore, the accurate identification of biological fluids is integral to the advancement of forensic investigations. In the context of sexual offenses, spectroscopic techniques have gained prominence for their capacity to analyze body fluids (such as semen and vaginal fluid) on substrates taken from the victims’ body or at the scene rapidly and non-destructively, without sample destruction or the need for extensive sample preparation. Among these techniques, Fourier Transform Infrared (FTIR) spectroscopy stands out due to its ability to provide rapid, cost-effective, and non-invasive analysis while preserving the integrity and quantity of the biological specimen.ResultsThe present study investigates the application of FTIR spectroscopy for the identification of seminal fluid, vaginal fluid, and their combinations on two fabric substrates: white cotton and patterned nylon. Spectral analysis enabled the identification of distinct functional groups associated with each type of biological fluid. Spectral markers specific to vaginal fluid and mixed samples were successfully distinguished.ConclusionA notable finding of the study was the identification of the PSA/Fructose functional group as a biochemical marker indicative of seminal fluid within mixed samples containing vaginal secretions. Although both seminal and vaginal fluids were found to share five common biomolecular components, the presence of a distinct PSA/Fructose peak was characteristic of seminal fluid, whereas the detection of a glycogen peak—attributed to epithelial shedding from the cervix—was indicative of vaginal fluid.This distinction facilitated the clear differentiation between seminal and vaginal constituents within mixed biological samples. These results highlight the efficacy and reliability of Fourier Transform Infrared (FTIR) spectroscopy as a forensic tool for the identification of biological fluids, particularly in the context of sexual assault investigations.

PSA is not suitable as a reliable marker for seminal fluid in rectal samples collected post-mortem

PSA is not suitable as a reliable marker for seminal fluid in rectal samples collected post-mortem

Ebola virus persistence: implications for human-to-human transmission and new outbreaks

Ebola virus (EBOV) infection usually leads to highly lethal EBOV disease (EVD) with associated viraemia. Viraemia is cleared in those that do survive, however, EBOV may remain hidden in the testes and other immune privileged niches (IPNs) where it can persist for years during asymptomatic convalescence. Viral shedding into seminal fluid may result in sexual transmission to naive contacts years after EBOV outbreaks have been declared over. This leads to flare-ups of cases, redefining our understanding of the shaping and origin of EBOV outbreaks. Such delayed sexual transmission eliminates the geographical boundaries which typically constrain EBOV outbreaks, thus posing a significant global health security threat. Despite hints of EBOV persistence dating over half a century, it was only until the unprecedented scale of the 2013–2016 Western Africa EBOV epidemic that the true importance of this phenomenon was revealed to scientists, public health officials and policy makers alike. This review summarises the evidence for EBOV persistence, suggests the possible underlying molecular mechanisms and proposes future directions for research in the field. A meta-analysis is presented to further investigate the duration of EBOV shedding in seminal fluid. The ultimate aim is to develop therapeutics that clear sites of persistence. Such therapeutics could prevent the re-emergence of the persistent virus, eliminating the chance of new outbreaks whilst alleviating the severe stigmatisation facing the EBOV survivor population.

Two neuropeptide signaling pathways regulate post-mating refractoriness and reproductive system in male crickets

After mating, some insects drastically reduce mating activity, termed post-mating refractoriness, due to physiological restrictions. Male crickets, Gryllus bimaculatus, characteristically exhibit a 1-hour post-mating refractory stage, which is controlled by terminal abdominal ganglion. The molecular mechanisms underlying the male-specific precisely timed refractory stage remain unclear. Here, we show that among 28 neuropeptide precursors expressed in the terminal abdominal ganglion, myosuppressin, allatotropin, and sNPF exhibited male-specific expression based on RT-qPCR and in situ hybridization. RNA interference experiments showed that only the knockdown of allatotropin and sNPF extended the refractoriness duration. Furthermore, allatotropin and sNPF knockdown influenced the male reproductive system by inhibiting seminal fluid secretion from the male accessory gland and decreasing spermatozoon storage in seminal vesicles, respectively. Knockdown of allatotropin and sNPF receptors caused similar phenotypes to their ligands. In conclusion, this study demonstrated the regulation of post-mating refractoriness and reproductive system by Allatotropin and sNPF signaling pathways in male crickets.

Male reproductive tract extracellular vesicles display region-specific heterogeneity in mice

In briefMale reproductive tract extracellular vesicles play a critical role in regulating sperm quality and male fertility. This study shows that extracellular vesicles from distinct regions of the male reproductive tract differ in their size, abundance and composition.As sperm transit the male reproductive tract, they undergo a series of dynamic changes, gaining motility, modifying lipid and protein content and refining their epigenetic composition. Extracellular vesicles are central to this post-testicular maturation and changes in their composition could directly impact male reproductive health, sperm quality and post-fertilisation development. This study aimed to characterise and compare extracellular vesicles isolated from distinct regions of the male reproductive tract. Extracellular vesicles were isolated from adult, male C57BL/6J cauda and caput epididymis (epididymosomes) and seminal vesicle fluid by precipitation and size exclusion chromatography. Isolated vesicles were characterised using nanoparticle tracking analysis, transmission electron microscopy, Western blotting and imaging flow cytometry. Epididymosomes and seminal fluid vesicles ranged from 110.26 to 121.26 nm in diameter, had a concentration of 109 to 1010 particles/cm3 and had a typical round, cup-shaped morphology. The size and concentration of extracellular vesicles from the caput were significantly larger than those from the cauda and seminal fluid. Imaging flow cytometry revealed that all isolated extracellular vesicles expressed CD81 and CD9 tetraspanins; however, CD63 was detected only in caput epididymosomes. Furthermore, there were significantly fewer CD9+ vesicles in seminal fluid EVs compared to epididymosomes. Using a range of bulk- and single-vesicle analytical approaches, we show that different regions of the male reproductive tract display distinct vesicle compositional phenotypes. However, additional studies are warranted to define the significance of this heterogeneity, their roles in regulating male reproductive health and the development of their offspring.

Evidence for Transcriptomic Conservation Between the Main Cells of the Drosophila Prostate-Like Accessory Gland and Basal Cells of the Mammalian Prostate.

The Drosophila accessory gland performs functions analogous to the mammalian prostate in production of seminal fluid components that are essential for male fertility. The mammalian prostate and Drosophila accessory glands share a similar tissue organization and structure. Both organs contain secretory epithelial cells forming a gland lumen, surrounded by a stroma with extracellular matrix enveloped by innervated muscle for organ contraction and fluid release. However, the Drosophila accessory gland secretory epithelium is postmitotic, polyploid and binucleate, and lacks a known stem cell population. By contrast, the mammalian prostate epithelium is made up of diploid luminal secretory cells and diploid basal cells that are maintained by at least two stem cell populations. Despite the differences in the tissues, it has been argued these tissues may share a 'deep homology' based on the expression of conserved genes during development. Here we performed a cross-species comparative analysis using single-cell RNA sequencing data from adult tissues using data from the Drosophila Fly Cell Atlas and mammalian adult prostate single-cell datasets. Our analysis provides additional evidence of transcriptomic similarity between the main epithelial cells of the Drosophila prostate-like accessory gland and the basal epithelial cells of the mammalian prostate. While we do not know whether these similarities reflect shared evolutionary homology, or independently derived features due to shared tissue functions, our results strengthen the arguments that the Drosophila accessory gland can be used to effectively model aspects of human prostate biology and disease.

A window of cell cycle plasticity enables imperfect regeneration of an adult postmitotic organ in Drosophila

The Drosophila ejaculatory duct (ED) is a secretory tissue of the male somatic reproductive system responsible for producing components of the seminal fluid which support fertility, serve antimicrobial functions and influence the physiological changes in the female after mating. The ED is a simple organ made up of secretory epithelial cells that are encased by extracellular matrix and a layer of innervated contractile muscle. These secretory cells are post-mitotic and lack known stem cells or progenitors in the adult, but they are not fully quiescent. They undergo a variant cell cycle called endoreplication immediately post-eclosion to increase organ size and protein synthesis capacity. Polyploid and post-mitotic tissues often face unique challenges in response to cell loss due to their inability to proliferate. Here, we show that the adult ED is capable of significant recovery after cell loss due to a combination of increased nuclear and cellular hypertrophy that partially restores tissue mass and organ function. The early cell cycle plasticity of this adult tissue is critical for this recovery, as older tissues that have few or no endocycles exhibit reduced capacity for recovery after cell loss. Together, our findings establish the Drosophila ED as a model to study post-mitotic polyploid tissue repair and highlight a combination of endocycles and hypertrophy as a key mechanism for functional regeneration in the absence of mitosis.

P-051 Antibodies in Seminal Fluid: A Potential Marker of Blood-Testis Barrier Integrity? A Prospective Clinical Trial

Abstract Study question Can the presence of specific acquired antibodies (Cytomegalovirus (CMV) IgG) or total IgG antibodies in seminal fluid, predict the outcomes in assisted reproductive technology (ART)? Summary answer IgG antibodies in seminal fluid did not correlate with ART outcomes, but did reflect distinct mechanisms: production within the genital tract or testicular barrier disruption. What is known already The blood-testis barrier is a crucial structure within the seminiferous tubules, maintaining an immunologically protected environment. Regulation of the constant creation and preservation of the barrier is hormonally mediated, particularly by testicular testosterone. When intact, the barrier prevents the passage of large molecules, such as IgG antibodies. However, recent studies suggest that IgG may be locally produced within the male genital tract. Study design, size, duration A prospective clinical study, conducted between March 2022 and September 2023 in a single university affiliated IVF unit. Sixty male patients were enrolled. Patients’ seminal fluid and serum samples were provided on ovum pickup (OPU) day. Participants/materials, setting, methods Participants were divided into three study groups: 24 (40%) diagnosed with male infertility, 18 (30%) with unexplained infertility, and 18 (30%) were presumed fertile (diagnosed female factor or treatment for pre gestational testing (PGT)). CMV IgG antibodies (CLIA) and total IgG levels (ELISA) were assessed in serum and seminal fluid. Patients' demographic data, including sperm parameters baseline and on the OPU day, serum testosterone levels and embryological outcomes, were recorded. Main results and the role of chance Mean male partner age was 34.5±7 and similar between study groups (P = 0.12). CMV IgG antibodies were present in 83.3% (50/60) of sera. All CMV seronegative patients were found to be negative in their seminal fluid. Out of the seropositive patients, 4 cases were excluded due to technical failure. 11/46 patients (23.9%) had detectable CMV IgG in their seminal fluid: 5 (29.4%) in male infertility patients, 5 (33.3%) in unexplained infertility patients, and 1 (7.1%) among presumed fertile men (P = 0.2). The presence of CMV IgG in seminal fluid showed no significant correlation with age, sperm concentration, fertilization rate, embryo quality, or testosterone levels (all P > 0.4). A moderate correlation was observed between CMV IgG in serum and seminal fluid (r = 0.56, P < 0.01). Total IgG levels were assessed in sera and seminal fluid from 58 patients. No significant differences in their levels were found between study groups (P > 0.5), and a weak correlation was seen between serum and seminal total IgG levels (r = 0.2, P = 0.14). Total IgG in seminal fluid did not correlate with ART outcomes, sperm motility, or testosterone levels (all P > 0.5). Limitations, reasons for caution This study was limited by its single center design and relatively small sample size, which may impact generalizability. However, our findings provide novel insights into the immunological environment of seminal fluid, particularly regarding IgG presence and testicular barrier integrity, warranting further investigation in larger studies. Wider implications of the findings The different correlations may reflect distinct mechanisms: infiltration through damaged barrier, or local production independent of serum levels. Hence, IgG presence in seminal fluid could serve as a biomarker of genital tract immune activity or barrier dysfunction. This in the future may carry diagnostic and therapeutic strategies in male infertility. Trial registration number Yes

O-280 Unveiling the hidden danger: detection and characterisation of microplastics in human follicular and seminal fluids

Abstract Study question Can we detect and characterise the presence and composition of microplastics (MPs) in human follicular (FF) and seminal fluids (SF)? Summary answer Various types of MPs were identified in both male and female reproductive systems, providing essential evidence to evaluate their potential risks to human reproductive health. What is known already Microplastics (MPs), defined as plastic particles smaller than 5 mm, have emerged as a pervasive form of pollution in consumer products and the environment. They represent a critical global environmental challenge with significant public health implications. Concerns regarding their impact on the human reproductive system have grown. However, limited data exist on the presence of MPs in human reproductive tissues and their potential effects on oocyte and sperm quality. Study design, size, duration This study evaluated the presence of MPs in the reproductive systems of donors and patients at our centre by analysing their FF and SF. FF samples were obtained from 25 women undergoing follicular aspiration, stored in sterile glass containers, and subsequently frozen. SF samples were collected from 18 men undergoing semen analysis and were similarly stored frozen in glass tubes. Participants/materials, setting, methods The detection of MPs in FF and SF was performed using direct laser infrared microscopy (LDIR). Prior to analysis, the samples were subjected to a mineralisation process with 10% (w/v) potassium hydroxide (KOH) and incubated at 40 °C for 48 hours. As a control, the containers used for sample collection and storage were analysed to confirm the absence of MP contamination. Main results and the role of chance A range of MPs was detected in both FF and SF, including polyamide (PA), polytetrafluoroethylene (PTFE), polyethylene (PE), polyurethane (PU), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS), polyvinyl chloride (PVC), and polylactic acid (PLA). The overall concentration of MPs was relatively low compared to non-plastic particles but was notably higher in FF than in SF. Specifically, over 50% of FF samples contained PA, PU, and PE, while PTFE and PET were detected in more than 30% of samples. PP, PVC, and PLA were identified in over 20%, with PS observed less frequently. In SF samples, MPs were detected in less than 30% of cases, except for PTFE, which was identified in 56% of samples. Limitations, reasons for caution The limited number of samples analysed, due to the challenges associated with obtaining them, is a key limitation. Expanding the sample size in future studies would strengthen the findings. Additionally, incorporating detailed information on the participants’ lifestyles and consumption habits would provide valuable context. Wider implications of the findings This study demonstrates the presence of MPs in both male and female reproductive systems, likely attributable to their easy entry into the body through ingestion, inhalation, and skin contact. Further research is urgently needed to elucidate the potential effects of MPs on human reproductive health and fertility. Trial registration number No

A multiomic approach for detecting clinically significant prostate cancer in seminal fluid: Results from a large multicenter clinical study.

e17135 Background: While Prostate Specific Antigen (PSA) is the standard of care for prostate cancer screening, it is highly controversial due to the performance characteristics of the test. Magnetic Resonance Imaging (MRI) is becoming increasingly used as a reflex test for high PSA, but there are limitations with cost, disparities in access, variations in interpretation, and moderate sensitivities/specificities. Given that the prostate is a reproductive organ and a large portion of seminal emissions are generated from the prostate, this study aimed to investigate whether a novel prostate cancer screening platform using cell free nucleic acids from seminal plasma could improve the detection of clinically significant prostate cancer. Methods: Over a 4-month period, 276 patients scheduled to undergo a prostate biopsy were enrolled across 12 sites. Semen samples were collected at home by men aged 40 yrs and older and shipped to the Fellow Health laboratory. Seminal plasma was prepared by centrifugation and cell free nucleic acids extracted. DNA methylation libraries were prepared using the Twist methylome target enrichment panel and RNA libraries were prepared using Watchmaker kits. All libraries were sequenced on a NovaSeq X. Clinical data (including PI-RAD score, cancer status and Grade Group (GG)) were merged with the sequencing results and machine learning tools employed for modeling predictive algorithms. Results: Median age was 64 (IQR=10 years), median PSA was 6.1 (IQR=3.925ng/ml), 79% of subjects were white, 45% had BPH, 12.5% were vasectomy positive and 66% received MRI prior to biopsy. 180 valid semen samples were received prior to the cutoff date, of which 146 were evaluable for both RNA and DNA analysis. The well-known prostate tissue markers PSA and TMPRSS2 were highly abundant in seminal fluid with mean normalized transcripts per million of 1597 and 397.5 respectively. Using healthy controls to set the limit of blank, 45.1% of cancer patients harbored an ETS fusion (ERG, ETV1, ETV4, ETV5, FLI1). The data was split into a training set (n=115) and an independent test set (n=31). With a panel of 66 expression markers, 26 methylation markers and PSA, the AUC in the test set was 0.83 for differentiating clinically significant prostate cancer (GG2+) from no cancer (<=GG1). Conclusions: In this cohort, the use of MRI still resulted in unnecessary biopsies 69.4% of the time. Meanwhile, we demonstrate that our multiomic approach using cell free DNA and RNA achieves high accuracy for identifying clinically significant prostate cancer. At-home semen collection offers a convenient alternative to in-lab testing and may have the potential to augment MRI in post-PSA reflex testing.

O-071 A non-invasive multi-omics panel on cell free seminal plasma to predict the success of testicular sperm retrieval

Abstract Study question Can multi-omics analysis of seminal plasma of men with idiopathic non-obstructive azoospermia (iNOA) predict residual spermatogenesis? Summary answer Multi-omics provides a non-invasive approach to reliably predict the outcome of microdissection testicular sperm extraction (micro-TESE). What is known already Testicular biopsies are typically performed to retrieve spermatozoa for men diagnosed with NOA, but it is only successful in about 60% of the cases. Therefore, to predict the success of sperm retrieval prior to surgery, diagnostic biopsy and histopathology have been inconclusive and invasive. Moreover, several parameters have been proposed, including serum FSH, inhibin B, and genetics. Some have even suggested algorithms utilizing multiple parameters, but unfortunately, there is no specific assay to reliably predict the successful sperm retrieval. Study design, size, duration Over the past five years, seminal plasma was collected from thirty-one consenting men with repeated failed extensive semen analyses. Transcriptomic analysis was performed by RNAseq on seminal fluid and the specific gene expression were evaluated and compared to a fertile donor control. Subsequently, DNAseq and proteomics were carried out to confirm our findings. These men underwent micro-TESE in synchrony with ICSI cycle. Participants/materials, setting, methods RNA and DNA from 31 men were isolated from seminal plasma using a commercially available kit and sequenced by Illumina HiSeq3000/4000 platform to browse the expression profile and detect mutations, comparing to a fertile donor control. An absolute log2fold change of > 1 and a P-value of < 0.0005 were considered statistically significant. The proteomic profiles were then obtained by liquid chromatography and tandem mass spectrometry. Main results and the role of chance Transcriptomic analysis of 31 ejaculates from iNOA men were assessed for a total of 21,855 genes against a fertile donor control. 3,505 imbalanced genes were identified, with 12 genes consistently imbalanced among 15 of the men, while 19 genes were imbalanced among the other 16 men. These men were split into two cohorts, and when comparing the DEG profiles between them, eight shared genes were found. Of note, TPTE2, a testis-specific gene that regulates spermatogenesis, was overexpressed in 13 of the 15 men in the first group, while being underexpressed in all men from the other cohort. DNAseq showed that the gene was conserved in the first group, while exhibiting a more severe frameshift mutation in second group. Most interestingly, NEU1, a gene involved in acrosome development and fertilization, was found to be clearly overexpressed in the first cohort, while being underexpressed in all the men in the other cohort. DNAseq confirmed the NEU1 gene exhibited a synonymous mutation in the first group and a frameshift mutation in the other group. While proteomics analysis did not seem to confirm the findings, discrepancies in the gene product (LGALS3BP, IDH1, and PATE4) were observed, following the same trend as the previous analyses. Limitations, reasons for caution Although our study revealed specific candidate genes that can be used to predict the outcome of micro-TESE, it is still unclear whether these genes are causative of failed spermatogenesis or simply associate to. Additionally, the RNAseq can be somewhat extensive and costly, and more expedited assays would be preferable. Wider implications of the findings Analysis of differentially expressed genes in seminal plasma represents a non-invasive tool that may predict presence of residual spermatogenesis and eventually spermatozoa. This may present a precision medicine tool that can guide physicians to better manage expectations and spare patients from unnecessary cost and distress related to failed sperm retrieval. Trial registration number No

P-041 Impact of ionizing radiation on human spermatozoa integrity

Abstract Study question Do ionizing radiations affect human sperm integrity? If so, at what dose? Summary answer In vitro exposure of human ejaculated spermatozoa significantly reduces vitality, motility, and chromatin integrity without causing DNA fragmentation, telomeric damage, or altering capacitation ability. What is known already Exposure to ionizing radiation (IR) is a public health concern. However, its impact on human spermatozoa remains poorly described, particularly regarding nuclear damage. Our objective is to assess the impact of IR on functional parameters, as well as on nuclear quality. To achieve this, we expose human spermatozoa in vitro to increase doses of IR, reflecting various diagnostic and therapeutic medical scenarios. This study will help elucidate the mechanisms by which IR affects male fertility and, consequently, optimize fertility preservation strategies. Study design, size, duration We exposed human spermatozoa in vitro to increase IR doses mimicking epididymis irradiation during medical procedure: diagnostic imaging (scanner CT: 14–70 mGy) and radioiodine therapy (140 mGy). Sperm vitality, motility, DNA fragmentation, chromatin decondensation, telomere length (STL), nuclear morphometry, oxidative stress and capacitation markers post-IR were compared to their non-irradiated controls for each patient to address inter-individual variability. Participants/materials, setting, methods We used surplus semen from patient having consented to research (Germethèque Biobank). Gamma radiation conditions mimicking CT doses of 14 (n = 35), 70 (n = 39), and 140 mGy (n = 73) were experimentally calibrated. Sperm parameters (WHO 2021 guidelines), DNA fragmentation (TUNEL/SCD assays), chromatin decondensation (Chromomycin A3), STL (qPCR), nuclear morphometry (3D bioimage analysis), oxidation (Oxiselect/MDA kits) and capacitation markers -velocity (SCA© CASA), [AMPc] (ELISA), phosphotyrosine profile (Western blot) and acrosome integrity (PSA-FITC)- were evaluated. Main results and the role of chance The three dose groups showed no significant differences in initial sperm parameters or bio-clinical criteria, confirming the absence of biases. We observed a significant decrease in sperm vitality across all three radiation doses compared to the non-irradiated control condition (-3.4% for 14 mGy, -4.2% for 70 mGy, and -5.6% for 140 mGy; p < 0.001). Progressive motility was reduced at 140 mGy (37.0 ± 2.3% versus (vs.) 33.1 ± 2.0% after IR exposure; -3.9%, p < 0.001). IR induced chromatin decondensation at 70 mGy (23.6 ± 1.9% vs. 26.0 ± 1.8%, +2.3%, p < 0.05) and 140 mGy (23.5 ± 1.3% vs. 26.1 ± 1.5%, +2.5%, p < 0.01), with greater decondensation observed in samples with pre-existing sperm parameter alterations (+3.5%, p < 0.05). No significant changes were detected in DNA fragmentation levels (TUNEL or SCD assays) or sperm telomere length (STL). Exposure to 140 mGy did not significantly alter oxidative levels in seminal fluid or sperm cells, nor did it affect capacitation markers, although motility alterations were identified using SCA®. Finally, no significant global impact on 3D nuclear morphometric parameters has been observed to date. Limitations, reasons for caution This model provides an opportunity to analyze IR-induced alterations without patients/pathology’s interference. Differences in dose rate, exposure duration, and the spermatozoa environment (seminal instead of epididymal fluid) represented limitations. In vivo studies are essential to fully understand the impacts of IR on the regulation pathways of spermatogenesis and sperm maturation. Wider implications of the findings We show for the first time the impact of IR on human sperm quality using three radiations doses used for clinical diagnosis or treatment. Significant but minor effects on conventional parameters and chromatin decondensation were observed. Ongoing work includes epigenomic alterations from sperm cells and seminal fluid to further investigate. Trial registration number Yes

P-016 Can we predict sperm DNA fragmentation values using patients’ semen parameters and clinical characteristics combined with machine learning?

Abstract Study question Which clinical factors and semen parameters best predict sperm DNA fragmentation percentage (DF%) using machine learning, and which model performs best for the prediction? Summary answer Progressive sperm motility, sperm morphology, primary or secondary infertility, varicocelectomy, and smoking predicted DF% across models, with Gamma Generalized Linear Model providing the best fit. What is known already Numerous studies have shown that sperm DNA fragmentation has a potential clinical significance in assisted reproduction outcomes. However, current testing methods are non-standardised, time-consuming and expensive. Predicting DF% during clinical consultations could inform clinical decision-making. No reliable pipelines exist to predict sperm DNA fragmentation from clinical and seminal fluid data. Therefore, we investigated the best predictors of DF% using machine learning, which can handle complex datasets and detect non-linear relationships. By integrating clinical and semen parameters, a prediction tool could be built to enable clinicians to quickly and objectively predict sperm DNA fragmentation. Study design, size, duration This cross-sectional study analyzed data from 236 men undergoing seminal fluid analysis recruited from two private IVF centres. In-person interviews collected demographic (age, body mass index), clinical (primary/secondary infertility, varicocelectomy), and smoking status between July 2023 and April 2024. All participants provided informed consent following ethical approval. Participants/materials, setting, methods Among the male participants (mean age: 34.72 years), 138 (58.5%) had primary infertility, 36 (15.3%) had secondary infertility, and 62 (26.3%) were fertile. Semen analysis followed World Health Organization (WHO) 2010 guidelines, assessing volume, motility, concentration, and morphology. DF% was assessed using the Sperm Chromatin Dispersion (SCD) test. Six statistical models, including linear and generalized linear models (GLM) with and without bootstrapping, were applied to identify DF% predictors. Main results and the role of chance Linear regression identified progressive sperm motility (B = -0.171, p = 0.031), normal sperm morphology (B = -1.195, p = 0.004), infertility status (B = -3.925, p = 0.005), and varicocelectomy (B = -6.686, p = 0.001) as significant predictors, explaining ∼20% of DF% variability (R² = 0.199). Lower DF% was observed in primary infertility cases compared to secondary infertility across all models. Internal validation through bootstrapping (1,000 resamples) reinforced the stability of identified predictors and uncovered smoking as a significant factor (B = -3.343, p = 0.046), minimizing potential sampling bias. The GLM validated these predictors, showing slightly improved explanatory power (R² = 0.209) and consistency for progressive sperm motility (B = -0.199, p = 0.014), normal sperm morphology (B = -1.162, p = 0.005), infertility status (B = -7.131, p = 0.012), and varicocelectomy (B = -6.642, p = 0.001). The Gamma GLM further refined predictions, addressing DF’s skewed distribution and delivering superior fit indices (AIC = 1791.53, BIC = 1829.49, deviance = 83.495, χ²(9) = 103.19, p < 0.001). Predictor consistency across models highlights robust relationships, while the Gamma GLM’s improved fit and tailored assumptions make it the most reliable approach for predicting DF% in clinical contexts. Limitations, reasons for caution The study is limited by the small sample size and restricted demographics, reducing generalizability and diagnostic power. Wider implications of the findings These preliminary findings contribute to developing machine learning models for predicting sperm DF%, utilising clinical factors and semen analysis parameters to support clinical decision-making in fertility assessments. Trial registration number No

The screening and validation of lnc-SLC25A39 and lnc-LINC00279-202 for distinguishing tissue origins of peripheral blood and semen samples by RT-qPCR.

The screening and validation of lnc-SLC25A39 and lnc-LINC00279-202 for distinguishing tissue origins of peripheral blood and semen samples by RT-qPCR.

O-296 Paternal exposure to nicotine and abstinence: post-fertilization effects of the spermatozoa and their epigenetic impact on offspring; what have we learned from the animal models?

Abstract Study question Can abstinence from nicotine contribute to the recovery of the nicotine-induced damage in the male reproductive organs (MROs)? What are the effects on the offspring? Summary answer Abstinence, by decreasing the nicotine-induced oxidative-stress(OS) in MROs, may contribute to production of sperm with better quality that can further affect the offspring development epigenetically. What is known already According to the WHO 1.3 billion people are tobacco-users worldwide (36.7% of men and 7.8% of women). Nicotine exposure can impair the steroidogenesis and spermatogenesis, resulting in decreased sperm concentration and motility. By increasing the OS nicotine may induce damage to the chromatin structure of sperm and impair the fertilizing capacity. In the present study, nicotine was selected as the primary addictive component in cigarettes and electronic cigarettes to investigate its effects in the MROs and to further identify the epigenetic impact of paternal exposure to nicotine on the offspring’s development. Additionally, the role of abstinence was investigated. Study design, size, duration Sixty male Wistar rats were separated equally into three groups: a) the Nico-group (n = 20) that was treated with nicotine (100 μg/ml) orally for 10 weeks, b) the Abstinence-group (n = 20) that was treated with nicotine (100 μg/ml) orally for 7 weeks followed by 3 weeks of abstinence from nicotine and c) the Control group (n = 20) that had free access to fresh water daily for the same period of time (10 weeks). Participants/materials, setting, methods Five days before the completion of the 10-week treatment, each male rat was placed in the same cage with two female rats and mating studies were performed. Subsequently all male rats were sacrificed and OS parameters were measured in MROs and histological evaluation was performed. Offspring-development was recorded up to the 28th day. Main results and the role of chance Nicotine treatment significantly increased OS-markers levels and their expression in the testis, epididymis and seminal vesicles (SV) in Nico−group compared to Abstinence and Control groups. Total antioxidant capacity was significantly decreased both in the epididymal and testicular tissues of the Nico group compare to Abstinence or Control groups. Cotinine, the predominant metabolite of nicotine, was present at high levels in urine, serum and seminal vesicular fluid(SVF) of Nico−group compared to Abstinence-group (P<0.05). Testosterone levels were significantly lower in Nico-group compared to Control−group, while abstinence partially corrected this abnormality. Cytochrome P450(CYP2A6), the primary enzyme responsible for the oxidation of nicotine and cotinine was strongly expressed and localized in the epithelial cells of the epididymis in Nico−group. 8-hydroxy-2-deoxyguanosine (8-OHdG), one of the major products of DNA-oxidation was strongly expressed in the epididymal and seminal vesicular tissues of the Nico-group compared to the Abstinence and the Control group. The number of offspring in Nico-group was slightly decreased compared to the other two groups. The development of offspring and their body weights were recorded on 2nd, 3rd, 5th, 14th and 28th postnatal days. The body weight of offspring of the Nico-group was significantly lower compared to Abstinence or Control groups at all time-points measured. Limitations, reasons for caution Extending the abstinence period up to one spermatogenic cycle could probably give better results in terms of tissue morphology, but even 3-week abstinence was beneficial. Wider implications of the findings Nicotine exposure increases OS in MROs and creates a toxic environment in which spermatozoa are produced in the testis, become mature in the epididymis and are transported in the SVF until they reach the female reproductive system. Accumulated OS damages the sperm-DNA, which affects epigenetically the development of the offspring. Trial registration number No

Effect of two doses of coenzyme Q10 on spermogram parameters, sperm chromatin integrity and partner pregnancy rate in men with idiopathic oligoasthenozoospermia: A prospective study

Effect of two doses of coenzyme Q10 on spermogram parameters, sperm chromatin integrity and partner pregnancy rate in men with idiopathic oligoasthenozoospermia: A prospective study

P-074 Evaluation of the impact of SARS-CoV-2 vaccine on semen quality, sex hormones, and pregnancy outcome: a systematic review and meta-analysis

Abstract Study question Does the SARS-CoV-2 vaccine adversely affect semen quality, sex hormones, and pregnancy outcomes? Summary answer The SARS-CoV-2 vaccine affected sperm parameters and hormone levels in men and women but had no significant impact on fertility or pregnancy outcomes. What is known already The available data on the impact of the SARS-CoV-2 vaccine on fertility indices are conflicting; hence this topic remains controversial. Additionally, previous meta-analyses on the effect of the SARS-CoV-2 vaccine on human reproduction evaluated quite a limited number of available studies. Study design, size, duration This study provides a robust systematic review and meta-analysis of the impact of the SARS-CoV-2 vaccine on semen quality, sex hormones, and pregnancy outcomes. This study was registered on PROSPERO (CRD42024533909). Participants/materials, setting, methods The study was performed according to PRISMA guidelines. 5529 studies were collected, employing a pre-defined systematic approach. Upon screening, 44 studies were adjudged eligible for inclusion, these studies were conducted in Iraq (1), Jordan (1), Israel (10), Greece (1), Germany (1), USA (5), China (11), Russia (4), Ukraine (1), Saudi Arabia (1), Indonesia (1), Italy (3), Spain (1), India (1), Turkey (1), and Ireland (1). Main results and the role of chance The SARS-CoV-2 vaccine was associated with a significant increase in sperm concentration, total sperm count, and serum LH in men, these changes persisted after sensitivity analyses. On the other hand, the vaccine markedly reduced progressive sperm motility and circulating testosterone /LH ratio (an index of Leydig cell function), which also remained upon sensitivity analyses. Nonetheless, no significant alteration in ejaculate volume, seminal fluid pH, sperm viability, total motility, the percentage of sperm cells with normal morphology, and sperm DNA fragmentation was observed. Additionally, the SARS-CoV-2 vaccine did not alter serum testosterone, FSH, estrogen, and prolactin. Furthermore, in women, the SARS-CoV-2 vaccine increased AMH and LH levels but decreased FSH and estrogen levels. However, it showed no significant effect on fertilization, implantation, chemical and clinical pregnancy, or live birth rates. Finally, the odds of preterm birth, stillbirth, low birth weight, and very low birth weight were similar between the control and vaccinated groups. Limitations, reasons for caution The eligible studies, being from few countries, may not fully represent the global population. Since SARS-CoV-2 infection is relatively novel and the vaccine was introduced after the pandemic, the included studies evaluated the impact of the vaccine at short-term. Well-designed studies, which investigate the long-term effect of SARS-CoV-2 are necessary. Wider implications of the findings This extensive meta-analysis updates previous findings on the effect of SARS-CoV-2 vaccine on human reproduction. While it alters some hormone levels and semen parameters, it does not impair fertility or pregnancy outcomes. Our findings support vaccine safety, though individuals planning conception should be monitored for potential medical interventions if needed. Trial registration number No

Sperm length and seminal fluid proteins promote male reproductive success in D. melanogaster.

Spermatozoal morphology is highly variable both within and among species, often corresponding to variation in the shape of the female sperm storage organs in ways that can significantly impact fertilization success. In an effort to understand genetic mechanisms of sperm length variation, we compared gene expression patterns in the testes of Drosophila melanogaster males that produce either long or short sperm. We found that genes upregulated in long sperm testes are enriched for long noncoding RNAs (lncRNAs) and seminal fluid proteins (Sfps). Transferred in seminal fluid to the female during mating, Sfps are secreted by the male accessory glands and affect female remating rate, physiology, and behavior with concomitant advantages for male reproductive success. While sperm and Sfps are both critical for male reproductive success, they are largely considered to be functionally, genetically, and developmentally independent and despite being upregulated in long sperm testes, Sfps have no known function in testis tissue. We found that knockouts of two Sfps upregulated in long sperm males, Sex Peptide (SP) and ovulin (Acp26Aa) resulted in shorter sperm, which altogether suggests that Sfps may play a role in the development of sperm length during spermatogenesis. Consistent with this, knockout of accessory gland function did not affect sperm length, suggesting that accessory gland expression had no influence on spermatogenic processes. We also found that long sperm males were better able to delay female remating. These results might suggest that long sperm males have a double advantage in sperm competition by both delaying female remating, likely through transfer of more Sfps, and by resisting sperm displacement. However, we found that the delay in female remating does not necessarily translate to more progeny or higher paternity success. Thus, we found that multiple components of the ejaculate promote male reproductive success at different stages of reproduction, but the realized fitness advantages in sperm competition are uncertain.

Stage-specific transcriptomic analysis reveals insights into the development, reproduction and biological function of allergens in the European house dust mite Dermatophagoides pteronyssinus

BackgroundHouse dust mites (HDMs) such as Dermatophagoides pteronyssinus are major allergy elicitors worldwide, yet their gene expression across developmental stages remains underexplored. Herein, we report a comprehensive RNAseq analysis of larvae, nymphs, and adult males and females, mapped to a recently published high-quality genome with extended functional annotations.ResultsAnalysis of differentially expressed genes (DEG) revealed that female-biased expression was the most prevalent profile (16% of genes), while males exhibited the highest fold-change differences. DEG data, combined with network clustering and functional enrichment analysis, highlighted distinct genes and biological processes for each stage and sex: females showed upregulation of genes related to cell division and oogenesis, with vitellogenins among the most abundant transcripts; males exhibited increased expression of genes encoding putative seminal fluid proteins (e.g. endopeptidases, serpins, antimicrobial peptides), and those involved in reproductive regulation (e.g. testis-specific serine kinases); while juveniles displayed enhanced expression of genes related to energy metabolism and growth. Further analysis of endocrine pathways revealed non-canonic mechanisms compared to insect models, particularly in ecdysteroid and sesquiterpenoid biosynthesis and regulation. Expression patterns in genes involved in cuticle formation were also identified, reflecting their role in developmental transitions and sexual differentiation. Allergen and allergen-related gene expression showed an overall increase in feeding juveniles, as well as sex-biased expression, with Der p 27 upregulated in females. These findings provide insight into the physiological roles of allergens in digestion, immunity, and muscle formation, among other functions. Additionally, seven new horizontally transferred genes, including a DNA-repair photolyase linked to females, and novel multigene families (e.g. 119 male-specific beta-propeller proteins, 70 hypothetical cuticular proteins, 23 tetraspanin-like proteins, 5 female-associated putative odorant-binding proteins) were identified.ConclusionsThis study provides the first genome-wide transcriptomic analysis of a HDM across life stages and sexes, expanding our understanding of the molecular mechanisms underlying mite development, sexual reproduction, and allergen expression. The generated data, fully available via supplementary spreadsheet and the ORCAE online platform, provide a valuable foundation for future allergy research and the development of new mite control strategies.

Prostate and urinary microbiomes in prostate cancer development: focus on Cutibacterium acnes.

Prostate cancer (PCa) is one of the most prevalent malignancies among men, with its incidence steadily increasing worldwide. Recent advances in microbiome research have opened new avenues for understanding and treating PCa; however, studies focusing specifically on the prostate tissue microbiome remain limited. Evidence suggests that the microbial communities within PCa tissues exhibit significant diversity and regional variability, with certain bacteria potentially contributing to PCa initiation and progression through chronic inflammation. The prostate microbiome comprises not only bacteria but also viruses, fungi, and parasites, and its diversity is influenced by a complex interplay of genetic, environmental, and lifestyle factors. Methodological limitations and sample contamination further complicate the interpretation of microbiome data. The urinary microbiome is similarly diverse and shaped by multiple overlapping influences. Although urine, prostatic fluid, and prostate tissue are anatomically and functionally connected, whether urine and prostatic fluid can accurately reflect the prostate tissue microbiome remains to be conclusively determined. Among the microorganisms detected, Cutibacterium acnes is frequently identified in prostate tissue, urine, and prostatic fluid from PCa patients. This bacterium is known to elicit inflammatory responses through various pathways, potentially impacting tumorigenesis and cancer progression. Nevertheless, findings across studies remain inconsistent. Further research is necessary to elucidate the underlying mechanisms by which the microbiome influences PCa. Such efforts may offer novel insights and strategies for the diagnosis, treatment, and prevention of this disease.