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Abstract: Seminal fluid extracellular vesicles (SFEVs) have previously been shown to interact with spermatozoa and influence their fertilisation capacity. Here, we sought to extend these studies by exploring the functional consequences of SFEV interactions with human spermatozoa. SFEVs were isolated from the seminal fluid of normozoospermic donors prior to assessing the kinetics of sperm-SFEV binding in vitro, as well as the effects of these interactions on sperm capacitation, acrosomal exocytosis, and motility profile. Biotin-labelled SFEV proteins were transferred primarily to the flagellum of spermatozoa within minutes of co-incubation, although additional foci of SFEV biotinylated proteins also labelled the mid-piece and head domain. Functional analyses of high-quality spermatozoa collected following liquefaction revealed that SFEVs did not influence sperm motility during incubation at pH 5, yet SFEVs induced subtle increases in total and progressive motility in sperm incubated with SFEVs at pH 7. Additional investigation of sperm motility kinematic parameters revealed that SFEVs significantly decreased beat cross frequency and increased distance straight line, linearity, straightness, straight line velocity, and wobble. SFEVs did not influence sperm capacitation status or the ability of sperm to undergo acrosomal exocytosis. Functional assessment of both high- and low-quality spermatozoa collected prior to liquefaction showed limited SFEV influence, with these vesicles inducing only subtle decreases in beat cross frequency in spermatozoa of both groups. These findings raise the prospect that, aside from subtle effects on sperm motility, the encapsulated SFEV cargo may be destined for physiological targets other than the male germline, notably the female reproductive tract., Lay Summary: A male's influence over the biological processes of pregnancy extends beyond the provision of sperm. Molecular signals present in the ejaculate can influence the likelihood of pregnancy and healthy pregnancy progression, but the identity and function of these signals remain unclear. In this study, we wanted to understand if nano-sized particles present in the male ejaculate, called seminal fluid extracellular vesicles, can assist sperm in traversing the female reproductive tract to access the egg. To explore this, we isolated seminal fluid extracellular vesicles from human semen and incubated them with sperm. Our data showed that seminal fluid extracellular vesicles act to transfer molecular information to sperm, but this resulted in only subtle changes to the movement of sperm.

Mating and the transfer of seminal fluid components including male accessory glands (MAGs) proteins can affect oviposition behavior in insects. After oviposition, some species of fruit flies deposit a host-marking pheromone (HMP) on the fruit that discourages oviposition by other females of the same or different species or genus and reduces competition between larvae. However, we know very little about how mating, receiving seminal fluid, or male condition can affect female host marking behavior. Here, we tested how the physiological state of females (mated or unmated), the receipt of seminal fluid, and the condition of the male (wild or sterile) affect oviposition and host-marking behavior (HMB) in Anastrepha ludens (Diptera: Tephritidae). We also determined the efficiency of the host-marking pheromone from mated or unmated females in deterring oviposition. In a further examination of how seminal fluid may be affecting HMB we assessed if there were differences in the size of wild or sterile MAGs and the protein quantity transferred during mating. Our results indicate that receiving seminal fluid increased egg laying and increased time invested in host-marking (HM). Unmated females laid fewer eggs than mated females but invested the same amount of time in depositing host-marking pheromone, which had similar effectiveness in deterring oviposition as that of mated females. Females that mated with sterile males laid the same number of eggs as females that mated with wild males but spent less time depositing host-marking pheromone, which suggests that females detect the condition of the male and invest less in marking hosts. Finally, sterile males had larger accessory glands and transferred more MAGs proteins during mating compared to wild males. Seminal proteins could be manipulating HM behavior and female investment into their current reproductive effort. We are only beginning to understand how male condition and seminal fluid can affect female physiology and maternal investment in HMP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Inadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated in situations of so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unprotected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed., Methods: To probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chromatography-tandem mass spectrometry. Further, drug-residue testing using patients' ejaculate was conducted by utilizing liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples (i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry., Results: In one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence of seminal fluid in one of the female athlete's doping control urine samples, and the analysis of clinical ejaculate specimens provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid., Conclusions: The combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments., (© 2024. The Author(s).)

Introduction: A decrease in sperm cell count has been observed along the last several decades, especially in the most developed regions of the world. The use of metabolomics to study the composition of the seminal fluid is a promising approach to gain access to the molecular mechanisms underlying this fact., Objectives: In the present work, we aimed at relating metabolomic profiles of young healthy men to their semen quality parameters obtained from conventional microscopic analysis., Methods: An untargeted metabolomics approach focusing on low- to mid-polarity compounds was used to analyze a subset of seminal fluid samples from a cohort of over 2700 young healthy men., Results: Our results show that a broad metabolic profiling comprising several families of compounds (including acyl-carnitines, steroids, and other lipids) can contribute to effectively distinguish samples provided by individuals exhibiting low or high absolute sperm counts., Conclusion: A number of metabolites involved in sexual development and function, signaling, and energy metabolism were highlighted as being distinctive of samples coming from either group, proving untargeted metabolomics as a promising tool to better understand the pathophysiological processes responsible for male fertility impairment., (© 2023. The Author(s).)

Male infertility has been recognized as a global health problem. Semen analysis, although considered the golden standard, may not provide a confident male infertility diagnosis alone. Hence, there is the urgent request for an innovative and reliable platform to detect biomarkers of infertility. The rapid expansion of mass spectrometry (MS) technology in the field of the 'omics' disciplines, has incredibly proved the great potential of MS-based diagnostic tests to revolutionize the future of pathology, microbiology and laboratory medicine. Despite the increasing success in the microbiology area, MS-biomarkers of male infertility currently remain a proteomic challenge. In order to address this issue, this review encompasses proteomics investigations by untargeted approaches with a special focus on experimental designs and strategies (bottom-up and top-down) for seminal fluid proteome profiling. The studies reported here witness the efforts of the scientific community to address these investigations aimed at the discovery of MS-biomarkers of male infertility. Proteomics untargeted approaches, depending on the study design, might provide a great plethora of biomarkers not only for a male infertility diagnosis, but also to address a new MS-biomarkers classification of infertility subtypes. From the early detection to the evaluation of infertility grade, new MS-derived biomarkers might also predict long-term outcomes and clinical management of infertility.

Seminal plasma (SP) mirrors the local pathophysiology of the male reproductive system and represents a non-invasive fluid for the study of infertility. Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) provides a high-throughput platform to rapidly extrapolate the diagnostic profiles of information-rich patterns. In this study, dispersive solid phase extraction ( d -SPE) combined with MALDI-TOF-MS was applied for the first time to the human SP, with the aim of revealing a diagnostic signature for male infertility. Commercially available octadecyl (C 18 )-, octyl (C 8 )-bonded silica sorbents and hexagonal mesoporous silica (HMS) were tested and the robustness of MALDI-TOF peptide profiling was evaluated. Best performances were obtained for C 18 -bonded silica with the highest detection of peaks and the lowest variation of spectral features. To assess the diagnostic potential of the method, C 18 -bonded silica d -SPE and MALDI-TOF-MS were used to generate enriched endogenous peptide profiles of SP from 15 fertile and 15 non-fertile donors. Principal component analysis (PCA) successfully separated fertile from non-fertile men into two different clusters. An array of seven semenogelin-derived peptides was found to distinguish the two groups, with high statistical significance. These findings, while providing a rapid and convenient route to selectively enrich native components of SP peptidome, strongly reinforce the prominent role of semenogelins in male infertility.

A central paradigm in evolutionary biology is that the fundamental divergence in the fitness interests of the sexes ('sexual conflict') can lead to both the evolution of sex-specific traits that reduce fitness for individuals of the opposite sex, and sexually antagonistic coevolution between the sexes. However, clear examples of traits that evolved in this way - where a single trait in one sex demonstrably depresses the fitness of members of the opposite sex, resulting in antagonistic coevolution - are rare. The Drosophila seminal protein 'sex peptide' (SP) is perhaps the most widely cited example of a trait that appears to harm females while benefitting males. Transferred in the ejaculate by males during mating, SP triggers profound and wide-ranging changes in female behaviour and physiology. Early studies reported that the transfer of SP enhances male fitness while depressing female fitness, providing the foundations for the widespread view that SP has evolved to manipulate females for male benefit. Here, we argue that this view is (i) a simplification of a wider body of contradictory empirical research, (ii) narrow with respect to theory describing the origin and maintenance of sexually selected traits, and (iii) hard to reconcile with what we know of the evolutionary history of SP's effects on females. We begin by charting the history of thought regarding SP, both at proximate (its production, function, and mechanism of action) and ultimate (its fitness consequences and evolutionary history) levels, reviewing how studies of SP were central to the development of the field of sexual conflict. We describe a prevailing paradigm for SP's evolution: that SP originated and continues to evolve to manipulate females for male benefit. In contrast to this view, we argue on three grounds that the weight of evidence does not support the view that receipt of SP decreases female fitness: (i) results from studies of SP's impact on female fitness are mixed and more often neutral or positive, with fitness costs emerging only under nutritional extremes; (ii) whether costs from SP are appreciable in wild-living populations remains untested; and (iii) recently described confounds in genetic manipulations of SP raise the possibility that measures of the costs and benefits of SP have been distorted. Beyond SP's fitness effects, comparative and genetic data are also difficult to square with the idea that females suffer fitness costs from SP. Instead, these data - from functional and evolutionary genetics and the neural circuitry of female responses to SP - suggest an evolutionary history involving the evolution of a dedicated SP-sensing apparatus in the female reproductive tract that is likely to have evolved because it benefits females, rather than harms them. We end by exploring theory and evidence that SP benefits females by functioning as a signal of male quality or of sperm receipt and storage (or both). The expanded view of the evolution of SP that we outline recognises the context-dependent and fluctuating roles played by both cooperative and antagonistic selection in the origin and maintenance of reproductive traits., (© 2022 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Purpose: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid., Methods: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital., Results: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with C t values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction., Conclusion: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation., (© 2021. The Author(s).)

Evidence collected from biological fluids obtained from a crime scene is essentially important in forensic cases. A potential profile can be generated from these obtained samples and this can help in identifying the victims and/or suspects of sexual assault. The water environments selected for this study are all related to the potential crime scenes from which there is a possibility of finding a dead body or clothing of a sexual assault victim. Tap water, River water, Swimming pool water, and Canal water were selected. Fabric types selected were khaddar, linen, silk, polyester, and chiffon. Detection of seminal stains was carried out by three methods; Alternate Light Source (ALS), Acid phosphatase (AP) testing, and Kernechtrot-Picro-indigo-carmine (KPIC) testing. These tests were performed for each fabric type in each water environment after regular intervals, 24 h, 48 h, 72 h, 4 days, 7 days, and 14 days. This study aimed to compare the ability of five types of fabrics to retain seminal material after immersion in four different types of water environments. Fluorescence was only detected in tap water-soaked silk fabric after 14 days of immersion. Seminal fluid was detected in khaddar, chiffon, silk, and polyester in samples immersed for 14 days in tap water. Spermatozoa were retained by khaddar and silk immersed in tap water, Polyester fabric in tap and river water, Chiffon in only river water and Linen in swimming pool water when immersed for 14 days. However, fluorescence, seminal fluid or spermatozoa were not detected in linen fabric regardless of all the afore mentioned variables., Competing Interests: Conflict of interests The authors declare that they have no competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Paternal experiences and exposures before conception can influence fetal development and offspring phenotype. The composition of seminal plasma contributes to paternal programming effects through modulating the female reproductive tract immune response after mating. To investigate whether paternal obesity affects seminal plasma immune-regulatory activity, C57Bl/6 male mice were fed an obesogenic high-fat diet (HFD) or control diet (CD) for 14 weeks. Although HFD consumption caused only minor changes to parameters of sperm quality, the volume of seminal vesicle fluid secretions was increased by 65%, and the concentrations and total content of immune-regulatory TGF-β isoforms were decreased by 75% to 80% and 43% to 55%, respectively. Mating with BALB/c females revealed differences in the strength and properties of the postmating immune response elicited. Transcriptional analysis showed >300 inflammatory genes were similarly regulated in the uterine endometrium by mating independently of paternal diet, and 13 were dysregulated by HFD-fed compared with CD-fed males. Seminal vesicle fluid factors reduced in HFD-fed males, including TGF-β1, IL-10, and TNF, were among the predicted upstream regulators of differentially regulated genes. Additionally, the T-cell response induced by mating with CD-fed males was blunted after mating with HFD-fed males, with 27% fewer CD4+ T cells, 26% fewer FOXP3+CD4+ regulatory T cells (Treg) cells, and 19% fewer CTLA4+ Treg cells, particularly within the NRP1+ thymic Treg cell population. These findings demonstrate that an obesogenic HFD alters the composition of seminal vesicle fluid and impairs seminal plasma capacity to elicit a favorable pro-tolerogenic immune response in females at conception., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The latest vaccination campaign has actualized the potential impact of antigenic stimuli on reproductive functions. To address this, we mimicked vaccination's effects by administering keyhole limpet hemocyanin (KLH ) to CD1 male mice and used their sperm for in vitro fertilization (IVF). Two-cell embryos after IVF with spermatozoa from control (C) or KLH-treated (Im) male mice were transferred to surrogate mothers mated with vasectomized control (C) or KLH-treated (Im) male mice, resulting in four experimental groups: C-C, Im-C, C-Im, and Im-Im. The pre-implantation losses were significantly lower in the Im-C group than in the C-Im group. At the same time, the resorption rates reduced markedly in the C-Im compared to the Im-C group. Embryo and placenta weights were significantly higher in the Im-Im group. Although the GM-CSF levels were lower in the amniotic fluid of the gestating surrogate mothers in the Im-Im group, they were strongly correlated with embryo mass. The number-size trade-off was only significant in the Im-Im group. This suggests a positive, cooperative effect of spermatozoa and seminal fluid from immune-primed males on embryo growth and the optimal distribution of surrogate mother maternal resources despite the negative impact of males' antigenic challenge on the IVF success rate.

Background: Viral detection in seminal fluid indicates their potential for both sexual transmission and impairment of reproductive health. Review of the mechanistic entry, sexual transmission and viral impacts for patients during major recent viral outbreaks of Zika virus (ZIKV), Ebola virus (EBOV), severe acute respiratory syndrome (SARS)-coronavirus (CoV), and SARS-coronavirus 2 (CoV-2) (the virus which causes COVID-19) provides a framework to discuss this potential., Aim: Comparative analysis of prior viral presence on seminal fluid against current (preliminary) findings for SARS-CoV-2 to predict biological implications of the novel coronavirus upon current sexual transmissibility, viral presence, and reproductive health., Methodology and Findings: Literature review was conducted using PubMed and Google Scholar databases. ZIKV and EBOV were found to be present in semen and to be sexually transmitted, leading the World Health Organization (WHO) to update their guidelines on prevention of the two viruses to include refraining from sexual contact. There are conflicting studies regarding the presence of SARS-CoV in male reproductive tissue, but it has been linked to testicular atrophy and orchitis. To date, two studies have detected SARS-CoV-2 RNA in semen, while seven studies have reported no positive detection., Conclusions: Though unlikely in the majority of cases, SARS-CoV-2 can potentially be present in seminal fluid, although there are no reports of sexual transmission to date. Prior epidemics raise significant concerns regarding the long-term reproductive health capacity for patients who are affected by entry of Sars-CoV-2 into the reproductive tract, therefore more study is needed to clarify the impacts to reproductive health.

Serological screening of sexual assault evidence has traditionally focused on enzyme activity and immunochromatographic assays that provide only a presumptive indication of seminal fluid and have limited sensitivity relative to DNA testing. Seminal fluid detection based on protein mass spectrometry represents a "Next Gen" serological technology that overcomes the specificity and sensitivity limitations of traditional serological screening but requires time-consuming sample preparation protocols. This paper describes a novel "peptidomics" approach to seminal fluid detection that eliminates the need for lengthy trypsin digestion. This streamlines sample preparation to a one-step process followed by high-resolution mass spectrometry to identify naturally occurring seminal fluid peptides and low-molecular weight proteins. Multiple protein biomarkers of seminal fluid were consistently and confidently identified based on the multiplexed detection of numerous endogenous peptides. These included Semenogelin I and II (90% and 86% sequence coverage, respectively); Prostate Specific Antigen/p30 (29% sequence coverage); and Prostatic Acid Phosphatase (24% sequence coverage). The performance of this streamlined peptidomics approach to seminal fluid identification in a forensic context was also assessed using simulated casework samples of the type typically collected as part of a sexual assault examination (e.g., oral and vaginal swabs stained with semen). The resulting data demonstrate that sub-microliter quantities of seminal fluid on cotton swabs can be recovered and reliably detected. This supports the forensic applicability of a peptidomic assay for seminal fluid identification with same-day sample preparation and analysis. Future development and streamlined multiplex peptidomic assays for additional biological stains can easily be envisaged., (© 2020 American Academy of Forensic Sciences.)

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA-the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

Great concerns have been raised on SARS-CoV-2 impact on men's andrological well-being, and one of the critically unanswered questions is whether it is present or not in the seminal fluid of infected subjects. The expression of ACE2 and TMPRSS2 in the testis and in the male genital tract allows speculations about a possible testicular involvement during the infection, possibly mediated by local and/or systemic inflammation that might allow a high viral load to overcome the hemato-testicular barrier. To date, few investigations have been carried out to ascertain the presence of SARS-CoV-2 in the seminal fluid with contrasting results. Furthermore, the cumulative number of subjects is far too low to answer the question unambiguously. Therefore, great caution is still needed when evaluating this data; otherwise, we risk unleashing unmotivated concerns in the scientific world with troublesome consequences in reproductive medicine., (© 2020 American Society of Andrology and European Academy of Andrology.)

Sperm production and allocation strategies have been a central concern of sperm competition research for the past 50 years. But during the 'sexual cascade' there may be strong selection for alternative routes to maximizing male fitness. Especially with the evolution of internal fertilization, a common and by now well-studied example is the accessory ejaculate investment represented by seminal fluid, the complex mixture of proteins, peptides and other components transferred to females together with sperm. How seminal fluid investment should covary with sperm investment probably depends on the mechanism of seminal fluid action. If seminal fluid components boost male paternity success by directly enhancing sperm function or use, we might often expect a positive correlation between the two forms of male investment, whereas trade-offs seem more likely if seminal fluid acts independently of sperm. This is largely borne out by a broad taxonomic survey to establish the prevailing patterns of seminal fluid production and allocation during animal evolution, in light of which I discuss the gaps that remain in our understanding of this key ejaculate component and its relationship to sperm investment, before outlining promising approaches for examining seminal fluid-mediated sperm competitiveness in the post-genomic era. This article is part of the theme issue 'Fifty years of sperm competition'.

Sperm competition theory predicts that males should tailor ejaculates according to their social status. Here, we test this in a model vertebrate, the house mouse ( Mus musculus domesticus ), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our analyses reveal that both sperm production and the composition of proteins found in seminal vesicle secretions differ according to social status. Dominant males invested more in ejaculate production overall. Their epididymides contained more sperm than those of subordinate or control males, despite similar testes size between the groups. Dominant males also had larger seminal vesicle glands than subordinate or control males, despite similar body size. However, the seminal vesicle secretions of subordinate males had a significantly higher protein concentration than those of dominant males. Moreover, detailed proteomic analysis revealed subtle but consistent differences in the composition of secreted seminal vesicle proteins according to social status, involving multiple proteins of potential functional significance in sperm competition. These findings have significant implications for understanding the dynamics and outcome of sperm competition, and highlight the importance of social status as a factor influencing both sperm and seminal fluid investment strategies. This article is part of the theme issue 'Fifty years of sperm competition'.

Tolerogenic dendritic cells (tDCs) play a central role in the development of paternal antigen-specific regulatory T cells (Tregs) during pregnancy. We examined whether uterine CD11c + antigen presenting cells (APC) induced paternal antigen-specific tolerance in allogeneic pregnant mice. Female BALB/c mice were mated with male DBA/2 mice, and their surface markers of APCs were studied using flow cytometry. After allogeneic mating, the uterine APCs exhibited significantly decreased expression of major histocompatibility complex (MHC) class II on day 3.5 post-coitus (pc) and day 5.5 pc. To analyze how seminal fluid affects surface markers of APCs, female BALB/c mice were mated with male mice that had undergone seminal vesicle excision (SVX). No reductions of MHC class II expression on APCs were seen in these mice. To analyze APC functions, a mixed lymphoid reaction (MLR) assay to paternal splenocytes was performed. Uterine APCs from allogeneic pregnant mice significantly suppressed the MLR reaction, but APCs from SVX mated mice did not suppress the MLR reaction Uterine APCs induced paternal antigen (Mls1a)-specific Treg development in vitro, but not in mice that mated with allogeneic SVX mice. These findings suggest that seminal fluid priming expands the paternal antigen-specific Treg population by inducing APCs development., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Seminal fluid is often assumed to have just one function in mammalian reproduction, delivering sperm to fertilize oocytes. But seminal fluid also transmits signaling agents that interact with female reproductive tissues to facilitate conception and .pregnancy. Upon seminal fluid contact, female tissues initiate a controlled inflammatory response that affects several aspects of reproductive function to ultimately maximize the chances of a male producing healthy offspring. This effect is best characterized in mice, where the female response involves several steps. Initially, seminal fluid factors cause leukocytes to infiltrate the female reproductive tract, and to selectively target and eliminate excess sperm. Other signals stimulate ovulation, induce an altered transcriptional program in female tract tissues that modulates embryo developmental programming, and initiate immune adaptations to promote receptivity to implantation and placental development. A key result is expansion of the pool of regulatory T cells that assist implantation by suppressing inflammation, mediating tolerance to male transplantation antigens, and promoting uterine vascular adaptation and placental development. Principal signaling agents in seminal fluid include prostaglandins and transforming growth factor-β. The balance of male signals affects the nature of the female response, providing a mechanism of ‟cryptic female choiceˮ that influences female reproductive investment. Male-female seminal fluid signaling is evident in all mammalian species investigated including human, and effects of seminal fluid in invertebrates indicate evolutionarily conserved mechanisms. Understanding the female response to seminal fluid will shed new light on infertility and pregnancy disorders and is critical to defining how events at conception influence offspring health.

Purpose: The purpose of this proof-of-concept study was to determine whether delta-9-tetrahydrocannabinol (THC) and THC metabolites (11-OH THC and THC-COOH) can be detected in semen., Methods: Twelve healthy men aged 18-45 years who identified as chronic and heavy users of inhaled cannabis were recruited. THC and THC metabolite levels were measured in serum, urine, and semen of the participants. Semen analyses were performed. Serum reproductive hormones were measured., Results: The median age and BMI of participants were 27.0 years and 24.7 kg/m 2 , respectively. Over half the participants were daily users of cannabis for over 5 years. Serum reproductive hormones were generally within normal ranges, except prolactin, which was elevated in 6 of 12 participants (mean 13.9 ng/mL). The median sperm concentration, motility, and morphology were 75.5 million/mL, 69.5%, and 5.5%, respectively. Urinary THC-COOH was detected in all 12 participants, and at least one serum THC metabolite was present in 10 of 12 participants. Two semen samples had insufficient volume to be analyzed. THC was above the reporting level of 0.50 ng/mL in the semen of two of the remaining participants. Seminal THC was moderately correlated with serum levels of THC (r = 0.66), serum 11-OH THC (r = 0.57), and serum THC-COOH (r = 0.67). Seminal delta-9 THC was not correlated with urinary cannabinoid levels or semen analysis parameters., Conclusion: This is the first study to identify and quantify THC in human semen, demonstrating that THC can cross the blood-testis barrier in certain individuals. Seminal THC was found to be moderately correlated with serum THC and THC metabolites.

This study was designed to investigate the metabolic and transcriptional alterations in seminal fluid caused by asthenozoospermia (AS). To address these issues, a method of metabonomics based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and real-time quantitative PCR (RT-qPCR) was performed to identify some crucial biomarkers and transcription levels of the enzymes in seminal fluid. Seminal fluid samples were collected from 87 AS patients and 73 healthy males with normozoospermia. The quantitative analysis by UPLC-MS/MS showed that 19 metabolites in seminal plasma were associated with AS, and they were involved in several metabolic pathways, such as energy metabolism, purine metabolism, methionine cycle, and branched chain amino acid metabolism. Among these metabolites, the levels of citric acid, malic acid, succinic acid, and pyruvic acid, which are related to energy metabolism, were collectively reduced in the AS group, whereas the lactic acid level was enhanced. These results indicated that lesser energy source (adenosine triphosphate) was produced through the anaerobic glycolysis pathway rather than via aerobic catabolism of suger and tricarboxylic acid cycle, resulting in reduced power of sperms. Meanwhile, partial least squares discriminant analysis showed significant differences in metabolic profiles between the AS and control groups. In addition, RT-qPCR results revealed that the expression levels of four genes encoding fructokinase citrate synthase, succinate dehydrogenase, and spermine synthase, which were related to energy metabolism, were decreased in the AS group. The 23 descriptors with differential expression in AS may be valuable for the diagnosis and sequential study on AS. These results will help highlight the role of sperm inactivity in AS pathogenesis., (© 2019 John Wiley & Sons, Ltd.)

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals., (© 2020 Blackwell Verlag GmbH.)

Steroids are essential hormones that play a crucial role in homeostasis of many biological processes including sexual development, spermatogenesis, sperm physiology and fertility. Although steroids have been largely studied in many biological matrices (such as urine and plasma), there is very limited information of the steroid content and their study as potential indicators of the quality of the seminal fluid. In this study, a LC-HRMS (liquid chromatography-high resolution mass spectrometry) strategy has been developed in order to obtain the extended steroid profile of human seminal fluid. A comparison between supported liquid extraction (SLE) and solid liquid extraction (SPE) was carried out and the chosen SPE method was further optimized to evidence the largest possible number of compounds. Steroids were automatically annotated by using DynaStI, a publicly available retention time prediction tool developed in our lab, to match the experimental data (i.e. accurate mass and t R ). Altogether, these resources allowed us to develop a post-targeted approach able to consistently detect 41 steroids in seminal fluid (with half of them being androgens). Such steroid pattern was found to be stable across different extraction times and injection days. In addition to accurate mass and retention time, the identity of 70% of the steroids contained in such steroid profile was confirmed by comparing their fragmentation patterns in real samples to those of pure commercial standards. Finally, the workflow was applied to compare and distinguish the steroid profile in seminal fluid from healthy volunteers (n = 7, with one of them being a vasectomized subject). In all, the developed steroidomics strategy allows to reliably monitor an extended panel of 41 steroids in human seminal fluid., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study aims to describe seminal plasma characteristics, detect changes during and between two consecutive spawning seasons (SS), and compare plasma features between two important South American fish species. Prochilodus lineatus and Brycon orbignyanus sperm was collected over two (SS1; SS2). Each season was divided into first and second sampling periods (P1; P2). Thus, the four experimental periods were referred to as SS1P1, SS1P2, SS2P1, and SS2P2. Seminal plasma was analyzed for osmolality, pH, and Na + , K + , and Ca 2+ concentration. Additionally, sperm concentration, motility rate, and velocities (curvilinear = VCL; straight line = VSL) were determined and correlated with plasma features. In P. lineatus, plasma osmolality was lower in SS1P2, pH was higher in SS2P2, Na + was higher and K + and Ca 2+ were lower in SS2P1 compared with other experimental periods. Positive correlations were observed between motility and plasma osmolality, motility and Na + , and VCL and Na + . In B. orbignyanus, plasma osmolality was higher in SS2P1 and SS2P2 and K + concentration was higher in SS1P1 compared with other experimental periods; no correlation was observed. Seminal plasma parameters change during SS; therefore, the composition of a sperm extender and artificial fertilization methods should be adapted to maximize fertilization rates., (© 2019 Wiley Periodicals, Inc.)

Study Objective: The ANRS163-ETRAL study showed that etravirine 200 mg/raltegravir 400 mg twice-daily dual therapy was highly effective in the treatment of human immunodeficiency virus (HIV)-infected patients older than 45 years, with virologic and therapeutic success rates at week 48 of 99.4% and 94.5%, respectively. The objective of this study was to determine whether a clinically significant pharmacokinetic interaction between etravirine and raltegravir exists by assessing steady-state total and unbound etravirine, raltegravir, and inactive raltegravir-glucuronide concentrations 12 hours after last intake (C 12h ) in blood plasma (BP) and seminal plasma (SP)., Design: Pharmacokinetic analysis of data from the ANRS163-ETRAL study., Patients: One hundred forty-six HIV-1-infected patients (of the 165 patients included in the ANRS-163 ETRAL study) who were receiving etravirine 200 mg and raltegravir 400 mg twice daily., Measurements and Main Results: Blood was collected from all 146 patients at weeks 2-4, 12, 24, and 48, and semen was collected from 21 patients at week 48. The extent of BP and SP protein binding was determined by using ultrafiltration assay. Total and unbound etravirine, raltegravir, and raltegravir-glucuronide C 12h were determined by ultra high performance liquid chromatography coupled with tandem mass spectrometry and interpreted by using the in vitro calculated protein-bound 95% inhibitory concentration (PBIC 95 ) for wild-type (WT) HIV: etravirine (116 ng/ml) and raltegravir (15 ng/ml). Median (interquartile range [IQR]) total BP etravirine C 12h (536 ng/ml [376-719]) and raltegravir (278 ng/ml [97-690]) were adequate in 99% and 96% of patients, respectively. Median (IQR) SP:BP C 12h ratio and BP unbound fraction were etravirine 0.3 (0.2-0.5) and < 1%, respectively, raltegravir 1.8 (1.3-3.3) and 12%, respectively, and raltegravir-glucuronide 12.0 (6.5-17.7) and > 99%, respectively. The BP raltegravir metabolic ratio (raltegravir glucuronide:raltegravir ratio) was 1.7, suggesting only weak induction of raltegravir glucuronidation by etravirine. Only three patients had etravirine and raltegravir C 12h < PBIC 95 simultaneously., Conclusion: No clinically significant pharmacokinetic interaction between etravirine and raltegravir was detected. Total etravirine and raltegravir BP concentrations were adequate in most patients, favoring virologic efficacy and confirming good treatment adherence (> 95%), despite twice-daily administration. The long half-life of etravirine and higher unbound fraction SP of raltegravir (57%) ensured adequate concentrations of dual therapy in genital compartments. Our results indicate that etravirine and raltegravir have good, complementary pharmacokinetic profiles, suggesting that they could be used in a dual-treatment strategy., (© 2019 Pharmacotherapy Publications, Inc.)

Reproductive organs play a pivotal role in asthma development and progression, especially in women. Endocrine environment changes associated with the menstrual cycle, pregnancy, and menopause can exacerbate the clinical features of asthma. Factors secreted by reproductive organs may be responsible for the gender difference and age-related changes in adult asthma. Here, we show that mammalian seminal fluid has anti-asthma effects exclusively in females. Exposure to murine seminal fluid markedly reduced eosinophilic airway inflammation in 2-month-old female mice upon ovalbumin inhalation. The anti-asthma effect with seminal fluid from 10-month-old males was double that with fluid from 2-month-old males, suggesting that it depended on male sexual maturation. We further found that seminal fluid from middle-aged human volunteers had beneficial effects in asthmatic female mice; these effects were associated with transcriptional repression of osteopontin and IL-17A, which are poor prognostic factors for asthma. In 2-month-old male mice, however, human seminal fluid failed to decrease asthmatic features and even enhanced osteopontin and IL-17A transcription. Our data demonstrate that age-related seminal fluid exerts opposing effects in asthmatic male and female mice. These findings may help the development of novel approaches to control the prevalence and age-related progression of asthma in women.

The male ejaculate contains a multitude of seminal fluid proteins (SFPs), many of which are key reproductive molecules, as well as sperm. However, the identification of SFPs is notoriously difficult and a detailed understanding of this complex phenotype has only been achieved in a few model species. We employed a recently developed proteomic method involving whole-organism stable isotope labelling coupled with proteomic and transcriptomic analyses to characterize ejaculate proteins in the seed beetle Callosobruchus maculatus. We identified 317 proteins that were transferred to females at mating, and a great majority of these showed signals of secretion and were highly male-biased in expression in the abdomen. These male-derived proteins were enriched with proteins involved in general metabolic and catabolic processes but also with proteolytic enzymes and proteins involved in protection against oxidative stress. Thirty-seven proteins showed significant homology with SFPs previously identified in other insects. However, no less than 92 C. maculatus ejaculate proteins were entirely novel, receiving no significant blast hits and lacking homologs in extant data bases, consistent with a rapid and divergent evolution of SFPs. We used 3D micro-tomography in conjunction with proteomic methods to identify 5 distinct pairs of male accessory reproductive glands and to show that certain ejaculate proteins were only recovered in certain male glands. Finally, we provide a tentative list of 231 candidate female-derived reproductive proteins, some of which are likely important in ejaculate processing and/or sperm storage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In forensic investigations, the identification of the cellular or body fluid source of biological evidence can provide crucial probative information for the court. Messenger RNA (mRNA) profiling has become a valuable tool for body fluid and cell type identification due to its high sensitivity and compatibility with DNA analysis. However, using a single marker to determine the somatic origin of a sample can lead to misinterpretation as a result of cross-reactions. While false positives may be avoided through the simultaneous detection of multiple markers per body fluid, this approach is currently limited by the small number of known differentially expressed mRNAs. Here we characterise six novel mRNAs, partly identified from RNA-Seq, which can supplement existing markers for the detection of circulatory blood, semen (with and without spermatozoa), and menstrual fluid: HBD and SLC4A1 for blood, TNP1 for spermatozoa, KLK2 for seminal fluid, and MMP3 and STC1 for menstrual fluid. Respective expression profiles were evaluated by singleplex endpoint reverse transcription polymerase chain reaction (RT-PCR). HBD, SLC4A1, and KLK2 were specific to their target body fluids. TNP1, MMP3, and STC1 each cross-reacted with two non-target samples; however, these signals were below 350RFU, not reproducible, and likely resulted from large body fluid inputs. All candidates were more sensitive for the detection of their target body fluids than corresponding well-known mRNAs, in particular those for menstrual fluid. The increased sensitivities were statistically significant, except for KLK2. Thus, the new mRNAs introduced here are promising new targets for improved body fluid profiling., (Copyright © 2017 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.)

In many species, males can make rapid adjustments to ejaculate performance in response to sperm competition risk; however, the mechanisms behind these changes are not understood. Here, we manipulate male social status in an externally fertilising fish, chinook salmon ( Oncorhynchus tshawytscha ), and find that in less than 48 hr, males can upregulate sperm velocity when faced with an increased risk of sperm competition. Using a series of in vitro sperm manipulation and competition experiments, we show that rapid changes in sperm velocity are mediated by seminal fluid and the effect of seminal fluid on sperm velocity directly impacts paternity share and therefore reproductive success. These combined findings, completely consistent with sperm competition theory, provide unequivocal evidence that sperm competition risk drives plastic adjustment of ejaculate quality, that seminal fluid harbours the mechanism for the rapid adjustment of sperm velocity and that fitness benefits accrue to males from such adjustment.

A change in social status can quickly lead to a change in the quality of the seminal fluid produced by a male Chinook salmon as he responds to increased reproductive competition from higher-status males.

There is considerable evidence that males will increase the number of sperm ejaculated in response to sperm competition risk. However, whether they have the capacity to adjust seminal fluid components of the ejaculate has received less attention. Male crickets ( Teleogryllus oceanicus ) have been shown to adjust the viability of sperm in their ejaculate in response to sperm competition risk. Here we show that socially mediated plasticity in sperm viability is probably due, at least in part, to male adjustments in the protein composition of the seminal fluid. Seven seminal fluid protein genes were found to have an increased expression in males exposed to rival calls. Increased expression of these genes was correlated with increased sperm viability in whole ejaculates, and gene knockdown confirmed that at least one of these proteins promotes sperm viability. Our results lend support for recent theoretical models that predict complex responses in male allocation to seminal fluid composition in response to sperm competition risk., (© 2017 The Author(s).)

Semen is a complex body fluid containing an admixture of spermatozoa suspended in secretions from the testes and epididymis which are mixed at the time of ejaculation with secretions from other accessory sex glands such as the prostate and seminal vesicles. High-throughput technologies have revealed that, contrary to the idea that sperm cells are simply a silent delivery vehicle of the male genome to the oocyte, the sperm cells in fact provide both a specific epigenetically marked DNA together with a complex population of proteins and RNAs crucial for embryogenesis. Similarly, -omic technologies have also enlightened that seminal fluid seems to play a much greater role than simply being a medium to carry the spermatozoa through the female reproductive tract. In the present review, we briefly overview the sperm cell biology, consider the key issues in sperm and seminal fluid sample preparation for high-throughput proteomic studies, describe the current state of the sperm and seminal fluid proteomes generated by high-throughput proteomic technologies and provide new insights into the potential communication between sperm and seminal fluid. In addition, comparative proteomic studies open a window to explore the potential pathogenic mechanisms of infertility and the discovery of potential biomarkers with clinical significance., Significance: The review updates the numerous proteomics studies performed on semen, including spermatozoa and seminal fluid. In addition, an integrative analysis of the testes, sperm and seminal fluid proteomes is also included providing insights into the molecular mechanisms that regulate the generation, maturation and transit of spermatozoa. Furthermore, the compilation of several differential proteomic studies focused on male infertility reveals potential pathways disturbed in specific subtypes of male infertility and points out towards future research directions in the field., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The seed beetle Callosobruchus maculatus is a significant agricultural pest and increasingly studied model of sexual conflict. Males possess genital spines that increase the transfer of seminal fluid proteins (SFPs) into the female body. As SFPs alter female behaviour and physiology, they are likely to modulate reproduction and sexual conflict in this species. Here, we identified SFPs using proteomics combined with a de novo transcriptome. A prior 2D-sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis identified male accessory gland protein spots that were probably transferred to the female at mating. Proteomic analysis of these spots identified 98 proteins, a majority of which were also present within ejaculates collected from females. Standard annotation workflows revealed common functional groups for SFPs, including proteases and metabolic proteins. Transcriptomic analysis found 84 transcripts differentially expressed between the sexes. Notably, genes encoding 15 proteins were highly expressed in male abdomens and only negligibly expressed within females. Most of these sequences corresponded to 'unknown' proteins (nine of 15) and may represent rapidly evolving SFPs novel to seed beetles. Our combined analyses highlight 44 proteins for which there is strong evidence that they are SFPs. These results can inform further investigation, to better understand the molecular mechanisms of sexual conflict in seed beetles., (© 2016 The Royal Entomological Society.)

Sexual assault samples are some of the most common samples encountered in forensic analysis. These samples can require a significant time investment due to differential extraction processes. We report on the first record of successful direct amplification of semen for STR analysis. Neat seminal fluid, dilutions ranging from 1:5 to 1:160 and GEDNAP samples were successfully amplified using a direct method. A mild differential isolation technique to enrich spermatozoa was developed and successfully implemented to separate and directly amplify a mixture of semen and female epithelial cells. Aliquots of samples subjected to the differential isolation protocol were stained with Haemotoxylin and Eosin for sperm scoring. Samples stained after PCR showed a complete lack of intact spermatozoa demonstrating that the cells are lysed during the PCR process. This paper demonstrates the potential to incorporate direct PCR in cases of sexual assault to more rapidly obtain results and achieve a higher sensitivity.

We report on an Ebola virus disease (EVD) survivor who showed Ebola virus in seminal fluid 531 days after onset of disease. The persisting virus was sexually transmitted in February 2016, about 470 days after onset of symptoms, and caused a new cluster of EVD in Guinea and Liberia., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objective: Prostaglandins present in seminal fluid are actively involved in vascular and non-vascular smooth muscle maintenance, reproduction, and inflammatory processes. Seminal plasma contains molecules, such as oxylipins, which possess cell signaling functions. Several studies have shown that specific molecules in seminal fluid can increase passive diffusion, and cause interactions in the female reproductive tract. This may provoke a cascade of cellular and molecular changes in general health and certain diseases. This study examines the hypothesis that the molecules in seminal fluid are involved in platelet activity. The molecules diffuse through cells and membranes, affecting Hoxa 10, binding ganglioside pathways, and acting over platelet function. When these molecules are at low levels, they may trigger prothrombotic states, explaining the pathophysiology of haemostatic response, such as preeclampsia, and increased risk of cardiovascular disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The role of the seminal fluid antioxidant system in protection against damage to spermatozoa during in vitro sperm storage is unclear. This study investigated the effect of in vitro storage of sterlet Acipenser ruthenus spermatozoa together with seminal fluid for 36 h at 4 °C on spermatozoon motility rate and curvilinear velocity, thiobarbituric acid reactive substance level, and components of enzyme and non-enzyme antioxidant system (superoxide dismutase and catalase activity and uric acid concentration) in seminal fluid. Spermatozoon motility parameters after sperm storage were significantly decreased, while the level of thiobarbituric acid reactive substances, activity of superoxide dismutase and catalase, and uric acid concentration did not change. Our findings suggest that the antioxidant system of sterlet seminal fluid is effective in preventing oxidative stress during short-term sperm storage and prompt future investigations of changes in spermatozoon homeostasis and in spermatozoon plasma membrane structure which are other possible reasons of spermatozoon motility deterioration upon sperm storage.

Aims: Denosumab is a fully human monoclonal immunoglobulin G2 antibody that inhibits bone resorption and increases bone mass and strength. The present clinical study assessed serum and seminal fluid pharmacokinetics following a single denosumab dose in healthy men, and evaluated whether denosumab in seminal fluid poses any risk to a fetus in the event of unprotected sexual intercourse with a pregnant partner., Methods: An open-label, single-dose study in 12 healthy men was conducted over a 106-day period. Subjects received a single subcutaneous dose of 60-mg denosumab on day 1. Serum and seminal fluid samples were collected at specified time points to assess denosumab pharmacokinetics. Adverse events were recorded., Results: Denosumab was measurable at low concentrations in seminal fluid (~2% of serum concentrations). The mean [standard deviation (SD)] maximum observed drug concentration (Cmax ) was 6170 (2070) ng ml(-1) (serum) and 100 (81.9) ng ml(-1) (seminal fluid). The median time to Cmax (tmax ) was 8 days (serum) and 21 days (seminal fluid). The mean (SD) area under the plasma concentration-time curve (AUC) from time zero to the time of the last quantifiable concentration (AUClast ) was 333 000 (122 000) day•ng ml(-1) (serum) and 5220 (4880) day•ng ml(-1) (seminal fluid). The mean (SD) Cmax and AUC ratios between seminal fluid and serum were 0.0217 (0.0154) and 0.0170 (0.0148), respectively. Using conservative assumptions for ejaculate volume (6 ml), vaginal absorption (100%) and placental transfer (100%), the measured mean denosumab seminal fluid Cmax would result in fetal exposure that was more than 110 times below the preclinically derived 'no effect level' for denosumab., Conclusions: These results indicate a negligible risk to a fetus exposed to denosumab via seminal fluid transfer to a pregnant partner., (© 2015 The British Pharmacological Society.)

The increasing use of "-omics" (genomic, transcriptomic, proteomic, epigenomic, and metabolomic) high-throughput measurement technologies over the past decade is beginning to reveal the complexity of human biology and physiology through the interactions of DNA, RNA, related proteins and small molecules. In reproductive medicine, the majority of this work, has thus far focused on the female factors, e.g., the oocyte, since they provide both the environment and the majority of elements required for embryogenesis. State-of-the-art sequencing and computational analyses have enabled a deeper understanding of the underlying components. Contrary to being simply a silent delivery vehicle to the oocyte of the packaged male DNA, sperm provide both a specific epigenetically marked genome together with a complex population of RNAs and proteins that are crucial for early embryogenesis. In addition to the sperm, seminal fluid appears to serve multiple roles providing a supplementary series of components that allow the sperm to successfully reach and fertilize the oocyte and prepare the female immune system to tolerate the semiallosteric embryo. A global analysis and review of what is presently known regarding the unique role of each component of the male factor and their associated interactions begins to shed light on this emergent field.

Seminal fluid induces pro-inflammatory cytokines and elicits an inflammation-like response in the cervix. Here, Affymetrix microarray and qPCR was utilised to identify activin A (INHBA) and its inhibitor follistatin (FST) amongst the cytokines induced by seminal plasma in Ect1 ectocervical epithelial cells, and a similar response was confirmed in primary ectocervical epithelial cells. TGFB is abundant in seminal plasma and all three TGFB isoforms induced INHBA in Ect1 and primary cells, and neutralisation of TGFB in seminal plasma suppressed the INHBA response. Bacterial lipopolysaccharide in seminal plasma also elicited INHBA, but potently suppressed FST production. There was moderate reciprocal inhibition between FST and INHBA, and cross-attenuating effects were seen. These data identify TGFB and potentially LPS as factors mediating seminal plasma-induced INHBA synthesis in cervical cells. INHBA and FST induced by seminal fluid in cervical tissues may thus contribute to regulation of the post-coital response in women., (Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.)

Parameters of LPO reflecting primary and end stages of oxidation and total antioxidant activity were evaluated in the seminal fluid of young fertile men living in Novosibirsk. By the results of andrological examination and spermogram analysis, various reproductive disorders and abnormal spermogram (pathospermia) were found in 48 patients. Shifts towards activation of prooxidant processes in LPO-antioxidant defense metabolic system were observed in men with reproductive disorders and pathospermia. The results of individual estimation of coefficient of oxidative stress, an integral parameter reflecting the balance in the LPO-antioxidant defense system, attest to the development of oxidative stress in men with reproductive disorders and pathospermia.

Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce expression of proinflammatory cytokines and chemokines, initiating immune tolerance mechanisms to facilitate pregnancy. TGFB cytokines are key signaling agents in seminal plasma but do not fully account for the female response to seminal fluid. We hypothesized that additional molecular pathways are utilized in seminal fluid signaling. Affymetrix microarray was employed to compare gene expression in the endometrium of mice 8 h after mating with either intact males or seminal fluid deficient (SVX/VAS) males. Bioinformatics analysis revealed TLR4 signaling as a strongly predicted upstream regulator activated by the differentially expressed genes and implicated TGFB signaling as a second key pathway. Quantitative PCR and microbead data confirmed that seminal fluid induces endometrial synthesis of several TLR4-regulated cytokines and chemokines, including CSF3, CXCL1, CXCL2, IL1A, IL6, LIF, and TNF. In primary uterine epithelial cells, CSF3, CXCL1, and CXCL2 were strongly induced by the TLR4 ligand LPS but suppressed by TGFB, while IL1A, TNF, and CSF2 were induced by both ligands. TLR4 was confirmed as essential for the full endometrial cytokine response using mice with a null mutation in Tlr4, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 contributes to seminal fluid modulation of the periconception immune environment. Activation of TLR4 signaling by microbial or endogenous components of seminal fluid is thus implicated as a key element of the female tract response to seminal fluid at the outset of pregnancy in mice., (© 2015 by the Society for the Study of Reproduction, Inc.)

Forensic DNA testing is grounded in molecular biology and population genetics. The technologies that were the basis of restriction length polymorphism testing (RFLP) have given way to PCR based technologies. While PCR has been the pillar of short tandem repeat (STR) methods and will continue to be used as DNA sequencing and analysis of single nucleotide polymorphisms (SNPs) are introduced into human identification, the molecular biology techniques in use today represent significant advances since the introduction of STR testing. Large forensic laboratories with dedicated research teams and forensic laboratories which are part of academic institutions have the resources to keep track of advances which can then be considered for further research or incorporated into current testing methods. However, many laboratories have limited ability to keep up with research advances outside of the immediate area of forensic science and may not have access to a large university library systems. This review focuses on filling this gap with respect to areas of research that intersect with selected methods used in forensic biology. The review summarizes information collected from several areas of the scientific literature where advances in molecular biology have produced information relevant to DNA analysis of sexual assault evidence and methods used in presumptive and confirmatory identification of semen. Older information from the literature is also included where this information may not be commonly known and is relevant to current methods. The topics selected highlight (1) information from applications of proteomics to sperm biology and human reproduction, (2) seminal fluid proteins and prostate cancer diagnostics, (3) developmental biology of sperm from the fertility literature and (4) areas where methods are common to forensic analysis and research in contraceptive use and monitoring. Information and progress made in these areas coincide with the research interests of forensic biology and cross-talk between these disciplines may benefit both., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

From many endangered or threatened species which are expected to profit from assisted reproduction techniques, mainly epididymal sperm of dead or freshly castrated males are available. These sperm had contact to epididymal secretion products but not to seminal fluid components. Notably, products of accessory sex glands have been shown in domestic animals to condition sperm for fertilization, in particular by mediating sperm-oviduct interaction. We report for the first time that motile epididymal sperm from domestic cats are able to bind to fresh oviduct epithelial cell explants from preovulatory females (median [min, max] of 10 [8, 16] and 10 [8, 17] sperm per 0.01 mm(2) explant surface from both isthmic and ampullar regions, respectively). More sperm attach to the explants when epididymal sperm were preincubated for 30 minutes with seminal fluid separated from electroejaculates of mature tomcats (median [min, max] of 17 [13, 25] and 16 [12, 21] sperm per 0.01 mm(2) explant surface from isthmus and ampulla, respectively). The proportion of bound sperm increased from a median of 54% to 62% by seminal fluid treatment. Sperm-oviduct binding could be facilitated by the decelerated sperm motion which was observed in seminal fluid-treated samples or supported by seminal fluid proteins newly attached to the sperm surface. Seminal fluid had no effect on the proportion of sperm with active mitochondria. Extent and pattern of sperm interaction in vitro were independent of explant origin from isthmus or ampulla. Sperm were attached to both cilia and microvilli of the main epithelial cell types present in all explants. In contrast to published sperm-binding studies with porcine and bovine oviduct explants where predominantly the anterior head region of sperm was attached to ciliated cells, the tails of some cat sperm were firmly stuck to the oviduct cell surfaces, whereas the heads were wobbling. Whether this response is a preliminary step toward phagocytosis or a precondition to capacitation and fertilization remains to be determined. In conclusion, treatment of epididymal sperm with seminal fluid or particular protein components should be considered in future investigations for its potential to improve the outcome of artificial insemination in felids., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Paternal antigen-specific regulatory T (PA-specific Treg) cells play an important role in feto-maternal tolerance. To detect the PA-specific Tregs, female BALB/c mice were mated with male DBA/2 mice. Mls Ia antigen on DBA/2 mice is recognized by the T-cell receptor Vβ6; thus, CD4(+)Foxp3(+)Vβ6(+) cells are recognized as PA-specific Treg cells. CD4(+)CD25(+)Vβ6(+) cells effectively suppressed the allo-reactive proliferation of lymphocytes compared with that of CD4(+)CD25(+)Vβ6(-) cells. Vβ6(+) PA-specific Treg cells expressed CCR4 and CCR5 on their surface. The frequency of Ki67(+) PA-specific Treg cells among Treg cells was significantly increased in draining lymph nodes on day 3.5 post-coitus (pc; 6.8±1.1%, p<0.05) and day 5.5 pc (7.2±1.1%, p<0.05) in allogeneic pregnant mice compared with that in nonpregnant mice (2.7±0.2%). The frequency of Ki67(+) PA-specific Treg cells in the uterus increased significantly after day 5.5 pc in allogeneic pregnant mice compared with that in nonpregnant mice (8.8±2.8% vs. 1.2±1.3%, p<0.05). However, Ki67(-)PA-specific Tregs did not change during pregnancy. To analyze the role of seminal fluid or sperm in Treg expansion, female BALB/c mice were mated with vasectomized DBA/2 male mice (VAS) or seminal vesicle-excised DBA/2 male mice (SVX). The frequency of Ki67(+) PA-specific Treg cells did not increase in draining lymph nodes or uterus in BALB/c×DBA/2 (SVX) allogeneic mating mice. These findings suggest that the priming by seminal fluid is important for the induction of proliferating PA-specific Tregs in uterine-draining lymph nodes just before implantation and pregnant uterus after implantation, resulting in successful implantation and the maintenance of allogeneic pregnancy., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Introduction: Postorgasmic illness syndrome (POIS) is a rarely described syndrome characterized by transient flu-like symptoms and cognition disorders. Recent studies suggest that immunogenic reactivity to autologous semen is the underlying mechanism in POIS. However, there are no data published on immunoglobulin E (IgE)-mediated allergy to autologous semen in men without POIS., Aim: The purpose of the current work was to characterize the first diagnosed POIS patient in China and to study the allergic response of autologous semen in the affected patient and in three healthy males., Methods: Specific IgE was tested with seminal fluid and common perennial aeroallergens in vitro. Skin prick tests and intracutaneous tests with autologous diluted semen were performed in the patient and three healthy donors. The pattern of IgE reactivity to patient's semen was identified using immunoblotting and ELISA., Main Outcome Measure: Clinical features of POIS, skin reactions with autologous diluted seminal fluid, and the IgE reactivity patterns of immunoblotting and ELISA in vitro., Results: A patient was diagnosed with POIS. The patient complained of lifelong premature ejaculation symptoms and allergic rhinitis. Routine laboratory and hormonal assessments were generally within normal range. The patient had a positive skin test with his own semen. Three healthy donors also showed positive skin tests. No semen-specific IgE to autologous semen was detected in the serum of the affected patient or healthy males., Conclusions: This is the first report of a man with POIS in China. He had positive skin reactions after injection of autologous seminal fluid but no detectable serum concentrations of specific IgE antibodies. IgE-mediated semen allergy in men may not be the potential mechanism of POIS., (© 2015 International Society for Sexual Medicine.)

Reproductive interference through mating between related species can cause fitness reduction and affect population dynamics of the interacting species. In experimental matings between two seed beetles, Callosobruchus chinensis and Callosobruchus maculatus, C. maculatus females, but not C. chinensis females, suffer from significant loss of fecundity when conspecific mating is followed by heterospecific mating. We hypothesized that male traits associated with sexual conflict, which are often harmful to females, pleiotropically affect fitness of heterospecific females through interspecific mating. We examined the effect of ejaculate of C. chinensis males on C. maculatus females as the cause of the fecundity loss in C. maculatus females due to interspecific copulation. We found that frequent interspecific copulation occurred between C. maculatus females and C. chinensis males, but not between C. chinensis females and C. maculatus males, resulting in frequent interspecific ejaculate transfer from C. chinensis males to C. maculatus females. However, injection of the extract from C. chinensis male reproductive organs into C. maculatus females did not significantly affect C. maculatus fecundity compared with saline injection, indicating that the effect of the heterospecific ejaculate transfer on fecundity is negligible. We suggest that other harmful male traits such as genital spines of C. chinensis males are mainly responsible for the fecundity reduction in C. maculatus females that have experienced interspecific mating., (Copyright © 2014 Elsevier Ltd. All rights reserved.)