Special Issue "Molecular Immunology of the Male Reproductive System".

The immunological aspects of male infertility have gradually become the focus of both basic and clinical research [...].

Corrigendum: Editorial: Spermatogenesis: from stem cells to spermatozoa.

[This corrects the article DOI: 10.3389/fendo.2023.1224313.].

Myd88 Signaling Is Involved in the Inflammatory Response in LPS-Induced Mouse Epididymitis and Bone-Marrow-Derived Dendritic Cells.

Epididymitis is an epididymal inflammation that may lead to male infertility. Dendritic cells (DCs) and myeloid differentiation primary response gene 88 (Myd88) were associated with epididymitis in rodents. However, the functions of Myd88 on epididymal DCs remain unclear. This study investigated the role of Myd88 in DCs for epididymitis. The Myd88 signaling pathway, phenotypes of DC subsets, and cytokines were investigated in lipopolysaccharide (LPS)-induced epididymitis in mice. CRISPR-Cas9 was used to knockout Myd88 in bone-marrow-derived dendritic cells (BMDCs) and immortalized mouse epididymal (DC2) cell line. In the vivo experiments, levels of the proinflammatory cytokines IL-1α, IL-6, IL-17A, TNF-α, IL-1ß, MCP-1, and GM-CSF, mRNA for MyD88 related genes, and the percentages of monocyte-derived DCs (Mo-DCs) were significantly elevated in mice with epididymitis. In the vitro experiments, LPS significantly promoted the apoptosis of BMDCs. In addition, the concentration of inflammatory cytokines in BMDCs and DC2s were increased in the LPS group, while decreasing after the knockout of Myd88. These findings indicate that Myd88 on DCs is involved in the inflammation of epididymitis in mice, which may be a potential target for better strategies regarding the treatment of immunological male infertility.

Natural killer and NKT cells in the male reproductive tract.

Natural killer (NK) cells are important effector lymphocytes that play a pivotal role in the innate and adaptive immune responses to tumors and viral infection. NKT cells are a heterogeneous group of T cells that share properties with both T cells and NK cells. They display immunoregulatory properties as they facilitate the cell-mediated immune response to tumors and infectious diseases, and inhibit cell-mediated immunity associated with autoimmune diseases and allograft rejection. However, the roles of NK and NKT cells in the male reproductive tract remain largely unexplored, in particular, NKT cells, tissue distribution, and state of health or disease. Infection and inflammation of the male genital tract are thought to be the primary etiological factors of male infertility. In this review, we considered this complex and rapidly growing field. We summarize the recent findings and the characterization and roles of NK and NKT cells in the male reproductive tract, including the testis, epididymis, prostate, seminal vesicle, and semen, to enhance our understanding of the immunological mechanisms of male infertility and for the design effective vaccines for male reproductive health in the future.

Human spermatozoa of male patients with subfertility express the interleukin-6 receptor.

Male subfertility has been associated with bacterial infections and chronic inflammation. In this context, several studies investigated cytokine levels in seminal plasma, whereas interleukin-6 (IL-6) appears to be crucial. However, little is known about its receptor, the IL-6R expression on human spermatozoa. Thus, the aim of the present study was to screen spermatozoa for IL-6R expression and to identify its localisation. Semen samples of 137 patients (median age 37.69, SD ± 7.82) with subfertility were analysed. Sperm analysis including determination of IL-6 was performed following the World Health Organization criteria. Also, flow cytometry was performed for sperm IL-6R expression. IL-6R+ cells were used for immunofluorescence staining to identify receptor localisation. The results showed positive staining for IL-6R in the midpiece of spermatozoa. Furthermore, a significant correlation between sperm IL-6R expression, seminal plasma IL-6 and total sperm count could be demonstrated, whereas a negative correlation was observed in sperm IL-6R expression and motility. However, no statistical significance could be observed between IL-6R expression, vitality and morphology. Moreover, incubation of spermatozoa with IL-6 led to a slight but significant decrease in motility after 24 hr. These data suggest that IL-6R expression may play a role in impaired sperm function during inflammation.

Significance of mast cells in spermatogenesis, implantation, pregnancy, and abortion: Cross talk and molecular mechanisms.

Both subsets of MCs including MCTC (tryptase-positive, chymase-positive) and MCT (tryptase-positive, chymase-negative) are present in the testis and epididymis. Increased number of MCs, higher levels of MC-released tryptase in testis and seminal plasma of males with fertility problems, and promoting sperm motility in individuals with oligozoospermia after using MC blockers provide evidence that MCs may play a role in male infertility/subfertility disturbances. MC-released tryptase and histamine contribute to the fibrosis and may disrupt spermatogenesis. MCs not only influence the process of spermatogenesis but also have effects on the function of other testis-residing cells. MC-derived histamine may influence the steroidogenesis of Leydig cells by acting through H1R and H2R receptors. Additionally, the interaction between MC-released ATP and P2X receptors expressed on the peritubular cells may induce the production of the pro-inflammatory mediators by peritubular cells. Further investigations showed that MCs may be involved in the pathology of female infertility during implantation, pregnancy, and abortion. In the uterus, MCT subtype is abundant in myometrium and adjacent basal layer while MCTC subtype is distributed in all layers. MCs in response to hormones mainly estradiol and progesterone become activated and release a wide range of mediators including histamine, VEGF, proteases, and metalloproteinases (MMPs) that have a role in different stages of pregnancy. An increasing influx of MCs to the cervix during the pregnancy occurs that helps to the physiologic cervical ripening. While MMPs degrade the extracellular matrix (ECM), VEGF modulates neovascularization and histamine influences the embryo implantation. MC-derived histamine may have a positive effect during implantation due to its participation in tissue remodeling. MC proteases including tryptase and chymase activate the precursors of MMP2 and MMP9 to mediate ECM degradation during the physiologic menstrual cycle. There is a line of evidence that MCs have a role in abortion by releasing TNF-α.

Low serum testosterone levels in male psoriasis patients correlate with disease severity.

Psoriasis is a chronic inflammatory skin disease with a genetic and autoimmune background. The involvement of sex hormones as a trigger factor for psoriasis has been suspected. Recently, low serum testosterone has been associated with autoimmune diseases in males, and the role of testosterone in psoriasis is unknown. To investigate serum testosterone levels in male psoriasis patients compared to control individuals with regards to the severity of psoriasis. A total of 121 male psoriasis patients and 217 control individuals were enrolled. The severity of psoriasis was documented using the Psoriasis Area Severity Index (PASI). Serum testosterone, sex hormone binding globulin (SHBG), and albumin were analysed. Moreover, psoriasis medication and the incidence of metabolic syndrome were recorded. In 52.1% psoriasis patients, low total testosterone values were detected. Compared to the control cohort, total testosterone (tT) and free testosterone (fT) in psoriasis patients were significantly lower. Despite psoriasis-specific medication, there was a significant inverse correlation between tT or fT and PASI, irrespective of age above or below 40 years. Low tT levels also correlated with the prevalence of metabolic syndrome. Nevertheless, in psoriasis patients without metabolic syndrome, higher PASI (≥10) was associated with significantly lower tT values. In addition, low tT was associated with clinical symptoms of testosterone deficiency. Severe psoriasis is associated with low serum testosterone. However, further studies are required to investigate whether this observation is an epiphenomenon and whether testosterone substitution might decrease the severity of psoriasis.

Male Oxidative Stress Infertility (MOSI): Proposed Terminology and Clinical Practice Guidelines for Management of Idiopathic Male Infertility.

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm's potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause.

Therapeutic options in male genital tract inflammation.

Chronic inflammatory conditions of the genital tract are still unsatisfactorily recognised in the workup of male infertility due to inappropriate definitions and inconsistent diagnostic criteria. The most popular term used for description of both, infections and inflammation in the genital tract is MAGI (male accessory gland infection). In asymptomatic patients, the diagnosis is primarily based on leucocytospermia (i.e., more than 1 million peroxidase-positive leucocytes per ml ejaculate), although ongoing infections should be identified and distinguished from post-infectious or non-infectious inflammatory disease. In addition to alterations of the basic semen parameters, sperm functions -and DNA integrity may be affected by chronic inflammation of the male genital tract. Despite considerable diagnostic drawbacks and a rather limited database concerning evidence-based therapy, adequate management of affected patients appears mandatory. Antibiotic treatment aims at the eradication or reduction of pathogenic bacteria in the ejaculate. Available studies suggest, that NSAID are effective in chronic inflammatory conditions. Moreover, low-dose corticosteroids, mast cell blockers, and other immune-modulatory compounds as well as a sequential adjuvant treatment with antioxidants can be considered as therapeutic options.

[Skin diseases and fertility/hormone disorders]. / Hauterkrankungen und Fertilitäts­/Hormonstörungen.

Male fertility can be impaired by a multitude of factors. In addition to environmental and life style factors, such as stress, noise, smoking and overweight, diverse diseases can also have a negative effect on the ability to father a child and the hormone balance, particularly the testosterone level. In many diseases the currently available data do not go beyond observations of limited fertility. In this article the focus is on diseases in the treatment field of dermatology. Special attention is paid to chronic inflammatory and autoimmune skin diseases. Data from recent years show that the excessive inflammatory reaction that these diseases have in common, most probably also has an influence on fertility and interacts with the testosterone concentration in serum. In addition, the impact of hereditary skin diseases on male fertility is discussed, which can have a direct influence on the ability to father a child due to disruption of the hypothalamus-pituitary-gonad axis.

Detection of invariant natural killer T cells in ejaculates from infertile patients with chronic inflammation of genital tract.

Chronic inflammation of genital tract is thought to play a major role in male fertility disorder. Natural killer (NK) T cells are a heterogeneous group of T cells that share properties of both T cells and NK cells which display immunoregulatory properties. However, little is known regarding the presence and function of NK T cells in ejaculates from patients with chronic inflammation of genital tract. Invariant NK T (iNK T) cells were detected by invariant (Vα24-JαQ) TCR chain in ejaculates from patients suffering from chronic inflammation of genital tract (CIGT) using flow cytometry and immunofluorescence of double staining (n=40). Inflammatory cytokines interleukin (IL)-6, IL-17, and IFN-γ were detected in cell-free seminal plasma using an enzyme-linked immunosorbent assay (ELISA). The correlation between the percentage of iNK T cells and spermatozoa count, motility, vitality, seminal IL-6, IL-17, and IFN-γ was investigated. Significant percentages of iNK T cells above 10% were detected in 50% (CIGT-NKT+ group). A negative correlation was detected between the percentage of iNK T cells and spermatozoa count (r=-.5957, P=.0056), motility (r=-.6163, P=.0038), and vitality (r=-.8032, P=.0019) in CIGT-NKT+ group (n=20). Interestingly, a significant correlation of iNK T cells to seminal IL-6 (r=.7083, P=.0005), IFN-γ (r=.9578, P<.0001) was detected whereas lack of correlation between iNK T cells and IL-17 (r=-.1557, P=.5122) in CIGT-NKT+ group. The proliferative response of iNK T cells could accompany an inflammatory response to spermatozoa and consequently influence sperm quality through secretion of IFN-γ but not IL-17 under chronic inflammatory condition.

Correction: Methylation Markers for the Identification of Body Fluids and Tissues from Forensic Trace Evidence.

[This corrects the article DOI: 10.1371/journal.pone.0147973.].

Methylation Markers for the Identification of Body Fluids and Tissues from Forensic Trace Evidence.

The identification of body fluids is an essential tool for clarifying the course of events at a criminal site. The analytical problem is the fact that the biological material has been very often exposed to detrimental exogenous influences. Thereby, the molecular substrates used for the identification of the traces may become degraded. So far, most protocols utilize cell specific proteins or RNAs. Instead of measuring these more sensitive compounds this paper describes the application of the differential DNA-methylation. As a result of two genome wide screenings with the Illumina HumanMethylation BeadChips 27 and 450k we identified 150 candidate loci revealing differential methylation with regard to the body fluids venous blood, menstrual blood, vaginal fluid, saliva and sperm. Among them we selected 9 loci as the most promising markers. For the final determination of the methylation degree we applied the SNuPE-method. Because the degree of methylation might be modified by various endogenous and exogenous factors, we tested each marker with approximately 100 samples of each target fluid in a validation study. The stability of the detection procedure is proved in various simulated forensic surroundings according to standardized conditions. We studied the potential influence of 12 relatively common tumors on the methylation of the 9 markers. For this purpose the target fluids of 34 patients have been analysed. Only the cervix carcinoma might have an remarkable effect because impairing the signal of both vaginal markers. Using the Illumina MiSeq device we tested the potential influence of cis acting sequence variants on the methylation degree of the 9 markers in the specific body fluid DNA of 50 individuals. For 4 marker loci we observed such an influence either by sole SNPs or haplotypes. The identification of each target fluid is possible in arbitrary mixtures with the remaining four body fluids. The sensitivity of the individual body fluid tests is in the same range as for the forensic STR-analysis. It is the first forensic body fluid protocol which considers the exogenic and endogenic parameters potentially interfering with the true results.

Autoantibodies against protein disulfide isomerase ER-60 are a diagnostic marker for low-grade testicular inflammation.

STUDY QUESTION: Is there a non-invasive biomarker for the diagnosis of testicular inflammatory lesions? SUMMARY ANSWER: In sera from infertile azoospermic patients with histologically confirmed low-grade testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60) were found. WHAT IS KNOWN ALREADY: Infection and inflammation of the genital tract are supposed to be responsible for up to 15% of cases among infertile males. However, specific seminal or serological markers are not available to assess subacute or chronic inflammatory conditions in the testis. STUDY DESIGN, SIZE, DURATION: This study consisted of the identification of autoantibodies for testicular antigens in sera of patients with low-grade testicular inflammation, validation of candidates, development of an ELISA for the most promising target antigen and measurement of autoantibodies titers in healthy normozoospermic men (n = 20); male blood donors (n = 14); men with impaired semen quality without (n = 14) or with (n = 26) symptoms of genital tract infection/inflammation; azoospermic men with histologically confirmed testicular inflammatory lesions (n = 16); men after pharmacotherapy of genital tract infection/inflammation (n = 15) and men with acute epididymo-orchitis (n = 30). PARTICIPANTS/MATERIALS, SETTING, METHODS: Proteins in lysates of normal testicular tissue were separated by high-resolution 2D gel electrophoresis and probed with sera of 13 patients with histologically confirmed chronic testicular inflammation. There were 14 proteins that immunoreacted with a majority of these sera and could be identified by mass spectrometry. Of these 14 proteins, disulfide isomerase family A, member 3 (ER-60), transferrin and chaperonin containing TCP1 complex, subunit 5 (epsilon) (CCT5) were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed. MAIN RESULTS AND THE ROLE OF CHANCE: The newly established ELISA detected significantly elevated titers of autoantibodies against ER-60 in the sera from infertile men with histologically confirmed chronic testicular inflammation (median 8.6; P < 0.01) compared with the control groups. Moreover, elevated levels of anti-ER-60 titers were detected in patients suffering from acute epididymo-orchitis (median 3.3; P < 0.05) as compared with healthy normozoospermic men (median 2.13; P < 0.001), male blood donors with unknown fertility status (median 2.72; P < 0.01), patients with impaired semen quality but no infection/inflammation (median 2.59; P < 0.001) and patients with symptoms of genital tract infections and/or inflammation (median 2.18; P < 0.001). Significantly lower levels of anti-ER-60 antibodies were measured in sera from patients after application of anti-inflammatory pharmacotherapy (median 1.9; P < 0.01) compared with those with histologically confirmed chronic testicular inflammation. The cut-off value of the assay was set to 6.6 U/ml based on a calculated sensitivity of 100% and a specificity of 81.2%. LIMITATIONS, REASONS FOR CAUTION: The results obtained in this study showed statistically significant elevated titers of ER-60 antibodies in sera from patients with histologically confirmed testicular inflammatory lesions and from a few patients with acute epididymo-orchitis. However, the number of serum samples tested was limited. Severe testicular damage seen in azoospermic patients could represent a bias towards ER-60 reactivity, while the assay does not allow for different etiologies of the lesions to be distinguished. Due to ethical reasons, the prevalence of testicular inflammatory lesions among controls and non-azoospermic men cannot be studied at the histological level. WIDER IMPLICATIONS OF THE FINDINGS: Measurement of ER-60 autoantibody titers in serum could be a novel non-invasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant of the Deutsche Forschungsgemeinschaft (ME 1323/4-4) and the Translational Science Fund (Wirtschafts-und Strukturbank Hessen-WI Bank). M.F., A.P., W.W., H.-C.S. and A.M. are supported by the LOEWE focus group 'MIBIE' (Male infertility during infection and inflammation). The ER-60 ELISA is protected by a patent to the Justus-Liebig-University of Giessen with A.M. and M.F. as inventors (patent no. DE 10 2008 053 503). T.Z. as employee of the DRG Company was responsible for the ELISA development.

Influence of reactive oxygen species on human sperm functions and fertilizing capacity including therapeutical approaches.

BACKGROUND: Reactive oxygen species (ROS) are an array of molecules including oxygen-centered radicals, which are endowed with one or more unpaired electrons and non-radical oxygen derivatives such as hydrogen peroxide, which behave, to a large extent, like a double-edged sword in human sperm biology. This study aimed to overview the current knowledge of ROS in sperm physiology and pathology, as well as related therapies in spermatozoal dysfunction. METHODS: We performed this study by searching for keywords from PUBMED, including reactive oxygen species, oxidative stress, sperm function, and antioxidant therapy. RESULTS AND CONCLUSIONS: Low levels of ROS exert critical function in normal sperm physiology, such as fertilizing ability (acrosome reaction, hyperactivation, capacitation, and chemotaxis) and sperm motility; while increased ROS generation and/or decreased antioxidant capacity leads to the imbalance between oxidation and reduction in living systems, which is called sperm oxidative stress. This condition was widely considered to be a significant contributory factor to sperm DNA damage/apoptosis, lipid peroxidation, and reduced motility, which in turn, increased risk of male factor infertility/subfertility and birth defects. Under the current status quo, numerous subsequent studies have concentrated on antioxidant therapy. Although utility of such a therapeutic strategy significantly improved sperm function and motility in a myriad of experimental and clinical reports, the overall effectiveness still remains controversial mainly due to non-standardized assay to measure the level of ROS and sperm DNA damage, various antioxidant supplementation strategies, and inadequate fertilization and pregnancy data after clinical treatment. Therefore, standardized assessment and evaluation of ROS and total antioxidant capacity in semen should be established to keep ROS in a physiological level and prevent over-treatment of antioxidants toward reductive stress, which should be kept in mind, especially in assisted reproductive procedure. Moreover, the significance of large sample size populations, double-blind randomized, placebo-controlled clinical trials of antioxidant therapies is emphasized in this review to achieve optimal ingredients and dosage of antioxidants for patients with reactive oxygen-induced male fertility/subfertility.