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Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Experimental autoimmune-orchitis (EAO), a rodent model of chronic testicular inflammation and fibrosis, replicates pathogenic changes seen in some cases of human spermatogenic disturbances. During EAO, increased levels of pro-inflammatory and pro-fibrotic mediators such as TNF, CCL2, and activin A are accompanied by infiltration of leukocytes into the testicular parenchyma. Activin A levels correlate with EAO severity, while elevated CCL2 acting through its receptor CCR2 mediates leukocyte trafficking and recruits macrophages. CCR2 + CXCR4 + macrophages producing extracellular matrix proteins contribute widely to fibrogenesis. Furthermore, testicular macrophages (TMs) play a critical role in organ homeostasis. Therefore, we aimed to investigate the role of the activin A/CCL2-CCR2/macrophage axis in the development of testicular fibrosis. Following EAO induction, we observed lower levels of organ damage, collagen deposition, and leukocyte infiltration (including fibronectin+, collagen I+and CXCR4+TMs) inCcr2−/−mice than inWTmice. Furthermore, levels ofIl-10,Ccl2, and the activin A subunitInhbamRNAs were lower inCcr2−/−EAO testes. Notably, fibronectin+TMs were also present in biopsies from patients with impaired spermatogenesis and fibrotic alterations. Overexpression of the activin A antagonist follistatin reduced tissue damage and collagen I+TM accumulation inWTEAO testes, while treating macrophages with activin A in vitro increased the expression ofCcr2,Fn1,Cxcr4,andMmp2and enhanced migration along a CCL2 gradient; these effects were abolished by follistatin. Taken together, our data indicate that CCR2 and activin A promote fibrosis during testicular inflammation by regulating macrophage function. Inhibition of CCR2 or activin A protects against damage progression, offering a promising avenue for therapeutic intervention.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a high prevalence of up to 15% and accounts for 90-95% of prostatitis diagnoses, and yet its etiopathogenesis and link to prostate cancer (PCa) are still unclear. Here, we investigated microRNAs in exosomes isolated from blood and post-prostatic-massage urine of CP/CPPS type IIIb patients and healthy men. THP-1 monocytes (human leukemia monocytic cell line) were treated with exosomes and subjected to mRNA arrays "Cancer Inflammation and Immunity Crosstalk" and "Transcription Factors." Using The Cancer Genome Atlas, the expression of CP/CPPS-associated microRNAs was analyzed in PCa and normal prostate tissue. In silico functional studies were carried out to explore the disease ontology of CP/CPPS. In CP/CPPS, urine exosomes exhibited significant upregulation of eight PCa-specific microRNAs (e.g., hsa-miR-501, hsa-miR-20a, and hsa-miR-106), whose target genes were significantly enriched for GO terms, hallmark gene sets, and pathways specific for carcinogenesis. In THP-1 monocytes, CP/CPPS-derived urine exosomes induced upregulation of PCa-associated proinflammatory genes (e.g., CCR2 and TLR2) and proto-oncogene transcription factors (e.g., MYB and JUNB). In contrast, CP/CPPS-derived blood exosomes exhibited molecular properties similar to those of healthy men. Thus, CP/CPPS exhibits molecular changes that constitute a risk for PCa and should be considered in the development of PCa biomarkers and cancer screening programs.

The identification of potential environmental hazards is of clinical relevance for the diagnosis of male infertility. Knowledge about these factors will improve prevention of fertility disorders. Apart from drugs or factors related to lifestyle such as alcohol and tobacco smoke, various environmental and occupational agents, both chemical and physical, may impair male reproduction. Reproductive toxicity may evolve at the hypothalamic-pituitary, testicular, or posttesticular level; endpoints comprise deterioration of spermatogenesis and sperm function as well as endocrine disorders and sexual dysfunction. However, due to the complex regulation of the male reproductive system, information regarding single exogenous factors and their mechanisms of action in humans is limited. This is also due to the fact that extrapolation of results obtained from experimental animal or in vitro studies remains difficult. Nevertheless, the assessment of relevant exposures to reproductive toxicants should be carefully evaluated during diagnostic procedures of andrological patients.Bei der Erfassung kausaler Faktoren männlicher Fertilitätsstörungen spielen Lebensstil und Umweltfaktoren eine wichtige Rolle. Das Wissen um die Wechselwirkungen zwischen derartigen Einflüssen und reproduktiven Funktionen des Mannes eröffnet zudem die Möglichkeit eines präventiven Ansatzes in der Andrologie. Relevante exogene Noxen sind Genussgifte, Pharmaka einschließlich Lifestyle-Medikamenten, Berufsstoffe, Umweltchemikalien sowie physikalische Faktoren. Reproduktionstoxische Effekte können sich prätestikulär, testikulär oder posttestikulär entfalten und neben einer Beeinträchtigung der Spermatogenese oder Spermienfunktion auch Hormon- und Sexualstörungen verursachen. Aufgrund der komplexen Regulation des männlichen Reproduktionssystems sowie der begrenzten Übertragbarkeit tierexperimenteller Befunde und In-vitro-Daten auf die Situation beim Menschen liegen allerdings nur für wenige Noxen gesicherte Erkenntnisse vor. In der andrologischen Sprechstunde sollten bei Erhebung der Anamnese potenzielle Expositionsrisiken erfasst und die Patienten über eine gesicherte oder mögliche Relevanz für ihre Fertilität aufgeklärt werden.

To study the impact of Doublesex and mab-3-related transcription factor 1 (DMRT1) gene variants on the encoded protein's function and the variants' pathogenic relevance for isolated male infertility caused by azoospermia.This study established a novel luciferase assay for DMRT1 missense variants using 2 different target promotors and validated the assay by analyzing previously published variants associated with differences in sex development.University genetics research institute and tertiary referral center for couples' infertility.Eleven infertile men with severely impaired spermatogenesis resulting in crypto- or azoospermia and carrying rare heterozygous missense variants in DMRT1 were identified within the Male Reproductive Genomics study.Luciferase assays with human DMRT1 variants to test functional effects on the CYP19A1 and Stra8 target promoters.We first developed and refined luciferase assays to reliably test the functional impact of DMRT1 missense variants. Next, the assay was validated by analyzing 2 DMRT1 variants associated with differences in sex development, of which c.240GC p.(Arg80Ser) displayed highly significant effects on both target promoters compared with the wild-type protein (-40% and +100%, respectively) and c.331AG p.(Arg111Gly) had a significant effect on the Stra8 promoter (-76%). We then systematically characterized 11 DMRT1 variants identified in infertile men. The de novo variant c.344TA p.(Met115Lys) showed a pronounced loss of function in both DMRT1 target promoters (-100% and -86%, respectively). Variants c.308AG p.(Lys103Arg) and c.991GC p.(Asp331His) showed a significant gain of function exclusively for the CYP19A1 promoter (+15% and +19%, respectively). Based on these results, 3 variants were reclassified according to clinical guidelines.The present study highlights the importance of functionally characterizing DMRT1 variants of uncertain clinical significance. Using luciferase assays for diagnostic purposes enables an improved causal diagnosis for isolated male infertility.

Sexual health is becoming increasingly important for many HIV-positive men undergoing highly effective antiretroviral therapy (ART) but remains frequently unaddressed in routine clinical consultation.To comprehensively evaluate sexual health in male patients with HIV on stable ART over a 12-month period.The prospectively registered cohort study comprising 87 HIV-positive men on stable ART (median age: 43 years) was conducted between 2011 and 2015 at a university hospital. Patients were enrolled from the outpatient infectious disease unit and underwent an extensive andrological workup to assess parameters of sexual health (questionnaires, sex hormones, ultrasound, 2-glass urine test including semen analysis with microbiological and viral diagnostics). The study period per patient lasted 12 months.The primary endpoint was the impact of chronic HIV infection on sexual health.Although, on average, sexual health was fine at baseline, 56% of the patients reported erectile dysfunction, 28% experienced reduced libido, 5% had hypogonadism, 36% showed at least 1 atrophic testicle with a volume of10 ml, 8% suffered bacterial sexually transmitted infections, 35% had seminal inflammation, and up to 47% showed reduced sperm quality. Sexual satisfaction was linked to mental health (12-Item Short Form Health Survey questionnaire) and International Index of Erectile Function scores. During the study period, the collected parameters on sexual health were generally stable. However, 35% of patients had new sex partners (median: 5 partners), 7% had fathered a child or were planning procreation, 47% reported changed libido, 17% suffered bacterial sexually transmitted infections in the urogenital tract, 16% revealed a positive HIV viral load in blood, 11% had a positive HIV viral load in semen, and 28% were treated for andrological disorders.Sexual ill-health exists in about one third of patients. This manifests itself in sexual dysfunction, sexually transmitted infections, urogenital tract inflammation, and abnormal sperm parameters, all of which require adequate counseling and therapy.The strength of this study is its comprehensive analysis of male sexual health over a 12-month period of stable ART treatment. Limitations are a heterogeneous patient cohort and a rather small percentage of patients with a positive HIV viral load in blood or semen, which prevented multivariate risk analysis.Our study provides evidence that sexual health should be actively taken into account in the routine consultation by infectious disease specialists, and an interdisciplinary approach is desirable in the case of symptoms or signs of sexual ill-health. Pilatz A, Maresch CC, Discher T, et al. Sexual Health in HIV-Positive Men Under Stable Antiretroviral Therapy During a 12-Month Period. J Sex Med 2021;18:284-294.

Bei der Erfassung kausaler Faktoren mannlicher Fertilitatsstorungen spielen Lebensstil und Umweltfaktoren eine wichtige Rolle. Das Wissen um die Wechselwirkungen zwischen derartigen Einflussen und reproduktiven Funktionen des Mannes eroffnet zudem die Moglichkeit eines praventiven Ansatzes in der Andrologie. Relevante exogene Noxen sind Genussgifte, Pharmaka einschlieslich Lifestyle-Medikamenten, Berufsstoffe, Umweltchemikalien sowie physikalische Faktoren. Reproduktionstoxische Effekte konnen sich pratestikular, testikular oder posttestikular entfalten und neben einer Beeintrachtigung der Spermatogenese oder Spermienfunktion auch Hormon- und Sexualstorungen verursachen. Aufgrund der komplexen Regulation des mannlichen Reproduktionssystems sowie der begrenzten Ubertragbarkeit tierexperimenteller Befunde und In-vitro-Daten auf die Situation beim Menschen liegen allerdings nur fur wenige Noxen gesicherte Erkenntnisse vor. In der andrologischen Sprechstunde sollten bei Erhebung der Anamnese potenzielle Expositionsrisiken erfasst und die Patienten uber eine gesicherte oder mogliche Relevanz fur ihre Fertilitat aufgeklart werden.

Eine Azoospermie findet sich bei 10–15 % der Manner, die wegen eines unerfullten Kinderwunschs untersucht werden. Differenzialdiagnostisch sind die obstruktive (OA) und nichtobstruktive Azoospermie (NOA) zu unterscheiden, wobei jeweils kongenitale bzw. genetisch bedingte sowie erworbene Ursachen zugrunde liegen konnen. Am haufigsten fuhren primar den Hoden betreffende Schaden zu einer NOA, hypothalamisch-hypophysar bedingte endokrine Storungen kommen vergleichsweise selten vor. Bei OA sind neben iatrogenen Ursachen wie der Vasektomie vor allem kongenitale Fehlbildungen der ableitenden Samenwege zu berucksichtigen. Diagnosestellung und Therapieplanung erfordern eine umfassende andrologische Untersuchung; soweit keine erworbenen Ursachen einer NOA bzw. OA vorliegen, gehoren zur Abklarung auch eine gezielte genetische Diagnostik und Beratung. Haufig ist eine Einordnung des Krankheitsbilds erst anhand der histopathologischen Untersuchung von Hodenbiopsaten moglich, die auch die Identifizierung einer Keimzellneoplasie in situ erlaubt. Unter therapeutischen Aspekten kommt der Hodenhistologie der groste prognostische Stellenwert im Hinblick auf eine erfolgreiche operative Spermiengewinnung (testikulare Spermienextraktion) zu; bei 40–60 % der Patienten mit NOA lassen sich eine fokal erhaltene Spermatogenese und damit Spermien fur eine assistierte Fertilisation finden. Abgesehen von den kongenitalen, operativ nicht korrigierbaren Fehlbildungen besteht bei OA die Moglichkeit mikrochirurgisch-rekonstruktiver Verfahren. Bei der Versorgung betroffener Patienten durfen keinesfalls konservativ-medikamentos behandelbare Formen einer Azoospermie ubersehen werden; dies gilt insbesondere fur die Therapie des hypogonadotropen Hypogonadismus.

Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth (post-natal day 0; PND0), were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages (F4/80, CD206, MHCII), T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80+ cells were the most abundant, comprising 90% of CD45+ cells at E13.5 and declining to 65% at PND0. Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80+ cells have different origins during embryonic life. CD3+ cells and F4/80−/MHCII+ were absent to rare until PND0. Ly6G+ cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45+ cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80+ and Ly6G+ cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80+ cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80Hi/CD206+) were different from macrophages at PND0 (F4/80Dim/CD206−), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.

Germ cells provide the cellular basis for sexual reproduction in multicellular animals. In males, germ cells differentiate into sperm, one of the most morphologically diverse eukaryotic cell types. Owing both to this remarkable diversity and to the rapid evolution of reproduction-related genes, the transcriptional program of male germ cells is widely regarded as divergent across species1,2. However, the possibility that these cells retain a distinctive evolutionarily-conserved genetic basis remains largely unexplored. Here we show, using phylostratigraphy, that the complex male germ cell transcriptome has an old evolutionary origin shared between vertebrate and invertebrate species. Through multilayer network analysis of the human, mouse and fruit fly meiotic transcriptome, we establish that old genes serve as a genetic scaffold from which complexity has evolved, and identify a core set of 79 ancient functional interactions at the heart of male germ cell identity. By silencing a cohort of 920 candidate genes likely to affect the acquisition and maintenance of this identity, we uncover 164 previously unknown spermatogenesis genes. Integrating this information with whole-exome sequencing data from azoospermic men reveals three novel genetic causes of human male infertility shared between species that have diverged for more than 600 million years. Our results redefine the importance of evolutionary history on human reproductive disease and illustrate the translational potential of a new synergy between comparative network biology and medical genetics. We anticipate that our open-access and easily-adaptable interdisciplinary research platform will be harnessed in the context of other cell types and diseases.

Das Ausmas der Fertilitatsschadigung infolge einer Chemo- und/oder Radiotherapie maligner Erkrankungen ist nicht sicher vorherzusagen, die Chancen einer Erholung der Spermatogenese sind von Therapieregime, Dosis der Noxen und individuellen Faktoren abhangig. Die einzige gesicherte Praventivmasnahme zur Fertilitatsprotektion stellt deshalb die Kryokonservierung von Spermien vor einer gonadotoxischen Behandlung bzw. vor die Reproduktionsorgane betreffenden operativen Eingriffen dar. Diese Option sollte grundsatzlich allen betroffenen Patienten zuganglich sein. Fur prapubertare Jungen mit malignen Erkrankungen kommt die Durchfuhrung einer Hodenbiopsie zur Asservierung testikularer Stammzellen als derzeit noch experimentelles Verfahren in Betracht.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German-language interdisciplinary S2k guideline on “Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)” was published in February 2019. The guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aim In one third of cases, the cause of involuntary childlessness remains unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. There is no standard treatment concept for these patients at present, as there are currently no standard multidisciplinary procedures for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnosis and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations This second part of the guideline describes the hematological workup for women as well as additional diagnostic procedures which can be used to investigate couples and which are carried out in cooperation with physicians working in other medical fields such as andrologists, geneticists and oncologists.

Infertilitat besteht, wenn trotz regelmasigen, ungeschutzten Geschlechtsverkehrs nach 12 Monaten keine Schwangerschaft eingetreten ist. Die Ursachen fur eine ungewollte Kinderlosigkeit liegen zu etwa gleichen Teilen mit je bis zu je 40 % bei Mann und Frau. In den ubrigen Fallen finden sich bei beiden Partnern Faktoren, die die Fertilitat beeintrachtigen konnen. Der Mann sollte daher gleichzeitig mit seiner Partnerin abgeklart werden. Ziel der andrologischen Diagnostik ist es, behandelbare Ursachen fruhzeitig zu erkennen und – wenn moglich – zu behandeln. Gleichzeit kann durch eine rechtzeitige Untersuchung des Mannes eine Verzogerung von Methoden der assistierten Reproduktion verhindert werden. Die intrazytoplasmatische Spermieninjektion erfolgt mehrheitlich aus andrologischer Indikation. Eine andrologische Diagnostik vor Ablauf der 12 Monate kann notwendig werden, wenn die Partnerin uber 35 Jahre alt ist oder Faktoren beim Mann vorliegen, die auf eine eingeschrankte Fertilitat hindeuten.

Eine andrologische Abklarung ist sinnvoll, wenn trotz regelmasigen ungeschutzten Geschlechtsverkehrs nach 12 Monaten keine Schwangerschaft eingetreten ist. Eine andrologische Diagnostik vor Ablauf der 12 Monate kann notwendig werden, wenn die Partnerin uber 35 Jahre alt ist oder Faktoren beim Mann vorliegen, die auf eine eingeschrankte Fertilitat hindeuten. Die Ursachen fur eine ungewollte Kinderlosigkeit liegen zu etwa gleichen Teilen bei Mann und Frau. Der Beginn einer andrologischen Abklarung ist die Erhebung der Anamnese. Erste Angaben dazu konnen bereits abgefragt werden, wenn sich die Partnerin zur Beratung an ihren behandelnden Gynakologen wendet. Der Gynakologe sollte daher Kenntnisse zu relevanten Aspekten der andrologischen Krankenvorgeschichte haben und mit den wesentlichen Fragen vertraut sein, die er selbst stellen kann. Berucksichtigung von Empfehlungen aus Leitlinien und der Weltgesundheitsorganisation. Die andrologische Anamnese umfasst Fragen zur allgemeinen Krankenvorgeschichte, zu Sexualstorungen, spezifischen andrologischen Aspekten, Medikamenteneinnahme, Lebensfuhrung und Berufsleben. Gynakologen haben eine Lotsenfunktion fur das Paar und tragen dazu bei, dass sich die Manner fruhzeitig und gleichzeitig mit ihren Partnerinnen einer Abklarung unterziehen.

STUDY QUESTION Does activin A contribute to testicular fibrosis under inflammatory conditions? SUMMARY ANSWER Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts. WHAT IS KNOWN ALREADY Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease. STUDY DESIGN, SIZE, DURATION This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n = 6), 50 (n = 6) and 80 (n = 5) days after first immunization. Age-matched mice injected with adjuvant alone (n = 14) and untreated mice (n = 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n = 3) or vector with an empty cassette (empty vector controls; n = 2) 30 days prior to the first immunization, as well as appropriate adjuvant (n = 2) and untreated (n = 2) controls, were also examined. Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n = 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n = 7). Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE Production of activin A by SC was stimulated by 25 and 50 ng/ml TNF (P LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts. WIDER IMPLICATIONS OF THE FINDINGS Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government’s Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1–2) on `Molecular pathogenesis on male reproductive disorders’ funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests.

The presence of bacteria and/or leukocytes can alter semen quality resulting in low sperm quality and infertility. Inflammation or infection increases the numbers of PMN or macrophages/monocytes in male genital tract. Release of extracellular traps (ETs) by leukocytes has been recognized as a novel mechanism of early host innate immunity, in response to invasive pathogens. This is the first work that evaluated the mechanism of triggered ETs in monocytes co-incubated with spermatozoa or bacteria and the effect on sperm function. Selected spermatozoa and human monocytes isolated from peripheral blood were obtained by healthy donors. Two experimental models were developed, one aseptic (non-infectious) incubating spermatozoa and monocytes, and septic models (infectious) incubating spermatozoa with monocytes and uropathogenic Escherichia coli (E. coli). ETs of monocytes (METs) (DNA, global histone and citrullinated histones) were visualized by scanning electron microscopy (SEM) and immunofluorescence analyses. Progressive motility was performed at 0, 10, 30, 60, and 180 min after co-incubation with CASA system. SEM- and immunofluorescence-analyses revealed human spermatozoa alone or in the presence of E. coli as strong inducers METs. In aseptic model, the motility decreased to 65.2 ± 3.5% at 10 min of incubation and 29.3 ± 3.3% at 30 min (p < 0.001). In septic model, motility decreased to 44.5 ± 5.9% (10 min) and 12.7 ± 2.2% (30 min) (p < 0.001). MET-derived small spermatozoa aggregations were observed in both models. METs might physically block spermatozoa and decrease motility after a brief contact. This may impair male fertility, especially in patients with genital tract infections or chronic inflammation. Abbreviations: PMN: polymorphonuclear; ETs: extracellular traps; E. coli: Escherichia coli; METs: ETs of monocytes; SEM: scanning electron microscopy; NE: neutrophil elastase; MPO: myeloperoxidase; MAGI: male accessory gland infection; PBMC: peripheral blood mononuclear cells; RT: room temperature; CFU: colony forming units; CASA: computer-aided sperm analysis; H4Cit3: histone H4 citrullinated 3.

Background: Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a frequent disease affecting men of every age and accounting for a great number of consultations at urology departments. Previous studies suggested a negative impact of CP/CPPS on fertility. As increasing attention has been attributed to additional aspects, such as sperm DNA integrity and sperm protein alterations, besides the WHO standard semen analysis when assessing male fertility, in this prospective study, we aimed to further characterize the fertility status in CP/CPPS patients with a focus on these parameters. Methods: Sperm DNA fragmentation measured by sperm chromatin structure assay (SCSA) and protamine 1 to protamine 2 mRNA ratio assessed by RT-qPCR were analyzed along with conventional ejaculate parameters and inflammatory markers in 41 CP/CPPS patients and 22 healthy volunteers. Results: We found significant differences between the groups concerning multiple conventional ejaculate parameters. A significant increase in sperm DNA fragmentation was shown in CP/CPPS patients with association to other sperm parameters. The majority of CP/CPPS patients exhibited protamine mRNA ratios out of the range of regular fertility. Conclusions: This is a pioneering study with a strong practical orientation revealing that CP/CPPS leads to increased sperm DNA damage and changes in sperm protamine levels, emphasizing an unfavorable impact of CP/CPPS on fertility.

PURPOSE: To study the effector mechanism against pathogens of polymorphonuclear neutrophils (PMN) and macrophages, called ETosis, involving the release of extracellular traps (ETs) in patients with acute epididymitis. To assess the different ET phenotypes present in semen samples and to identify correlations between ETosis and clinical parameters. MATERIALS AND METHODS: Samples from patients diagnosed with acute epididymitis were examined and compared with samples from uninfected controls. Biochemical analyses of seminal fluid included determination of peroxidase, α-glucosidase, fructose, and elastase levels. ETosis in semen was determined through presence of citrullinated histones, global histones, and extracellular DNA. Different ETosis phenotypes such as spread ETs, aggregated ETs, and diffuse ETs were identified by co-localisation of extruded DNA with myeloperoxidase and global histones. Anti-CD15(+) and anti-CD68(+) antibodies were used to identify different cell lines. RESULTS: Revealed a high number of ETs compared with the control group. The mean number of CD15(+)PMN and CD68(+) macrophages was higher in the acute epididymitis group. ETosis increase in ejaculates correlated with clinical parameters such as enhancement of elastase concentrations and diminution of fructose in the semen. CONCLUSIONS: This work shows for the first time the presence of ETs and their components in semen from patients with acute epididymitis. The presence of infections is an important factor for induction of ETs in semen. Furthermore, the presence of ETosis in ejaculates is suggestive of developing infectious processes and might possibly have a diagnostic value.

Detailed morphological characterization of testicular leukocytes in the adult CX3CR1 gfp/+ transgenic mouse identified two distinct CX3CR1 + mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX3CR1gfp/+ cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX3CR1, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3+ (T lymphocytes) and NK1.1+ (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206+MHCII+ sub-population (12% of total CD45+ cells). Rare testicular subsets of CX3CR1 +CD11c+F4/80+ (4.3%) mononuclear phagocytes and CD3+NK1.1+ (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba+/−) or elevated activin A (Inha+/−) were assessed using flow cytometry. Although the proportion of F4/80+CD11b+ leukocytes (macrophages) was not affected, the frequency of CD206+MHCII+cells was significantly lower and CD206+MHCII− correspondingly higher in Inha+/− testes. This shift in expression of MHCII in CD206+ macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is associated with urinary tract symptoms and hormonal imbalances amongst others. The heterogeneous clinical presentation, unexplored molecular background and lack of prostate biopsies complicate therapy. Here, using liquid biopsies, we performed a comprehensive translational study on men diagnosed with CP/CPPS type III (n= 50; median age 39.8, range 23-65) and age-matched controls (n= 61; median age 36.8, range 20-69), considering biochemical parameters of blood and ejaculates, and epigenetic regulation of the estrogen receptor genes (ESR1 and ESR2) in leukocytes isolated from blood (systemic regulation) and in somatic cells isolated from ejaculates (local regulation). We found elevated 17β-estradiol (E2) levels in seminal plasma, but not in blood plasma, that was significantly associated with CP/CPPS and impaired urinary tract symptoms. In ejaculated somatic cells of CP/CPPS patients we found that ESR1 and ESR2 were both significantly higher methylated in CpG-promoters and expressionally down-regulated in comparison to controls. Mast cells are reported to contribute to CP/CPPS and are estrogen responsive. Consistent with this, we found that E2 -treatment of human mast cell lines (HMC-1 and LAD2) resulted in altered cytokine and chemokine expression. Interestingly, in HMC-1 cells, possessing epigenetically inactivated ESR1 and ESR2, E2 -treatment led to a reduced transcription of a number of inflammatory genes. Overall, these data suggest that elevated local E2 levels associate with an epigenetic down-regulation of the estrogen receptors and have a prominent role in CP/CPPS. Investigating E2 levels in semen could therefore serve as a promising biomarker to select patients for estrogen targeted therapy.

Background Infection and inflammation of the reproductive tract are significant causes of male factor infertility. Ascending infections caused by sexually transmitted bacteria or urinary tract pathogens represent the most frequent aetiology of epididymo-orchitis, but viral, haematogenous dissemination is also a contributory factor. Limitations in adequate diagnosis and therapy reflect an obvious need for further understanding of human epididymal and testicular immunopathologies and their contribution to infertility. A major obstacle for advancing our knowledge is the limited access to suitable tissue samples. Similarly, the key events in the inflammatory or autoimmune pathologies affecting human male fertility are poorly amenable to close examination. Moreover, the disease processes generally have occurred long before the patient attends the clinic for fertility assessment. In this regard, data obtained from experimental animal models and respective comparative analyses have shown promise to overcome these restrictions in humans. Objective and rationale This narrative review will focus on male fertility disturbances caused by infection and inflammation, and the usefulness of the most frequently applied animal models to study these conditions. Search methods An extensive search in Medline database was performed without restrictions until January 2018 using the following search terms: 'infection' and/or 'inflammation' and 'testis' and/or 'epididymis', 'infection' and/or 'inflammation' and 'male genital tract', 'male infertility', 'orchitis', 'epididymitis', 'experimental autoimmune' and 'orchitis' or 'epididymitis' or 'epididymo-orchitis', antisperm antibodies', 'vasectomy'. In addition to that, reference lists of primary and review articles were reviewed for additional publications independently by each author. Selected articles were verified by each two separate authors and discrepancies discussed within the team. Outcomes There is clear evidence that models mimicking testicular and/or epididymal inflammation and infection have been instructive in a better understanding of the mechanisms of disease initiation and progression. In this regard, rodent models of acute bacterial epididymitis best reflect the clinical situation in terms of mimicking the infection pathway, pathogens selected and the damage, such as fibrotic transformation, observed. Similarly, animal models of acute testicular and epididymal inflammation using lipopolysaccharides show impairment of reproduction, endocrine function and histological tissue architecture, also seen in men. Autoimmune responses can be studied in models of experimental autoimmune orchitis (EAO) and vasectomy. In particular, the early stages of EAO development showing inflammatory responses in the form of peritubular lymphocytic infiltrates, thickening of the lamina propria of affected tubules, production of autoantibodies against testicular antigens or secretion of pro-inflammatory mediators, replicate observations in testicular sperm extraction samples of patients with 'mixed atrophy' of spermatogenesis. Vasectomy, in the form of sperm antibodies and chronic inflammation, can also be studied in animal models, providing valuable insights into the human response. Wider implications This is the first comprehensive review of rodent models of both infectious and autoimmune disease of testis/epididymis, and their clinical implications, i.e. their importance in understanding male infertility related to infectious and non-infectious/autoimmune disease of the reproductive organs.