RESUMO
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.
Assuntos
Epididimite/genética , Armadilhas Extracelulares/genética , Leucócitos/metabolismo , Sêmen/metabolismo , Adulto , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Linhagem Celular , Citrulinação/genética , Epididimite/diagnóstico , Epididimite/metabolismo , Epididimite/patologia , Armadilhas Extracelulares/metabolismo , Feminino , Frutose/metabolismo , Histonas/genética , Humanos , Leucócitos/patologia , Antígenos CD15/genética , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/metabolismo , Peroxidase/metabolismo , Projetos Piloto , alfa-Glucosidases/metabolismoRESUMO
Leucocytospermia has been associated with loss of sperm function. Extracellular traps (ETs) of leucocytes are produced during innate immune response. ETs can be activated by spermatozoa in contact with polymorphonuclear (in vitro), inducing sperm entrapment and decrease motility. In this pilot study, we describe the results of ETosis ex vivo, in seminal fluid (SF) smear of infertile patients, associating ETs with leucocytospermia and bacteriospermia. In 21 infertile patients, semen parameters (WHO, 2010), microbiological study, leucocytospermia and presence of ETs in SF were determined. Leucocytes (CD45, CD15 and CD68) were evaluated by immunostaining in SF smears. Indirect immunofluorescence (global histone and H4-citrullinated 3) and scanning electron microscopy (SEM) were used to determine ETs morphology. In 28.6% of patients presented leucocytospermia without bacteriospermia, all of them presented a large number of ETs in the SF smears examined. About 76.6% of the patients without leucocytospermia were positive for ETs. Samples with leucocytospermia have a higher number of ETs and would be related to the amount of leucocytes in the SF. The morphological predominant ETs were diffuse (diffETs) and spread (sprETs). The formation of ETs indicates leucocyte activation in semen, and it was observed that ETosis does not depend exclusively on the presence of bacterial contamination.
Assuntos
Armadilhas Extracelulares/imunologia , Infertilidade Masculina/imunologia , Leucócitos/imunologia , Sêmen/citologia , Adulto , Bactérias/isolamento & purificação , Humanos , Morte Celular Imunogênica/imunologia , Infertilidade Masculina/microbiologia , Leucócitos/citologia , Leucócitos/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Oligopeptídeos , Projetos Piloto , Sêmen/imunologia , Sêmen/microbiologia , Análise do Sêmen/métodosRESUMO
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.