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Problem Biological significance of increased number of leukocytes in ejaculate remains a subject of controversy. The aim of this research was to describe the morphological characteristics of the sperm elimination by leukocytes in in vivo and in vitro conditions using natural stimulator of the immune system—uropathogenic Escherichia coli, O75:HNT, isolated from semen. Method of study The study was performed on ejaculated spermatozoa from normozoospermic men with leukocytospermia (n = 10, in vivo experiment) or without leukocytospermia (n = 15, in vitro experiment). Morphological observations were performed using light and scanning electron microscopy. Results Sperm removal by active leukocytes mediated by traditional phagocytosis and generation of extracellular traps were observed in in vivo and in vitro experiments. Conclusion Our morphological data suggest that human germ cells activate leukocytes triggering both traditional phagocytosis and a novel trapping mechanism, followed by extensive sperm elimination.

The study was aimed at evaluating an in vitro induction of DNA damage in three sperm subpopulations exposed to selected inflammatory mediators, such as leukocytes, two combinations of pro-inflammatory cytokines (interleukin [IL]-6+IL-8 and IL-12+IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus). Semen samples from normozoospermic volunteers were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). Leukocytes were isolated from the whole heparinized blood using the density gradient centrifugation technique. DNA fragmentation in sperm fractions was evaluated using flow cytometry with TUNEL labeling and Comet assay. Out of the inflammatory factors tested, bacteria were found to have a greatest toxic effect on sperm DNA, especially in fractions isolated by Percoll gradient, compared with untreated cells (P

To evaluate whether ejaculated human spermatozoa undergo complete apoptosis or necrosis during experimental semen bacterial infection in vitro.Apoptotic markers, including mitochondrial transmembrane potential (ΔΨm), phosphatidylserine (PS) externalization, and DNA fragmentation, have been detected simultaneously in ejaculated human sperm after their incubation with a known pathogenic (Escherichia coli), as well as with conditionally pathogenic bacterial strains (Staphylococcus haemolyticus, Bacteroides ureolyticus) and/or leukocytes. The ΔΨm and translocation of PS was evaluated using the JC-1 and Annexin V binding tests, respectively. A modified TUNEL assay with additional staining for sperm viability was used to detect the DNA fragmentation level.The exposure of ejaculated spermatozoa to bacterial strains was associated with a simultaneous decrease in the percentage of sperm with normal ΔΨm and an increase in the proportion of Annexin V-positive sperm. Additionally, in the presence of S. haemolyticus, B. ureolyticus and/or leukocytes, a significant increase in the percentage of live TUNEL-positive (apoptotic) as well as dead TUNEL-positive (necrotic) sperm cells was also observed.The cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.

Objective To assess the in vitro effect of three bacterial isolates ( Escherichia coli , serotype O75:HNT, Staphylococcus haemolyticus , and Bacteroides ureolyticus ) and/or leukocytes on sperm motility, subcellular changes in sperm plasma membranes, and sperm fertilizing potential. Design An in vitro model of semen bacterial infection. Setting Basic research laboratory. Patient(s) Healthy normozoospermic volunteers and healthy blood donors. Intervention(s) None. Main Outcome Measure(s) Sperm plasma membrane stability was evaluated with a LIVE/DEAD Sperm Viability Kit and with the merocyanine 540 (M540) test both performed using flow cytometry. An oxiSelect TBARS Assay Kit was used for quantitative measurement of malondialdehyde content. Functional ability of spermatozoa was assessed by hypo-osmotic swelling (HOS) test and sperm penetration assay (SPA). Result(s) The incubation of sperm with bacteria and/or leukocytes was associated with the reduction of their fertilizing potential demonstrated in both the HOS test and SPA, and this effect can be considered as a natural consequence of diminished motility and sperm membrane injury of lipid bilayers. Bacteroides ureolyticus demonstrated the most significant detrimental effect on sperm structure and function. Conclusion(s) Sperm motility and lipid sperm membrane status might be the earliest and the most sensitive indicators of sperm damage with negative consequences for male factor fertility, which can be attributed to both bacteria and leukocytes action.