In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation.

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 < 0.05). The results indicate that inflammatory mediators can be a direct cause of DNA fragmentation in ejaculated spermatozoa, which can ultimately lead to limited fertilizing abilities of the germ cells. In contrast to the swim-up technique, the selection of spermatozoa by gradient procedures increases the vulnerability of mature spermatozoa to the harmful effects of infectious agents on DNA integrity. This observation may have some meaning for recommendations concerning laboratory techniques used in assisted reproductive therapy.

Proinflammatory cytokines as an intermediate factor enhancing lipid sperm membrane peroxidation in in vitro conditions.

We have examined the effect of white blood cells (WBCs), various proinflammatory cytokines, or a combination of the two on the peroxidation of human sperm membrane lipids in in vitro conditions. Six recombinant cytokines, such as interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, IL-18, and tumor necrosis factor alpha (TNF-alpha), used singly or in combinations, were analyzed. WBCs were isolated from the whole heparinized blood using a density gradient technique (Histopaque 1.077). Spermatozoa were isolated from semen samples with normal sperm parameters by both the swim-up technique (swim-up fraction) and by a discontinuous Percoll gradient centrifugation (90% and 47% Percoll fractions). Peroxidative damage to sperm membrane lipids was assessed by determining the concentration of malondialdehyde (MDA) in lysates of spermatozoa using high-performance liquid chromatography (HPLC). There were no statistically significant differences in MDA concentrations between sperm fractions incubated with cytokines and respective controls (spermatozoa alone). In spermatozoa isolated by the swim-up technique, the MDA level was significantly higher only after incubation with IL-6 and IL-8 plus WBCs when compared to sperm incubated with leukocytes alone (0.62 +/- 0.21 micromol/L and 0.42 +/- 0.22 micromol/L, respectively; P < .05). In spermatozoa recovered from the 47% Percoll, only a combination of IL-12 and IL-18 used together with WBCs was linked with a significant increase in MDA concentration (from 0.41 +/- 0.13 micromol/L to 0.65 +/- 0.19 micromol/L; P < .05). The results obtained suggest that cytokines produced during the inflammatory process intensify the level of oxidative stress caused by leukocytes, which may have serious consequences for sperm membrane integrity.

Male genital tract infection: an influence of leukocytes and bacteria on semen.

We have studied the oxidative status of 155 semen samples, 95 originating from healthy individuals and 60 from infertile patients, which were subdivided into two groups: (a) normozoospermic with genitourinary tract infection (GTI); and (b) with pathological spermiogram and GTI. Several phases of infection were observed: with bacterial presence only, bacteria and leukocytes, and leukocytes only, following the routine inflammatory pattern. Leukocyte numbers, bacterial strains, pro- and anti-oxidants, and selected pro-inflammatory cytokines (IL-1 beta, IL-6, IL-8 and TNF-alpha) were studied. Additionally, two oxido-sensitive indices were created (SOD/XO and CAT/XO) in order to follow particular phases of semen infection in two subgroups of patients. Different patterns of activities of pro- and anti-oxidant substances, as well as cytokines, were observed in the studied populations. It was reflected mainly by elevated XO activity in a group of patients with a pathological spermiogram while, in a group of patients with GTI and normozoospermia, xanthine oxidase was normal. In the latter group, oxido-sensitive indices were elevated in favour of anti-oxidants; similarly, this occurred with IL-6 levels in comparison to healthy controls. It appears therefore that normozoospermic semen recovers better after infection than pathological semen. Perhaps, IL-6 secretion might be helpful in the observed recovery?

Interaction between leucocytes and human spermatozoa influencing reactive oxygen intermediates release.

The relationship between the presence of white blood cells (WBCs) and the fertilizing potential of human semen is still an open question. It is well known that the presence of leucocytes in human semen can be related to the production of reactive oxygen intermediates (ROI). Semen samples were obtained from 15 normozoospermic men and leucocytes were isolated from heparinized blood drawn from 15 volunteers. Lucigenin and luminol-mediated chemiluminescence assays were used to determine reactive oxygen species (ROS) generation by non-activated or activated leucocytes through 12-myristate-13-acetate or N-formyl-methionyl-leucyl-phenyalanine (FMLP) before the addition of spermatozoa isolated by swim-up or Percoll procedures. All spermatozoal fractions used in this study were characterized by defining their motility, morphology and viability. The levels of ROS formation by non-activated as well as stimulated leucocytes were significantly decreased after addition of swim-up separated spermatozoa (p < 0.01). The ability to inhibit the basal chemiluminescence was of lower degree for spermatozoa isolated from 90% Percoll fractions than for swim-up sperm. However, addition of sperm cells from 47% Percoll fraction was found to increase both lucigenin and luminol signals. Moreover, the determined ROI levels changed depending on the type of inducing factor used for oxidative burst. Then, spermatozoa selected by swim-up procedure although with only slightly higher viability and morphology than sperm obtained from 90% Percoll fraction clearly exhibited much higher capacity to inhibit ROI secretion by receptor-stimulated leucocytes (FMLP-activation) than Percoll fractionated sperm. Such results may indicate that within normal semen may exist sperm subpopulations with different biochemical mechanisms controlling the interaction between spermatozoa and contaminating leucocytes. When ROI levels contained in normozoospermic semen are dependent on the WBCs activation, it seems that spermatozoa with preserved normal functional competence are able to defend themselves against leucocytes-derived ROI. Also for normozoospermic ejaculates, swim-up sperm may improve semen antioxidant characteristics when comparing with Percoll (90%) separated sperm. It may help for optimal sperm preparation when assisting to infertility treatment.

Reactive oxygen species and sperm cells.

There is a dynamic interplay between pro- and anti-oxidant substances in human ejaculate. Excessive reactive oxygen species (ROS) generation can overwhelm protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membranes. Additionally, changes in DNA can be induced. The essential steps of lipid peroxidation have been listed as well as antioxidant substances of semen. A variety of detection techniques of lipid peroxidation have been summarized together with the lipid components of sperm membranes that can be subjected to stress. It is unsolved, a threshold for ROS levels that may induce functional sperm ability or may lead to male infertility.

Male genital tract inflammation: The role of selected interleukins in regulation of pro-oxidant and antioxidant enzymatic substances in seminal plasma.

Human semen contains spermatozoa as well as populations of round nonspermatozoal cells primarily consisting of leukocytes. Activation of white blood cells present in the seminal plasma during genital tract inflammation and cellular reactions against microbial agents may provoke a release of a variety of products such as cytokines and reactive oxygen species. The aim of this study was to evaluate whether a panel of selected cytokines (interleukin [IL]-1 beta, IL-6, IL-8, and tumor necrosis factor-alpha [TNF alpha]) detectable in seminal plasma during male genital tract inflammation could be considered as mediators between altered semen parameters and changed levels of pro-oxidant and antioxidant substances. Studies using chemiluminometric, spectrophotometric, and enzyme-linked immunosorbent assay methods indicate that proinflammatory cytokines such as IL-1 beta, IL-6, IL-8, and TNF alpha may modulate pro-oxidant and antioxidant activities in the male genital tract. The data also suggest that the function of pro-oxidant and antioxidant systems in semen may directly influence basic semen parameters. The elevated numbers of leukocytes present in semen during male genital tract inflammation without an associated contribution of cytokines and semen antioxidant capacity appear to be of little prognostic value in evaluating male fertilization potential.

Infertility in Poland--present status, reasons and prognosis as a reflection of Central and Eastern Europe problems with reproduction.

Poland has been considered a representative country for the population of Central and Eastern Europe as it has genetically homogenous population and reveals profound similarities in economical and historical processes common for all countries of the region. According to our observations there is approximately 20% of infertile couples in Poland (lack of concentration after at least 12 month of unprotected intercourse) and 40-60% of them are infertile because of the male factor. However, studies of infertility in Poland that have been conducted so far need to be verified. The changes connected with the style of life and new tendencies observed during the tedious political changes had considerable influence on childbearing rate in Poland. Additional factors diminishing the fertility rate are: the change of the status of women in a society, raising amount of everyday stress, the high rate (17%) of unemployment and the low average income. Furthermore, in Poland, the diagnostics and treatment (assisted reproductive technology) are entirely financed from the private funds of a patient. Summing up, the problem of childlessness in Central and Eastern Europe is a complex one and very alarming therefore it is necessary to work out and to implement research, preventative and therapeutic programmes strengthened through the international cooperation of specialized centers of excellence.

[Pro- and antioxidant system activity in cervical mucosa]. / Aktywnosc ukladów pro- i antyoksydacyjnego w sluzie szyjkowym szyjki macicy.

The aim of the study was to evaluate the activity of the pro- and antioxidant systems in cervical mucus of healthy women and women with pathological of disorders cervical. After cervical mucus liquefaction, the activity of superoxide dismutase, catalase and xanthine oxidase was determined by means of chemiluminescence assays. The activity of the antioxidant system in cervical mucus of healthy women was higher as compared to the group with pathological cervical disorders. Moreover, in mucus samples obtained from women with dysplasia, the significantly heightened activity of xanthine oxidase (strong pro-oxidant) was observed. The antioxidant system present in cervical mucus may protect both the cells of the uterine cervix and the viable sperm.