Scholarly article on topic 'In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation'

In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation Academic research paper on "Veterinary science"

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{"Semen inflammation" / "DNA fragmentation" / "TUNEL assay" / "Comet assay"}

Abstract of research paper on Veterinary science, author of scientific article — Monika Fraczek, Anna Szumala-Kakol, Grzegorz Dworacki, Dorota Sanocka, Maciej Kurpisz

Abstract 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.

Academic research paper on topic "In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation"

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Journal of Reproductive Immunology

journal homepage: www.elsevier.com/locate/jreprimm

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

Monika Fraczek3, Anna Szumala-Kakolb, Grzegorz Dworackic, Dorota Sanockad, Maciej Kurpisza*

a Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland b Unit of Microbiology, Hospital Medical College, Poznan, Poland c Department of Clinical Immunology, University of Medical Sciences, Poznan, Poland d Clinic ofInfertility, Cork, Ireland

ARTICLE INFO ABSTRACT

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 nor-mozoospermic 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.

© 2013 Published by Elsevier Ireland Ltd.

Article history: Received 4 April 2013 Received in revised form 17 September 2013 Accepted 19 September 2013

Keywords:

Semen inflammation DNA fragmentation TUNEL assay Comet assay

1. Introduction

The 'poor' sperm DNA quality appears to be one of the important factors affecting male reproductive ability, both in natural and assisted procreation (Simon et al., 2011;

* This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Corresponding author at: Strzeszynska 32, 60-479 Poznan, Poland. Tel.: +48 61 6579 212; fax: +48 61 8233 235.

E-mail addresses: framon@man.poznan.pl (M. Fraczek), kurpimac@man.poznan.pl, kurpimac@rose.man.poznan.pl (M. Kurpisz).

Aitken et al., 2009; Carrell et al., 2006; Comhaire et al., 1999; Sergerie et al., 2005). This has been confirmed by numerous reports in which a higher percentage of spermatozoa with fragmented DNA has been found in infertile men compared with fertile individuals (Baccetti et al., 1996; Hughes et al., 1996; Lopes et al., 1998; Smit et al., 2010; LaVignera et al., 2012). Sperm DNA fragmentation can be attributed to various pathological conditions including: local and systemic diseases, environmental factors, sperm preparation protocols and infection/inflammation in the male reproductive tract (Erenpreiss et al., 2006; Muratori et al., 2006). Three mechanisms described in the literature can disrupt sperm DNA integrity, such as defective chromatin packaging, apoptosis and oxidative stress (Aitken and De Iuliis,

0165-0378/$ - see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.jri.2013.09.005

2007; Schulte et al., 2010; Tamburrino et al., 2012). In the case of inflammation of the genitourinary tract, the redox imbalance is probably one of the etiological factors responsible for the destructive effects of the inflammatory process on male gametes, which is mainly associated with peroxidation of sperm macromolecules (Comhaire et al., 1999; Aitken and Baker, 2006; Fraczek and Kurpisz, 2007).

Patients with semen urogenital infection/inflammation have more frequently shown a higher number of spermatozoa with DNA fragmentation than fertile controls (Allam et al., 2008; La Vignera et al., 2012). Moreover, many authors claim that the percentage of DNA-fragmented spermatozoa in semen is connected to semen contamination with bacterial species (Moskovtsev et al., 2009; Domes et al., 2012), although others have not found any relationship between bacteriospermia and sperm DNA integrity (Rybar et al., 2012). Published studies also reported conflicting results on the harmful impact of leuko-cytospermia on sperm DNA integrity as measured by DNA fragmentation assays (Ochsendorf, 1999; Henkel et al., 2003; Moskovtsev et al., 2007; Fariello et al., 2009; Domes et al., 2012). These contradictory opinions may be connected to the specific site of infection/inflammation within the reproductive tract, with a different diagnostic profile for semen microbial culture, and colonization of the male genital tract by specific bacterial strains. Furthermore, bacteriospermia and/or leukocytospermia do not necessarily mean an infection/inflammation with negative consequences for fertility (Merino et al., 1995; Kohn et al., 1998; Rodin et al., 2003; Lackneret al., 2006; Gdoura et al., 2008).

The direct association between the presence of infectious factors in semen and sperm fertilizing potential has been intensely discussed and constitutes a significant problem in contemporary andrology. It is impossible to avoid the issue of sperm DNA integrity when analyzing the influence of male genitourinary tract inflammation on oxygen metabolism, and its effect on sperm structure and function. Thus, we decided to reconstruct semen inflammation in vitro and to analyze the effect of selected inflammatory mediators on an in vitro DNA fragmentation of different sperm subpopulations. Out of the many factors participating in the inflammatory process, previously studied regarding lipid sperm membrane peroxidation vulnerability, we chose peripheral blood mononuclear cells (PBMC), two combinations of human recombinant pro-inflammatory cytokines (interleukin (IL)-6 + IL-8 and IL-12 + IL-18) and two pathogenic bacterial strains isolated from semen samples (Escherichia coli and Bacteroides ure-olyticus) for the present study.

2. Materials and methods

2.1. Sample collection and preparation

Semen specimens were obtained from ten healthy volunteers attending the Outpatient Andrology Clinic, Poznan, Poland after four days of sexual abstinence. Following 30 min of sample liquefaction at room temperature, sperm parameters were assessed according to the World Health Organization criteria (WHO, 1999) and Kruger's strict

criteria for morphology (Kruger et al., 1986). Each semen specimen was also checked for the presence of peroxidase-positive cells by Endtz test (Endtz, 1974). All samples tested were subjected to extended microbiological examination, including aerobic, anaerobic, and atypic bacteria (BioMerieux, Marcy-L'Etoile, France). Only normozoosper-mic semen samples with leukocytes <0.2 x106/mL and negative bacterial culture were utilized for further experiments (Table 1). Semen samples selected forthe study were fractionated by the swim-up technique and the Percoll gradient procedure as previously reported (Fraczek et al., 2004, 2007). The cells from swim-up, 90% and 47% Percoll sperm fractions, were finally washed in phosphate-buffered saline (PBS) and adjusted to a density of 2 x 107 spermatozoa/mL.

Heparinized venous blood samples were collected from ten healthy adults donating to the Regional Blood Centre, Poznan, Poland. Leukocytes were isolated using the density gradient centrifugation technique (Histopaque-1.077 (Sigma, St. Louis, MO, USA)) as described elsewhere (Fraczek et al., 2004, 2007). The peripheral blood mononuclear cells (PBMC) suspensions were diluted to a concentration of 1 x 107 cells/mL for further use.

The bacterial isolates used in this study were obtained from the Outpatient Clinic of Poznan Hospital Medical University, using the following biochemical test kits(BioMerieux, Marcy-L'Etoile, France): ID 32 E for Gram-negative rods and API 20 A for anaerobic bacteria. The bacterial strains were isolated from semen samples, with significant bacteriospermia (>3 x105cells/mL and >1 x 106 cells/mL of semen for E. coli and B. ureolyti-cus, respectively) and leukocytospermia, of our infertile patients. Suspensions of all isolates containing 3000 bacteria per mL were prepared in a sterile 0.85% saline no more than 3 h before the experiment in which they were to be used. A specific anaerobic atmosphere generator system (GenBag Anaer, BioMerieux) was used for the transport of anaerobic strain.

2.2. Incubation of sperm fractions with inflammatory mediators

One million spermatozoa of all the three sperm fractions resuspended in PBS were then incubated with PBMC (1 x 106 per mL of sperm suspension), human recombinant proinflammatory cytokines (200 pg, 500 pg, 50 pg, and 500 pg per mL of sperm suspension respectively for IL-6, IL-8, IL-12, and IL-18) or bacteria (1 x 103 cells per mL of sperm cells, for both E. coli and B. ureolyticus) for 1 h at 37 °C. Leukocytes were then removed from the co-incubated mixtures using a Dynal MPC-1 immunomagnetic cell isolation system (Fraczek et al., 2004, 2007, 2008).

2.3. Tunel labeling

Once induced in spermatozoa, DNA fragmentation was evaluated using the TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assay (Flow-TACS Apoptosis Detection Kit, R&D Systems, Minneapolis, MN, USA) following the manufacturer's instruction. Sperm samples were fixed with 3.7% formaldehyde solution and permeabilized with Cytonin. Next, biotinylated nucleotides

Table 1

Standard semen parameters (n =10 samples).

Semen parameter Median Min.-max. Mean ±SD

Volume (mL) 3.00 1.50- 7.50 3.62 ± 1.65

Concentration (x106/mL) 110.00 71- 180 112.48 ± 32.36

Total number of spermatozoa (x106) 345.60 106.5- 756 400.68 ± 207.10

Progressive motility (%) 63.00 50.00- 78.00 63.31 ± 10.09

Total motile (progressive + nonprogressive) (%) 68.00 52.00- 92.00 69.08 ± 13.56

Immotile (%) 31.00 20.00- 44.00 30.92 ± 8.20

Vitality (% alive) 83.00 71.00- 92.00 82.62 ± 6.99

Spermatozoa with normal morphology (%) 25.00 7.00- 42.00 24.50 ± 8.52

Peroxidase-positive cells (x106/mL) 0.04 0.00- 0.12 0.07 ± 0.05

Round cells of spermatogenic lineage (x106/mL) 1.00 0.28- 1.92 1.15 ± 0.46

were added to the free 3'-ends of the DNA fragments in the presence of terminal deoxynucleotidyl transferase (TdT). After the creation of a complex between biotinylated DNA fragments and streptavidin-conjugated fluorescein (FITC) spermatozoa were analyzed by means of a FAC-Scan flow cytometer (Becton Dickinson, SanJose, CA, USA). A minimum of 10,000 events were acquired for each evaluated sample. The results were analyzed using Facs-Diva software (Becton Dickinson, San Jose, CA, USA). The percentage of TUNEL-positive cells was determined. The background fluorescence was assessed in comparison to both negative (sperm exposed to the reaction mixture without TdT) and positive (sperm pre-treated with DNase I) controls. In addition, the TUNEL-FITC-labeled spermatozoa were observed under a fluorescent microscope (BX41, Olympus, Tokyo, Japan) to monitor the morphology of TUNEL-positive spermatozoa.

2.4. Comet assay

For evaluation of the DNA status of individual spermatozoon, single cell gel electrophoresis (Comet assay) was performed using the CometAssay Apoptosis Detection Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's specification with some own modifications. Sperm cells were immobilized in melted agarose (LMAgarose) on slides supplied in the Kit and lysed for 24 h at 4°C. The lysis buffer consisted of 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% Triton X-100, 200 ^g/mL protease K. After lysis, the slides were left in an alkaline solution for 1 h at room temperature in the dark to allow sperm DNA to unwind. Next, horizontal electrophoresis was carried out for 20 min at 25 V and 300 mA (0.714 V/cm). After electrophoresis, slides were immersed in ice cold 100% methanol for 5 min, followed by 100% ethanol for a further 5 min. Fixed and dried samples were stored at room temperature prior to staining with a fluorescent dye DAPI (2 ^g/mL; SERVA, Heidelberg, Germany). Samples of untreated sperm cells were used as controls for each sperm fraction, respectively. The tail length measured from the edge of the comet head was evaluated using an Olympus BX41 microscope and analyzed using Isis (in situ imaging system) software (MetaSystems, Altlussheim, Germany). A minimum of 50 comets was scored per slide. All the samples were run twice - each on a separate slide.

2.5. Statistical analysis

Statistical analysis was performed using STATISTICA, version 7.0 (StatSoft, Tulsa, OK, USA) with parametric as well as non-parametric tests. The data obtained from the Comet assay were normally distributed and subjected to one-way analysis of variance (ANOVA), and followed by the Dunnett and Tukey multiple comparisons tests. For the percentage of TUNEL-positive cells non-parametric analysis of variances (Kruskal-Wallis test) was used followed by the Dunnett and Dunn multiple comparisons tests. The Mann-Whitney U test was used to assess differences between swim-up and 90% Percoll sperm fractions. Data obtained using parametric tests were presented as mean ± standard deviation (SD). In turn, data analyzed using a non-parametric test were presented as median ± average deviation (AD). Differences were regarded as significant if P< 0.05, P< 0.01 or P< 0.001.

3. Results

3.1. Characteristics of sperm fractions

The detailed characteristics of all three sperm fractions applied in the present study are presented in Table 2. Spermatozoa isolated by the swim-up procedure exhibited the best seminological parameters, specifically in respect of high motility and good morphology. Spermatozoa from the 90% Percoll fraction had slightly (but statistically significant) worse morphology and viability compared with the swim-up separated germ cells (P<0.05). The 47% Percoll fraction contained spermatozoa with statistically significant poorer sperm quality concerning the motility, morphology, and viability (P<0.001, compared with the swim-up sperm fraction).

3.2. Tunel-positive spermatozoa in sperm fractions

The overall results (irrespective of inflammatory factor applied) for TUNEL-positive spermatozoa in tested sperm fractions are shown in Fig. 1. The percentage of TUNEL-positive cells was significantly higher in spermatozoa from the 90% Percoll fraction (13.08%) than in the swim-up selected sperm (9.84%; P< 0.05).

Table 2

Characteristics of spermatozoa isolated by swim-up or by Percoll gradient centrifugation (n = 10 samples in each sperm fraction).

Sperm parameter Swim-up sperm fraction (%) Median, min.-max., mean ±SD 90% sperm Percoll fraction (%) Median, min.-max., mean ±SD 47% sperm Percoll fraction (%) Median, min.-max., mean ± SD

Progressive motility 76.52 67.53 5.84***

68.45-88.25 57.45-75.62 2.82-9.24

77.2 ±5.82 67.28 ±5.02 5.80 ±2.14

Morphology 27.22 20.24* 9.13***

18.59-35.76 14.32-25.32 6.39-12.65

26.40 ±5.02 20.17 ± 3.49 9.28 ± 2.06

Vitality (% alive) 83.99 69.98* 60.34***

73.56-96.72 63.56-75.83 46.35-66.47

84.31 ±7.58 70.5 ±4.81 59.53 ± 6.44

* P< 0.05, in comparison with swim-up sperm fraction. " P<0.001, in comparison with swim-up sperm fraction.

Fig. 1. The percentage of TUNEL-positive spermatozoa in tested sperm fractions (n = 60 in each sperm fraction). The results are expressed as median ± AD; P<0.05 calculated using the Kruskal-Wallis test.

3.3. Effect of inflammatory mediators on the percentage of TUNEL-positive cells in sperm fractions

The effect of selected inflammatory mediators on the percentage of TUNEL-positive cells in different sperm fractions is summarized in Fig. 2. Untreated control spermatozoa isolated using the swim-up technique had the lowest percentage of the TUNEL-positive cells. When

Fig. 2. Influence of selected inflammatory mediators on the percentage of TUNEL-positive spermatozoa in the sperm fractions tested (n = 10 in each sperm fraction). The results are presented as median± AD; P<0.05 calculated using the Kruskal-Wallis test, and compared with respective controls.

compared with the swim-up sperm fraction, approximately two to three times as many elevated TUNEL-positive cells were observed for the Percoll sperm fractions (90% and 47%) analyzed. In general, the presence of leukocytes was associated with a decrease in the number of TUNEL-positive cells. However, this effect was not statistically significant compared with sperm incubated alone (controls). Incubation of spermatozoa with IL-6 combined with IL-8 was connected to an increased percentage of TUNEL-positive cells, especially in the 90% Percoll sperm fraction. However, this increase was also statistically insignificant compared with the respective control. The combination of IL-12 with IL-18 caused an increase in the percentage of TUNEL-positive cells, especially in sperm fractions isolated by the Percoll gradient, although this increase was also insignificant compared with untreated cells. When spermatozoa were exposed to E. coli, a much higher percentage of TUNEL-positive cells was noted only in cases of spermatozoa recovered from the 47% Percoll fraction (P<0.05). Anaerobic bacteria represented by B. ureolyticus had a statistically significant influence on the percentage of TUNEL-positive cells in sperm from the 90% Percoll fraction (P<0.05). Moreover, this increase was the highest among all the inflammatory factors applied in this study. Representative sperm samples observed in the TUNEL assay are presented in Fig. 3.

3.4. Effect of inflammatory factors on comet length in sperm fractions

The results of comet length in all the sperm fractions incubated with leukocytes that were examined, pro-inflammatory cytokines, as well as bacteria are presented in Fig. 4 and representative photographs of sperm comets are shown in Fig. 5. Regardless of the type of inflammatory factor applied, the shortest comet length was found in swim-up-isolated spermatozoa; it was longer in spermatozoa recovered from the 90% Percoll fraction, and the longest in the 47% Percoll sperm fraction. The presence of leukocytes increased the comet length in spermatozoa recovered from the swim-up and 90% Percoll fractions (P<0.01, in comparison to untreated cells). Incubation of spermatozoa from all the fractions examined with pro-inflammatory cytokines was connected to a significant increase in DNA strand breaks compared with appropriate sperm controls (P<0.01). When the bacterial strains were

FITC-A

Fig. 3. Representative sperm samples observed with the TUNEL assay (histogram in the left panel and the same sperm as observed under a fluorescent microscope in the right panel) showing: (A) the negative control (sperm from the 90% Percoll fraction exposed to the reaction mixture without TdT); (B) the positive control (sperm from the 90% Percoll fraction pretreated with DNase I); (C) sperm from the 90% Percoll fraction incubated alone; (D) sperm from the 90% Percoll fraction incubated with B. ureolyticus.

Fig. 4. Influence of selected inflammatory mediators on comet length in the sperm fractions tested (n =10 in each sperm fraction). The results are presented as mean ±SD; P<0.01 calculated using the one-way analysis of variance (ANOVA), and compared with respective controls.

used, a high and significant increase in comet length was found in spermatozoa isolated both by the swim-up and the 90% Percoll gradient (P<0.01, compared with control spermatozoa), but not from the 47% Percoll fraction.

3.5. Comparison of the percentage ofTUNEL-positive cells and comet length between swim-up and 90% Percoll sperm fractions

Table 3

Comparison of the percentage ofTUNEL-positive cells and comet length in swim-up and 90% Percoll sperm fractions applied to selected inflammatory mediators; calculated using the Mann-Whitney U test.

Inflammatory factor % ofTUNEL-positive cells Comet length

No factor (control) NS NS

Leukocytes NS NS

IL-6 + IL-8 P <0.01 NS

IL-12 + IL-18 NS NS

E. coli NS NS

B. ureolyticus P <0.01 NS

NS - not statistically significant.

Higher% ofTUNEL-positive cells in favor ofthe 90% Percoll sperm fraction ifP<0.01.

the swim-up and 90% Percoll fractions, incubated (both alone) and with particular inflammatory mediators, are presented in Table 3. The presence of the combination of IL-6 with IL-8 or B. ureolyticus was associated with a significant increase in the percentage of TUNEL-positive cells of sperm from the 90% Percoll fraction in comparison with sperm separated by the swim-up technique (P<0.01). As regards comet length, there were no statistical differences between these sperm fractions, irrespective of the inflammatory mediator applied.

4. Discussion

Results aimed at comparing the percentage of TUNEL-positive cells and comet length between spermatozoa from

Fig. 5. Representative photographs of sperm examined with the Comet assay, showing: (A) sperm from the 90% Percoll fraction incubated alone; (B) sperm from the 90% Percoll fraction incubated with B. ureolyticus.

The present study is a continuation of our previous reports concerning the influence of the inflammatory process on structural, metabolic, and functional disorders of three various sperm subpopulations using an in vitro system (Fraczek et al., 2007, 2008). The use of both the TUNEL and Comet assays for the evaluation of DNA fragmentation in ejaculated spermatozoa seemed to strengthen the value of our findings, particularly because the results obtained by these two methods corroborated each other to some degree. As for the percentage of DNA-fragmented spermatozoa in motile fraction after the swim-up procedure, we expected to obtain results similar to those reported by other authors (Ramos and Wetzels, 2001; Muratori et al., 2003; Lachaud et al., 2004; Aziz et al., 2007). Indeed, we observed the lowest number ofTUNEL-positive cells and the shortest comet length in this fraction. In turn, the highest percentage of cells with spontaneously fragmented DNA was noted in the fraction of spermatozoa with the poorest seminological parameters (47% Percoll fraction). These findings are also in agreement with other reports showing the significantly higher proportion of sperm with DNA damage in the fraction of sperm with low motility, compared with the fraction with high sperm motility (Barroso et al., 2000; Mahfouz et al., 2010).

It is known that through in vitro manipulation with semen samples DNA fragmentation can be forced upon spermatozoa because of the induction of exogenous stresses (Toro et al., 2009; Jackson et al., 2010). It turned out that even morphologically normal human spermatozoa from the swim-up population may demonstrate DNA fragmentation (Avendano et al., 2009). According to some authors sperm samples prepared using the gradient centrifugation technique may be even more stable, in terms of DNA fragmentation, than samples prepared by a

swim-up procedure (O'Connell et al., 2003; Zhang et al., 2011). In the present study, normal spermatozoa isolated using the swim-up technique were assessed as being the most resistant to inflammatory agent-induced DNA fragmentation; in contrast to spermatozoa obtained from the 90% Percoll sperm fraction, which appeared to be the most susceptible (Fig. 1). These results have confirmed that the structural and functional differences between the spermatozoal fractions studied (Table 2) may influence the frequency and intensity of sperm DNA fragmentation, when subjected to various inflammatory mediators. In the light of the present data, the selection of spermatozoa by gradient procedures increases the susceptibility of mature spermatozoa to harmful effects toward DNA quality exerted by inflammatory factors. Such findings may help with optimal sperm preparation used in assisted reproductive therapy, especially in infertile patients with urogenital tract infection/inflammation.

The most critical mechanism to explain the origin of DNA degeneration occurring in ejaculated spermatozoa indicated in the present study can be related to apopto-sis and/or necrosis as indicated by others (Lachaud et al., 2004; Aitken et al., 2009). In this study, we applied two tests commonly used to detect DNA strand breaks in human spermatozoa. In our view, the flow cytometric TUNEL assay could be potentially useful for the diagnosis of sperm DNA apoptotic fragmentation, while the Comet assay can be applied to detect DNA degeneration. It cannot be excluded that the noted differences among sperm fractions, such as the number of dead and/or dying sperm, were a reason for the observed differences between the percentage of TUNEL-positive cells and comet length values, particularly when spermatozoa were exposed to inflammatory mediators (Figs. 2 and 4). Moreover, the higher specificity of the TUNEL assay allowed us to find some differences in sperm DNA fragmentation between sperm separated by the swim-up technique and those obtained from the 90% Percoll gradient, while this was not the case when evaluating Comet assay data (Table 3). Probably, the use of additional techniques for detecting apoptotic-like changes in ejaculated spermatozoa, such as the Annexin V/PI binding assay or electron microscopy technique, could dispel doubts regarding the nature of detected DNA breaks during male genital infection/inflammation. Moreover, the use of a modified TUNEL assay as proposed recently by a group from Cleveland (with staining for live sperm and dithiothreitol for decondensation of chromatin) should better define the status of DNA damage and may offer more comprehensive information on the understanding of sperm DNA (apoptotic and/or necrotic) fragmentation occurring under in vivo and in vitro inflammatory reactions in semen (Mitchell et al., 2011).

Most clinical studies concerning infections of the male genitourinary tract have reported that well-established causative pathogens, such as Gram-negative rods (e.g., E. coli) and genital Ureaplasmas and Mycoplasmas, are connected to decreased sperm DNA quality (Reichart et al., 2000; Sanocka-Maciejewska et al., 2005; Gallegos et al., 2008; Domes et al., 2012). There are many experimental data that have revealed the induction of apoptotic-like changes in human ejaculated spermatozoa by bacteria

in respect to mitochondrial membrane potential, phos-phatidylserine translocation, sperm viability and motility; although none of them have exclusively focused on sperm DNA quality (Diemeret al., 1996; Kohn et al., 1998; Villegas et al., 2005; Berktasetal., 2008; Schulzetal., 2010; Fraczek et al., 2012). However, one group has recently demonstrated an increase in bovine sperm DNA fragmentation due to bacterial growth (González-Marín et al., 2011). Interestingly, we observed in our study, that pathogenic bacteria may directly induce DNA fragmentation in ejaculated spermatozoa (Fig. 2). Based on our previous study, we can postulate that co-incubation of human ejaculated spermatozoa with pathogenic, as well as conditionally pathogenic bacterial strains may diminish sperm plasma membrane integrity. Of all the bacterial strains applied, the greatest increase in Pl-positive sperm cells had previously been found in the presence of B. ureolyticus (Fraczek et al., 2012). Out of the two bacterial strains used in the current study, the greatest influence on sperm DNA was also caused by this anaerobic bacterial strain. Taking into consideration our previous data regarding sperm viability, we cannot exclude the possibility that direct contact of nonpathogenic bacteria with spermatozoa might also be an initial signal for germ cell DNA fragmentation.

ln the present study, we applied two pathogens representing the different groups of bacteria that are most often isolated at a significant bacteriospermia level in the semen of infertile men. Differences in metabolism and pathogenicity between the two bacterial strains used in the present study also deserve consideration during the interpretation of the results obtained. lt has been documented that there is a relationship between the presence of E. coli in the male reproductive tract and a decrease in sperm motility (Diemer et al., 1996), as well as good sperm morphology (Menkveld and Kruger, 1998). Our data confirm those observations and indicate the strongest increase in TUNEL-positive cells in the 47% sperm Percoll fraction, a fraction rich in male gametes with abnormal morphology and altered motility. As for B. ureolyticus, it is known to produce superoxide dismutase (SOD), which allows them to survive under oxygen conditions. lt is therefore possible that free radical species may mediate the cytotoxic effects of these bacteria toward spermatozoa, especially those recovered from the 90% Percoll fraction, which was shown, not for the first time, to be extra-susceptible to peroxidative damage (Fraczek et al., 2007).

The main potential mechanism in which genital tract inflammation/infection might affect male germ cells could be the impact of leukocytes infiltrating the inflammatory site (Ochsendorf, 1999). However, there is an ongoing controversy concerning the relationship between sperm DNA integrity and leukocytospermia (Alvarez et al., 2002; Erenpreiss et al., 2002; Henkel et al., 2005; Moskovtsev et al., 2007; Domes et al., 2012). In our study we observed a lower percentage of TUNEL-positive cells after the incubation of spermatozoa with leukocytes. One potential hypothesis for this paradox is the rapid removal of damaged germ cells by leukocytes due to phagocytosis. This mechanism of sperm selection can be an important part of the elimination of nonviable or damaged spermatozoa (unpublished data). Thus, the observed decrease in the

number of germ cells with fragmented DNA, especially in the 47% Percoll sperm fraction with altered morphology and motility, as well as with an increased proportion of dead spermatozoa, partially provides evidence for the positive role of leukocytes in ejaculate, which has occasionally been mentioned in the literature (Tomlinson et al., 1992; Ricci et al., 2002; Henkel, 2011; Barraud-Lange et al., 2011).

The relationship between cytokines participating in urogenital infections and human sperm apoptotic-like changes has been recently identified as a central area of interest (Feldmann and Saklatvala, 2001; Perdichizzi et al., 2007). For example, TNF-a-induced apoptosis in ejaculated spermatozoa measured by an increase in the percentage of the spermatozoa with phosphatidylserine (PS) exter-nalization on the cell membrane surface and/or by an increase in the TUNEL-positive spermatozoa has been confirmed in some experimental and clinical studies (Said et al., 2005; Perdichizzi et al., 2007; Allam et al., 2008). Of a large group of pro-inflammatory cytokines, IL-6, IL-8, and IL-18 have been most frequently mentioned in the literature as diagnostic markers for male genitourinary tract infections (Depuydt et al., 1996; Eggert-Kruse et al., 2001; Sanocka et al., 2003; Matalliotakis et al., 2006). In our previous in vitro report, two combinations of pro-inflammatory cytokines (IL-6 + IL-8, IL-12 +IL-18) turned out to be an important factor enhancing sperm membrane lipid peroxi-dation primarily caused by leukocytes (Fraczek et al., 2008). In the present study, we were able to demonstrate that in in vitro conditions some pro-inflammatory cytokines tend to affect the DNA integrity of ejaculated spermatozoa and this effect was visible, especially in sperm from the 90% Percoll fraction (Fig. 2). It might be that in the case of IL-6 concomitantly applied with IL-8, the observed effect occurred through binding to its receptor, whose presence in spermatozoa has already been reported (Laflamme et al., 2005). It is well known that some proinflammatory cytokines, such as IL-1P, TNF-a, or IL-18, participate in the regulation of the apoptotic process via the induction of the Fas/Fas ligand (FasL) system (Dinarello, 2000; Feldmann and Saklatvala, 2001). Some reports have described the harmful effects of IL-18 on sperm quality in infertile men with urogenital infections (Matalliotakis et al., 2006). The possible cooperation of IL-18 with IL-12 in the apoptosis induction of mature spermatozoa via the Fas/Fas ligand (FasL) system cannot, therefore, be excluded. Moreover, it is possible that the population of sperm obtained from the 90% Percoll gradient (with a relatively high percentage of morphologically damaged gametes or dead cells), in which we observed a high sperm fragmentation index, contained spermatozoa rich in Fas that earlier escaped apoptosis (Sakkas et al., 1999) The explanation of the mechanisms of the influence of cytokines on sperm DNA and apopto-sis during male reproductive tract infection/inflammation needs further research.

In conclusion, the data obtained in this experimental study revealed that sperm DNA fragmentation can be a consequence of inflammatory reactions occurring during semen infection. An increase in DNA damage observed in the ejaculated spermatozoa in the presence of mediators of the inflammatory process in vitro seems to be in line with the observed DNA fragmentation during

inflammation in situ (Aitken and De Iuliis, 2007; Domes et al., 2012; La Vignera et al., 2012). The present study supports the view that during male urogenital inflammation microbial pathogens are the most prominent agents responsible for damage to both sperm membranes and DNA, with potential consequences for sperm function. Although this study provides a better understanding of the harmful effects of particular inflammatory factors on DNA status in specific sperm subpopulations with different fertilizing potentials, further investigations using morphological and molecular tests determining sperm membrane status, mitochondrial function, and DNA integrity should be applied to achieve a clear picture of subcellular changes in ejaculated spermatozoa (including the native, unprocessed sperm) occurring in the course of semen inflammation/infection under in vivo and in vitro conditions.

Acknowledgements

Study financed by grants no. NR 13006606, NN 407283539.

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