Fertilizing potential of ejaculated human spermatozoa during in vitro semen bacterial infection

Fertilizing potential of ejaculated human spermatozoa during in vitro semen bacterial infection Monika Fraczek, Ph.D.,a Ewa Wiland, Ph.D.,a Malgorzata Piasecka, Ph.D.,b Magdalena Boksa, M.S.,c Dariusz Gaczarzewicz, Ph.D.,d Anna Szumala-Kakol, Ph.D.,e Tomasz Kolanowski, M.S.,a Lothar Beutin, Ph.D.,f and Maciej Kurpisz, M.D., Ph.D.a a Institute of Human Genetics Polish Academy of Sciences, Poznan; b Laboratory of Histology and Developmental Biology, Pomeranian Medical University, Szczecin; c Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Poznan; d Department of Animal Reproduction, Biotechnology and Environmental Hygiene, West Pomeranian University of Technology, Szczecin; and e Unit of Microbiology, Hospital Medical College, Poznan, Poland; and f National Reference Laboratory for Escherichia coli, Federal Institute for Risk Assessment (BfR), Berlin, Germany

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, Use your smartphone which can be attributed to both bacteria and leukocytes action. (Fertil SterilÒ 2014;102:711–9. to scan this QR code Ó2014 by American Society for Reproductive Medicine.) and connect to the Key Words: Semen bacterial infection, sperm plasma membranes, sperm fertilizing potential Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/fraczekm-ejaculated-human-spermatozoa-bacterial-infection/

T

he potential negative influence of genitourinary tract inflammation/infection on sperm fertilizing ability has been long debated

(1). Recently, some investigators have discussed the long-term effects of the local inflammatory process and have postulated its role as the one of possible

Received January 20, 2014; revised and accepted June 3, 2014; published online July 17, 2014. M.F. has nothing to disclose. E.W. has nothing to disclose. M.P. has nothing to disclose. M.B. has nothing to disclose. D.G. has nothing to disclose. A.S.-K. has nothing to disclose. T.K. has nothing to disclose. L.B. has nothing to disclose. M.K. has nothing to disclose. This work was financed by Ministry of Science and Higher Education grant no. NN 407283539 and the National Centre for Research and Development grant no. NR 13006606. Tomasz Kolanowski is a scholarship recipient of EU 8.2.2 OP-Innovative Economy. Reprint requests: Maciej Kurpisz, M.D., Ph.D., Strzeszynska Strasse 32, 60-479 Poznan, Poland (E-mail: [email protected]). Fertility and Sterility® Vol. 102, No. 3, September 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.06.002 VOL. 102 NO. 3 / SEPTEMBER 2014

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reasons for impaired reproductive quality in middle-aged men (2). In the context of these reports, the appropriate diagnostic algorithm of urogenital inflammation/infection and rapid initiation of anti-inflammatory treatment (before the reproductive potential of the sperm is severely affected) became important issues in contemporary andrology. Attention has been focused on the search for new biomarkers of semen inflammation/infection, which are often beyond the scope of routine seminological 711

ORIGINAL ARTICLE: ANDROLOGY analysis. The direction of research has been determined by the kinetics of the inflammatory process showing the relationship among the infectious factor, leukocytes, and proinflammatory cytokines (3). Data concerning a significantly increased number of leukocytes and/or bacteria in semen are inconsistent. The presence of seminal peroxidase-positive leukocytes at an amount R1
106/mL of ejaculate, defined by the World Health Organization (WHO) (4) as leukocytospermia, is still considered to be pathological for sperm, although this threshold value has been repeatedly questioned in the literature (lower concentrations have been proposed), particularly with regard to monitoring the intensity of inflammatory reaction, which differs in time (5–7). Regardless of the number of leukocytes in the semen, most investigators agree that the final effects of the cells of the immune system on spermatozoa may depend on their activity (1, 3, 8). As for bacteriospermia, it may represent semen contamination, colonization, or infection (9, 10). The problem is further complicated by the fact that the same common bacterial strains are often isolated from the semen of both fertile and infertile patients. Moreover, there is a lack of uniformity in defining the critical number of bacteria, above which a decrease in sperm fertilizing potential occurs. On the one hand, according to WHO recommendations (11), a concentration R1
103 colony-forming unit (CFU)/mL of common uropathogenic bacteria per milliliter of ejaculate is regarded as bacteriospermia. On the other hand, a concentration R1
104 CFU of pathogenic and nonpathogenic bacterial strain per milliliter of ejaculate has been suggested by most investigators as significant bacteriospermia requiring diagnostics, although practically during active in situ bacterial infection elevated concentrations of bacteria have been often observed (5, 10, 12). It seems that the location of infection in the male genitourinary tract and the diagnostic profile of routine semen microbial culture are of great importance in the assessment of the clinical significance of bacteria in semen. Among the accepted pathogenic microbial strains causing genitourinary infections associated with suspected male factor infertility, Escherichia coli, Enterococcus faecalis, Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma hominis are the most often mentioned (13). However, there are also reports of harmful effects on sperm induced by conditionally pathological bacterial strains belonging to Staphylococci, Streptococci, and anaerobic Gram-negative and Gram-positive rods and other groups (14–16). It seems that an unequal effect of individual species and types of bacteria on germ cells could be significant in the personal assessment of inflammation and the monitoring of antiinflammatory therapy. Experimental approaches have shown a possible mechanism of interaction between sperm and single inflammatory mediator, which obviously cannot be observed in situ. There are many experimental data that have revealed the induction of structural changes in purified sperm suspensions by a single bacterial strain, as compared with uninfected sperm, with respect to sperm viability and motility (17–20), mitochondrial membrane potential (20), 712

and phosphatidylserine translocation (20–23); however, none of them have been exclusively focused on sperm fertilizing potential. In the present study, we have attempted to compare the changes in sperm plasma membrane integrity observed during classic experimental in vitro bacterial semen infection with results from functional sperm tests, such as the hypo-osmotic swelling (HOS) test and the sperm penetration assay (SPA). Moreover, these functional results would complement the findings of our earlier molecular, morphological, and cytochemical studies regarding an experimental model of semen infection (16).

MATERIALS AND METHODS Semen and blood sample collection was approved by the Local Bioethical Committee, Medical University of Poznan. Experiments with animals were approved by the Local Ethical Committee for the Animal Experiments, Poznan University of Life Sciences.

Reagents and Chemicals Phosphate buffered saline (PBS) was purchased from Biomed. The LIVE/DEAD Sperm Viability Kit (SYBR-14 and propidium iodide [PI]) was from Molecular Probes, and the OxiSelect TBARS Assay Kit for malondialdehyde (MDA) quantitation was from Cell Biolabs. Pregnant mare serum gonadotropin (Folligon, 200 IU) was purchased from Intervet International B.V., and hCG (Pregnyl, 1,500 IU) was from Organon. The remaining chemicals used were purchased from Sigma Chemical Co.

Flow Cytometry Measurements Flow cytometry analysis of sperm samples was performed using a Beckman Coulter flow cytometer (Cell LabQuanta SC MPL) equipped with a 488-nm argon-ion laser. For each sample, 10,000 events, at a rate of 150–250 events per second, were recorded within the characteristic flame-shaped region in the electronic volume (parameter depends on the cell size) and side scatter (parameter depends on cellular granules) dot plot corresponding to the sperm population. The green (480–550) and red (590–670) fluorescence were detected using the FL1 and FL3 channels, respectively. The fluorescence data were obtained at a fixed gain setting in logarithmic (FL1, FL3) mode. Data were analyzed using Cell LabQuanta SC MPL Analysis software (Beckman Coulter). Fluorescence reading was repeated 2 times from distinct samples.

Semen Sample Collection and Preparation The study population included healthy volunteers (n ¼ 15), between 20 and 35 years of age, recruited at an andrology outpatient clinic in Poznan, Poland. Selected donors were asymptomatic for genitourinary inflammations and varicocoele. Freshly ejaculated semen samples were collected in sterile containers after 3–5 days of sexual abstinence. Within 60 minutes after ejaculation and liquefaction, the conventional semen analyses were performed according to the WHO 2010 criteria (4). The peroxidative test as originally VOL. 102 NO. 3 / SEPTEMBER 2014

Fertility and Sterility® described by Endtz (24) was used for the assessment of leukocytes in ejaculate. Microbial cultures of semen were also performed for each semen sample, including aerobic, anaerobic, and atypical bacteria (Bio Merieux). Additionally, all the tested samples were checked for the presence of sperm antibodies using the direct immunobead test (Irvine Scientific). For all the experiments, only semen samples from normozoospermic donors with absence of antisperm antibodies and with no sign of semen infection (leukocytes <0.2
106/mL and negative semen bacterial culture) were used. The characteristics of the selected semen samples are presented in Supplemental Table 1. Spermatozoa from semen samples were separated from seminal plasma by centrifugation at 600 g for 8 minutes. After washing with warm PBS by centrifugation at 600 g for 8 minutes the sperm pellets were used for further experiments.

Blood Sample Collection and Preparation Fresh heparinized venous blood samples were obtained from healthy adults attending the Regional Blood Center, Poznan, Poland. The density gradient centrifugation technique (Histopaque-1077 g/cm3) was used for peripheral blood leukocyte isolation (400 g, 20 minutes, room temperature). Cells from the interface were washed twice with Hanks balanced salt solution, checked for viability via trypan blue staining, and counted. The cell pellet was finally resuspended to a concentration of 1
107/mL in PBS.

Bacteria Collection and Preparation Suspensions of three bacterial strains, that is, Escherichia coli (serotype O75:HNT), Staphylococcus haemolyticus, and Bacteroides ureolyticus, were obtained from the Outpatient Andrology Clinic/Unit of Microbiology of Poznan Hospital Medical College. The strains were isolated from semen samples of infertile patients with active infection in the urogenital tract (leukocytospermia and significant bacteriospermia >3
105 CFU/mL, >6
104 CFU/mL, and >1
106 CFU/mL of semen for E. coli, S. haemolyticus, and B. ureolyticus, respectively). The isolates were identified using the following biochemical test kits (Bio Merieux): ID 32 E for gram-negative rods, ID 32 Staph for staphylococci, and API 20 A for anaerobic bacteria. Fresh subcultures were prepared by streaking thawed stock cultures on Columbia agar containing 5% sheep blood cells and incubated at 37 C, under aerobic or anaerobic systems, respectively. On the day of the experiment, bacterial suspensions containing approximately 3
106 CFU/mL were prepared in sterile 0.85% saline. A specific anaerobic atmosphere generator system (GenBag Anaer, Bio Merieux) was used for transport of B. ureolyticus. After incubation, the viability of bacteria applied was checked by inoculation of the samples taken from stock bacterial suspensions to Columbia agar. All strains were alive during the experiments.

E. coli Serotyping Serotyping of O (lipopolysaccharide) and H (flagellar) antigens was performed using specific monovalent rabbit antisera as described elsewhere (25). VOL. 102 NO. 3 / SEPTEMBER 2014

Coincubation of Sperm with Bacteria and/or Leukocytes Twenty million spermatozoa resuspended in PBS were incubated with bacteria and/or leukocytes by means of an orbital shaker with a speed of 200 rpm for 2 hours at 37 C. To animate real and pathognomic in situ semen inflammation, bacterial strains were added at a concentration of 1
105 CFU/mL, and leukocytes were added at a concentration of 1
106 cells/mL to coincubated mixtures. As a control, sperm cells were incubated in the absence of both bacteria and leukocytes, while in a second control, sperm were incubated with leukocytes alone. After the incubation, spermatozoa were washed in PBS at 600 g for 5 minutes at room temperature, and sperm pellets were used for further experiments. For the assessment of lipid peroxidation, coincubated cell suspensions were selectively depleted from leukocytes to obtain pure sperm samples.

Sperm and Leukocyte Separation CD-45-positive cells were removed from cell suspensions using magnetic M-450 Dynabeads (Dynal). After 30 minutes of incubation at 4 C, supernatants with spermatozoa were removed, washed, and resuspended in PBS to a final concentration of 1
107 sperm/mL. Sperm pellets were then lysed with isotonic 10 mmol/L potassium buffer phosphate, pH 7.2, containing 0.05% butylated hydroxytoluene, and stored at 80 C until they were used for MDA measurement. All of the lysate was used in the assay.

Sperm Motility Assessment Sperm motility was assessed under phase-contrast microscopy using
400 magnification at room temperature according to recent WHO guidelines (4).

SYBR/PI Staining To assess cell viability, sperm suspensions (2
106/mL) were stained with 100 nM/L SYBR-14 (Ex/Em 490/516 nm, emission of green fluorescence) and incubated in the dark at 37 C for 5 minutes. Then the cells were incubated with 12 mM/L PI (Ex/Em 535/617 nm, emission of red fluorescence) in the dark at room temperature. Immediately after incubation, samples were analyzed via flow cytometry. The green (FL1) and red (FL3) fluorescence were detected simultaneously. Cells were divided into [1] FL1-negative and FL3-negative (SYBR-14-negative and PI-negative sperm), [2] FL1-positive and FL3-negative (SYBR-14-positive and PI-negative sperm), [3] FL1-positive and FL3-positive (SYBR-14 and PI-positive sperm), and [4] FL1-negative and FL3-positive (PI-positive sperm) populations. Only sperm positive for PI were analyzed statistically.

M540 Staining The level of scrambling of the phospholipids in the sperm plasma membrane lipid bilayer was determined using a lipophilic fluorescent probe merocyanine 540 (M540). Sperm suspensions (2
106/mL) were incubated with 4.9 mM/L M540 713

ORIGINAL ARTICLE: ANDROLOGY (Ex/Em 563/607 nm, emission of red fluorescence) for 15 minutes at 37 C in the dark and then evaluated via flow cytometry for detection of red fluorescence through the FL3 channel. Cells were divided into FL3-positive (M540positive sperm) and FL3-negative (M540-negative sperm) populations. Sperm negative for M540 were analyzed statistically.

Determination of MDA Samples containing 100 mL of sperm lysate, water (for a blank sample), or the known MDA concentration, 100 mL SDS lysis solution and 250 mL thiobarbituric acid solution, were heated in a water bath for 1 hour at 95 C. After cooling, the samples were centrifuged at 3,000 rpm for 15 minutes. Supernatants were then mixed with n-butanol (1:1, v/v) and centrifuged at 10,000 g for 15 minutes. The butanol fraction of each sample was transferred to a 96-well microplate. Duplicates of each standard and test sample were read using a spectrophotometric reader plate at 532 nm (ELx808, Bio Tek Instruments).

HOS Test Vitality test using the HOS was carried out as originally described by Jeyendran et al. (26). At least 200 spermatozoa treated with swelling solution for 30 minutes were evaluated with a phase-contrast microscope at
200 magnification. The percentage of swollen cells was calculated. Each sample was examined in duplicate. The lower reference limit for membrane-intact spermatozoa according to WHO is set for 58% (4).

SPA of Human Spermatozoa to Hamster Oocytes The test was performed including Syrian hamsters according to the method described by Martin et al. (27), with some of own modifications (28). After incubation with bacteria and/ or leukocytes (described above), spermatozoa were washed twice in BWW buffer (Biggers, Whitten, Whittingham), supplemented with human serum albumin (3.5 mg/mL), adjusted to a concentration of 5
106/mL, and subjected to capacitation at 37 C for 5–6 hours. Syrian hamster females used for the experiments were superovulated (using a standard protocol dependent on their estrous cycle) with an IP injection of 30 IU Folligon, followed by an IP injection of 25 IU Pregnyl. Zona-free eggs were obtained by dissecting the oviducts, preparing the cumulus oophorus, treating with 0.1% hialuronidase, and finally digesting with a 0.1% trypsin solution. For each tested sample, at least 100 zona-free oocytes were placed into a drop (200 mL) of capacitated spermatozoa and incubated at 37 C (5%CO2, 95% humidity) for 3.5–4 hours. After incubation, all the oocytes were evaluated under a phasecontrast light microscope, and the proportion of positively penetrated oocytes was calculated. An oocyte was considered to be positively penetrated when at least two pronuclei were visible: female and male. The assay was performed with a positive control semen sample with a penetration level over 75%. The mean control value of SPA for fertile males in our laboratory is 57%  10% (29). 714

Statistical Analysis All statistical calculations were carried out with the STATISTICA software package, version 10.0 (StatSoft). The ShapiroWilk test was used for checking the normal distribution in the studied groups. Any significant differences were assessed using a nonparametric analysis of variances (Kruskal-Wallis test) followed by the Dunn multiple comparisons test. The nonparametric Mann-Whitney U-test was used to compare the results obtained without leukocytes and after leukocyte incubation applied to selected bacterial strains. The correlations were calculated using the Spearman rank test. P< .05 was considered significant, P< .01 very significant, and P< .001 most significant.

RESULTS Effect of Bacteria on Sperm Motility Among bacterial strains applied, E. coli and B. ureolyticus decreased sperm motility, as compared with control sperm (P< .05; Table 1). None of the three analyzed bacterial strains used together with leukocytes decreased sperm motility, in comparison with sperm incubated with leukocytes alone.

Effect of Bacteria on Sperm Viability—SYBR-14/PI Test The incubation of sperm with bacteria was associated with an increased proportion of dead cells, especially in the presence of B. ureolyticus, as compared with untreated cells (P< .001; Table 1). In general, the addition of leukocytes to the coincubated mixtures resulted in an elevated proportion of PI-positive sperm, irrespective of the bacteria applied. A significant increase in dead cells was observed only with respect to B. ureolyticus used together with leukocytes, in comparison with sperm incubated with leukocytes alone (P< .05; Table 1).

Effect of Bacteria on Sperm Membrane Architecture—M540 Test All of the applied bacterial strains had a destructive effect on sperm membrane architecture, and the percentage of M540negative sperm with normal membrane architecture was significantly lower compared with untreated samples (P< .01 for each bacterial strain; Table 1). There were no marked differences between the percentage of M540negative sperm incubated with particular bacterial strains together with leukocytes and sperm incubated with leukocytes alone.

Effect of Bacteria on MDA Concentration The incubation of spermatozoa with bacteria resulted in a significant increase in MDA concentrations when compared with control sperm (P< .001; Table 1). This was true for each tested bacterial strain. In spermatozoa treated with bacteria together with leukocytes, the increase in MDA content was significant with S. haemolyticus and B. ureolyticus (P< .05; Table 1). VOL. 102 NO. 3 / SEPTEMBER 2014

Effect of Bacteria on Sperm Membrane Integrity— HOS Test

20.50; 15.24–35.61; 20.86  6.81 25.93a; 10.99–40.18; 26.23  7.59 62.47; 39.09–79.00; 61.26  9.45 14.25a; 6.42–22.88; 13.97  3.96

The decrease in the percentage of swollen sperm was highly significant with E. coli and S. haemolyticus (P< .01), although the greatest harmful effect on sperm membrane functional integrity was found in the presence of B. ureolyticus, as compared with untreated cells (P< .001; Table 2). Moreover, the incubation of sperm with anaerobic bacteria and leukocytes also significantly decreased the proportion of swollen sperm, as compared with sperm incubated with leukocytes alone (P< .01; Table 2).

Effect of Bacteria on Sperm Penetration—SPA Data obtained from the SPA were confirmed by the results obtained with the use of the HOS test. The inhibition of sperm-to-oocyte penetration by bacteria was observed, and statistically significant differences were found in the presence of all bacterial strains applied compared with untreated sperm (P< .001; Table 2). The addition of leukocytes to the coincubated mixtures was generally associated with a further lowering of the ability of sperm to penetrate the oocyte. Incubation of spermatozoa with leukocytes and bacterial strains significantly decreased the percentage of penetrated oocytes compared with sperm incubated with leukocytes alone (P< .01; Table 2).

Fraczek. Semen bacterial infection and sperm function. Fertil Steril 2014.

Effect of Leukocytes on Sperm Motility, Plasma Membrane Changes, and Sperm Function

Note: Data are median; min–max; mean  SD. a P< .05 calculated using Kruskal-Wallis test, compared with untreated sperm. b P< .001 calculated using Kruskal-Wallis test, compared with untreated sperm. c P< .01 calculated using Kruskal-Wallis test, compared with untreated sperm.

26.35; 19.43–43.08; 27.88  8.85 24.41; 7.89–39.12; 24.74  8.19 62.63; 34.70–79.45; 61.47  9.89 13.88a; 5.69–25.20; 13.75  4.17 24.19; 16.23–34.86; 23.46  5.28 25.15; 9.14–39.86; 25.01  7.88 62.99; 43.75–77.22; 62.49  8.92 12.32; 5.33–22.17; 12.95  4.22 26.50; 17.41–40.54; 26.75  7.55 20.24; 8.51–35.67; 20.62  7.12 62.87; 44.34–75.90; 61.52  9.39 10.80; 4.54–19.89; 10.80  3.81

Test

Progressive motility (%) PI-positive sperm (%) M540-negative sperm (%) MDA concentration (mM/mL)

Leukocytes D S. haemolyticus Leukocytes D E. coli Leukocytes (no bacteria)

VOL. 102 NO. 3 / SEPTEMBER 2014

Leukocytes D B. ureolyticus

B. ureolyticus

24.26a; 16.45–45.12; 26.12  9.85 25.92b; 8.46–39.49; 26.00  7.77 62.08c; 44.80–79.04; 60.75  9.95 6.60b; 2.47–12.97; 7.05  2.47

S. haemolyticus

28.33; 23.31–50.28; 32.25  10.35 22.93; 9.18–32.69; 21.31  7.25 58.26b; 39.60–74.08; 58.64  10.55 4.36b; 2.09–9.55; 5.06  1.91

E. coli

21.27a; 17.14–42.07; 24.62  9.13 22.37; 6.18–33.70; 20.98  7.99 60.16c; 40.37–75.81; 59.91  10.19 5.85b; 1.17–11.37; 5.81  2.44

No bacteria

43.50; 30.45–59.53; 41.75  10.22 16.13; 7.88–27.05; 16.89  6.27 70.86; 50.37–81.98; 69.50  8.26 2.79; 1.01–5.69; 2.68  1.00 Progressive motility (%) PI-positive sperm (%) M540-negative sperm (%) MDA concentration (mM/mL)

Test

Effect of bacteria and/or leukocytes on sperm motility and membrane integrity (PI-positive sperm, M540-negative sperm, and MDA concentration).

TABLE 1

Fertility and Sterility®

The Mann-Whitney test discriminated the leukocyte effect on the percentage of motile, PI-positive, HOS test–positive, M540-negative sperm, MDA concentration, and the percentage of fertilized oocytes in SPA, in respect to the particular bacterial strain used in the study (Table 3). In the absence of bacteria, leukocytes themselves decreased sperm progressive motility, induced lipid disorders in sperm plasma membranes, and reduced the functional integrity of the sperm's plasma membrane in the HOS test, and the effect was statistically significant (P< .01 for motility and P< .001 for MDA concentration, M540 test, and HOS test). The presence of all bacterial strains together with leukocytes was associated with a highly significant increase in lipid sperm membrane peroxidation (P< .001). Additionally, in the case of S. haemolyticus and B. ureolyticus, leukocytes had a statistically significant influence on the ability of sperm to penetrate oocytes (P< .001 and P< .05, respectively).

Spearman Rank Order Correlations between Sperm Plasma Membrane Changes and Sperm Function Table 4 shows the correlation values between sperm plasma membrane changes and sperm function. Most significant interesting correlations were observed between the percentage of M540-negative sperm and the percentage of swollen sperm. This was true for B. ureolyticus, leukocytes alone, and all bacterial strains used together with leukocytes. Additionally, in the case of leukocytes, the percentage of swollen 715

716

55.00a; 34.00–66.00; 53.94  8.61 51.40a; 46.30–61.30; 51.67  5.11

DISCUSSION

Fraczek. Semen bacterial infection and sperm function. Fertil Steril 2014.

Note: Data are median; min–max; mean  SD. a P< .01 calculated using Kruskal-Wallis test, compared with untreated sperm. b P< .001 calculated using Kruskal-Wallis test, compared with untreated sperm.

Leukocytes D B. ureolyticus Leukocytes D S. haemolyticus Test

sperm positively correlated with the percentage of fertilized oocytes in the SPA. A strong negative correlation between MDA levels and the percentage of fertilized oocytes was also observed for the combination of E. coli and leukocytes. Moreover, in sperm incubated with anaerobes, the percentage of penetrated oocytes was positively associated with the percentage of M540-negative sperm.

58.00; 43.00–75.00; 58.68  8.97 50.90a; 43.60–58.09; 50.54  5.70

Leukocytes D E. coli

62.00; 35.00–75.00; 60.45  10.45 55.70a; 44.40–65.90; 56.09  6.11

Leukocytes (no bacteria)

63.00; 43.00–87.00; 64.13  11.60 68.30; 60.60–81.60; 69.79  6.28

63.00 ; 28.00–83.00; 60.45  12.33 57.4b; 51.60–69.9; 59.04  8.11 65.00 ; 38.00–79.00; 61.55  11.53 58.2b; 49.5–68.10; 58.61  7.96 73.00; 59.00–89.00; 72.26  9.30 70.5; 64.60–82.50; 71.81  7.48 HOS test (%) SPA (%)

HOS test (%) SPA (%)

56.00 ; 31.00–73.00; 56.53  10.64 54.7b; 48.70–63.7; 55.25  7.05

S. haemolyticus

a

No bacteria Test

Effect of bacteria and/or leukocytes on sperm fertilizing potential (HOS test, SPA).

TABLE 2

a

E. coli

b

B. ureolyticus

ORIGINAL ARTICLE: ANDROLOGY

This study is the first one in which we attempted to correlate the observed sperm membrane alterations with sperm fertilization potential during in vitro semen bacterial infection. Because of the unavailability of human oocytes, we used the zona-free hamster oocyte penetration assay, which despite its obvious limitations, provides useful information on the ability of previously capacitated, acrosome-reacted sperm to fuse with the egg membrane and to form male pronuclei. Many years ago several investigators reported that the addition of mycoplasma organisms into sperm suspension resulted in reduced penetration of zona-free oocytes by sperm (30), and these experimental results were later confirmed in clinical research (31). In the present study, we analyzed the sperm penetration in hamster oocytes by E. coli, S. haemolyticus, or B. ureolyticus-treated sperm, and we were also able to observe a clear reduction in the ability of spermatozoa to interact with eggs compared with uninfected sperm. Moreover, the results obtained in the SPA corresponded well with the results obtained in the HOS test (Table 2). Such similarities can confirm the widely accepted view that the HOS test may be a useful indicator of the fertilizing potential of spermatozoa, although the recommendation of this test in the diagnosis and treatment of semen bacterial infection would be too far-reaching of a proposal at the present stage of research. In the light of the data obtained, bacteria appear as the important inducers of sperm membrane alterations, which in turn may lead to subfertility. The prognosis can probably be reduced drastically in cases of men who are subfertile at the start of the inflammation/infection. It is well-known that the functional cell membrane is absolutely necessary for the fertilizing potential of spermatozoa, as it plays an integral role at the start of the fertilization process. Our earlier fluorescence microscopy studies with the use of lipophilic M540 dye showed an increase of sperm with low and high red fluorescence over the head and/or the midpiece and principal piece, which corresponded to discrete and strong changes in plasma membrane architecture, respectively (16). Data obtained using flow cytometry and presented here (Table 1) confirmed those observations and revealed that coincubation of human spermatozoa with all studied bacterial strains resulted in sperm membrane changes associated with an increase in the scrambling of the plasma membrane phospholipids. According to some investigators (32, 33), the loss of lipid asymmetry observed with the help of the M540 test, especially in human spermatozoa, may reflect degenerative membrane modifications occurring during apoptotic-like events rather than capacitation-related membrane changes. In turn, Grunewald et al. (34) showed significantly opposite relationships between sperm apoptosis signaling and the VOL. 102 NO. 3 / SEPTEMBER 2014

Fertility and Sterility®

TABLE 3 Comparison of the percentage of motile, PI-positive, HOS test-negative, M540-negative sperm, and MDA concentrations and the percentage of fertilized oocytes in SPA without leukocytes and after incubation with leukocytes applied to selected bacterial strains. No bacteria vs. leukocytes

Progressive motility (%) PI-positive sperm (%) M540-negative sperm (%) MDA concentration (mM/mL) HOS test (%) SPA (%)

Z

P

2.635 1.959 3.343 6.485 2.696 1.224

.008252 NS .000828 .000000 .006970 NS

E. coli vs. E. coli D leukocytes Z

S. haemolyticus vs. S. haemolyticus D leukocytes

P

0.316 NS 1.726 NS 0.960 NS 5.754 .000000 0.450 NS 1.037 NS

B. ureolyticus vs. B. ureolyticus D leukocytes

Z

P

Z

P

0.947 1.539 1.027 6.174 1.077 3.671

NS NS NS .000000 NS .000241

1.427 0.016 0.316 5.668 1.211 2.302

NS NS NS .000000 NS .021291

Note: Z ¼ mann-whitney test values; NS ¼ nonstatistically significant. Fraczek. Semen bacterial infection and sperm function. Fertil Steril 2014.

percentage of penetrated oocytes in SPA. In this context, numerous correlations between the percentage of M540negative sperm and the percentage of either swollen sperm or penetrated oocytes, as demonstrated in our study (Table 4), seem to provide a clear although indirect support for the proapoptotic effect of local bacterial infections suggested by other investigators (22, 23, 35). Moreover, in light of these results, sperm lipid asymmetry level measured by the M540 test rather than by lipid peroxidation seems to be a precise predictor of sperm membrane destabilization caused by pathogenic as well as conditionally pathogenic bacteria. However, the suggestion to use the M540 test as a tool to detect sperm functional membrane modifications during bacterial semen infection needs further investigation. According to data presented in numerous reports, there is a relationship between the rate of lipid peroxidation and fertilizing potential in human spermatozoa (36–40). An association between the presence of some known pathogens, such as Ureaplasma urealyticum and Chlamydia trachomatis, and the induction of sperm lipid peroxidation, as judged by the MDA levels, has been suggested (41, 42). In our previous in vitro study regarding sperm MDA detection, we were able to demonstrate that bacteria tend to damage the sperm membrane lipids (43). In the current study, increasing the number of samples and applying bacteria at a pathognomic bacteriospermia (1
105 CFU/ mL) titre have enabled us to obtain statistically significant differences in MDA levels between sperm coincubated with bacterial strains and control samples (Table 1). Additionally, these results indirectly corresponded to our previous clinical

study, in which negative correlation between MDA levels and the number of fertilized oocytes in the IVF program was clearly documented (44). Taking into consideration MDA concentration and findings regarding M540-stained spermatozoa, it can be assumed that the phospholipid status of sperm membranes is an important issue from the point of view of the relationship between bacterial semen infection and sperm fertilizing potential. The pathological properties of the bacterial strains used in our experiments deserve some attention during the interpretation of the results. Among bacteria applied in the study, E. coli has the best documented direct negative impact on sperm quality, although the ability of E. coli to adhere to, agglutinate, and immobilize human spermatozoa depends on its serotype (20, 45–47). Our previous morphological observations using both light and scanning electron microscopy have indicated E. coli serotype O75:HNT, S. haemolyticus, and B. ureolyticus adhesion to the apical part of the acrosome, midpiece, and flagellum of spermatozoa (16). Taking into account our previous and recent data, we can state that the decrease in sperm motility and/or the molecular changes in sperm membrane lipid status documented here were the consequence of the direct contact of all bacteria with sperm cells. Another possible mechanism by which some kind of bacteria might affect the fertilizing potential of sperm is through its cytotoxic activity. This is evidenced by the results obtained in the SYBR14/PI test, in which we observed a significant increase in the percentage of dying or dead cells with disintegrated plasma membranes only in sperm samples treated with

TABLE 4 Spearman rank order correlations between sperm membrane integrity and sperm function in relation to inflammatory factor applied. Correlation

R spearman

P

HOS test and M540 test; SPA and M540 test HOS test and M540 test; HOS test and SPA HOS test and M540 test; SPA and MDA HOS test and M540 test HOS test and M540 test

0.488780; 0.529670 0.417355; 0.550000 0.461634; 0.657497 0.430352 0.489021

.007134; .051417 .024286; .033655 .011708; .014595 .019790 .007102

Inflammatory factor B. ureolyticus Leukocytes E. coli þ leukocytes S. haemolyticus þ leukocytes B. ureolyticus þ leukocytes

Fraczek. Semen bacterial infection and sperm function. Fertil Steril 2014.

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717

ORIGINAL ARTICLE: ANDROLOGY B. ureolyticus, as compared with the controls, irrespective of whether it was used alone or together with leukocytes (Table 1). These findings are in agreement with our previously published reports, where the induction by this anaerobic strain of apoptotic-like changes in human ejaculated spermatozoa was documented with respect to membrane stability, mitochondrial membrane potential, and DNA fragmentation (16, 48). However, the association between the anaerobes present in ejaculates and sperm apoptosis/ necrosis requires further study. Other proposed mechanisms for how semen bacterial infections might interfere with human ejaculated spermatozoa involve leukocytes infiltrating the inflammatory site (49). In certain studies, the increased number of leukocytes in the ejaculate during semen inflammation/infection is considered to be the dominant factor associated with deterioration in sperm parameters, most probably by the specific actions of ROS and proinflammatory cytokines and/or the production of antisperm antibodies (49–54). In the current experimental study, the physical presence of leukocytes in sperm suspensions was found to decrease sperm motility and the sperm fertilizing potential (Table 3). These results are in agreement with other experimental and clinical reports in which the detrimental effect of leukocytospermia on sperm motility and sperm function reflected in SPA, the HOS test, or acrosome reaction was observed (55–57). Interestingly, in the present study, we were also able to demonstrate that in in vitro conditions leukocytes can alter the sperm membrane architecture in the MDA test. Moreover, this effect was similar in sperm incubated with leukocytes alone and combined with bacteria (Table 2). Such results indicate that leukocytospermia per se can damage spermatozoa, independent of bacteriospermia. Moreover, the molecular mechanism by which leukocytes interfere with the fertilizing capacity of spermatozoa may be related to the lipid content in sperm membranes. This hypothesis can be based on the numerous correlations observed between the sperm membrane architecture and membrane functional integrity in all cases when leukocytes were present (Table 4). It should also be noted that the joint action of leukocytes and bacteria intensified lipid sperm membrane alterations, particularly those associated with the peroxidative process. Moreover, the highest MDA levels observed in the samples incubated with leukocyte and bacterial strains (particularly with conditionally pathogenic bacteria) were associated with a significant reduction in the number of penetrated oocytes, as reflected by SPA results (Table 3). This is not the first time that we have suggested the complementary role of these two inflammatory mediators in deepening the harmful effect of bacterial semen infection on sperm membranes (43), but this is the first study in which we have reported the combined effect of infectious factors and leukocytes in triggering both structural and functional defects in human germ cells. Based on the results obtained, it can be concluded that bacterial semen infection may be an important factor negatively influencing fertility status and worsening reproductive potential. Sperm motility and lipid sperm membrane status might be the earliest and the most sensitive indicators of 718

sperm damage, which can be attributed to both bacteria and leukocytes. Bacteria may be damaging to ejaculated spermatozoa independent of leukocyte contamination, although the concomitant presence of bacteria clearly enhanced the harmful effects of leukocytes by inducing the peroxidative damage of sperm membranes to a level above which their ability to penetrate and fuse with an oocyte may be significantly reduced. The present findings provide further evidence that positive microbial semen culture must be treated with attention, especially in patients consulting for infertility and qualifying for assisted reproduction techniques. In the light of these results, extended semen microbiological diagnostics should be recommended in all cases of leukocytospermia, and this assumption has been supported by some earlier observations (44, 58, 59). Acknowledgments: The authors thank Anna Czernikiewicz, M.Sc., for blood sample processing.

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SUPPLEMENTAL TABLE 1 Standard semen parameters (n [ 15 samples). Semen parameter

Median

Min–Max

Mean ± SD

Volume (mL) pH Sperm concentration (
106/mL) Total no. of spermatozoa (
106/ejaculate) Progressive motility of spermatozoa (%) Total sperm motility (%) Immotile sperm (%) Sperm vitality (% alive) Spermatozoa with normal morphology (%) Peroxidase-positive cells (
106/mL) Round cells of spermatogenic lineage (
106/mL) Antisperm antibodies (IgG, IgA, IgM) (%)

3.50 8.0 100.00 345.60 57.00 66.00 34.00 87.00 18.00 0.04 0.96 0

2.50–7.50 7.5–8.2 58–128 187.5–675.0 45.00–78.00 51.00–80.00 20.00–49.00 73.00–92.00 11.00–26.00 0.00–0.12 0.36–2.04 0

3.85  1.65 8.0  0.2 94.45  21.47 343.81  134.92 58.64  11.01 65.64  9.22 34.36  9.22 84.73  6.12 17.30  4.14 0.05  0.03 1.11  0.49 0

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