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Biology of Reproduction 63, 1041-1048 (2000)
© 2000 Society for the Study of Reproduction, Inc.

ARTICLES

In Vivo and In Vitro Impairment of Human and Ram Sperm Nuclear Chromatin Integrity by Sexually Transmitted Ureaplasma urealyticum Infection1

Malka Reicharta, Itzhak Kahane3,,b, and Benjamin Bartoov2,,a

a Male Fertility Laboratory, Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel b Department of Membrane and Ultrastructure Research, The Hebrew University Hadassah Medical School, Jerusalem, 91120, Israel


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of Ureaplasma urealyticum infection in the semen of infertile men is variable (7%–42%). Evidence has accumulated through routine semen analysis to suggest that this infection can cause embryo loss without necessarily affecting sperm quality. The aim of this study was to specifically investigate the effects of U. urealyticum infection on sperm chromatin stability and DNA integrity, which are known to be correlated to pregnancy outcome. Sperm cells isolated from human semen infected in vivo with U. urealyticum exhibited a low percentage of stable chromatin as determined by nuclear chromatin decondensation assay (42% ± 4.8%, n = 8) and a high percent of denatured DNA as determined by sperm chromatin structure assay (60.9% ± 9.1%, n = 7). After doxycyclin treatment, a significant improvement in both parameters was observed (73.7% ± 3.6%, P < 0.001 and 30.1% ± 3.5%, P < 0.008, respectively). Sperm cells infected in vitro exhibited higher rates of viability and motility than uninfected cells. In contradistinction, U. urealyticum caused significant dose- and time-dependent chromatin decondensation and DNA damage. The percentage of human sperm cells with denatured DNA increased significantly by 54.9% ± 23.9% and 47.9% ± 12.1%, after 30 min infection with serotypes 8 and 3, respectively, at a multiplicity of infection of 100 ureaplasmas per sperm compared with uninfected control cells. The damage to DNA was significantly more pronounced in infected ram sperm (180.9% ± 21.5%). These results indicate that preserved sperm activity post U. urealyticum infection resulted in damage to paternal DNA, although a high fertilization rate was maintained, and embryonic development may, therefore, be impaired.

implantation/early development, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ureaplasma urealyticum is a self-replicating prokaryote belonging to the taxonomic class Mollicutes, which lack a cell wall [1]. Genital U. urealyticum infection is considered to be a sexually transmitted infection and therefore occurs more frequently during fertile ages. The bacterium has been found to be involved in prostatitis [2] and epididymitis [3]. The question of whether the presence of U. urealyticum in semen may be a possible cause of infertility has long been debated [4–6] especially because the incidence of this organism in semen of fertile and infertile men was found to be similar, in the range of 7% to 42% [7, 8].

During the past decade, evidence has accumulated of damage caused by U. urealyticum to the development and viability of human embryos. Colonization of the upper female genital tract with U. urealyticum was found to be associated with adverse pregnancy outcomes [9]. In human in vitro fertilization systems, the presence of U. urealyticum in either semen or the female genital tract resulted in a decrease in pregnancy rate per embryo transfer [4]. Shalika et al. [6] reported that pregnancies were not achieved at all when U. urealyticum was present in semen. Specific evidence that the presence of U. urealyticum in semen causes inhibition of embryonic development to blastocysts was reported in a mouse in vitro fertilization system in which sperm cells were preincubated in vitro with this bacterium [10].

The mechanism by which U. urealyticum affects sperm quality has not been elucidated. Some investigators were unable to correlate the presence of U. urealyticum with any alteration in semen characteristics [8, 11, 12], others have reported that the presence of U. urealyticum in semen was related to a decrease in sperm density [13, 14], motility [7, 13], and morphology [15, 16].

On one hand, these sperm parameters, although somewhat related to fertilization potential, are less relevant to embryonic development; on the other hand, the integrity of the sperm nucleus and its chromatin content are apparently more related to pregnancy outcome [17]. Indeed, the following sperm nuclear parameters were found to be related to pregnancy achievement: ultrastructural examination of nuclear shape and chromatin texture [18], chromatin decondensation induced in vitro [19], sperm chromatin packaging quality using chromomycin A3 [20] or aniline blue staining [21], DNA staining after in situ denaturation of chromatin [22], in situ nick translation [20], TUNEL [23], and oxidized nucleoside (8-hydroxy-2'-deoxyguanosine) content in total DNA [24].

Because the integrity of sperm nuclear chromatin may be related to embryonic development, the aim of this study was to examine whether this parameter could be the underlying mechanism by which U. urealyticum causes a male factor adverse pregnancy outcomes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Sperm In Vivo Infection Study

Eight men with semen cultures positive only to U. urealyticum in two consecutive semen analyses at an interval of 1 mo were included in this study. Six of the men belonged to 14 randomly selected couples with no previous history of infertility, who achieved spontaneous pregnancy within the last 5 yr. Two men had unexplained infertile normozoospermia. All patients were referred to the Bar-Ilan Male Fertility Laboratory from Meir Hospital, Kfar Saba, for this specific study, which was authorized by the hospital's Helsinki Committee. Routine semen analysis was performed as described previously [25]. Nuclear chromatin integrity tests (i.e., nuclear chromatin decondensation assay [NCDA] and sperm chromatin structure assay [SCSA]) were performed before and after doxycyclin treatment (100 mg per day for 10 days).

Human Sperm In Vitro Infection Study

Semen from normozoospermic men according to the definitions of the World Health Organization [26], who were referred to the Bar-Ilan Male Fertility Laboratory, were obtained after 4 days of sexual abstinence. The semen was diluted 1:3 with Hams F-10 buffer (Biological Industries, Beit Haemek, Israel) pH 7.6 and centrifuged for 15 min at 400 x gmax at 25°C. The sperm pellet was resuspended in 3 ml Hams F-10 buffer and washed by similar centrifugation. The washed sperm pellet was resuspended in the same buffer to a standard concentration of 50 x 106/ml. Duplicate aliquots of 0.2 to 1 ml sperm suspension were infected with a multiplicity of infection (m.o.i.) of 10 to 200 U. urealyticum colony-forming units per sperm at 37°C under hypoxic conditions (in 2 ml closed Eppendorf tubes flushed with nitrogen gas whenever exposed to air). Aliquots for postinfection tests were taken at 30-min intervals as follows: viability (100 µl), motility (50 µl), adherence and penetration using light and electron microscopy (250 µl), as well as NCDA (200 µl) and SCSA (300 µl). The results presented are the mean values of the duplicates.

Ram Sperm In Vitro Infection Study

Ram semen was collected from Assaf breed rams using a sterile artificial vagina after male stimulation by a rutted female. Sperm cell preparation, infection, and analytical procedures were as described earlier. This specific investigation was authorized by the Ethics Committee of the Bar-Ilan University and that of the State of Israel.

Preparation of U. urealyticum Specimens

U. urealyticum serotypes 3 and 8, representing the two different biovars [27], were grown in 1-L batches, harvested, and washed as previously described [28]. The final ureaplasma pellet was resuspended in 5 ml Pleuropneumonia-like organism broth (Difco, Detroit, MI) pH 6.5 containing 5% dimethyl sulfoxide (Sigma, St. Louis, MO). The colony-forming units titer was evaluated and found to be approximately 1010/ml. Aliquots of 0.5 ml were frozen in liquid nitrogen and stored at -70°C. For every in vitro experiment, 1 to 2 aliquots were thawed in a 37°C incubator for 30 min, then diluted 1:20 in Hams F-10 and centrifuged for 10 min at 26 890 x gmax at 4°C. The pellet was resuspended in Hams F-10 buffer to the original volume.

Efficacy of Infection

The efficacy of infection was evaluated by adherence and penetration of bacteria to sperm subcellular organelles using three microscopy analyses.

Light Microscopy

Adherence was determined using an inverted microscope (Olympus 1X-70) equipped with Nomarsky optics, Uplan Apo x100/1.35 lens and 0.55 NA condensor. Adherence of at least one ureaplasma to a specific location on a spermatozoon was defined as a positive result. Two parameters were determined in 250 sperm cells examined in each sample: percent of cells with at least one positive result, and percent of positive results on sperm head and tail regions.

Transmission Electron Microscopy

Sperm cells were separated from seminal plasma by centrifugation for 15 min at 1650 x gmax at room temperature. The washed sperm pellet was resuspended in 0.1 M phosphate buffer, pH 7.4. The sperm suspension was fixed, dehydrated, and embedded as previously described [29–31]. Thin sections were cut using an LKB ultratome (Bromma, Stockholm, Sweden) and observed using a Jeol JEM 1200EX transmission electron microscope (Jeol Ltd., Tokyo, Japan).

Scanning Electron Microscopy

Sperm cells were separated from their plasma and fixed as they were for the transmission electron microscope preparation. The samples were dehydrated with graded alcohol and freon solutions, critical point-dried with CO2, and coated with gold [32]. Sperm cells were examined with a Jeol JSM 840 scanning electron microscope.

Sperm Activity Tests

Viability Sperm cell viability was assessed using Eosin nigrosin staining as described previously [33]. Four hundred sperm cells were assessed for each sample.

Motility Detailed motility changes of different sperm cell samples, which occurred during the experiments, were determined by the sperm motility index, which was obtained by using the sperm quality analyzer [34]. The sperm motility index was measured on duplicate samples for 20 sec at 30-min intervals. For the sake of comparison between samples, the intensity of sperm motility was calculated using the sum of the sperm motility index values obtained throughout the experiment, until motility ceased. This value was defined as the integral of motility.

Sperm Chromatin Structure Assay

Sperm sample aliquots from in vitro-infected sperm cells were resuspended in 2 ml TNE buffer solution (0.01 M Tris, 0.15 M NaCl, 0.001 M EDTA pH 7.4) and centrifuged for 20 min at 400 x gmax at 25°C. Semen samples from in vivo-infected sperm cells were centrifuged twice as described earlier. The final pellet was resuspended in 2 ml of TNE buffer. Separation of sperm heads from their tails was achieved by sonication at an amplitude of 9 for 75 sec at 4°C, using a 150-watt ultrasonic disintegrator (MSE, Ltd., Crawley, UK) equipped with a titanium microprobe. The suspension was centrifuged at 3000 x gmax for 15 min at 4°C. The obtained nuclei pellet was suspended in 0.1 ml TNMg buffer (0.02 M Tris, 0.15 M NaCl, 0.005 M MgCl2 pH 7.4), then fixed by forceful pipetting into 0.9 ml of an acetone:70% ethanol (1:1 v/v) solution. All steps of this procedure were performed at 4°C. Induction of acid DNA denaturation in situ, acridine orange staining procedure, and flow cytometry measurement were performed according to the method of Evenson and Jost [35] using a Becton-Dickinson FACSort (San Francisco, CA) flow cytometer equipped with ultrasense and a 15 mW argon ion laser with an excitation wavelength of 488 nm. The internal standard for calibration was a stock of fixed ram sperm nuclei prepared as described earlier. The {alpha}t was calculated using a ratio time 1.1 software package, which was written by Jan van der Aa (Becton-Dickinson, Erembodegem, Belgium). Five thousand sperm cells were examined for each sample. SCSA parameters were computed on the basis of the distribution of {alpha}t values. First, mean channel of the {alpha}t distribution, and second, percent of cells outside the main {alpha}t peak (%COMP{alpha}t). The distribution of {alpha}t values of nuclei isolated from sperm of fertile men exhibiting relatively high stability to the acid induction of DNA denaturation was designated as the main {alpha}t peak [35]. The area of the main peak was obtained by visually inspecting the nuclei {alpha}t distribution on the computer screen and setting a fixed region. The two parameters computed from the {alpha}t distribution indicated relative stability of sperm nuclei to acid denaturation. All measured parameters were computed using WinMDI 2.0.5 software (URL: http://Facs.scripps.edu/).

The reliability of the SCSA parameter, mean {alpha}t value and %COMP{alpha}t, were assessed by testing 10 aliquots of fixed sperm nuclei that had been prepared from ejaculates of 3 different patients (total of 30 samples). The stability reliability, as analyzed by Cronbach {alpha}, was very high: mean {alpha}t value = 0.99, and %COMP{alpha}t = 0.98, indicating that the values are stable.

Assessment of In Vitro Nuclear Chromatin Decondensation

Cytometric analysis Sperm nuclei were prepared, fixed, denatured, and stained with acridine orange as described earlier. Five thousand sperm cells were examined for each sample. The degree of sperm nuclei decondensation caused by the detergent (Triton; BDH, Poole, Dorset, England) and acid treatment [22, 36, 37] was assessed by relative mean nuclear size, determined by forward light scatter; and mean green fluorescence emission, indicating the penetration ability of the dye into nucleic DNA. The measured parameters were computed using WinMDI 2.0.5 software. The reliability of these parameters was assessed by testing 10 aliquots of fixed sperm nuclei prepared from ejaculates of 3 different patients (total of 30 samples). An average coefficient of variation (SD/mean) of 3% ± 1.4% for the relative nuclear size and 3.3% ± 1.7% for the mean green emission indicate that these parameters are reliable.

Cytological analysis The NCDA procedure for in vivo-infected sperm cells was performed essentially as described by Lipitz et al., Kvist, and Bedford et al. [19, 38, 39]. The same procedure was used for in vitro-infected sperm cells except that the first step of sperm cell separation from seminal plasma using Ficoll (Sigma, St. Louis, MO) was omitted. The staining procedure was modified as follows: 3-µl samples of the fixed sperm cells were layered onto glass slides, air-dried, and stained with spermac stain (Stain Enterprises, Wellington, South Africa). Nuclear chromatin decondensation of 500 sperm cells from each sample was semiquantitatively assessed with a light microscope at a magnification of 1500x. Four degrees of decondensation were assessed: stable condensed cells; and slightly, intermediate, and highly decondensed cells. The reliability of the measured percentage of stable cells was assessed by evaluating 3 identical aliquots from 15 different experimental samples (a total of 45 samples). An average coefficient of variation (SD/mean) of 2.7% ± 1.6% indicated that this method is reliable.

Statistical Analysis

Statistical analysis was performed on the raw values obtained for the different sperm parameters measured using the SPSS-X package [40]. A separate multivariate analysis of variance (MANOVA) for repeated measures design was performed for each of the sperm parameters to analyze the significance of the dose curves and time courses, followed by separate contrasts between the control uninfected dose and each m.o.i. level (some of the values were missing in the repeated measures analysis, which thus reduced the degrees of freedom [df]). For percent of live sperm cells, a two-factorial repeated measures MANOVA design (time [0, 30, 60, 120 min] x dose [0, 10, 50 m.o.i.]) was performed. A mix design ANOVA (species [ram, human] by dose [0, 10, 50, 100 m.o.i.]), with the latter being a repeated measure, was executed for comparing the differences between U. urealyticum effects on human and ram chromatin decondensation and DNA integrity parameters. The same mix design ANOVA (serotype [3, 8] by dose [0, 10, 50, 100 m.o.i.]) was executed for comparing the effects of the two U. urealyticum serotypes on human chromatin and DNA parameters. The significance of the analyses of variance was determined by F ratio = explained variance/residual. A P value < 0.05 was considered statistically significant. Means are listed with standard errors for in vitro-infected sperm cells and with standard deviations for in vivo-infected sperm cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Infection Study

Adherence of U. urealyticum to sperm cells Both serotypes 8 and 3 of U. urealyticum adhere to washed ejaculated ram and human sperm cells after 15 to 30 min of exposure in vitro (Fig. 1, A and B). In ram, after 2 h of infection, ureaplasmas were found to adhere to 56% of sperm cells. The percent of adherence to the sperm head was four times higher than to the sperm tail (81.5% and 18.5%, respectively). After 60 min of infection, bacteria were found in different cytoplasmic spaces within the sperm cells (Fig. 1C).



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  		FIG. 1. U. urealyticum adherence and penetration to human and ram sperm cells. Sperm cells were infected with U. urealyticum for 15–30 min in vitro under hypoxic conditions in Hams F-10 at 37°C, at an m.o.i. of 10–100 per sperm. Adherence to the acrosomal region of ram (A, light microscope) and human (B, scanning electron microscope) sperm cells. U. urealyticum in the cytoplasmic droplet in the middle piece region of human sperm cells after 60 min of infection (C, transmission electron microscope) and normal uninfected sperm cell with cytoplasmic droplet (D, transmission electron microscope). Bar = 10 µm in A, 1 µm in B, and 0.5 µm in C and D. M, Mitochondria; U, U. urealyticum.

Sperm activity Viability and motility of uninfected human sperm cells decreased spontaneously during 120 min of incubation under hypoxic conditions (Fig. 2a). A similar decrease in both parameters was observed in infected sperm cells. The decrease in both uninfected and infected sperm cell viability with time was found to be significant (F = 11.4; df = 2, 10; P < 0.003, n = 6). Necrotic effect was significantly lower in infected sperm cells. This decrease was dose-dependent (F = 30.4; df = 2, 10; P < 0.001). Reduction in motility of infected ram sperm cells was also significantly lower than in uninfected cells (Fig. 2b). Integral of motility of infected and uninfected sperm cells was 1111.2 ± 414 and 1411 ± 452, respectively (F = 5.0; df = 1, 10; P < 0.048).



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  		FIG. 2. Maintenance of human sperm cell viability (a) and ram sperm cell motility expressed by the sperm motility index (b) by U. urealyticum. Duplicate sperm cell suspensions were infected in vitro with U. urealyticum serotype 8 under hypoxic conditions in Hams F-10 at 37°C, at an m.o.i. of 10 and 50 ureaplasmas per sperm. Values are mean ± SEM of six different experiments in a and a representative experiment out of six performed in b. Uu, U. urealyticum.

Sperm Chromatin Condensation

As determined by NCDA, ureaplasmas of serotypes 8 and 3 induced decondensation of human sperm cells after 30 min of infection. This induction was dose-dependent up to an m.o.i. of 200 ureaplasmas per sperm (F = 13.6; df = 4, 16; P < 0.001 for serotype 8 and F = 12.9; df = 4, 20; P < 0.001 for serotype 3) and reached maximum levels of 33% ± 4.3%, and 31.2 ± 6.6%, respectively (Fig. 3a). Fifty percent of the maximal effect was observed at an m.o.i. of approximately 50 ureaplasmas per sperm. Decrease in chromatin stability was directly related to duration of infection, when measured at an m.o.i. of 50 ureaplasmas per sperm cell (F = 4; df = 4, 8; P < 0.045, Fig. 3b).



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  		FIG. 3. Dose response (a) and time kinetics (b) of U. urealyticum on human sperm chromatin stability, observed by NCDA. a) Duplicate sperm cell suspensions were infected for 30 min in vitro with U. urealyticum serotype 8 (diamonds), and serotype 3 (squares), under hypoxic conditions in Hams F-10 at 37°C, at an m.o.i. of 10 to 100 ureaplasmas per sperm. Values are mean ± SEM of the percent of the elevation obtained in the percent of decondensed nuclei compared with control uninfected nuclei (n = 7 and 8 different experiments for serotype 8 and 3, respectively). b) Infection with serotype 8 at an m.o.i. of 50 (n = 4 different experiments). *Significantly different from control, P < 0.05. Uu, U. urealyticum.

A significant dose-dependent elevation in values of mean relative nuclear size was found after 30 min of infection, at a multiplicity of >=10 ureaplasmas per sperm cell, with serotype 8 in ram sperm cells and serotypes 8 and 3 in human sperm cells (F = 9.2; df = 4, 16; P < 0.001 for ram sperm and F = 21.5; df = 4, 20; P < 0.001 and F = 7.1; df = 4, 16; P < 0.002 for human sperm infected with serotypes 8 and 3, respectively; Fig. 4, a and b). At an m.o.i. of 100, the mean relative nuclear size of human sperm increased by 30% ± 3% and by 28.7% ± 5.2% for serotypes 8 and 3, respectively. Although the dose curve of the mean intensity of green fluorescence emission was significant only for human sperm cells infected by U. urealyticum serotype 3 (F = 3.51; df = 4, 12; P < 0.041) at an m.o.i. of 100, mean green fluorescence emission exhibited a significant (19.8% ± 8.8%) increase (F = 10; df = 1, 5; P < 0.025) and 14.4% ± 0.4% (F = 36.7; df = 1, 5; P < 0.002) for serotypes 8 and 3, respectively, compared with uninfected control cells. The decondensation effect induced by U. urealyticum serotype 8 on ram sperm cells was approximately half of that obtained in human sperm cells: a 15.5% ± 2.1% increase in mean relative nuclear size (F = 31.3; df = 1, 4; P < 0.005) and an 8.5% ± 1.9% increase in mean green fluorescence emission (F = 7.5; df = 1, 4; P < 0.052). The difference between ram and human sperm cells was significant only for mean relative nuclear size (F = 6.9; df = 1, 17; P < 0.018).



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  		FIG. 4. Dose response of U. urealyticum serotype 3 (squares, n = 7 different experiments) and 8 (diamonds, n = 6 different experiments) on human and serotype 8 on ram (triangles, n = 5 different experiments) sperm chromatin decondensation (a, b) and susceptibility to acid denaturation (c, d) measured by flow cytometry using SCSA after 30 min of incubation. a) Relative nuclear size, b) green fluorescence emission, c) mean {alpha}t value, d) percent of cells stable to acid denaturation. Duplicate sperm cell suspensions were infected in vitro with U. urealyticum serotypes 8 and 3, for 30 min under hypoxic conditions in Hams F-10 at 37°C, at an m.o.i. of 10 to 100 ureaplasmas per sperm. Values are mean ± SEM of the percent of the change obtained in the value of the examined parameter compared with control uninfected cells. *Significantly different from control, P < 0.05. Uu, U. urealyticum.

Integrity of Sperm Nuclear DNA

A decrease in the integrity of nuclear DNA of human and ram sperm cells was observed following a 30-min infection with U. urealyticum at a multiplicity of >=10 ureaplasmas per sperm cell. This effect was observed with infection of ram sperm cells by serotype 8 and of human sperm cells by serotypes 8 and 3 (Fig. 4, c and d). Human sperm cells infected with U. urealyticum exhibited increased {alpha}t values and an increase in percent of sperm cells with denatured DNA (%COMP{alpha}t), which was dose-dependent at a multiplicity of >=10 ureaplasmas per sperm cell (F = 3.2; df = 4, 12; P < 0.05 and F = 6.16; df = 3, 15; P < 0.006 for {alpha}t values and F = 8.33; df = 4, 12; P < 0.002 and F = 5.83; df = 3, 15; P < 0.008 for the increase in the %COMP{alpha}t, of sperm cells infected by U. urealyticum serotypes 8 and 3, respectively; Fig. 4, c and d). After 30 min of infection with U. urealyticum serotypes 8 and 3, at an m.o.i. of 100, the percent of sperm cells with denatured DNA increased significantly (54.9% ± 23.9% and 47.9% ± 12.1%, respectively) compared with uninfected control cells (F = 37.5; df = 1, 4; P < 0.004 and F = 16.7; df = 1, 6; P < 0.006, respectively; Fig. 4d). Ram sperm cells exhibited a 180.9% ± 21.5% increase in percent of sperm cells with denatured DNA upon infection with U. urealyticum serotype 8 at an m.o.i. of 100 (F = 8.8; df = 1, 3; P < 0.06; Fig. 4d). The dose-dependent increase in %COMP{alpha}t of ram sperm cells caused by U. urealyticum infection was significantly higher than that caused to human sperm cells (F = 2.7; df = 4, 36; P < 0.046).

In Vivo Infection Study

U. urealyticum-infected sperm cells isolated from human semen exhibited an average low percent of stable chromatin as determined by NCDA (42% ± 4.8%, n = 8; Fig. 5a) and an average high percent of cells with denatured DNA (%COMP{alpha}t) determined by SCSA (60.9% ± 9.1%, n = 7; Fig. 5b). After doxycyclin treatment, which resulted in eradication of ureaplasmas from semen (judged by semen culture), the average percents of the former parameter increased significantly to 73.7% ± 3.6% (F = 21.8; df = 1, 14; P < 0.001) and that of the latter parameter decreased significantly to 30.1% ± 3.5% (F = 10; df = 1, 12; P < 0.008). These values were close to those obtained in normozoospermic semen samples (Fig. 5, a and b). Sperm viability, motility, and morphology remained unchanged (Table 1).



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  		FIG. 5. The effect of successful antibiotic treatment against U. urealyticum infection on chromatin condensation (a) determined by NCDA (n = 8) and DNA integrity (b) determined by SCSA (n = 7). Each line represents a different patient. The value of the percent of chromatin stable cells was the mean of two consecutive analyses with a 1-mo interval before treatment (BEFORE) and of two consecutive analyses, after a 10-day course of doxycycline treatment, which resulted in eradication of the bacteria (AFTER). Average = the mean percent of stable cells in all semen examined before and after treatment. *Significantly different from the mean value before treatment (P < 0.001). **Significantly different from the mean value before treatment (P < 0.008)


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  		TABLE 1. Routine sperm parameters of in vivo-infected sperm cells before and after doxycycline treatment


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Semen quality, especially chromatin and DNA integrity, was investigated for a possible link between U. urealyticum infection of semen and embryo development and abortion. Because semen of in vivo-infected males may not represent the direct effect of infection and because other etiologies of male factor infertility cannot be ruled out, the present study focused mainly on in vitro infection by U. urealyticum, with in vivo infection serving a complementary role.

Small changes in human sperm quality are difficult to assess because human sperm cells differ widely in their quality, as expressed by motility and morphology. Ram sperm cells were therefore used as an animal model. The question arose whether ram sperm cells can be infected by the human pathogen, U. urealyticum [41]. In the first part of this study it was shown that indeed, ram sperm cells are infected in vitro by U. urealyticum, as expressed by adherence and penetration after a short exposure time, similarly to that observed in human sperm cells infected in vitro.

The results of nuclear chromatin analysis in in vitro-infected sperm cells indicate that U. urealyticum causes premature chromatin decondensation and damage to DNA integrity of both human and ram sperm cells in a dose- and time-dependent fashion. Although the energy metabolism of the sperm cells in this study was directed to the glycolytic pathway, as induced by hypoxic conditions as described in Materials and Methods, the deleterious effect of U. urealyticum infection on sperm chromatin was also observed under normal aerobic conditions. This effect was observed after a short infection time (30 min). These results confirm the observation that U. urealyticum causes mitotic alteration, especially chromatid gaps and chromatid breaks, in cultures of human lymphocytes [42, 43].

No significant differences were observed in the degree of chromatin condensation and DNA integrity caused by both U. urealyticum serotypes 8 and 3, which represent the two U. urealyticum biovars. This indicates that these effects are probably not serotype-dependent.

Some differences between human and ram were observed in the deleterious effect of U. urealyticum on sperm chromatin. Ram sperm nuclear chromatin is apparently more stable to the decondensation effect induced by U. urealyticum than human sperm is. These observations are supported by the findings of Perreault et al. [44] that bull sperm chromatin is more stable to decondensation induced either by dithiothreitol treatment or by hamster oocytes, than human sperm cells. In contradistinction, the integrity of ram sperm nuclear DNA was found to be more sensitive to U. urealyticum than human sperm cells (Fig. 4d).

Infection of sperm cells by U. urealyticum did not increase their necrosis. On the contrary, reduced cell death and reduced loss of activity were observed, which usually occurs spontaneously in control uninfected sperm cells under hypoxic conditions. Indeed, induction of sperm motility upon in vitro infection with U. urealyticum was reported previously by Talkington et al. [45]. It can be speculated that the metabolites obtained from the degradation of nuclear DNA (i.e., deoxyribose), can serve as a substrate for the Embden-Meyerhof pathway of sperm cells as well as for the energy metabolism of U. urealyticum. This effect emphasizes the dilemma of a possible relation between semen quality and impaired embryo development associated with U. urealyticum infection. Our finding of the deleterious effect of U. urealyticum infection on sperm nuclear chromatin may resolve this dilemma because the integrity of sperm chromatin is not necessarily directly related to the integrity of the sperm tail structure or function or to the integrity of its plasma membrane. On the other hand, integrity of chromatin could have implications for embryo development [23] and abortion [22, 46]. It should be noted that the dual effect of U. urealyticum on sperm nuclear chromatin and metabolism may emphasize the concern raised by Chan et al. [47] that exogenous DNA can be efficiently transferred to a developing embryo by injecting an infected sperm cell into the ovum during IVF-intracytoplasmic sperm injection procedure.

In the literature there are reports describing a link between nonspecific infection in vivo and damage to sperm chromatin. Evenson et al. [48] reported that patients suffering from prostatitis had increased nuclear DNA damage in sperm cells. The problem was corrected after appropriate antibiotic treatment. A possible link between the appearance of leukocytes in an ejaculate and an increase in hypocondensed sperm cells [49] and an increased percentage of DNA-damaged sperm cells as observed by SCSA has also been reported [50]. However, other etiologies may be involved in in vivo studies. Furthermore, nonspecific infection may cause necrosis, which leads to chromatin degradation [48, 49]. Indeed, these two authors reported a correlation between necrosis and chromatin hypocondensation and DNA damage. In our in vivo-infected sperm cells, no difference in sperm motility and viability was observed before and after treatment, although a significant difference in chromatin integrity was observed. It can therefore be assumed that U. urealyticum, which was the only bacterium isolated from the investigated semens, has a direct effect on sperm chromatin. The mechanisms by which U. urealyticum causes damage to chromatin stability is not yet understood and is now under investigation.

In conclusion, the results obtained in this study following in vivo and in vitro infection of both human and ram sperm cells by U. urealyticum provide specific and conclusive evidence that U. urealyticum has a direct deleterious effect on sperm nuclear chromatin and DNA. The fact that this deleterious effect was not correlated with sperm activity may shed light on phenomena reported in the literature of a possible link between U. urealyticum infection and an impairment in embryo development [4, 6, 10, 51]. The preserved sperm activity post U. urealyticum infection may enable a high fertilization rate; however, damaged paternal DNA will impair embryonic development.


   ACKNOWLEDGMENTS
 
We thank Dr. Peri Kedem for statistical assistance and Ms. Sharon Victor for typing the manuscript.


   FOOTNOTES
 
First decision: 30 December 1999.

1 Research was supported by the Ihel, Haim et Sara Bessinover Dragonster Fund. This paper is part of the Ph.D. thesis of M.R., as submitted to the Senate of Bar-Ilan University, Ramat Gan, Israel. Back

2 Correspondence. FAX: 972 3 5343679; bartoob{at}mail.biu.ac.il Back

3 Deceased July 1997. Back

Accepted: May 16, 2000.

Received: November 29, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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