| BackgroundIt is reported about15%-20%couples in the worldwide suffering from the infertile problem at the child-bearing age. Due to the influence of environment, psychological and social factors, the male sperm quality and quantity showed obvious downward trend, and the ratio of male and female infertility have changed from3:7to5:5. Ureaplasma Urealyticum (U.urealyticum), belonging to the conditions of pathogenic microorganism, can adhere to genitourinary tract and cause urinary tract infections. It can also affect male fertility in several ways, such as interfering with spermatogenesis, affecting sperm metabolism and sperm-oocyte binding, as well as triggering immune mechanisms in the genital tract. But the mechanism of male infertility caused by the U.urealyticum infection is not fully clear. Meanwhile some studies have shown that no significant effects of U.urealyticum were seen on male infertility. Because various reagent and method were used in the laboratories, the prevalences of U.urealyticum differed from each other. It varied from5%to58%in the infertile male seminal fluids and3%to31%in the fertile male seminal fluids. It still remains controversy about the influence of U.urealyticum on semen quality. Some studies showed that UU infections may alter various characteristics of semen, such as sperm motility, density, pH and morphology, and that antibiotic treatment can lead to the improvement of semen quality. While other studies show no influence of U.urealyticum infection on semen quality. On the basis of genotypic characteristics, U. urealyticum is divided into two biovars-biovar I (ureaplasma parvum, parvo) composed of serovars1,3,6and14, and biovar II (ureaplasma urealyticum, T960) composed of serovars2,4,5,7,8,9,10,11,12and13. Infection by biovar II is more closely involved with several inflammatory pathologies, such as nongonococcal urethritis (NGU), prostatitis and pelvic inflammatory disease than is biovar I. There are two main sources for production of free radicals in the semen:leukocytes and sperm. The same reactive oxygen species (ROS) that under physiological conditions are an inseparable element of the fertilization process, as well as being important regulatory factors in the control of spermatogenesis efficiency, may under pathological conditions (ROS excess) be responsible for structural, metabolic, and functional disorders of the male germ cells. ROS can damage the sperm membrane which in turn reduces the sperm’s motility and ability to fuse with the oocyte. It can attack the integrity of DNA in the sperm nucleus by causing base modifications, DNA strand breaks, and chromatin cross-linking. High levels of ROS may destroy the mitochondrial membrane, the most important organelle mediating cell apoptosis, release cytochrome c, trigger the cysteine protease cascade and induce cell apoptosis at last.Objective:1. Investigate the prevalence of U. urealyticum and its different biovars in the infertile and fertile men.2. Reveal the impacts of two biovars of U. urealyticum infection on sperm parameters, movement parameters and morphous of sperm cells. Investigate the relationship between U. urealyticum infection and seminal leukocytes as well as the role of seminal leukocytes in the progress of U. urealyticum infection in semen. 3. Study the effects of two biovars of U. urealyticum on ROS generation in semen as well as the lipid peroxidation of sperm membrane and DNA damage. Discuss the relationship among U. urealyticum infection, ROS generation and leukucytes elevation in semen.Methods:1. The prevalence of U. urealyticum and different biovars in the infertile and fertile men.From July2012to February2014,223patients with infertility and146fertile whose wives had non-assisted pregnancies in the past and were clinically asymptomatic were selected from the Infertility Center, the First Affiliated Hospital of Shantou University Medical College (SUMC), China. All the subjects were screened strictly by the exclusion criteria for participants. Traditional culture method was used to detect U. urealyticum infection in the semen of infertile male and fertile male. Then the biovar species were identified by real-time PCR with U. urealyticum positive specimen.2. The impacts of two biovars of U. urealyticum infection on sperm parameter and the role of leukocytes in the progressSemen samples were obtained by masturbation. After semen production, the samples were liquefied at37℃for about30minutes. Routine semen analysis, including volume, pH, spermatozoa count, motility, morphology and leukocyte counts was carried out according to WHO guidelines by using a WLJY-9000TYPE WEILI Color Sperm Analysis System. The computer sperm morphology analysis software combined with artificial judgement was used to analysis.3. The effects of different biovars of U. urealyticum on ROS generation, lipid peroxidation of sperm membrane and DNA damageROS production was measured using luminol as the probe by the chemiluminescence method. Semen malondial-dehyde content in semen was measured by the sulfo-barbitone acid method; total superoxide dismutase in semen was measured by the xanthine oxidase method. Sperm nuclear DNA damage was assessed of by sperm chromatin structure assay (SCSA) and single-cell gel electrophoresis (SCGE).Statistical Analysis:Data was analyzed by SPSS version13.0for Windows. Differences in proportions were compared using chi-square test. For more than two groups, one-way analysis of variance (ANOVA) was used. After ANOVA, a LSD test was applied to analyze the difference between two groups. The Levene’s test was used for homogeneity of the variance analysis, and in the case of non-homogeneity, a Welch test was used. Statistical significance was set at P<0.05. Results were expressed as mean±SD.Results:1. The prevalence of U. urealyticum and different biovars in the infertile and fertile men.The prevalence of U. urealyticum-positive in semen specimens from infertile men (75/223,33.6%) tended to be higher than that in the fertile men (36/146,24.7%), but it did not reach statistical significance (χ2=3.379, P=0.066). Among U. urealyticum-positive semen, biovar II was isolated from32/75(42.7%) infertile cases and8/36(22.2%) fertile cases (χ2=4.411, P=0.036). Eight (10.7%) infertile and three (8.3%) fertile men harbored mixed pathogens, but no significant difference existed between two groups (χ2=0.148, P=0.700).2. The impacts of two biovars of U. urealyticum infection on sperm parameter and the role of leukocytes in the progress (1) The impacts of two biovars of U. urealyticum infection on sperm parameters, motion parameters and leukocyte counts in semen.In order to identify the effects of biovar I and biovar II on semen, we compared semen parameters between infected and uninfected groups. Among the biovar I, biovar II, mixed infection and uninfected groups, only the total motility showed significant difference between infected and uninfected individuals (P=0.001). Although other parameters including spermatozoa concentration and progressive motility in the positive groups tended to be lower than those in the uninfected group, the differences were statistically insignificant (P>0.05). The spermatozoa concentration (59.25±49.21) in the biovar II group, and the total motility in the biovar II (54.95±16.21) and mixed infection (51.26±20.88) groups were significantly lower than the uninfected group separately (P=0.038; P<0.001, P=0.005). When compared with uninfected group, the sperm motion parameters showed significant decreases of VCL(P<0.001), VSL(P=0.002), VAP(P=0.024), LIN(P=0.009), WOB(P=0.004) in Biovar II and VCL(P=0.017), VSL(P=0.007), LIN(P=0.016) in mixed infection group, but there was no difference between the biovar I and uninfected groups (P>0.05). A total of12/71subjects in biovar I,12/40subjects in biovar II,3/11subjects in mixed infection and25/258subjects in uninfected group were found Leukocytes elevated (≥106/ml). There were significant differences among the four groups(χ2=14.822, P=0.002).When compared with uninfected group separately, the leukocytes in biovar II increased significantly (χ2=13.136, P<0.001), but the leukocytes showed no significant difference in the biovar I and mixed infection groups (χ=2.901,P=0.089;χ2=3.498, P=0.61). The results indicated that biovar II infection had more close relationship with increased leukocyts in semen.(2) The impacts of leukocytes on sperm parameters, motion parameters and sperm morphology.Subsequently we compared the semen parameters between the normal leukocytes group and abnormal leukocytes group. The results showed that the semen parameters including total motility, progressive motility and normal forms in the abnormal leukocytes group decreased significantly compared with normal leukocytes group (P=0.008; P=0.005; P=0.003). The sperm motion parameters including VCL (P=0.023)、VSL (P=0.010)、VAP (P=0.011)、WOB (P<0.001) were showed significant lower in the abnormal leukocytes group than abnormal leukocytes group. The ratio of sperm abnormality in the abnormal leukocytes group increased significantly compared with the normal leukocytes group (P=0.008),in which the head abnormal increased significantly (P=0.005). The above results suggested increased leukocytes can lead to a decline in semen quality and increased rates of sperm abnormality.3. The effects of different biovars of U. urealyticum on ROS generation, lipid peroxidation of sperm membrane and DNA damage(1) The effects of different biovars of U. urealyticum on ROS generation and lipid peroxidation.To further explore the effect of abnormal increase of leukocyte caused by the biovar II on semen redox system, we compared the ROS, MDA and T-SOD levels in the semen of four groups(including71cases in biovar Ⅰ,40cases in biovar II,11cases in mixed infection and80cases in uninfected group). The results showed ROS levels (using the log-transformation) were significantly higher in biovar II (2.66±0.85) and mixed infection (2.82±1.37) groups than that in controls (1.94±0.79)(P<0.001, P=0.003; respectively), but no significant difference was observed between the biovar I group (2.20±0.94) and uninfected group(P=0.075). MDA in the biovar II (1.25±0.61nmol/l) and mixed infection (1.30±0.61nmol/l) groups were approximately35%higher than that in the negative group (0.92±0.12nmol/l)(P=0.004,P=0.040), but no significant difference was observed between the biovar I group (1.05±0.55) and uninfected group (P=0.226). Although T-SOD levels in biovar I (63.57±8.95), biovar II infection (62.71±7.10), mixed infections group (61.81±7.62) showed lower than the uninfected group(64.49±7.23), the differences among the four groups were not statistically significant (P=0.556).(2) The effects of different biovars of U. urealyticum on sperm DNA damage.Data of SCSA indicated there were significant increases of DFI%and HDS%in the biovar II (18.32±7.62;8.06±4.23) and DFI%in mixed infection groups (23.44±4.89) compared to the negative group (18.32±7.62;8.06±4.23)(P=0.003; P=0.001, P=0.032respectively), but no significant difference was observed between the biovar I group (20.03±7.21,8.92±3.60) and uninfected group (P=0.155; P=0.175). SCGE analysis, in terms of tail moments, showed higher motility in the biovar II (1.06±0.15) than in the uninfected group (0.94±0.12)(P<0.001), but no significant difference was observed between the biovar I group (0.96±0.11), mixed infection groups (1.05±0.15) and uninfected group (P=0.257, P=0.224).Conclusion:1. The infection rate by U. urealyticum showed a tendency to be higher in the infertile group than in the fertile group, but it did not reach statistical significance. However, stratification into different biovar infections revealed that the prevalence of biovar II in infertile men was significantly higher than that in fertile men. These results demonstrated the biovar II infection was more frequent in infertile men, suggesting the role in male infertility.2. In this study, the semen volume, pH, spermatozoa count, motility, and morphology with the presence of biovar I and biovar II were compared. Only the total motility showed a significantly decrease between infected and uninfected individuals. Spermatozoa concentration in the biovar II group, total motility in the biovar II and mixed infection groups were significantly reduced (P<0.05).The above results suggested that compared with biovar I, biovar II had more significant impact on sperm parameters, expecially in sperm motility. The results of sperm movement parameters also showed that VSL, VCL, VAP, LIN and WOB in biovar II were significantly lower than the uninfected group. At the same time, leukocyte counts in the biovar II were significantly higher than that in the negative group, but no significant difference was observed between the biovar I group, mix infection group and uninfected group. The mechanisms of sperm motility decrease include U. urealyticum’s strong ability of adhesion to sperm; ROS increase which can attack and induce lipid peroxidation, which disrupts the integrity of the plasmamembrane and impairs sperm motility. In addition, ROS can change the mitochondrial membrane potential, which may reduce sperm energy supply and eventually slow sperm movement. Combined with the results of semen parameters change and leukocytes elevation, increased ROS seemed to play a more important role in this process, because the significant reduction in sperm motility and increased leukocytes occurred in biovar II group at the same time, and leukocytes have been considered to be an important source of ROS in semen.3. To verify the effect of leukocytes elevation on the spermatozoa quality, we compared the semen parameters, sperm motion parameters and sperm morphology between the normal leukocytes group and abnormal leukocytes group. The results showed that the semen parameters in the abnormal leukocytes group including total motility, progressive motility and normal forms, the sperm motion parameters including VCL, VSL, VAP and WOB decreased significantly compared with normal leukocytes group. Meanwhile the ratio of sperm abnormality (especially the head abnormality) in the abnormal leukocytes group increased significantly compared with the normal leukocytes group. All these data suggested that excessive leukocytes may result in the decrease of spermatozoa quality.4. Further, we explored the effect of abnormal increase of leukocyte caused by the biovar II infection on semen redox system. We compared the ROS, MDA and T-SOD levels in the semen of four groups. The results showed ROS levels (using the log-transformation) were significantly higher in biovar II and mixed infection groups than that in uninfected group, but no significant difference was observed between the biovar I and uninfected group. The data indicated that the biovar II infection may increase the leukocyte counts in the semen and subsequently produce excessive ROS. Two mechanisms were involved in the excessive ROS generation caused by U urealyticum infection. First, the most toxic agents for spermatozoa are the metabolic products of U urealyticum, which include H2O2and ammonia (NH3). Although H2O2is itself harmful to sperm, it is also a source of hydroxide anion (OH), which is a highly toxic radical for cell membranes. Furthermore, Ureaplasma phospholipases A and C may influence changes in the lipid composition of the cell membranes of male gametes, leading to loss of integrity and increased permeability. Second, U urealyticum can induce ROS generation through increasing leukocyte concentration in semen. Our results suggested that leukocytes elevation may be more responsible for the ROS increase. MDA results also supported this view, and indicated biovar II was more likely to cause sperm membrane lipid peroxidation. But results of T-SOD showed no significant difference between infected and non-infected group.5. Our study showed the biovar II and mixed infection groups displayed elevated DFI%and HDS%values in the SCSA assay. The data of significant increase compared to the negative group strongly suggested DNA damage caused by biovar II infection may damage sperm DNA integrity and interfere with male fertility. Innovation points:1. Investigated the prevalence of U. urealyticum and its different biovars in the infertile and fertile men, demonstrated the biovar II infection was more frequent in infertile men.2. Studied the impacts of two biovars of U. urealyticum infection on sperm parameter and the role of leukocytes in the progress, found spermatozoa concentration and total motility in biovar II and mix infection groups were lower significantly than in the U. urealyticum negative group; cases with leukocytes increasing in biovar II were significantly higher.3. Explored the effects of two biovars of U. urealyticum on semen redox system(including ROS, MDA and T-SOD) as well as sperm DNA damage, suggested that leukocyte-induced ROS elevation owing to the biovar II infection may be more responsible for spermatozoa membrane and DNA damage. |
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