Establishment of a rapid detection method of Ureaplasma urealyticum based on recombinant polymerase amplification

Ureaplasma urealyticum(UU),is one of the most vital pathogens causing genitourinary tract infections of the body,and it can result in poor maternal and perinatal outcomes.The aim of this study was to establish a method to detect Ureaplasma urealyticum based on recombinant polymerase amplification(RPA)technique.Specific primers and probes were designed according to the 16sRNA gene sequence of Ureaplasma urealyticum.Six pathogens were detected for real-time fluorescence RPA specificity verification,including Mycoplasma hominis(MH),Chlamydia trachomatis(CT),Neisseria gonorrhoeae(NG),Staphylococcus aureus,Escherichia coli,and Lactobacillus vaginalis.The sensitivity of the method was performed by gradient dilution of the extracted template.A total of 60 clinical samples were detected by the established real-time fluorescence RPA.Detection of Ureaplasma urealyticum can be completed within 20 minutes at 39°C using established RPA method.The minimum detection limit of Ureaplasma urealyticum by real-time fluorescence RPA was 3 pg.The evaluation of 60 clinical samples proved that RPA method was feasible.A high specificity,sensitivity,simplicity and rapidity method for Ureaplasma urealyticum detection was successfully established based on the real-time fluorescence RPA method.

解脲支原体(Ureaplasma urealyticum,Uu)感染对雄性大鼠生殖系统的影响

目的 研究解脲支原体 (Ureaplasmaurealyticum ,Uu)感染对雄性大鼠生殖系统的影响 ,探究Uu感染引起雄性不育的途径与机制。方法 采用模拟性交的自然方式使雄性SD大鼠感染Uu ,反复感染。 8周末处理大鼠 ,解剖泌尿生殖系统 ,观察有无异常 ;取附睾精液 ,涂片 ,瑞氏染色 ,显微镜下观察 ;取睾丸、前列腺、输精管等组织进行石腊切片 ,H·E染色 ,光镜观察 ;取血 ,检测血清中细胞因子 (IL - 6 ,TNF -α ,IFN -α ,IL - 8)水平。结果 Uu感染后大鼠的精子头尾卷曲 ,尾部断裂明显、肿胀 ;血清中细胞因子IL - 6、IL - 8水平明显升高 ,TNF -α、IFN -α无明显变化 ;而睾丸、输精管、前列腺等组织的结构没有明显改变。结论 Uu感染使精子头尾卷曲 ,尾部断裂、肿胀 ;影响细胞因子的分泌 ,使血清中细胞因子IL - 6、IL - 8含量有明显升高 ,但不能使TNF -α、IFN -α明显变化 ;Uu感染对睾丸、输精管、前列腺的形态组织结构未有明显的影响。

Do Ureaplasma urealyticum infections in the genital tract affect semen quality?

Aim： To investigate the relationship between Ureaplasma urealyticum （UU） infection and semen quality. Methods： From 2001 to 2003, 346 eligible patients aged 20-45 years were invited from two hospitals in Shanghai, China, to participate in an investigation which included questionnaires about general and reproductive health, an external genital tract examination, UU culture and semen analysis. Multiple linear regression models were used to examine whether UU had a significant effect on semen quality after adjustment for confounding factors. Results： Findings suggested that UU infection was associated with higher semen viscosity and lower semen pH value. Sperm concentration was lower in UU positive subjects than that in UU negative subjects （54.04 × 10^6/mL vs.70.58 × 10^6/mL）. However, UU did not significantly affect other semen quality indexes. Conclusion： UU infection of the male genital tract could negatively influence semen quality.

Germ cell apoptosis induced by Ureaplasma urealyticum infection

Aim: To study the effect of Ureaplasma urealyticum (UU) infection on germ cell apoptosis of male rats. Meth-ods: Male rats were infected artificially with UU serotype 8 (T_(960)). Morphological changes of germ cells in the sem-iniferous tubules and the lumen of the epididymides were observed under the light microscope. Fluorescence-conjugatedpolyclonal antibodies to Fas and Fas ligand (FasL) were used to localize Fas and FasL. TUNEL staining of germ cellsand Sertoli cells was performed by the AKPase method. TUNEL-positive rate (% positive cells) and TUNEL-positivearea (area occupied by stained cells) were analysed by KS400 Image Analysis System. The DNA laddering analysiswas performed by agarose gels electrophoresis. Results: In those rats infected with UU; (1) Exfoliated germ cellswere dramatically increased. Many multinucleated giant cells were found in the seminiferous tubules and the lumen ofthe epididymides. (2) The number of TUNEL-positive cells and the TUNEL-positive area were significantly increased.(3) The expression of Fas and FasL in germ cells and Sertoli cells was up-regulated. (4) Discrete bands of fragmentedDNA were found in the testicular cells. Conclusion: In male rats, germ cell apoptosis was increased in UU infec-tion . (Asian J Androl 2001 Sep; 3: 199 - 204)

Ureaplasma urealyticum infection and apoptosis of spermatogenic cells

Aim: To study the relationship between Ureaplasma urealyticum (UU) infection and apoptosis of human spermato-genic cells. Methods: Spermatogenic cells were observed under light microscope with Wright-Giemsa staining andby means of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling(TUNEL) technique. Results: Apoptotic rate of UU-infected males (15.5 % ± 6.8 % ) was significantly higherthan that of controls (5.2 % ± 2.3 % ). Conclusion: Apoptosis of spermatogenic cells can be caused by UU in-fection, which provides further evidence for UU-induced male infertility. (Asian J Androl 1999 Sep ; 1: 127 - 129)

Evaluation of patients with dry eye disease for conjunctival Chlamydia trachomatis and Ureaplasma urealyticum

AIM：To determine the possibility of the development of dry eye disease（DED） as a result of persistent infection with Chlamydia trachomatis and Ureaplasma urealyticum in the conjunctiva of patients.METHODS： This study was conducted on 58 patients of age range 20-50 y,diagnosed with DED confirmed by Schirmer I test and tear breakup time.The non-dry eye control group included 27 subjects of the same age.Ocular specimens were collected as conjunctival scrapings and swabs divided into three groups： the first used for bacterial culture,the second and third taken to detect Chlamydia trachomatis and Ureaplasma urealyticum by direct fluorescent antibody（DFA） assay and polymerase chain reaction（PCR） method. RESULTS： Chlamydia trachomatis was detected in 65.5% and 76% of DED patients by DFA and PCR methods respectively.Ureaplasma urealyticum was found in 44.8% of DED infected patients using the PCR method.Both organisms were identified in only 37.9% of DED patients found to be infected.Control subjects had a 22%detection rate of Chlamydia trachomatis by DFA assay versus a 7% detection rate by PCR; while Ureaplasma urealyticum was detected in 3.7% of the controls by PCR method.The conjunctival culture revealed that gram positive microorganisms represented 75% of isolates with coagulase negative Staphylococci the most common（50%） followed by Staphylococcus aureus（20%）,whereas gram negative microorganisms occurred in 25% of cases,isolating Moraxella spp.as the most frequent organism. CONCLUSION： Our results tend to point out that Chlamydia trachomatis and Ureaplasma urealyticum were detected in a moderate percentage of patients with DED,and could be a fair possibility for its development.PCR is more reliable in detecting Chlamydia trachomatis than DFA technique.The presence of isolated conjunctival bacterial microflora can be of some potential value.

Association of Ureaplasma urealyticum Colonization with Development of Bronchopulmonary Dysplasia: A Systemic Review and Meta-analysis

There is controversy regarding the roles of Ureaplasma urealyticum （U. urealyticum） colo- nization in the development of hronchopulmonary dysplasia （BPD）. This study explored the association between U. urealyticum and bronchopulmonary dysplasia at 36 weeks post-menstrual age （BPD36）. Studies published before December 31, 2013 were searched from Medline, Embase, Ovid, Web of Sci- ence, and Cochrane databases, with the terms ＂Ureaplasma urealyticum＂, ＂chronic lung disease＂, or ＂BPD36＂ used, and English language as a limit. The association between U. urealyticum colonization and BPD36 was analyzed with RevMan 4.2.10 software, using the odds ratio （OR） and relative risk （RR） for dichotomous variables. Out of the enrolled 81 studies, 11 investigated the BPD36 in total 1193 in- fants. Pooled studies showed no association between U. urealyticum colonization and subsequent de- velopment of BPD36, with the OR and RR being 1.03 （95% CI=0.78-1.37; P=-0.84） and 1.01 （95% CI= 0.88-1.16, P=-0.84）, respectively. These findings indicated no association between U. urealyticum colo- nization and the development of BPD36.

Ureaplasma Urealyticum or Mycoplasma Hominis Infections and Semen Quality of Infertile Men in Abidjan

Objective To determine the prevalence of U. urealyticum and M. hominis in semen samples collected from men admitted in clinic for infertility, and to compare the quality of these semen samples.Methods A total of 1 058 semen samples collected were investigated. Sperm semiological assays were performed according to the guidelines of the World Health Organisation （WHO）. Semen were examined by Mycoplasma IST for the detection of mycoplasma. Semen culture on agar media was used to detect other microorganisms. Chlamydia was detected using direct fluorescent assay （DFA） of Clamydia Trachomatis.Results Among 1 058 semen samples, microorganisms were detected in 638 （60.3%）. The infected sperms consisted of mycoplasma alone in 507 cases （47.9%）, mycoplasma and other microorganisms in 98 （9.3%）, giving in all 605 （57.2%） samples infected with mycoplasma. The last 33 （3.1%） consisted of other microorganisms alone. The frequency of U. urealyticum, M. hominis and mixed genital infections detected in semen samples of infertile men were 39%, 23.8% and 5.6%, respectively. The rates of abnormal semen parameters recorded among patients infected with mycoplasma were for volume （22.2%-25%）, viscosity （29.6%-43.5%）, pH （64.7%-72.9%）, motility （80.8%-93.8%）, morphology （36.3%-47.9%）, sperm concentration （53.3%-58.3%） and leukocyte count （51.4%-58.3%）.Conclusion Frequency of U. urealyticum infection was higher than that of M. hominis. Mycoplasma infections were associated with disorders of pH, motility and sperm concentration. In addition M. hominis infection affected spermatozoa morphology. Therefore, screening of U. urealyticum clinically relevant in Abidjan. and M. hominis for routine semen analysis is

Microdilution inhibition test of Chinese herbs to assess their effect against clinical strains of Ureaplasma urealyticum in vitro

Objective：To explore the antibacterial effect of Chinese crude drugs against clinical strains of Ureaplasma urealyticum（UU）, including eight pure herbs and three compound herbs, and determine their minimal inhibitory concentrations（MICs）. Methods：Isolates were collected from clinical patients with UU infection, and cultured in UU broth. In order to test the different effects on clinical strains of UU, the assays were performed by microdilution inhibition tests, and MICs of the herbs against the clinical strains of UU were calculated. Results：The MICs of eight pure herbs against clinical strains of UU were as follows： Galla Chinensis：0.313-1.25 g/L; Fructus Forsythiae： 1.25-5.00 g/L; Cortex Phellodendri： 1.25-5.00 g/L; Radix Paeoniae Rubra： 1.25-2.50 g/L; Semen Plantaginis：2.50-10.00 g/L; Herba Lysimachiae： 5.00-20.00 g/L; Rhizoma Coptidis： 5.00-20.00g/L, Herba Houttuyniae： 10.00-20.00 g/L. The MICs of compound herbs were： Liuheji： 0.625-2.50 g/L; Bazhengsan： 5.00-20.00 g/L; Wulinsan：2.50-20.0 g/L.Conclusion：Galla Chinensis, Fructus Forsythiae, Cortex Phellodendrim, Radix Paeoniae Rubra, and Semen Plantaginis, exerted the stronger antibacterial effect against clinical strains of UU, whereas Herba Lysimachiae, Rhizoma Coptidis and Herba Houttuyniae, had relatively weaker activity against UU. Compound herbs, Bazhengsan and Wulinsan, and particularly Liuheji, also had antibacterial effects against UU. Further studies of the effects and mechanisms of action of Chinese crude drugs against UU infections are worthwhile.

Effects of Ureaplasma Urealyticum on Fas Ligand Expression in Rat Sertoli Cell

To investigate the effect of ureaplasma urealyticum (UU) on the expression of Fas ligand (FasL) on rat Sertoli cell Materials & Method Isolated rat Sertoli cells were infected by living UU, UU super- natants, inactivated UU, then Fluorescence Activated Cell Sorter and observed fluores- cence microscopy were used to assay for the FasL expression on the surface of Sertoli cells. Results UU infection could increase the expression of FasL in Sertoli cell. Conclusion The functional expression of FasL is related to the immune privilege and can give the immune regulation on the testis.

Influence of Ureaplasma urealyticum infection on the sperm egg binding associated molecule,sulfogalactosylglycerolipid

Aim: To study the influence of Ureaplasma urealyticum (Uu) infection on the sperm egg binding associated molecule, sulfoga-lactosylglycerolipid (SGG). Methods: Epididymal sperm was collected from adult mice. The sperm suspension was randomly divided into 4 groups: the Uu group (coincubated with Uu suspension), the medium group (coincubated with Uu medium), the normal group and the PRS group. Indirect immunofluorescence technique was used to localize SGG on the sperm membrane and to observe the influence of Uu on SGG. Results: In the epididymal sperm, SGG was localized at the head plasma membrane overlying the acroso-mal region. The SGG positive rate of the sperm coincubated with Uu medium was 82.0 %, while the SGG positive rate of those coincubated with Uu suspension was reduced to 39.0 % (P = 0.001). Conclusion: Uu can adhere to the sperm surface. SGG might be a membrane receptor on the sperm surface for Uu infection of the mammalian male genital tract. The blockage of SGG by Uu might be one of the molecular mechanisms correlated with male infertility induced by Uu infection.

Studies on relationship between semen viscosity and other semen parameters, Ureaplasma urealyticum infection and seminal plasma antisperm antibody

Objective: To study the relationship between semen viscosity and other semen parameters, Ureaplasma urealyticum (UU) infection and seminal plasma antisperm antibody (AsAb) in male infertiles. Methods: Semen parameters, Ureaplasma urealyticum (UU) infection and antisperm antibody (AsAb) were measured and analyzed in 4337 infertile men. Results: The seminal viscosity was higherr than normal in 65.02 % of 4337 male infertiles. The sperm motility and grade (a, b) motile sperm were significantly lower in the high viscosity group than in the normal viscosity group (P<0.05-0.01). The rate of abnormal morphology sperm was higher and duration of semen liquefaction was longer in the high viscosity than in the normal viscosity group (P<0.01). The seminal volume, sperm concentration and semen pH were not significantly different between the two groups. The semen viscosity is significantly higher in subjects with higher seminal WBC (>5/ HP) than in those with lower WBC (<5/HP). The positive AsAb and UU infection rates were significantly higher in the high viscosity group (P<0.01). Conclusion: The semen viscosity is related to other seminal parameters, as well as to UU infection and seminal AsAb.

A Study on Location of Ureaplasma Urealyticum in Spermatogenic Cells

A study on the location of Ureaplasma Urealyticum (UU) in spermatogenic cells in seminal fluid from infertile males was made using rabbit anti-UU antibodies labelled with horse peroxidase (HRP). The results showed that the positive rates of UU in spermatogenic cells were 39.8% (113/284) and 42.2% (35/83)in the infertile males and the males whose wives had history of habitual abortion, respectively. The positive rates of UU were 45.8% (38/83) and 31.6% (30/95)in the patients with positive and negative anti-sperm antibodies, respectively. A high positive rate of UU (73.3%) was found in the infertile males with oligospermia (<20×109/L). The medicines of tetracyclines, such as minomycin, could effectively inhibit the growth of UU, thus converting the UU-positive into negative and improving the patients'fertility.

Effect of Ureaplasma Urealyticum Infection on the Interleukin-1 Secretion in Rat Sertoli Cells

Objective To investigate the effect of Ureaplasma Urealyticum(UU) on the expression of IL 1 secretion in rat Sertoli cells Material & Methods Isolated rat Sertoli cells were infected by living UU, UU supernatants, inactivated UU of different dosage respectively. IL 1 secretion by rat Sertoli cells is determined by using group t test and F test. Results UU infection, both living UU and supernatant without UU, could inhibit the IL 1 secretion in rats Sertoli cells (P<0.01). Conclusion The infection of Sertoli cells by UU inhibit the biological function of the Sertoli cells in rat.

THE RELATION OF UREAPLASMA UREALYTICUM INFECTION TO UROGENITAL STONE FORMATION

Objective To study the mechanism of Ureaplasma urealyticum (UU) infection to the genesis of urogenital stone. Methods One hundred male SD rats were randomly divided into 3 groups: group A (n=45) rats were inoculated intravesically with 1ml (105 CCU/ml) of UU serotype 8 (T960) broth. Group B (n=22) rats were treated as the group A, 3 months later, administration of Minomycin (20~100mg/kg, oral daily for 14d) was given to each rat. Group C (n=33) rats were given sterile broth intravesically as control. After UU inoculation UU were collected from different parts of urogenital tract and cultured. In 3 testes positive in UU culture, UU specilic DNA fragments were tested by PCR technique. Results The percentages of positive UU culture in groop A animals were 87.5% in urinary bladders, 62.5% in testes, 55% in ePididymides, 65% in seminal vesicles, and 57.5% in prostates. The corresponding percentages in group B were significantly lower than that in group A (P<0.01). The incidences of bladder stones in groop A and B were 40% vs 35% (P>0.05). The main comPonent of stone was struvite (MgNH4PO4 6H2O). MicroscOPically, microlithes were found in seminderous tubules in 4 infected rats. In microlithes containing tubules, germinal cells and Sertoli cells were exfoliated or even absent. In some tubules, only membrana limitans remained intact. No microlithes found in testicular interstitial tissue. Cooclusion UU infection does induce urogenital stones and Minomycin can inhibit UU growth. With interlerence on spermatogenesis, UU may be one of the causes of male inlertility.

Localization of Ureaplasma Urealyticum on Human Spermatozoa

Semen specimens were collected from 20 normal fertile men and 20 unexplained infertile men with Ureaplasma urealyticum (U.U.)in semen.SPermatozoa Of both groups were examined by immunogold technique and immunofiuorescence test. A number of gold particles of the U.U.adhered to the sperm surface of infertile men irers observed.Strong special fluorescence was noticed on the sperm surface of infertile men,mostly on the midpiece and/or postacrosomal region.A significant increase of sperm molphological abnormality,especially the swollen midpiece,the coiled lail,and head-tail angulation sperms was observed in infertile group.The sperm motility in inferlile grouP is dramatically lower than that infertile group.It is hypolhesized that teratospermia,poor motility and interference with sperm-ovum interaction might be the possible mechanisms for male infertility caused by Ureaplasma urealyticum.

In Vitro Efficacy of Crataegus oxycantha L. (Hawthorn) and Its Major Components against ATCC and Clinical Strains of Ureaplasma urealyticum

Crataegus oxycantha L., commonly known as hawthorn, has traditionally been used for its beneficial effect on cardiovascular health, which is related to its flavonoid content. The aim of the present study was to evaluate the antibacterial properties of a fluid extract and a hydro-ethanolic macerate from buds of Crataegus oxycantha against clinical isolates of Ureaplasma urealyticum. The major purified flavonoids present in the extracts were also tested against ATCC strains and clinical isolates. Both the fluid extract and the hydro-ethanolic macerate were active against thirty-due clinical strains of U. urealyticum, with MIC ranges between 15.6 and 250 μg/ml and 15.6 and 62.5 μg/ml, respectively. All pure organic compounds, with the exception of rutin, showed activity against the strains tested, luteolin 3,7-diglucoside being the most active compound (MICs in the range of 0.48 and 1.95 μg/ml), followed by apigenin-7-O-glucoside (MICs in the range of 0.48 and 3.9 μg/ml). The activity of the pure flavonoids was greater against the clinical isolates compared to the ATCC strains. The data presented here demonstrate that flavonoids present in Crataegus oxycantha are effective against clinical isolates of U. urealyticum and could be used in combination with antibiotics in order to combat resistance.

Investigation on Biovars and Serotypes of Ureaplasma Urealyticum in Cervix in Chinese Gynecologic Check-up Population, Sexually Transmitted Infections Clinic Clients and Sex Workers

Objectives: To characterize the distribution pattern biovars and serotypes of Ureaplasma urealyticum in normahealthy women, sexually transmitted infections clinic clienand in sex workers. Methods: We cultured cervical swabs taken from 26physical check-up clients, 599 STI clinic outpatients and 9sex workers using commercial selective medium. Sompositive cultures were further biotyped and serotyped bPCR. Results: (1) Biovar 1 of U urealyticum (95.0%), especiallsingle infection of serotype 1, 3, and 6 of biovar 1,commonly found in healthy women. (2) U urealyticummore commonly isolated in sex workers (90.8%) than iphysical check-up group (60.9%) and STI outpatients grou(61.3%) (P<0.001). (3) Biovar 2 infection of U urealyticuris more prevalent in sex workers (28.1%) and SToutpatients group (26.6%) than that in physical check-ugroup (4.9%) (P<0.001). (4) Mixed infection caused bmore than one serotype of U. urealyticum is increasing fromphysical check-up group (8.6%) to STI outpatients (12.4%and to sex wokers (23.9%) (P<0.01). (5) There is nstatistic difference in the distribution of serotype 1, 3, and of biovar 1 among these three groups (P=0.763). (6) ThPCR method described here is relatively simple, rapid anspecific for the biotyping and serotyping of biovar 1 of Uurealyticum. Conclusion: we should pay more attention to biovarand mixed infection than single infection of biovar 1 of Uurealyticum in clinic practice. PCR is a good method ibiotyping and serotyping.

Biovars and Serotypes of Ureaplasma Urealyticum among Chinese Women Undergoing Routine Gynecologic Exam, Women with Sexually Transmitted Infections, and Female Sex Workers

Objectives: To characterize the distribution pattern of biovars and scrotypes or Ureaplasma urealyyicum in normalhealthy women, sexually transmitted infections clinic clients,and in sex workers. Methods: We cultured cervical swabs taken from 261physical check-up clients, 599 STI clinic outpatients and 98 sexworkers using commercial selective medium. Some positivecultures were further biotyped and serotyped by PCR. Results: (1) U. urealyticum is more commonly isolated in sexworkers (90.8%) than in the physical check-up group (60.9%)or the STI outpatient group (61.3%) (P<0.001). (2) Biovar 1of U. 'realyticum (95.0%), especially single infection ofserotype 1. 3, and 6 of biovar 1, is commonly found in healthywomen. (3) Biovar 2 infection of U urealyticum is moreprevalent in sex workers (28.1%) and STI outpatients group(26.6%) than that in the physical check-up group (4.9%) (P<0.001). (4) Mixed infection caused by more than one serotypeof U urealyticum increased from physical check-up group(8.6%) to STI utpatients (12.4%) to sex workers (23.9%) (P<0.01). (5) There is no statistically significant difference in thedistribution of serotype 1, 3, and 6 of biovar 1 among thesethree groups (P=0.763). (6) The PCR method described here isrelatively simple, rapid and specific for the biotyping andserotyping of biovar 1 of U urealyticum. Conclusion: We should pay more attention to biovar 2 andmixed infections of U. urealyticum than single infection ofhiovar 1 in clinic practice. PCR is a good method for biotypingand serotvping.

Expression of inducible nitric oxide synthase induced by lipid-associated membrane proteins of Ureaplasma urealyticum is regulated by nuclear factor κB-mediated mechanism in murine macrophages

The aim was to investigate the molecular mechanisms responsible for the inducible nitric oxide synthase （iNOS） gene expression stimulated by lipid associated membrane proteins （LAMPs） of Ureaplasma urealytictan （ U. urealyticum ）. Detection of NO, the expression of iNOS and the activation of nuclear factor κB （NF-κB） in direct response to U. urealyticum LAMPs in a murine macrophages, the effects of pyrrolidine dithiocarbamate （PDTC）, an inhibitor of NF-κB and of cycloheximide （CHX）, a protein synthase inhibitor were available. The results indicated that U. urealyticum LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose- and time-dependent manner by activating NF-κB. The expression of iNOS, NO production and the activation of NF-κB were inhibited by U. urealyticum LAMPs combination with PDTC or CHX. In conclusion, our findings suggest that U. urealyticum may be an etiological factor to certain diseases due to its ability to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.