Mycoplasma hominis attaches to and locates intracellularly in human spermatozoa

BACKGROUND: The study of sperm-mycoplasma interaction has been focused on the effects of infection on sperm quality, but few studies have reported the direct interaction of this bacterium with spermatozoa. METHODS: Selected populations of viable, motile and infection-free human spermatozoa from three healthy men were incubated with 15-480 multiplicity of infection (MOI) units of DiIC18-labelled Mycoplasma hominis. Cells were analyzed by means of confocal microscopy and by the eosin-Y dye exclusion test between 10 min and 24 h post-infection. RESULTS: As early as 10 min post-infection, clusters of M. hominis were seen attached to the sperm head, midpiece or tail. Mycoplasma showed an approximately 2.5-4.5-fold higher interaction with sperm head or tail than with midpiece. Sequential sectioning of infected spermatozoa revealed the intracellular location of M. hominis within cytosolic spaces of head and midpiece regions. A minor proportion of infected spermatozoa showed bent or coiled tails, and/or midpiece thickening. Sperm viability was not altered by M. hominis infection. CONCLUSIONS: These results provide specific and conclusive evidence of M. hominis attachment and invasiveness towards human sperm cells, which seems not to affect their viability, suggesting that a short-term M. hominis interaction with spermatozoa results in non-apparent or subtle damage, but might have implications for long-term male or couple's fertility.     

Serological investigation of Mycoplasma genitalium in infertile women

BACKGROUND: The role of Mycoplasma genitalium in the pathogenesis of pelvic inflammatory disease has not been characterized. METHODS: Sera from 308 infertile women were investigated for antibodies to M. genitalium by immunoblotting. Women with tubal factor infertility (TFI) made up 132 of the patients, 67 of the women had an infertile male partner and 109 were infertile for unknown reasons. RESULTS: Of the TFI patients 29 (22.0%) were seropositive to the major adhesin, MgPa, of M. genitalium versus 11 (6.3%) in the group of women with normal tubes. No cross-reactions between MgPa and P1 of the related Mycoplasma pneumoniae were found. Besides, MgPa positive sera were confirmed by immunoblotting using a cloned fragment of the C-terminal part of MgPa specific to M. genitalium. Chlamydia trachomatis is known to be able to cause infertility as a result of salpingitis. Therefore, the sera were tested against C. trachomatis using a commercial ELISA test. Seventy-five (56.8%) of the TFI patients were seropositive to C. trachomatis. Eight (27.6%) TFI patients seropositive to MgPa were negative to C. trachomatis. CONCLUSIONS: This study indicates that M. genitalium may be an independent risk factor in the development of an inflammatory process leading to scarring of the uterine tubes in women and thereby causing infertility.     

Morphology of human Fallopian tubes after infection with Mycoplasma genitalium and Mycoplasma hominis--in vitro organ culture study

BACKGROUND: Female infertility can be caused by scarring and occlusion of the Fallopian tubes. Sexually transmitted bacteria can damage the delicate epithelial layer of human Fallopian tubes (HFT). Genital mycoplasmas are associated with human reproductive failure. Yet, there is not enough evidence that mycoplasmas can cause tubal factor infertility. We analysed the effects of infections with Mycoplasma hominis and Mycoplasma genitalium on the HFT epithelium and compared them with the effects of infections with genital pathogens: Chlamydia trachomatis and Neisseria gonorrhoeae. METHODS: We used an in vitro model in which pieces of normal HFT were infected with different bacteria, and the outcome of the infections was analysed by scanning electron microscopy (SEM) and confocal microscopy. RESULTS: The presence of M. hominis did not cause any morphological changes of the epithelium of HFT. Noticeable changes in the morphology of the ciliated cells were observed in M. genitalium-infected tissue. Five days post-infection, the cilia were abnormally swollen and some of the ciliated cells fell off the epithelium. These effects could be inhibited by pre-incubation of M. genitalium with antibody directed against the C-terminal part of the adhesion protein MgPa before infection of HFT organ culture. CONCLUSION: We have shown that the presence of M. genitalium, but not M. hominis, in the HFT organ culture affected the epithelium and resulted in cilia damage. The effect of infection with M. genitalium on the HFT was, however, very moderate when compared with the extensive damage of the epithelium caused by N. gonorrhoeae or C. trachomatis.     

ELISA法检测江门地区性病门诊患者生殖支原体抗原

目的应用酶联免疫吸附试验（ELISA）检测性病门诊患者泌尿生殖道生殖支原体（Mg）抗原，探讨江门地区性传播疾病（STD）门诊患者Mg感染状况。方法采用Mg多克隆抗体，标记酶结合物，建立检测Mg抗原的双抗体夹心ELISA法，对性病门诊患者泌尿生殖道分泌物进行MIg抗原检测。结果双抗体夹心ELISA法可检测到5μg／ml蛋白浓度，除肺炎支原体外与其他泌尿生殖道常见支原体和细菌无交叉反应；共检测了174例标本，Mg抗原阳性33例，阳性率为18．97％，其中男性和女性阳性率分别为19．88％和7．69％；男性患者中，非淋菌性尿道炎（NGU）、淋病、前列腺炎患者的Mg抗原阳性率分别为13．43％、62．5％和4．84％。结论STD门诊患者存在Mg感染，应用双抗体夹心ELISA法检测Mg抗原具有一定的灵敏度，且具有快速、简便的特点，适合临床筛查Mg抗原。     

X-ray microscopy of human spermatozoa shows change of mitochondrial morphology after capacitation

Using X-ray microscopy two morphologically distinct states were observed of the human spermatozoan mitochondria: (i) compact and tightly wrapped around the axoneme, and (ii) morphologically transformed, i.e. with circular areas of high X-ray transmission, either loosely wrapped around the axoneme or distended. The spermatozoa were examined at two stages of their post-ejaculation maturation process, i.e. as present in fresh ejaculated semen and after in-vitro capacitation. X-ray microscopy allowed sample preparation that was as simple as for conventional light microscopy whilst giving high resolution (30 nm) imaging of samples in liquid media compatible with the requirements of live biological specimens. The specimens were not fixed, stained or metal coated. These features make X-ray microscopy useful in the study of cells, particularly cells in suspension. The relative frequencies of the two morphological states of the mitochondria in seminal plasma and after in-vitro capacitation were compared. In seminal plasma, almost all spermatozoa had compact and tightly wrapped mitochondria. After harvesting by swim-up technique, an increase in the morphologically transformed state had occurred. However, the greatest increase in the morphologically transformed state occurred when the sample had been incubated under capacitating conditions. In this case almost all spermatozoa had morphologically transformed mitochondria.     

Expression and functional identification of the hypothetical adhesin P32 from Mycoplasma genitalium

Mycoplasma genitalium is the main causative agent for non-gonococcal and non-chlamydial urethritis. P32 is the putative surface-exposed membrane protein of M. genitalium and it has substaintial identity in amino acid sequence with adhesin protein P30 from M. pnewnoniae. Since M. pneumoniae mutants lacking P30 protein is defective in cytadherence, P32 protein has been proposed to be an essential adhesin implicated in the adherence of M. genitalium to host cells. The prokaryotic expression vector pET-30 ( )/p32 was constructed in the present study, and the recombinant protein was expressed in E. coli and purified under denaturing condition. As demonstrated by the immunoblotting analysis, the recombinant protein could react with rabbit antisera against M. genitalium, and adherence inhibition assays were petformed with antisera against this recombinant protein. It was demonstrated that P32 protein apperared to be an adhesion protein of M. genitalium, thus providing the experimental basis for better understanding of the pathogenesis of M. genitalium infection and for the development of the related vaccines against the infection.     

生殖支原体和解脲支原体感染与自然流产的关系

目的：探讨生殖支原体和解脲支原体感染与自然流产的关系。方法：收集自然流产患者胚胎组织54份作为流产组;另取40例人工流产者胚胎组织为对照组。采用套式PCR方法对两组标本进行了生殖支原体和解脲支原体的检测。结果：流产组54例中Mg阳性9例阳性率16．7％;Uu阳性21例,阳性率38．9％Mg与Uu合并阳性4例,占7．4％。Mg和Uu分别与对照组比较,结果有显著性差异。结论：自然流产与Mg和UU感染有密切关系。     

Cervicitis and genitourinary symptoms in women culture positive for Mycoplasma genitalium

PROBLEM: Mycoplasma genitalium has been associated with male urethritis. We sought to relate M. genitalium to genitourinary signs and symptoms in women. METHOD OF STUDY: We compared 26 culture-positive women (group 1), 257 additional polymerase chain reaction-positive women (group 2), and 107 negative control women. We used logistic regression to evaluate signs and symptoms, controlling for co-infections, pregnancy, age, and intervention group assignment. RESULTS: Comparing group 1 with controls, we found significantly elevated odds ratios (ORs) for intermediate vaginal discharge (OR = 5.4; 95% confidence interval 1.01, 29.2) and action in response to discharge [3.9 (1.1, 13.5)]. Non-significant increases were observed for pathologic vaginal discharge [3.8 (0.78, 18.2)], pathologic dyspareunia [1.5 (0.25, 9.0)], vaginal odor [2.1 (0.75, 5.7)], and cervical mucopus [4.1 (0.74, 22.4)]. Group 2 results were similar, but showed no increase in cervical mucopus relative to controls. CONCLUSION: Infection with M. genitalium in women is independently related to increased genitourinary symptomatology.     

应用噬菌体展示肽库筛选生殖支原体黏附蛋白的模拟表位

目的 利用纯化的兔抗重组生殖支原体黏附素蛋白(rMgPa)的多克隆抗体(pAb)从噬菌体展示随机12肽库筛选MgPa的模拟表位.方法 用纯化的兔抗rMgPa的pAb对噬菌体随机12肽库进行生物淘洗,随机挑取噬菌体克隆进行DNA测序与分析,并用MIMOX对噬菌体克隆所展示的肽序列进行生物信息学分析.用ELISA、竞争性结合试验和Western blot检测噬菌体与pAb结合的特异性.结果 4轮生物淘洗后特异性噬菌体克隆得到了明显的富集.依据氨基酸组成的不同,74个噬菌体克隆所展示的肽序列可大致分为3组.经对肽序列进行比较分析以及用MIMOX进行生物信息学分析,结果表明这3组的核心序列分别为:P-S-A-A/V-X-R-F/W-E/S-L-S-P、A-K-I/L-T/Q-X-T-L-X-L和K-S-L-S-R-X-D-X-I.ELISA、竞争性结合试验和Western blot方法检测的结果表明噬菌体能与pAb发生特异性结合,说明其可能是MgPa的模拟表位.结论 成功筛选到MgPa的3个可能的模拟表位,为研制基于Mg抗原表位的诊断试剂与多肽疫苗奠定了一定的实验基础.     

Study of apoptotic DNA fragmentation in human spermatozoa

The aim of our work was to define and better understand apoptosis in the spermatozoa of normal subjects, infertile patients and patients affected by specific tumoral diseases employing the method of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and confirming the results by electron microscopy. We studied 23 healthy, normozoospermic subjects (group A), 29 oligoasthenoteratozoospermic patients, affected by various andrological pathologies (group B), 28 patients with Hodgkin's disease (C1) and 30 patients with testicular cancer (C2). Our data demonstrate that the percentage of apoptosis in normozoospermic subjects (group A) is significantly lower than in all the other groups (B, C1, C2) (P < 0.001). This confirms that high DNA fragmentation is one of the characteristics of spermatogenetic failure. The induction of apoptosis, which can also be a basic response to neoplastic disease, can even act right up to the mature male gamete. Our results suggest that apoptosis could be the final result of various pathologies and of a deregulation of spermatogenesis control systems.     

Identifying a consensus sample type to test for Chlamydia trachomatis,Neisseria gonorrhoeae,Mycoplasma genitalium,Trichomonas vaginalis and human papillomavirus

ObjectivesSexually transmitted infections (STIs) are a global cause of acute illness. Early detection plays a crucial role in interrupting transmission and preventing complications. However, the accessibility of STI testing is curbed by the lack of an overall preferred sample type. By means of a prospective study in female sex workers (FSW), we compared the sensitivity of samples from different anatomical sites in detecting Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium and human papillomavirus. Besides, we documented the prevalence of each STI in this high-risk population.MethodsWe selected 303 FSW and tested them for each STI by nucleic acid amplification testing on two vaginal and cervical swabs from different manufacturers, cervical smear and first-void urine. The sensitivity of each sample type was compared for each infectious agent in order to identify a consensus sample type.ResultsVaginal swabs were superior to all other sample types, with an overall sensitivity of 86%. The sensitivity was the lowest for first-void urine, detecting only 63% of positive cases. The prevalence was 3.3% (10/299) for Neisseria gonorrhoeae; 9.0% (27/299) for Chlamydia trachomatis; 7.4% (22/298) for Trichomonas vaginalis; 10.8% (32/296) for Mycoplasma genitalium and 55.6% (158/284) for human papillomavirus.ConclusionsWhen testing for STIs, vaginal swabs are the sample of choice and first-void urine should be avoided. Designating (self-sampled) vaginal swabs as a consensus sample type enables harmonization of STI testing and extension of testing to large numbers of unscreened females.     

Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism

Objectives: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Materials and Methods: Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium) and a 559 bp fragment (U. urealyticum). Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. Results: Of the 210 samples, a total of 100 (47.6%) samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%), and coinfections with both species were detected in four samples (1.9%). The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. Conclusion: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.     

Variable kinematics of capacitating human spermatozoa

Human spermatozoa were prepared by swim-up fromsemen into in-vitrofertilization (IVF) culture medium, and their movement recordedby videomicrography using NTSC video to give 60 images/s onfreeze-frame playback. Trajectoriesin which spermatozoa appearedto switch fromone type of motility pattern to another (fromnon-hyperactivatedto transitional or star-spin hyperactivated),were reconstructed manually and the movement characteristicsdetermined for short consecutive sequences along eachtrack.The spermatozoa were able to switch between allof these motilitypatterns, apparently at random. The seobservations illustratedthat hyperactivated motility is areversible state in human spermatozoa,with phase changesfrom hyperactivated to non-hyperactivatedmotility patternsalong trajectories. It was not necessary forspermatozoato pass through the transitional hyperactivated motilityphasewhen switching from non-hyperactivated to starspinhyperactivatedmotility or vice versa.     

Ultrastructural abnormalities of human spermatozoa

The observation of sperm cells in the scanning and electronmicroscope reveals that malformed spermatozoa show either asingle anomaly of each of their structural components—acrosome,nucleus, axoneme and accessory structures—or a combinationof these anomalies. A defined semen profile can be establishedwhen the majority of ejaculated spermatozoa present a predominantlysingle anomaly or the same combination of associated anomalies.Only in these case can there be a relationship between morphologicaldefects and impairment of the fertilizing ability. The validityof the ultrastructural examination of sperm cells depends uponthe data obtained by light microscopy, and quantification ofthe frequencies of the ultrastructural alterations is necessaryto define clearly the limits of abnormality.     

Preliminary characterization of a factor in human follicular fluid that stimulates human spermatozoa motion

Follicular fluid alters the physiology and behaviour of spermatozoaby increasing acrosome reaction, accelerating capacitation,attracting the spermatozoon and enhancing vigorous motion ofthe cell. The objective of this study was to characterize thefactor(s) in human follicular fluid that causes vigorous spermatozoamotion. Follicular fluid and its fractions were tested for stimulationof spermatozoa motion using a standardized assay which employsa computerized digital imaging system. Our results show thatboth follicular fluid and its methanol extract stimulatevigorousspermatozoa motion. To determine the characteristics of theactive factor(s), the methanol extract was subjected to molecularweight fractionation, protease digestion, microcrystalline thinlayerchromatography (TLC) and C18 reverse-phase high-pressure liquidchromatography (HPLC). The spermatozoa motion stimulator inthe methanol extract was dialysable against a low molecularweight membrane (1000 Da), insensitive to boiling and low pH(3.5) and was largely inactivated by proteinase K digestion.The activity was detected near the solvent front on TLC. Usingreverse-phase HPLC monitored at 254 nm (UV), the activity elutedas a single peak of activity at low methanol concentration,indicating that the activity was relatively hydrophilic. Theactivity in the HPLC peak lost most of its motion-stimulatingability after digestion with proteinase K. The motion stimulatorcould be a peptide analogous to the egg-associated peptidescharacterized in echinoderms which stimulate spermatozoa motion,respiration and chemotaxis.     

Hyperactivation may assist human spermatozoa to detach from intimate association with the endosalpinx

The behaviour of human spermatozoa was observed during incubationwith epithelial cells isolated from the isthmic and ampullarysections of human uterine (Fallopian) tubes.During incubation,spermatozoa were observed to bind to the epithelial cells ofthe tube (the endosalpinx), and individual spermatozoa attachedand detached at intervals.The kinematic characteristics of spermatozoaduring these behaviour patterns were determined. The resultsshowed that detached spermatozoa typically had an increasedcurvilinear velocity and amplitude of lateral head displacement,accompaniedby a decrease in their linearity. Significantly (P<0.01)more of the detaching spermatozoa were hyperactivated than werespermatozoa prior to attachment for both isthmic (35.3 ±5.5 versus 4.0 ± 3.3%mean ± SEM) and ampullary(26.0 ± 7.0 versus 2.0 ±1.4%) regions. Incubationwith epithelial cells from either region produced no differencesin any category of sperm behaviour. Furthermore, there was nosignificant difference between regions in the amount of timespermatozoa spent bound (33.6 ± 12.9 and 20.6 ±3.0 s for isthmic and ampullary tissue respectively). Theseresults support the hypothesis that hyperactivation may assistspermatozoa in breaking connections with epithelial cells.     

The gamete membrane fusion test to assay the fertilizing ability of human spermatozoa

The value of the gamete fusion test for the assessment of humanspermatozoa is assessed. The factors influencing the test arediscussed, including the conditions of heterologous fertilizationusing zona-free oocytes such as the nature of sperm preparation,sperm concentration and capacitation time, and the importanceof albumin in the medium. The oocytes can be examined beforeor after fixation, and the spermatozoa around the eggs assessedfor acrosome changes, while the number of sperm tails on theoocyte can be related to the number of chromatin decondensations.The test provides information on the fertilizing abilty of spermatozoaof given individuals, and may prove of value in testing contraceptives.     

The evaluation of morphological characteristics of human spermatozoa according to stricter criteria

The evaluation of the morphology of human spermatozoa varies widely between and sometimes even within laboratories. The purpose of this study was to determine whether the method that has been developed in our laboratory and which resulted in the use of stricter criteria for the evaluation of sperm morphology is a practical, reliable and repeatable method and to establish the within and between observer variations. The criteria used for a 'normal' spermatozoon are based on the appearance of spermatozoa found in the mucus of the upper endocervical canal. The results of the morphological evaluations of 26 samples by four observers were statistically analysed by various methods. The method of Barnett showed a high degree of relative accuracy between observers with error variances of between 2.89 and 19.67 as well as high Spearman rank correlation coefficients of between 0.8675 and 0.6537 (P less than 0.0003). The Spearman correlation coefficient for 15 duplicate evaluations by one observer was 0.9650 (P less than 0.0001) while the coefficients of variation for repeated evaluations of single samples were also within acceptable limits. Based on these results, the method described in this article allows comparable and reliable results between and within observers to be obtained. From this and other studies it can be concluded that the method also has a good prognostic value for the prediction of expected IVF fertilization, the hamster test and hemizona assay.     

Laser-induced immobilization and plasma membrane permeabilization in human spermatozoa

We evaluated the potential use of a non-contact, 1.48 microm wavelength diode laser for immobilization of human spermatozoa and permeabilization of the sperm membrane in different culture media. When we applied a single laser shot near to the middle region of the sperm tail, spermatozoa could be immobilized either temporarily or permanently, depending on the energy used. Above an energy of 2 mJ in polyvinylpyrrolidone and 2-3 mJ in culture medium, a reliable permanent immobilization was achieved by permeabilization of the sperm tail membrane. We then explored the use of a double laser shot technique. Spermatozoa were temporarily immobilized by a first laser shot applied near to the sperm tail followed by permeabilization with a second laser shot aimed directly at the sperm tail. This sequential approach yielded permanent immobilization at much lower energy values compared with the single shot technique. Following the injection of laser-treated spermatozoa, mouse oocytes underwent normal activation and pronuclear formation. We conclude that a non-contact 1.48 microm diode laser system can be used for immobilization of spermatozoa and for permeabilization of the sperm tail membrane. This laser procedure may offer an alternative to currently used sperm pretreatment prior to intracytoplasmic sperm injection.     

Apoptosis and necrosis in human ejaculated spermatozoa

BACKGROUND: Features of both apoptosis and necrosis have been reported in ejaculated human spermatozoa. This study examines the relative contribution of these two modes of cell death to the demise of these terminally differentiated cells. METHODS: Sperm fractions were prepared from aliquots of semen samples from young normozoospermic donors by simple washing from seminal plasma, by discontinuous density gradient centrifugation or by swim-up technique. They were subsequently incubated in vitro at 37 degrees C for 24 h. Sperm motility, viability, and the presence of two apoptotic markers, phosphatidylserine externalization (annexin-V binding) and DNA fragmentation (TUNEL), were examined before incubation and again after 4 and 24 h of incubation. RESULTS: The swim-up technique was the most efficient in terms of the recovery of viable, motile and non-apoptotic spermatozoa, followed by density gradient centrifugation and finally simple washing. No changes in the parameters tested were observed after 4 h of incubation, but a significant decrease in sperm motility and viability was detected after 24 h irrespective of the sperm preparation technique employed. However, these changes were not accompanied by any increase in the incidence of spermatozoa showing markers of an active apoptotic process. CONCLUSIONS: Healthy human ejaculated spermatozoa appear incapable of initiating apoptosis, at least under in vitro conditions.