Ureaplasma spp. in male infertility and its relationship with semen quality and seminal plasma components

Objective

We investigated the prevalence of Ureaplasma spp. in semen samples of infertile men in Shanghai, China and evaluated the correlation between the sperm parameters (seminal volume, sperm concentration, progressive motility and non-progressive) and the secretary function in these infectious populations.

Development of a multilocus sequence typing scheme for Ureaplasma

Ureaplasma is a commensal of the human urogenital tract but is always associated with invasive diseases such as non-gonococcal urethritis and infertility adverse pregnancy outcomes. To better understand the molecular epidemiology and population structure of Ureaplasma, a multilocus sequence typing (MLST) scheme based on four housekeeping genes (ftsH, rpL22, valS, thrS) was developed and validated using 283 isolates, including 14 serovars of reference strains and 269 strains obtained from clinical patients. A total of 99 sequence types (STs) were revealed: the 14 type strains of the Ureaplasma serovars were assigned to 12 STs, and 87 novel and special STs appeared among the clinical isolates. ST1 and ST22 were the predominant STs, which contained 68 and 70 isolates, respectively. Two clonal lineages (CC1 and CC2) were shown by eBURST analysis, and linkage disequilibrium was revealed through a standardized index of association (I _A ^S). The neighbor-joining tree results of 14 Ureaplasma serovars showed two genetically significantly distant clusters, which was highly congruent with the species taxonomy of ureaplasmas [Ureaplasma parvum (UPA) and Ureaplasma urealyticum (UUR)]. Analysis of the biotypes of 269 clinical isolates revealed that all the isolates of CC1 were UPA and those of CC2 were UUR. Additionally, CC2 was found more often in symptomatic patients with vaginitis, tubal obstruction, and cervicitis. In conclusion, this MLST scheme is adequate for investigations of molecular epidemiology and population structure with highly discriminating capacity.     

Suppression of Antimicrobial Peptide Expression by Ureaplasma Species

Ureaplasma species commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome of Ureaplasma infections. THP-1 cells, a human monocytoid tumor line, were cocultured with Ureaplasma parvum and U. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR. In vitro antimicrobial activities of synthetic AMPs against Ureaplasma spp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation with U. parvum and U. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1, DEFA5, DEFA6, and CAMP), was significantly downregulated compared to that of TNFA and IL-8, which were upregulated. In vitro flow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity against Ureaplasma spp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation with U. parvum infection. No DNA methylation status changes were detected upon Ureaplasma infection. In conclusion, AMPs have in vitro activity against Ureaplasma spp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization.     

Treatment of genital mycoplasma in colonized pregnant women in late pregnancy is associated with a lower rate of premature labour and neonatal complications

Mycoplasma hominis and Ureaplasma spp. may colonize the human genital tract and have been associated with adverse pregnancy outcomes such as preterm labour and preterm premature rupture of membranes. However, as these bacteria can reside in the normal vaginal flora, there are controversies regarding their true role during pregnancy and so the need to treat these organisms. We therefore conducted a retrospective analysis to evaluate the treatment of genital mycoplasma in 5377 pregnant patients showing symptoms of potential obstetric complications at 25-37 weeks of gestation. Women presenting with symptoms were routinely screened by culture for the presence of these bacteria and treated with clindamycin when positive. Compared with uninfected untreated patients, women treated for genital mycoplasma demonstrated lower rates of premature labour. Indeed preterm birth rates were, respectively, 40.9% and 37.7% in women colonized with Ureaplasma spp. and M. hominis, compared with 44.1% in uncolonized women (Ureaplasma spp., p 0.024; M. hominis, p 0.001). Moreover, a reduction of neonatal complications rates was observed, with 10.9% of newborns developing respiratory diseases in case of Ureaplasma spp. colonization and 5.9% in the presence of M. hominis, compared with 12.8% in the absence of those bacteria (Ureaplasma spp., p 0.050; M. hominis, p <0.001). Microbiological screening of Ureaplasma spp. and/or M. hominis and pre-emptive antibiotic therapy of symptomatic pregnant women in late pregnancy might represent a beneficial strategy to reduce premature labour and neonatal complications.     

Clonal diversity of <Emphasis Type="Italic">Ureaplasma</Emphasis> species and its relationship with oligozoospermia and semen quality in Chinese infertile males

Whether Ureaplasma spp. are a causative agent of male infertility remains controversial. Previous studies concerning Ureaplasma spp. and male infertility have been confined to the species level of Ureaplasma. Currently, an expanded multilocus sequence typing (eMLST) scheme has been established with high discriminatory power. The aim of this study was to use eMLST to explore the distribution of Ureaplasma spp. and to analyze its role in oligozoospermia and semen quality. A total of 480 semen samples were obtained from Chinese infertile males. The associations between Ureaplasma spp. with oligozoospermia and semen characteristics were further evaluated. Phylogenetic analysis revealed that 102 Ureaplasma spp. could be separated into two clusters and seven sub-groups. Within cluster I (U. parvum), eST16 and eST41 were the most frequent clones. For cluster II (U. urealyticum), eST82 and eST147 were the most prevalent clones. Sub-groups A and C belonging to cluster I and sub-group 1 belonging to cluster II showed an association with oligozoospermia, in contrast with the Ureaplasma spp. negative group (P <?0.05). Compared with the negative group, semen motility decreased in sub-group 2, especially for non-progressive motility (P <?0.05). These results indicated that sub-groups A and C belonging to cluster I (U. parvum) and sub-group 1 belonging to cluster II (U. urealyticum) were shown to be associated with oligozoospermia. Sub-group 2 belonging to cluster II may have the ability to impair semen motility, especially for non-progressive motility.     

Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid

While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models.     

High-Resolution Melt PCR Analysis for Genotyping of Ureaplasma parvum Isolates Directly from Clinical Samples

Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.     

唑来膦酸抑制U937细胞增殖及诱导凋亡

目的:研究唑来膦酸(ZOL)对人类急性髓系白血病细胞U937的增殖抑制及促凋亡作用。方法:CCK-8法检测不同时间ZOL对U937细胞的生长抑制率;流式细胞术检测ZOL对U937细胞周期的影响;Annexin V-PI法及Hoechst 33342法检测ZOL作用前后细胞凋亡情况变化,JC-1检测ZOL对U937细胞线粒体膜电位变化的影响;克隆形成实验检测U937细胞克隆形成能力;Western blot法检测ZOL对U937细胞周期和凋亡相关蛋白的变化。结果:CCK-8结果显示ZOL可以抑制U937细胞的活力,并呈时间-剂量依赖性;Annexin V-PI及Hoechst33342结果显示ZOL可以促进U937细胞凋亡,且呈时间-剂量依赖性;JC-1结果显示ZOL可以明显降低U937细胞线粒体膜电位;PI法证实ZOL将U937细胞周期阻滞在S期,克隆形成实验证实0.2 mmol/L ZOL可以完全抑制U937细胞的克隆形成能力;Western blot结果显示ZOL作用于U937细胞48 h后细胞周期相关蛋白p21表达显著增强,促凋亡蛋白Bax表达增强,抑凋亡蛋白Bcl-2表达明显减弱。结论:ZOL抑制U937细胞的增殖和克隆形成主要是由于抑制了细胞周期相关蛋白表达,同时ZOL可以促进U937细胞凋亡,这种作用主要是通过调节线粒体凋亡途径相关蛋白来实现的。     

Pseudomonas aeruginosa Quorum Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine-Lactone Induces HLA-G Expression in Human Immune Cells

HLA-G is a nonclassical class I human leukocyte antigen (HLA) involved in mechanisms of immune tolerance. The objective of this study was to determine whether N-(3-oxododecanoyl)-l-homoserine lactone (3O-C₁₂-HSL), a quorum sensing molecule produced by Pseudomonas aeruginosa, could modify HLA-G expression to control the host immune response. We evaluated the ability of 3O-C₁₂-HSL to induce HLA-G expression in primary immune cells, monocytes (U937 and THP1), and T-cell lines (Jurkat) in vitro and analyzed the cellular pathway responsible for HLA-G expression. We studied the HLA-G promoter with a luciferase assay and interleukin-10 (IL-10) and p38/CREB signaling with enzyme-linked immunosorbent assay and immunofluorescence, respectively. We observed that 3O-C₁₂-HSL is able to induce HLA-G expression in human monocytes and T cells. We showed that the induction of HLA-G by 3O-C₁₂-HSL is p38/CREB and IL-10 dependent. 3O-C₁₂-HSL treatment is able to arrest only the U937 cell cycle, possibly due to the peculiar expression of the ILT2 receptor in the U937 cell line. Our observations suggest HLA-G as a mechanism to create a protected niche for the bacterial reservoir, similar to the role of HLA-G molecules during viral infections.     

Senescence of human fibroblasts induced by oncogenic Raf

The oncogenes RAS and RAF came to view as agents of neoplastic transformation. However, in normal cells, these genes can have effects that run counter to oncogenic transformation, such as arrest of the cell division cycle, induction of cell differentiation, and apoptosis. Recent work has demonstrated that RAS elicits proliferative arrest and senescence in normal mouse and human fibroblasts. Because the Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins, we examined the ability of conditionally active forms of Raf-1 to elicit cell cycle arrest and senescence in human cells. Activation of Raf-1 in nonimmortalized human lung fibroblasts (IMR-90) led to the prompt and irreversible arrest of cellular proliferation and the premature onset of senescence. Concomitant with the onset of cell cycle arrest, we observed the induction of the cyclin-dependent kinase (CDK) inhibitors p21^Cip1 and p16^Ink4a. Ablation of p53 and p21^Cip1 expression by use of the E6 oncoprotein of HPV16 demonstrated that expression of these proteins was not required for Raf-induced cell cycle arrest or senescence. Furthermore, cell cycle arrest and senescence were elicited in IMR-90 cells by the ectopic expression of p16^Ink4a alone. Pharmacological inhibition of the Raf/MEK/MAP kinase cascade prevented Raf from inducing p16^Ink4a and also prevented Raf-induced senescence. We conclude that the kinase cascade initiated by Raf can regulate the expression of p16^Ink4a and the proliferative arrest and senescence that follows. Induction of senescence may provide a defense against neoplastic transformation when the MAP kinase signaling cascade is inappropriately active.     

CDK11p58 kinase activity is required to protect sister chromatid cohesion at centromeres in mitosis

The cyclin-dependent kinase CDK11^p58 is specifically expressed at G2/M phase. CDK11^p58 depletion leads to different cell cycle defects such as mitotic arrest, failure in centriole duplication and centrosome maturation, and premature sister chromatid separation. We report that upon CDK11 depletion, loss of sister chromatid cohesion occurs during mitosis but not during G2 phase. CDK11^p58 depletion prevents Bub1 and Shugoshin 1 recruitment but has no effect on the dimethylation of histone H3 lysine 4 at centromeres. We also report that a construct expressing a kinase dead version of CDK11^p58 fails to prevent CDK11 depletion-induced sister chromatid separation, showing that CDK11^p58 kinase activity is required for protection of sister chromatid cohesion at centromeres during mitosis. Thus, CDK11^p58 kinase activity appears to be involved in early events in the establishment of the centromere protection machinery.     

Honokiol Inhibits Tumor Necrosis Factor-α-Stimulated Rat Aortic Smooth Muscle Cell Proliferation via Caspase- and Mitochondrial-Dependent Apoptosis

This study aims to investigate the effects of honokiol on proliferation, cell cycle, and apoptosis in tumor necrosis factor (TNF)-α-induced rat aortic smooth muscle cells (RASMCs). We found that honokiol treatment showed potent inhibitory effects on TNF-α-induced RASMC proliferation, which were associated with G0/G1 cell cycle arrest and downregulation of cell cycle-related proteins, including cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2 and CDK4. Furthermore, honokiol treatment led to the release of cytochrome c into cytosol and a loss of mitochondrial membrane potential (ΔΨm), as well as a decrease in the expression of Bcl-2 and an increase in the expression of Bax. Treatment with honokiol also reduced TNF-α-induced phosphorylation of p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase. Taken together, our results suggest that honokiol suppresses TNF-α-stimulated RASMC proliferation via caspase- and mitochondria-dependent apoptosis and highlight the therapeutic potential of honokiol in the prevention of cardiovascular diseases.     

CDK inhibitor p18(INK4c) is required for the generation of functional plasma cells

B cell terminal differentiation is associated with the onset of high-level antibody secretion and cell cycle arrest. Here the cyclin-dependent kinase (CDK) inhibitor p18(INK4c) is shown to be required within B cells for both terminating cell proliferation and differentiation of functional plasma cells. In its absence, B cells hyperproliferate in germinal centers and extrafollicular foci in response to T-dependent antigens but serum antibody titers are severely reduced, despite unimpaired germinal center formation, class switch recombination, variable region-directed hypermutation, and differentiation to antibody-containing plasmacytoid cells. The novel link between cell cycle control and plasma cell differentiation may, at least in part, relate to p18(INK4c) inhibition of CDK6. Cell cycle arrest mediated by p18(INK4C) is therefore requisite for the generation of functional plasma cells.     

Vaginal <Emphasis Type="Italic">Ureaplasma</Emphasis> species increase chorioamnionitis in very preterm infants with preterm premature rupture of the membranes at &lt; 28 weeks of gestation

Our aim was to investigate the association between vaginal Ureaplasma species (spp.) and the subsequent occurrence of chorioamnionitis (CAM), perinatal death, neonatal morbidity, and long-term neurodevelopmental impairments (NDIs) at 3 years of age. We analyzed 55 pregnant women with singleton pregnancy who had preterm premature rupture of the membranes (pPROM) at <?28⁺⁰ weeks of gestation, and delivered between 22⁺⁰ and 31⁺⁶ weeks at our tertiary hospital in 2007–2016. NDIs were defined as either cerebral palsy or developmental delay evaluated at 1.5 and/or 3 years old. The presence of Ureaplasma spp. and Mycoplasma hominis were evaluated using urea-arginine broth and Mycoplasma PPLO Agar. The presence of Ureaplasma spp. in the vagina was positive in 41%. Vaginal Ureaplasma spp. was a significant risk factor for CAM; however, it was not significantly associated with the occurrence of perinatal death, pulmonary hypoplasia, respiratory distress syndrome, transient tachypnea of the newborn, intraventricular hemorrhage, periventricular leukomalacia, bronchopulmonary dysplasia defined as oxygen required and occasional ventilatory assistance required at week 36 as modified (BPD₃₆), or NDIs. The crude odds ratio (95% confidence interval) of Ureaplasma spp. for the occurrence of CAM was 9.5 (1.10–82) (p?=?0.041). In very preterm birth infants with pPROM, CAM, BPD₃₆, and NDIs occurred in 78, 60, and 36%, respectively. Vaginal Ureaplasma spp. was a significant risk factor for CAM in very preterm birth infants with pPROM. The incidences of BPD₃₆ and NDIs in such infants were very high, nearing 3/5 and 1/3, respectively.     

Juniperus communis extract induces cell cycle arrest and apoptosis of colorectal adenocarcinoma in vitro and in vivo

Juniperus communis (JCo) is a well-known traditional Chinese medicinal plant that has been used to treat wounds, fever, swelling, and rheumatism. However, the mechanism underlying the anticancer effect of JCo extract on colorectal cancer (CRC) has not yet been elucidated. This study investigated the anticancer effects of JCo extract in vitro and in vivo as well as the precise molecular mechanisms. Cell viability was evaluated using the MTT assay. Cell cycle distribution was examined by flow cytometry analysis, and cell apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Protein expression was analyzed using western blotting. The in vivo activity of the JCo extract was evaluated using a xenograft BALB/c mouse model. The tumors and organs were examined through hematoxylin-eosin (HE) staining and immunohistochemistry. The results showed that JCo extract exhibited higher cytotoxicity against CRC cells than against normal cells and showed synergistic effects when combined with 5-fluorouracil. JCo extract induced cell cycle arrest at the G₀/G₁ phase via regulation of p53/p21 and CDK4/cyclin D1 and induced cell apoptosis via the extrinsic (FasL/Fas/caspase-8) and intrinsic (Bax/Bcl-2/caspase-9) apoptotic pathways. In vivo studies revealed that JCo extract suppressed tumor growth through the inhibition of proliferation and induction of apoptosis. In addition, there was no obvious change in body weight or histological morphology of normal organs after treatment. JCo extract suppressed CRC progression by inducing cell cycle arrest and apoptosis in vitro and in vivo, suggesting the potential application of JCo extract in the treatment of CRC.     

Apigenin,a flavonoid constituent derived from P. villosa,inhibits hepatocellular carcinoma cell growth by CyclinD1/CDK4 regulation via p38 MAPK-p21 signaling

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide. Apigenin was widely used in HCC treatment; however, the detailed mechanisms have not been clarified. We isolated, characterized, and identified Apigenin from the P. villosa plant using ethanol-extracted, semi-preparative HPLC and NMR. MTT was used to detect the cytotoxicity of Apigenin in HepG2, SMMC-7721 and Huh-7 cell lines. The cell cycle changes of Apigenin on HepG2 using flow cytometry and the key molecules of cell cycle regulation by RT-qPCR and Western blot. Apigenin was ethanol-extracted and semi-preparative HPLC was used for isolation and purification. The compounds were identified and the results showed Apigenin was one of the bioactive compounds. Apigenin exhibited relatively high cytotoxicity in HepG2, SMMC-7721, and Huh-7. Cell cycle analysis showed that Apigenin could induce G1 arrest in HepG2 in a dose-dependent manner. CyclinD1 was up-regulated and CDK4 was down-regulated upon Apigenin treatment, which indicated that Apigenin could block cell cycle progression at the G1 phase though the regulation of CDK4 and CyclinD1 expression. In conclusion, the present findings might provide new insights about the implication of Apigenin and P. villosa in cancer therapy.     

Mycoplasma bovis-derived lipid-associated membrane proteins activate IL-1β production through the NF-κB pathway via toll-like receptor 2 and MyD88

Mycoplasma bovis causes pneumonia, otitis media, and arthritis in young calves, resulting in economic losses to the cattle industry worldwide. M. bovis pathogenesis results in part from excessive immune responses. Lipid-associated membrane proteins (LAMPs) can potently induce host innate immunity. However, interactions between M. bovis-derived LAMPs and Toll-like receptors (TLRs), or signaling pathways eliciting active inflammation and NF-κB activation, are incompletely understood. Here, we found that IL-1β expression was induced in embryonic bovine lung (EBL) cells stimulated with M. bovis-derived LAMPs. Subcellular-localization analysis revealed nuclear p65 translocation following EBL cell stimulation with M. bovis-derived LAMPs. An NF-κB inhibitor reversed M. bovis-derived LAMP-induced IL-1β expression. TLR2 and myeloid differentiation primary response gene 88 (MyD88) overexpression increased LAMP-dependent IL-1β induction. TLR2-neutralizing antibodies reduced IL-1β expression during LAMP stimulation. LAMPs also inhibited IL-1β expression following overexpression of a dominant-negative MyD88 protein. These results suggested that M. bovis-derived LAMPs activate IL-1β production through the NF-κB pathway via TLR2 and MyD88.