| Background and objective:Chlamydia trachomatis(C.trachomatis)leads to multiple diseases such as trachoma,cervicitis and infertility,which was reported being able to increase the risk of HPV subtypes infection and cervical cancer.Genital tract infection with C.trachomatis also causes markedly vaginal microbiota change,however,whether this change has effect on C.trachomatis infection was not elucidated.As a valuable pathogen causing of infertility,there are few epidemiological data about C.trachomatis infection among infertile women.Therefore,this study has collected clinical samples from infertile female patients to investigate C.trachomatis epidemiological characteristics including prevalence,genotype distribution,co-infection of C.trachomatis and HPV and their correlation with cervical intraepithelial neoplasia.On this basis,16S rRNA sequencing was employed to determine the diversity of vaginal microbiota in infertile women with C.trachomatis infection,and to explore the effects of dominant bacteria and their metabolites on the infectivity of C.trachomatis in vitro.Consequently,mice model was constructed to identify the effects of this dominant bacteria on Chlamydia clearance both in lower genital tract and intestinal tract,pathological damage of reproductive system and intestinal tract tissues and immune response induced by Chlamydia.Briefly,this study has systematically demonstrated the epidemiology of C.trachomatis infection among infertile women,diversity of vaginal flora and their effects on Chlamydia genital tract infection from the perspectives of population,cellular and animal model,shedding light on the treatment of C.trachomatis infection via micro-ecological regulation intervention,and additionally providing an experimental basis on enrichment of C.trachomatis pathogenic mechanism.Methods:1.C.trachomatis plasmid ORF2 was amplified from 5006 clinical samples for screening C.trachomatis infection.Then,nested PCR was employed to amplify Omp1 VS1～VS2 gene from C.trachomatis-positive samples,the PCR products of which were sequenced for C.trachomatis genotyping.2.Combination of low-through hybridization with PCR technique were utilized to detect 21 HPV genotypes,including 14 high-risk HPV types(HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68),6 low-risk HPV types(HPV 6,11,42,43,44,CP8304)and suspected high-risk HPV53.3.Administrating 666 female patients from the reproductive center as the case group,a ratio of 1:1 case-control study matched by age was carried out to investigate the prevalence of C.trachomatis,HPV and their co-infections.Additionally,based on 236 C.trachomatis-positive or 778 HPV-positive patients,a ratio of 1:1 case-control study matched by age and clinical departments was carried out to analyze the correlation between C.trachomatis and HPV infection.4.DNA libraries were constructed using NEB Next(?)UltraTM DNA Library Prep,and second-generation high-throughput bacterial 16S rRNA amplicon sequence technology was applied to perform bacterial genome sequence from the vaginal secretions of female infertility with C.trachomatis-positive or negative,followed by analyzing the diversity of vaginal flora and exploring the dominant bacteria causing the vaginal flora changes via QIIME、MUSCLE、SILVA 132、Cytoscape and Graphviz software.5.UPLC-MS/MS was used to detect succinylacetone,non-derivatized various amino acids and carnitine in the metabolites of Lactobacillus,electrode method for lactic acid levels and spectrophotometric method for L(+)lactic acid and D(-)lactic acid respectively.6.The Lactobacillus cultured supernatants or 0～20 mM lactic acid isomers were incubated with C.trachomatis at different pH,indirect immunofluorescence was applied to measure Chlamydia IFUs and trypan blue staining method for identification of HeLa cell viability.7.L(+)lactic acid or D(-)lactic acid with a final concentration of 10 mM were added to Lactobacillus iners metabolites,together with inorganic HCL as a parallel control to stimulate C.trachomatis respectively.Chlamydia IFUs were then detected via IFA.8.Constructing mouse model for Chlamydia muridarum genital tract infection,2×109 CFU Lactobacillus crispatus,Lactobacillus reuteri,Lactobacillus iners and their mixtures at a ratio of 1:1:1 were inoculated via genital tract 3 days after C.muridarum infection.The intervention with Lactobacillus was confirmed by qRT-PCR.9.Thirty-five female BALB/c mice aged 4～6 weeks were randomly divided into SPG、Cm、Cm+LC、Cm+LR、Cm+LN、Cm+Mix and Cm+Mix-P groups.Collecting mouse vaginal and anal swabs every 3 or 7 days,indirect immunofluorescence was used for detecting Chlamydia IFUs,and ELISA method employed for measuring the production of TNF-α、IFN-γ、IL-1β、IL-6 and IL-10 in vaginal swabs.10.Eighty days after C.muridarum infection,reproductive system and intestinal tissues were isolated to evaluate the pathological damage following H&E staining;intracellular CD4+,CD8+,CD8+/IFN-y,CD4+/IFN-y,CD8+/IL-4 and CD4+/IL-4 cell levels were detected by flow cytometry,and TNF-α、IFN-y and IL-10 production in supematant were measured using ELISA.Results:1.A total of 5006 clinical samples were employed.The overall prevalence of C.trachomatis was 4.7%(236/5006),while the prevalence of that was statistically significant(P=0.043)among women from the assisted reproductive technology center,physical examination center and gynecology clinic,which was 3.5%(23/666),3.8%(38/1006)and 5.2%(175/3334)respectively.The main distribution of C.trachomatis was E(30.1%,69/229),F(23.1%,53/229)and J type(17.9%,41/22)in this study,which revealed no significant association with HPV genotype distribution(r=0.08,P=0.67).2.The C.trachomatis prevalence was higher in the group of ≤25 years than in those of>25 years(10.4%vs 4.0%,P<0.001).Both age and HPV are the risk factors for C.trachomatis infection(OR=0.496,P<0.001;OR=1.710,P<0.001).C.trachomatis infection is a risk factor for low-grade cervical lesions in women(OR=3.25,P=0.018)while HPV infection is a risk factor for low-grade and high-grade cervical lesions or cancer in women(OR=6.89,P<0.001;OR=15.86,P<0.001).3.Twenty-five samples were sequenced via the Illumina Nova sequencing platform,obtaining 1,949,887 valid original sequences.Following quality control screening,1,639,766 high-quality sequences were obtained.An average of 65,591 sequences per sample can be used for following data analysis,the Goods overage index value of which exceeds 98%,suggesting the sequencing depth covered over 98%of the bacterial phylotypes,and reflecting exactly the situation of the microorganisms contained in each sample.4.Both Chlamydia and Actinobacteria among the infertility female with C.trachomatis-positive were higher than those with C.trachomatis-negative ones or other group(P<0.05).While the Lactobacillus in the vagina among the infertility female with C.trachomatis-positive was lower than that in other group(P<0.01),which,however,was significantly increased after treatment with azithromycin.5.The average concentration of C.trachomatis and C.muridarum in this study were 3.39×108 IFU/mL,5.15×108 IFU/mL respectively.Lactic acid in the metabolites of Lactobacillus crispatus,Lactobacillus jensenii,Lactobacillus salivarius,Lactobacillus iners,Lactobacillus gasseri,Lactobacillus mucosa and Lactobacillus reuteri were 2～20 fold higher than that in MRS(P<0.01),which were furthermore robust higher than other metabolites components of that such as succinylacetone,non-derivatized various amino acids and carnitine(P<0.01),indicating lactic acid comprises the main component of Lactobacillus metabolites.6.Lactobacillus metabolites inhibited C.trachomatis infectivity in a dose,time and pH-dependent manner.Lactobacillus crispatus metabolites were able to reduce about 85%of C.trachomatis infectivity,indicating the robustest effect on the inhibition of that compared to other Lactobacillus metabolites(P<0.01),while Lactobacillus iners metabolites conducted a relatively weak inhibition(reduced by about 45%).7.The concentration of D(-)lactic acid in Lactobacillus crispatus metabolites was significantly higher than that in Lactobacillus iners metabolites(9.8 mM VS 0.6 mM,P<0.01),the antagonistic effects of Lactobacillus iners metabolites added with a final concentration of 10 mM D(-)lactic acid was significantly stronger than that with L(+)lactic acid or single Lactobacillus iners metabolites(P<0.01).Furthermore,D(-)lactic acid showed a stronger inhibition of C.trachomatis infectivity than that of L(+)lactic acid(P<0.01),suggesting that D(-)lactic acid represents the key component to inhibit C.trachomatis infectivity.8.Compared to mice in Cm group,the incidence of hydrosalpinx and the clearance of Chlamydia in intestinal tract have not significantly been reduced in mice from Cm+LC、Cm+LR、Cm+LN、Cm+Mix and Cm+Mix-P(P>0.05);no significant difference observed in Chlamydia loads in the lower genital tract between Cm+Mix and Cm+Mix-P(P>0.05),however,which was markedly lower in Cm+LC and Cm+Mix compared with Cm(P<0.05);dilation and inflammation scores of oviducts in Cm+Mix were markedly lower than that in Cm(P<0.05).9.Production of TNF-α、IFN-y、IL-1β and IL-6 in vaginal discharge of Cm、Cm+LC、Cm+LR、Cm+LN、Cm+Mix and Cm+Mix-P was significant higher than that in SPG(P<0.05),but not IL-10(P>0.05).In addition,with the exception of IL-6,the production of TNF-α、IFN-γ and IL-1β in vaginal discharge of Cm+LC、Cm+LR、Cm+LN、Cm+Mix and Cm+Mix-P was significant lower than that in Cm(P<0.01).10.All mice in Cm+LC、Cm+LR、Cm+LN、Cm+Mix and Cm+Mix-P groups have produced a high titer of Chlamydia-specific Ig G、Ig G1 and Ig G2a,and the ratio of Ig G2a/Ig G1>1,however,which proved no significant difference with Cm(P>0.05);CD4+/IFN-y and CD8+/IFN-y cell in Cm,Cm+Mix and Cm+Mix-P was statically higher than that in SPG(P<0.05),nevertheless,no significant difference of CD4+/IL-4 and CD8+/IL-4 cell was observed among groups(P>0.05).Conclusions:1.The prevalence of C.trachomatis infection among young infertile women is high.C.trachomatis E,F and J is the main genotype distributed in this study,and the genotype distribution of which revealed no relation to the HPV genotype distribution.While both C.trachomatis and HPV are associated with varying degrees of cervical intraepithelial neoplasia.2.Infertile women with C.trachomatis infection have a Lactobacillus iners-dominated vaginal microbiota,whose relative abundance of Lactobacillus in the vagina of which has been significantly decreased.After treatment with azithromycin,most lactobacilli in the vagina have been restored to differing degrees.3.Lactobacillus metabolites were able to inhibit C.trachomatis infectivity in varying degrees,among which D(-)lactic acid comprise the key component.Furthermore,proper pH was equally essential for inhibition of C.trachomatis infectivity by D(-)lactic acid or Lactobacillus metabolites.4.Lactobacillus intervention did not significantly reduce the incidence of hydrosalpinx caused by C.muridarum,the clearance rate of intestinal Chlamydia,and alter the type of cellular immune response induced by C.muridarum.However,mixed Lactobacillus intervention was capable of promoting the clearance of Chlamydia in the lower genital tract and attenuating its hydrosalpinx dilation and inflammation reaction. |
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