Effects And Mechanisms Of Vaginal Lactobacillus Crispatus In Improving Chronic Endometritis And Promoting Embryonic Implantation

Background and aims:With a growing infertile population,infertility has become a serious social concern that cannot be ignored nowadays.Genital tract infections are the most common cause of female infertility,with chronic endometritis（CE）showing the highest prevalence.CE is significantly associated with embryo implantation rates and pregnancy outcomes of assisted reproductive therapy.Although antibiotics remain the main treatment strategies against CE,their beneficial effects are limited due to the appearance of drug resistance and high recurrence rates.Therefore,it is urgent to find a novel and effective method in order to improve the therapeutic efficacy of CE,thus enhancing the implantation rate and pregnancy outcome of infertile patients.Many studies have suggested that the female vaginal microbiota dominated by Lactobacillus play an active role in the treatment of genital tract infectious diseases.However,most researches focused on the therapeutic benefits of probiotic in vaginitis and cervicitis,and there are few in regard to the treatment of CE.This project isolated Lactobacillus crispatus（L.crispatus）from the vagina of healthy women.After verifying probiotic properties of L.crispatus through in vitro nature assays,we investigate mechanisms by which it improves the therapeutic effect of CE through constructing CE female mice models,and further explored the potential mechanism of its impact on embryo implantation.The results showed that L.crispatus exhibited excellent growth performance and probiotic properties in vitro.In addition,L.crispatus intervention was effective in inhibiting the colonization and growth of CE-related pathogens on endometrium,as well as improving the phenotype of the endometrium of CE female mice.Also,an increase in the possibility of CE female mice pregnancy and embryo implantation was observed after L.crispatus therapy.Methods:1.Evaluation of probiotic properties of L.crispatus in vitroIncubated L.crispatus in a corresponding liquid culture medium at 37℃in a constant temperature incubator for 24-36 h,and preserved the strain for subsequent experiments.The in vitro probiotic properties of L.crispatus were evaluated by plotting growth curves,conducting acid resistance experiments,antioxidant experiments(detection of DPPH free radical,hydroxyl free radical,and superoxide free radical scavenging capacity,Fe²⁺chelating ability,and total reducing power),bacteriostatic experiments,cell adhesion tests,and other in vitro probiotic assays.2.Effects of L.crispatus on CE female mice model and the exploration of related mechanisms（1）The establishment of animal models and experimental groups:65 female BALB/c mice aged around 6-8 weeks were randomly divided into:blank control group（C group）,endometritis model group（M group）,antibiotic treatment group（Abx group）,L.crispatus treatment group（L group）,and antibiotic combined with L.crispatus treatment group（L+Abx group）,with 13 mice in each group.After drinking water with mixed antibiotics for 24 h,mice in M group were given a single intrauterine injection of the pathogenic bacteria mixture to establish CE female mice models,and mice in C group were given an injection of sterile PBS as a control.After3 weeks,histopathological examination was conducted to ensure the success of model establishment.（2）Intervention with vaginal administration:After the model establishment,following treatments were given.C and M groups:vaginal insertion of gelatin sponges containing 20μL PBS;Abx group:vaginal insertion of gelatin sponges containing 10μL 30mg/m L metronidazole solution;L group:vaginal insertion of gelatin sponges containing 10μL 1×10⁵CFU/m L L.crispatus liquid;L+Abx group:vaginal insertion of gelatin sponges containing 10μL 30mg/m L metronidazole solution for 3 days,followed by the vaginal insertion of the gelatin sponges containing 10μL 1×10⁵CFU/m L L.crispatus liquid for 4 days after 12 h of antibiotics discontinuation.At the end of treatment,4 female mice in each group were reserved.Collected blood and vaginal secretions from other mice,and obtained their uterine tissues for pathological examinations to evaluate the treatment efficacy.（3）Mechanism analysis:Used H&E staining of uterine tissue to observe histopathological changes in mice endometrium,then compared the endometrial damage and inflammatory cell infiltration in CE mice before and after treatment;Used western blotting to detect the expression of inflammatory pathway related proteins TLR4,Myd88,NF-κB and IκB in endometrial tissues;Used q-PCR technique measure the expression of inflammatory cytokines including IL-6,IL-8,IL-1β,TNF-α,and the abundance of Staphylococcus aureus,Beta hemolytic streptococcus,Escherichia coli,and Lactobacillus in mice endometrium.3.Effects of L.crispatus on embryo implantation and the exploration of related mechanisms（1）Experimental animals and groups:After the treatment of CE mice,the mice in C group and the reserved CE mice in different intervention groups were caged with normal male mice in a ratio of 4:1 for female to male.Checked the vaginal suppository in female mice the next morning,and recorded it as day 0 of pregnancy once observed（the caging period do not exceed 2 weeks）.After caging,the mice were grouped into:blank control group（C group）,normal implantation group（CIM group）,endometritis model group（M group）,antibiotic treatment group（Abx group）,L.crispatus treatment group（L group）,and antibiotic combined with L.crispatus treatment group（L+Abx group）.On the 5^thday after pregnancy（implantation period）,collected tissue samples to observe the number of embryos implanted,and compared the differences in embryo implantation among groups.Organ tissues were taken for histopathological examination and molecular biological test.（2）Mechanism analysis:Used immunohistochemistry to detect the expression of implantation related factors including vascular endothelial growth factor（VEGF）and matrix metalloproteinase 9（MMP9）.Used q-PCR to detect the differences in the transcription of implantation related factors including VEGF,Indian hedgehog（IHH）,bone morphogenetic protein 2（BMP2）,secretory glycoprotein 4（Wnt4）,human growth factor（HGF）,colony stimulating factor（CSF）,insulin like growth factor（IGF）,and epidermal growth factor（EGF）.Used Western blotting to detect the expression of implantation related cytokines including VEGF and MMP9,as well as the expression of key proteins in implantation related signaling pathways such as Wnt4,BMP2 andβ-catenin.Results:1.Evaluation of probiotic properties of L.crispatus in vitro（1）The growth cycle of L.crispatus was about 24 h and with a logarithmic growth phase from 4 h to18 h.The counting results showed that the number of viable bacteria reached 1.9×10¹⁰CFU/m L at 18 h,indicating that the strain exhibited an excellent growth performance.（2）Culture conditions with different p H values had no significant impact on the colony count of L.crispatus.The highest viable count demonstrated at p H=5.0 with approximately 5.7×10⁹CFU/m L indicating that the strain possessed a favorable resistance to acidic environments.（3）The scavenging rates of oxidizing substances by L.crispatus were 32.2%,41.49%and 96.08%for DPPH,OH and O^2-,respectively.The chelating ability of L.crispatus to Fe²⁺was about 86%,and the total reducing power was measured at OD₇₀₀to be about 1.54,indicating that L.crispatus had a good antioxidant activity in vitro.（4）L.crispatus can significantly inhibit the growth of genital tract pathogenic bacteria,with an average bacteriostatic ring diameter>1.5 cm indicating an excellent antimicrobial property of the strain.（5）The number of L.crispatus attached to VK2/E6E7（immortalized human vaginal epithelial cell line）cells was about 7000 CFU/100 cells,and this adhesion effect presented no significant impact on the normal growth of VK2/E6E7 cells,indicating that the strain had a strong cellular adhesive capacity for vaginal epithelium.2.Effect of L.crispatus on CE female mice and mechanism exploration（1）After 3 weeks of modeling,the endometrial tissue of female mice was heavily infiltrated with lymphocytes and plasma cells together accompanied by structural alterations in the endometrium,which conformed to the histopathological changes of CE,indicating that the CE model was successfully constructed in this project and can be utilized for subsequent experiments.（2）Compared with M group,L.crispatus supplement could significantly alleviate the inflammatory response in endometrial tissue,reduce the infiltration of lymphocytes and plasma cells in endometrial stroma,and reverse the histopatholo-gical damage in the endometrium.These alternations indicated that L.crispatus could effectively improve CE phenotype and exert synergized effects with antibiotic treatment to promote endometrial repair.（3）Compared with M group,L group and L+Abx group evidently down-regulated the expression of inflammatory proteins TLR4,p-p65/p65 and My D88（p<0.01）,while also strongly reduced the transcription levels of pro-inflammatory factors IL-1β,TNF-α,IL-6 and IL-8（p<0.01）in endometrial tissues.These results demonstrated that L.crispatus could significantly reduce the inflammatory response in the endometrium of CE female mice by inhibiting the expression of inflammatory signaling proteins.（4）With increased relative abundance of pathogenic bacteria in the endometrium of female mice in M group,the pathogenic abundance in Abx,L,and L+Abx groups were significantly decreased（p<0.01）and moreover the relative abundance of Lactobacillus in endometrium of female mice in L group（p<0.05）and L+Abx group（p<0.01）were on contrary significantly increased.3.Effect and potential mechanism of L.crispatus on embryo implantation in CE female mice（1）Compared to the complete absence of pregnancy and embryo implantation observed in M group,the pregnancy rate in both L group and L+Abx group was100%.And the average number of implantations being 7 in L group and 10 in L+Abx group.These results demonstrated that L.crispatus could effectively improve the pregnancy rate and the number of implantations in CE modeling female mice.（2）Compared with group C,the expression of VEGF and MMP9 in CIM group was significantly increased,while that in M group was significantly lower than in other groups.L.crispatus in L group and L.crispatus combined with antibiotics intervention in L+Abx group effectively increased the expression of above factors,indicating that the strain can significantly increase the expression of implantation related cytokines and promote embryo implantation.（3）The transcription level of implantation related factors（VEGF,IHH,BMP2,Wnt4,HGF,CSF,IGF,EGF）in endometrium of female mice in CIM group were increased,while their expression in group M was inhibited to varying degrees.L.crispatus or L.crispatus combined with antibiotics treatment both could promote the expression of implantation-related factors on transcription level,in which the expression of BMP2 and Wnt4 differed most significantly among different groups（p<0.01）.These results suggested that the strain may play a crucial role in promoting embryo implantation through influencing the expression of signal proteins or molecules related to implantation pathways.（4）The results from Western blot showed that the expression of Wnt/β-catenin classical signaling pathway related molecules Wnt4,BMP2 andβ-catenin was markedly inhibited in group M,while significantly increased in L and L+Abx groups（p<0.05）,revealing that the role of L.crispatus in promoting embryo implantation may be achieved through the activation of Wnt/β-catenin classical signaling pathway.Conclusions:In summary,the research results of this project confirmed that L.crispatus exhibited an excellent probiotic property in vitro and can markedly inhibited the proliferation of common pathogenic bacteria in genital tract.L.crispatus intervention could reduce endometrial inflammation and effectively improve histopathological phenotype of the endometrium in CE female mice by inhibiting the expression of inflammatory pathway signaling proteins,while could simultaneously inhibit the growth of pathogens in the endometrium through endometrial colonization resistance.In addition,this strain might regulate endometrial decidualization,increase endometr-ial tolerance and regulate embryo implantation by activating the Wnt/β-catenin classical signaling pathway,thereby increasing pregnancy rate and promoting embryo implantation in CE female mice.This study can present new ideas for the future clinical treatment of CE and the improvement of pregnancy outcomes,at the same time provide data support for the subsequent clinical application of L.crispatus.