Research About The Diversity Of Vaginal Flora In Patients With Candida Vaginitis In The First Trimester

Objective:1.To explore the occurrence condition of vaginitis in early pregnancy through analyzing the results of vaginal secretions of pregnant women in the first trimester and non-pregnant women in the child-bearing age;2.ITS rDNA and 16S rDNA amplification sequencing technologies were used to analyze the diversity of vaginal bacteria and fungus from pregnant women with Vulvovaginal candidiasis(VVC)in early pregnancy,and to explore the differences in bacterial distribution between the two groups;3.Provide a relatively reliable theoretical basis for clinical diagnosis and treatment of candida vaginitis in early pregnancy by analyzing the distribution characteristics of vaginal flora in patients with candida vaginitis in early pregnancy.Methods:Part one:the analysis of the incidence condition of vaginitis in early pregnancy1.The subjects:(1)Group P:totally 274 singleton early pregnancy women who visited the outpatient department of Obstetrics and Gynecology of the second hospital of Lanzhou University from May 2018 to May 2019 were selected as the experimental group,their age ranged from19 to 35 years old,with an average age of(28.58±4.40).(2)Group N:totally 127 non-pregnancy women in the outpatient department of obstetrics at the same time as Group P were selected as the control group,they aged 18-35 years old,with an average age of(29.48±4.23).2.The experiment content:After informed of the purpose of the study and agreed,the subjects who met the inclusion criteria were carefully questioned about their medical history and whether they had vaginitis related symptoms,followed by gynecological examination,and sterile cotton swab was used to take the vaginal lateral dome secretion,which was immediately numbered after removal.The serial number was sent to the laboratory for vaginal excretion examination(the main analysis contents includes:vaginal cleanliness,white blood cell,miscellaneous bacteria,gram-negative diplococcus,mold,trichomoniasis,pH,clue cells,etc.).According to the diagnostic criteria of vaginitis in department of obstetrics and gynecology,the incidence of vaginitis in the two groups was statistically analyzed.Part two:the study on the diversity of vaginal flora in patients with candida vaginitis in early pregnancy1.The subjects:(1)The normal group:totally 10 singleton early pregnancy women who visited the obstetrical clinic of the second hospital of Lanzhou University from May 2018 to May2019 were selected,their ages range from 25 to 32 years old,average(29.00+2.45)years old,for in May 2018 to December 2018 in the second hospital of Lanzhou University,70-97 days of gestation maternity clinics single pregnancy pregnant.(2)The VVC group:totally 10 singleton early pregnancy women,aged 25-33 years old,with an average age of(30.20±3.19)years old,were selected at the same period with the normal group.2.The experiment content:2.1 Extraction of total DNA of the samples:HiPure Soil DNA Kit B(Magen,Guangdong,China)was used to extract the total DNA from vaginal secretions according to its instructions strictly(two copies of collected vaginal secretions from each patient were taken for DNA extraction and divided into two sets).2.2 Amplification of samples’16S rDNA and ITS rDNA gene fragments:a set of sample DNA extracted with the above steps(20 copies in total)was taken,and the highly variable regions of v3-v4 on 16S rRNA of each sample were amplified using primer 338F-806R.The ITS2 variable region on ITS rDNA of samples was amplified through using ITS primers 5’-GTGAATCGARTC-3’and 5’-TCCTCCGCTTATTGAT-3’.2.3 Illumina MiSeq sequencing and bioinformatics analysis:the concentration of PCR products of 16S rDNA and ITS rDNA in the above samples was detected.After calibration to 10nM,PE250 was sequenced on the Illumina MiSeq(Illumina,San Diego,CA,USA)platform.The information after sequencing was read and analyzed by Qiime1.9.1 software,and be analyzed for further OTUs extraction,OTUs overlapping,OTUs clustering analysis,Alpha diversity analysis,Beta diversity analysis and significance analysis of inter-group community structure differences were conducted.Results:Part one:the analysis of the incidence condition of vaginitis in early pregnancy1.The pH of vaginal secretions of women in early pregnancy was lower than the women without pregnancy in the same age group,and the results showed statistical difference(P<0.05).2.The common types of vaginitis were bacterial vaginitis,candida vaginitis and trichomoniasis vaginitis in women in early pregnancy and non-pregnancy.There was no statistically significant difference in the incidence of vaginitis between the two groups(χ~2=0.009,P=0.924).3.The incidence of bacterial vaginitis and candida vaginitis in early pregnancy is slightly higher than that of women who are not pregnant,while the incidence of trichomoniasis vaginitis is slightly lower than that of women who are not pregnant.Part two:the study on the diversity of vaginal flora in patients with candida vaginitis in early pregnancy1.The vaginal pH of candida vaginitis patients in early pregnancy was significantly higher than that of the normal pregnant group;2.Lactobacillus was still the dominant bacterium in the vaginal bacterial community of early pregnancy patients with VVC,accounting for 70.99%,but the relative abundance of lactobacillus was significantly lower than that of normal women(accounting for 97.99%)in early pregnancy.3.Through Alpha diversity analysis of biological information after sequencing,it was found that the vaginal bacterial community diversity of patients with VVC in early pregnancy increased compared with that of women in early pregnancy,while that of women in early pregnancy decreased,and the differences were statistically significant(P<0.05).4.LEfSe analysis of the biological information of ITS rDNA sequencing of the two groups indicated that the characteristic fungus in the VVC group was Candida,while the characteristic fungus in the normal group was Malassezia.Conclusion:1.The pH of patients with VVC in early pregnancy is higher than that of normal women in early pregnancy;2.The vaginitis in early pregnancy according their incidence from high to lower are:bacterial vaginitis,candida vaginitis and trichomoniasis vaginitis;3.The bacterial community diversity of patients with VVC in the first trimester increased and the fungal community diversity decreased compared with that of women in the first trimester.4.There were differences in community structure between the VVC group in the first trimester and the normal pregnant women in the first trimester.The characteristic fungus in the VVC group was Candida,while the characteristic fungus in the normal group was Malassezia.5.Lactobacillus was still the dominant bacterium in the vaginal bacterial community of the patients with VVC in the first trimester,but its abundance was significantly lower than that of normal women in early pregnancy.