| Objective:Infertility due to reproductive tract infections can reach20%-60%of couples in the world each year.The main pathogens causing infertility were Ureaplasma urealyticum（UU）,Chlamydia trachomatis（CT）and Neisseria gonorrhoeae（NG）.The infection rate of genitourinary tract caused by Chlamydia trachomatis and Ureaplasma urealyticum increased.In addition,studies have found that anti-sperm antibodie is also one of the factors that affect infertility.This study examined the blood and vaginal secretions from patients and healthy people,to explored the distribution characteristics of microflora in urogenital,in order to provide a more scientific basis for the diagnosis of infertility.Method:1.Research objects 50 female patients with infertility and no other underlying diseases diagnosed in the First Affiliated Hospital of Hebei North University from January,2017 to January,2018,aged 22 to 35 years old（27.45±2.54）.The control group selected 50 patients with normal fertility in our hospital.The age ranged from 22 to 37 years（28.65±2.94）.2.Inclusion criteriaMarried for more than two years,no contraceptive measures were taken,don’t have trichomonas vaginitis、mycotic vaginitis and bacterial vaginosis,excluding genetic factors、anatomical factors、endocrine factors and abnormal immune function,have fertility requirements。3.Real-time PCR:50 female patients with infertility diagnosed in the First Affiliated Hospital of Hebei North University from January 2017 to January 2018 and no other underlying diseases were selected,and selected for the same age at the same time.50 in the control group.Collect the vaginal secretions of the patient in a sterile tube,add 1 ml of sterile physiological saline to the sterile tube,shake well,shake the cotton swab,transfer all the liquid to a 1.5 ml centrifuge tube,centrifuge at 12000 rpm for 5 min,discard the supernatant..50μl of DNA extract was added to the pellet,shaken for 5 to10 s,metal bath at 100°C for 10±1 min,and centrifuged at 12,000 rpm/5min.Take appropriate amount of centrifuged sample to be tested,place a sample into the 8 tube,cover the tube,and centrifuge for a few seconds to get on the machine.PCR amplification conditions:93°C 2 min,93°C 45s→55°C60s 10 cycles,93°C 30s→55°C 45s 30 cycles.Automatically read the UU and CT quantitative values of the real-time PCR instrument.Enzyme-linked immunosorbent assay（ELISA）:Subjects were given appropriate venous blood 3 to 5 ml in the early morning fasting state,and did not do any anticoagulation treatment.After 5 min 3000 rpm centrifugation,the serum was separated and stored in-20 In the°C environment,the serum was thawed before the test and allowed to stand at room temperature until the temperature was equilibrated.The assay was carried out by enzyme-linked immunosorbent assay（ELISA）.The method of detecting the operation and the method of judging the results are strictly in accordance with the instructions of the analyzer and the kit.Among them,each group of antibodies was tested with a negative result and a positive result control group.The absorbance value of the microplate well under the wavelength of 450 nm was higher than the standard value,or the negative test result was 2.1 times the standard value.The test results were positive.Sequencing of the lon PGMTM sequencing system:Collecting vaginal secretions from patients and healthy childbearing control subjects to extract DNA from feces or vaginal secretions.Take appropriate samples and centrifuge tubes and dilute the sample to 1 ng/ul using sterile water.PCR amplification,detection and purification were performed.The samples were mixed after purification.The library was constructed and the samples were sequenced and analyzed.4.Statistical analysisSPSS21.0 software was used for statistical analysis.The count data are represented by numbers or percentage,chi-square test is used to compare differences between groups,P<0.05 is statistically different.Pearson correlation analysis was used for Correlation.the Pearson value of>0 represents a positive correlation,Pearson value<0 represents a negative correlation.P<0.05 indicates significant correlation.Results:1 Lon PGMTM Sequencing:The Chlamydia trachomatis library was diluted20-fold and the fragment size was 370 bp with a concentration of 253.40 pg/ul.The ureaplasma urealyticum library was diluted 50-fold and the fragment size was 429 bp with a concentration of 97.9 pg/ul.Chlamydia trachomatis mate-pair library fragment size is 299bp and the concentration is 48.57pg/ul.The ureaplasma urealyticum mate-pair library fragment size was 299bp and the concentration was 28.10pg/ul.The strains were sequenced to generate 993Mbp data from Chlamydia trachomatis,a total of 4737434 reads,and the average fragment length was 210 bp.Ureaplasma urealyticum produced642Mbp data,a total of 3461853 reads,and the average fragment length was186bp.Comparing the whole genome with the genes of Chlamydia trachomatis and Ureaplasma urealyticum,the results were consistent.2.Real-time PCR results:1)The number of UU-positive cases in 50 infertility patients was 19cases,the positive rate was 38%,and the number of CT-positive cases was 17cases,the positive rate was 34%.50 cases of normal control group were detected,including 5 cases of UU positive,10%positive,4 cases of CT positive,and 8%positive.The statistical analysis of the UU and CT positive rates in the two groups were all P<0.005,and the difference was statistically significant.The positive rate of UU and CT in the infertile group was significantly higher than that of the control group.2)UU and CT after quantification in infertility group were 5.78±1.01after quantification and 3.01±1.13 in control group（P<0.05）.There was a statistically significant difference between the two groups in the mean value of quantitative data,and the UU quantitative data in infertility group were significant.Higher than the control group.Results and analysis of CT quantitative data showed that CT in the infertile group was 5.39±0.93,while in the control group it was 2.76±0.69,t=1.25,P<0.05.There was a statistically significant difference between the two groups.3)The correlation coefficients between UU,CT and infertility were:0.251,0.604（P<0.05）.There is a correlation between UU and CT and infertility.3 Detection of antinuclear antibodie,antisperm antibodie in serum:1)The positive rates of antinuclear antibody,antisperm antibody in infertility group were 40%and 44%respectively.The positive rate in the control group was 6%and 8%respectively,and there was a significant difference between the control group and infertile patients（P<0.01）.The content of two antibodies in blood samples of infertile patients was significantly higher than that of normal controls.2)The level of antibody in blood.The contents of antinuclear antibodie,antisperm antibodie in serum of infertile patients were 2.36±0.23 and2.89±0.34 respectively.It is 1.08±0.35 and 1.35±0.19 in the control group.Serum antibody levels in the infertile group were higher than those in the control group（P<0.05）.3)The correlation coefficients between anti-nuclear antibody,anti-sperm antibody of infertility were 0.451,0.701 respectively.The occurrence of infertility was related to anti-nuclear antibody and anti-sperm antibody in the serum of patients（P<0.05）.Conclusion:1.Vaginal infection with chlamydia trachomatis and ureaplasma urealyticum may be the cause of infertility.2.The body’s own immune factors,such as antinuclear antibodie,antisperm antibodie are also factors causing infertility.3.Vaginal infection and autoimmune factors work together to increase the risk of infertility.4.Test of Vaginal infection factors and the immune factors at the same time can more accurately determine the occurrence of infertility. |
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