| Objective: Fluorescence in situ hybridization technology(fluorescence in situ hybridization, FISH) were used to detect 13, 16, 18, 21, 22, X and Y chromosome abnomality in 100 cases fetal tissues of spontaneous abortion from our college of obstetrics and gynecology clinic, including chromosome probe for CSP18 / CSPX/CSPY group, GLP13 / GLPZI group, GLP16 / GLP22 group. To explore chromosome aneuploidy incidence and abnormal types of spontaneous abortion fetal tissues; to compare the chromosome abnormality in the diffrent pregnancy abortion period; to explore the relationship between history of spontaneous abortion, age and incidence of abnormal chromosome. This study is of great significance for clear spontaneous abortion reasons, and provide genetic counseling data for the next pregnancy.Methods:1 B ultrasonic diagnosis of spontaneous abortion or stillbirthResearch object's main symptoms were vaginal bleeding, cervical pain, uterine tenderness, accessories area tenderness, lower abdomen ache or acute lower quadrant abdominal pain with syncope shock, etc. The ALOKA ECHO GE-E8 type ultrasonic diagnostic instrument was used to detect uterine size, outline, intrauterine, accessories area, pelvic cavity, and so on. Determine whether it was abnormal pregnancy according to the gestational sac implantation site, size, shape, embryo bud, heart beat, etc.2 Cleaning up the uterus by B ultrasonic monitoring and curettageAbortion: intravenous anesthesia was done using fentanyl combined propofol, using conventional mask to provide oxygen and monitoring of blood pressure, blood oxygen saturation, heart rate and breathing during surgen..Then surgical instrument direction and depth were closely observed and guided with longitudinal section of the uterus, the whole process of operation was dynamically observed by screen.Curettage: the same anesthesia and monitoring as abortion. Misoprostol 200 mg should be administered in vagina of Patient 3 hours before surgery. By monitoring the whole surgery process, the surgen can clear all pregnant tissues.Dead fetus abortion: a: ethacridine Injection into uterine cavity : the patient lie in supine position, locating by ultra-sound, inserting lumbar spinal needle into amniotic cavity vertically. Inject 50-100 mg ethacridine into amniotic cavity and pull out the needle. b. Misoprostol abortion: use Misoprostol25-50 ug at posterior fomix. Repeat every 3-4 hours according to the uterine contraction, less than 150 ug inside 24 hours. Observe the stage of labor, check carefully the placenta, umbilical cord and fetus.3 Samples processingVillis were choosed as experimental samples in early abortion cases and muscle groups preferred in stillbirth cases. Under aseptic conditions, decidual tissues were removed with scissors and tweezers. Villis or muscle tissue were selected about 0.5 cm x 0.5 cm size and cut up as far as possible. Avoid maternal cells pollution, the specimen not be able to cut up would removed. Fresh samples were put and washed with 0.9% saline solution, and saved no more than four days in 4?, while fixed fluid samples were saved no more than a month in-20?.4 Slides preparationProcessed samples were selected about 5 mg, 37?0.075 M KCL solution were added, then in low permeability for 15 min at 37?. Gently blowing suspension cells 2-3 times, and abandoning the supernatant; 2 ml fixed liquid(methanol: ice acetic acid=3:1) were slowly added and fixed in advance. Abandoning supernatant after the slow blowing; 5 ml liquid was added for repeated fixed, abandoning supernatant; 1 ml glacial acetic acid was added and digested about 1 min, make individual cells never cut up in the organization, then 3 ml methanol was added to termination of digestion, abandoning supernatant; Cell suspension liquid was made according to the amount of cells, suitable amount of fixed liquid was drop for FISH hybridization.5 FISH detectionFISH probe is provided by Beijing golden pu jia company, instructions were according to the fluorescence in situ hybridization kit provided. Preparation slides were rinsed in the 2xSSC(pH7.0) solution, digested in 0.08 mg/ml pepsin, dehydrated and degenerated in ethanol, hybridizated in 42?incubator for 16 h. slides was washed, redyed with DAPI, and then examined using ECLIPSE801 type fluorescent microscope, imaged by JENOPTIK(ProgRes MFcool) cold CCD acquisition, hybridization signal were analysed by VideoTest- FISH2.0 software.6 Result interpretationIn normal nuclei, for CSP18 / CSPX/CSPY probe group, one red signal, one green and two blue signal(men), or two blue, two green signal(female) were shown; for GLP16 / GLP22 probe group, two red and two green signals; for GLP13 / GLP21 probe group, two red and two green signals. At least 100 cells were counted in each probe group, and each index were individual judged. If normal cells ratio were > 90%, abnormal cells ratio < 10%, the index was normal; If abnormal cells ratio > 60%, the index was abnormal; If some abnormal cells ratio between 10% to 60%, the prompt was chimeras, the number of cells were increased for observation, 100 cells were counted again, and calculated abnormal rate.7 Statistical analysisStatistical analysis was done by SPSS 18.0 software, measurement data was shown using the mean and standard deviation(? ?), group comparison was matched using t-test, count data was tested by chi-square test, the difference was statistically significant by P < 0.05.Results:1 Case diagnosis and results of surveyBy ultrasound diagnosis and monitoring, spontaneous abortion samples group of 100 patients were are obtained, which were all singletons and intrauterine pregnancy. Muscle and villis samples were separatively 96 and 4 cases, mean age 28.24±5.24, mean gestational age 11.24±4.06, the previous spontaneous abortion times 1.74±0.96.2 FISH detection cases of chromosomal abnormalities100 cases of chromosomal abnormalities occurred in spontaneous miscarriage or stillbirth were analyzed by FISH. The success detection rate is 100%, the abnormal rate 45%, including 45 positive cases and 55 negative cases.3 Chromosomal abnormalities type45 cases of chromosome abnormality are mainly 16 trisomy(9 cases, 20.00%), 22 trisomy(4 cases, 8.89%), 13 trisomy(4 cases, 8.89%), XO(8 cases, 17.78%) and triploid XXY(8 cases, 17.78%).4 Chromosome abnormal results in different period for spontaneous miscarriage or stillbirth100 abortion embryonic tissue cases are divided into the first, middle and last three months groups of pregnancy. There are 68 cases, 28 cases and 4 cases in the first, middle and last three months of pregnancy respectively, and chromosome aneuploidy rate are 60%, 37.78 and 2.22% respectively. There are no statistically significant difference about incidence of chromosomal abnormalities among the three groups(P > 0.05).5 Relationship between the incidence of abnormal chromosome and history of spontaneous abortion100 abortion embryonic tissue cases are divided into 3 groups according to the times of pregnant abortion, 58 cases with no history of spontaneous abortion, 21 cases one times, 21 cases two or more spontaneous abortion times respectively. Chromosome abnormality cases are 22, 11 and 11 cases, incidence of chromosomal abnormalities are 37.93%, 57.14% and 57.14% respectively. Tthere was no statistically significant difference about incidence of chromosomal abnormalities among the three groups(P > 0.05).6 Relationship between the incidence of abnormal chromosome and age100 abortion embryonic tissue cases are divided into 2 groups according to the age. 86 cases are < 35 years old, 14 cases > 35 years old, 35 and 10 cases are in two groups of chromosomal abnormalities, and incidence of chromosomal abnormalities were 40.70%, 71.43% respectively. Incidence of chromosomal abnormalities differences between groups was statistically significant(P < 0.05).Conclusion:1 All samples of 100 cases were detected successfully by FISH, 45 cases of abnormal chromosome was detected and abnormality rate was 45%, abnormal types mainly for 16 trisomy(20.00%), XO(17.78%) and XXY(17.78%).2 Incidence of natural miscarriage or stillbirth during pregnancy at first, middle, and last three months are 60%, 37.78% and 60% respectively. Chromosome abnormality rate between groups was no statistical significance differences(P > 0.05).3 Chromosome abnormality rate among groups at different pregnant abortion times(0, 1 and 2 times or higher) was no statistical significance differences(P > 0.05).4 Chromosome abnormality rate in different age groups(< 35 years old and > 35 years old) was statistically significant differences(P < 0.05).5 Abnormal chromosome number is one of the important factors that lead to abortion. FISH technique is a rapid accurate molecular diagnostic techniques, and clear on the causes of spontaneous abortion, diagnosis, and has important application value for the again pregnancy instruction. |
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