The Study Of Rapid Prenatal Diagnosis Using Transcervical Cells In First Trimester

Part I The Study of Methods for Fetus Trophoblastic Cell to Obtain through the Cervix in the First TrimesterObjective: To selecting the appropriate methods and applied to the experiments of prenatal diagnosis through the comparison of several methods for obtaining fetus trophoblastic cells through the cervix in the first trimester.Methods: Sixty pregnant women who want to request termination of pregnancy were recruited to the study.After informed these pregnant women,transcervical cell samples were collected from women whose fetuses were between 6 and 10 weeks'gestation. These women were divided into three groups on random,20 cases in one group: GroupA using cervical canal cotton swab, GroupB using endocervical mucus aspiration and Group C using cytobrush sampling of the endocervix. As the control group,cell suspesion were collected from 20 non-pregnant women by uterine lavage. These samples were stained by HE and immunocytochemistry, then were observed under the microscope to watch the presence of trophoblast cells and to count the cells. We used monoclonal antibodies cytokeratin -7 (CK-7) which is a specific maker on the trophoblast cells to count the positive expression cells under the microscope.Results: 1.Method of HE stained:All the three groups of the collected specimens have trophoblast cells existed in group A, group B and group C.The positive rates of the three kinds of methods were 40% (8 cases), 55% (11 cases) and 90% (18 cases). There was no trophoblast cells were observed in control group.The frequency of trophoblast cells in group A and B have no statistical differences ( p>0.05). The frequency in group C is more than group A and B , they have the statistical differences (p<0.01, p<0.01).2. Method of immunocytochemical stained: The positive rates of the three kinds of methods were 10% (2 cases), 25% (5 cases) and 80% (16 cases). There were no trophoblast cells were observed in control. The frequency of trophoblast cells in group A and B have no statistical differences ( p>0.05). The frequency in group C is more than group A and B , they have the statistical differences (p<0.01,p<0.01).3. The comparison of the amout of trophoblast cells by the three kinds of methods.(1). HE stained: The method of cytobrush get the largest quantity number of cells. There were no significant difference observed compared with the amout between the two groups of cervical canal cotton swab and endocervical mucus aspiration (P=0.605). The amout of the cytobrushe group compared with the other two groups were statistically significant (P=0.002,p=0.002).(2). Immunocytochemical stained: The method of brush get the largest quantity number of positive cells. There were only two cases in the swab group, so it can not compare with others. The amout positive cells of the cytobrush group compared with the mucus aspiration group were statistically significant (P=0.000). 4. The number of the collected trophoblast cells have a certain relationship with gestational age. In this study, we observed that with the gestational age increased, the number of trophoblast cells obtained greater from 7 week to 10 week of gestation (r=0.548,p=0.005). 5. The number of pregnant history and blood contamination have a certain degree of relationship with the positive rate of trophoblast cells (p = 0.033, p = 0.048), and maternal age and previous deliveries had no correlation with it(p=0.122,p=0.228).Conclusions: 1.It is feasible to obtain the trophoblastic cells through the cervix in the first-trimester pregnancy.2.The method of cytobrush sampling of the endocervix can obtain larger cells and with higher success rate. 3.It is effective to retrieve transcervical trophoblastic cells after 7 week of gestation. Part II The Application of Fluorescence in situ Hybridization in Rapid Detection for Cervical TrophoblastObjective: To investigate the application of fluorescence in situ hybridization (FISH) to the rapid detection of fetal chromosomes, and to explore its sensitivity as well as specificity in the early diagnosis for transcervical trophoblast cells , in order to get an evaluation of its further application.Methods: Transcervical trophoblastic cells through the enrichment and purification were tested by FISH,as the experimental group;at the same time, the corresponding villis were obtained under sterile conditions as the control group,then were tested by routine karyotype analysis.(1) 20 cases pregnant women who want to request termination of pregnancy were recruited to the study.After informed these pregnant women,transcervical cell samples were collected by cytobrush sampling from women whose fetuses were between 8 and 10 weeks'gestation. After the exclusion of semen contamination,the specimens were cultured in short-term by the method of differential adhesion as much as possible to purify and enrich the cells,then were identified by CK-7 and were counted the positive-stained cells per section on the slide.The specimens of experimental group went through a series of FISH procedures including preprocessing, degeneration of cells, degeneration of probes, hybridization and postprocessing, till the final step that the results were observed under the fluorescence microscopy; Whereas control group was taken into ordinary routine examine including cell incubation, screening and karyotype analysis of chromosomes.(2) Results from two groups were compared in both identity and difference. Furthermore, the strengths and limitations of each method were analyzed according to the rapidity, safety, accuracy as well as specificity. This is of key importance in guiding the clinical practice as well as choosing the appropriate method to those women who are in urgent need of getting rapid and early diagnosis.(3) The fluorescence probes used in our research were simultaneously tested in several characteristics in terms of requirements of laboratory facilitates, accuracy and stability, etc.Results:(1)These specimens were cultured in short-term by the method of differential adhesion,18 out of 20 from experiment group were cultured successful.The cells cultured were counted under microscope after identified by immunocytochemistry. The number of positive-stained cells reached 103/ml-104/ml.Ten of cultured specimens were tested by FISH.They were all well-hybridized with respective nucleus in dark background, as well as distinct fluorescence signals manifestation under fluorescence microscopy.The results of FISH were six cases of male fetus and four cases of female fetus,the number of 13/18/21 fetal chromosomes were normal.The results of control group were seven cases of male fetus and three cases of female fetus,the number of 13/18/21 fetal chromosomes were normal.(2)The coincident rate of experimental group and control group results was 90%.Every specimen costed 24h in average,the number of cells were limited in spite of culured for 4 days. Through the differential adhesion method we can remove some of maternal cells as much as possible, enriched within a relatively short period of time, so the number of trophoblast cells was suitable for the purpose of prenatal diagnosis.(3) Under the process of testing by domestic probe,we modify some steps to delete the appliance of toxiferous reagent,the results of experiment were also good.Conclusions:(1)The transcervical specimens collected by using cytobrush can be enriched and purified by the method of differential adhesion.(2)Fluorescent in situ hybridization using cultured trophoblast is fast and accurate in diagnosis of number of fetal chromosomes, thus has potential significance in future clinical application. (3) The clinical sampling methods and easy-derived enrichment method are still need to improve continuously in the first trimester. In this way it can build up the foundation for future application.