Study On The Separation And Purification Of Maternal Peripheral Blood Trophoblast Cells For Prenatal Diagnosis

Background: With the gradual implementation of the national "two child" policy,the number of elderly mothers is increasing gradually.Moreover,the number of fetuses with "problems" will increase significantly due to the role of environment,lifestyle,genetic factors and other factors.Birth defects incidence rate,morbidity and mortality are relatively high.The birth defects in China are as high as 5.6%.According to the U.S.survey,every 33 babies have a birth defect.In 2013,the cost of treatment related to birth defects reached 22.9 billion US dollars,causing serious economic and social burden.Genetic diseases are the main cause of birth defects.At present,the prenatal diagnosis of genetic diseases can be divided into invasive prenatal diagnosis and noninvasive prenatal diagnosis according to whether the diagnostic methods have adverse effects on the fetus.Invasive prenatal diagnosis is harmful to the fetus and may lead to abortion.Prenatal diagnosis with maternal peripheral blood is non-invasive.It is currently used for the diagnosis of aneuploidy(such as trisomy 21,trisomy 18,trisomy 13 and chromosomal abnormalities)and monogenic genetic diseases.At present,the non-invasive prenatal testing(NIPT)or NIPT plus,which have been widely used in clinical practice,is based on the presence of fetal free DNA(cff DNA)in maternal peripheral blood.cff DNA in maternal peripheral blood is analyzed to reflect the situation of fetal chromosomes 13,18 and 21,mainly used for down’s syndrome screening,the disadvantage is that the technique relies on the measured chromosome values for conversion,which will produce certain errors.In addition,the time of cff DNA in maternal peripheral blood is late,and the content is quite different,in addition,the interference of maternal DNA cannot be completely excluded.A large number of studies have shown that the trophoblast cells in maternal peripheral blood are completely derived from the fetus.It has been reported that trophoblast cells exist in maternal blood at the 6th week of pregnancy(fertilization age)or even at the early stage of pregnancy,and the genetic material of trophoblast cells is completely consistent with that of fetus,so it is possible to separate trophoblast cells from maternal peripheral blood for the diagnosis of chromosomal diseases and monogenetic diseases.However,the prenatal diagnosis technology of peripheral blood trophoblast cells has not been developed.The purpose of this study is to isolate trophoblast cells from maternal peripheral blood and sequence them for analysis,and compare the analysis results with the conventional non-invasive prenatal diagnosis results,in order to develop the diagnostic technology of chromosomal diseases and single gene genetic diseases with higher diagnostic accuracy and shorter detection time,we hope to provide strong technical support for the national neonatal malformation screening,in order to reduce the birth of defective children and provide guarantee for human health.Objective: Study on the development of prenatal diagnosis technology by the separation and purification of trophoblast cells from maternal peripheral blood Method: On the basis of informed consent,From September 2017 to January 2019,we selected pregnant women who were 5-7 weeks pregnant in the outpatient department of Obstetrics and Gynecology of the First Affiliated Hospital of Dali University,and took 5ml of their peripheral blood into the EDTA anticoagulation tube,then,the blood cell components of pregnant women were removed by chemrus ? disposable filter funnel filtration,and the separated cell smear was stored on the microsection for future use.According to the same pregnant woman in 12-22 weeks of pregnancy to carry out NIPT detection and routine pregnancy examination,NIPT detection problems with amniocentesis diagnosis.If any one of the two screening methods is clear that the fetus has "problems",take the microsection made by the cells obtained from the same pregnant woman in the early pregnancy,and take the trophoblast cells from the fetus under the microdissector for single cell sequencing and analysis.At the same time,the whole genome of fetal umbilical vein blood was detected when the fetus was born.Results:(1)Separation and purification of trophoblast cells:The trophoblast cells were isolated and purified in 14 samples at 5-7 weeks of gestation.Then,the cells were identified.The number of trophoblast cells was 0-1 / HP.There were 8-10 trophoblast cells per 5ml of blood.(2)Routine non-invasive prenatal test results: The results of NIPT in cases 1,2,3,4,5,6 and 8 showed a low risk.Except for normal B-ultrasonic examination in case 2 and abnormal skin after birth,the other cases showed abnormal fetal development or deformity by B-color Doppler ultrasound.The results of NIPT in No.9 case showed that there was a micro duplication of chr1:200400000-207150000(1q32.1-q32.1,6.8m) with a size of 6.8m in fetal chromosome 1,and no abnormality was found in B-mode color Doppler ultrasound.The serological screening of case No.7 indicates the critical risk of Down’s syndrome,the NIPT results indicate the high risk of fetal chromosome 21 abnormality,and the color Doppler ultrasound in the outside hospital indicates the fetal malformation,but the color Doppler ultrasound in our hospital does not indicate the malformation.The normal control cases were screened by serology,NIPT test showed low risk,and no abnormality was found by color ultrasonography.(3)Traditional invasive examination results:Among the 9 patients with abnormal embryo performance,except for the patients with No.7 and No.9 who agreed to have amniocentesis,the other 7 patients refused to have amniocentesis.(4)Sequencing results:A total of 9 embryos with "abnormal" manifestations were collected.In addition,5 cases with normal NIPT and CDU were collected as control cases.The results of sequencing showed that the variation of chromosome level was detected in 9 patients with abnormal manifestations,which was consistent with the detection of umbilical vein blood.It is suggested that trophoblasts derived from maternal peripheral blood can be used to detect the abnormal development of embryo in early stage.Conclusion:The detection rate or detection range of trophoblast sequencing and whole genome sequencing were higher than that of NIPT or NIPT plus;The detection rate or detection range of trophoblast sequencing is basically the same as that of whole genome sequencing.However,due to the small sample size and short follow-up time,further study is needed to detect its reliability and accuracy.