The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing(NIPT).The fetal DNA molecules have significant clinical potential in determining the overall performance of NIP...The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing(NIPT).The fetal DNA molecules have significant clinical potential in determining the overall performance of NIPT and clinical interpretation.It is important to measure fetal DNA fraction before NIPT.However,there is still little research on how to calculate the concentration of female fetuses.Two estimation approaches were proposed to calculate fetal DNA fraction,including the fragments size-based approach,aneuploid-based approach,which are all approaches based on chromosome segments.Based on high-throughput sequencing data,two approaches to calculate the DNA fraction of male fetuses were tested and obtained the experiment values,which were close to the actual values.The correlation coefficient of fragments size-based approach was 0.9243(P<0.0001)and the aneuploid-based approach reached 0.9339(P<0.0001).We calculated the concentration of female fetuses and obtained remarkable experimental results.We came up with two approaches for calculating the fetal DNA fraction of female fetuses.It provides an important theoretical basis for the detection of female fetal concentration in future clinical diagnosis.展开更多
To investigate the feasibility of using free fetal DNA from maternal plasma as the source of fetal material in non invasive prenatal diagnosis, SRY gene of free DNA in maternal blood of 65 samples were analyzed by us...To investigate the feasibility of using free fetal DNA from maternal plasma as the source of fetal material in non invasive prenatal diagnosis, SRY gene of free DNA in maternal blood of 65 samples were analyzed by using primer extension preamplication (PEP) and probe microplate hybridization techniques. The results showed that the detection rate of SRY gene in maternal blood from women carrying male fetuses detected by probe microplate hybridization alone and probe microplate hybridization with PEP were 76.09 % (35/46) and 95.65 % (44/46) respectively, and there was a significant difference between them. The non detection rate of SRY gene in blood samples from women carrying female fetus was 100 % (19/19). It is indicated that probe microplate hybridization was an effective method in detecting trace fetal DNA from maternal plasma and the sensitivity could be substantially improved by combined use of the two techniques. Analysis of fetal DNA in maternal plasma can serve as an alternative for non invasive prenatal diagnosis.展开更多
To explore the application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, the DNA template was extracted by hydroxybenzene chloroform from 44 maternal (7-41 weeks) plasma. The Fetus derived Y se...To explore the application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, the DNA template was extracted by hydroxybenzene chloroform from 44 maternal (7-41 weeks) plasma. The Fetus derived Y sequence DYZ 1 gene (149bp) was chosen to be amplified by PCR. The fragment was identified in all the plasma of male bearing pregnant women with the diagnostic accordance rate being 100.00 %. Two of the 22 female bearing pregnant women had false positive results. Among the 44 pregnant women, the diagnostic accordance rate was 88.89 % at early pregnant stage, 100.00 % at medium pregnant stage, and 96.55 % at late stage respectively. The final accuracy of 95.45 % was obtained in all cases. It was concluded that by means of hydroxybenzene chloroform extraction the authors of this article promoted the concentration and purity of the DNA template, and diagnosed more accurately. The results showed that free fetal DNA in the maternal plasma could be regarded as the gene resource for noninvasive prenatal diagnosis.展开更多
In order to search for a more reliable method of sorting fetal nucleated red blood cells (NRBCs) and DNA from maternal peripheral blood and to identify origin of NRBCs and DNA, NRBCs were isolated from peripheral bloo...In order to search for a more reliable method of sorting fetal nucleated red blood cells (NRBCs) and DNA from maternal peripheral blood and to identify origin of NRBCs and DNA, NRBCs were isolated from peripheral blood of 88 pregnant women by density gradient centrifugation and fluorescence activated cell sorter (FACS) respectively. Nested polymerase chain reaction was used to detect normal male SRY gene from blood plasma DNA of 65 pregnant women. The results revealed that fetal NRBCs were found in 14 of 27 maternal samples by density gradient centrifugation. The number of cells was from 1 to 10. Using FACS, CD71 + cells were identified among all 61 samples. The frequency was (0.35±0.25)×10 -2; The detectable rate of the SRY gene of blood plasma DNA from 46 women carrying male fetuses was 65.22 % (30/46). Non-detectable rate for 19 women carrying female fetuses was 94.74 % (18/19). It was concluded that the methods of sorting fetal NRBCs and DNA have already made great progress. The methods for fetal NRBCs and plasma DNA from maternal peripheral blood to diagnose genetic diseases seem to be the best methods of noninvasive prenatal diagnosis.展开更多
Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also ...Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.展开更多
The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for noninvasive diagnosis. By RT-PCR, circulating fetal DNA can be detected in the plasma of pregnant women, even in the ...The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for noninvasive diagnosis. By RT-PCR, circulating fetal DNA can be detected in the plasma of pregnant women, even in the first trimester of pregnancy, and thus can be used for noninvasive prenatal diagnosis of sex-linked disorders, the RhD status of fetuses, and single gene disorders such as beta-thalassaemia, congenital adrenal hyperplasia, and achondroplasia. In addition, quantitative aberrations of circulating fetal DNA may indicate various pregnancy-associated disorders, including preeclampsia, preterm labor and fetal trisomy 21.展开更多
Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simp...Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction （QF-PCR） to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat （STR） fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93％ （26/28; confidence interval,CI：77％-98％） and the specificity was 100％ （26/26; CI：88％-100％）.The determination rate of the origin of the extra chromosome was 69％.The sensitivity and the specificity of the assay in the euploid were 100％ （27/27）.Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.展开更多
基金supported by the National Key Research and Development Program of China 2016YFC1000307The sub-project of National Key Research and Development Program of China 2016YFC1000307-10the Program of National Research Institute for Family Planning(2017GJM04,2018CNV).
文摘The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing(NIPT).The fetal DNA molecules have significant clinical potential in determining the overall performance of NIPT and clinical interpretation.It is important to measure fetal DNA fraction before NIPT.However,there is still little research on how to calculate the concentration of female fetuses.Two estimation approaches were proposed to calculate fetal DNA fraction,including the fragments size-based approach,aneuploid-based approach,which are all approaches based on chromosome segments.Based on high-throughput sequencing data,two approaches to calculate the DNA fraction of male fetuses were tested and obtained the experiment values,which were close to the actual values.The correlation coefficient of fragments size-based approach was 0.9243(P<0.0001)and the aneuploid-based approach reached 0.9339(P<0.0001).We calculated the concentration of female fetuses and obtained remarkable experimental results.We came up with two approaches for calculating the fetal DNA fraction of female fetuses.It provides an important theoretical basis for the detection of female fetal concentration in future clinical diagnosis.
基金This project was supported by a grant from ScienceFoundation of Health Ministry(No.96 .2 - 112 ) and HubeiProvincial Natural Science Foundation(No.96 J0 6 8)
文摘To investigate the feasibility of using free fetal DNA from maternal plasma as the source of fetal material in non invasive prenatal diagnosis, SRY gene of free DNA in maternal blood of 65 samples were analyzed by using primer extension preamplication (PEP) and probe microplate hybridization techniques. The results showed that the detection rate of SRY gene in maternal blood from women carrying male fetuses detected by probe microplate hybridization alone and probe microplate hybridization with PEP were 76.09 % (35/46) and 95.65 % (44/46) respectively, and there was a significant difference between them. The non detection rate of SRY gene in blood samples from women carrying female fetus was 100 % (19/19). It is indicated that probe microplate hybridization was an effective method in detecting trace fetal DNA from maternal plasma and the sensitivity could be substantially improved by combined use of the two techniques. Analysis of fetal DNA in maternal plasma can serve as an alternative for non invasive prenatal diagnosis.
文摘To explore the application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, the DNA template was extracted by hydroxybenzene chloroform from 44 maternal (7-41 weeks) plasma. The Fetus derived Y sequence DYZ 1 gene (149bp) was chosen to be amplified by PCR. The fragment was identified in all the plasma of male bearing pregnant women with the diagnostic accordance rate being 100.00 %. Two of the 22 female bearing pregnant women had false positive results. Among the 44 pregnant women, the diagnostic accordance rate was 88.89 % at early pregnant stage, 100.00 % at medium pregnant stage, and 96.55 % at late stage respectively. The final accuracy of 95.45 % was obtained in all cases. It was concluded that by means of hydroxybenzene chloroform extraction the authors of this article promoted the concentration and purity of the DNA template, and diagnosed more accurately. The results showed that free fetal DNA in the maternal plasma could be regarded as the gene resource for noninvasive prenatal diagnosis.
文摘In order to search for a more reliable method of sorting fetal nucleated red blood cells (NRBCs) and DNA from maternal peripheral blood and to identify origin of NRBCs and DNA, NRBCs were isolated from peripheral blood of 88 pregnant women by density gradient centrifugation and fluorescence activated cell sorter (FACS) respectively. Nested polymerase chain reaction was used to detect normal male SRY gene from blood plasma DNA of 65 pregnant women. The results revealed that fetal NRBCs were found in 14 of 27 maternal samples by density gradient centrifugation. The number of cells was from 1 to 10. Using FACS, CD71 + cells were identified among all 61 samples. The frequency was (0.35±0.25)×10 -2; The detectable rate of the SRY gene of blood plasma DNA from 46 women carrying male fetuses was 65.22 % (30/46). Non-detectable rate for 19 women carrying female fetuses was 94.74 % (18/19). It was concluded that the methods of sorting fetal NRBCs and DNA have already made great progress. The methods for fetal NRBCs and plasma DNA from maternal peripheral blood to diagnose genetic diseases seem to be the best methods of noninvasive prenatal diagnosis.
文摘Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.
文摘The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for noninvasive diagnosis. By RT-PCR, circulating fetal DNA can be detected in the plasma of pregnant women, even in the first trimester of pregnancy, and thus can be used for noninvasive prenatal diagnosis of sex-linked disorders, the RhD status of fetuses, and single gene disorders such as beta-thalassaemia, congenital adrenal hyperplasia, and achondroplasia. In addition, quantitative aberrations of circulating fetal DNA may indicate various pregnancy-associated disorders, including preeclampsia, preterm labor and fetal trisomy 21.
文摘Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction （QF-PCR） to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat （STR） fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93％ （26/28; confidence interval,CI：77％-98％） and the specificity was 100％ （26/26; CI：88％-100％）.The determination rate of the origin of the extra chromosome was 69％.The sensitivity and the specificity of the assay in the euploid were 100％ （27/27）.Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.
