Rapid clearance of fetal DNA from maternal plasma.

Fetal DNA has been detected in maternal plasma during pregnancy. We investigated the clearance of circulating fetal DNA after delivery, using quantitative PCR analysis of the sex-determining region Y gene as a marker for male fetuses. We analyzed plasma samples from 12 women 1-42 d after delivery of male babies and found that circulating fetal DNA was undetectable by day 1 after delivery. To obtain a higher time-resolution picture of fetal DNA clearance, we performed serial sampling of eight women, which indicated that most women (seven) had undetectable levels of circulating fetal DNA by 2 h postpartum. The mean half-life for circulating fetal DNA was 16.3 min (range 4-30 min). Plasma nucleases were found to account for only part of the clearance of plasma fetal DNA. The rapid turnover of circulating DNA suggests that plasma DNA analysis may be less susceptible to false-positive results, which result from carryover from previous pregnancies, than is the detection of fetal cells in maternal blood; also, rapid turnover may be useful for the monitoring of feto-maternal events with rapid dynamics. These results also may have implications for the study of other types of nonhost DNA in plasma, such as circulating tumor-derived and graft-derived DNA in oncology and transplant patients, respectively.     

Detection of Y STR markers of male fetal dna in maternal circulation

BACKGROUND:

Circulating fetal cells and cell free DNA in the maternal blood has been shown to help in prenatal diagnosis of genetic disorders without relying on invasive procedures leading to significant risk of pregnancy loss.

AIM:

The current study was undertaken to detect the male fetal population using Y STR markers DYS 19, DYS 385 and DYS 392 and also to study the extent of persistence of fetal DNA in the mother following delivery.

MATERIALS AND METHODS:

Blinded study was conducted on 50 mothers delivering male and female babies. Cellular and cell free DNA was extracted from maternal and fetal cord blood and amplified for Y STR markers by PCR.

RESULTS:

The amplification sensitivity of Y specific STR, DYS19 was 100% (22/22) in the male fetal DNA samples. The incidence of other STRs, i.e., DYS385 and DYS392 were 91% (20/22) each. Analysis of results revealed that thirteen of the twenty six women had detectable male fetal DNA at the time of delivery. However fetal DNA was not detectable twenty four hours after delivery.

CONCLUSION:

Preliminary results show that the separation of fetal cell-free DNA in the maternal circulation is a good low-cost approach for the future development of novel strategies to provide non-invasive techniques for early prenatal diagnosis.     

Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer

The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new opportunities for noninvasive prenatal diagnosis (NIPD). However, the extremely low levels of cfDNA within a high background of the maternal DNA in maternal circulation necessitate highly sensitive molecular techniques for its reliable use in NIPD. In this proof of principle study, we evaluated the earliest possible detection of cfDNA in the maternal plasma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, amplification, magnetics). Blood samples were collected from in vitro fertilization (IVF) patients at 2 to 6 weeks following embryo transfer (i.e., 4 to 8 week pregnancies) and plasma DNA was extracted. The genomic regions of both X and Y chromosome-specific sequences (AMELX and AMELY) were concurrently amplified in two sequential PCRs; first by conventional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene sequences were counted by a flow cytometer. Our results showed that the pregnancies yielding boys had significantly higher plasma AMELY gene fractions (0.512 ± 0.221) than the ones yielding girls (0.028 ± 0.003) or non-pregnant women (0.020 ± 0.005, P= 0.0059). Here, we clearly demonstrated that the BEAMing technique is capable of reliably detecting cfDNA in the blood circulation of 4-week-pregnant women, which is only two weeks after the embryo transfer. BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders.     

Evaluation of bidirectional transfer of plasma DNA through placenta

To clarify the origin of cell-free fetal DNA in maternal plasma, we analyzed bidirectional transfer of plasma DNA between fetus and mother. We analyzed maternal and fetal plasma DNA obtained from 15 pregnant women at the time of Cesarean section. The subjects were five patients with preeclampsia and 10 gestational-age-matched normal controls. DNA was extracted from 1.5-ml plasma samples and the cellular fraction of maternal and umbilical blood. Seven polymorphic marker genes were analyzed. The relative concentration of fetal DNA in maternal plasma and maternal DNA in cord blood were evaluated. The relative concentration of maternal DNA in fetal circulation (median, 0.9%; range, 0.2–8.4%) was significantly lower than that of fetal DNA in maternal blood (14.3%, 2.3–64%), with P=0.007. The relative concentration of maternal DNA in fetal blood was not affected by preeclampsia. These findings indicate that cell-free DNA is unequally transferred through the placenta. The structural characteristics of the placenta suggest that the majority of cell-free fetal DNA in maternal plasma is derived from villous trophoblasts.     

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.     

Fetal cells in the maternal circulation during first trimester in pregnancies

To investigate the presence of fetal cells in the maternal circulation during early pregnancy, the polymerase chain reaction was used to test the presence of human Y chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood specimens from 19 women carrying male fetuses and 12 women carrying female fetuses. The presence of fetal cells was suggested as early as 6 weeks gestation in 1 of the 19 women bearing male fetuses. Fetal cells were present in the maternal circulation of 15 of the 19 women by 9 weeks gestation, and in only 1 of the 19 were fetal cells not detected until the 12th week after conception. These results suggest that identification of fetal cells in the maternal circulation is possible with a properly designed and executed polymerase chain reaction. However, there was considerable variation with respect to when these fetal cells first became detectable during pregnancy. These fetal cells are potentially a valuable source of material for biochemical and genetic studies of the fetuses.     

利用孕妇血浆DNA检测胎儿性别的研究

本文探讨应用孕妇血浆中游离DNA进行无创性产前性别诊断的可行性。用柱分离法提取73例孕妇血浆中DNA,用巢式PCR技术检测其胎儿SRY基因。 结果73位孕妇血浆DNA含量为0.0062~0.3399μg/μL。巢式PCR检测胎儿SRY基因的灵敏度为97.37%（37/38）,假阴性率2.86%(1/35),特异度85.71%（30/35）,假阳性率13.16%(5/38),总符合率91.78%（67/73）。采用孕妇血浆胎儿DNA和巢式PCR技术可以快速简便的进行无创性产前性别诊断,诊断结果的准确率为91.8%,对性连锁遗传病的预防具有重要意义。 Abstract:To investigate the feasibility and possibility of application of fetal DNA from maternal plasma for noninvasive prenatal diagnosis of fetal sex,plasma DNAs in blood samples of 73 pregnant women at the gestational period of 26 to 41 weeks were extracted by column separation and nested polymerase chain reaction were employed to amplify the SRY gene.A comparison was made between the amplification results and the real sex of the fetus after their delivery.The concordance rate of SRY gene amplification results of plasma free DNA with real fetal sex was 91.78% (67/73),the sensitivity rate was 97.37% (37/38),and the specific rate was 85.71% (30/35).The cell-free fetal DNA in maternal blood can be one of the valuable material sources for noninvasive prenatal diagnosis and the method of nested PCR could be useful for fetal sex determination.The specific rate of the test was 91.78%.It is of significance to prevent sex-linked inheritant diseases.     

Non-invasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in RhD-negative pregnancies.

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.     

Bovine fetal microchimerism in normal and embryo transfer pregnancies and its implications for biotechnology applications in cattle

Fetal cells and DNA have been detected in the maternal circulation during and after pregnancy in a few mammalian species. The incidence of similar microchimerism in cattle could have repercussion for the application of modern biotechnologies such as the transfer of transgenic embryos. To determine if feto-maternal leakage can occur in pregnant cows, we have analyzed maternal blood samples for the presence of fetal DNA during gestation and post-partum periods. Y chromosome-specific DNA was detected in up to 73% of blood samples from naturally mated heifers carrying conventional bull calves and a transgene-specific sequence in up to 50% of recipient cows carrying transgenic fetuses. These findings document for the first time that transplacental leakage of fetal DNA into the maternal circulation can occur in cattle despite the epitheliochorial placenta of ruminants, with potential implications for the utilization of recipient cows in the food chain.     

Fetal DNA in maternal serum: does it persist after pregnancy?

Fetal DNA and cells present in maternal blood have previously been used for non-invasive prenatal diagnosis. However, some fetal cells can persist in maternal blood after a previous pregnancy. Fetal rhesus status and sex determination have been performed by using amplification by real-time polymerase chain reaction (PCR) of fetal DNA sequences present in maternal circulation; no false-positive results related to persistent fetal DNA from a previous pregnancy have been reported. This idea has recently been challenged. An SRY real-time PCR assay was performed on the serum of 67 pregnant women carrying a female fetus but having previously given birth to at least one boy and on the serum of 30 healthy non-pregnant women with a past male pregnancy. In all cases, serum was negative for the SRY gene. These data suggest that fetal DNA from a previous pregnancy cannot be detected in maternal serum, even by using a highly sensitive technique. Therefore, non-invasive prenatal diagnosis by fetal sex determination for women at risk of producing children with X-linked disorders, and fetal RHD genotyping is reliable and secure as previously demonstrated.     

A noninvasive approach to studying fetal cells for prenatal diagnosis of chromosomal aneuploidies

Fetal cells isolated from maternal peripheral blood during the second trimester of pregnancy were analyzed. Blood samples were centrifuged in a Ficoll-Paque gradient, the mononuclear cell fraction was isolated and stained with fluorescent monoclonal antibodies against glycophorine A (GPA + PE), transferrin (CD71 + FITC), and Hoechst 33342. Fluorescence-activated cell sorting (FACS) was conducted on a Vantage flow cytofluorimeter (Becton Dickinson). Fluorescence in situ hybridization (FISH) with Y chromosome-specific DNA probe revealed fetal cells that exhibited Y signal in all 20 blood samples obtained from women pregnant with healthy male fetuses. The concentration of these fetal cells averaged about 1.34% and ranged from 0.1 to 4.2% in different blood samples. In six cases, blood samples were obtained from pregnant women, in which prenatal cytogenetic analysis revealed various fetal aneuploidies. Using FISH with DNA probes specific for chromosomes X, 18, and 13/21, Fetal cells with chromosomal aberrations were detected in these six maternal blood samples at a concentration from 1.5 to 5.6% (on average 3.7%). These results indicate the possibility of a new noninvasive approach, which is safe for both mother and fetus when used for isolation of fetal cells from pregnant women's blood samples and prenatal diagnosis of a broad spectrum of fetal cell chromosomal aberrations.     

Elevated Plasma Levels of Hypermethylated RASSF1A Gene Sequences in Pregnant Women with Intrahepatic Cholestasis

Intrahepatic cholestasis of pregnancy (ICP) is associated with increased perinatal mortality and morbidity. Circulating cell-free fetal DNA has been a useful parameter for monitoring of pregnancy-associated diseases. The purpose of this study was to determine the concentrations of hypermethylated RAS-association domain family 1, isoform A (RASSF1A) gene sequences in the plasma of pregnant women with intrahepatic cholestasis. This study included 56 women in their third trimester of pregnancy, of whom 26 had ICP (study group) and 30 were healthy (control group). Real time PCR was performed to detect RASSF1A concentrations after methylation-sensitive restriction digestion with HinpII and HhaI to measure cell-free fetal DNA. Beta-actin was detected as an internal control to confirm complete enzyme digestion. The data show a significant increase in the circulating hypermethylated RASSF1A levels regarding the pregnancies complicated with ICP as compared with normal pregnancies. Circulating hypermethylated RASSF1A levels in maternal plasma related to total bile acid. Based on these observations, we suggest that the circulating hypermethylated RASSF1A levels in maternal plasma may be used as a diagnostic marker for ICP.     

Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin

The nucleated erythrocyte (NRBC) is one of the target fetal cell types for noninvasive genetic diagnosis using maternal peripheral blood. However, it is now known that pregnancy can stimulate the production of maternal NRBCs. When isolating female gamma-positive NRBCs, fluorescence in situ hybridization (FISH) analysis may show two X chromosome signals per nucleus, and therefore it cannot be conclusively determined whether the isolated cells are fetal or maternal in origin. The purpose of this study was to develop a means of verifying that a female cell is fetal on the basis of polymorphic short tandem repeat markers. Peripheral blood samples were obtained from women who had just undergone termination of pregnancy. Nucleated candidate fetal cells were isolated by flow-sorting using antibody to the gamma-chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed using X and Y chromosome specific probes. Female gamma-positive cells and leukocytes were micromanipulated separately and subjected to fluorescent polymerase chain reaction amplification of chromosome 21 and/or 18 STR markers (D21S11, D21S1411, D21S1412, and D18S535). In all ten cases analyzed, the gamma-positive female candidate fetal cells were determined to be fetal in origin by the presence of shared and nonshared DNA polymorphisms when compared with maternal leukocytes. These results show that genetic analysis can be performed on all fetal NRBCs, including female fetal cells that cannot be distinguished from maternal cells based on FISH analysis alone.     

High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing

Background

Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine.

Methodology

We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine.

Principal Findings

Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length.

Conclusions

With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.     

Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18

Cell-free fetal DNA in the maternal circulation is a potential noninvasive marker for fetal aneuploidies. In previous studies with Y DNA as a fetal-specific marker, levels of circulating fetal DNA were shown to be elevated in women carrying trisomy 21 fetuses. The goal of this study was to determine whether cell-free fetal DNA levels in the serum of pregnant women carrying fetuses with trisomies 13 or 18 are also elevated. Archived maternal serum samples from five cases of male trisomy 13 and five cases of male trisomy 18 were studied. Each case was matched for fetal gender, gestational age, and duration of freezer storage to four or five control serum samples presumed to be euploid after newborn medical record review. Real-time quantitative polymerase chain reaction amplification of DYS1 was performed to measure the amount of male fetal DNA present. Unadjusted median serum fetal DNA concentrations were 97.5 GE/ml (genomic equivalents per milliliter; 29.2-187.0) for the trisomy 13 cases, 31.5 GE/ml (18.6-77.6) for the trisomy 18 cases, and 40.3 GE/ml (3.7-127.4) for the controls. Fetal DNA levels in trisomy 13 cases were significantly elevated ( P=0.016) by analysis of variance of the ranks of values within each matched set. In contrast, fetal DNA levels in trisomy 18 cases were no different from the controls ( P=0.244). Second trimester maternal serum analytes currently used in screening do not identify fetuses at high risk for trisomy 13. Fetal DNA may facilitate noninvasive screening for trisomy 13 provided that a gender-independent fetal DNA marker can be developed.     

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Analysis of cell-free fetal DNA in plasma and serum of pregnant women.

Sixty blood samples from pregnant women during gestational weeks 9-28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.     

Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age

To determine the frequency of fetal nucleated cells in maternal peripheral blood during different stages of pregnancy, 50 primigravidas were investigated by determining the frequency of cells with the Y chromosome using fluorescence in situ hybridization (FISH) of Y-specific repetitive sequences of the DYZ1 family. Polymerase chain reaction (PCR) amplifying the same part of the DYZ1 used as the probe in FISH and a single-copy Y-specific fragment was also carried out for genomic DNA from the same samples. Cells with the hybridization signal were detected by FISH at and after 15 weeks of pregnancy in all pregnant women who gave birth to boys. The ratio of cells with the signal to those without the signal ranged from 1 in 144,000 to 1 in 4,000 with a tendency to increase as the pregnancy advanced. The frequency of fetal cells estimated by the PCR experiments was significantly and positively correlated with that found by FISH. The present study suggests that fetal nucleated cells increase in maternal peripheral blood with advancing gestation, from less than 1 in 100,000 nucleated cells in the first trimester to around 1 in 10,000 at term. These frequencies were much lower than those reported by cytological methods.     

PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies.

Fetal cells in maternal blood are a noninvasive source of fetal genetic material for prenatal diagnosis. We determined the number of fetal-cell DNA equivalents present in maternal whole-blood samples to deduce whether this number is affected by fetal karyotype. Peripheral blood samples were obtained from 199 women carrying chromosomally normal fetuses and from 31 women with male aneuploid fetuses. Male fetal-cell DNA-equivalent quantitation was determined by PCR amplification of a Y chromosome-specific sequence and was compared with PCR product amplified from known concentrations of male DNA run simultaneously. The mean number of male fetal-cell DNA equivalents detected in 16-ml blood samples from 90 women bearing a 46,XY fetus was 19 (range 0-91). The mean number of male fetal-cell DNA equivalents detected in 109 women bearing a 46,XX fetus was 2 (range 0-24). The mean number of male fetal-cell DNA equivalents detected when the fetus was male compared with when the fetus was female was highly significant (P = .0001). More fetal cells were detected in maternal blood when the fetus was aneuploid. The mean number of male fetal-cell DNA equivalents detected when the fetal karyotype was 47,XY,+21 was 110 (range 0.1-650), which was significantly higher than the number of male fetal-cell DNA equivalents detected in 46,XY fetuses (P = .0001). Feto-maternal transfusion of nucleated cells appears to be influenced by fetal karyotype. The sixfold elevation of fetal cells observed in maternal blood when the fetus had trisomy 21 indicates that noninvasive cytogenetic diagnosis of trisomy 21 should be feasible.     

Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield

Objective

Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used.

Methods

Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples.

Results

Total cell-free DNA quantity increased significantly with time in samples stored in K₃EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4°C did not prevent these changes.

Conclusion

When samples can be processed within eight hours of blood draw, K₃EDTA tubes can be used. Prolonged transfer times in K₃EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.