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.     

Evaluation of a Novel Assay for Detection of the Fetal Marker RASSF1A: Facilitating Improved Diagnostic Reliability of Noninvasive Prenatal Diagnosis

Background

Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results.

Methods

cfDNA was extracted from maternal plasma (n = 90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A, SRY and DYS14 was performed by real-time PCR.

Results

Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY.

Conclusion

Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.     

Quantitation of fetal DNA in maternal serum in normal and aneuploid pregnancies

We investigated whether the amount of circulating cell-free fetal DNA in maternal serum is influenced by fetal karyotype, using real-time quantitative polymerase chain reaction assay. Serum samples were obtained from pregnant women at gestational ages ranging from 15 to 17 weeks, prior to their undergoing amniocentesis. In total, we examined 70 samples consisting of 55 cases of pregnancy with 46,XY, 5 cases with 47,XY,+21, 3 cases with 47,XY,+18, a single case with 46,XY,dup(1) and 2 cases with twins of 46,XY, and 4 cases with 46,XX which were used as negative controls. We measured the concentration of the SRY sequence as a molecular marker for fetal DNA. The SRY sequence was detectable and measurable when the fetuses were male except for one case with 47,XY,+18. This case showed fetal growth retardation and bradycardia. No amplification signals of the SRY sequence were detected when the fetuses were female. The mean concentration of fetal DNA in maternal serum was 31.5 copies/ml in the pregnancy with 46,XY, 23.5 copies/ml in the pregnancies with 47,XY,+21 and 21.5 copies/ml in the pregnancies with 46,XY,+18. There were no significant differences in the concentration of fetal DNA between pregnancies with fetuses of normal karyotype and those with fetuses of abnormal karyotype.     

Presence of fetal DNA in maternal plasma decades after pregnancy

Cells of fetal origin and cell-free fetal DNA can be detected in the maternal circulation during pregnancy, and it has recently been shown that fetal cells can persist long after delivery. Given the various biological and clinical implications of this fact, we tested the hypothesis that cell-free fetal DNA can be present in maternal plasma decades after pregnancy. We extracted DNA from plasma samples and nucleated blood cells of 160 healthy women with male offspring at different time intervals after delivery (range 1-60 years). All of the samples were tested by means of a real-time quantitative PCR assay for a specific Y chromosome sequence (the SRY gene). Y chromosome-specific DNA was detected in 16 peripheral blood cell samples (10%) and 35 plasma samples (22%). The women with male sequences in the cell fraction had significantly greater total parity ( P=0.018). The proportion of women with detectable Y sequences in the plasma or cell samples was not related to the time since delivery. The fetal DNA concentrations in the genomic material extracted from plasma samples were significantly higher than those extracted from the Y-positive cell samples (149+/-140 vs 20+/-13 genome-equivalents/ml; P<0.001). There was no relationship between the concentration of fetal DNA and the time since delivery. Not only fetal cells, but also fragments of fetal DNA can be present in the maternal circulation indefinitely after pregnancy. This finding has practical implications for non-invasive prenatal diagnoses based on maternal blood, and may be considered for possible pathophysiological correlations.     

The Reversed Feto-Maternal Bile Acid Gradient in Intrahepatic Cholestasis of Pregnancy Is Corrected by Ursodeoxycholic Acid

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder associated with an increased risk of adverse fetal outcomes. It is characterised by raised maternal serum bile acids, which are believed to cause the adverse outcomes. ICP is commonly treated with ursodeoxycholic acid (UDCA). This study aimed to determine the fetal and maternal bile acid profiles in normal and ICP pregnancies, and to examine the effect of UDCA treatment. Matched maternal and umbilical cord serum samples were collected from untreated ICP (n = 18), UDCA-treated ICP (n = 46) and uncomplicated pregnancy (n = 15) cases at the time of delivery. Nineteen individual bile acids were measured using HPLC-MS/MS. Maternal and fetal serum bile acids are significantly raised in ICP compared with normal pregnancy (p = <0.0001 and <0.05, respectively), predominantly due to increased levels of conjugated cholic and chenodeoxycholic acid. There are no differences between the umbilical cord artery and cord vein levels of the major bile acid species. The feto-maternal gradient of bile acids is reversed in ICP. Treatment with UDCA significantly reduces serum bile acids in the maternal compartment (p = <0.0001), thereby reducing the feto-maternal transplacental gradient. UDCA-treatment does not cause a clinically important increase in lithocholic acid (LCA) concentrations. ICP is associated with significant quantitative and qualitative changes in the maternal and fetal bile acid pools. Treatment with UDCA reduces the level of bile acids in both compartments and reverses the qualitative changes. We have not found evidence to support the suggestion that UDCA treatment increases fetal LCA concentrations to deleterious levels.     

Fetal DNA detection in maternal plasma throughout gestation

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.     

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.     

Recent advances in fetal nucleic acids in maternal plasma.

The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for noninvasive prenatal diagnosis. Circulating fetal DNA molecules have been detected in maternal plasma from the first trimester onwards and can be robustly detected using a variety of molecular methods. This approach has been used for the prenatal investigation of sex-linked diseases, fetal RhD status, and prenatal exclusion of beta-thalassemia major. Recently, fetal RNA has also been found in maternal plasma. Such fetal RNA has been shown to originate from the placenta and to be remarkably stable. The use of microarray-based approaches has made it feasible to rapidly generate new circulating RNA markers. It is hoped that further developments in this field will make the routine and widespread practice of noninvasive nucleic acid-based prenatal diagnosis for common pregnancy-associated disorders feasible in the near future.     

Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains

BackgroundCirculating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors.ResultsWe perform whole genome bisulfite sequencing on a set of unmatched samples including circulating cell-free DNA from non-pregnant and pregnant female donors and genomic DNA from maternal buffy coat and placenta samples. We find CpG cytosines within longer fragments are more likely to be methylated. Comparison of the methylomes of placenta and non-pregnant circulating cell-free DNA reveal many of the 51,259 identified differentially methylated regions are located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases. We find these placenta hypomethylated domains are consistently located within regions exhibiting low CpG and gene density. Differentially methylated regions identified when comparing placenta to non-pregnant circulating cell-free DNA are recapitulated in pregnant circulating cell-free DNA, confirming the ability to detect differential methylation in circulating cell-free DNA mixtures.ConclusionsWe generate methylome maps for four sample types at single-base resolution, identify a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0645-x) contains supplementary material, which is available to authorized users.     

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.     

Non-Invasive Prenatal Testing of Trisomy 18 by an Epigenetic Marker in First Trimester Maternal Plasma

Background

Quantification of cell-free fetal DNA by methylation-based DNA discrimination has been used in non-invasive prenatal testing of fetal chromosomal aneuploidy. The maspin (Serpin peptidase inhibitor, clade B (ovalbumin), member 5; SERPINB5) gene, located on chromosome 18q21.33, is hypomethylated in the placenta and completely methylated in maternal blood cells. The objective of this study was to evaluate the accuracy of non-invasive detection of fetal trisomy 18 using the unmethylated-maspin (U-maspin) gene as a cell-free fetal DNA marker and the methylated-maspin (M-maspin) gene as a cell-free total DNA marker in the first trimester of pregnancy.

Methodology/Principal Findings

A nested case-control study was conducted using maternal plasma collected from 66 pregnant women, 11 carrying fetuses with trisomy 18 and 55 carrying normal fetuses. Median U-maspin concentrations were significantly elevated in women with trisomy 18 fetuses compared with controls (27.2 vs. 6.7 copies/mL; P<0.001). Median M-maspin concentrations were also significantly higher in women with trisomy 18 fetuses than in controls (96.9 vs. 19.5 copies/mL, P<0.001). The specificities of U-maspin and M-maspin concentrations for non-invasive fetal trisomy 18 detection were 96.4% and 74.5%, respectively, with a sensitivity of 90.9%.

Conclusions

Our results suggest that U-maspin and M-maspin concentrations may be useful as potential biomarkers for non-invasive detection of fetal trisomy 18 in the first trimester of pregnancy, irrespective of the sex and genetic variations of the fetus.     

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.     

Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach

Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods’ performance parameters—standard curve linearity, detection limit and measurement precision—were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438).     

Effective detection of fetal sex using circulating fetal DNA in first-trimester maternal plasma

The aim of this study was to develop a simple and effective method for noninvasively detecting fetal sex using circulating fetal DNA from first-trimester maternal plasma. A study was conducted with maternal plasma collected from 203 women between 5 and 12 wk of gestation. The presence of circulating fetal DNA was confirmed by a quantitative methylation-specific polymerase chain reaction of the unmethylated-PDE9A gene (U-PDE9A). Multiplex real-time PCR was used to simultaneously quantify the amount of DYS14 and GAPDH in maternal plasma. The results were confirmed by phenotype at birth. Pregnancy outcomes and U-PDE9A concentrations were obtained in all cases, including 99 male-bearing and 104 female-bearing participants. At equivalent specificity (100%), the false-negative rate was 9.1% for DYS14 quantification cycle, 7.1% for DYS14 concentration, and 0.0% for the concentration ratio of DYS14/GAPDH, respectively. In male-bearing participants, DYS14, U-PDE9A, and GAPDH concentrations were significantly lower in the false-negative case than in correct case (P<0.001 in all). Moreover, DYS14, U-PDE9A, and GAPDH concentrations showed significantly positive associations with each other (P≤0.001 in all). The ratio of DYS14/GAPDH in maternal plasma was an effective biomarker for noninvasive fetal sex detection during the first trimester, indicating that it could be useful for clinical application.     

Recognition of asymptomatic hypercholanemia of pregnancy: Different clinical features,fetal outcomes and bile acids metabolism from intrahepatic cholestasis of pregnancy

ObjectiveTo explore the clinical features, fetal outcomes and serum bile acids (BAs) metabolism in asymptomatic hypercholanemia of pregnancy (AHP), as well as the comparison with those in intrahepatic cholestasis of pregnancy (ICP) and normal pregnancies.MethodsA study containing 676 pregnant women was performed to investigate the clinical informations, routine biochemical features and obstetric outcomes of AHP by the comparison with ICP and normal pregnancies. Within the study subjects, 203 pregnant women received prospective determination for 55 serum individual BAs based on a validated UPLC-QTOF-MS/MS method. The differences in clinical features and serum BAs metabolism among the three groups were then investigated.ResultsThe risk of adverse fetal outcomes in AHP (28.3%) was significantly higher than that in normal pregnancies (8.9%, p < 0.001), but lower than that in ICP group (52.1%, p < 0.001). Multivariate statistics analysis indicated a distinctive serum BAs metabolic profiling among the three groups (PLS-DA, R²Y = 0.580, Q² = 0.537). Levels of serum BAs especially for deoxycholic acid species were found remarkably elevated in AHP as compared to those in ICP.ConclusionsAHP group had distinguished clinical features and serum BAs metabolism as compared to ICP group and normal pregnancies.     

No evidence of fetal DNA persistence in maternal plasma after pregnancy

Short- and long-term persistence of fetal DNA in maternal plasma has been investigated. Short-term persistence at very low concentration was detected in 47 out of 105 women within two days after delivery. Twelve out of 13 samples re-tested within three days scored negative. No long-term persistence was detected in 172 women who had previous sons or abortions. Molecular microchimerism due to circulating fetal DNA persisting from previous pregnancies should not hamper non-invasive plasma-based prenatal testing.     

Fetal outcomes in pregnancies complicated by intrahepatic cholestasis of pregnancy in a Northern California cohort

Background

Intrahepatic cholestasis of pregnancy (ICP) has important fetal implications. There is increased risk for poor fetal outcomes, including preterm delivery, meconium staining of amniotic fluid, respiratory distress, fetal distress and demise.

Methods

One hundred and one women diagnosed with ICP between January 2005 and March 2009 at San Francisco General Hospital were included in this study. Single predictor logistic regression models were used to assess the associations of maternal clinical and biochemical predictors with fetal complications. Clinical predictors analyzed included age, race/ethnicity, gravidity, parity, history of liver or biliary disease, history of ICP in previous pregnancies, and induction. Biochemical predictors analyzed included serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, direct bilirubin, albumin, total protein, and total bile acids (TBA).

Results

The prevalence of ICP was 1.9%. Most were Latina (90%). Labor was induced in the majority (87%) and most were delivered by normal spontaneous vaginal delivery (84%). Fetal complications occurred in 33% of the deliveries, with respiratory distress accounting for the majority of complications. There were no statistically significant clinical or biochemical predictors associated with an increased risk of fetal complications. Elevated TBA had little association with fetal complications until reaching greater than 100 µmoL/L, with 3 out of 5 having reported complications. ICP in previous pregnancies was associated with decreased risk of fetal complications (OR 0.21, p = 0.046). There were no cases of late term fetal demise.

Conclusions

Maternal clinical and laboratory features, including elevated TBA, did not appear to be substantial predictors of fetal complications in ICP.     

Fetomaternal adrenomedullin levels in diabetic pregnancy.

We investigated whether maternal and fetoplacental adrenomedullin, a newly discovered hypotensive peptide involved in the insulin regulatory system, is modified in diabetic pregnancy. We studied its correlation with pregnancy complications associated with this disease. Thirty-six pregnant women with diabetes (13 with type I and 23 with gestational diabetes mellitus) and in 40 uncomplicated pregnancies were included. 10 out of 36 diabetic pregnancies were complicated by gestational hypertension. In each woman, adrenomedullin concentration in maternal and fetal plasma and in amniotic fluid was assessed by specific radioimmunoassay. We found that overall mean amniotic fluid adrenomedullin concentration was higher (p < 0.05) in diabetic (14.7 +/- 1.6 fmol/ml) than in uncomplicated pregnancies (10.8 +/- 0.9 fmol/ml), whereas no differences were present in maternal and fetal plasma adrenomedullin levels between diabetic and uncomplicated pregnant women. High levels of amniotic fluid adrenomedullin were found in both type I and gestational diabetes mellitus pregnancies (13.7 +/- 1.4 and 15.6 +/- 2.2 fmol/ml, respectively). Diabetic pregnancies complicated by gestational hypertension showed lower (p < 0.05) amniotic fluid adrenomedullin concentrations than normotensive diabetic patients. These findings suggest that placental adrenomedullin production is upregulated in diabetic pregnancy, and it may be important to prevent excessive vasoconstriction of placental vessels.     

A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection

Objective

The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure.

Methods

Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction.

Results

A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B.

Conclusion

A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.