Prenatal sex determination from maternal peripheral blood using the polymerase chain reaction

We have investigated the use of a nested polymerase chain reaction assay for the detection of a fetal-specific Y-chromosomal sequence (DYS14) from DNA extracted from unsorted maternal peripheral blood. Serial dilutions of male DNA into female cord blood DNA indicated that the assay could detect an equivalent of a single male cell in 300000 female cells. The assay exhibited absolute specificity for male DNA with no amplification from a DNA panel obtained from 10 female cord blood samples. When used on DNA extracted from unsorted peripheral blood from a series of pregnant women, the predictive values of a positive test for a male fetus were 86%, 67% and 87% in the first, second and third trimesters, respectively. We have also demonstrated that retesting the samples allows the detection of a proportion of male-bearing pregnancies with a high degree of accuracy, in that all 15 women who gave positive signals in two consecutive amplifications had male fetuses. We have also applied the test at 8 weeks post-partum to eight women who had previously delivered male babies; no Y-specific signal could be detected in any of them, suggesting that most women have cleared their circulation of fetal cells by 8 weeks after parturition.     

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.     

孕牛外周血中胎儿Y-特异性序列的检测

根据公牛Sry特异性序列设计两对寡核苷酸引物。并对20份妊娠晚期母牛外周血DNA样品进行PCR扩增,结果有9份样品检测出有Sry片断(阳性),其余11份为阴性。经分娩牛犊性别验证,在20份被检测的样品中,有16份PCR检测结果与犊牛实际性别相符,其余4例不符。我们的实验结果说明,胎儿细胞可以进入到孕牛的外周血中去。     

Cattle fetal sex determination by polymerase chain reaction using DNA isolated from maternal plasma

The objective of this study was to evaluate the use of polymerase chain reaction analysis (PCR) of fetal cells/DNA in the maternal plasma of pregnant cows to determine the sex of the fetus. Plasma was harvested from 35 cows of mixed genotype at different stages of pregnancy ranging from 5 to 35 weeks. A male calf and a heifer calf provided the control samples. Fetal sex was determined by amplification of Y-specific sequences. For the 35 cows, the fetal sex predicted by this technique was in accordance with the sex of the calf at birth in 88.6% of cases. The agreement between predicted and observed fetal sex was less for cows with a gestational length of 35-48 days (63.6%). Regression analysis showed that there was a strong relationship between the probability of correctly predicting fetal sex and the stage of gestation. It was estimated that the test performed at 43.8 days post fertilization would have 95% accuracy, increasing to 99% accuracy for testing at 48.4 days and 99.9% accuracy for tests at 55.0 days or later. It was concluded that PCR analysis of fetal cells in maternal plasma can be used to predict successfully the sex of the fetus in cattle.     

Fetal gender determination and BclI polymorphism using nucleated erythrocytes in maternal blood.

This study demonstrated determination of fetal gender from nucleated red blood cells (NRBCs) in maternal blood and attempted to apply prenatal diagnosis of hemophilia A using BclI DNA polymorphism. Venous blood was drawn from 20 pregnant women, and NRBCs were recovered by magnetic activated cell sorting and anti-GPA (glycophorin A) immunostaining. After microdissector isolation of the NRBCs, primer extension preamplification (PEP) and nested PCR of the amelogenin gene were performed to determine fetal gender. We also performed PEP and nested PCR of BclI polymorphism to verify the validity of prenatal diagnosis of hemophilia A. DNA amplification was achieved in 107 cells (51.9%) and fetal gender determined with 65.0% accuracy. Unfortunately, we could not verify the validity within the scope of this study. However, in a larger number of cases that are informative in BclI polymorphism, we will be able to identify patients affected by hemophilia A using fetal NRBCs in maternal blood.     

Fetal gender determination in early first trimester pregnancies of rhesus monkeys (Macaca mulatta) by fluorescent PCR analysis of maternal serum

Non-human primate fetal gender determination can be a powerful tool for research study design and colony management purposes. The recent discovery of the presence of fetal DNA in maternal serum has offered a new non-invasive approach for identification of fetal gender. We present a rapid and simple method for the sexing of developing rhesus monkeys in the first trimester by polymerase chain reaction (PCR) analysis of maternal serum. Serum samples were obtained from 72 gravid rhesus monkeys during 20-32 days of gestation (term 165 +/- 10 days). Fetal gender and the quantity of circulating fetal DNA were determined by real-time PCR analysis of the rhesus Y-chromosomal DNA sequences. The sensitivity for identifying a male fetus was 100% by 30 days gestation, and no false-positive results were observed. This study demonstrates that fetal gender can be reliably determined in the early first trimester from maternal serum samples, a non-invasive method for routine gender screening.     

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.     

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.     

Fetal gender determination in early pregnancy through qualitative and quantitative analysis of fetal DNA in maternal serum

Fetal DNA in maternal plasma and serum has been shown to be a useful material for fetal gender determination and for screening tests for abnormal pregnancies except during early gestational ages. Maternal serum samples were obtained from 81 pregnant women during the 5th-10th weeks of gestation. Fetal gender was determined by conventional polymerase chain reaction (PCR) to detect a Y-chromosomal sequence (DYS14) in maternal serum during early gestation and confirmed by examination of the newborns after delivery. Real-time quantitative analyses of the SRY and beta-globin genes were also performed in order to determine fetal gender and to quantify fetal DNA concentration in maternal serum during early gestation. When using conventional PCR, the total sensitivity of identifying a male fetus was 95%, but its sensitivity after the 7th week was 100%, whereas in real-time quantitative PCR, the total sensitivity after the 5th week was 100%. Quantitative analyses of the SRY gene revealed that the mean concentration of fetal DNA in maternal serum was 30.55 copies/ml, that fetal DNA concentration showed a tendency to increase with the progression of pregnancy, and that it had a wide normal range. Thus, we could confidently determine fetal gender by using maternal serum samples taken as early as the 7th week.     

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.     

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.     

Direct PCR of washed blood cells.

We report a simple and rapid method for direct DNA amplification of washed blood cells by PCR. Small samples (2-100 microliters) of blood were washed, the cells resuspended in a buffer and used directly for PCR after boiling. Amplification of a specific DNA sequence of the human transthyretin gene, directed by the primers, was successfully performed. The method gives comparable results to amplifications made by purified DNA from blood.     

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.     

Akonni TruTip? and Qiagen? Methods for Extraction of Fetal Circulating DNA - Evaluation by Real-Time and Digital PCR

Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed - a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.     

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.     

Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing

PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.     

A test of the multiplex pre-amplification approach in microsatellite genotyping of wolverine faecal DNA

Recently, a two-step PCR approach, referred to as multiplex pre-amplification, was proposed to improve microsatellite amplification from non-invasive samples such as faecal DNA. Here, we compare this new approach to standard PCR with respect to amplification success and genotyping error rates in microsatellite analysis (18 markers) of wolverine faecal DNA (48 extracts initially shown to contain amplifiable DNA). The multiplex pre-amplification approach was clearly advantageous both in terms of successful PCR amplifications (91% vs. 80%) and allelic dropout rate (2.4% vs. 12.5%). However, dropouts were to a high extent repeated in all second-step amplifications following multiplex pre-amplification, indicative of being generated during the initial PCR. Analysing more than one PCR from the initial multiplex PCR product may thus be of limited value. We instead suggest to perform two initial multiplex PCRs and to analyse a single second-step PCR from each of them. This was tested for 22 extracts at 18 loci and proved to be an effective way to obtaining a correct genotype.     

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.     

Trophoblasts isolated from the maternal circulation: in vitro expansion and potential application in non-invasive prenatal diagnosis.

Prenatal diagnosis based on rare fetal cells in maternal blood is currently not a feasible option. An effort was made to improve cell yields by targeting trophoblast cells. After sorting, the HLA-G-positive cell fraction was analyzed directly or after culture. In situ hybridization technology was applied to prove fetal cell source in samples from women carrying a male fetus and to predict gender in samples without previous knowledge of fetal sex. In vitro culture led to a significant increase in fetal cells and accurate gender prediction in 93% of these samples. This approach might be useful for non-invasive prenatal diagnosis.     

Detection of fetal RHD pseudogene (RHDΨ) and hybrid RHD-CE-Ds from RHD-negative pregnant women with a free DNA fetal kit

Hemolytic disease of the newborn is a clinical condition in which maternal and paternal Rh blood group antigens are incompatible and the mother is negative for the antigen whereas the father is positive. Analysis of fetal cells recovered from maternal plasma can provide a highly sensitive prenatal diagnosis. The fetal RHD gene in plasma DNA is detected by real-time PCR amplification of two different segments of the RHD gene (exons 7 and 10). Each amplicon is revealed with specific probes. We examined 40 female blood samples to verify the specificity of RHD exons (7 and 10) amplified by real-time PCR. Thirty fetuses were predicted to be RHD-positive based on analysis of plasma DNA. Seven fetuses were predicted to be RHD-negative. One fetus was negative for RHD on exon 10, and positive for RHD on exon 7 (early gestation age); two fetuses were RHD-negative on exon 7, and RHD-positive on exon 10 (RHD-CE-D(s) or RHDΨ), indicative of a maternal RHD allele. We conclude that it is necessary to analyze at least two exon regions in the RHD gene.