Free Fetal DNA Research Articles

Droplet digital PCR, a new approach to analyze fetal DNA from maternal blood: application to the determination of fetal RHD genotype.

The discovery of free fetal DNA in the maternal circulation has inaugurated the era of non-invasive prenatal diagnosis. The latter has the advantage of avoiding the use of conventional obstetric procedures, such as chorionic villus sampling or aspiration of amniotic fluid, thus limiting the risks of miscarriage they induce. However, as free fetal DNA accounts for about 10% of cell-free DNA in maternal plasma, the presence of ambient maternal DNA can make it difficult to detect fetal alleles of paternal origin. Digital Droplet PCR (ddPCR) is a very sensitive method derived from quantitative real-time PCR (qPCR) for the detection of rare alleles and their absolute quantification by removing the necessity of standards. Here we show that this new technology can be applied in routine prenatal fetal RHD genotyping from maternal blood. In conclusion, the use of quantitative properties of digital PCR, in terms of accuracy, sensitivity and specificity, allows one to consider extending the applications of this new technology in non-invasive prenatal diagnosis of many diseases such as autosomal monogenic diseases, either dominant or recessive.

Detection of Fetal Fraction During Noninvasive Prenatal Screening (NIPS) in HIV-Infected Pregnant Women [22

INTRODUCTION: To describe the reliability of Non Invasive Prenatal Screening (NIPS) in HIV infected pregnant women. NIPS analyzes cell free fetal DNA which circulates in maternal plasma but originates from the placenta and is quantitated as “fetal fraction” (FF). Sensitivity of NIPS increases with FF. In certain populations NIPS reliability decreases, with obesity as the strongest factor negatively associated with FF. Additional variables including PAPP-A and smoking have been shown to affect FF. Our study aims to determine if immune system alterations such as HIV or antiretroviral medications impact FF. METHODS: A 2:1 matched case-control study was carried out comparing 15 HIV infected pregnant women to 30 without HIV undergoing NIPS. Variables such as BMI, ethnicity, medical comorbidities, toxic habits, fetal CRL, serum analytes, FF, and pregnancy outcomes were compared. Immune status and antiretroviral medications were evaluated in correlation with FF in HIV infected patients. RESULTS: HIV infected patients receiving integrase inhibitors (INI) had lower mean FF than those without INI—4.93 (SD 2.20) versus 15.7 (SD 5.43) (P=<.01). NIPS was invalid for three HIV infected patients – all of whom were receiving INI - due to low FF. HIV infected patients with an undetectable viral load (VL) at NIPS had a significantly lower median FF compared to those with detectable VL (P=.01). CONCLUSION/IMPLICATIONS: Integrase inhibitors may play a role in reliability of NIPS in HIV infected women, as reflected by lower FF for patients taking INI. Further study is needed to fully elucidate the mechanism for INI and decreased FF.

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Fetal RHD Genotyping Using Real-Time Polymerase Chain Reaction Analysis of Cell-Free Fetal DNA in Pregnancy of RhD Negative Women in South of Iran.

Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions. In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA (cffD- NA) was extracted from maternal plasma. Real-time quantitative polymerase chain reaction (qPCR) for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 (RASSF1A) gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively. Out of 48 fetuses between 8 and 32 weeks (wks) of gestational age (GA), we correctly diagnosed 45 cases (93.75%) of RHD positive fetuses and 2 cases (4.16%) of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative. Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration.

Epsilon globin gene expression in developing human fetal tissues.

The discovery of free fetal DNA in plasma of pregnant women has opened a new avenue for non-invasive prenatal diagnosis. We hypothesized that epsilon (ɛ)-globin gene expression could serve as a positive control for the presence of fetal nucleic acid. We measured ɛ-globin mRNA in human fetal tissues and compared concentrations with that measured in adult non-pregnant and pregnant samples. Total RNA was isolated from fetal marrow, liver, blood, and placenta (10-24 weeks gestation), from adult peripheral blood mononuclear cells, and from maternal plasma. RNA was reverse transcribed and quantitative polymerase chain reaction performed for ɛ-globin expression. ɛ-globin gene expression was detected in all fetal samples, was detected in plasma of pregnant women, but was negligible in non-pregnant samples. Relative ɛ-globin gene expression was significantly greater in fetal blood compared to fetal liver, and was minimally expressed in placenta. ɛ-globin gene expression decreased at the highest gestational ages in fetal blood, while expression was greatest at 15-19 weeks in fetal marrow. Fetal ɛ-globin gene expression is significantly greater than adult expression and is increased in maternal plasma compared to non-pregnant samples. ɛ-globin gene expression might serve as a positive control when determining the presence of fetal nucleic acid in total nucleic acid isolated from maternal plasma.

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Algorithm-Based Fetal Gender Determination Using X and Y Mini-Short Tandem Repeats at Early Gestational Ages

Background: Detection of fetal DNA in maternal blood has been examined by many research groups for a few years; thereby, scientists have a shorter way to take to approach prenatal diagnosis of abnormal pregnancies. The Y chromosome sequences have recently become the most common applicable indices for fetal sex determination. Objectives: We conducted an algorithmic X and Y mini-Short Tandem Repeats (STRs) genotyping method that could solve the problem of false negative (no detection of Y sequences) results of previous methods. Patients and Methods: Blood samples were obtained from 106 pregnant women and their spouses. Conventional PCR amplified 19 mini-Short Tandem Repeats (STRs) and three non-STR markers, which were subsequently genotyped by the means of Polyacrylamide gel electrophoresis (PAGE). Results: Sensitivity and specificity of the mini-STR genotyping method for fetal DNA detection were calculated (95.9% and 98%, respectively) with a confidence interval of 95%. In addition, sensitivity and informativeness were computed for each of the single mini-STR markers in our conventional PCR method. We also introduced the minimum number of mini-STRs needed to reach maximum validity for fetal gender determination in clinical settings. Conclusions: Algorithm-based mini-STR genotyping method significantly increases the reliability (sensitivity and specificity) of gender determination using free fetal DNA and could be applied in prenatal clinical testing.

Rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations by free fetal cell DNA analysis; Gaucher disease as a proof of principle

Rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations by free fetal cell DNA analysis; Gaucher disease as a proof of principle

Non-invasive preimplantation genetic screening (PGS) – free embryonic DNA in embryo culture media exists and may offer the potential for comprehensive chromosomal screening of human blastocysts

Non-invasive prenatal testing via free fetal DNA has revolutionized the way physicians manage their pregnant patients in the first trimester. Currently, the only way to assess embryonic genetic competency prior to implantation is to perform embryo biopsy, an invasive procedure. If free fetal DNA exists, does free embryonic DNA?

Noninvasive Prenatal Detection of a Partial Trisomy 4 Using Whole Genome Semiconductor Sequencing

Massively parallel sequencing of cell free fetal DNA (cffDNA) obtained from maternal plasma is used to detect fetal trisomies and selected sex chromosomal aneuploidies. Different technologies can be used to detect fetal chromosomopathies noninvasively, such as Next Generation sequencing and microarrays. In this case report, we show a procedure for detecting chromosomal imbalances as a result of balanced translocations inherited from parents, using noninvasive prenatal detection of common aneuploidies based protocol. This case study illustrates the potential power of whole-genome semiconductor sequencing when used to augment the diagnostic spectrum of noninvasive prenatal testing to detection of copy number variants.

323: A comparison between PAPP-A and free fetal DNA in maternal serum to predict adverse pregnancy outcomes

323: A comparison between PAPP-A and free fetal DNA in maternal serum to predict adverse pregnancy outcomes

174: Cell free fetal DNA fraction and inflammatory conditions: is there a correlation?

174: Cell free fetal DNA fraction and inflammatory conditions: is there a correlation?

Cell free fetal DNA in the plasma of pregnant women with preeclampsia

Objective: Insufficient cytotrophoblast invasion to the myometrium is associated with preeclampsia, especially with the early-onset preeclampsia (before 34 gestational weeks). Several investigations have marked changes in the concentration of cell free fetal DNA in the maternal circulation of women with preeclampsia. However, these studies were not performed for early or late preeclampsia subgroups individually. The present authors planned to determine the levels of the cell free both fetal and maternal DNA in the maternal circulation in early preeclampsia subgroup and compare it with normotensive control cohort. Materials and Methods: A total of 16 women; eight of these with preeclampsia and eight normotensive control cohorts with singleton male pregnancy between 28 and 32 gestational weeks were included in the study. Real-time PCR analysis was performed for determining the circulating cell free DNA levels. Results: Cell free fetal DNA concentrations were higher in early preeclamptic women than control subjects. The authors found no statistically significant difference in each levels of maternal and total DNA between hypertensive and normotensive groups. Conclusions: The present findings suggest that the levels of cell free fetal DNA in maternal circulation were higher in pregnancies which are complicated with early preeclampsia than normotensive controls.

Current controversies in prenatal diagnosis 3: is there still a value in a nuchal translucency screening ultrasound in conjunction with maternal plasma non-invasive cell-free DNA testing?

What's already known about this topic? Nuchal translucency is an ultrasound measurement of the fetal physiological lymphatic development as it can identify both normal and abnormal lymphatic collections. Abnormal or enlarged nuchal translucency measurements are nonspecific and heterogeneous in origin. The source of maternal plasma cell free fetal DNA is placental apoptosis. The embryonic and cellular development of the placenta can have true chromosomal mosaicism (involves fetus and placenta) orconfined placental mosaicism (CPM) (placenta only). CPM is present in 1–2% of placentas and can cause false positive results for diagnostic chorionic villus sampling and maternalplasma cell free DNA prenatal aneuploidy screening resultswhile false negative placental results are much less common at 1 per 3000. What does this study add? This report summarizes an oral debate presented at the 19th International Conference on Prenatal Diagnosis and Therapy inWashington, DC, USA, on 14 July 2015. This study compares the fetal aneuploidy screening and detection process with the ultrasound nuchal translucency aneuploidy-geneticpathology screening test/measurement and the more recent maternal plasma cell free DNA aneuploidy only screening test when used in conjunction or separately. Informed consent and patient understanding of the aneuploidy screening options and techniques is an important counselling principle for the variety of aneuploidy and genetic screening protocols.

The usage and current approaches of cell free fetal DNA (cffDNA) as a prenatal diagnostic method in fetal aneuploidy screening

Prenatal diagnosis of invasive and noninvasive tests can be done in a way (NIPT), but because of the invasive methods have risks of infection and abortion, diagnosing non-invasive procedure increasing day by day. One of the widespread cell free fetal DNA in maternal blood test (cffDNA) that is increasing in clinical use has been drawing attention. The incidence of aneuploidy chromosomal anomaly of the kind in which all live births; Trisomy 21 (Down Syndrome) 1/800, trisomy 13 (Patau syndrome) 1 /10,000, trisomy 18 (Edwards syndrome) is a form of 1/6000. Because of the high mortality and morbidity, it is vital that congenital anomalies should be diagnosed in prenatal period. Aneuploidy testing for high-risk pregnant women after the 10th week of pregnancy in terms of the blood sample is taken and free fetal DNA in maternal plasma is based on the measurement of the relative amount. Knowledge of the current criteria for use by healthcare professionals in the field test will allow the exclusion of maternal and fetal risks. In this study, it is aimed to demonstrate current international approaches related to the positive and negative sides of non-invasive that is one of the prenatal diagnostic methods of cffDNA test.

Detection of paternally inherited fetal point mutations for β-thalassemia in maternal plasma using simple fetal DNA enrichment protocol with or without whole genome amplification: an accuracy assessment

Objective: To design and evaluate a noninvasive protocol for prenatal diagnosis (PND) of β-thalassemia, using cell free fetal DNA (cff-DNA) in maternal circulation. Traditional current PND which is mainly based on chorionic villous sampling (CVS), amplification refractory mutation system and sequencing holds as gold standard.Methods: Ten thalassemia trait couples with distinct mutations for the husband and wife were included in this study. The mutations in carrier fathers were IVSI-1, IVSI-5, FR8/9 and CD44. After maternal plasma isolation and free DNA extraction, all samples subjected to designed protocol including DNA size separation on agarose gel, elution of DNA from the gel slices using a simple and efficient manual purification method, with or without whole genome amplification and the detection method was allele-specific real-time PCR.Results: Presence or absence of the paternal mutant allele was correctly determined in all of cases and the accuracy of designed protocol was determined 100%.Conclusions: The protocol described here is very simple, inexpensive and easy to perform, but with satisfactory accuracy in detection of paternal mutations in cff-DNA. Due to the risk of fetal loss with current invasive sampling for PND, a noninvasive alternative is highly demanded in clinical setting.

The effect of chorionic villus sampling on the fraction of cell-free fetal DNA in maternal plasma

Objective: This study aims to assess whether the fraction of cell-free fetal DNA (cffDNA) is different at 24 h or 7 days after chorionic villus sampling (CVS), compared to subjects that do not undergo CVS.Methods: Pregnant women undergoing CVS for genetic testing and matched subjects undergoing first trimester combined screening alone were enrolled between 110/7 and 136/7 weeks gestation. The fractions of cffDNA were compared before the procedure, 24 h after and 7 days after between CVS patients and ultrasound-only patients.Results: Forty-five women underwent CVS and 45 had ultrasound alone. The women undergoing CVS were, on average, older (36.8 years versus 28.5 years, p=0.001) and had a higher baseline fraction of cffDNA than women in the comparison group (11.4% versus 9.8%, p=0.033). Both groups had a decrease in the mean fraction of cffDNA after 24 h. After 7 days, the trend of the mean fraction of cffDNA continued to decline in the CVS group but began to trend toward an increase in the ultrasound only group.Conclusions: CVS does not significantly increase the fraction of cell free fetal (placental) DNA in the maternal plasma. A downward trend in cffDNA in maternal plasma is seen at 24 h and 7 days following CVS compared to baseline.

Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA--Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis.

Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated CpG sites to be used with methyl-sensitive restriction enzymes (MSRE) in PCR-based NIPD, we furthermore refined the list of selected sites, containing a restriction sites for one of 16 different methylation-sensitive restriction enzymes. We present a list of markers on chromosomes 13, 18 and 21 with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes.

Use of free fetal DNA from maternal blood in screening for aneuploidies: advantages and limitations

Use of free fetal DNA from maternal blood in screening for aneuploidies: advantages and limitations

Non-invasive prenatal diagnosis of fetal RhD by using free fetal DNA

Anti-D immunoglobulin is applied to all pregnant women having RhD incompatibility to prevent hemolytic disease of the newborn. The aim of this study is to determine fetal RhD status in the Rh incompatible pregnancies with an non-invasive technique; free fetal DNA isolation from maternal circulation. In the case of Rh incompatibility especially with a history of previous fetal anemia, it can be beneficial to know Rh status antenatally in terms of monitoring fetuses with Rh positive [RhD(+)] status consciously.Total free DNA was isolated in 50 Rh negative [RhD(-)] pregnant women, who had RhD alloimmunisation with their husbands. The gene in isolated DNA was investigated with TagMan prob and real time PCR by using primers belonging to exon 7 of the RhD gene.The authors analyzed 50 RhD(-) women by using quantitative real time PCR technique. Five of them were RhD(-) and the rest of them were found to be RhD(+). After birth one of the infants who were analyzed as RhD(+) were found to be RhD(-).The detection of fetal RhD status by using a non-invasive method from maternal circulation was found to be possible. Assessing fetal RhD status non-invasively by using free fetal DNA in maternal blood will be cost-efficient, avoiding unnecessary indirect Coombs test and unnecessary Rhogam applications that is used in RH incompatible pregnancies. This study will throw a fresh light on prenatal diagnosis.