mRNA and Protein Expression in Human Fetal Membrane Cells: Potential Biomarkers for Preterm Prelabor Rupture of the Fetal Membranes?

Clinically, unique markers in fetal membrane cells may contribute to the search for biomarkers for preterm prelabor rupture of the fetal membranes (pPROM) in maternal blood. pPROM is associated with overwhelming inflammation and premature cellular senescence causing "biological microfractures" of the fetal membranes. We hypothesize that these pathological processes are associated with the shedding of fetal membrane cells into the maternal circulation. The aim of this study was to identify markers expressed exclusively in fetal membrane cells to facilitate their isolation, characterization, and determination of biomarker potential in maternal blood. We have (1), by their transcriptomic profile, identified markers that are upregulated in amnion and chorion tissue compared to maternal white blood cells, and (2), by immunohistochemistry, confirmed the localization of the differentially expressed proteins in fetal membranes, placenta, and the placental bed of the uterus. RNA sequencing revealed 31 transcripts in the amnion and 42 transcripts in the chorion that were upregulated. Among these, 22 proteins were evaluated by immunohistochemistry. All but two transcripts were expressed both on mRNA and protein level in at least one fetal membrane cell type. Among these remaining 20 proteins, 9 proteins were not significantly expressed in the villous and extravillous trophoblasts of the placenta.

Clinical interpretation of cell-based non-invasive prenatal testing for monogenic disorders including repeat expansion disorders: potentials and pitfalls.

Introduction: Circulating fetal cells isolated from maternal blood can be used for prenatal testing, representing a safe alternative to invasive testing. The present study investigated the potential of cell-based noninvasive prenatal testing (NIPT) for diagnosing monogenic disorders dependent on the mode of inheritance. Methods: Maternal blood samples were collected from women opting for prenatal diagnostics for specific monogenic disorders (N = 7). Fetal trophoblasts were enriched and stained using magnetic activated cell sorting and isolated by fluorescens activated single-cell sorting. Individual cells were subject to whole genome amplification, and cells of fetal origin were identified by DNA-profiling using short tandem repeat markers. The amplified fetal DNA was input for genetic testing for autosomal dominant-, autosomal recessive-, X-linked and repeat expansion disorders by direct variant analysis and haplotyping. The cell-based NIPT results were compared with those of invasive testing. Results: In two cases at risk of skeletal dysplasia, caused by variants in the FGFR3 gene (autosomal dominant disorders), cell-based NIPT correctly stated an affected fetus, but allelic dropout of the normal alleles were observed in both cases. Cell-based NIPT gave an accurate result in two cases at risk of autosomal recessive disorders, where the parents carried either different diastrophic dysplasia causing variants in the SLC26A2 gene or the same cystic fibrosis disease-causing variant in the CFTR gene. Cell-based NIPT accurately identified an affected male fetus in a pregnancy at risk of Duchenne muscular dystrophy (DMD gene, X-linked recessive disorders). In two cases at risk of the myotonic dystrophy type 1 (DMPK gene, repeat expansion disorder), cell-based NIPT correctly detected an affected and an unaffected fetus, respectively. Discussion: Circulating fetal cells can be used to detect both maternally- and paternally inherited monogenic disorders irrespective of the type of variant, however, the risk of allelic dropout must be considered. We conclude that the clinical interpretation of the cell-based NIPT result thus varies depending on the disorders' mode of inheritance.

How does cell-based non-invasive prenatal test (NIPT) perform against chorionic villus sampling and cell-free NIPT in detecting trisomies and copy number variations? A clinical study from Denmark.

OBJECTIVES: We aimed to compare cell-based NIPT (cbNIPT) to chorionic villus sampling (CVS) and to examine the test characteristics of cbNIPT in the first clinical validation study of cbNIPT compared to cell-free NIPT (cfNIPT). MATERIAL AND METHODS: Study 1: Women (N = 92) who accepted CVS were recruited for cbNIPT (53 normal and 39 abnormal). Samples were analyzed with chromosomal microarray (CMA). Study 2: Women (N = 282) who accepted cfNIPT were recruited for cbNIPT. cfNIPT was analyzed using sequencing and cbNIPT by CMA. RESULTS: Study 1: cbNIPT detected all aberrations (32/32) found in CVS: trisomies 13, 18 and 21 (23/23), pathogenic copy number variations (CNVs) (6/6) and sex chromosome aberrations (3/3). cbNIPT detected 3/8 cases of mosaicism in the placenta. Study 2: cbNIPT detected all trisomies found with cfNIPT (6/6) and had no false positive (0/246). One of the three CNVs called by cbNIPT was confirmed by CVS but was undetected by cfNIPT, two were false positives. cbNIPT detected mosaicism in five samples, of which two were not detected by cfNIPT. cbNIPT failed in 7.8% compared to 2.8% in cfNIPT. CONCLUSION: Circulating trophoblasts in the maternal circulation provide the potential of screening for aneuploidies and pathogenic CNVs covering the entire fetal genome.

Diagnosis of hydatidiform moles using circulating gestational trophoblasts isolated from maternal blood.

INTRODUCTION: Identifying hydatidiform moles (HMs) is crucial due to the risk of gestational trophoblastic neoplasia. When a HM is suspected on clinical findings, surgical termination is recommended. However, in a substantial fraction of the cases, the conceptus is actually a non-molar miscarriage. If distinction between molar and non-molar gestations could be obtained before termination, surgical intervention could be minimized. METHODS: Circulating gestational trophoblasts (cGTs) were isolated from blood from 15 consecutive women suspected of molar pregnancies in gestational week 6-13. The trophoblasts were individually sorted using fluorescence activated cell sorting. STR analysis targeting 24 loci was performed on DNA isolated from maternal and paternal leukocytes, chorionic villi, cGTs, and cfDNA. RESULTS: With a gestational age above 10 weeks, cGTs were isolated in 87% of the cases. Two androgenetic HMs, three triploid diandric HMs, and six conceptuses with diploid biparental genome were diagnosed using cGTs. The STR profiles in cGTs were identical to the profiles in DNA from chorionic villi. Eight of the 15 women suspected to have a HM prior to termination had a conceptus with a diploid biparental genome, and thus most likely a non-molar miscarriage. DISCUSSION: Genetic analysis of cGTs is superior to identify HMs, compared to analysis of cfDNA, as it is not hampered by the presence of maternal DNA. cGTs provide information about the full genome in single cells, facilitating estimation of ploidy. This may be a step towards differentiating HMs from non-HMs before termination.

Noninvasive prenatal screening for cystic fibrosis using circulating trophoblasts: Detection of the 50 most common disease-causing variants.

OBJECTIVES: Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders. Prenatal or preconception CF screening is offered in some countries. A maternal blood sample in early pregnancy can provide circulating trophoblasts and offers a DNA source for genetic analysis of both the mother and the fetus. This study aimed to develop a cell-based noninvasive prenatal test (NIPT) to screen for the 50 most common CF variants. METHODS: Blood samples were collected from 30 pregnancies undergoing invasive diagnostics and circulating trophoblasts were harvested in 27. Cystic fibrosis testing was conducted using two different methods: by fragment length analysis and by our newly developed NGS-based CF analysis. RESULTS: In all 27 cases, cell-based NIPT provided a result using both methods in agreement with the invasive test result. CONCLUSION: This study shows that cell-based NIPT for CF screening provides a reliable result without the need for partner- and proband samples.

Cell-Based NIPT Detects 47,XXY Genotype in a Twin Pregnancy.

Background: The existing risk of procedure-related miscarriage following invasive sampling for prenatal diagnosis is higher for twin pregnancies and some women are reluctant to test these typically difficultly obtained pregnancies invasively. Therefore, there is a need for noninvasive testing options that can test twin pregnancies at an early gestational age and ideally test the twins individually. Case presentation: A pregnant woman opted for cell-based NIPT at GA 10 + 5. As cell-based NIPT is not established for use in twins, the test was provided in a research setting only, when an ultrasound scan showed that she carried dichorionic twins. Materials and Methods: Fifty mL of peripheral blood was sampled, and circulating fetal cells were enriched and isolated. Individual cells were subject to whole-genome amplification and STR analysis. Three fetal cells were analyzed by chromosomal microarray (aCGH). Results: We identified 20 fetal cells all sharing the same genetic profile, which increased the likelihood of monozygotic twins. aCGH of three fetal cells showed the presence of two X chromosomes and a gain of chromosome Y. CVS from both placentae confirmed the sex chromosomal anomaly, 47,XXY and that both fetuses were affected. Conclusion: NIPT options can provide valuable genetic information to twin pregnancies that help the couples in their decision-making on prenatal testing. Little has been published about the use of cell-based NIPT in twin pregnancies, but the method may offer the possibility to obtain individual cell-based NIPT results in dizygotic twins.

Screening for Fetal Aneuploidy and Sex Chromosomal Anomalies in a Pregnant Woman With Mosaicism for Turner Syndrome-Applications and Advantages of Cell-Based NIPT.

Background: Cell-free NIPT and cell-based NIPT are risk-free testing options using maternal blood samples to screen for fetal aneuploidies, but the methods differ. For cell-free NIPT, the fetal fraction of cell-free DNA in plasma is analyzed with a high background of maternal DNA. In contrast, for cell-based NIPT, a limited number of the rare, intact fetal cells are isolated for the genetic analysis. This case demonstrates the differences regarding testing for fetal sex-chromosomes anomalies (SCAs) between these two tests. Materials and Methods: A pregnant woman with mosaicism for Turner syndrome opted for NIPT in first trimester. For the cell-free NIPT analysis, DNA extraction, genome-wide massive parallel sequencing, and data analysis were carried out as described by the kit manufacturer (Illumina©, San Diego, CA, USA). For cell-based NIPT, the first sample gave no result, but the woman consented to repeat cell-based NIPT. After whole genome amplification and STR analysis, fetal DNA from three individual fetal cells was subjected to chromosomal microarray (aCGH, Agilent oligoarray, 180 kb). Results: Fetal fraction was 7%, and cell-free NIPT showed 2 copies of chromosomes 13, 18, and 21 and a decreased proportion of chromosome X, suggestive of fetal Turner syndrome. In contrast, the cell-based NIPT result showed no aneuploidy and two X-chromosomes in the fetus. Conclusion: cell-based NIPT may provide a non-invasive testing option to screen for SCAs in women with mosaicism for monosomy-X in blood, where cell-free NIPT cannot discriminate whether the X-loss is maternal or fetal.

Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing.

PURPOSE: Proof of concept of the use of cell-based non-invasive prenatal testing (cbNIPT) as an alternative to chorionic villus sampling (CVS) following preimplantation genetic testing for monogenic disorders (PGT-M). METHOD: PGT-M was performed by combined testing of short tandem repeat (STR) markers and direct mutation detection, followed by transfer of an unaffected embryo. Patients who opted for follow-up of PGT-M by CVS had blood sampled, from which potential fetal extravillous throphoblast cells were isolated. The cell origin and mutational status were determined by combined testing of STR markers and direct mutation detection using the same setup as during PGT. The cbNIPT results with respect to the mutational status were compared to those of genetic testing of the CVS. RESULTS: Eight patients had blood collected between gestational weeks 10 and 13, from which 33 potential fetal cell samples were isolated. Twenty-seven out of 33 isolated cell samples were successfully tested (82%), of which 24 were of fetal origin (89%). This corresponds to a median of 2.5 successfully tested fetal cell samples per case (range 1-6). All fetal cell samples had a genetic profile identical to that of the transferred embryo confirming a pregnancy with an unaffected fetus, in accordance with the CVS results. CONCLUSION: These findings show that although measures are needed to enhance the test success rate and the number of cells identified, cbNIPT is a promising alternative to CVS. TRIAL REGISTRATION NUMBER: N-20180001.

Cell-based non-invasive prenatal diagnosis in a pregnancy at risk of cystic fibrosis.

OBJECTIVE: We aimed to develop cell-based NIPT for cystic fibrosis (CF) and test a pregnancy at risk of two common pathogenic variants. METHOD: A pregnant woman carrying monozygotic twins opted for prenatal testing as she and her partner were heterozygote carriers of F508del (c.1521:1523del). The partner was also positive for the CFTR-related variant R117H (c.350G>A). Fetal trophoblasts from maternal blood were enriched and isolated using antibodies and a capillary-based cell-picking instrument. Multiplex PCR-based fragment length analysis was performed on the extracted fetal DNA for STR-genotyping, fetal gender and F508del variant status. The R117H variant status was tested using SNaPshot analysis. RESULTS: The fetal origin of the isolated cells was verified by detection of two paternally inherited STR alleles and an Y chromosome marker, while no maternal DNA contamination was detected. The direct variant analysis detected F508del heterozygosity and the SNaPshot analysis for R117H detected only the normal allele. Thus, the results showed that the fetuses were healthy carriers of F508del, concordant with the findings of conventional prenatal testing. CONCLUSION: Cell-based NIPT could accurately state the fetal variant status and distinguish fetal trophoblasts from maternal cells. In the future, cell-based NIPT may provide an accurate less invasive alternative to chorionic villous sampling.

Hydatidiform mole diagnostics using circulating gestational trophoblasts isolated from maternal blood.

BACKGROUND: In gestational trophoblastic disease, the prognosis is related to the genetic constitution. In some cases, taking a biopsy is contraindicated. METHODS: In a pregnant woman, ultrasound scanning suggested hydatidiform mole. To explore if the genetic constitution can be established without taking a biopsy (or terminating the pregnancy), cell-free DNA and circulating gestational trophoblasts were isolated from maternal blood before evacuation of the uterus. The evacuated tissue showed the morphology of a complete hydatidiform mole. Without prior whole-genome amplification, short tandem repeat analysis of 24 DNA markers was performed on the samples, and on DNA isolated from evacuated tissue, and from the blood of the patient and her partner. RESULTS: Identical genetic results were obtained in each of three circulating gestational trophoblasts and the evacuated tissue, showing that this conceptus had a diploid androgenetic nuclear genome. In contrast, analysis of cell-free DNA was less informative and less specific due to the inherent presence of cell-free DNA from the patient. CONCLUSION: Our results show that it is possible to isolate and analyze circulating gestational trophoblasts originating in a pregnancy without maternal nuclear genome. For diagnosing gestational trophoblastic diseases, genotyping circulating gestational trophoblasts appears to be superior to analysis of cell-free DNA.

Cell-based noninvasive prenatal testing (cbNIPT) detects pathogenic copy number variations.

In two cases, cell-based noninvasive prenatal testing (cbNIPT) detected pathogenic copy number variations (CNVs) in the fetal genome. cbNIPT may potentially be an improved noninvasive alternative for the detection of smaller CNVs.

The Number of Circulating Fetal Extravillous Trophoblasts Varies from Gestational Week 6 to 20.

Cell-based non-invasive prenatal testing (cbNIPT) based on circulating fetal extravillous trophoblasts (fEVTs) has shown to be possible in gestational week (GW) 10-13. Prenatal testing is relevant for a wider time period than GW 10-13, but it is unclear if fEVTs are present in sufficient numbers for cbNIPT at other time points during pregnancy. We present the first longitudinal study where the number of circulating fEVTs was determined from the mid first trimester to the mid second, specifically GW 6-8, 12-13, and 19-20. Blood samples from 13 women opting for assisted reproduction were collected at GW 6-8, 12-13, and 19-20. fEVTs were enriched using a magnetic-activated cell sorting system, stained with anti-cytokeratin antibodies, and fEVTs were identified with the use of a MetaSystem fluorescence microscope scanner. Blood samples drawn at GW 6-8 yielded an average of 5.5 fEVTs per 30 mL of blood. This increased significantly to an average of 11.8 in GW 12-13 (P value: 0.0070, Mann-Whitney test), and decreased significantly to an average of 5.3 in GW 19-20 (P value: 0.0063, Mann-Whitney test). In 9 out of 13 cases, the number of fEVTs peaked in GW 12-13 compared to GW 6-8 and GW 19-20. For the majority of cases, fEVTs can be identified at GW 6-8 and GW 19-20, but the highest number of fEVTs is observed at GW 12-13 indicating this is the optimal time point for cbNIPT.

Do fetal extravillous trophoblasts circulate in maternal blood postpartum?

INTRODUCTION: Circulating fetal extravillous trophoblasts may offer a superior alternative to cell-free fetal DNA for noninvasive prenatal testing. Cells of fetal origin are a pure source of fetal genome; hence, unlike the cell-free noninvasive prenatal test, the fetal cell-based noninvasive prenatal test is not expected to be affected by maternal DNA. However, circulating fetal cells from previous pregnancies may lead to confounding results. MATERIAL AND METHODS: To study whether fetal trophoblast cells persist in maternal circulation postpartum, blood samples were collected from 11 women who had given birth to a boy, with blood sampling at 1-3 days (W0), 4-5 weeks (W4-5), around 8 weeks (W8) and around 12 weeks (W12) postpartum. The existence of fetal extravillous trophoblasts was verified either by X and Y chromosome fluorescence in situ hybridization analysis or by short tandem repeat analysis. To exclude technological bias in isolating fetal cells, blood samples were also collected from 10 pregnant women between a gestational age of 10 and 14 weeks, the optimal time frame for cell-based noninvasive prenatal test sampling. All the samples were processed according to protocols established by ARCEDI Biotech for fetal extravillous trophoblast enrichment and isolation. RESULTS: Fetal extravillous trophoblasts were found in all the 10 samples from pregnant women between a gestational age of 10 and 14 weeks. However, only 4 of 11 blood samples taken from women at 1-3 days postpartum rendered fetal extravillous trophoblasts, and only 2 of 11 samples rendered fetal extravillous trophoblasts at 4 weeks postpartum. CONCLUSIONS: In this preliminary dataset on few pregnancies, none of the samples rendered any fetal cells at or after 8 weeks postpartum, showing that cell-based noninvasive prenatal testing based on fetal extravillous trophoblasts is unlikely to be influenced by circulating cells from previous pregnancies.

Does Maternal Body Mass Index Affect the Quantity of Circulating Fetal Cells Available to Use for Cell-Based Noninvasive Prenatal Test in High-Risk Pregnancies?

We present the first study that investigates the effect of maternal body mass index (BMI) on the quantity of circulating fetal cells available to use in cell-based noninvasive prenatal test (cbNIPT). cbNIPT has been proposed as a superior alternative to noninvasive prenatal test from cell-free fetal DNA. Kølvraa et al. [Prenat Diagn. 2016 Dec; 36(12): 1127-34] established that cbNIPT can be performed on as few as one fetal cell, and Vestergaard et al. [Prenat Diagn. 2017 Nov; 37(11): 1120-4] demonstrated that these fetal trophoblast cells could be used successfully in cbNIPT to detect chromosomal and sub-chromosomal abnormalities. This study on 91 pregnant women with high-risk pregnancies suggests that cbNIPT should not be hampered by an increased BMI because every pregnancy, irrespective of the BMI, has rendered fetal cells for downstream genetic analysis. The mean number of fetal cells per sample was 12.6, with a range of 1-43 cells in one sample. ANOVA showed that increasing maternal BMI tends to decrease the number of fetal cells, but not significantly.

On the road to replacing invasive testing with cell-based NIPT: Five clinical cases with aneuploidies, microduplication, unbalanced structural rearrangement, or mosaicism.

OBJECTIVE: Trophoblastic fetal cells harvested from maternal blood have the capacity to be used for copy number analyses in a cell-based non-invasive prenatal test (cbNIPT). Potentially, this will result in increased resolution for detection of subchromosomal aberrations due to high quality DNA not intermixed with maternal DNA. We present 5 selected clinical cases from first trimester pregnancies where cbNIPT was used to demonstrate a wide range of clinically relevant aberrations. METHOD: Blood samples were collected from high risk pregnancies in gestational week 12 + 1 to 12 + 5. Fetal trophoblast cells were enriched and stained using fetal cell specific antibodies. The enriched cell fraction was scanned, and fetal cells were picked using a capillary-based cell picking instrument. Subsequently, whole genome amplification (WGA) was performed on fetal cells, and the DNA was analyzed blindly by array comparative genomic hybridization (aCGH). RESULTS: We present 5 cases where non-invasive cell-based prenatal test results are compared with aCGH results on chorionic villus samples (CVS), demonstrating aneuploidies including mosaicism, unbalanced translocations, subchromosomal deletions, or duplications. CONCLUSION: Aneuploidy and subchromosomal aberrations can be detected using fetal cells harvested from maternal blood. The method has the future potential of being offered as a cell-based NIPT with large high genomic resolution.

Genome-wide copy number analysis on DNA from fetal cells isolated from the blood of pregnant women.

OBJECTIVE: Non-invasive prenatal testing (NIPT) based on fetal cells in maternal blood has the advantage over NIPT based on circulating cell-free fetal DNA in that there is no contamination with maternal DNA. This will most likely result in better detection of chromosomal aberrations including subchromosomal defects. The objective of this study was to test whether fetal cells enriched from maternal blood can be used for cell-based NIPT. METHODS: We present a method for enriching fetal cells from maternal blood, subsequent amplification of the fetal genome and detection of chromosomal and subchromosomal variations in the genome. RESULTS: An average of 12.8 fetal cells from 30 mL of maternal blood were recovered using our method. Subsequently, whole genome amplification on fetal cells resulted in amplified fetal DNA in amounts and quality high enough to generate array comparative genomic hybridization as well as next-generation sequencing profiles. From one to two fetal cells, we were able to demonstrate copy number differences of whole chromosomes (21, X-, and Y) as well as subchromosomal aberrations (ring X). CONCLUSION: Intact fetal cells can be isolated from every maternal blood sample. Amplified DNA from isolated fetal cells enabled genetic analysis by array comparative genomic hybridization and next-generation sequencing. © 2016 John Wiley & Sons, Ltd.

Microarray-Based Analysis of Methylation of 1st Trimester Trisomic Placentas from Down Syndrome, Edwards Syndrome and Patau Syndrome.

Methylation-based non-invasive prenatal testing of fetal aneuploidies is an alternative method that could possibly improve fetal aneuploidy diagnosis, especially for trisomy 13(T13) and trisomy 18(T18). Our aim was to study the methylation landscape in placenta DNA from trisomy 13, 18 and 21 pregnancies in an attempt to find trisomy-specific methylation differences better suited for non-invasive prenatal diagnosis. We have conducted high-resolution methylation specific bead chip microarray analyses assessing more than 450,000 CpGs analyzing placentas from 12 T21 pregnancies, 12 T18 pregnancies and 6 T13 pregnancies. We have compared the methylation landscape of the trisomic placentas to the methylation landscape from normal placental DNA and to maternal blood cell DNA. Comparing trisomic placentas to normal placentas we identified 217 and 219 differentially methylated CpGs for CVS T18 and CVS T13, respectively (delta ß>0.2, FDR<0.05), but only three differentially methylated CpGs for T21. However, the methylation differences was only modest (delta ß<0.4), making them less suitable as diagnostic markers. Gene ontology enrichment analysis revealed that the gene set connected to theT18 differentially methylated CpGs was highly enriched for GO terms related to"DNA binding" and "transcription factor binding" coupled to the RNA polymerase II transcription. In the gene set connected to the T13 differentially methylated CpGs we found no significant enrichments.

The Number of Endovascular Trophoblasts in Maternal Blood Increases Overnight and after Physical Activity: An Experimental Study.

INTRODUCTION: Fetal cells in maternal blood may be used for noninvasive prenatal diagnostics, although their low number is a challenge. This study's objectives were to evaluate whether physical activity, transabdominal and transvaginal ultrasound scans of the uterus, as well as overnight or day-to-day variation affect the number of isolated fetal cells, more specifically the presumed endovascular trophoblast (pEVT). MATERIAL AND METHODS: In each of 3 different experiments, 10 normal singleton pregnant women (gestational age 10+4-14+4 weeks) participated. The number of pEVTs was assessed in 30-36 ml blood using specific markers for enrichment and identification. RESULTS: The number of pEVTs increased overnight (p = 0.001) from a median of 1.5 to 3.5 and even further to a median of 6.0 after 30 min of physical activity (p = 0.04) but was not affected by transabdominal and transvaginal ultrasound scans. Repeated sampling showed that the interindividual variation of pEVTs was higher than the intraindividual variation (p < 0.001). However, even in pregnant women with a consistently low number of pEVTs, isolation of the pEVTs for prenatal diagnoses was possible in all cases by doing 2 separate blood samplings a few days apart. DISCUSSION: The number of pEVTs identified in maternal blood can be increased by presampling conditions or repeated sampling.

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.