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.

Cervical collagen is reduced in non-pregnant women with a history of cervical insufficiency and a short cervix.

INTRODUCTION: Preterm cervical shortening and cervical insufficiency may be caused by a constitutional weakness of the cervix. The aim of this study was to assess the cervical collagen concentration in non-pregnant women with a history of cervical insufficiency or of a short cervix in the second trimester of pregnancy. MATERIAL AND METHODS: In this case-control study we included non-pregnant women one year or more after pregnancy: 55 controls with a history of normal delivery; 27 women with a history of cervical insufficiency; and 10 women with a history of a short cervix (<5th percentile) and 10 women with a history of a long cervix (>95th percentile) at gestational weeks 18-20. We obtained biopsies (3 × 3-4 mm) from the ectocervix and determined the collagen concentration by measuring the hydroxyproline concentration. RESULTS: Women with cervical insufficiency had lower collagen concentrations (63.5 ± 5.1%; mean ± SD) compared with controls (68.2 ± 5.4%; p = 0.0004); area under the ROC curve 0.73 (95% CI 0.62-0.84). A cut-off value at 67.6% collagen resulted in a positive likelihood ratio of 3.2, a sensitivity of 60%, and a specificity of 81%. Also, women with a short cervix in the second trimester had lower collagen concentrations in a non-pregnant state (62.1% ± 4.9%) compared with women with a long cervix (67.8% ± 5.0%; p = 0.02). CONCLUSIONS: Both cervical insufficiency and a short cervix in the second trimester of pregnancy are associated with low cervical collagen concentrations in a non-pregnant state more than one year after pregnancy.

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.

Polymorphisms in Genes Coding for Cytokines, Mannose-Binding Lectin, Collagen Metabolism and Thrombophilia in Women with Cervical Insufficiency.

OBJECTIVE: To study the association between cervical insufficiency and single nucleotide polymorphisms in seven genes coding for pro- and anti-inflammatory cytokine-related factors, mannose-binding lectin 2 (MBL2), collagen1α1 (COL1A1), factor II and factor V Leiden genes. METHODS: In a case-control study, potential maternal biomarkers for cervical insufficiency were investigated in 30 women with a history of second-trimester miscarriage or preterm birth due to cervical insufficiency and in 70 control women. RESULTS: Homozygous carriers of the interleukin 6 (IL6) -174 genotype GG had an odds ratio (OR) of 3.1 [95% confidence interval (95% CI) 1.3-7.4, p = 0.01] and MBL2 genotypes coding for low or intermediate levels of plasma MBL had an OR of 3.3 (95% CI 1.2-9.0, p = 0.01) for cervical insufficiency compared with controls. Serum MBL levels were lower in women with cervical insufficiency than in controls (median 408 and 1,985 ng/ml, respectively, p < 0.01). CONCLUSIONS: Single nucleotide polymorphisms in the IL6 gene and the MBL2 gene and low MBL levels related to the latter polymorphism may increase the risk of preterm birth due to cervical insufficiency.

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.

The Danish Fetal Medicine Database: establishment, organization and quality assessment of the first trimester screening program for trisomy 21 in Denmark 2008-2012.

OBJECTIVE: To describe the establishment and organization of the Danish Fetal Medicine Database and to report national results of first-trimester combined screening for trisomy 21 in the 5-year period 2008-2012. DESIGN: National register study using prospectively collected first-trimester screening data from the Danish Fetal Medicine Database. POPULATION: Pregnant women in Denmark undergoing first-trimester screening for trisomy 21. METHODS: Data on maternal characteristics, biochemical and ultrasonic markers are continuously sent electronically from local fetal medicine databases (Astraia Gmbh software) to a central national database. Data are linked to outcome data from the National Birth Register, the National Patient Register and the National Cytogenetic Register via the mother's unique personal registration number. First-trimester screening data from 2008 to 2012 were retrieved. MAIN OUTCOME MEASURES: Screening performance was assessed for the years 2008-2012 by calculating detection rates and screen-positive rates. RESULTS: A total of 268 342 first-trimester risk assessments for trisomy 21 were performed in singleton pregnancies. Participation rate in first-trimester screening was >90%. The national screen-positive rate increased from 3.6% in 2008 to 4.7% in 2012. The national detection rate of trisomy 21 was reported to be between 82 and 90% in the 5-year period. CONCLUSION: A national fetal medicine database has been successfully established in Denmark. Results from the database have shown that at a national level first-trimester screening performance for trisomy 21 is high with a low screen-positive rate and a high detection rate.

Optimisation of the coagulation zone for thermal ablation procedures: a theoretical approach with considerations for practical use.

PURPOSE: This paper outlines a theoretical approach for optimisation of the coagulation zone for thermal ablation procedures and considerations for its practical application. METHODS: The theoretical approach is outlined in the Cartesian coordinate system. Considerations for practical application are implemented. The optimised coagulation zone is defined as the bare coverage of tumour mass plus a safety margin. The eccentricity of coagulation centre (ECC) is defined as the distance between the coagulation centre and the tumour centre. The direction of the applicator shaft is determined based on the x-axis direction. The tumour centre and coagulation centre are defined within the x/y-plane. The distance between coagulation margin (applicator tip) and tumour margin is called parallel offset (PAO). RESULTS: For spherical coagulation shapes, a linear relationship exists between optimised coagulation diameter and ECC. An exponential relationship exists between optimised coagulation volume and ECC. A complex relationship was found between PAO and determinants of ECC, which are ex and ey. PAO is an extremely important parameter, which allows for determination of the optimal applicator tip position in relation to the tumour margin. It can be calculated in such a manner that the optimised coagulation zone is minimised by neutralising dislocation of the coagulation centre in applicator shaft direction. The latter can be realised by withdrawing or further inserting the applicator shaft. CONCLUSIONS: The presented concept can be used to optimise the extent of the coagulation zone for thermal ablation procedures after positioning of the applicator. Its inherent advantage is the simple adjustment of the applicator shaft, which obviates the need for a repuncture.

Quantification of tissue shrinkage and dehydration caused by microwave ablation: experimental study in kidneys for the estimation of effective coagulation volume.

PURPOSE: To quantify the extent of tissue shrinkage and dehydration caused by microwave (MW) ablation in kidneys for estimation of effective coagulation volume. MATERIALS AND METHODS: MW ablations were carried out in ex vivo porcine kidneys. Six study groups were defined: groups 1A, 2A, and 3A for MW ablation (90 W for 5 min, 7.5 min, or 10 min), and groups 1B, 2B, and 3B for control (without MW ablation). Pre- and postinterventional volume analyses were performed. Effective coagulation volumes (original tissue included in coagulation) were determined. Postinterventional dehydration analyses were performed with calculation of mean mass fractions of water. RESULTS: Mean deployed energies were 21.6 kJ ± 1.1 for group 1A, 29.9 kJ ± 1.0 for group 2A, and 42.1 kJ ± 0.5 kJ for group 3A, and were significantly different (P < .0001). Differences between pre- and postinterventional volumes were -3.8% ± 0.6 for group 1A, -5.6% ± 0.9 for group 2A, and -7.2% ± 0.4 for group 3A, and -1.1% ± 0.3 for group 1B, -1.8% ± 0.4 for group 2B, and -1.1% ± 0.4 for group 3B. Postinterventional volumes were significantly smaller than preinterventional volumes for all groups (P < .01). Underestimations of effective coagulation volume from visualized coagulation volume were 26.1% ± 3.5 for group 1A, 35.2% ± 11.2 for group 2A, and 42.1% ± 4.9 for group 3A, which were significantly different (P < .01). Mean mass fractions of water were 64.2% ± 1.4 for group 1A, 63.2% ± 1.7 for group 2A, and 62.6% ± 1.8% for group 3A, with significant differences versus corresponding control groups (P < .01). CONCLUSIONS: For MW ablation in kidneys, underestimation of effective coagulation volume based on visualized coagulation volume is significantly greater with greater deployed energy. Therefore, local dehydration with tissue shrinkage is a potential contributor.

Identification of circulating fetal cell markers by microarray analysis.

OBJECTIVE: Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester. METHOD: Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem cell microarray analysis. RESULTS: Twenty-eight genes were identified as fetal cell marker candidates. CONCLUSION: Of the 28 fetal marker candidate genes, five coded for proteins, which are located on the outer surface of the cell membrane and not expressed in blood. The protein product of these five genes, MMP14, MCAM, KCNQ4, CLDN6, and F3, may be used as markers for fetal cell enrichment.

Cervical collagen concentration within 15 months after delivery.

OBJECTIVE: Cervical collagen concentration decreases during pregnancy. The increased risk of preterm birth after a short interpregnancy interval may be explained by an incomplete remodeling of the cervix. The objective of this study was to describe the changes in cervical collagen concentration over 15 months after delivery. STUDY DESIGN: The collagen concentrations were determined in cervical biopsy specimens that were obtained from 15 women at 3, 6, 9, 12, and 15 months after delivery. RESULTS: The mean cervical collagen concentrations were 50%, 59%, 63%, 65%, and 65% of dry weight (SD, 4.2-6.5). This increase was statistically significant until month 9, but not between months 9 and 12. CONCLUSION: Low collagen concentrations in the uterine cervix may contribute to the association between a short interpregnancy interval and preterm birth.

[Down's syndrome risk assessment in Denmark--secondary publication]. / Risikovurdering for Downs syndrom i Danmark--sekundaerpublikation.

In 2004 The Danish National Board of Health introduced a new guideline regarding prenatal screening. All pregnant women are now offered a Down's syndrome risk assessment. The new guideline has had an impact on the number of invasive early prenatal procedures. The number of procedures fell by 50% from 2000 to 2006. 90% of the foetuses with Down's syndrome are detected prenatally. Denmark is one of the first countries in the world in which risk assessment for Down's syndrome has been successfully implemented at a national level.