Cell-free DNA analysis in maternal plasma in cases of fetal abnormalities detected on ultrasound examination.

OBJECTIVE: To evaluate the utility of noninvasive prenatal testing using cell-free circulating fetal DNA for detection of the three main autosomal fetal trisomies in the setting of ultrasonographically identified fetal anomalies. METHODS: Nine hundred patients at risk for fetal aneuploidy with or without ultrasonography anomalies and who underwent invasive procedures were included in the study. Cell-free DNA analysis was performed by massive parallel sequencing during a multicenter, noninterventional, prospective study and the results were compared with a fetal karyotype. RESULTS: Among all 900 pregnancies, cell-free DNA identified 76 of 76 (100%) fetal Down syndrome, 22 of 25 (88%) trisomy 18, and 12 of 12 (100%) trisomy 13. In those with a normal ultrasonogram and normal cell-free DNA analysis, karyotype identified 2 of 483 (0.4%) additional aneuploidies other than trisomies 13, 18, and 21. In those with an abnormal ultrasonogram and a normal cell-free DNA analysis, there were 23 of 290 (7.9%) additional pathogenic karyotypes. These additional aneuploidies included sex chromosome abnormalities and triploidy. The rates of additional aneuploidies not identifiable by standard cell-free DNA screening in the two groups is significantly different at P<.01. CONCLUSION: In women with fetal abnormalities by ultrasonography, the rate of pathogenic chromosome abnormalities missed by cell-free DNA was 8%. Noninvasive prenatal testing should not be offered to women with fetal abnormalities because a negative result is falsely reassuring. LEVEL OF EVIDENCE: III.

De novo monosomy 9p24.3-pter and trisomy 17q24.3-qter characterised by microarray comparative genomic hybridisation in a fetus with an increased nuchal translucency.

OBJECTIVES: Increased nuchal translucency (NT) during the first trimester of pregnancy is a useful marker to detect chromosomal abnormalities. Here, we report a prenatal case with molecular cytogenetic characterisation of an abnormal derivative chromosome 9 identified through NT. METHODS: Amniocentesis was performed because of an increased NT (4.4 mm) and showed an abnormal de novo 46,XX,add(9)(p24.3) karyotype. To characterise the origin of the small additional material on 9p, we performed a microarray comparative genomic hybridisation (microarray CGH) using a genomic DNA array providing an average of 1 Mb resolution. RESULTS: Microarray CGH showed a deletion of distal 9p and a trisomy of distal 17q. These results were confirmed by FISH analyses. Microarray CGH provided accurate information on the breakpoint regions and the size of both distal 9p deletion and distal 17q trisomy. The fetus was therefore a carrier of a de novo derivative chromosome 9 arising from a t(9;17)(p24.3;q24.3) translocation and generating a monosomy 9p24.3-pter and a trisomy 17q24.3-qter. CONCLUSION: This case illustrates that microarray CGH is a rapid, powerful and sensitive technology to identify small de novo unbalanced chromosomal abnormalities and can be applied in prenatal diagnosis.

Prenatal diagnosis of megacystis-microcolon-intestinal hypoperistalsis syndrome: contribution of amniotic fluid digestive enzyme assay and fetal urinalysis.

OBJECTIVES: Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a usually lethal disease during the first year of life. There is no specific ultrasound prenatal diagnosis and no identified genetic locus. The value of amniotic fluid digestive enzyme assay and fetal urine biochemistry in the prediction of MMIHS was analysed. METHODS: Retrospective study of 14 MMIHS cases. Amniotic fluid digestive enzymes and fetal urine biochemistry were compared in MMIHS and megabladder (63 and 264 cases respectively). RESULTS: Abnormal amniotic fluid digestive enzyme profile (vomiting of bile or digestive secretion leakage) was observed in 8/10 MMIHS cases. These profiles were observed in 7/63 controls; 80% sensitivity (95%CI = 55%-100%); 89% specificity (95%CI = 81%-96%). Fetal urinalysis was normal in 12/12 MMIHS cases except high calcium (>0.6 mmol/l). This profile was observed in 33/264 megabladder control cases; 100% sensitivity; 98.7% specificity (95%CI = 83.5%-91.5%). CONCLUSION: For the first time, we propose a prenatal diagnosis of MMIHS based on amniotic fluid digestive enzyme assay and on fetal urinalysis.

Fetal RhD genotyping by maternal serum analysis: a two-year experience.

OBJECTIVE: The purpose of this study was to determine the accuracy of the none-invasive prenatal determination of polymerase chain reaction (PCR)-based fetal RhD genotyping. STUDY DESIGN: A prospective case series was undertaken on all RhD-negative pregnant women presenting for genetic counseling in our prenatal diagnosis center from January 2001 until December 2002. Results were compared with serologic RhD typing of the newborns. RESULTS: Among the 285 pregnant women who participated in the study, fetal RhD status could be determined for 283 patients. In 2 cases, the RhD-negative phenotype of the mother was not the result of a complete RHD gene deletion, and therefore, the status of the fetus could not be determined. Neither false-negative nor false-positive results were observed. CONCLUSION: The present report demonstrates that a reliable fetal RHD genotype determination can be achieved with 100% accuracy. It is therefore possible to consider that such an assay could be systematically proposed to all RhD-negative pregnant women in order to more effectively utilize RhD prophylaxis.

Fetal DNA in maternal serum: does it persist after pregnancy?

Fetal DNA and cells present in maternal blood have previously been used for non-invasive prenatal diagnosis. However, some fetal cells can persist in maternal blood after a previous pregnancy. Fetal rhesus status and sex determination have been performed by using amplification by real-time polymerase chain reaction (PCR) of fetal DNA sequences present in maternal circulation; no false-positive results related to persistent fetal DNA from a previous pregnancy have been reported. This idea has recently been challenged. An SRY real-time PCR assay was performed on the serum of 67 pregnant women carrying a female fetus but having previously given birth to at least one boy and on the serum of 30 healthy non-pregnant women with a past male pregnancy. In all cases, serum was negative for the SRY gene. These data suggest that fetal DNA from a previous pregnancy cannot be detected in maternal serum, even by using a highly sensitive technique. Therefore, non-invasive prenatal diagnosis by fetal sex determination for women at risk of producing children with X-linked disorders, and fetal RHD genotyping is reliable and secure as previously demonstrated.

Fetal RHD genotyping in maternal serum during the first trimester of pregnancy.

Fetal RHD genotype determination is useful in the management of sensitized RhD-negative pregnant women. It can be ascertained early during pregnancy by chorionic villus sampling (CVS) or amniocentesis. However, these procedures are invasive, resulting both in an increased risk of fetal loss and in an increased severity of immunization due to fetomaternal haemorrhage. A reliable determination of RHD genotype by fetal DNA analysis in maternal serum during the first trimester of pregnancy is reported in this study. One hundred and six sera from RhD-negative pregnant women were obtained during the first trimester of pregnancy. These sera were tested for the presence of RHD gene using a new real-time polymerase chain reaction assay and the results compared with those obtained later in pregnancy on amniotic fluid cells and by RHD serology of the new-born. All sera from women carrying a RhD-positive fetus (n = 62) gave positive results for RHD gene detection and sera from women carrying a RhD-negative fetus (n = 40) were negative. The high level of accuracy of fetal RHD genotyping obtained in this study could enable this technique to be offered on a routine basis for the management of RhD-negative patients during the first trimester of pregnancy.