Bernadette Bossers

Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia

OBJECTIVE To determine first‐trimester fetal sex by isolating free fetal DNA from maternal plasma. METHODS The index case was a pregnant woman who previously delivered a girl with congenital adrenal hyperplasia. The SRY gene as a marker for the fetal Y chromosome was detected in maternal serum and plasma by quantitative polymerase chain reaction analysis. Simultaneously, we performed the same test in 25 and 19 women in the first and second trimester, respectively, and compared plasma results with fetal gender as assessed by prenatal karyotyping or as seen at ultrasound or birth. RESULTS In 44 of 45 patients at gestational ages ranging from 8 3/7 to 17 3/7 weeks, we correctly predicted fetal sex using quantitative polymerase chain reaction analysis of the SRY gene in maternal plasma. In one case, the test result was inconclusive. Overall, fetal sex was correctly predicted in 97.8% of cases (95% confidence interval 88.2%, 99.9%). CONCLUSION Amplification of free fetal DNA in maternal plasma is a valid technique for predicting fetal sex in early pregnancy. In case of pregnancies at risk for congenital adrenal hyperplasia, the technique allows restriction of dexamethasone treatment to female fetuses resulting in a substantial decrease of unnecessary treatment and invasive diagnostic tests.

Reliability of Fetal Sex Determination Using Maternal Plasma

OBJECTIVE: To determine the diagnostic accuracy of noninvasive fetal sex determination in maternal plasma. METHODS: All consecutive patients for whom fetal sex determination in maternal plasma was performed in our laboratory from 2003 up to 2009 were included in the study. Real-time polymerase chain reaction was performed for the SRY gene and multicopy DYS14 marker sequence. A stringent diagnostic algorithm was applied. In the case of a positive result for both Y chromosome–specific assays, a male-bearing pregnancy was reported. In the case of a negative result, the presence of fetal DNA was ascertained through the use of 24 biallelic insertion/deletion polymorphisms or paternally inherited blood group antigens. Only if the presence of fetal DNA was confirmed was a female-bearing pregnancy reported. Results were compared with the pregnancy outcomes. RESULTS: A total of 201 women were tested. The median gestational age was 9 0/7 weeks (interquartile range 8 0/7 to 10 0/7 weeks). In 189 of 201 cases (94%), a test result was issued; in 10 cases, the presence of fetal DNA could not be confirmed; in two cases, an early miscarriage was observed. Pregnancy outcome was obtained in 197 cases, including 105 male-bearing and 81 female-bearing pregnancies and 11 miscarriages. Sensitivity and specificity of the test were 100% (95% confidence intervals 96.6–100% and 95.6–100%, respectively). In all 10 cases in which the presence of fetal DNA could not be confirmed, a female was born. CONCLUSION: Noninvasive fetal sex determination in maternal plasma is highly reliable and clinically applicable. LEVEL OF EVIDENCE: III

Use of bi-allelic insertion/deletion polymorphisms as a positive control for fetal genotyping in maternal blood: first clinical experience

Abstract:  Amplification of fetal DNA in maternal plasma is a new way for non‐invasive fetal genotyping in pregnancies at risk for disorders where the presence of a paternal DNA sequence contributes to the risk status of the fetus. We describe the use of a panel of 10 bi‐allelic highly polymorphic markers to ascertain the presence and amplification of fetal DNA in case the fetus is negative for the targeted paternal “disease” sequence.

Analysis of false-positive results of fetal RHD typing in a national screening program reveals vanishing twins as potential cause for discrepancy.

We aim to elucidate causes of false‐positive fetal RHD screening results obtained with cell‐free DNA.

Frequency and characterization of known and novel RHD variant alleles in 37 782 Dutch D-negative pregnant women

To guide anti‐D prophylaxis, Dutch D‐ pregnant women are offered a quantitative fetal‐RHD‐genotyping assay to determine the RHD status of their fetus. This allowed us to determine the frequency of different maternal RHD variants in 37 782 serologically D‐ pregnant women. A variant allele is present in at least 0·96% of Dutch D‐ pregnant women The D‐ serology could be confirmed after further serological testing in only 54% of these women, which emphasizes the potential relevance of genotyping of blood donors. 43 different RHD variant alleles were detected, including 15 novel alleles (11 null‐, 2 partial D‐ and 2 DEL‐alleles). Of those novel null alleles, one allele contained a single missense mutation (RHD*443C>G) and one allele had a single amino acid deletion (RHD*424_426del). The D‐ phenotype was confirmed by transduction of human D‐ erythroblasts, consolidating that, for the first time, a single amino acid change or deletion causes the D‐ phenotype. Transduction also confirmed the phenotypes for the two new variant DEL‐alleles (RHD*721A>C and RHD*884T>C) and the novel partial RHD*492C>A allele. Notably, in three additional cases the DEL phenotype was observed but sequencing of the coding sequence, flanking introns and promoter region revealed an apparently wild‐type RHD allele without mutations.

Fetal RHD genotyping after bone marrow transplantation

Fetal RHD genotyping allows targeted diagnostic testing, fetal surveillance, and eventually intrauterine treatment to D‐alloimmunized pregnant women who carry an RHD+ fetus. However, false‐positive and false‐negative results of noninvasive prenatal fetal RHD genotyping have been described due to a variety of causes. In this case report we present two cases where noninvasive fetal RHD typing was complicated by a previous bone marrow transplantation (BMT).