Very early prenatal diagnosis of genetic diseases based on coelomic fluid analysis: a feasibility study.

BACKGROUND: Coelocentesis may represent the ideal technique for very early prenatal diagnosis. Although cell density in coelomic fluid (CF) is very low, the results of analyses on the cellular compartment have been proposed for prenatal diagnosis. METHODS AND RESULTS: We aimed to evaluate the amount of total DNA (i.e. cellular and cell-free) in 14 samples (0.4-0.8 ml) of CF, taken from women at 8- to 9-week gestation, who are about to undergo termination of pregnancy, and to assess the feasibility of multiple single-gene analyses using multiplex real-time PCR. We found that the amount of total DNA in the CF was very low and varied widely. Genetic testing using multiplex real-time PCR was successfully achieved in 10 of 14 samples (71%). However, when considering samples that could provide a reliable prenatal diagnosis (i.e. successful PCR analysis and no marked maternal contamination), reliable CF-DNA-based prenatal diagnoses were obtained in only 8 of the 14 (58%) samples. CONCLUSION: The development of highly reliable procedures adapted to pauci-cellular CF is crucially needed before coelocentesis could be proposed for early prenatal diagnosis of genetic diseases before 10 weeks.

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.

Cytomegalovirus (CMV) glycoprotein B genotype and CMV DNA load in the amniotic fluid of infected fetuses.

OBJECTIVE: To assess the value of both cytomegalovirus (CMV) DNA quantification in amniotic fluid (AF) and CMV glycoprotein B (gB) genotype as prognostic factors in CMV congenital infection. METHODS: Quantification of CMV DNA was performed prospectively by real-time PCR and gB genotypes were analysed by gene sequencing analysis in the amniotic fluid of 42 fetuses infected by CMV. These were correlated with clinical data on fetal anomalies and outcome. RESULTS: The proportion of severely symptomatic fetuses was similar in each gB genotype group. Median CMV DNA load was higher in the group of symptomatic fetuses but this difference was not significant and high and low viral loads were found in both groups. CONCLUSION: Neither gB genotype nor CMV DNA load in AF correlate with the severity of CMV congenital infection and these markers are unlikely to prove useful for the management of congenital infection.

Chimeric LNA/DNA probes as a detection system for real-time PCR.

OBJECTIVE: Evaluation of the use of short probes as a detection system in real-time PCR. DESIGN AND METHOD: Comparison of results obtained with hybridization probes with and without locked nucleic acid (LNA) residues in the detection of fetal DNA in maternal serum. RESULTS: The use of chimeric LNA/DNA probes led to a slight but significantly higher level of sensitivity as well as a higher level of fluorescence signal. CONCLUSION: Chimeric LNA/DNA probes offer an interesting alternative detection method in real-time PCR.

Detection and quantification of Mycoplasma genitalium in male patients with urethritis.

Detection and quantification of Mycoplasma genitalium were evaluated in 83 patients with urethritis (group 1), 60 patients with urethral symptoms but no urethritis (group 2), and 50 asymptomatic men (group 3). Quantification of M. genitalium was carried out using real-time polymerase chain reaction (PCR) analysis of first-pass urine samples. The rate of detection of M. genitalium was significantly higher in group 1 than in groups 2 and 3 (P<.0001). The mean observed concentration of M. genitalium was 1.2x10(4) equivalent genomes/mL of urine (range, 50 to 8x10(4) equivalent genomes/mL). Analysis of M. genitalium load in serial urine samples collected before and after the administration of antibacterial treatment showed an association between clinical and microbiological responses, with a shift to negative PCR results in symptom-free patients. Our results illustrate the usefulness of monitoring the M. genitalium load in evaluating the susceptibility of M. genitalium to antibacterial treatment.

Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes.

BACKGROUND: Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. METHODS: Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. RESULTS: Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. CONCLUSION: We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

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.

Clinical applications of fetal sex determination in maternal blood in a preimplantation genetic diagnosis centre.

BACKGROUND: Couples with a risk of transmitting X-linked diseases who are included in a preimplantation genetic diagnosis (PGD) programme need early and rapid fetal sex determination in two situations. The first situation is for the control of embryo sexing after PGD and the second situation is for those couples having a spontaneous pregnancy before the start of their PGD cycle. Among invasive techniques, chorionic villus sampling is the earliest procedure for fetal sex determination and molecular analysis of X-linked genetic disorders during the first trimester but it is associated with a risk of fetal loss. Non-invasive procedures such as ultrasound examination allow reliable fetal sex determination only during the second trimester. Reliable fetal sex determination can now be realised by using SRY gene amplification in maternal blood. METHODS AND RESULTS: We report the use of fetal sex determination from maternal serum as a diagnostic tool for the control of embryo sexing (two cases) and to manage spontaneous pregnancies in couples included in a PGD programme for X-linked diseases (five cases). Fetal sex determination using SRY gene amplification in maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis or ultrasound examination and at birth. This novel strategy allowed the PGD results to be controlled precociously and avoided the performance of invasive procedures in four cases of female fetus. CONCLUSIONS: This rapid fetal sex determination during the first trimester provides advantages to both clinicians and patients in a PGD centre.