Lucia Tului

Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples

We report the results of the first major study of applying quantitative fluorescence polymerase chain reaction (QF-PCR) assays for the detection of major chromosome numerical disorders. The QF-PCR tests were performed on a total of 247 chorionic villus samples, which were analysed blind, without any knowledge of the results obtained using conventional cytogenetic analysis. The aims of this investigation were to evaluate the detection power and accuracy of this approach by testing a large number of fetal samples and to assess the diagnostic value of each of the chromosome specific small tandem repeat (STR) markers used. In addition, we introduced three more markers specific for chromosomes 13, 18, and X to allow an accurate analysis of samples homozygous for a particular STR. Fluorescent labelled primers were used to amplify 12 STRs specific for chromosomes 21, 18, 13, X, and the amylogenin-like DNA sequence AMXY, expressed on the X and Y chromosomes. In this blind study of 247 fetal samples, 222 were correctly diagnosed by QF-PCR as normal for each of the five chromosomes investigated; 20 were diagnosed by QF-PCR as trisomic for chromosomes 21, 18, or 13, in agreement with the cytogenetic tests. Only one false negative result was observed, probably owing to the mishandling of the sample, which had been transferred through three laboratories before being analysed by QF-PCR. The 247 samples also included four cases of mosaicism or translocation; one case of mosaic trisomy 21 was detected by QF-PCR and the other cases were not identified by QF-PCR. The results of this investigation provide clear evidence that the QF-PCR assays are powerful adjuncts to conventional cytogenetic techniques and can be applied for the rapid and accurate prenatal diagnosis of the most frequent aneuploidies.

International, collaborative experience of 1789 patients having multifetal pregnancy reduction: a plateauing of risks and outcomes.

Objective: To develop the most up-to-date, complete data base of multifetal pregnancy reduction (MFPR) from cases, and to provide the best counseling for couples with multifetal pregnancies. Methods: From nine centers in five countries, 1789 completed MFPR cases were collected and outcomes evaluated. Pregnancy losses were defined as through 24 weeks and deliveries categorized in groups of 25-28, 29-32, 33-36, and 37 or more weeks. Results: Overall, the pregnancy loss rate was 11.7% but varied from a low of 7.6% for triplets to twins and increased with each additional starting number to 22.9% for sextuplets or higher. Early premature deliveries (25-28 weeks) were 4.5% and varied with starting number. Loss rates by finishing number were highest for triplets and lowest for twins, but gestational age at delivery was highest for singletons. Conclusions: Multifetal pregnancy reduction has been shown to be a safe and effective method to improve outcome in multifetal pregnancies. Outcomes are worse with higher-order gestations and support the need for continued vigilance of fertility therapy.

Chorionic villus sampling and amniocentesis.

Purpose of review The advantages and disadvantages of common invasive methods for prenatal diagnosis are presented in light of new investigations. Recent findings Several aspects of first-trimester chorionic villus sampling and mid-trimester amniocentesis remain controversial, especially fetal loss rate, feto-maternal complications, and the extension of both sampling methods to less traditional gestational ages (early amniocentesis, late chorionic villus sampling), all of which complicate genetic counseling. A recent randomized trial involving early amniocentesis and late chorionic villus sampling has confirmed previous studies, leading to the unquestionable conclusion that transabdominal chorionic villus sampling is safer. The old dispute over whether limb reduction defects are caused by chorionic villus sampling gains new vigor, with a paper suggesting that this technique has distinctive teratogenic effects. The large experience involving maternal and fetal complications following mid-trimester amniocentesis allows a better estimate of risk for comparison with chorionic villus sampling. Summary Transabdominal chorionic villus sampling, which appears to be the gold standard sampling method for genetic investigations between 10 and 15 completed weeks, permits rapid diagnosis in high-risk cases detected by first-trimester screening of aneuploidies. Sampling efficiency and karyotyping reliability are as high as in mid-trimester amniocentesis with fewer complications, provided the operator has the required training, skill and experience.

RhD genotyping by quantitative fluorescent polymerase chain reaction: a new approach

Objective To develop a new method of RhD/d genotype determination using a quantitative fluorescent PCR (QF‐PCR) assay.

Prenatal diagnosis by chorionic villus sampling

Chorionic villus sampling (CVS) retains its great advantage over mid-trimester amniocentesis by producing early results. Moreover, rapid analytical techniques reduce significantly the waiting time between sampling and diagnosis, while recombinant DNA technology and human gene mapping progress amplify enormously the spectrum of the indications. The recent inclusion in the prenatal diagnosis package of screening tests based on DNA analysis for the major genetic diseases (i.e. cystic fibrosis, fragile-X mental retardation syndrome) may efficiently contribute to prevent the genetic disease. The role of CVS in twin pregnancy has been investigated and compared to amniocentesis. Although these techniques are equally safe, CVS should be considered the approach of choice for a number of technical advantage and in relation to selective fetal reduction in discordant twins. Recent reports have substantially contributed on the hypothetical relationship between limb reduction defects (LRDs) and chorion biopsy. The analysis of LRDs among more than 130,000 CVS reported to WHO CVS-Registry has been unable to find out any relationship between sampling and fetal malformations, including LRDs. In conclusion, first trimester CVS should be considered the gold standard procedure for prenatal diagnosis of genetic diseases.

The Use of Non-physiological Conditions to Isolate Fetal Cells From Maternal Blood

Although fetal cells are consistently present in maternal blood from the first trimester on, there is no simple, rapid, and reliable procedure for isolating these cells so that genetic studies can be done noninvasively. The small number of circulating fetal cells and their loss during enrichment procedures create problems in recovering them. The investigators describe a single-step method for isolating fetal cells from maternal blood, relying on nonphysiological conditions to alter cell density before separating them using density gradient centrifugation and a new cell separation device. The procedure is grounded in findings that fetal nucleated red blood cells (NRBCs) from cord blood can be isolated up to purity. Isolated fetal cells were examined by cytochemistry, Soret band absorption microscopy, fluorescence in situ hybridization (FISH), and immunostaining with monoclonal antibodies for e- and γ-chain-hemoglobin and for i-antigen. Fetal cells were identified in all 105 maternal blood samples examined. The mean number of nucleated cells isolated from maternal blood was 3.2 X 10 6 . All samples studied were positive for benzidine-stained nucleated cells, for e- and γ-stained NRBCs, and for I-antigen. Fetal gender was correctly predicted in all but 1 of 38 samples analyzed by the polymerase chain reaction technique. Trisomic cells were present in all 8 aneuploid pregnancies. They also were identified in cases in which blood was sampled before any invasive procedure. It is possible to consistently isolate fetal cells from maternal blood if sophisticated equipment or technically demanding procedures are available. The present study shows that fetal cells also can be obtained from maternal blood in numbers sufficient for noninvasive prenatal study using simple laboratory equipment and procedures.

Transabdominal and Transcervical Chorionic Villus Sampling: Efficiency and Risk Evaluation of 2411 Cases

Efficacy and risks of transcervical and transabdominal chorionic villus sampling (CVS) methods were evaluated in 1,501 and 910 cases, respectively. We reported a success rate of more than 99% for both sampling procedures and obtained an adequate amount of chorionic tissue in more than 96% of cases on the first attempt. However, the transcervical method was more difficult to learn and was contraindicated by a higher number of unfavorable clinical and anatomical conditions. Transabdominal sampling proved to be a less time-consuming procedure and could also be used after the 12th week of gestation. Bleeding/spotting (10.1%) and uterine cramping (2.5%) were the most frequent early complications following transcervical and transabdominal sampling, respectively. Uterine infection was rare (0.13%) and was reported only after cervical aspiration. No significant effects on fetal growth, preterm delivery, placenta disorders, congenital defects, and perinatal mortality following transcervical and transabdominal sampling, and for both procedures total fetal loss rate was less than 4%. This experience suggests that transabdominal needle sampling is the method of choice; however, transcervical aspiration appears more likely to succeed in a limited number of well-defined conditions.