Late first-trimester invasive prenatal diagnosis: results of an international randomized trial.

OBJECTIVE: To assess, in a randomized trial, the safety and accuracy of amniocentesis and transabdominal chorionic villus sampling (CVS) performed at 11-14 weeks of gestation, given that this time frame is increasingly relevant to early trisomy screening. METHODS: We compared amniocentesis with CVS from 77 to 104 days of gestation in a randomized trial in a predominantly advanced maternal age population. Before randomization, the feasibility of both procedures was confirmed by ultrasonography, and experienced operators performed sampling under ultrasound guidance; conventional cytogenetic analysis was employed. The primary outcome measure was a composite of fetal loss plus preterm delivery before 28 weeks of gestation in cytogenetically normal pregnancies. RESULTS: We randomized 3,775 women into 2 groups (1,914 to CVS; 1,861 to amniocentesis), which were comparable at baseline. More than 99.6% had the assigned procedure, and 99.9% were followed through delivery. In contrast to previous thinking, in the cytogenetically normal cohort (n = 3,698), no difference in primary study outcome was observed: 2.1% (95% confidence interval 1.5, 2.8) for CVS and 2.3% (95% confidence interval, 1.7, 3.1) for amniocentesis. However, spontaneous losses before 20 weeks and procedure-related, indicated terminations combined were increased in the amniocentesis group (P =.07, relative risk 1.74). We found a 4-fold increase in the rate of talipes equinovarus after amniocentesis (P =.02) overall and in week 13 (P =.03, relative risk = 4.65), but data were insufficient to determine this risk in week 14. CONCLUSION: Amniocentesis at 13 weeks carries a significantly increased risk of talipes equinovarus compared with CVS and also suggests an increase in early, unintended pregnancy loss. LEVEL OF EVIDENCE: I

The effect of the elapsed time between blood draw and processing on the recovery of fetal cells from maternal blood.

OBJECTIVE: To test the hypothesis that a delay in initial fetal cell enrichment processing of maternal blood samples (defined as the time between blood draw and the initial density gradient centrifugation step) compromises the ability to recover fetal cells, we performed a randomized comparison of immediate (within 4 hours of draw) versus delayed (between 18-24 hours of draw) processing. METHODS: Four centers participated: two centers utilized flow cytometry (FLOW), and two centers utilized magnetic-activated cell sorting (MACS) techniques. Each center collected 34 samples. The outcome was the percentage of gamma positive (gamma(+)) cells for FLOW or the number of nucleated red blood cells (NRBCs) for MACS, found in the final enriched cell population. Both outcomes reflect cell properties that are potentially fetal in origin, thus making them representative of the ability to recover fetal cells. RESULTS: Our results did not support our hypothesis that delay in processing compromises fetal cell recovery. Instead, in MACS processing, we observed an increase in recovered NRBCs when blood sample processing was delayed compared with immediate processing. There was no significant difference in gamma(+) cells with FLOW. CONCLUSION: Time-related changes in the density of target cells, perhaps associated with their progress towards apoptosis during the delay period, may result in increased intact fetal cells with the study methods utilized.

Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study.

OBJECTIVES: The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) is a prospective, multicenter clinical project to develop non-invasive methods of prenatal diagnosis. The initial objective was to assess the utility of fetal cells in the peripheral blood of pregnant women to diagnose or screen for fetal chromosome abnormalities. METHODS: Results of fluorescence in situ hybridization (FISH) analysis on interphase nuclei of fetal cells recovered from maternal blood were compared to metaphase karyotypes of fetal cells obtained by amniocentesis or chorionic villus sampling (CVS). After the first 5 years of the study we performed a planned analysis of the data. We report here the data from 2744 fully processed pre-procedural blood samples; 1292 samples were from women carrying singleton male fetuses. RESULTS: Target cell recovery and fetal cell detection were better using magnetic-based separation systems (MACS) than with flow-sorting (FACS). Blinded FISH assessment of samples from women carrying singleton male fetuses found at least one cell with an X and Y signal in 41.4% of cases (95% CI: 37.4%, 45.5%). The false-positive rate of gender detection was 11.1% (95% CI: 6.1,16.1%). This was higher than expected due to the use of indirectly labeled FISH probes in one center. The detection rate of finding at least one aneuploid cell in cases of fetal aneuploidy was 74.4% (95% CI: 76.0%, 99.0%), with a false-positive rate estimated to be between 0.6% and 4.1%. CONCLUSIONS: The sensitivity of aneuploidy detection using fetal cell analysis from maternal blood is comparable to single marker prenatal serum screening, but technological advances are needed before fetal cell analysis has clinical application as part of a multiple marker method for non-invasive prenatal screening. The limitations of the present study, i.e. multiple processing protocols, are being addressed in the ongoing study.

First-trimester trisomy screening: nuchal translucency measurement training and quality assurance to correct and unify technique.

OBJECTIVE: To describe the process of training for measuring nuchal translucency at five clinical centers in North America and to evaluate methods of quality assurance and feedback. DESIGN: Throughout a period of 18 months, the performance of sonographers in measuring fetal nuchal translucency was monitored using qualitative and quantitative methods of review. After 12 months, different approaches (written and personal feedback) were used to inform sonographers of technical aspects that needed to or could be improved. RESULTS: On initial qualitative review, discrepancies in judgment from different reviewers coincided with suboptimal magnification, failure to visualize the amniotic membrane and/or use of cross-shaped calipers. At subsequent global review, 13 (29%) images of nuchal translucency measurements were considered unacceptable. Quantitative assessment revealed that, during the first part of the study, the means from four sonographers were significantly smaller and the mean from the fifth sonographer was significantly larger than expected on the basis of findings from The Fetal Medicine Foundation (P < 0.0001). Following feedback, sonographers who underestimated nuchal translucency and who received a written report only did not change measurements overall (P = 0.9759). In contrast, those who received additional intervention showed a marked difference (P < 0.0001). CONCLUSIONS: Global qualitative review of images from one sonographer may be preferable to assessment of individual aspects of images. Results from global qualitative review correspond well with findings from quantitative analysis, indicating that the latter can be applied for ongoing audit. Observation of divergent results should prompt extensive personal feedback, rather than a written report, to prevent sonographers from settling in their own, inappropriate technique.

Extra structurally abnormal chromosomes (ESACs)--presentation of 10 new cases.

BACKGROUND: The goal of the presented studies as a retrospective reliability assessment of classical banding cytogenetic studies and of prognosing epicrises in a group of 14 cases, affected with additional marker chromosomes. MATERIAL AND METHODS: Having collected the study material from peripheral blood, by means of trophoblast biopsy or amniocentesis, cytogenetic preparations were obtained, allowing for pre- or postnatal evaluation of the karyotype. A panel of auxiliary cytogenetic techniques accompanied the routine CTG protocol. RESULTS: In a group of 6875 persons with recommendations to pre- or postnatal cytogenetic diagnostics, 14 (0.2%) cases of ESACs were diagnosed. In 5 cases of DA/DAPI(+) inv dup (15) as observed. A presence of polymorphic interstitial RHG(+) band was found within the marker chromosome. The measured size of that band allowed associating it with either the presence or the absence of pathological signs. In 9 cases of ESACs, DA/DAPI(-), the application of banding techniques (NOR and CBG) allowed to discover bisatellite heterochromatic ESACs in 6 cases (2 non-mosaic and 4 mosaic). In three other mosaic and non-satellite cases of ESACs, a 'genetic inactivity' of the marker chromosome was observed in one case, while a 'genetic activity' was ascertained in two cases. The 'activity' of marker chromosomes was studied by means of replication banding techniques. CONCLUSIONS: At the time of the outburst of molecular techniques, still up-to-date is the use of classical banding techniques and of the replication techniques, allowing DNA replication kinetics studies at the level of single band.

Phenotypic analysis of adhesion molecules in first-trimester decidual tissue from chorion villus samples.

PROBLEM: First-trimester decidua contain CD56bright/CD16neg lymphocytes that phenotypically resemble a subset of peripheral blood natural killer (NK) cells. Factor controlling their localization to decidua are unknown, but they may relate to trophoblast invasion, endometrial decidualization, and adhesion molecule expression. METHOD OF STUDY: Ninety-one chorion villous samples (CVS) were screened for the presence of decidua, and selected specimens were analyzed for the expression of adhesion molecule pairs using a panel of monoclonal antibodies. RESULTS: Lymphoid cells in CVS-derived decidua expressed CD56, PECAM, intracellular adhesion molecule-1 (ICAM-1), and the integrins LFA-1, alpha E beta 7, and alpha 4 beta 1, and co-receptors for these molecules were expressed by decidual stromal cells, invasive trophoblasts, and venule endothelium. Some endothelium expressed a cuboidal phenotype. CONCLUSIONS: The expression of complimentary pairs of adhesion molecules by maternal lymphoid cells and by either extravillous cytotrophoblast or decidual stromal cells supports the hypothesis that these cells interact within decidua. Also, both LFA-1 and alpha 4 beta 1 may contribute to decidual localization of CD56bright NK cells.

Ring chromosome 4 mosaicism coincidence of oligomeganephronia and signs of Seckel syndrome.

We present a patient with features suggestive of Seckel syndrome who was found to be mosaic for ring 4 chromosome. Seckel syndrome is a rare entity characterized by marked growth retardation, microcephaly, facies characterized by receding forehead and chin, large beaked nose, and severe retardation, usually thought to be inherited as an autosomal recessive condition. In addition, our patient had oligomeganephronia, a rare and usually sporadic renal malformation, previously reported in two other patients with abnormalities of chromosome 4. Besides pointing out the overlap between the Seckel phenotype and Wolf-Hirschhorn syndrome, our patient illustrates the need to consider cytogenetic studies in patients with the Seckel phenotype, so that accurate diagnoses can be given to families. Also, the case suggests that there may be a locus for oligomeganephronia distal to the Wolf-Hirschhorn critical region on 4p.

Prenatal diagnosis using fetal cells isolated from maternal peripheral blood: a review.

Many questions remain about the feasibility of using fetal cells from maternal blood for prenatal diagnosis. Although recently there has been more focus on clinically relevant methods, many studies have been performed using blood drawn after invasive procedures, and over a wide range of gestational ages. For methods to be applicable to clinical use, more work is needed on isolating cells early in pregnancy, when termination is still an option for parents who are found to have an affected pregnancy. It is generally agreed that fetal nucleated erythrocytes are the most efficacious cell type for prenatal diagnosis, but it has not yet been shown definitively whether there is an ideal gestational age for sampling, whether ABO incompatibility might limit availability of fetal cells, or whether the number of cells present might be different in normal versus abnormal pregnancies. PCR has been shown to be a powerful tool in allowing amplification and identification of very small amounts of fetal DNA. However, this is limited to cases in which a specific and unique gene from the father is sought. This means that there is the potential to diagnose many paternally inherited autosomal dominant diseases and some autosomal recessive diseases, in which the parents have different and identifiable mutations. However, when parents are both carriers of the same autosomal recessive mutations, or when the disease is X linked, PCR will not aid in prenatal diagnosis. Cytogenetic analysis of fetal cells by FISH after cell sorting is another potentially useful method of prenatal diagnosis, but requires relatively pure samples of fetal cells or an independent marker that allows easy microscopic identification. The latter might be accomplished by identifying fetal cells through their expression of embryonic hemoglobins or because they contain HLA-G mRNA. In addition, current techniques of cell sorting must be improved so that a higher percentage of fetal cells can be isolated. Currently, the best cell sorting techniques usually produce a maximum purity of 10% fetal cells. Commonly, in normal pregnancies, fewer than 0.1% of the cells isolated after sorting are fetal in origin. Improving the concentration and quantity of fetal cells will improve the accuracy of FISH. Methods such as immunophenotyping that allow the selective identification of fetal cells by microscopy, and can be used in conjunction with FISH, may be extremely valuable because they may allow the genetic analysis of only the few fetal cells within a background preponderance of maternal cells. Although the retrieval of fetal cells from maternal blood is an attractive concept, it must be clearly stated that presently it is only in the investigational phase because of the low sensitivity and specificity. There is no current application for these methods in clinical practice. It remains to be determined whether testing maternal blood for fetal cells or DNA will be used as a screening tool, similar to the maternal serum screening currently in use, or whether the accuracy can be improved to a level such that the techniques can be used diagnostically. Although there are many questions that remain unanswered at this time, the outlook for noninvasive prenatal genetic testing in the future is optimistic.

Voice abnormalities in short stature syndromes.

Patients with short stature have a high incidence of voice and laryngeal abnormalities. In sixteen patients with short stature of various etiologies, these abnormalities appeared to be unrelated to hearing loss or other otolaryngologic problems. However, in many patients they were associated with typical symptoms and signs of voice abuse. Physicians and speech-language pathologists caring for patients with short stature should be alert for voice problems, and should consider instituting early voice education, diagnosis and treatment.

Isolation of decidual lymphocytes from chorionic villus samples: phenotypic analysis and growth in vitro.

PROBLEM: Giemsa stained cell isolates prepared from chorionic villus samples (CVS) contain granulated cells morphologically similar to large granular lymphocytes. METHOD: Phenotypic characterization of these cellular isolates by two-color immunofluorescence and subsequent in vitro culture in the presence of recombinant interleukin-2 (rIL-2) were done in order to determine whether CVS could serve as a source of decidual lymphocytes. RESULTS: A major fraction of the CVS-derived lymphocytes were characterized as decidual NK cells, exhibiting high levels of CD56 expression (CD56+bright), without concomitant expression of CD16. The T cell population present in CVS-derived lymphocytes contained both CD4+ and CD8+ cells in a ratio somewhat reduced compared to that found in peripheral blood. While both T cells and CD56+bright cells from CVS proliferate in vitro in response to rIL-2 alone, preferential growth of CD56+bright cells was accomplished using a selective culture technique wherein co-culture with an irradiated, B lymphoblastoid cell line promoted the growth of CD56+ cells. CONCLUSION: CVS contains decidual NK cells and T cells that proliferate in response to rIL-2 and/or third party stimulator cells. These culture techniques will allow investigations into the maturation and/or activation of decidual NK cells and T cells.

Growth manifestations in the Brachmann-de Lange syndrome.

We have obtained serial measurements on 180 patients with clinically confirmed Brachmann-de Lange syndrome (BDLS) in order to derive standard growth curves. The patients were evaluated in our genetics department and through meetings of the Cornelia de Lange Syndrome Foundation, a support group for families of affected individuals. The data were obtained from the records of pediatricians, other physicians, schools and parents, as well as from personal examination on each of these patients at least once, often periodically. The growth curves include height, weight and head circumference measurements from birth through adulthood. Prenatal growth and birth weights are below the 5th centile in most (68%) cases, with an average birth weight of 2,277 g. Growth persists below the normal curves in most of the patients throughout life. Height velocity is equal to the normal range but there is slower pubertal growth. Weight velocity is below the normal range throughout life until late adolescence. Average head circumference remains below the second centile. Thin body habitus coupled with slow growth and proportionate small stature is a manifestation of the syndrome, but is commonly mistaken for failure to thrive.

Developmental data on individuals with the Brachmann-de Lange syndrome.

One hundred twenty-two patients with clinically confirmed Brachmann-de Lange syndrome (BDLS) were evaluated developmentally. Recruitment was made from our genetics department and through meetings of the Cornelia de Lange Syndrome Foundation parent support group. Developmental information was obtained from records of physicians, schools and developmental centers, or from parents on each of the 122 individuals, allowing division into four groups for study: group 1 (n = 48) underwent formal developmental assessments, which generated intelligence or developmental quotients, and had a completed parental questionnaire with specific developmental questions regarding ages of skills mastered; group II (n = 23) had additional developmental records available without formal testing, as well as the questionnaire; group III (n = 22) had only a completed questionnaire; and group IV (n = 29) had formal developmental testing or other developmental records but no available questionnaire. These data were analyzed in order to be able to predict attainable psychomotor development. Average scores on formal testing were found to be in the mild to moderate level of mental retardation, ranging from below 30 to 85, with an average intelligence quotient of 53, higher than previously reported. Visual-spatial memory and perceptual organization skills were found to be strengths. Younger individuals born before 1980 demonstrated higher scores on testing. Early intervention appears to play a major role in the level of developmental achievement.

Cytokine production in first trimester chorionic villi: detection of mRNAs and protein products in situ.

Growth factor cytokines must play important roles in placental growth and differentiation. We used cDNA-mRNA in situ hybridization to examine whether human first trimester chorionic villi could produce these substances locally. IL-1 beta and IL-4 mRNA was found in both trophoblast layers, whereas CSF-1 and TNF alpha were found mostly in syncytiotrophoblast with much less localization to cytotrophoblast areas. IFN-gamma message was exclusively found in the cytotrophoblast. Immunohistochemical analysis confirmed that cytokines are produced by placental tissue, and the patterns of expression correlated well with the data obtained by in situ hybridization. These data are discussed regarding possible roles of cytokines in regulation of MHC antigen expression by trophoblasts and immunomodulation of decidual lymphocytes.

Genetic syndromes and uniparental disomy: a study of 16 cases of Brachmann-de Lange syndrome.

Uniparental disomy is responsible for a proportion of cases in Prader-Willi, Angelman, and Wiedemann-Beckwith syndromes. In these syndromes, the chromosomes involved are thought to contain one or more imprinted genes. When two copies of the imprinted (inactivated) gene are inherited from a single parent through uniparental disomy or the active gene is deleted, the phenotype of the syndrome results. Our goal is to identify additional syndromes caused by uniparental disomy. Our approach is to select syndromes that appear to have more than one mode of inheritance and are occasionally associated with a cytogenetic abnormality. Given this criterion, we have chosen Brachmann-de Lange Syndrome (BDLS) to investigate since the phenotype is similar to that found in patients with dup(3q). We have studied 16 probands with BDLS and their parents using a multiplex of four PCR-based polymorphic loci on chromosome 3. None of the probands studied had uniparental disomy for chromosome 3 and all demonstrated normal biparental inheritance for at least one locus. Given these results, uniparental disomy of chromosome 3 does not appear to be a major contributor to the syndrome. Additionally, both maternally and paternally derived chromosome abnormalities have resulted in the dup(3q) phenotype and dominant inheritance of BDLS from both mildly affected mothers and fathers have been reported which suggests that imprinting is not involved in these syndromes.

Chromosome errors as a cause of spontaneous abortion: the relative importance of maternal age and obstetric history.

OBJECTIVE: To evaluate the likelihood of obtaining a chromosome diagnosis in cases of spontaneous abortion (SAB) and of the relative importance of maternal age versus obstetric history in predicting the fetal karyotype. DESIGN: Obstetric history was obtained from all 100 cases of miscarriage in 1 year when products of conception were sent for chromosome studies. Multiple logistic regression analysis was used to calculate odds ratio and statistical significance for correlations between historical factors and the probability of any chromosomal abnormality or trisomy. RESULTS: A chromosome diagnosis was made in 84% of cases. Maternal age was a more important predictor of chromosome abnormality, specifically trisomy, than history of previous livebirths or miscarriages. CONCLUSION: Results from chromosome studies using chorionic villi from SABs are diagnostically useful, even when the patient has a history of repeated miscarriages.

A randomized comparison of transcervical and transabdominal chorionic-villus sampling. The U.S. National Institute of Child Health and Human Development Chorionic-Villus Sampling and Amniocentesis Study Group.

BACKGROUND: Chorionic-villus sampling is done in early pregnancy to obtain fetal cells for the prenatal diagnosis of genetic and chromosomal defects. Transcervical chorionic-villus sampling has been shown to be safe and effective in national trials. Recently, an alternative transabdominal technique has been suggested as potentially easier and safer. METHODS: From April 1987 through September 1989, we prospectively compared transcervical and transabdominal chorionic-villus sampling in 3999 women with singleton pregnancies in whom the risk of a genetically abnormal fetus was increased. Women between 7 and 12 weeks of gestation underwent ultrasonographic evaluation of placental and uterine position. Those with active vaginal infections, active bleeding, or cervical polyps were excluded. If the obstetrician thought either sampling procedure was acceptable, the woman was asked to consent to random assignment to one of the two procedures. Both groups were followed to determine the outcome of pregnancy and the rate of spontaneous fetal loss after chorionic-villus sampling. RESULTS: Among the 3999 women who entered the study, sampling was attempted in 3873 (97 percent), 1944 of whom had been assigned to undergo transcervical sampling and 1929 to undergo transabdominal sampling. Of these 3873 women, sampling was eventually successful in 3863. Sampling was successful after a single insertion of the sampling instrument in 94 percent of the transabdominal procedures and 90 percent of the transcervical procedures. Among the women with cytogenetically normal pregnancies who had sampling because of maternal age, the rate of spontaneous fetal loss through 28 weeks of pregnancy was 2.5 percent in the transcervical-sampling group and 2.3 percent in the transabdominal-sampling group (difference, 0.26 percent; 95 percent confidence interval, -0.5 to 1.0 percent). CONCLUSIONS: Transabdominal and transcervical chorionic-villus sampling appear to be equally safe procedures for first-trimester diagnosis of fetal abnormalities.