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Biomedical subjects

D Van Opstal

Publications and source records attributed to D Van Opstal.

At least 19 recordsLinked to original sources
PubMed2007

FISH analysis of 15 chromosomes in human day 4 and 5 preimplantation embryos: the added value of extended aneuploidy detection.

OBJECTIVE: Screening for an increased number of chromosomes may improve the detection of abnormal embryos and thus contribute to the capability of preimplantation genetic screening (PGS) to detect the embryo(s) for transfer in IVF with the best chance for a healthy child. Good-quality day 4 and 5 embryos were analyzed after cryopreservation for the nine chromosomes mostly recommended for screening (13, 14, 15, 16, 18, 21, 22, X and Y), next to six additional chromosomes which are less well studied in this context (1, 2, 7, 6, 10 and 17). METHOD: The copy numbers of 15 chromosomes were investigated by fluorescence in situ hybridization (FISH) in three consecutive rounds. The proportion of aneuploid and mosaic embryos was determined and compared in retrospect to results in case only the recommended probe set had been analyzed. RESULTS: A total of 52 embryos from 29 infertile women were analyzed. Screening the embryos for six additional chromosomes increased the proportion of abnormal embryos from 67 to 81% (P = 0.03), owing to an increase in mosaic embryos. CONCLUSION: All but one of the meiotic aneuploidies found in this study would have been detected by the probe set most frequently used in PGS clinics. However, aneuploid cell lines originating from mitotic errors could be detected for almost all chromosomes, so screening of six additional chromosomes mainly increased the proportion of mosaic embryos. The added value of screening for six additional chromosomes in PGS for clinical practice will remain undetermined as long as the fate of mosaic embryos after transfer is unclear.

Aneuploidy↗
PubMed2005

Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF.

BACKGROUND: In order to assess the frequency of aneuploidy and mosaicism in embryos obtained from IVF patients aged <38 years, preimplantation genetic screening (PGS) was performed after biopsy of two blastomeres. Furthermore, the reliability of this diagnosis was assessed by performing reanalysis of the embryo on day 5. METHOD: The copy numbers of 10 chromosomes (1, 7, 13, 15, 16, 18, 21, 22, X and Y) were investigated by fluorescence in situ hybridization (FISH) analysis. Embryos that were found to be abnormal or of insufficient morphological quality were cultured until day 5 and reanalysed. Results obtained were compared to the day 3 blastomere analysis. RESULTS: After analysis of 196 embryos (one cell in 38% and two cells in 62%), only 36% of the embryos were found to be normal on day 3. After analysis of two blastomeres, 50% showed chromosomal mosaicism. Comparison of the FISH results from day 3 blastomeres and day 5 embryos yielded an overall cytogenetic confirmation rate of 54%. CONCLUSIONS: The rates of mosaicism and aneuploidy in these embryos from young IVF patients are similar to those published for older women. We found the best confirmation rate after a diagnosis based on two cells, where both blastomeres showed the same chromosomal abnormality. In contrast, after a mosaic diagnosis the confirmation rate was low. The present study provides the first detailed reanalysis data of embryos analysed by PGS and clearly demonstrates the impact of mosaicism on the reliability of the PGS diagnosis.

Adult↗
PubMed2004

Screening for aneuploidies of ten different chromosomes in two rounds of FISH: a short and reliable protocol.

OBJECTIVE: To develop a DNA labelling protocol for the simultaneous detection of five different fluorescent chromosomal DNA probes within one round of hybridisation. In combination with a commercial five-colour probemix for the second round of hybridisation, this results in a fast and reliable Fluorescence in situ Hybridization (FISH) protocol, enabling the detection of 10 chromosomes within a working day. This is especially of use for Preimplantation Genetic Screening (PGS), when only single interphase nuclei are available for analysis and when time is restricted. METHOD: DNA probes were labelled with four different fluorochromes (Pacific Blue, Alexa Fluor 350, Alexa Fluor 594 and Alexa Fluor 488) using an ARES labelling kit, based on a two-step method. Aminoallyl-dUTPs were incorporated by nick translation, followed by chemical linking of the amino-modified fluorescent dye. The fifth colour was achieved by using two fluorescent dyes in the chemical reaction, resulting in dual labelling of the DNA probe and a fluorescence detectable with a specific filter set. These five probes were simultaneously hybridised in a first FISH round, followed by a second hybridisation with a commercial five-colour probemix, thus allowing the detection of chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. The fixation and pre-treatment procedures of the blastomere nuclei were further optimised. RESULTS: Using this labelling and FISH protocol, probe hybridisation efficiency, when tested on lymphocyte nuclei, is 95 to 99%. With the fixation protocol, blastomere nuclei maintain good morphology and show condensed and clear signals even after the second round of hybridisation. CONCLUSION: This labelling method in combination with specific epifluorescence filters, enables an independent detection of five different chromosomes in one round of FISH. The whole process of biopsy, fixation and two rounds of hybridisation with the analysis of ten chromosomes can be completed within a day.

Aneuploidy↗
PubMed2004

Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5.

BACKGROUND: Chromosomal mosaicism in human embryos may give rise to false positive or false negative results in preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Therefore, we have investigated whether the results obtained from a 2-cell biopsy of frozen-thawed embryos and fluorescence in situ hybridization (FISH) analysis are representative for the chromosome constitution of the remaining embryo on day 5. METHODS: Cryopreserved day 3 embryos were thawed and from surviving embryos two blastomeres were biopsied. FISH analysis was performed for chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. After biopsy, the embryos were cultured until day 5 and further analysed using the same probe panels. RESULTS: In all, 17 embryos were available with a diagnosis based on two blastomeres on day 3 and confirmatory studies on day 5. In 10 of these 17 cases the initial diagnosis could be confirmed. However, in only six cases cytogenetic results were concordant. Besides the 10 cases with a 'correct' diagnosis, there were six false positive results and one false negative, all involving mosaicism. CONCLUSIONS: Investigating the chromosomal constitution of two blastomere nuclei offers a good opportunity to study the incidence of chromosomal mosaicism in early embryo development. The confirmation rate of the results obtained on day 3 depends on the interpretation and is higher when considered from a clinical than from a cytogenetic point of view.

Aneuploidy↗
PubMed2001

Follow-up investigations in uncultured amniotic fluid cells after uncertain cytogenetic results.

Fluorescence in situ hybridization (FISH) on uncultured amniotic fluid (AF) cells is a widespread technique for the rapid prenatal detection of specific chromosome aberrations. During a 6-year period (1993-1998) we used FISH for quick follow-up investigations in uncultured AF cells after finding an uncertain chromosome aberration in a first chorionic villus (CV) or AF sample in 79 cases. These FISH results were compared with conventional cytogenetic results of the AF cell cultures in all cases. We found discrepant FISH and cytogenetic results in four instances. In general, FISH on uncultured AF cells proved to be a reliable technique for the rapid differentiation between confined placental mosaicism and true fetal mosaicism, and between pseudomosaicism and true mosaicism, respectively. Uncultured cells may sometimes even better reflect chromosomal mosaicism than cultured cells, since they are not subject to culture induced selection mechanisms. However, we found evidence that exceptional cases of tissue confined mosaicism may go undetected in uncultured cells.

Amniocentesis↗
PubMed2001

The diagnostic performance of cytogenetic investigation in amniotic fluid cells and chorionic villi.

First-trimester chorionic villus sampling has not reached the popularity of second-trimester amniocentesis in prenatal cytogenetic diagnosis, in contrast to initial expectations. We investigated whether a difference in the diagnostic performances of cytogenetic investigation in amniotic fluid (AF) cells and chorionic villi in favour of AF-cells might justify this. Diagnostic performance was measured as laboratory failure rate, karyotype quality (G-band score, rate of follow-up samples, rate of wrong diagnoses), and karyotype representativity (rate of follow-up samples, rate of wrong diagnoses). From 1993-1999, 11 883 AF-samples were investigated (AF-cells). In chorionic villi, short term culture preparations solely were karyotyped from 1993-1996 (n=3499) (STC-villi), short and long-term culture preparations simultaneously provided a sufficient amount of tissue being available from 1997 onwards (n=1829) ((STC+LTC)-villi). Laboratory failure rates were the same after amniocentesis (0.40%) and chorionic villus sampling (0.50%). G-band scores (mean+/-SD) were equal in AF-cells (373+/-38.1) and LTC-villi (364+/-32.6) but significantly lower in STC-villi (311+/-34.6) (p=0.001). Follow-up sampling rates because of quality reasons were the same in AF-cells (0.14%), STC- villi (0.13%) and (STC+LTC)-villi (0.11%). Two wrong diagnoses turned up among AF-cells. Follow-up sampling rates because of representativity reasons differed significantly between AF-cells (0.10%), (STC+LTC)-villi (1.31%), and STC-villi (1.99%) (p<0.001). However, the ratios of the total numbers of follow-up samples and uncertain or abnormal cytogenetic results in STC, and (STC+LTC)-villi at cytogenetic risks > or =3% (0.132 and 0.160, respectively) were equal to that in AF-cells at risks <3% (0.155). Two wrong diagnoses were made in STC-villi. Diagnostic performance improved in the rank order of STC-villi, (STC+LTC)-villi and AF-cells. At cytogenetic risks > or =3%, (STC+LTC)-villi showed a diagnostic performance equal to that in AF-cells. This might justify a selective use of chorionic villus sampling.

Amniocentesis↗
PubMed2001

Management of prenatally detected trisomy 8 mosaicism.

We report on ten pregnancies with trisomy 8 mosaicism. Nine cases were prenatally detected in chorionic villi (n=6), amniotic fluid (AF) cells (n=2) or fetal blood (FB) lymphocytes (n=1). Follow-up laboratory investigations showed confined placental mosaicism (CPM) or pseudomosaicism in eight cases. In one case with ultrasound abnormalities, trisomy 8 mosaicism was detected in FB cells although cultured AF cells showed normal cells only. Another case of mosaic trisomy 8 was prenatally missed; cytogenetic analysis of short-term cultured villi revealed a normal male karyotype, while postnatally, trisomy 8 mosaicism was detected in peripheral blood lymphocytes and skin fibroblasts of the affected child. These findings indicate the difficulties in the prenatal diagnosis of trisomy 8 mosaicism. When found in chorionic villi, it mostly represented CPM, while in a case of true fetal trisomy 8 mosaicism, the cytotrophoblast cells showed a normal karyotype. So, the cytotrophoblast compartment of chorionic villi is a poor indicator of the presence or absence of fetal trisomy 8 mosaicism. Follow-up investigations including amniocentesis and especially fetal blood sampling are required to come to a definite prenatal diagnosis of trisomy 8 mosaicism.

Amniocentesis↗
PubMed2000

Case of 45,X/46,XY mosaicism with non-mosaic discordance between short-term villi (45,X) and cultured villi (46,XY).

We report on a prenatally detected case of discordant non-mosaic karyotypes following chorionic villus sampling. A 45,X karyotype was found in cytotrophoblast cells and a 46,XY karyotype in mesenchymal core cells. A subsequent amniocentesis showed a true 45,X/46,XY mosaicism. Confirmatory studies, including fluorescence in situ hybridization (FISH) in various fetal and placental tissues as well as in the original villi preparations changed the presumed condition of generalized mosaicism with culture confined normality to that of generalized mosaicism with absolute concordance. This case underscores the importance of the investigation of both short-term and cultured villi preparations, the implementation of prenatal FISH studies, and the need for thorough follow-up investigation in cases of discrepant results.

Chorionic Villi Sampling↗
PubMed2000

Accuracy of abnormal karyotypes after the analysis of both short- and long-term culture of chorionic villi.

We report in detail the cytogenetic results of 1838 consecutive chorionic villus samples with the availability of both short-term culture (STC-villi) and long-term culture (LTC-villi) preparations in 1561 cases (84.9%). A high degree of laboratory success (99.5%) and diagnostic accuracy (99.8%) was observed; in four cases of low mosaicism, all four associated with the final birth of a normal child, a small risk of uncertainty was accepted. The combined analysis of STC- and LTC-villi reduced follow-up amniocenteses by one-third in comparison with the analysis of STC-villi alone. We believe that the desired level of quality and accuracy of prenatal cytogenetics in chorionic villi can only be achieved when both STC- and LTC-villi are available. We conclude that CVS might then be the mode of prenatal diagnosis of first choice in pregnancies with a high (cytogenetic) risk.

Amniocentesis↗
PubMed1999

Nasopharyngeal teratoma and mosaic tetrasomy 1q detected at amniocentesis. A case report and review of the literature.

The occurrence of nasopharyngeal teratomas (NPT) is an infrequent event and prenatal detection of such tumors is even rarer. We present a case report and review of the literature (N = 78 cases), in which we describe the cytogenetic, DNA, and pathological findings of a fetus with a mature NPT which was detected prenatally by ultrasound investigation following complaints of severe polyhydramnios by the mother.

Adult↗
PubMed1999

Amniocentesis or chorionic villus sampling in multiple gestations? Experience with 500 cases.

500 women with multiple pregnancies underwent amniocentesis or chorionic villus (CV) sampling at our department between January 1988 and July 1997. The aim of this retrospective study was to evaluate the laboratory aspects and the consequences of discordant results in these pregnancies in relation to the method of sampling. Uncertain results in one or both samples, requiring further investigation were more frequent in CV samples (eight times in 163 paired samples, 5 per cent) than in amniotic fluid (AF) samples (once in 298 paired samples, 0.3 per cent). Sampling one fetus twice (erroneous sampling) was seen only once among 163 pregnancies with two CV samples in our study. Cross contamination due to mixed sampling was discovered in two of seven pregnancies that underwent DNA diagnosis in CV and might be a rather regular occuring phenomenon. In none of the 500 pregnancies mixed sampling caused diagnostic dilemmas. A third sampling problem, maternal cell contamination caused a diagnostic problem once among the AF samples. Selective fetal reduction appeared safer after CV sampling than after amniocentesis. Subsequently, CV sampling instead of amniocentesis has become the method of choice for prenatal diagnosis in multiple pregnancies in our department.

Amniocentesis↗
PubMed1998

Abnormal karyotypes in semi-direct chorionic villus preparations of women with different cytogenetic risks.

Among 3499 cytogenetically investigated semi-direct chorionic villus samples, 219 (6.3 per cent) abnormal karyotypes were encountered. The karyotypes were considered certainly abnormal (generalized abnormal with high probability) in 109 cases (3.1 per cent), and in 110 cases (3.1 per cent) uncertainly abnormal (potentially confined to the placenta), requiring further investigation. Of these 110 uncertain abnormalities, the cytogenetic result turned out to be finally abnormal representing generalized abnormality in 36 cases (32.7 per cent), finally normal representing confined placental mosaicism (CPM) in 69 cases (62.7 per cent), and remained undetermined in 5 instances (4.5 per cent). The rate of the numbers of certainly abnormal and all (certainly + uncertainly) abnormal results, the certainty rate, and that of generalized abnormalities and all abnormalities (generalized abnormalities + CPM cases), the predictive value, are strongly correlated with the cytogenetic risk. Therefore, we advise chorionic villus sampling for cytogenetic investigation only in women with a cytogenetic risk equal to or exceeding that of a 40-year-old pregnant woman. Because of the high rate of prenatal follow-up investigations after the finding of uncertain results in semi-direct villi, semi-direct and cultured villi should be karyotyped simultaneously.

Adult↗
PubMed1998

Prospective prenatal investigations on potential uniparental disomy in cases of confined placental trisomy.

In most reported cases of uniparental disomy (UPD) associated with confined placental mosaicism (CPM), a high level of mosaicism or a full trisomy was found in chorionic villi. At the time that we started our investigations, it was not quite clear whether fetal UPD also existed in the more frequently occurring low levels of mosaicism. During a 4-year period, a follow-up amniocentesis was performed in all cases of mosaic or non-mosaic trisomy detected in chorionic villus (CV) semi-direct preparations and suspected to be confined to the placenta. We performed fluorescent in situ hybridization (FISH) on uncultured amniotic fluid cells to differentiate between generalized mosaicism and CPM. We found 29 cases of CPM and we determined the incidence of UPD in 23 of these cases. Normal biparental chromosome contributions were found in 22 cases. In one case, we detected a maternal heterodisomy for chromosome 16. UPD appeared to be a rare phenomenon in the cases of CPM (type I and/or type III) that we encountered in 3958 consecutively investigated CV samples, and is not the cause of the pregnancy complications found in seven out of 23 cases with CPM.

Amniocentesis↗
PubMed1997

Full monosomy 21, prenatally diagnosed by fluorescent in situ hybridization.

We describe a case of full monosomy 21 which was prenatally diagnosed in chorionic villi by fluorescent in situ hybridization (FISH). Because of intrauterine fetal death, a curettage was performed and cytogenetic analysis of skin fibroblasts confirmed the presence of monosomy 21 in fetal cells. DNA investigations showed a paternal origin of the single chromosome 21. Inspection and autopsy of the fetus revealed several congenital malformations. Some of them have been reported in earlier studies of monosomy 21; others concern new observations. Regarding the eye, the following abnormalities were microscopically observed: absence of the anterior and posterior eye chambers, aniridy, a hypoplastic ciliary body, Peter's anomaly, and a double retina with secondary dysplasia. In addition, malformations of the extremities were seen: partial, proximal syndactyly of digits 3 and 4 of the right hand; pes varus position of the right foot; and transverse reduction defect at the tarsals of the left foot. To our knowledge, this is the first case in which full monosomy 21 has been proven.

Abnormalities, Multiple↗
PubMed1997

Prenatal diagnosis of trisomy 9: cytogenetic, fish, and DNA studies.

A cytogenetic survey and follow-up studies were performed in eight cases of full, mosaic, and pseudomosaic trisomy 9 prenatally diagnosed among 36,213 prenatal samples in our department between August 1970 and July 1996. Besides conventional chromosome analysis, interphase fluorescent in situ hybridization (FISH) was employed. FISH turned out to be a rapid and accurate method for verification of trisomy cell lines and could provide additional information to the prenatal cytogenetic results. FISH also enables the study of uncultured specimens of amniotic fluid, not accessible for traditional cytogenetic analysis. In three cases, retrospective DNA analysis showed the supernumerary chromosome 9 to be of maternal origin. The disomic cell lines in both mosaic trisomy 9 cases showed maternal uniparental disomy.

Alleles↗
PubMed1997

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection.

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.

Chromosome Aberrations↗
PubMed1996

Ring chromosome 18 in a fetus with only facial anomalies.

We report on a prenatally detected case of ring chromosome 18 [46,XX,r(18)] in amniotic fluid cells of a fetus with an abnormal facial profile on ultrasound as the only malformation. The chromosome 18 origin of the ring chromosome, of a supernumerary marker chromosome in some cells, and of micronuclei was demonstrated by fluorescent in situ hybridization with a whole chromosome 18 paint (Cambio) and 18 centromere probe L1.84. DNA investigations showed deletions of 18p as well as 18q material of r(18), which turned out to be of paternal origin. Autopsy of the fetus after termination of pregnancy at 20 weeks of gestation showed no additional malformations, in agreement with the previous ultrasound findings.

Abortion, Induced↗