Amniotic Fluid Cell Cultures Research Articles

Limited additional value of karyotyping cultured amniotic fluid cell colonies in addition to microarray on uncultured cells for confirmation of abnormal non-invasive prenatal testing results.

Non-invasive prenatal testing (NIPT) allows the detection of placental chromosome aberrations. To verify whether the fetus also has the chromosome aberration, diagnostic follow-up testing is required. The aim of this retrospective study was to assess the added value of analyzing amniotic fluid (AF) cell cultures in addition to uncultured AF cells for the detection of fetal mosaicism. NIPT was performed as part of the Dutch TRIDENT study. Cytogenetic studies in uncultured AF were performed using single nucleotide polymorphism (SNP)-array. Cultured AF cell colonies (in situ method) were investigated with fluorescent in situ hybridization and/or karyotyping. Clinical outcome data were collected in cases with discordant results. Between April 2014 and December 2021, 368 amniocenteses were performed after a chromosomal aberration was detected with NIPT. Excluding 134 cases of common aneuploidies (confirmed by quantitative fluorescence polymerase chain reaction), 29 cases with investigation of uncultured cells only and 1 case without informed consent, 204 cases were eligible for this study. In 196 (96%) cases, the results in uncultured and cultured cells were concordant normal, abnormal or mosaic. Five cases (2%) showed mosaicism in cultured AF cells, whereas uncultured AF cells were normal. Two (1%) of these, one mosaic trisomy 13 and one mosaic trisomy 16, were considered true fetal mosaics. The added value of investigating AF cell cultures in addition to uncultured cells is limited to two of 204 (1%) cases in which true fetal mosaicsm would otherwise be missed. The clinical relevance of one (trisomy 13) remained unknown and the other case also showed ultrasound anomalies, which determined pregnancy management. This seems to justify limiting prenatal cytogenetic confirmatory testing to SNP arrays on uncultured AF cells, considerably shortening the reporting time.

Application of flat-sided culture tubes during prenatal diagnosis

To assess the value of using flat-sided culture tubes for preparing chromosomes through chorionic villi (CV) and amniotic fluid (AF) cell cultures during prenatal diagnosis. From February to March 2020, 157 CV samples and 147 AF samples subjected to prenatal diagnosis at the Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region were selected as the study subjects. For each sample, one flat-sided tube and one flask culture were set up by following the standard protocols. The methods were evaluated by comparing the cell growth, experimental process, quality of chromosome preparation and costs. The success rates for the culturing of CV and AF samples by the flat-sided culture tube method were 97.45% (153/157) and 97.96% (144/147), respectively. By contrast, the success rates for the conventional flask method were 98.72% (155/157) for CV and 98.64% (145/147) for AF samples. No significant difference was found between the two methods (P > 0.05). The average harvest time required by the flat-sided culture tube method was 8.45 days for CV and 9.43 days for AF cultures, whilst the average harvest time for conventional flask method was 9.05 days and 9.54 days, respectively. The flat-sided culture tube method for CV had required significantly shorter average harvest time than the conventional method (P < 0.001). No statistical significant difference was found in the average harvest time for AF by the two methods (P > 0.05). The conventional culturing method had required three containers with two sample transfers. By contrast, the flat-sided culture tube method was carried out in one tube without any sample transfer. The average total amount of medium used was 3.91 mL for each flat-sided culture tube and 6.26 mL for each conventional flask. The flat-sided culture tube method can provide a simple, cost-effective and error-reducing procedure for the CV and AF samples culture during prenatal diagnosis.

Application of Complete Prenatal Diagnosis in Fetal Ultrasound Abnormalities.

To investigate the value of prenatal diagnosis in fetal ultrasound abnormalities. Amniotic fluid samples of pregnant women who underwent invasive amniocentesis for ultrasound examination suggestive of fetal ultrasound structural abnormalities and soft markers of abnormalities during pregnancy from January 2016 to June 2021 at the Prenatal Diagnostic Center of Putian College Hospital were collected for amniotic fluid cell culture and G manifest band karyotype analysis. (1) Most pregnant women with ultrasound abnormalities were delivered at the age of 23 to 32 years. (2) There were 66 cases of abnormal karyotype with an abnormal incidence of 9.64% (66/685), with chromosomal number abnormalities predominating. (3) Fetal ultrasound abnormalities are mainly soft markers of abnormalities. (4) The comparison of ultrasound abnormalities in the low and high maternal-fetal age groups was not statis-tically significant. (1) Fetal ultrasound abnormalities are mainly soft markers of abnormalities. (2) The rate of fetal chromosomal abnormalities was the highest among the soft ultrasound indicators with NT thickening. (3) The incidence of fetal ultrasound abnormalities did not increase with increasing age. There was no statistical difference in the incidence of chromosomal abnormalities between the low age group and the high age group. (4) Fetal ultrasound testing provides a scientific and accurate basis for clinical heritage counseling and rebirth counseling, especially for chromosomal aneuploidy number abnormalities.

Ultrasound findings in prenatal diagnosis of trisomy 18 associated with elevated levels of maternal serum alpha-fetoprotein

Rationale: Trisomy 18, also referred to as Edwards syndrome, is the second most common autosomal trisomy syndrome. Trisomy 18 can be identified during prenatal screening by the detection of abnormal maternal serum results and one or more structural abnormalities on ultrasound. Previous studies confirmed that levels of pregnancy-related plasma protein A, alpha-fetoprotein, and free β-subunit of human chorionic gonadotropin in the serum of pregnant women carrying fetuses with trisomy 18 were lower than those in women with normal pregnancies. Patient concerns: A 29-year-old pregnant woman with a high risk of trisomy 18 underwent maternal serum screening at 16 weeks of gestation. The patient exhibited an elevated level of maternal serum alpha-fetoprotein (125 U/mL; 3.5225-fold higher than the multiple of the median). Ultrasonography revealed multiple abnormalities. Diagnoses: Culture of amniotic fluid cells revealed a karyotype of 47, XY, +18. Interventions and outcomes: The pregnancy was terminated. Lessons: Trisomy 18 can be identified prenatally by detection of abnormal levels of key proteins in the maternal serum, and detection of one or more structural abnormalities by ultrasound screening. Prenatal serological screening combined with ultrasound can effectively diagnose fetuses with trisomy 18 in the second trimester.

Cytogenetic analysis of 3387 umbilical cord blood in pregnant women at high risk for chromosomal abnormalities

BackgroundCordocentesis in our practice is most commonly indicated for rapid karyotyping in the second or third trimester and is regarded as the gold standard for foetal chromosomal aberration diagnosis in pregnancies at high risk for chromosomal abnormalities. In this study, we investigated 3387 umbilical cord blood samples for karyotyping from pregnant women who underwent cordocentesis and explored the pregnancy outcomes of foetal sex chromosome mosaicism and chromosomal polymorphism.ResultsOut of the 3387 samples, 182 abnormal karyotypes were detected. Ultrasound soft markers were the most common prenatal diagnostic indication, but the detection rate of abnormal karyotypes was 2.02%, while it was 46.97% in the genome-wide NIPT-positive group. The rate of aneuploidy was lower in the soft marker group than in the other groups. Out of 16 cases with sex chromosome mosaicism, three pregnant women with foetuses with a lower proportion of sex chromosome mosaicism delivered healthy foetuses; the foetus with karyotype 46,X,i(Y)(q10)[20]/45,X[6] showed unclear genitals. Three foetuses with chromosomal polymorphisms had postnatal disorders.ConclusionsNIPT should not be recommended as the first-tier screening for chromosomal aberration for pregnancies with ultrasound soft markers or pathological ultrasound findings, but NIPT can be considered an acceptable alternative for pregnancies with contraindications to cordocentesis or the fear of procedure-related foetal loss. Mosaicism found in amniotic fluid cell culture requires further cordocentesis for karyotype confirmation, and the continuation of pregnancy is safe when a normal karyotype is identified in foetal blood culture. Further genetic testing and parental karyotype analysis are needed for foetal chromosomal polymorphisms.

Application of amniotic fluid cell inheritance and re-culture in cases of amniotic fluid with abnormal traits

Objective To evaluate amniotic fluid cell inheritance and re-culture in cases of amniotic fluid with abnormal traits. Methods From January 2014 to December 2018, 15 cases of amniotic fluid with abnormal traits in the First Affiliated Hospital of Anhui Medical University were selected in amniocentesis.Amniotic fluid cells were routinely seeded and cultured for 10 days, then subcultured into other culture bottles.The number of cells and karyotyping after harvesting were counted. Results Seven of 15 cases of amniotic fluid color were slightly darker, 3 cases were pink as water washed meat, and 5 cases were light brown or brown.The average number of cells in original bottles was (2.40±5.87)×105/mL, the average number of cells in inheritance bottles was (2.76±0.64)×106/mL.All 15 samples in the cell inheritance bottles got satisfactory results in cell karyotype analysis. Conclusion Amniotic fluid cell inheritance and re-culture can increase the number of cells in amniotic fluid cell culture and improve the success rate of karyotyping. Key words: Amniocentesis; Amniotic fluid; Prenatal diagnosis; Cell culture techniques; Chromosomes; Cytogenetic analysis; Karyotyping

Fetal chromosomal anomalies in southeast Serbia - single center cohort retrospective study

Congenital anomalies are the cause of prenatal death in 20-25% of the cases, while 3% of children are born with a malformation of varying size. Many of these anomalies can be detected before birth using different non-invasive and invasive prenatal diagnostic tests. This study was used to determine the distribution of genetic disorders in relation to the age of the mother, the frequency of aberrations and to study the effects and importance of prenatal diagnosis in South Serbia. Prenatal diagnostics was performed at the Pediatric Clinic within the Clinical Center of Nis. This retrospective study included a group of 8830 pregnant women, aged between 18 and 47 years during the period from 2004 to 2017. Amniocentesis was performed between the 16th and 18th week of pregnancy and involved the aspiration of 20 ml of amniotic fluid. Isolated cells were cultured in a medium that stimulates cell growth for 10 days. After cytogenetic processing, the obtained karyotype was analyzed using G-banding techniques. In 8830 samples of amniotic fluid cell cultures, 198 karyotypes with chromosomal aberrations were found - 179 with numerical aberrations and 19 with structural aberrations such as translocations, inversions and deletions. There were 85 karyotypes with autosomal numerical aberrations and 32 karyotypes with sex chromosome numerical aberrations. The most frequent one was trisomy 21 (106 cases). The highest number of autosomal numerical aberrations, 84%, was found in pregnancies where maternal age was above 30 years. Preventive action, advice, education and availability of prenatal diagnosis can lead to a significant reduction in the number of children born with various malformations.

Effects of Pharmacological Agents on Human Amniotic Fluid-Derived Stem Cells in Culture.

Amniotic fluid contains multipotent cells and could be a source of stem cells for clinical use. Amniotic fluid cells (AFCs) are made up of a heterogeneous population of fetal cells that can be retrieved during pregnancy without ethical concerns as it is a standard clinical procedure. Studies of proliferation, multipotent marker expression, differentiation, and gene expression were performed after culturing with dexamethasone, valproic acid, and magnesium sulfate. There were correlations between known drug effects on the human fetus and changes seen in human AFCs in culture, as well as previously undescribed observations in neural and chondrogenic inducibility. Gene expression profiles confirmed these observations. AFC culture may provide a novel method to evaluate pharmacological agents before clinical use in pregnancy.

Prenatal diagnosis of complete ring chromosome 22 without phenotypical abnormalities

A healthy 34-year-old, para 1, Taiwanese woman presented for genetic amniocentesis at 18 weeks of gestation due to advanced maternal age. Her previous medical history was unremarkable except for a cesarean delivery following prolonged labor with a failed induction. Prenatal ultrasonography did not detect any structural abnormalities, except for borderline ventriculomegaly (Fig. 1). The karyotype of amniotic fluid cell culture was identified as 46,XY,r(22)(p13q13) (Fig. 2), which was further confirmed by cord blood sampling. Following detailed consultation and nondirective genetic counseling with physicians, the patient and her husband chose to terminate the pregnancy via hysterotomy. The procedure was performed uneventfully at 21 weeks of gestation. A dead male fetus weighing 520 g was delivered from breech presentation without any significant phenotypical abnormalities (Fig. 3) and no structural anomalies were found in an autopsy. Fetal skin and umbilical cord cultures were used to confirm the initial karyotype report postdelivery. Ring chromosome 22 [r(22)] is a rare cytogenetic abnormality first identified by Weleber et al in 1968 [1]. Since that initial report, more than 60 cases have been described in medical literature. Ring chromosomes usually occur when a terminal break in both arms of the chromosome and the broken ends fuse together, or one broken chromosome end joins with the opposite telomeric region. This condition results in the loss of genetic material. Alternatively, ring chromosomes can be formed by the fusion of subtelomeric sequences or telomere-telomere fusion with no deletion, resulting in complete ring chromosomes. Ring chromosomes often lead to developmental anomalies but are

Neuroprotective Effects of GDNF-expressing Human Amniotic Fluid Cells

Brain injury continues to be one of the leading causes of disability worldwide. Despite decades of research, there is currently no pharmacologically effective treatment for preventing neuronal loss and repairing the brain. As a result, novel therapeutic approaches, such as cell-based therapies, are being actively pursued to repair tissue damage and restore neurological function after injury. In this study, we examined the neuroprotective potential of amniotic fluid (AF) single cell clones, engineered to secrete glial cell derived neurotrophic factor (AF-GDNF), both in vitro and in a surgically induced model of brain injury. Our results show that pre-treatment with GDNF significantly increases cell survival in cultures of AF cells or cortical neurons exposed to hydrogen peroxide. Since improving the efficacy of cell transplantation depends on enhanced graft cell survival, we investigated whether AF-GDNF cells seeded on polyglycolic acid (PGA) scaffolds could enhance graft survival following implantation into the lesion cavity. Encouragingly, the AF-GDNF cells survived longer than control AF cells in serum-free conditions and continued to secrete GDNF both in vitro and following implantation into the injured motor cortex. AF-GDNF implantation in the acute period following injury was sufficient to activate the MAPK/ERK signaling pathway in host neural cells in the peri-lesion area, potentially boosting endogenous neuroprotective pathways. These results were complemented with promising trends in beam walk tasks in AF-GDNF/PGA animals during the 7day timeframe. Further investigation is required to determine whether significant behavioural improvement can be achieved at a longer timeframe.

Detection of trisomy 21 by quantitative fluorescent PCR in clinical samples undergoing prenatal diagnosis for hereditary hearing loss

To establish the genetic test technique of trisomy 21 concurrently conducts with prenatal diagnosis for hereditary hearing loss. Fifty-four pregnant women who underwent prenatal diagnosis for hearing loss of their fetuses in Chinese People's Liberation Army General Hospital from March 2009 to May 2010 were enrolled in this study. All probands from the deaf families have confirmed the causative mutation for hearing loss in Genetic Testing Center in Chinese People's Liberation Army General Hospital. The mean age of 54 pregnant women is 31 years at pregnancy of 18 - 26 weeks, 5 cases > pregnancy of 23 weeks, 9 cases ≥ 35 years. All subjects did not conduct the serologic tests for trisomy 21 before. Fifteen to twenty ml amniotic fluid was drawn from 49 cases at pregnancy of 18 - 23 weeks and 5 cases > pregnancy of 23 weeks. One to two ml umbilical blood was drawn from 5 cases > pregnancy of 23 weeks. For 9 cases ≥ 35 years, amniotic fluid cell culture and karyotyping analysis were conducted concurrently. A multiple quantitative fluorescent (QF) PCR and six microsatellite markers were applied to diagnosis trisomy 21. The samples with peaks of 1:1:1 or 2:1 at two microsatellite markers can be diagnosed as trisomy 21. (1) Fifty-four fetuses were successfully conducted prenatal genetic diagnosis for hearing loss (included GJB2 and SLC26A4). Ten fetuses copied the exactly same genotypes as the probands. The other 44 cases fetuses did not copy the same genotypes as the probands and won't develop hearing loss. The hearing test showed normal hearing for the neonates. (2) All the 54 fetuses were excluded of trisomy 21 by QF-PCR and were verified after birth. Five fetuses with advanced maternal age were performed karyotyping analysis and showed normal. The diagnostic results of QF-PCR can be obtained in 1-3 days without misdiagnosed. QF-PCR is an efficient, rapid and accurate technique for detection of trisomy 21 without increasing sample amount. It can be used for fetuses who were undertaken hearing loss gene test or other prenatal gene test.

Array-CGH characterization of a prenatally detected de novo 46,X,der(Y)t(X;Y)(p22.3;q11.2) in a male fetus

We report on an apparently normal 5-month-old boy with a X;Y complex rearrangement identified first on prenatal diagnosis and found on array-CGH to have a 7.6 Mb duplication of Xp22.3 chromosome and a deletion of Yq chromosome, distal to the AZFa locus. Karyotype analysis on amniotic fluid cell cultures revealed a de novo homogenous chromosome marker that we interpreted as an isochromosome Yp. FISH analysis using SRY probe revealed only one signal on the derivative Y chromosome. The final karyotype was interpreted as 46,X,der(Y)t(X;Y)(p22.31;q11.22). Translocation Xp22;Yq11 in male are very rare event and only 4 cases have been published, all showing mental retardation and malformations. Herein we discussed some possible explanation for this apparent phenotypic variability.

A novel method to derive amniotic fluid stem cells for therapeutic purposes

BackgroundHuman amniotic fluid stem (hAFS) cells have become an attractive stem cell source for medical therapy due to both their ability to propagate as stem cells and the lack of ethical debate that comes with the use of embryonic stem cells. Although techniques to derive stem cells from amniotic fluid are available, the techniques have limitations for clinical uses, including a requirement of long periods of time for stem cell production, population heterogeneity and xeno-contamination from using animal antibody-coated magnetic beads. Herein we describe a novel isolation method that fits for hAFS derivation for cell-based therapy.Methods and ResultsWith our method, single hAFS cells generate colonies in a primary culture of amniotic fluid cells. Individual hAFS colonies are then expanded by subculturing in order to make a clonal hAFS cell line. This method allows derivation of a substantial amount of a pure stem cell population within a short period of time. Indeed, 108 cells from a clonal hAFS line can be derived in two weeks using our method, while previous techniques require two months. The resultant hAFS cells show a 2-5 times greater proliferative ability than with previous techniques and a population doubling time of 0.8 days. The hAFS cells exhibit typical hAFS cell characteristics including the ability to differentiate into adipogenic-, osteogenic- and neurogenic lineages, expression of specific stem cell markers including Oct4, SSEA4, CD29, CD44, CD73, CD90, CD105 and CD133, and maintenance of a normal karyotype over long culture periods.ConclusionsWe have created a novel hAFS cell derivation method that can produce a vast amount of high quality stem cells within a short period of time. Our technique makes possibility for providing autogenic fetal stem cells and allogeneic cells for future cell-based therapy.

Telomere aggregates in amniocytes with karyotype of balanced chromosomal rearrangements

Telomeres are TTAGGG repetitions at the ends of chromosomes. Functioning telomeres are essential for normal segregation and maintenance of chromosomes during mitotic and meiotic divisions. Dysfunctional telomeres support the survival of aneuploid cells, a characteristic of many human malignancies. In contrast to the non-overlapping nature of telomeres in normal nuclei, telomeres of tumor nuclei tend to form aggregates. In this study, our objective was to evaluate the number of telomere aggregates (TAs) in karyotype-balanced structural rearrangements. This is an additional parameter of genetic instability, which might suggest a possible increased risk for diseases related to genomic instability, such as cancer. Twenty-six amniotic fluid cell cultures were established following genetic amniocentesis. Telomere FISH protocol was applied to the samples. Telomere aggregates were counted using a 2D microscope. The results were statistically tested by analysis of variance (ANOVA) and Kruskal-Wallis tests. More telomere aggregates in the structural balanced rearrangements were found in both study groups (balanced translocations and inversions) compared to the control group (P < 0.05). The persistence of TAs in cells is probably related to Breakage-Bridge-Fusion (BBF) cycles, a mechanism of TAs described by Muller and McClintock, resulting in end-to-end fusion that contributes to the onset of genomic instability. BBF cycles contribute to deletions, gene amplification, non-reciprocal translocations, and overall genetic changes associated with tumor genesis. According to our studies, the individuals who are carriers of balanced structural chromosomal rearrangements show some of the genetic instability parameters that appear in other circumstances, such as premalignant and malignant conditions.

Rapid diagnosis with FISH for chromosomal abnormality of fetal pyelectasia

Fluorescence in situ Hybridization (FISH) was used to investigate whether the chromosome of the fetus prenatally diagnosed as pyelectasis was normal or not. Amniotic fluid was taken from the pregnant woman whose fetus was detected with pyelectasia by prenatal examination. The chromosome of the amniotic fluid cell without culture was examined with FISH. The result shows that compared with the traditional amniotic fluid cell culture, FISH has the advantages of more rapid, higher sensitivity and specificity, and was 10–12 days earlier to complete the diagnosing than the traditional method. The fetuses detected chromosomal abnormality in each groups were induced during the middle and late trimester, while those fetuses with normal chromosome continued pregnancy, the rate of spontaneous disappearance of pyelectasia decreased as the severity of pyelectasia increased. FISH can satisfy the urgent need in the clinical prenatal diagnosis due to its rapidity to determine whether fetus with pyelectasia was accompanied with chromosomal.

Chromosome 11 segmental paternal isodisomy in amniocytes from two fetuses with omphalocoele: new highlights on phenotype–genotype correlations in Beckwith–Wiedemann syndrome

Background: The phenotypic variability in Beckwith–Wiedemann syndrome (BWS) reflects the genetic heterogeneity of the mechanism which by default leads to the deregulation of genes located at 11p15.5. Genotype–phenotype correlation studies...

Triploidies in First and Second Trimesters of Pregnancies in Turkey

Triploidies in First and Second Trimesters of Pregnancies in TurkeyTriploidy is the most common chromosomal abnormality in human conceptions that cause fetal loss before 20 weeks of gestation and has an incidence of 1 in 10,000 live births. Placental changes, fetal malformations and intrauterine growth retardation (IUGR) occur in triploid pregnancies. We performed karyotype analyses of 75 spontaneous abortions and 1,114 amniotic fluid cultures by conventional methods between 2002 and 2005 at our cytogenetics laboratory. Triploidy was detected in five spontaneous abortions and four amniotic fluid samples. Abortions were between the 9th and 13th weeks of pregnancy, and cytogenetic analyses revealed a 69, XXY karyotype in four of the aborted fetuses and a 69, XXX karyotype in one. Amniotic fluid cell cultures revealed a 69, XXX karyotype in three cases and 69, XXY in one. Amniocentesis was performed in four patients because of an increased trisomy 18 risk detected in maternal serum screening. In pregnant women undergoing second trimester amniocentesis, increased risk for trisomy 18 may be due to triploidy and should be taken into account during genetic counseling.

An absolute procedure to test the growth potential of medium and the influence of decreased oxygen tension in primary amniotic fluid cell cultures

For prenatal cytogenetic diagnosis, cell cultures should be maximally successful. When introducing a change in conditions, e.g. a new batch of medium, the growth potential of a culture is usually compared under both the new condition and the one already in use. Such a relative test is in principle subject to drift and may over time increasingly lead to rejection of new adequate conditions, c.q. good batches of medium. We therefore wanted to design an absolute test to assess the quality of a new condition for amniotic fluid (AF) in situ cell culturing. We tested batches of medium under sub-optimal (stress) conditions, expecting that differences in growth potential would thereby be more readily observed. In our stress test, we diluted the culture medium to the extent of achieving a 50% growth reduction. Thresholds for rejecting a new condition were empirically determined, based on the acceptance of a less than 1% probability of false rejection of a good condition. Testing three cultures per patient for ten patients, i.e. 30 cultures in total, in a medium diluted to 30% of the original concentration, showed that a minimal number of 23 successful cultures and an average number of three or more colonies per culture appeared as thresholds meeting our rejection criteria. Testing five different media resulted in the rejection of one. Using the same stress test to evaluate the effect of culturing under decreased oxygen tension showed that 2.5 and 5% oxygen tension caused a larger colony size. We designed a sensitive absolute test to assess the quality of culturing conditions for cells to be used in prenatal diagnosis in general and in particular to test the growth potential of different batches of culture medium.

Quality aspects of prenatal cytogenetic diagnosis: determining the effect of various factors involved in handling amniotic fluid and chorionic villus material for cytogenetic diagnosis

To investigate the effect of factors involved in cell culturing and slide preparation of amniotic fluid (AF) and chorionic villus biopsies (CVB) for prenatal cytogenetic diagnosis. The effect on the outcome of our standard AF cell culture procedure of volume and appearance of the submitted AF specimen, gynaecologist performing the amniocentesis, week of gestation in which the specimen was taken and culture medium was retrospectively investigated. In a prospective study controlled experimental variation was introduced in composition of fixative, relative humidity, temperature and airflow during slide preparation from primary CVB and AF in situ cultures. For evaluation, analysis of regression or variance was used. Provided that at least 0.8 mL AF per culture dish was admitted, none of the investigated factors appeared as critical resulting in unacceptable variation in outcome. Variation in appearance of the AF had a relatively major impact: bloody or brown AF resulted in a 3 days longer culture time. To a limited degree, metaphase quality of AF and CVB cells was affected by composition of fixative, relative humidity, ambient temperature and airflow during slide preparation. Current prenatal cytogenetic practice as described here appears in general to be robust and reliable. The investigated conditions are not critical within the investigated range. Expensive measures for fine control of these conditions are, therefore, not required.

Recurrent Trisomy 21 and Uniparental Disomy 21 in a Family

Objective: A 32-year-old pregnant woman was referred to our genetic counselling because of recurrent trisomy 21 in the family. Analysis of amniotic fluid cell culture revealed karyotype 47,XY+21 of the fetus. Methods: Karyotyping and molecular analysis were undertaken in the fetal and parental samples to determine the origin of the extra chromosome 21. Results: Both parents had a normal blood karyotype. Microsatellite marker analysis showed maternal origin of the fetal extra chromosome 21. As the mother showed homozygosity for all investigated markers on chromosome 21, we also tested her family. We detected the same homozygosity in some family members which was consistent with isodisomy of the chromosome 21 caused by uniparental disomy (UPD). Conclusions: Here we report on a family in which multiple aneuploid conceptions occurred with trisomy 21, and molecular analysis showed that the euploidy of the investigated healthy family members is due to UPD21. This observation stresses the importance of prenatal cytogenetic and molecular analysis in case of parental UPD.