Chorionic Villus Cultures Research Articles

572: The use of multiplex ligation-dependent probe amplification (MLPA) versus karyotype analysis in the investigation of pregnancy loss

amplification (MLPA) versus karyotype analysis in the investigation of pregnancy loss Danielle O’Leary, Keelin O’Donoghue Cork University Maternity Hospital, University College Cork, Obstetrics and Gynecology, Cork, Ireland OBJECTIVE: Karyotyping is important in pregnancy loss as about 6% of stillborn babies will have a chromosomal abnormality, while in couples with recurrent miscarriage, chromosomal abnormalities of the embryo account for 30-60% of further miscarriages. Some abnormalities are potentially recurrent and can be tested for in future pregnancies. Traditionally cytogenetic investigation of pregnancy loss is performed by culture of chorionic villi and karyotype analysis on fetal material. This technique has a high failure rate (reported at 1040%), associated with misdiagnosis due to overgrowth of maternal tissue and is also labour intensive. The technique of multiplex ligation-dependent probe amplification (MLPA) reportedly has a higher success rate, ranging from 85-95%, and a quicker reporting time. We aimed to compare the success rate and time taken for reporting with MLPA and karyotype analysis and to compare the number of normal and abnormal results and indications for testing for both techniques. STUDY DESIGN: Tissue samples sent for MLPA testing between October 2011-June 2012 and for karyotype analysis in the corresponding months in 2010-2011 were recorded. Reports were examined and time taken for processing and results calculated. RESULTS: 102 tissue samples were sent for MLPA testing, compared to 60 samples for karyotype analysis. Indications included investigation of unexplained stillbirth (26/162), late miscarriage (53/162) and recurrent miscarriage (76/162). The success rate of MLPA analysis was higher at 89%, in contrast to 73% for karyotype analysis. MLPA had a quicker reporting time, on average 9.2 days compared to 16.3 days. CONCLUSION: Our findings confirm the higher success rates and quicker reporting time with MLPA analysis versus karyotype by tissue culture. The lower cost per sample is also advantageous in times of sparse resources. From these data, we recommend the continued use of MLPA analysis as the cytogenetic investigation of choice in pregnancy loss. 573 Extremely high risk of fetal death and severe perinatal complications associated with simultaneously elevated AFP and very low unconjugated estriol David Krantz, Burton Rochelson, Jon Carmichael, Terrence Hallahan, Nidhi Vohra, Stephanie Augustine PerkinElmer, NTD Labs, Melville, NY, North Shore Long Island Jewish Heath System, Maternal Fetal Medicine, Manhassett, NY OBJECTIVE: To determine the risk of severe anomalies and perinatal complications in pregnancies associated with a simultaneously elevated AFP and very low unconjugated estriol (UE3). STUDY DESIGN: A total of 6,365 second trimester ONTD/Down syndrome screening tests from a single hospital performed between March 2010 and February 2012 were analyzed. Outcomes were obtained from pregnancies with simultaneously elevated AFP ( 2 MoM) and a very low UE3 ( 1 %ile). RESULTS: There were 139 elevated AFP results (2.2%) and 72 (1.1%) very low UE3 results. After excluding 1 case of anencephaly, 9 patients had simultaneously elevated AFP and very low UE3 results. There were 3 cases of fetal loss and 2 cases of pregnancy termination due to severe fetal anomalies or severe pregnancy complications. In the remaining 4 cases, 3 pregnancies were associated with IUGR and admittance to the NICU and one pregnancy was associated with oligohydramnios and small placenta but did not require admittance to NICU. CONCLUSION: Both elevated AFP and low UE3 are known to be associated with increased risk of fetal anomalies and pregnancy complications. Based on this case series, when both of these results occur simultaneously the pregnancy is at significant risk for severe fetal anomalies, fetal death and NICU admission at birth. Even if severe anomalies are not immediately diagnosed in these patients, continued close monitoring of the pregnancy until delivery is still warranted.

Protein Kinase Profiling in Miscarriage: Implications for the Pathogenesis of Trisomic Pregnancy

ObjectiveTrisomy in pregnancy increases risks of miscarriage, fetal anomalies, and perinatal complications, with trisomy 16 the most common trisomy in human conceptions. The pathogenesis and protein expression profiles in trisomic pregnancy have not been well elucidated. Our objective was to profile protein kinase expression in trisomic and chromosomally normal miscarriage. MethodsPlacental chorionic villus cultures were set up from first-trimester miscarriages (n = 73). Protein kinases (n = 75) were profiled using a 2D Western blot in cultures from trisomic miscarriages (n = 6) (trisomy 16 or trisomy 15) and compared to chromosomally normal (euploid) miscarriages (n = 4). ResultsDistinct patterns of protein kinase expression were seen in the two trisomic groups, including dosage-dependent overexpression of the chromosome 16-encoded ERK1 in trisomy 16. This supports a role for chromosome-specific effects in the pathogenesis of trisomy (gene dosage hypothesis). In addition, both trisomic groups had increased inter-individual variation in protein kinase expression, which supports a role for amplified sensitivity to environmental and genetic variation in the pathogenesis of trisomy (amplified instability hypothesis). ConclusionBoth gene dosage effects and amplified instability operate simultaneously on the expression of protein kinases in trisomic pregnancies ending in miscarriage.

Developmental Origin of Chorionic Villus Cultures From Spontaneous Abortion and Chorionic Villus Sampling

ObjectiveChorionic villus cultures from spontaneous abortions and chorionic villus sampling (CVS) are routinely used for clinical cytogenetic analysis. Although these cultures are assumed to represent the chorionic villus mesenchymal core, and therefore the inner cell mass (ICM) of the blastocyst, immunochemical studies using a true trophoblast-specific marker to definitively rule out trophoblast contamination have not been done. Therefore, we used cytokeratin-7 (CK7), a trophoblast-specific marker, to assess the developmental origin of these chorionic villus cultures. MethodsWe assessed chorionic villus cultures from CVS and spontaneous abortions for CK7 immunostaining (n = 20). ResultsCultures from both CVS and spontaneous abortions showed little or no CK7 staining (≤1%). ConclusionChorionic villus cultures from CVS and spontaneous abortions exhibit little or no trophoblast contamination. They are therefore representative of the villus mesenchymal core and ultimately originate from the ICM of the blastocyst.

A non‐mosaic tetraploidy in the long‐term culture of chorionic villi with a trisomy 13 in concomitant amniocytes

A non‐mosaic tetraploidy in the long‐term culture of chorionic villi with a trisomy 13 in concomitant amniocytes

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

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

Four years' cytogenetic experience with the culture of chorionic villi

Four years' cytogenetic experience with the culture of chorionic villi

Posttranscriptional regulation of human leukocyte antigen G during human extravillous cytotrophoblast differentiation.

Human maternal tolerance to a semiallogenic fetus may be maintained, in part, by the unusual expression pattern of antigen-presenting molecules in placental trophoblast cells. Extravillous cytotrophoblast (EVC) cells, which invade the maternal decidua, express high levels of human leukocyte antigen G (HLA-G), a nonclassical, major histocompatibility complex (MHC) class I molecule. HLA-G transcripts have been detected in tumors and other tissues, yet protein accumulation is rare. We show that, within EVC cells themselves, the mRNA is more broadly expressed than the protein. Specifically, accumulation of HLA-G protein was markedly delayed during EVC cell differentiation. To elucidate this mechanism, we performed a comprehensive analysis comparing the expression of HLA-G and proteins essential for MHC class I expression at the cell surface. The transporter for antigen processing proteins TAP1 and TAP2, as well as tapasin and beta(2)-microglobulin, appeared to be coordinately expressed throughout EVC cell columns. Strikingly, they all accumulated well in advance of the HLA-G protein but concurrently with its mRNA. A similar delay in the accumulation of the HLA-G protein was observed in vitro, using cultures of chorionic villi. We conclude that posttranscriptional regulation of HLA-G is fundamental to EVC cell development and is achieved independently of the peptide loading system. This represents a novel mechanism of MHC class I regulation.

Four years' cytogenetic experience with the culture of chorionic villi

In 1958 chorionic villus samples, investigated by culture method, we found 137 (7%) abnormalities. The abnormal results were classified in certain abnormal (generalised abnormal at high probability) and uncertain abnormal (potentially confined to the placenta) results. Certain abnormal were 73 cases (3.7%). Uncertain abnormal were 64 cases (3.3%), in which confirmation studies were done in 47 cases. In 12 cases of these 47, the abnormality was confirmed and in 35 cases (1.8%) the abnormality was confined to the placenta. Among the latter cases, poor pregnancy outcome [16% intrauterine death (IUD), 6% intrauterine growth retardation (IUGR)] was increased. Total maternal cell contamination was not seen. The positive predictive value of all confirmed abnormal cases was 66%. The positive predictive value was 100% for indications 'ultrasound abnormalities' and 'carrier' and between 50 and 60% for all other indications. Predictive value among uncertain abnormal cases was low (26%). However, the positive predictive value depends of the type of abnormality. Therefore we conclude that the culture method for chorionic villi is a good test for indications 'ultrasound abnormalities' and 'carrier' and reliable for all other indications. Whether or not follow-up investigations should be offered to the parents depends of the type of abnormality. We conclude that the culture method is reliable for prenatal diagnosis and can be used as the sole investigative method.

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.

Clinical Implications of Atypical Chromosome Abnormalities Diagnosed Prenatally

In Brief Objective To determine the frequency of atypical aneuploidy resulting from prenatal testing and assess the implications of these diagnoses on prenatal decision making. Methods We reviewed all amniotic fluid and chorionic villus samples obtained between January 1994 and September 1997 and grouped the abnormal cases into typical or atypical subcategories. This distinction was based upon whether the diagnosis provided a straightforward range of prognoses or an ambiguous clinical implication. Results were stratified by sample source to determine whether atypical aneuploidy was more commonly seen in cultures of chorionic villi or amniocytes. We also evaluated the influence of ultrasound findings on prenatal decision making in atypical aneuploid cases. Results Of 2960 samples, 134 were abnormal (4.4%), with 27 of 134 abnormalities (20%) representing atypical aneuploidies. The percentages of chorionic villus and amniocentesis cases complicated by atypical aneuploidy (22% and 78%, respectively) were consistent with the distribution of procedures in the entire study. Ultrasound abnormalities did not invariably prompt a decision to terminate pregnancy (only two terminations of six fetuses with congenital malformation), whereas atypical karyotypes led to termination even in the presence of normal-appearing fetal anatomy (five terminations of 21 without malformations; P =.63). Conclusion The frequency of atypical aneuploidy resulting from prenatal diagnosis was approximately 1.0%, and these cases represented 20% of all abnormal karyotypes observed. The ambiguity conferred by atypical aneuploidy can influence a family's decision making, even in the presence of normal ultrasound findings. Atypical aneuploidy, observed in 1.0% of prenatal karyotypes, confers sufficient ambiguity to render counseling and prenatal decision making difficult, even in the absence of associated structural malformations.

Clinical implications of atypical chromosome abnormalities diagnosed prenatally

Objective: To determine the frequency of atypical aneuploidy resulting from prenatal testing and assess the implications of these diagnoses on prenatal decision making. Methods: We reviewed all amniotic fluid and chorionic villus samples obtained between January 1994 and September 1997 and grouped the abnormal cases into typical or atypical subcategories. This distinction was based upon whether the diagnosis provided a straightforward range of prognoses or an ambiguous clinical implication. Results were stratified by sample source to determine whether atypical aneuploidy was more commonly seen in cultures of chorionic villi or amniocytes. We also evaluated the influence of ultrasound findings on prenatal decision making in atypical aneuploid cases. Results: Of 2960 samples, 134 were abnormal (4.4%), with 27 of 134 abnormalities (20%) representing atypical aneuploidies. The percentages of chorionic villus and amniocentesis cases complicated by atypical aneuploidy (22% and 78%, respectively) were consistent with the distribution of procedures in the entire study. Ultrasound abnormalities did not invariably prompt a decision to terminate pregnancy (only two terminations of six fetuses with congenital malformation), whereas atypical karyotypes led to termination even in the presence of normal-appearing fetal anatomy (five terminations of 21 without malformations; P = .63). Conclusion: The frequency of atypical aneuploidy resulting from prenatal diagnosis was approximately 1.0%, and these cases represented 20% of all abnormal karyotypes observed. The ambiguity conferred by atypical aneuploidy can influence a family’s decision making, even in the presence of normal ultrasound findings.

Unilateral Renal Hypoplasia And Contralateral Renal Agenesis: A New Association With 45,X/46,XY Mosaicism

The association of urinary anomalies with Turner's syndrome is well established. This report describes an unusual antenatal presentation of this cytogenetic disorder and the first reported case of unilateral renal hypoplasia and contralateral renal agenesis in a 45,X/46,XY fetus. The fetus presented with severe third trimester oligohydramnios and symmetrical intrauterine growth retardation at 29 weeks and 2 days' gestation. Chorionic villus cultures revealed a 45,X karyotype. A phenotypically male infant weighing 1833 g was delivered at 35 weeks and 2 days. Chromosomal analysis of the newborn showed a 45,X/46,XY mosaicism, and surgical exploration revealed absence of the left kidney and a hypoplastic right kidney. The infant died at 11 months of age from renal failure and peritonitis. This case demonstrates that monosomy X may be encountered in fetuses with marked growth delay and oligohydramnios. The etiology of the oligohydramnios in this case was a fetal renal malformation not previously described in Turner's syndrome. Antenatal cytogenetic findings should be confirmed postnatally, with a search for mosaicism, when monosomy X is encountered.

Immunophenotyping of mitotic cells from long-term cultures of chorionic villi

Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (beta-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture.

Direct analysis of uncultured cytotrophoblastic cells from second- and third-trimester placentas: An accurate and rapid method for detection of fetal chromosome abnormalities

Abstract Transabdominal chorionic villus sampling can be readily used for detection of fetal chromosome abnormalities in the second and third trimesters of pregnancy. Although culture of chorionic villi offers little advantage over cultured amniotic fluid cells with respect to time required to obtain results, cytogenetic analysis of chorionic villi by direct analysis of uncultured cytotrophoblastic cells offers clear advantages because of the very short time required to obtain results. To determine whether direct analysis of uncultured cytotrophoblastic cells from second- and third-trimester placentas can routinely provide rapid and accurate assessment of fetal status, we evaluated chorionic villus specimens obtained from 57 placentas; 49 placentas were sampled in the second trimester whereas eight were sampled in the third trimester. Direct preparations yielded karyotypes in 56 (98.2%) preparations; all results of direct analyses were available within 72 hours and, when requested, within 12 hours. All results were confirmed by chromosome analysis of cultured mesenchymal core cells or cultured fetal tissue. We conclude that direct analysis of cytotrophoblastic cells from second- and third-trimester placentas is a very rapid and accurate method for determining fetal chromosome status that is comparable with, if not superior to, percutaneous umbilical blood sampling. (AM J OesTET GYNECOL 1990;163:1606-9.)

Characterization of long-term cell cultures of human chorion villi and fibroblasts using antibodies to cytoskeletal proteins

In long-term cultures of chorion villi the expression of cytoskeletal proteins--vimentin, cytokeratin, actin, tropomyosin and vinculin--was studied using immunoreactivity to antibodies. Vimentin was detected in all cells of chorionic villi cultures despite their morphological appearance. In comparison to fibroblast cultures from human skin biopsies with bundle-like vimentin structures, chorionic villi cells showed fine ramified vimentin filaments. Some of the chorionic villi cells with typical fibroblastoid appearance also had cytokeratin filaments. No differences were observed in the distribution of vinculin. Tropomyosin structure and actin filaments could be detected in cultures of chorionic villi whereas fibroblasts showed only an intense diffuse staining.

The significance of trisomy 7 mosaicism in chorionic villus cultures

Two cases of mosaic trisomy 7 confined to the cultured cells and not found in direct preparation were detected from 200 consecutive first-trimester chorionic villus samples (CVS) analysed. The mosaicism was similar in the two cases, but the pregnancy outcome was different. In both cases, the direct metaphases from the CVS were 46,XY. Culture metaphases were mos46,XY/47,XY, +7; the trisomy 7 was seen in 34 per cent of cells from case 1 and 53 per cent from case 2. A sonogram at 15 1/2 weeks revealed fetal death in utero in case 1, and the patient declined amniocentesis. The fetal tissue failed to grow in culture, but the placental cultured cells were 47,XY, +7 in 28 (100 per cent) cells analysed. In the second case, all the amniotic fluid cells were 46,XY and the pregnancy resulted in a normal male with a 46,XY karyotype in the cord blood and foreskin fibroblast cultures. The term placenta was mosaic with 13/163 (8 per cent) trisomy 7 cells. Extensive cytogenetic studies on the placenta for the first time confirmed trisomy 7 mosaicism confined to the villus cultures.

Chorionic villus culture for prenatal diagnosis of chromosome defects: reduction of the long-term cultivation time.

We report in detail two series of chorionic villus cultivation for prenatal chromosomal diagnosis. Chorionic villi were sampled from both first- and second-trimester pregnancies. One hundred cultures were treated with trypsin-EDTA for 2 h and collagenase overnight, (method A) and 100 were treated with trypsin-EDTA for 1 h and collagenase for 2 h (method B). Using short-term enzymatic digestion, the cultivation time was reduced from 14 days to 6 days. Sufficient amounts of metaphases of good quality were present in 93 per cent of primary cultures harvested in situ, whereas enough metaphases of sufficiently good quality were in most cases present only after subcultivation of the cultures using method A. The decrease in cultivation time obtained is probably due to a higher yield of viable cells in monocellular suspension, an increased attachment efficiency, and a more rapid attachment of single cells (within 24 h).

Differentiation in human chorionic villus cultures: hCG and hla expression

The present report describes methods to separate, culture, and study syncytio-cytotrophoblast and mesenchymal core of the first-trimester human chorionic villus. The cultured outer layer cells (syncytio-cytotrophoblast) are multinucleated, pleomorphic, and active in the formation of human chorionic gonadotrophin (hCG). The mesenchymal core cells are more fibroblast-like in appearance, do not show multinucleation, and have less hCG in their culture media. Both cultured cell types express HLA (ABC) Class I histocompatibility antigens but not HLA (DR) Class II antigens. These and previous studies from this laboratory postulate different embryonic origins: (1) Syncytio-cytotrophoblast cultures of chorionic villus derive from differentiated trophoblast and preserve multinucleation as well as hCG hormone function. (2) Cells cultured from the chorionic villus core originate from extraembryonic mesenchyme. (3) Amniocytes (AF cells) cultured from amniotic fluid resemble the multipotential and early-stage trophoblast, retaining pleomorphism, multinucleation, and lacunae formation as well as production of hCG, progesterone, oestrogen, basement membrane glycoprotein, and Type IV collagen. These cell types cultured from the chorionic villus and amniotic fluid provide a means for in vitro study of specific embryonic cell lineages.

Prenatal diagnosis of rhizomelic chondrodysplasia punctata

Plasmalogen biosynthesis and phytanic acid oxidation activity were measured in cultured chorionic villus samples or amniocytes from four pregnancies at risk for the rhizomelic form of chondrodysplasia punctata (RCDP). Normal results were obtained in three of the samples and post-natal examination or fetal ultrasound studies confirmed that the fetuses were unaffected. Chorionic villus culture in one case demonstrated defective plasmalogen biosynthesis and lack of phytanic acid oxidation. Pregnancy was interrupted at 10 weeks. Immunoblot studies of post-mortem fetal tissues showed that peroxisomal 3-oxoacyl-coenzyme A thiolase was present in the unprocessed form, a finding we had previously demonstrated in RCDP. These results establish that RCDP can be identified prenatally.

In vitro characteristics of human fetal cells obtained from chorionic villus sampling and amniocentesis.

In vitro characteristics of human fetal cells have been investigated after chorionic villus sampling at the first trimester and amniocentesis at the second trimester of pregnancy. Light microscopy revealed heterogeneous morphology of cell types in both the chorionic villus culture and the amniotic fluid cultures. Based on the experiments performed, chorionic villus cells are more sensitive to pronase, trypsin, and versene during subculture and have a higher DNA content per single cell and release more [125I]-Beta-human chorionic gonadotropin into culture medium than those found in amniotic fluid cells. The practical applications of this study are discussed.