- Email: [email protected]
Analysis of placental transcriptome in patients with preeclampsia at preterm
S116 SMFM Abstracts 193
194
December 2003 Am J Obstet Gynecol
GESTATIONAL AGE–BASED PERFORMANCE OF DOWN SYNDROME SCREENING MARKERS: RESULTS FROM THE FASTER TRIAL JACOB A. CANICK1, NICHOLAS J. WALD2, FERGAL D. MALONE3, T. FLINT PORTER4, DAVID A. NYBERG5, CHRISTINE H. COMSTOCK6, GEORGE SAADE7, KEITH EDDLEMAN8, SUSAN KLUGMAN9, LORRAINE DUGOFF10, SABRINA D. CRAIGO11, ILAN E. TIMOR12, STEPHEN R. CARR1, HONOR M. WOLFE13, LISA SULLIVAN14, GERALYN LAMBERT-MESSERLIAN1, ALICJA RUDNICKA2, ALLAN HACKSHAW2, MARY E. D’ALTON3, 1 Brown University, Providence, RI 2Wolfson Institute of Preventive Medicine, London, United Kingdom 3Columbia University, New York, New York 4 University of Utah, Salt Lake City, UT 5Swedish Medical Center, Seattle, WA 6William Beaumont Medical Center, Royal Oak, MI 7University of Texas Medical Branch, Galveston, TX 8Mount Sinai School of Medicine, New York, NY 9Albert Einstein College of Medicine, New York, NY 10University of Colorado Health Sciences Center, Denver, CO 11Tufts University, Boston, MA 12 New York University, New York, NY 13UNC Medical Center, Chapel Hill, NC 14 DM-Stat, Boston, MA OBJECTIVE: To examine the median multiple of the median (MoM) values and screening performance of first-trimester Down syndrome (DS) markers by gestational week. STUDY DESIGN: 33,557 unselected patients with viable singleton pregnancies at 10 3/7 to 13 6/7 weeks were recruited at 15 centers in the United States. The median MoM was determined for each marker by completed week for 84 cases of DS having both first- and second-trimester data. Screening performance for each marker for a 5% false-positive rate (FPR) at weeks 11 (n = 17), 12 (n = 41), and 13 (n = 26) was estimated. RESULTS: The overall NT median in DS pregnancies was 1.68 MoM, though the estimate varied across the 3 weeks. As expected, the median PAPP-A MoM decreased and free beta hCG MoM increased over this period. Performance of NT increased from 11 to 13 weeks because of the decrease in standard deviation; analysis assumes a constant NT median for affected pregnancies. Performance of PAPP-A deteriorated markedly and that for free beta hCG increased (by regression analysis). In the second trimester, median marker levels were similar from week to week and performance characteristics were similar to those in previous studies. Median MoM values for DS pregnancies were 0.74 for AFP, 0.61 for uE3, 1.87 for hCG, and 2.00 for inhibin-A; detection rates (DR) for a 5% FPR were 28%, 50%, 32%, and 51%, respectively, without using maternal age. CONCLUSION: In the first trimester, gestational age–specific parameters may be necessary. In the second trimester this is not necessary.
NT
PAPP-A
Free beta hCG
DS median MoM at 11 weeks 12 weeks 13 weeks 11-13 weeks
1.75 1.96 1.33 1.68
0.32 0.48 0.78 0.51
2.20 1.93 2.57 2.20
DR for 5% FPR at 11 weeks 12 weeks 13 weeks
47% 53% 54%
61% 40% 18%
22% 29% 39%
ANALYSIS OF PLACENTAL TRANSCRIPTOME IN PATIENTS WITH PREECLAMPSIA AT PRETERM RAMEN CHMAIT1, DOUGLAS WOELKERS1, ANDREW HULL1, THOMAS MOORE1, IVAN LABAT2, MATHEW ARTERBURN2, SUE ANDARMANI2, BIRGIT STACHE-CRAIN2, JUHI LEE2, WALTER FUNK2, LUBI BOGIC3, 1University of California, San Diego, Reproductive Medicine, San Diego, CA 2Nuvelo, Sunnyvale, CA 3 University of California, San Diego, Reproductive Medicine, La Jolla, CA OBJECTIVE: To characterize differentially expressed genes and their secretory products in placentae and serum of patients with preterm preeclampsia (PPE). STUDY DESIGN: cDNA libraries from human placental transcriptome were screened by oligonucleotide hybridization. A total of 600,000 cDNA clones were derived from libraries of PPE patients and non-PPE gestational age-matched controls. Samples were collected from the basal and chorionic plate. Individual cDNA clones were assigned a signature obtained by hybridization with 200-300 oligonucleotide probes. cDNA clones were grouped into clusters by similarity of their signatures. Clusters of statistically significant difference in number of clones (PPE vs control) were selected for further analysis. The results generated from the mRNA expression studies were confirmed by mass spectrometry using Ciphergen proteomics. RESULTS: Approximately 18,000 placental genes were transcribed between 23 and 32 weeks’ gestation; 394 genes were differentially expressed in chorionic plate and 402 in basal plate. The difference in mRNA expression levels was confirmed by quantitative RT-PCR analysis using the original sample material (23-32 weeks of gestation) and an additional set of placental samples from 38-42 weeks of gestation. From the differentially expressed group of genes in the basal plate, 163 genes (96 upregulated in PPE, 67 downregulated in PPE) encode predicted extracellular protein products, of which 113 are secretory proteins and 50 transmembrane/shed proteins. Proteomic analysis confirmed the differential protein expression of several secreted and/or shed placental factors in the serum samples of PPE patients. CONCLUSION: The extracellular products of identified placental genes that are preferentially expressed in preeclamptic placentae may be useful as disease biomarkers. Diagnostic application of placental biomarkers identified in this study may enable greater efficiency of current treatments by targeting highrisk patients destined to develop PPE.
195
IS THERE A ROLE FOR RAPID PRENATAL DIAGNOSIS USING INTERPHASE FISH IN ALL AMNIOCENTESIS? BETTY HARRISSON1, CAMILLE KANAAN1, SCOTT DEXTER1, MICHAEL PLEVYAK1, JEANCLAUDE VEILLE1, 1Albany Medical College, Ob Gyn, Albany, NY OBJECTIVE: To determine the advantage of obtaining rapid results using FISH as compared to traditional chromosome analysis, thereby reducing anxiety in patients and allowing ample time for decision making when chromosome abnormalities are found. STUDY DESIGN: Over a 5-year period, 480 amniotic fluid samples were analyzed using interphase FISH in second- and third-trimester pregnancies. The primary indications were ultrasound suggestive of chromosome abnormality, late maternal age, and maternal serum associated risk. RESULTS: The detection rate for chromosomes 13,18,21,X and Y was high (88%) while only 12% contained a variety of other abnormalities including translocations, inversions, marker chromosomes, duplications, and deletions. 100% of the informative samples were concordant with cytogenetic results. The majority of tests were reported within 24 to 48 hours of receipt of the specimen. The finding of normal results with these five probes substantially reduces the risk of an abnormal karyotype. In the case of a fetal cardiac anomaly diagnosed late in utero (truncus arteriosus), FISH for 22q11.2 revealed a microdeletion in cultured fibroblasts from the amniotic fluid. Maternal blood also had a deletion for 22q11.2. The affected parent is at a 50% risk of transmitting the deletion. In published surveys thus far, the accuracy of detection of aneuploidy in informative cases is 99.9%. CONCLUSION: FISH has proven to be a highly accurate and informative test for pregnancy management and should be considered and offered to all patients at the time of genetic amniocentesis or in the presence of fetal anomaly detected on ultrasound.
196
AFAFP LEVEL AMONG FETUSES WITH GASTROSCHISIS, OMPHALOCELE, AND THORACO-ABDOMINAL VENTRAL WALL 1 2 DEFECTS NOAM LAZEBNIK , SUSAN DIMENT , STUART SCHWARTZ3, MICHAEL GRAF3, HEIDI FRASURE1, GEORGE VAN-BUREN1, 1Case Western Reserve University, Reproductive Biology, Cleveland, OH 2Henry Ford Health System, Genetics, Detroit, MI 3Case Western Reserve University, Genetics, Cleveland, OH OBJECTIVE: To characterize the AFP amniotic fluid (AFAFP) level of patients with various types of ventral wall defects. STUDY DESIGN: Cases with gastroschisis, omphalocele, and combined thoracic and abdominal ventral wall defects (CTAVWD) were included. All cases (n = 92) underwent ultrasound study and amniocentesis. Defect confirmation was obtained from autopsy and delivery records. AFAFP was assayed between 13 and 24 weeks’ gestation. Analysis of variance was used to compare the distribution of AFAFP values between the three types of defects. A t-test was used to compare the distribution of AFAFP between cases with abnormal versus normal chromosomes within each defect type. When the assumptions of ANOVA or the Student’s t-test were not met, the Kruskal-Wallis or Mann-Whitney non-parametric tests were used. Significance required P < 0.01. RESULTS: There were 65 cases with omphalocele, 14 cases with gastroschisis, and 13 with CTAVWD. There was a significant difference of the mean AFAFP MoM values between the three ventral wall defect types: omphalocele (range 0.5-18.8, mean 3.7 ± 3.4 MoM), gastroschisis (range 7.050.3, mean 23.0 ± 10.4 MoM), and CTAVWD (range 13.9-100.0, mean 44.0 ± 29.9 MoM). When pair-wise comparisons were done, each of the groups was different than the other two (P < 0.01). All cases of gastroschisis were of normal karyotype. Among cases with omphalocele, the distribution of AFAFP between fetuses with abnormal vs normal chromosomes did not differ. Borderline nonsignificant differences were found between fetuses with normal vs abnormal chromosomes among cases with CTAVWD, 55.74 ± 11.49 MoM vs 25.18 ± 10.74 MoM (P = 0.07). Cases with normal chromosomes had larger and more complex defects. CONCLUSION: We conclude that the AFAFP level differed between cases with gastroschisis, omphalocele, and CTAVWD. Abnormal chromosomes did not comprise higher AFAFP. Among cases with CTAVWD, the amniotic fluid AFP was higher in cases with normal chromosomes and larger defects.
194
December 2003 Am J Obstet Gynecol
GESTATIONAL AGE–BASED PERFORMANCE OF DOWN SYNDROME SCREENING MARKERS: RESULTS FROM THE FASTER TRIAL JACOB A. CANICK1, NICHOLAS J. WALD2, FERGAL D. MALONE3, T. FLINT PORTER4, DAVID A. NYBERG5, CHRISTINE H. COMSTOCK6, GEORGE SAADE7, KEITH EDDLEMAN8, SUSAN KLUGMAN9, LORRAINE DUGOFF10, SABRINA D. CRAIGO11, ILAN E. TIMOR12, STEPHEN R. CARR1, HONOR M. WOLFE13, LISA SULLIVAN14, GERALYN LAMBERT-MESSERLIAN1, ALICJA RUDNICKA2, ALLAN HACKSHAW2, MARY E. D’ALTON3, 1 Brown University, Providence, RI 2Wolfson Institute of Preventive Medicine, London, United Kingdom 3Columbia University, New York, New York 4 University of Utah, Salt Lake City, UT 5Swedish Medical Center, Seattle, WA 6William Beaumont Medical Center, Royal Oak, MI 7University of Texas Medical Branch, Galveston, TX 8Mount Sinai School of Medicine, New York, NY 9Albert Einstein College of Medicine, New York, NY 10University of Colorado Health Sciences Center, Denver, CO 11Tufts University, Boston, MA 12 New York University, New York, NY 13UNC Medical Center, Chapel Hill, NC 14 DM-Stat, Boston, MA OBJECTIVE: To examine the median multiple of the median (MoM) values and screening performance of first-trimester Down syndrome (DS) markers by gestational week. STUDY DESIGN: 33,557 unselected patients with viable singleton pregnancies at 10 3/7 to 13 6/7 weeks were recruited at 15 centers in the United States. The median MoM was determined for each marker by completed week for 84 cases of DS having both first- and second-trimester data. Screening performance for each marker for a 5% false-positive rate (FPR) at weeks 11 (n = 17), 12 (n = 41), and 13 (n = 26) was estimated. RESULTS: The overall NT median in DS pregnancies was 1.68 MoM, though the estimate varied across the 3 weeks. As expected, the median PAPP-A MoM decreased and free beta hCG MoM increased over this period. Performance of NT increased from 11 to 13 weeks because of the decrease in standard deviation; analysis assumes a constant NT median for affected pregnancies. Performance of PAPP-A deteriorated markedly and that for free beta hCG increased (by regression analysis). In the second trimester, median marker levels were similar from week to week and performance characteristics were similar to those in previous studies. Median MoM values for DS pregnancies were 0.74 for AFP, 0.61 for uE3, 1.87 for hCG, and 2.00 for inhibin-A; detection rates (DR) for a 5% FPR were 28%, 50%, 32%, and 51%, respectively, without using maternal age. CONCLUSION: In the first trimester, gestational age–specific parameters may be necessary. In the second trimester this is not necessary.
NT
PAPP-A
Free beta hCG
DS median MoM at 11 weeks 12 weeks 13 weeks 11-13 weeks
1.75 1.96 1.33 1.68
0.32 0.48 0.78 0.51
2.20 1.93 2.57 2.20
DR for 5% FPR at 11 weeks 12 weeks 13 weeks
47% 53% 54%
61% 40% 18%
22% 29% 39%
ANALYSIS OF PLACENTAL TRANSCRIPTOME IN PATIENTS WITH PREECLAMPSIA AT PRETERM RAMEN CHMAIT1, DOUGLAS WOELKERS1, ANDREW HULL1, THOMAS MOORE1, IVAN LABAT2, MATHEW ARTERBURN2, SUE ANDARMANI2, BIRGIT STACHE-CRAIN2, JUHI LEE2, WALTER FUNK2, LUBI BOGIC3, 1University of California, San Diego, Reproductive Medicine, San Diego, CA 2Nuvelo, Sunnyvale, CA 3 University of California, San Diego, Reproductive Medicine, La Jolla, CA OBJECTIVE: To characterize differentially expressed genes and their secretory products in placentae and serum of patients with preterm preeclampsia (PPE). STUDY DESIGN: cDNA libraries from human placental transcriptome were screened by oligonucleotide hybridization. A total of 600,000 cDNA clones were derived from libraries of PPE patients and non-PPE gestational age-matched controls. Samples were collected from the basal and chorionic plate. Individual cDNA clones were assigned a signature obtained by hybridization with 200-300 oligonucleotide probes. cDNA clones were grouped into clusters by similarity of their signatures. Clusters of statistically significant difference in number of clones (PPE vs control) were selected for further analysis. The results generated from the mRNA expression studies were confirmed by mass spectrometry using Ciphergen proteomics. RESULTS: Approximately 18,000 placental genes were transcribed between 23 and 32 weeks’ gestation; 394 genes were differentially expressed in chorionic plate and 402 in basal plate. The difference in mRNA expression levels was confirmed by quantitative RT-PCR analysis using the original sample material (23-32 weeks of gestation) and an additional set of placental samples from 38-42 weeks of gestation. From the differentially expressed group of genes in the basal plate, 163 genes (96 upregulated in PPE, 67 downregulated in PPE) encode predicted extracellular protein products, of which 113 are secretory proteins and 50 transmembrane/shed proteins. Proteomic analysis confirmed the differential protein expression of several secreted and/or shed placental factors in the serum samples of PPE patients. CONCLUSION: The extracellular products of identified placental genes that are preferentially expressed in preeclamptic placentae may be useful as disease biomarkers. Diagnostic application of placental biomarkers identified in this study may enable greater efficiency of current treatments by targeting highrisk patients destined to develop PPE.
195
IS THERE A ROLE FOR RAPID PRENATAL DIAGNOSIS USING INTERPHASE FISH IN ALL AMNIOCENTESIS? BETTY HARRISSON1, CAMILLE KANAAN1, SCOTT DEXTER1, MICHAEL PLEVYAK1, JEANCLAUDE VEILLE1, 1Albany Medical College, Ob Gyn, Albany, NY OBJECTIVE: To determine the advantage of obtaining rapid results using FISH as compared to traditional chromosome analysis, thereby reducing anxiety in patients and allowing ample time for decision making when chromosome abnormalities are found. STUDY DESIGN: Over a 5-year period, 480 amniotic fluid samples were analyzed using interphase FISH in second- and third-trimester pregnancies. The primary indications were ultrasound suggestive of chromosome abnormality, late maternal age, and maternal serum associated risk. RESULTS: The detection rate for chromosomes 13,18,21,X and Y was high (88%) while only 12% contained a variety of other abnormalities including translocations, inversions, marker chromosomes, duplications, and deletions. 100% of the informative samples were concordant with cytogenetic results. The majority of tests were reported within 24 to 48 hours of receipt of the specimen. The finding of normal results with these five probes substantially reduces the risk of an abnormal karyotype. In the case of a fetal cardiac anomaly diagnosed late in utero (truncus arteriosus), FISH for 22q11.2 revealed a microdeletion in cultured fibroblasts from the amniotic fluid. Maternal blood also had a deletion for 22q11.2. The affected parent is at a 50% risk of transmitting the deletion. In published surveys thus far, the accuracy of detection of aneuploidy in informative cases is 99.9%. CONCLUSION: FISH has proven to be a highly accurate and informative test for pregnancy management and should be considered and offered to all patients at the time of genetic amniocentesis or in the presence of fetal anomaly detected on ultrasound.
196
AFAFP LEVEL AMONG FETUSES WITH GASTROSCHISIS, OMPHALOCELE, AND THORACO-ABDOMINAL VENTRAL WALL 1 2 DEFECTS NOAM LAZEBNIK , SUSAN DIMENT , STUART SCHWARTZ3, MICHAEL GRAF3, HEIDI FRASURE1, GEORGE VAN-BUREN1, 1Case Western Reserve University, Reproductive Biology, Cleveland, OH 2Henry Ford Health System, Genetics, Detroit, MI 3Case Western Reserve University, Genetics, Cleveland, OH OBJECTIVE: To characterize the AFP amniotic fluid (AFAFP) level of patients with various types of ventral wall defects. STUDY DESIGN: Cases with gastroschisis, omphalocele, and combined thoracic and abdominal ventral wall defects (CTAVWD) were included. All cases (n = 92) underwent ultrasound study and amniocentesis. Defect confirmation was obtained from autopsy and delivery records. AFAFP was assayed between 13 and 24 weeks’ gestation. Analysis of variance was used to compare the distribution of AFAFP values between the three types of defects. A t-test was used to compare the distribution of AFAFP between cases with abnormal versus normal chromosomes within each defect type. When the assumptions of ANOVA or the Student’s t-test were not met, the Kruskal-Wallis or Mann-Whitney non-parametric tests were used. Significance required P < 0.01. RESULTS: There were 65 cases with omphalocele, 14 cases with gastroschisis, and 13 with CTAVWD. There was a significant difference of the mean AFAFP MoM values between the three ventral wall defect types: omphalocele (range 0.5-18.8, mean 3.7 ± 3.4 MoM), gastroschisis (range 7.050.3, mean 23.0 ± 10.4 MoM), and CTAVWD (range 13.9-100.0, mean 44.0 ± 29.9 MoM). When pair-wise comparisons were done, each of the groups was different than the other two (P < 0.01). All cases of gastroschisis were of normal karyotype. Among cases with omphalocele, the distribution of AFAFP between fetuses with abnormal vs normal chromosomes did not differ. Borderline nonsignificant differences were found between fetuses with normal vs abnormal chromosomes among cases with CTAVWD, 55.74 ± 11.49 MoM vs 25.18 ± 10.74 MoM (P = 0.07). Cases with normal chromosomes had larger and more complex defects. CONCLUSION: We conclude that the AFAFP level differed between cases with gastroschisis, omphalocele, and CTAVWD. Abnormal chromosomes did not comprise higher AFAFP. Among cases with CTAVWD, the amniotic fluid AFP was higher in cases with normal chromosomes and larger defects.