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First-trimester fetal MRNA of placental origin in maternal plasma
SMFM Abstracts S111
Volume 189, Number 6 Am J Obstet Gynecol 173
FIRST-TRIMESTER FETAL MRNA OF PLACENTAL ORIGIN IN MATERNAL PLASMA ATTIE T. J. I. GO1, A. VISSER2, M. A. M. MULDERS2, B. A. WESTERMAN2, M. A. BLANKENSTEIN2, C. B. M. OUDEJANS2, JOHN M.G. VUGT1, 1VU University Medical Center, OB/GYN, Amsterdam, Netherlands 2VU University Medical Center, Clinical Chemistry, Amsterdam, Netherlands OBJECTIVE: We recently demonstrated that chromosome 21-encoded mRNA of placental origin (LOC90625) can be detected reliably in maternal plasma during the first trimester of pregnancy. The intrinsic advantage of the plasma RNA–based strategy is that additional markers become available as analysis is not restricted to markers that only appear in the maternal circulation as a consequence of active secretion. In this study, we tested additional marker genes for this purpose. STUDY DESIGN: Intron-spanning primers were designed for genes with known and/or preferential expression in the early human placenta and either located on chromosome 21 (marker gene) or on different autosomes (reference gene). Tissue expression analysis was done by RT-PCR on total RNA isolated from first-trimester placental tissues. Plasma analysis (1.6 mL) was done by RTPCR on RNA from pregnant (week 9-13) females and non-pregnant controls following isolation of RNA using the QIAamp MinElute Virus Vacuum system. RESULTS: To identify marker and reference genes for the analysis of placental RNA in maternal plasma, genes with proven or suspected expression in the placenta were screened by RT-PCR and, if expressed, analyzed for their presence in maternal plasma and absence in plasma of non-pregnant controls. Out of 17 genes tested, all but 3 showed expression in early human placenta. Of the 14 genes with expression in early placenta, mRNA of 4 genes (CHS1, TNCRNA, LOC90625, and PTTG1IP) could be detected in the plasma of pregnant females. However, of these 4 genes, mRNA of TNCRNA and PTTG1P was seen in non-pregnant controls as well. In contrast, mRNA of LOC90625 (chrom 21) and of CHS1 (chrom 17) was seen in pregnant females only. CONCLUSION: The presence and detectability of placental RNA in the maternal plasma during the first trimester of pregnancy could permit novel screening assays for the prenatal detection of Down syndrome.
174
FAST PRENATAL DIAGNOSIS OF SEVERE BETA-THALASSEMIA JIAXUE WEI1, CAN LIAO1, HUIZHU ZHONG1, 1Guangzhou Maternal & Neonatal Hospital, Guangzhou, Guangdong, China OBJECTIVE: Evaluate a fast method of prenatal diagnosis of severe betathalassemia. STUDY DESIGN: All the pregnant couples both with beta-thalassemia identified by gene diagnosis were referred to prenatal diagnosis. 1 to 2 mL of fetal blood was collected by cordocentesis during 17-28 GW for gene diagnosis and hemoglobin analysis with high-performance liquid chromatography (HPLC). Results of gene diagnosis and hemoglobin analysis of fetus were compared. RESULTS: 77 fetal blood samples were analyzed by gene diagnosis and hemoglobin analysis. 20 cases were identified to be of beta-thalassemia major with beta-gene homozygotes or of compound heterozygotes for beta-gene mutations; 29 cases were identified to be beta-thalassemia mutation carriers, and 2 cases were of HbE mutation carriers. All 20 fetuses with thalassemia major had no HbA2 or less than 0.9%; 27 fetuses with beta-thalassemia intermedia had HbA2 concentrations between 1.4% and 3.9%, 2 fetuses with a-gene compound b-gene mutations were same as the remaining normal 26 fetuses with HbA2 levels more than 3.9%. In the condition without a-thalassemia gene compounding, there is no overlapping between the three clinical beta-thalassemia types. CONCLUSION: We recommend hemoglobin analysis by HPLC as a fast and highly effective method in prenatal diagnosis of beta-thalassemia major.
Results of gene diagnosis and hemoglobin analysis
a-Gene homozygotes (major) Compound heterozygotes for a-gene mutations (major) a-Gene mutation carriers(minor) a-Gene compound a-gene mutation (minor) HbE (intermedia) Normal
Number
HbA (%)
HbA2
4
0.0
0.0
16
0.0-0.9
0.0
27
1.4-3.8
0.0
2
4.2-4.0
0.0
2 16
2.7-3.0 3.9-13.7
.2, 1.5 (HbE+A2) 0.0
175
PREGNANCY LOSS RATES AFTER MIDTRIMESTER AMNIOCENTESIS: THE FASTER TRIAL KEITH EDDLEMAN1, RICHARD BERKOWITZ1, YARA KHARBUTLI1, FERGAL MALONE2, JOHN VIDAVER3, T. FLINT PORTER4, DAVID LUTHY5, CHRISTINE COMSTOCK6, GEORGE SAADE7, SUSAN KLUGMAN8, LORRAINE DUGOFF9, SABRINA CRAIGO10, ILAN TIMOR11, STEPHEN CARR12, HONOR WOLFE13, MARY D’ALTON2, 1Mount Sinai School of Medicine, Obstetrics, Gynecology and Reproductive Science, New York, NY 2Columbia University, Obstetrics and Gynecology, New York, NY 3 DM-Stat, Boston, MA 4University of Utah, Obstetrics and Gynecology, Salt Lake City, UT 5Swedish Medical Center, Obstetrics and Gynecology, Seattle, WA 6William Beaumont Medical Center, Obstetrics and Gynecology, Royal Oak, MI 7University of Texas Medical Branch at Galveston, Obstetrics and Gynecology, Galveston, TX 8Albert Einstein College of Medicine, Obstetrics & Gynecology and Women’s Health, Bronx, NY 9University of Colorado Health Sciences Center, Obstetrics and Gynecology, Denver, CO 10Tufts University, Obstetrics/Gynecology, Boston, MA 11New York University, Obstetrics and Gynecology, New York, NY 12Women and Infant’s Hospital, Obstetrics and Gynecology, Providence, RI 13University of North Carolina– Chapel Hill, Ob/Gyn/Maternal Fetal Med, Chapel Hill, NC OBJECTIVE: To quantify the contemporary procedure-related loss rate after midtrimester amniocentesis using the database generated from patients who entered the FASTER Trial. STUDY DESIGN: Unselected patients from the general population with viable singleton pregnancies were enrolled in the FASTER Trial between 10 3/7 and 13 6/7 weeks gestation and followed prospectively for complete pregnancy outcome information. Patients who either did (study group, n = 1605) or did not (control group, n = 26,187) undergo midtrimester amniocentesis were identified from the database. Amniocenteses were performed both by referring obstetricians and perinatologists. The rate of fetal loss < 24 weeks was compared between the two groups using Fisher’s exact test. Multiple logistic regression analysis was used to estimate the effect of midtrimester amniocentesis and to adjust for potential confounders such as maternal age, weight, parity, history of preterm delivery, prior miscarriage, substance abuse, medications, vaginal bleeding, chromosomal abnormalities, and FASTER screen positivity. RESULTS: The spontaneous fetal loss rate < 24 weeks in the study group was 0.62% (95% CI: 0.30, 1.14) and was not statistically different from the background 0.47% loss rate (95% CI: 0.39, 0.56) seen in the control group (P = .35, OR 1.3, 95% CI: 0.7, 2.5). The procedure-related loss rate after midtrimester amniocentesis was 0.15% (0.62% minus the background rate of 0.47%) with a 95% CI from ÿ0.15 to 0.68. In a model adjusting for significant covariates (fetal chromosomal abnormality, vaginal bleeding, and maternal antihypertensive medications), the difference between the study groups approached but still failed to reach statistical significance (P = .06, adjusted OR 0.2, 95% CI 0.03, 1.1). CONCLUSION: The procedure-related loss rate after midtrimester amniocentesis performed on patients who entered the FASTER Trial was 0.15%. This is less than the traditionally quoted procedure-related loss rate of 0.5%.
176
DETECTION OF MYCOPLASMA HOMINIS IN MIDTRIMESTER AMNIOTIC FLUID BY POLYMERASE CHAIN REACTION (PCR)-ELISA: RELATION TO AMNIOTIC FLUID CYTOKINE LEVELS AND PREGNANCY OUTCOME SRIRAM C. PERNI1, SANTOSH VARDHANA1, IRINA KORNEEVA1, SARA L. TUTTLE1, LILLY-ROSE PARASKEVAS1, STEPHEN T. CHASEN1, ROBIN B. KALISH1, STEVEN S. WITKIN1, 1Weill Medical College of Cornell University, Obstetrics & Gynecology, New York, NY OBJECTIVE: To determine the prevalence of M. hominis in midtrimester amniotic fluid by PCR and its relation to amniotic fluid cytokine concentrations and subsequent pregnancy outcome. STUDY DESIGN: Amniotic fluid from 163 asymptomatic pregnant women was tested blinded for M. hominis by a PCR assay incorporating digoxigenin (DIG)-labeled dUTP. To enhance specificity and sensitivity the PCR amplicons were hybridized to a biotinylated internal probe and detected by ELISA using enzyme-labeled anti-DIG antibody. Amniotic fluid supernatants were assayed for cytokines using ELISA kits. Pregnancy outcome data were retrospectively obtained from patients’ charts. RESULTS: M. hominis was detected in 11 (6.7%) of the amniotic fluids. The median amniotic fluid concentrations of interleukin (IL)-4 (10.2 pg/mL vs 0.0 pg/mL, P = 0.001), tumor necrosis factor-a (1.1 pg/mL vs 0.8 pg/mL, P = 0.04) and IL-6 (1.8 ng/mL vs 0.5 ng/mL, P = 0.09) were higher in amniotic fluids that contained M. hominis than in PCR-negative fluids. Pregnancy outcome data were available from 10 of the M. hominis–-positive patients. Detection of M. hominis was associated with an increased rate of subsequent spontaneous preterm birth (SPTB) as compared to PCR-negative women (2/10 vs 3/114, P = 0.05, OR = 9.00, 95% CI 1.308-61.908). M. hominis was almost fourfold more prevalent in women with a history of spontaneous abortion (5/43 [11.6%]) than in women with no prior abortions (5/130 [3.8%]) (P = 0.12). CONCLUSION: Utilization of a sensitive PCR-ELISA identified M. hominis in midtrimester amniotic fluid from 6.7% of healthy asymptomatic pregnant women. Its detection was associated with elevated amniotic fluid cytokine levels and an increased subsequent rate of SPTB. The association between M. hominis and spontaneous abortions in prior pregnancies suggests that this microorganism may have been in the uterus prior to the current conception.
Volume 189, Number 6 Am J Obstet Gynecol 173
FIRST-TRIMESTER FETAL MRNA OF PLACENTAL ORIGIN IN MATERNAL PLASMA ATTIE T. J. I. GO1, A. VISSER2, M. A. M. MULDERS2, B. A. WESTERMAN2, M. A. BLANKENSTEIN2, C. B. M. OUDEJANS2, JOHN M.G. VUGT1, 1VU University Medical Center, OB/GYN, Amsterdam, Netherlands 2VU University Medical Center, Clinical Chemistry, Amsterdam, Netherlands OBJECTIVE: We recently demonstrated that chromosome 21-encoded mRNA of placental origin (LOC90625) can be detected reliably in maternal plasma during the first trimester of pregnancy. The intrinsic advantage of the plasma RNA–based strategy is that additional markers become available as analysis is not restricted to markers that only appear in the maternal circulation as a consequence of active secretion. In this study, we tested additional marker genes for this purpose. STUDY DESIGN: Intron-spanning primers were designed for genes with known and/or preferential expression in the early human placenta and either located on chromosome 21 (marker gene) or on different autosomes (reference gene). Tissue expression analysis was done by RT-PCR on total RNA isolated from first-trimester placental tissues. Plasma analysis (1.6 mL) was done by RTPCR on RNA from pregnant (week 9-13) females and non-pregnant controls following isolation of RNA using the QIAamp MinElute Virus Vacuum system. RESULTS: To identify marker and reference genes for the analysis of placental RNA in maternal plasma, genes with proven or suspected expression in the placenta were screened by RT-PCR and, if expressed, analyzed for their presence in maternal plasma and absence in plasma of non-pregnant controls. Out of 17 genes tested, all but 3 showed expression in early human placenta. Of the 14 genes with expression in early placenta, mRNA of 4 genes (CHS1, TNCRNA, LOC90625, and PTTG1IP) could be detected in the plasma of pregnant females. However, of these 4 genes, mRNA of TNCRNA and PTTG1P was seen in non-pregnant controls as well. In contrast, mRNA of LOC90625 (chrom 21) and of CHS1 (chrom 17) was seen in pregnant females only. CONCLUSION: The presence and detectability of placental RNA in the maternal plasma during the first trimester of pregnancy could permit novel screening assays for the prenatal detection of Down syndrome.
174
FAST PRENATAL DIAGNOSIS OF SEVERE BETA-THALASSEMIA JIAXUE WEI1, CAN LIAO1, HUIZHU ZHONG1, 1Guangzhou Maternal & Neonatal Hospital, Guangzhou, Guangdong, China OBJECTIVE: Evaluate a fast method of prenatal diagnosis of severe betathalassemia. STUDY DESIGN: All the pregnant couples both with beta-thalassemia identified by gene diagnosis were referred to prenatal diagnosis. 1 to 2 mL of fetal blood was collected by cordocentesis during 17-28 GW for gene diagnosis and hemoglobin analysis with high-performance liquid chromatography (HPLC). Results of gene diagnosis and hemoglobin analysis of fetus were compared. RESULTS: 77 fetal blood samples were analyzed by gene diagnosis and hemoglobin analysis. 20 cases were identified to be of beta-thalassemia major with beta-gene homozygotes or of compound heterozygotes for beta-gene mutations; 29 cases were identified to be beta-thalassemia mutation carriers, and 2 cases were of HbE mutation carriers. All 20 fetuses with thalassemia major had no HbA2 or less than 0.9%; 27 fetuses with beta-thalassemia intermedia had HbA2 concentrations between 1.4% and 3.9%, 2 fetuses with a-gene compound b-gene mutations were same as the remaining normal 26 fetuses with HbA2 levels more than 3.9%. In the condition without a-thalassemia gene compounding, there is no overlapping between the three clinical beta-thalassemia types. CONCLUSION: We recommend hemoglobin analysis by HPLC as a fast and highly effective method in prenatal diagnosis of beta-thalassemia major.
Results of gene diagnosis and hemoglobin analysis
a-Gene homozygotes (major) Compound heterozygotes for a-gene mutations (major) a-Gene mutation carriers(minor) a-Gene compound a-gene mutation (minor) HbE (intermedia) Normal
Number
HbA (%)
HbA2
4
0.0
0.0
16
0.0-0.9
0.0
27
1.4-3.8
0.0
2
4.2-4.0
0.0
2 16
2.7-3.0 3.9-13.7
.2, 1.5 (HbE+A2) 0.0
175
PREGNANCY LOSS RATES AFTER MIDTRIMESTER AMNIOCENTESIS: THE FASTER TRIAL KEITH EDDLEMAN1, RICHARD BERKOWITZ1, YARA KHARBUTLI1, FERGAL MALONE2, JOHN VIDAVER3, T. FLINT PORTER4, DAVID LUTHY5, CHRISTINE COMSTOCK6, GEORGE SAADE7, SUSAN KLUGMAN8, LORRAINE DUGOFF9, SABRINA CRAIGO10, ILAN TIMOR11, STEPHEN CARR12, HONOR WOLFE13, MARY D’ALTON2, 1Mount Sinai School of Medicine, Obstetrics, Gynecology and Reproductive Science, New York, NY 2Columbia University, Obstetrics and Gynecology, New York, NY 3 DM-Stat, Boston, MA 4University of Utah, Obstetrics and Gynecology, Salt Lake City, UT 5Swedish Medical Center, Obstetrics and Gynecology, Seattle, WA 6William Beaumont Medical Center, Obstetrics and Gynecology, Royal Oak, MI 7University of Texas Medical Branch at Galveston, Obstetrics and Gynecology, Galveston, TX 8Albert Einstein College of Medicine, Obstetrics & Gynecology and Women’s Health, Bronx, NY 9University of Colorado Health Sciences Center, Obstetrics and Gynecology, Denver, CO 10Tufts University, Obstetrics/Gynecology, Boston, MA 11New York University, Obstetrics and Gynecology, New York, NY 12Women and Infant’s Hospital, Obstetrics and Gynecology, Providence, RI 13University of North Carolina– Chapel Hill, Ob/Gyn/Maternal Fetal Med, Chapel Hill, NC OBJECTIVE: To quantify the contemporary procedure-related loss rate after midtrimester amniocentesis using the database generated from patients who entered the FASTER Trial. STUDY DESIGN: Unselected patients from the general population with viable singleton pregnancies were enrolled in the FASTER Trial between 10 3/7 and 13 6/7 weeks gestation and followed prospectively for complete pregnancy outcome information. Patients who either did (study group, n = 1605) or did not (control group, n = 26,187) undergo midtrimester amniocentesis were identified from the database. Amniocenteses were performed both by referring obstetricians and perinatologists. The rate of fetal loss < 24 weeks was compared between the two groups using Fisher’s exact test. Multiple logistic regression analysis was used to estimate the effect of midtrimester amniocentesis and to adjust for potential confounders such as maternal age, weight, parity, history of preterm delivery, prior miscarriage, substance abuse, medications, vaginal bleeding, chromosomal abnormalities, and FASTER screen positivity. RESULTS: The spontaneous fetal loss rate < 24 weeks in the study group was 0.62% (95% CI: 0.30, 1.14) and was not statistically different from the background 0.47% loss rate (95% CI: 0.39, 0.56) seen in the control group (P = .35, OR 1.3, 95% CI: 0.7, 2.5). The procedure-related loss rate after midtrimester amniocentesis was 0.15% (0.62% minus the background rate of 0.47%) with a 95% CI from ÿ0.15 to 0.68. In a model adjusting for significant covariates (fetal chromosomal abnormality, vaginal bleeding, and maternal antihypertensive medications), the difference between the study groups approached but still failed to reach statistical significance (P = .06, adjusted OR 0.2, 95% CI 0.03, 1.1). CONCLUSION: The procedure-related loss rate after midtrimester amniocentesis performed on patients who entered the FASTER Trial was 0.15%. This is less than the traditionally quoted procedure-related loss rate of 0.5%.
176
DETECTION OF MYCOPLASMA HOMINIS IN MIDTRIMESTER AMNIOTIC FLUID BY POLYMERASE CHAIN REACTION (PCR)-ELISA: RELATION TO AMNIOTIC FLUID CYTOKINE LEVELS AND PREGNANCY OUTCOME SRIRAM C. PERNI1, SANTOSH VARDHANA1, IRINA KORNEEVA1, SARA L. TUTTLE1, LILLY-ROSE PARASKEVAS1, STEPHEN T. CHASEN1, ROBIN B. KALISH1, STEVEN S. WITKIN1, 1Weill Medical College of Cornell University, Obstetrics & Gynecology, New York, NY OBJECTIVE: To determine the prevalence of M. hominis in midtrimester amniotic fluid by PCR and its relation to amniotic fluid cytokine concentrations and subsequent pregnancy outcome. STUDY DESIGN: Amniotic fluid from 163 asymptomatic pregnant women was tested blinded for M. hominis by a PCR assay incorporating digoxigenin (DIG)-labeled dUTP. To enhance specificity and sensitivity the PCR amplicons were hybridized to a biotinylated internal probe and detected by ELISA using enzyme-labeled anti-DIG antibody. Amniotic fluid supernatants were assayed for cytokines using ELISA kits. Pregnancy outcome data were retrospectively obtained from patients’ charts. RESULTS: M. hominis was detected in 11 (6.7%) of the amniotic fluids. The median amniotic fluid concentrations of interleukin (IL)-4 (10.2 pg/mL vs 0.0 pg/mL, P = 0.001), tumor necrosis factor-a (1.1 pg/mL vs 0.8 pg/mL, P = 0.04) and IL-6 (1.8 ng/mL vs 0.5 ng/mL, P = 0.09) were higher in amniotic fluids that contained M. hominis than in PCR-negative fluids. Pregnancy outcome data were available from 10 of the M. hominis–-positive patients. Detection of M. hominis was associated with an increased rate of subsequent spontaneous preterm birth (SPTB) as compared to PCR-negative women (2/10 vs 3/114, P = 0.05, OR = 9.00, 95% CI 1.308-61.908). M. hominis was almost fourfold more prevalent in women with a history of spontaneous abortion (5/43 [11.6%]) than in women with no prior abortions (5/130 [3.8%]) (P = 0.12). CONCLUSION: Utilization of a sensitive PCR-ELISA identified M. hominis in midtrimester amniotic fluid from 6.7% of healthy asymptomatic pregnant women. Its detection was associated with elevated amniotic fluid cytokine levels and an increased subsequent rate of SPTB. The association between M. hominis and spontaneous abortions in prior pregnancies suggests that this microorganism may have been in the uterus prior to the current conception.