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526: Perinatal predictors of abnormal childhood neurodevelopment in early onset fetal growth restriction (FGR)
SMFM Abstracts
www.AJOG.org 524
EFFECT OF HYPOXIA AND HYPERTHERMIA ON ENOS PROTEIN CONCENTRATION IN CULTURED OVINE UMBILICAL VEIN ENDOTHELIAL CELLS JUAN A ARROYO1, RUSSELL V. ANTHONY2, BRADLEY T. ZIEBELL1, HENRY L. GALAN1, 1University of Colorado Health Sciences Center, Obstetrics and Gynecology, Aurora, Colorado, 2University of Colorado Health Sciences Center, Pedriatics, Aurora, Colorado OBJECTIVE: Nitric Oxide (NO) and endothelial nitric oxide synthase (eNOS), important regulators of placental blood flow, are reduced in a hyperthermic-induced (HT) ovine model of fetal growth restriction (FGR) with fetal hypoxia (HX) and hypertension. Our objective was to differentiate the effect of hypoxia and HT on NO and eNOS production in ovine umbilical vein endothelial cells (OUVEC). STUDY DESIGN: At 130 days gestational age (dGA), OUVEC were isolated with a collagenase B solution and cultured in 2% gelatin coated flasks for up to three passages. CD31 and Factor VIII staining confirmed endothelial cell type. Endothelial cells were treated with HX (2% O2) or HT (40°C) for 24, 48 and 72 hours (H). At each time point, OUVEC eNOS protein and NO from culture media were assessed by Western blot and a NOx assay kit, respectively. RESULTS: Compared to controls, hypoxia-exposed OUVEC showed increased eNOS protein concentration at 48H (2-fold, p⬍ 0.0005), but decreased at 72H (1.6-fold, p⬍ 0.03) while OUVEC media NOx concentration was only increased at 48H (figure) with otherwise no changes. In HT exposed OUVEC, eNOS protein was increased at 72H (2-fold, p⬍ 0.05) with no changes observed for NOx at any time point. CONCLUSION: Hypoxia and hyperthermia independently regulate endothelial eNOS protein production in OUVEC. Hypoxia induced upregulation of this protein is temporary as a concomitant increase in NO production is only seen at 48H, which may reflect ongoing cell death or a decrease in the eNOS substrate L-arginine. (Supported by NIH grant R01 HL071990-01A1).
526
PERINATAL PREDICTORS OF ABNORMAL CHILDHOOD NEURODEVELOPMENT IN EARLY ONSET FETAL GROWTH RESTRICTION (FGR) AHMET BASCHAT1, ROSE VISCARDI1, BRENDA HUSSEY-GARDNER1, NADEEM HASHMI2, CHRISTOPHER HARMAN1, 1 University of Maryland at Baltimore, Baltimore, Maryland, 2University of Maryland at Baltimore, Pediatrics, Baltimore, Maryland OBJECTIVE: Metabolic and cardiovascular deterioration in early onset FGR is prenatally detectable by ductus venosus (DV) Doppler and biophysical profile score (BPP). We analyzed if such surveillance predicts poor neurodevelopment at 2 years corrected age. STUDY DESIGN: Prospective observational study of FGR (Abdominal Circumference ⬍5%ile, high umbilical artery (UA) Doppler). UA, DV Doppler, BPP, birth acidemia (artery pH ⬍7.0 ⫹/or base deficit⬎12), gestational age (GA), birthweight (BW) and neonatal morbidity (NM⫽bronchopulmonary dysplasia, ⬎grade 2 intraventricular bleed, or necrotizing enterocolitis) were related to 2 year neurodevelopmental delay (NDD) as assesed by Best Beginnings Developmental Screen (BBDS), Bayley (BSID) and Clinical Adaptive/Clinical linguistic auditory milestone stage. BSID ⬍70, cerebral palsy (CP), abnormal tone, hearing loss ⫹/or blindness defined NDD. RESULTS: 52 / 87 enrolled patients completed analysis (10 stillbirth, 13 neonatal / 2 infant deaths, 9 no follow up. 15 fetuses had UA reversed diastolic flow (REDV), 20 abnormal DV Doppler, 22 a BPP ⬍6/10. Median delivery GA / BW were 30.1 / 890 g. 10 had acidemia and 25 NM. 28 (53.8%) of infants with NDD included 27 (51.9) with abnormal tone, 19 (36.5%) with speech delay, 14 (26.9%) with abnormal neurologic exam, 5 (9.6%) with hearing deficit, and 4 with CP (7.7%). UA REDV primarily determined hearing and speech deficit (LR 4.8 and 4.9 respectively, r2 ⫽ 0.16 p⫽0.006). While NM was strongly associated with abnormal tone (LR 9.6, p⬍0.0001), BW was the main determinant of this complication r2 ⫽ 0.36, p⬍0.001). Abnormal DV Doppler was the primary determinant of NDD, r2 ⫽ 0.13, p⫽0.03). An abnormal BPP had no impact on any of the study outcomes. CONCLUSION: Chronicity and severity of placental vascular dysfunction independently predict poor neurodevelopment in preterm FGR. Biophysical deterioration does not appear to have an additional impact. Birthweight and neonatal complications are important factors that appear to mediate the effects of prematurity on adverse neurodevelopment. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.528
527
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.526
525
HYPOXIC AND HYPERTHERMIC EFFECTS ON SIGNAL TRANSDUCTION AND PROLIFERATION IN OVINE UMBILICAL VEIN ENDOTHELIAL CELLS JUAN A ARROYO1, BRADLEY T. ZIEBELL1, HENRY L. GALAN1, 1University of Colorado Health Sciences Center, Obstetrics and Gynecology, Denver, Colorado OBJECTIVE: The hyperthermia (HT) induced ovine model of fetal growth restriction (FGR) is characterized by hypoxia and endothelial cell dysfunction (fetal hypertension; reduced umbilical blood flow and placental eNOS protein). Our goal was to assess the independent effects of hypethermia (HT) and hypoxia (HX) on signal transduction pathways affecting cellular proliferation (Ki-67, ERK), survival (AKT) or death (JNK) in ovine umbilical vein endothelial cells (OUVEC). STUDY DESIGN: At 130 days gestational age (dGA), OUVEC were isolated with a collagenase B solution and cultured in 2% gelatin coated flask for up to three passages. CD31 and Factor VIII staining confirmed endothelial cell type. OUVEC were treated with HX (2% O2) or HT (40°C) for 24, 48 and 72 hours (H). Staining for proliferation marker Ki-67 and Western blot analyses for pERK/ERK, pAKT/ AKT and pJNK/JNK were performed at each time point. RESULTS: HX significantly decreased cell proliferation (Ki-67 positive cells) at 24, 48, and 72H (figure), even though ERK (cell survival) was increased at 48H (p⬍0.0003) and AKT (cell survival/apoptosis) was increased at 48 (p⬍0.007) and 72H (p⬍6.0⫻10-5). HX also increased JNK protein at 24H (p⬍0.02) and 48H (p⬍0.0004). Conversely, HT did not affect cell proliferation, but increased ERK protein at all time points. CONCLUSION: HX stress decreases OUVEC proliferation (2Ki-67 staining; 1JNK protein) while attempting to survive (1ERK; 1AKT proteins) HT does not decrease proliferation suggesting that HX is the primary stressor in endothelial cell dysfunction in these experimental conditions. (Supported by NIH grant R01 HL071990-01A1).
HYPOXIC BRAIN DAMAGE AS MEASURED BY FETAL BLOOD AND AMNIOTIC FLUID LEVELS AND IMMUNOHISTOCHEMICAL BRAIN EXPRESSION OF S100 IN A RABBIT MODEL OF INTRAUTERINE GROWTH RESTRICTION ELISENDA EIXARCH1, EDGAR HERNANDEZ-ANDRADE1, FRANCESC FIGUERAS1, SILVIA OLIVEIRA1, FATIMA CRISPI1, EDUARD GRATACOS1, 1Hospital Clinic-University of Barcelona, Department of Maternal-Fetal Medicine (ICGON)), IDIBAPS and CIBER-ER, Barcelona, Spain OBJECTIVE: To evaluate the presence of fetal brain damage, as measured by fetal serum and amniotic fluid levels and brain expression of protein S100  in a rabbit model of placental restriction resembling human intrauterine growth restriction (IUGR) STUDY DESIGN: Fifteen New Zealand pregnant rabbits at gestational age 21 days were randomly assigned to moderate (ligature 20-30% of uteroplacental vessels,n⫽32 fetuses), or severe placental insufficiency (ligature 40-50%,n⫽40 fetuses). All gestational sacs in one uterine horn were treated while sacs from contralateral horn were used as controls (n⫽53). Doppler assessment of fetal vessels was performed before ligature and prior to delivery by caesarean section at 26 days. Fetal and brain weight,anterior-posterior head diameter (A-P diam) and fetal serum (S) and amniotic fluid (AF) samples were obtained. Random sections of the brain were immunostained for S100 RESULTS: Hemodynamic changes in Doppler were progressively abnormal across the experimental groups. Mortality rate showed a significant linear increase through the study groups (controls 6.4%, moderate 37.5%, severe 67.5%,p⬍.001). Immunostained sections showed a clear expression of S100  in glial cells,which was higher in the IUGR group CONCLUSION: IUGR is associated with signs of hypoxic-ischemic brain damage in a rabbit model resembling the hemodynamic situation of human growth restriction. This experimental model might be suitable for future studies on the type and distribution of brain damage at different degrees of severity of IUGR Control (n ⫽ 48) 20–30% (n ⫽ 20) 40–50% (n ⫽ 13) p Fetal weight (gr) A-P diam (mm) Brain weight (gr)
24,2 (7,6) 22,4 (5,3) 2,5 (,13) 2,4 (,6) 0,8 (,06) 0,8 (,07) Control (n ⫽ 10) 20–30% (n ⫽ 19) S100 S (gr/l) 5,6 (2) 4,3 (3,2) S100 AF (gr/l) 0,42 (,7) 1,22 (4,6)
18,3 (5,1) 2,3 (,33) 0,71 (,06) 40–50% (n ⫽ 10) 9,3 (4,6) 1,32 (25,2)
⬍,05 ⬍,05 ⬍,05 ⬍,05 ⬍,05
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.529
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.527
Supplement to DECEMBER 2007 American Journal of Obstetrics & Gynecology
S153
www.AJOG.org 524
EFFECT OF HYPOXIA AND HYPERTHERMIA ON ENOS PROTEIN CONCENTRATION IN CULTURED OVINE UMBILICAL VEIN ENDOTHELIAL CELLS JUAN A ARROYO1, RUSSELL V. ANTHONY2, BRADLEY T. ZIEBELL1, HENRY L. GALAN1, 1University of Colorado Health Sciences Center, Obstetrics and Gynecology, Aurora, Colorado, 2University of Colorado Health Sciences Center, Pedriatics, Aurora, Colorado OBJECTIVE: Nitric Oxide (NO) and endothelial nitric oxide synthase (eNOS), important regulators of placental blood flow, are reduced in a hyperthermic-induced (HT) ovine model of fetal growth restriction (FGR) with fetal hypoxia (HX) and hypertension. Our objective was to differentiate the effect of hypoxia and HT on NO and eNOS production in ovine umbilical vein endothelial cells (OUVEC). STUDY DESIGN: At 130 days gestational age (dGA), OUVEC were isolated with a collagenase B solution and cultured in 2% gelatin coated flasks for up to three passages. CD31 and Factor VIII staining confirmed endothelial cell type. Endothelial cells were treated with HX (2% O2) or HT (40°C) for 24, 48 and 72 hours (H). At each time point, OUVEC eNOS protein and NO from culture media were assessed by Western blot and a NOx assay kit, respectively. RESULTS: Compared to controls, hypoxia-exposed OUVEC showed increased eNOS protein concentration at 48H (2-fold, p⬍ 0.0005), but decreased at 72H (1.6-fold, p⬍ 0.03) while OUVEC media NOx concentration was only increased at 48H (figure) with otherwise no changes. In HT exposed OUVEC, eNOS protein was increased at 72H (2-fold, p⬍ 0.05) with no changes observed for NOx at any time point. CONCLUSION: Hypoxia and hyperthermia independently regulate endothelial eNOS protein production in OUVEC. Hypoxia induced upregulation of this protein is temporary as a concomitant increase in NO production is only seen at 48H, which may reflect ongoing cell death or a decrease in the eNOS substrate L-arginine. (Supported by NIH grant R01 HL071990-01A1).
526
PERINATAL PREDICTORS OF ABNORMAL CHILDHOOD NEURODEVELOPMENT IN EARLY ONSET FETAL GROWTH RESTRICTION (FGR) AHMET BASCHAT1, ROSE VISCARDI1, BRENDA HUSSEY-GARDNER1, NADEEM HASHMI2, CHRISTOPHER HARMAN1, 1 University of Maryland at Baltimore, Baltimore, Maryland, 2University of Maryland at Baltimore, Pediatrics, Baltimore, Maryland OBJECTIVE: Metabolic and cardiovascular deterioration in early onset FGR is prenatally detectable by ductus venosus (DV) Doppler and biophysical profile score (BPP). We analyzed if such surveillance predicts poor neurodevelopment at 2 years corrected age. STUDY DESIGN: Prospective observational study of FGR (Abdominal Circumference ⬍5%ile, high umbilical artery (UA) Doppler). UA, DV Doppler, BPP, birth acidemia (artery pH ⬍7.0 ⫹/or base deficit⬎12), gestational age (GA), birthweight (BW) and neonatal morbidity (NM⫽bronchopulmonary dysplasia, ⬎grade 2 intraventricular bleed, or necrotizing enterocolitis) were related to 2 year neurodevelopmental delay (NDD) as assesed by Best Beginnings Developmental Screen (BBDS), Bayley (BSID) and Clinical Adaptive/Clinical linguistic auditory milestone stage. BSID ⬍70, cerebral palsy (CP), abnormal tone, hearing loss ⫹/or blindness defined NDD. RESULTS: 52 / 87 enrolled patients completed analysis (10 stillbirth, 13 neonatal / 2 infant deaths, 9 no follow up. 15 fetuses had UA reversed diastolic flow (REDV), 20 abnormal DV Doppler, 22 a BPP ⬍6/10. Median delivery GA / BW were 30.1 / 890 g. 10 had acidemia and 25 NM. 28 (53.8%) of infants with NDD included 27 (51.9) with abnormal tone, 19 (36.5%) with speech delay, 14 (26.9%) with abnormal neurologic exam, 5 (9.6%) with hearing deficit, and 4 with CP (7.7%). UA REDV primarily determined hearing and speech deficit (LR 4.8 and 4.9 respectively, r2 ⫽ 0.16 p⫽0.006). While NM was strongly associated with abnormal tone (LR 9.6, p⬍0.0001), BW was the main determinant of this complication r2 ⫽ 0.36, p⬍0.001). Abnormal DV Doppler was the primary determinant of NDD, r2 ⫽ 0.13, p⫽0.03). An abnormal BPP had no impact on any of the study outcomes. CONCLUSION: Chronicity and severity of placental vascular dysfunction independently predict poor neurodevelopment in preterm FGR. Biophysical deterioration does not appear to have an additional impact. Birthweight and neonatal complications are important factors that appear to mediate the effects of prematurity on adverse neurodevelopment. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.528
527
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.526
525
HYPOXIC AND HYPERTHERMIC EFFECTS ON SIGNAL TRANSDUCTION AND PROLIFERATION IN OVINE UMBILICAL VEIN ENDOTHELIAL CELLS JUAN A ARROYO1, BRADLEY T. ZIEBELL1, HENRY L. GALAN1, 1University of Colorado Health Sciences Center, Obstetrics and Gynecology, Denver, Colorado OBJECTIVE: The hyperthermia (HT) induced ovine model of fetal growth restriction (FGR) is characterized by hypoxia and endothelial cell dysfunction (fetal hypertension; reduced umbilical blood flow and placental eNOS protein). Our goal was to assess the independent effects of hypethermia (HT) and hypoxia (HX) on signal transduction pathways affecting cellular proliferation (Ki-67, ERK), survival (AKT) or death (JNK) in ovine umbilical vein endothelial cells (OUVEC). STUDY DESIGN: At 130 days gestational age (dGA), OUVEC were isolated with a collagenase B solution and cultured in 2% gelatin coated flask for up to three passages. CD31 and Factor VIII staining confirmed endothelial cell type. OUVEC were treated with HX (2% O2) or HT (40°C) for 24, 48 and 72 hours (H). Staining for proliferation marker Ki-67 and Western blot analyses for pERK/ERK, pAKT/ AKT and pJNK/JNK were performed at each time point. RESULTS: HX significantly decreased cell proliferation (Ki-67 positive cells) at 24, 48, and 72H (figure), even though ERK (cell survival) was increased at 48H (p⬍0.0003) and AKT (cell survival/apoptosis) was increased at 48 (p⬍0.007) and 72H (p⬍6.0⫻10-5). HX also increased JNK protein at 24H (p⬍0.02) and 48H (p⬍0.0004). Conversely, HT did not affect cell proliferation, but increased ERK protein at all time points. CONCLUSION: HX stress decreases OUVEC proliferation (2Ki-67 staining; 1JNK protein) while attempting to survive (1ERK; 1AKT proteins) HT does not decrease proliferation suggesting that HX is the primary stressor in endothelial cell dysfunction in these experimental conditions. (Supported by NIH grant R01 HL071990-01A1).
HYPOXIC BRAIN DAMAGE AS MEASURED BY FETAL BLOOD AND AMNIOTIC FLUID LEVELS AND IMMUNOHISTOCHEMICAL BRAIN EXPRESSION OF S100 IN A RABBIT MODEL OF INTRAUTERINE GROWTH RESTRICTION ELISENDA EIXARCH1, EDGAR HERNANDEZ-ANDRADE1, FRANCESC FIGUERAS1, SILVIA OLIVEIRA1, FATIMA CRISPI1, EDUARD GRATACOS1, 1Hospital Clinic-University of Barcelona, Department of Maternal-Fetal Medicine (ICGON)), IDIBAPS and CIBER-ER, Barcelona, Spain OBJECTIVE: To evaluate the presence of fetal brain damage, as measured by fetal serum and amniotic fluid levels and brain expression of protein S100  in a rabbit model of placental restriction resembling human intrauterine growth restriction (IUGR) STUDY DESIGN: Fifteen New Zealand pregnant rabbits at gestational age 21 days were randomly assigned to moderate (ligature 20-30% of uteroplacental vessels,n⫽32 fetuses), or severe placental insufficiency (ligature 40-50%,n⫽40 fetuses). All gestational sacs in one uterine horn were treated while sacs from contralateral horn were used as controls (n⫽53). Doppler assessment of fetal vessels was performed before ligature and prior to delivery by caesarean section at 26 days. Fetal and brain weight,anterior-posterior head diameter (A-P diam) and fetal serum (S) and amniotic fluid (AF) samples were obtained. Random sections of the brain were immunostained for S100 RESULTS: Hemodynamic changes in Doppler were progressively abnormal across the experimental groups. Mortality rate showed a significant linear increase through the study groups (controls 6.4%, moderate 37.5%, severe 67.5%,p⬍.001). Immunostained sections showed a clear expression of S100  in glial cells,which was higher in the IUGR group CONCLUSION: IUGR is associated with signs of hypoxic-ischemic brain damage in a rabbit model resembling the hemodynamic situation of human growth restriction. This experimental model might be suitable for future studies on the type and distribution of brain damage at different degrees of severity of IUGR Control (n ⫽ 48) 20–30% (n ⫽ 20) 40–50% (n ⫽ 13) p Fetal weight (gr) A-P diam (mm) Brain weight (gr)
24,2 (7,6) 22,4 (5,3) 2,5 (,13) 2,4 (,6) 0,8 (,06) 0,8 (,07) Control (n ⫽ 10) 20–30% (n ⫽ 19) S100 S (gr/l) 5,6 (2) 4,3 (3,2) S100 AF (gr/l) 0,42 (,7) 1,22 (4,6)
18,3 (5,1) 2,3 (,33) 0,71 (,06) 40–50% (n ⫽ 10) 9,3 (4,6) 1,32 (25,2)
⬍,05 ⬍,05 ⬍,05 ⬍,05 ⬍,05
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.529
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.527
Supplement to DECEMBER 2007 American Journal of Obstetrics & Gynecology
S153