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755: Toll-like receptor expression in preeclampsia
Poster Session V
Fetus Diabetes, etc
elevated BP had the strongest association with placental abruption (OR ⫽ 5.82, 95% CI 4.87-6.77, p ⬍ 0.001) with a significantly lower strength of association with cocaine use (OR ⫽ 3.22, 95% CI 3.003.44, p ⬍ 0.01), smoking (OR ⫽ 1.89, 95% CI 1.4-2.38, p ⬍ 0.03), and history of placental abruption (OR ⫽ 2.8, 95% CI 2.65-2.95, p ⬍ 0.01). CONCLUSION: Elevated blood pressure is strongly associated with placental abruption. 0002-9378/$ – see front matter • doi:10.1016/j.ajog.2009.10.771
755 Toll-like receptor expression in preeclampsia Joshua Nitsche1, Dennis McWeeney1, Wendy White1, Norman Davies1, Carl Rose1, William Watson1, Brian Brost1 1
Mayo Clinic College of Medicine, Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Rochester, Minnesota
OBJECTIVE: In vitro, neutrophil Toll-like receptor (TLR) activation provokes an immunological response mimicking that seen in preeclampsia, such as cytokine release, altered integrin expression, and superoxide production. TLR4 expression is increased in the placenta during preeclampsia, but data on TLR expression in other tissues during preclampsia is lacking. This study uses real time PCR to determine if TLR mRNA expression in maternal neutrophils is altered in preeclampsia. STUDY DESIGN: After IRB approval, 20 preeclamptic patients, 10 with mild and 10 with severe disease, were enrolled in the study along with 22 normal pregnant controls at similar gestational ages. Venous blood was drawn, neutrophils isolated, total RNA extracted, and cDNA copies made. Real time quantitative PCR was then performed and counts standardized to GAPDH levels using the formula 2^-([TLR][GAPDH]). After logarithmic transformation, TLR expression was compared between preeclamptic patients and normal pregnant controls using a two-sample t-test.‘ RESULTS: The results are summarized in Table 1. Compared to normal pregnant controls there was a significant decrease in TLR2 and TLR4 expression whether the analysis included all preeclamptic women or was limited to women with mild or severe disease alone. CONCLUSION: Although increased in the placenta, neutrophil TLR2 and TLR4 expression is decreased in preeclampsia. Surprisingly the decrease in TLR expression was greater in women with mild compared to severe disease. Given the many immunological changes in preeclampsia, this may represent an adaptation to the increased inflammatory signals present in preeclampsia. Further study is needed to clarify the role of the TLR in preeclampsia. Table 1. TLR expression in Preeclampsia (Mean ⴙ/ⴚ SEM, * p<0.05)
Total Preeclampsia
TLR2
TLR4
0.61 ⫹/⫺ 0.11*
0.26 ⫹/⫺ 0.04*
..........................................................................................................................................................................................
Control 1.93 ⫹/⫺ 0.32 0.98 ⫹/⫺ 0.15 .......................................................................................................................................................................................... Mild Preeclampsia 0.35 ⫹/⫺ 0.09* 0.22⫹/⫺ 0.06* .......................................................................................................................................................................................... Control 1.92 ⫹/⫺ 0.50 0.96 ⫹/⫺ 0.22 .......................................................................................................................................................................................... Severe Preeclampsia 0.86 ⫹/⫺ 0.16* 0.31 ⫹/⫺ 0.05* .......................................................................................................................................................................................... Control 1.93 ⫹/⫺ 0.43 0.99 ⫹/⫺ 0.21 .......................................................................................................................................................................................... 0002-9378/$ – see front matter • doi:10.1016/j.ajog.2009.10.772
756 N-myc downstream regulated gene-2 (NDRG-2) is elevated in placentas with severe preeclampsia Gene Lamonica1, William Rayburn2, Kimberly Leslie3 1
American College of Obstetricians and Gynecologists, Albuquerque, New Mexico, 2University of New Mexico, Obstetrics and Gynecology, New Mexico, 3University of Iowa, Obstetrics and Gynecology, Iowa City, Iowa
OBJECTIVE: Elevated expression of N-myc Down Regulated Genes (NDRG) has been described in response by trophoblastic tissue to hypoxic injury. The NDRG family of factors is proposed to protect cells against apoptosis during hypoxia and stress, but the relationship
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www.AJOG.org of these proteins to the trophoblastic adaptation to preeclampsia has not been fully explored. Our objective was to use a novel, proprietary plate-based array to profile the expression of 23 Hif-regulated gene products (including NDRG – 1 and 2) in the presence of normal and preeclamptic placental tissue to identify factors that correlate with preeclampsia. STUDY DESIGN: We sampled 11 placentas immediately after delivery: 4 normal, 4 mild preeclamptic and 3 severe preeclamptic. Total RNA was isolated from 30 mg of placental tissue from each specimen. The total RNA was then reverse transcribed to cDNA and then applied to Signosis Human Hif-Regulated cDNA plate arrays and incubated per protocol. Captured cDNA was detected via a streptavidin-HRP chemiluminescent reaction. Relative chemiluninescence was detected with a plate reader. Results are reported as relative chemiluminescent units. RESULTS: A significant increase in relative chemiluminesence was detected in NDRG-2 in severe preeclamptic compared with normal placentas using ANOVA, p0.05. There was an increase in expression in mild preeclamptics compared to normals, but that difference was not significant. A definite trend in increased expression of NDRG-1, Hif-1, Hif-2, and PAI-1, was observed in preelamptic versus normal placentas. There was also a downward trend in the expression of VEGF comparing normals versus preeclamptics. However, both of these trends did not reach statistical significance. CONCLUSION: This pilot study demonstrates a significant elevation of NDRG-2 in placentas of severe preeclamptics. We propose that high NDGR-2 expression may be a marker for worsening disease and highlights a compensatory pathway through which trophoblastic cells attempt to escape hypoxic insult. 0002-9378/$ – see front matter • doi:10.1016/j.ajog.2009.10.773
757 Quantitative gene expression analysis of iNOS and eNOS isoform and NO production in placentas from HELLP syndrome patients Laura Mazzanti1, Monia Cecati2, Arianna Vignini3, Monica Emanuelli4, Stefano Raffaele Giannubilo5, Franca Saccucci6, Andrea Luigi Tranquilli7 1
Marche Polytechnic University, Biochemistry, Ancona, Italy, 2Marche Polytechnic University, Maternal and Child Sciences, Ancona, AN, Italy, 3 Polytechnic University of Marche, Biochemistry, Ancona, Italy, 4 Marche Polytechnic University, Biochemical biotechnologies, Ancona, Italy, 5Polytechnic University of Marche, Department of Clinica Sciences, Ancona, Marche, Italy, 6Polytechnic University of Marche, Biology and Genetics, Ancona, Italy, 7Marche Polytechnic University, Maternal and Child Sciences, Ancona, Italy
OBJECTIVE: To determine placental gene expression of endothelial and
inducible nitric oxide synthases (eNOS and iNOS) in patients with HELLP syndrome using Real Time quantitative PCR. To measure nitric oxide (NO) levels in the same samples. STUDY DESIGN: Term placentas were obtained from 30 normal pregnancies (controls) and 15 patients with HELLP syndrome. The levels of mRNA were evaluated by real-time PCR, while NO production was measured by means of a commercially available Kit by assay designs (Stressgen). RESULTS: Placental gene expression of iNOS and eNOS were significantly lower in the HELLP group than in controls (3.49-fold and 2.83fold lower respectively). Placental nNOS mRNA level in HELLP was too low to be determined by Real Time quantitative PCR. On the contrary NO production was higher in placentas from HELLP syndrome than in controls (38.29 ⫾ 5.87 nmol nitrite/mg prot vs. 23.98 ⫾ 5.14 nmol nitrite/mg prot; p⬍0.05). CONCLUSION: The reduced eNOS and iNOS gene expression in women with HELLP syndrome may indicate the extreme placental dysfunction that is unable to compensate the endothelial derangement and the related hypertension. The higher NO formation found in HELLP placentas could be explained as a counteraction to the impaired feto-
American Journal of Obstetrics & Gynecology Supplement to DECEMBER 2009
Fetus Diabetes, etc
elevated BP had the strongest association with placental abruption (OR ⫽ 5.82, 95% CI 4.87-6.77, p ⬍ 0.001) with a significantly lower strength of association with cocaine use (OR ⫽ 3.22, 95% CI 3.003.44, p ⬍ 0.01), smoking (OR ⫽ 1.89, 95% CI 1.4-2.38, p ⬍ 0.03), and history of placental abruption (OR ⫽ 2.8, 95% CI 2.65-2.95, p ⬍ 0.01). CONCLUSION: Elevated blood pressure is strongly associated with placental abruption. 0002-9378/$ – see front matter • doi:10.1016/j.ajog.2009.10.771
755 Toll-like receptor expression in preeclampsia Joshua Nitsche1, Dennis McWeeney1, Wendy White1, Norman Davies1, Carl Rose1, William Watson1, Brian Brost1 1
Mayo Clinic College of Medicine, Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Rochester, Minnesota
OBJECTIVE: In vitro, neutrophil Toll-like receptor (TLR) activation provokes an immunological response mimicking that seen in preeclampsia, such as cytokine release, altered integrin expression, and superoxide production. TLR4 expression is increased in the placenta during preeclampsia, but data on TLR expression in other tissues during preclampsia is lacking. This study uses real time PCR to determine if TLR mRNA expression in maternal neutrophils is altered in preeclampsia. STUDY DESIGN: After IRB approval, 20 preeclamptic patients, 10 with mild and 10 with severe disease, were enrolled in the study along with 22 normal pregnant controls at similar gestational ages. Venous blood was drawn, neutrophils isolated, total RNA extracted, and cDNA copies made. Real time quantitative PCR was then performed and counts standardized to GAPDH levels using the formula 2^-([TLR][GAPDH]). After logarithmic transformation, TLR expression was compared between preeclamptic patients and normal pregnant controls using a two-sample t-test.‘ RESULTS: The results are summarized in Table 1. Compared to normal pregnant controls there was a significant decrease in TLR2 and TLR4 expression whether the analysis included all preeclamptic women or was limited to women with mild or severe disease alone. CONCLUSION: Although increased in the placenta, neutrophil TLR2 and TLR4 expression is decreased in preeclampsia. Surprisingly the decrease in TLR expression was greater in women with mild compared to severe disease. Given the many immunological changes in preeclampsia, this may represent an adaptation to the increased inflammatory signals present in preeclampsia. Further study is needed to clarify the role of the TLR in preeclampsia. Table 1. TLR expression in Preeclampsia (Mean ⴙ/ⴚ SEM, * p<0.05)
Total Preeclampsia
TLR2
TLR4
0.61 ⫹/⫺ 0.11*
0.26 ⫹/⫺ 0.04*
..........................................................................................................................................................................................
Control 1.93 ⫹/⫺ 0.32 0.98 ⫹/⫺ 0.15 .......................................................................................................................................................................................... Mild Preeclampsia 0.35 ⫹/⫺ 0.09* 0.22⫹/⫺ 0.06* .......................................................................................................................................................................................... Control 1.92 ⫹/⫺ 0.50 0.96 ⫹/⫺ 0.22 .......................................................................................................................................................................................... Severe Preeclampsia 0.86 ⫹/⫺ 0.16* 0.31 ⫹/⫺ 0.05* .......................................................................................................................................................................................... Control 1.93 ⫹/⫺ 0.43 0.99 ⫹/⫺ 0.21 .......................................................................................................................................................................................... 0002-9378/$ – see front matter • doi:10.1016/j.ajog.2009.10.772
756 N-myc downstream regulated gene-2 (NDRG-2) is elevated in placentas with severe preeclampsia Gene Lamonica1, William Rayburn2, Kimberly Leslie3 1
American College of Obstetricians and Gynecologists, Albuquerque, New Mexico, 2University of New Mexico, Obstetrics and Gynecology, New Mexico, 3University of Iowa, Obstetrics and Gynecology, Iowa City, Iowa
OBJECTIVE: Elevated expression of N-myc Down Regulated Genes (NDRG) has been described in response by trophoblastic tissue to hypoxic injury. The NDRG family of factors is proposed to protect cells against apoptosis during hypoxia and stress, but the relationship
S272
www.AJOG.org of these proteins to the trophoblastic adaptation to preeclampsia has not been fully explored. Our objective was to use a novel, proprietary plate-based array to profile the expression of 23 Hif-regulated gene products (including NDRG – 1 and 2) in the presence of normal and preeclamptic placental tissue to identify factors that correlate with preeclampsia. STUDY DESIGN: We sampled 11 placentas immediately after delivery: 4 normal, 4 mild preeclamptic and 3 severe preeclamptic. Total RNA was isolated from 30 mg of placental tissue from each specimen. The total RNA was then reverse transcribed to cDNA and then applied to Signosis Human Hif-Regulated cDNA plate arrays and incubated per protocol. Captured cDNA was detected via a streptavidin-HRP chemiluminescent reaction. Relative chemiluninescence was detected with a plate reader. Results are reported as relative chemiluminescent units. RESULTS: A significant increase in relative chemiluminesence was detected in NDRG-2 in severe preeclamptic compared with normal placentas using ANOVA, p0.05. There was an increase in expression in mild preeclamptics compared to normals, but that difference was not significant. A definite trend in increased expression of NDRG-1, Hif-1, Hif-2, and PAI-1, was observed in preelamptic versus normal placentas. There was also a downward trend in the expression of VEGF comparing normals versus preeclamptics. However, both of these trends did not reach statistical significance. CONCLUSION: This pilot study demonstrates a significant elevation of NDRG-2 in placentas of severe preeclamptics. We propose that high NDGR-2 expression may be a marker for worsening disease and highlights a compensatory pathway through which trophoblastic cells attempt to escape hypoxic insult. 0002-9378/$ – see front matter • doi:10.1016/j.ajog.2009.10.773
757 Quantitative gene expression analysis of iNOS and eNOS isoform and NO production in placentas from HELLP syndrome patients Laura Mazzanti1, Monia Cecati2, Arianna Vignini3, Monica Emanuelli4, Stefano Raffaele Giannubilo5, Franca Saccucci6, Andrea Luigi Tranquilli7 1
Marche Polytechnic University, Biochemistry, Ancona, Italy, 2Marche Polytechnic University, Maternal and Child Sciences, Ancona, AN, Italy, 3 Polytechnic University of Marche, Biochemistry, Ancona, Italy, 4 Marche Polytechnic University, Biochemical biotechnologies, Ancona, Italy, 5Polytechnic University of Marche, Department of Clinica Sciences, Ancona, Marche, Italy, 6Polytechnic University of Marche, Biology and Genetics, Ancona, Italy, 7Marche Polytechnic University, Maternal and Child Sciences, Ancona, Italy
OBJECTIVE: To determine placental gene expression of endothelial and
inducible nitric oxide synthases (eNOS and iNOS) in patients with HELLP syndrome using Real Time quantitative PCR. To measure nitric oxide (NO) levels in the same samples. STUDY DESIGN: Term placentas were obtained from 30 normal pregnancies (controls) and 15 patients with HELLP syndrome. The levels of mRNA were evaluated by real-time PCR, while NO production was measured by means of a commercially available Kit by assay designs (Stressgen). RESULTS: Placental gene expression of iNOS and eNOS were significantly lower in the HELLP group than in controls (3.49-fold and 2.83fold lower respectively). Placental nNOS mRNA level in HELLP was too low to be determined by Real Time quantitative PCR. On the contrary NO production was higher in placentas from HELLP syndrome than in controls (38.29 ⫾ 5.87 nmol nitrite/mg prot vs. 23.98 ⫾ 5.14 nmol nitrite/mg prot; p⬍0.05). CONCLUSION: The reduced eNOS and iNOS gene expression in women with HELLP syndrome may indicate the extreme placental dysfunction that is unable to compensate the endothelial derangement and the related hypertension. The higher NO formation found in HELLP placentas could be explained as a counteraction to the impaired feto-
American Journal of Obstetrics & Gynecology Supplement to DECEMBER 2009