- Email: [email protected]
41: The effect of maternal pravastatin therapy on adverse neurologic outcomes of the offspring in a murine model of preeclampsia
www.AJOG.org
General
Oral Plenary Session II (Fellows)
vascular disease in the general population. We aim to estimate the incidence of cardiovascular disease among pregnant and reproductive aged women with OSA. STUDY DESIGN: 1008 consecutive women aged 15-40 who met criteria for OSA based on Apnea-Hypopnea Index (AHI) during nocturnal polysomnogram (PSG) from 2005-2012 at a tertiary care center were included. Sleep lab data and individual transthoracic echocardiogram (TTE) reports were reviewed. RESULTS: Among the cohort, 551 of 1,008 women (55%) had mild (AHI 5-15), 235(23%) had moderate (AHI 15-30), and 222(22%) had severe (AHI ⬎30) OSA. Those with mild-moderate and severe OSA were similar in age, race, and body mass index. During PSG, the severe OSA group had higher AHI scores (62⫾31 v. 12⫾6), lower O2 nadirs in desaturation (SaO2 78⫾12 v. 83⫾9 mmHg), and more frequent percent of total respiratory time below 90% SaO2 (10⫾18 v. 3⫾12 %), p⬍.001 for each. The frequency of cardiac arrhythmias during PSG increased with OSA severity (20% mild-moderate v. 29% severe, p⫽.006). Of 250 who underwent TTE, 64 (26%) had evidence of heart disease: 22 had left ventricular hypertrophy (LV mass index ⬎110g, 9%), 15 had pulmonary hypertension (systolic pulmonary artery pressure ⬎40 mmHg, 6%), 13 had valve disease (5%), 8 had heart failure (ejection fraction ⬍40%, 3%) and 50 (20%) had systolic or diastolic dysfunction. TTE abnormalities were similarly frequent within OSA severity groups (26% mild, 23% moderate, 28% severe OSA, p⫽.8). Among the 84 pregnant women, 38 had an echocardiogram. Pregnant and non-pregnant women had similar rates of severe OSA (19 v. 22%), cardiac arrhythmias (23 v. 22%) and abnormal TTE (28 v. 25%) (p⬎.5 for each). CONCLUSION: Pregnant gravidas with OSA have a 26% incidence of cardiac dysfunction. Echocardiogram should be considered during the clinical management of these women.
39 Predictors of recurrent preterm birth: secondary analysis of maternal body fluid proteins among women in recurrent preterm labor Jamie Lo1, Ashok Reddy2, Philip Wilmarth3, Amanda Kinhnarath1, Janice Snyder1, Monica Rincon1, Michael Gravett4, Srinivasa Nagalla5, Leonardo Pereira1 1 Oregon Health and Science University, Obstetrics and Gynecology, Portland, OR, 2Oregon Health and Science University, Proteomics Shared Resources, Portland, OR, 3Oregon Health and Science University, Department of Biochemistry and Molecular Biology, Portland, OR, 4University of Washington, Department of Obstetrics and Gynecology, Seattle, WA, 5 Oregon Health and Science University, Department of Pediatrics, Portland, OR
OBJECTIVE: A secondary analysis of 4 cervical vaginal fluid (CVF) proteins as predictors of recurrent preterm birth (RPTB) in at-risk women prior to onset of preterm labor (PTL). STUDY DESIGN: Patients with singleton gestations and a history of preterm birth (PTB) in all past pregnancies were enrolled prospectively from 2007-2011 and underwent serial CVF sampling at 2 week intervals. CVF proteomes were compared between subjects with and without RPTB. CVF proteins were taken within 2 weeks of SPTB prior to onset of PTL and matched for gestational age with subjects without RPTB. Liquid chromatography tandem mass spectroscopy was performed by prior established techniques. Protein matches were conducted with Sprot human PASTA protein database v.2012.06. Protein quantification was performed by spectral counting. Pair-wise comparisons used 2 test and Z-transformation of average spectral counts. RESULTS: 107 patients were enrolled and 97 were analyzed after exclusions. Of the 97, SPTB ⬍ 37 wga occurred in 26 (26.8%), and SPTB ⬍ 34 wga occurred in 11 (11.3%). Of 748 proteins identified, 72 were statistically significantly at p ⬍ 0.05 and 38 were highly differential at p ⬍0.01. Of those, 4 were highly abundant: Heat Shock Protein Beta-1 (HSPB1) and Cysteine-rich secretory protein 3 (CRISP3) were upregulated and Protein S100-A7 (Psoriasin) and Alpha-enolase (Enolase 1) were downregulated (Table 1). HSPB1 is in actin cystoskeleton
and expressed in environmental stress. Psoriasin is secreted and plays a role in inflammatory response. CRISP3 is secreted and involved with immune response. Enolase 1 is an enzyme that stimulates immunoglobulin production and participates in inflammatory response. CONCLUSION: We identified 4 highly abundant and differentially expressed proteins in the CVF of women with RPTB prior to the onset of PTL. These proteins are involved in inflammatory response and cystoskeletal remodeling and represent potential predictive biomarkers and therapeutic targets.
Protein differential expression (spectral counts)
40 Transplacental transfer of glyburide: is it clinically significant? Rachelle Schwartz1, Barak Rosenn1, Katarina Aleksa2, Gideon Koren2 1 St. Luke’s-Roosevelt Hospital Center, Obstetrics & Gynecology, New York, NY, 2Hospital for Sick Children, Division of Clinical Pharmacology/ Toxicology, Toronto, ON, Canada
OBJECTIVE: To determine the magnitude of transplacental transfer of glyburide (GLY) in women with gestational diabetes (GDM). STUDY DESIGN: A prospective, observational study was conducted including patients with signed informed consent and GDM on glyburide therapy. Upon delivery admission, the GLY dose and time of last dose were recorded. Immediately post-delivery, maternal and umbilical venous blood samples were obtained, centrifuged, and the plasma was frozen for future evaluation. The concentrations of GLY were determined by using previously described and validated methods employing high performance liquid chromatography-mass spectrometry. The limit of detection was 0.5 ng/mL and limit of quantification was 0.75 ng/mL. RESULTS: Twenty-two patient sets were analyzed. The mean and median total daily maternal GLY doses were 7.05 ⫾ 6.61 and 2.5mg/day, respectively. The mean and median times between last dose and sampling were 17.6 ⫾ 12.7 and 15.0 hours, respectively. Maternal GLY concentrations ranged from 0.93-70.7 ng/mL, with a mean and median of 13.6 ⫾ 19.8 ng/mL and 5.26 ng/mL, respectively. Umbilical vein GLY concentrations ranged from undetectable to 32.4 ng/mL, with a mean and median of 6.6 ⫾ 7.9 ng/mL and 3.54 ng/mL, respectively. The concentration of GLY was ⬍10ng/mL in 18 (82%) of the 22 umbilical samples. Thirty-two percent (7/22) of umbilical samples had a higher concentration than the maternal sample, but 86% (6/7) of those were ⬍10ng/ml. The mean ratio of umbilical GLY to maternal GLY was 0.77 ⫾ 0.49. Using natural log transformation and regression analysis, a 1% change in maternal GLY resulted in a 0.71% change in umbilical GLY (r2⫽0.57, p⬍0.0001). CONCLUSION: Transplacental transfer of glyburide does occur and there appears to be a relationship between maternal and umbilical glyburide concentrations. However, in most neonates, the concentration is well below therapeutic levels.
41 The effect of maternal pravastatin therapy on adverse neurologic outcomes of the offspring in a murine model of preeclampsia Alissa Carver1, Esther Tamayo1, J Regino Perez-Polo2, George Saade1, Maged Costantine1 1 University of Texas Medical Branch, Obstetrics & Gynecology, Galveston, TX, 2University of Texas Medical Branch, Biochemistry & Molecular Biology, Galveston, TX
OBJECTIVE: Preeclampsia alters fetal programming and results in long-term adverse outcomes in the offspring. Pravastatin, a hydro-
Supplement to JANUARY 2013 American Journal of Obstetrics & Gynecology
S25
Oral Plenary Session II (Fellows) philic drug that does not cross the placenta, prevents preeclampsia in animal models. This study aims to define the role of preeclampsia and pravastatin therapy on fetal programming of adult motor function using a mouse model. STUDY DESIGN: At day 8 of gestation, pregnant CD-1 mice were randomized to tail vein injection with adenovirus carrying soluble Fmslike tyrosine kinase 1 (sFlt-1) or murine immunoglobulin G2Fc (mFc; control). sFlt-1 dams received pravastatin (5mg/kg/d; sFlt-pra group) or water (sFlt group) until weaning, and mFc received water (mFc group). Offspring (n⫽13-20/group) were tested at 3 and 6 months of age using motor and coordination assays: righting reflex (RR), negative geotaxis (NG), balance beam (BB), and grid climbing (GC). 2-way ANOVA and ANCOVA were used for statistical analysis. RESULTS: All offspring retained RR at 3 and 6 months. At 6 months, sFlt offspring took longer to right themselves in NG compared to mFc and sFlt-pra (p⫽0.003). From 3 to 6 months, sFlt time to righting in NG worsened (p⫽0.03). Female sFlt offspring had double the paw slips (PS) at 3 months on BB compared with mFc and sFlt-pra (p⬍0.001). PS decreased in sFlt group at 6 months (p⫽0.005). BB falls were similar between groups at 3 and 6 months. No difference was seen in time to begin GC, but male sFlt offspring more than doubled their time to reach the top at 3 months (p⫽0.01). At 6 months, both time to start GC and to reach the top was higher in sFlt compared to mFc and sFlt-pra (p⫽0.01 and 0.04 respectively). Between 3 and 6 months, time to start GC worsened for sFlt (p⫽0.007). In all assays the offspring in the sFlt-pra group showed responses similar to control. CONCLUSION: Preeclampsia alters motor and coordination function in adult offspring. Maternal therapy with pravastatin prevents this altered developmental programming in this animal model of preeclampsia.
42 Progesterone supplementation during pregnancy does not alter signal transduction pathways involved in myometrial contractility Christopher Nold1, Monique Maubert1, Lauren Anton1, Michal Elovitz1 1 University of Pennsylvania Perelman School of Medicine, Maternal and Child Health Research Program, Department of Obstetrics and Gynecology, Philadelphia, PA
OBJECTIVE: Clinically, vaginal progesterone (VP) and 17 alpha-hydroxyprogestreone caproate (17P) have been shown to prevent preterm birth (PTB) in high risk populations. However, the molecular mechanisms by which progesterone supplementation prevents PTB remains undiscovered. This study sought to assess if vaginal or systemically administered progestational agents (as used clinically) altered signal transduction pathways implicated in myometrial activation and contractility. STUDY DESIGN: Using a mouse model, on days E14-E17 pregnant CD-1 mice were treated with either 0.1cc of 10mg/ml of 17P subcutaneously, 0.1cc of castor oil subcutaneously, 0.1 cc of 25 mg/ml of progesterone in Replens vaginally, or 0.1cc of Replens vaginally, with four dams per treatment group. Mice were sacrificed six hours after the last treatment on E17.5. Uterine tissue was collected from all treatment groups. QPCR was performed to assess differential gene expression in the following pathways: 1) contraction-associated proteins (CAPs): connexin-43, oxytocin receptor and cyclooxygenase-2; 2) progesterone mediated regulators of uterine quiescence: stat 5b, zeb 1, zeb 2, 20-aphahydroxysteroid dehydrogenase; and 3) microRNAs involved in uterine quiescence and contractility: mir-200a and mir-429. RESULTS: Neither vaginal progesterone nor systemically administered 17P significantly altered the signal transduction pathways investigated. Specifically, none of the individual targets assessed were altered by VP or 17P. CONCLUSION: These studies suggest that treatment with VP or 17P do not prevent preterm birth through modulation of signal transduction pathways involved in uterine quiescence or contractility. If these
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General
www.AJOG.org
agents are having a clinical effect in the prevention of preterm birth, than it is prudent that their mechanisms of action be elucidated so that this therapy can be used in the most appropriate patients at risk for preterm birth.
43 Smoking and preeclamspia protection: cigarette smoke increases placental adrenomedullin expression and improves trophoblast invasion via the adrenomedullin pathway Daniel Kraus1, Liping Feng1, R. Phillips Heine1, Haywood Brown1, Amy Murtha1, Chad Grotegut1 1 Duke University, Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Durham, NC
OBJECTIVE: Smoking reduces preeclampsia risk, but the mechanism of this effect is unknown. Adrenomedullin (ADM) is a vasoactive peptide highly expressed in the placenta. Placentas of preeclamptics exhibit decreased ADM expression. Furthermore, placentas of mice lacking ADM exhibit a preeclampsia phenotype of decreased villi branching and fewer decidual natural killer cells. Our aim was to determine the impact of cigarette smoke extract (CSE) on trophoblast invasion and if the effect was mediated by ADM. We also sought to determine whether placentas from smokers demonstrated enhanced ADM expression. STUDY DESIGN: HTR-8/SVneo trophoblast cells were incubated for 24 hours in Matrigel-invasion chambers with six treatment groups: nonstimulated (NS), ADM, ADM inhibitor (Inhibitor), 1% CSE, ADM ⫹ Inhibitor, and 1% CSE ⫹ Inhibitor. Cells that penetrated through the chambers were quantified, invasion indices were calculated, and compared using a one-way ANOVA with Bonferroni corrections. Next, term placental tissue was obtained from eleven smokers and eleven non-smokers and processed for immunohistochemistry. The intensity of ADM staining in the syncytiotrophoblasts was evaluated by three blinded observers using a four-point intensity scale and compared between the groups. RESULTS: Trophoblast cells treated with either ADM or CSE exhibited increased invasion compared to NS [1.5-fold (Figure, *p⬍0.01) and 1.45-fold (Figure, *p⬍0.01), respectively]. Co-treatment with the ADM inhibitor significantly attenuated the increased invasion seen with both ADM or CSE (Figure, ^p⬍0.01). Placentas from smokers demonstrated more intense ADM staining compared to placentas from non-smokers (p⬍0.004). CONCLUSION: CSE increases trophoblast cell invasion via an ADM-mediated process, and placental ADM expression is increased among term smokers. These findings provide evidence that the ADM pathway may play a role in the protection from preeclampsia seen in smokers.
Matrigel invasion assay
American Journal of Obstetrics & Gynecology Supplement to JANUARY 2013
General
Oral Plenary Session II (Fellows)
vascular disease in the general population. We aim to estimate the incidence of cardiovascular disease among pregnant and reproductive aged women with OSA. STUDY DESIGN: 1008 consecutive women aged 15-40 who met criteria for OSA based on Apnea-Hypopnea Index (AHI) during nocturnal polysomnogram (PSG) from 2005-2012 at a tertiary care center were included. Sleep lab data and individual transthoracic echocardiogram (TTE) reports were reviewed. RESULTS: Among the cohort, 551 of 1,008 women (55%) had mild (AHI 5-15), 235(23%) had moderate (AHI 15-30), and 222(22%) had severe (AHI ⬎30) OSA. Those with mild-moderate and severe OSA were similar in age, race, and body mass index. During PSG, the severe OSA group had higher AHI scores (62⫾31 v. 12⫾6), lower O2 nadirs in desaturation (SaO2 78⫾12 v. 83⫾9 mmHg), and more frequent percent of total respiratory time below 90% SaO2 (10⫾18 v. 3⫾12 %), p⬍.001 for each. The frequency of cardiac arrhythmias during PSG increased with OSA severity (20% mild-moderate v. 29% severe, p⫽.006). Of 250 who underwent TTE, 64 (26%) had evidence of heart disease: 22 had left ventricular hypertrophy (LV mass index ⬎110g, 9%), 15 had pulmonary hypertension (systolic pulmonary artery pressure ⬎40 mmHg, 6%), 13 had valve disease (5%), 8 had heart failure (ejection fraction ⬍40%, 3%) and 50 (20%) had systolic or diastolic dysfunction. TTE abnormalities were similarly frequent within OSA severity groups (26% mild, 23% moderate, 28% severe OSA, p⫽.8). Among the 84 pregnant women, 38 had an echocardiogram. Pregnant and non-pregnant women had similar rates of severe OSA (19 v. 22%), cardiac arrhythmias (23 v. 22%) and abnormal TTE (28 v. 25%) (p⬎.5 for each). CONCLUSION: Pregnant gravidas with OSA have a 26% incidence of cardiac dysfunction. Echocardiogram should be considered during the clinical management of these women.
39 Predictors of recurrent preterm birth: secondary analysis of maternal body fluid proteins among women in recurrent preterm labor Jamie Lo1, Ashok Reddy2, Philip Wilmarth3, Amanda Kinhnarath1, Janice Snyder1, Monica Rincon1, Michael Gravett4, Srinivasa Nagalla5, Leonardo Pereira1 1 Oregon Health and Science University, Obstetrics and Gynecology, Portland, OR, 2Oregon Health and Science University, Proteomics Shared Resources, Portland, OR, 3Oregon Health and Science University, Department of Biochemistry and Molecular Biology, Portland, OR, 4University of Washington, Department of Obstetrics and Gynecology, Seattle, WA, 5 Oregon Health and Science University, Department of Pediatrics, Portland, OR
OBJECTIVE: A secondary analysis of 4 cervical vaginal fluid (CVF) proteins as predictors of recurrent preterm birth (RPTB) in at-risk women prior to onset of preterm labor (PTL). STUDY DESIGN: Patients with singleton gestations and a history of preterm birth (PTB) in all past pregnancies were enrolled prospectively from 2007-2011 and underwent serial CVF sampling at 2 week intervals. CVF proteomes were compared between subjects with and without RPTB. CVF proteins were taken within 2 weeks of SPTB prior to onset of PTL and matched for gestational age with subjects without RPTB. Liquid chromatography tandem mass spectroscopy was performed by prior established techniques. Protein matches were conducted with Sprot human PASTA protein database v.2012.06. Protein quantification was performed by spectral counting. Pair-wise comparisons used 2 test and Z-transformation of average spectral counts. RESULTS: 107 patients were enrolled and 97 were analyzed after exclusions. Of the 97, SPTB ⬍ 37 wga occurred in 26 (26.8%), and SPTB ⬍ 34 wga occurred in 11 (11.3%). Of 748 proteins identified, 72 were statistically significantly at p ⬍ 0.05 and 38 were highly differential at p ⬍0.01. Of those, 4 were highly abundant: Heat Shock Protein Beta-1 (HSPB1) and Cysteine-rich secretory protein 3 (CRISP3) were upregulated and Protein S100-A7 (Psoriasin) and Alpha-enolase (Enolase 1) were downregulated (Table 1). HSPB1 is in actin cystoskeleton
and expressed in environmental stress. Psoriasin is secreted and plays a role in inflammatory response. CRISP3 is secreted and involved with immune response. Enolase 1 is an enzyme that stimulates immunoglobulin production and participates in inflammatory response. CONCLUSION: We identified 4 highly abundant and differentially expressed proteins in the CVF of women with RPTB prior to the onset of PTL. These proteins are involved in inflammatory response and cystoskeletal remodeling and represent potential predictive biomarkers and therapeutic targets.
Protein differential expression (spectral counts)
40 Transplacental transfer of glyburide: is it clinically significant? Rachelle Schwartz1, Barak Rosenn1, Katarina Aleksa2, Gideon Koren2 1 St. Luke’s-Roosevelt Hospital Center, Obstetrics & Gynecology, New York, NY, 2Hospital for Sick Children, Division of Clinical Pharmacology/ Toxicology, Toronto, ON, Canada
OBJECTIVE: To determine the magnitude of transplacental transfer of glyburide (GLY) in women with gestational diabetes (GDM). STUDY DESIGN: A prospective, observational study was conducted including patients with signed informed consent and GDM on glyburide therapy. Upon delivery admission, the GLY dose and time of last dose were recorded. Immediately post-delivery, maternal and umbilical venous blood samples were obtained, centrifuged, and the plasma was frozen for future evaluation. The concentrations of GLY were determined by using previously described and validated methods employing high performance liquid chromatography-mass spectrometry. The limit of detection was 0.5 ng/mL and limit of quantification was 0.75 ng/mL. RESULTS: Twenty-two patient sets were analyzed. The mean and median total daily maternal GLY doses were 7.05 ⫾ 6.61 and 2.5mg/day, respectively. The mean and median times between last dose and sampling were 17.6 ⫾ 12.7 and 15.0 hours, respectively. Maternal GLY concentrations ranged from 0.93-70.7 ng/mL, with a mean and median of 13.6 ⫾ 19.8 ng/mL and 5.26 ng/mL, respectively. Umbilical vein GLY concentrations ranged from undetectable to 32.4 ng/mL, with a mean and median of 6.6 ⫾ 7.9 ng/mL and 3.54 ng/mL, respectively. The concentration of GLY was ⬍10ng/mL in 18 (82%) of the 22 umbilical samples. Thirty-two percent (7/22) of umbilical samples had a higher concentration than the maternal sample, but 86% (6/7) of those were ⬍10ng/ml. The mean ratio of umbilical GLY to maternal GLY was 0.77 ⫾ 0.49. Using natural log transformation and regression analysis, a 1% change in maternal GLY resulted in a 0.71% change in umbilical GLY (r2⫽0.57, p⬍0.0001). CONCLUSION: Transplacental transfer of glyburide does occur and there appears to be a relationship between maternal and umbilical glyburide concentrations. However, in most neonates, the concentration is well below therapeutic levels.
41 The effect of maternal pravastatin therapy on adverse neurologic outcomes of the offspring in a murine model of preeclampsia Alissa Carver1, Esther Tamayo1, J Regino Perez-Polo2, George Saade1, Maged Costantine1 1 University of Texas Medical Branch, Obstetrics & Gynecology, Galveston, TX, 2University of Texas Medical Branch, Biochemistry & Molecular Biology, Galveston, TX
OBJECTIVE: Preeclampsia alters fetal programming and results in long-term adverse outcomes in the offspring. Pravastatin, a hydro-
Supplement to JANUARY 2013 American Journal of Obstetrics & Gynecology
S25
Oral Plenary Session II (Fellows) philic drug that does not cross the placenta, prevents preeclampsia in animal models. This study aims to define the role of preeclampsia and pravastatin therapy on fetal programming of adult motor function using a mouse model. STUDY DESIGN: At day 8 of gestation, pregnant CD-1 mice were randomized to tail vein injection with adenovirus carrying soluble Fmslike tyrosine kinase 1 (sFlt-1) or murine immunoglobulin G2Fc (mFc; control). sFlt-1 dams received pravastatin (5mg/kg/d; sFlt-pra group) or water (sFlt group) until weaning, and mFc received water (mFc group). Offspring (n⫽13-20/group) were tested at 3 and 6 months of age using motor and coordination assays: righting reflex (RR), negative geotaxis (NG), balance beam (BB), and grid climbing (GC). 2-way ANOVA and ANCOVA were used for statistical analysis. RESULTS: All offspring retained RR at 3 and 6 months. At 6 months, sFlt offspring took longer to right themselves in NG compared to mFc and sFlt-pra (p⫽0.003). From 3 to 6 months, sFlt time to righting in NG worsened (p⫽0.03). Female sFlt offspring had double the paw slips (PS) at 3 months on BB compared with mFc and sFlt-pra (p⬍0.001). PS decreased in sFlt group at 6 months (p⫽0.005). BB falls were similar between groups at 3 and 6 months. No difference was seen in time to begin GC, but male sFlt offspring more than doubled their time to reach the top at 3 months (p⫽0.01). At 6 months, both time to start GC and to reach the top was higher in sFlt compared to mFc and sFlt-pra (p⫽0.01 and 0.04 respectively). Between 3 and 6 months, time to start GC worsened for sFlt (p⫽0.007). In all assays the offspring in the sFlt-pra group showed responses similar to control. CONCLUSION: Preeclampsia alters motor and coordination function in adult offspring. Maternal therapy with pravastatin prevents this altered developmental programming in this animal model of preeclampsia.
42 Progesterone supplementation during pregnancy does not alter signal transduction pathways involved in myometrial contractility Christopher Nold1, Monique Maubert1, Lauren Anton1, Michal Elovitz1 1 University of Pennsylvania Perelman School of Medicine, Maternal and Child Health Research Program, Department of Obstetrics and Gynecology, Philadelphia, PA
OBJECTIVE: Clinically, vaginal progesterone (VP) and 17 alpha-hydroxyprogestreone caproate (17P) have been shown to prevent preterm birth (PTB) in high risk populations. However, the molecular mechanisms by which progesterone supplementation prevents PTB remains undiscovered. This study sought to assess if vaginal or systemically administered progestational agents (as used clinically) altered signal transduction pathways implicated in myometrial activation and contractility. STUDY DESIGN: Using a mouse model, on days E14-E17 pregnant CD-1 mice were treated with either 0.1cc of 10mg/ml of 17P subcutaneously, 0.1cc of castor oil subcutaneously, 0.1 cc of 25 mg/ml of progesterone in Replens vaginally, or 0.1cc of Replens vaginally, with four dams per treatment group. Mice were sacrificed six hours after the last treatment on E17.5. Uterine tissue was collected from all treatment groups. QPCR was performed to assess differential gene expression in the following pathways: 1) contraction-associated proteins (CAPs): connexin-43, oxytocin receptor and cyclooxygenase-2; 2) progesterone mediated regulators of uterine quiescence: stat 5b, zeb 1, zeb 2, 20-aphahydroxysteroid dehydrogenase; and 3) microRNAs involved in uterine quiescence and contractility: mir-200a and mir-429. RESULTS: Neither vaginal progesterone nor systemically administered 17P significantly altered the signal transduction pathways investigated. Specifically, none of the individual targets assessed were altered by VP or 17P. CONCLUSION: These studies suggest that treatment with VP or 17P do not prevent preterm birth through modulation of signal transduction pathways involved in uterine quiescence or contractility. If these
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www.AJOG.org
agents are having a clinical effect in the prevention of preterm birth, than it is prudent that their mechanisms of action be elucidated so that this therapy can be used in the most appropriate patients at risk for preterm birth.
43 Smoking and preeclamspia protection: cigarette smoke increases placental adrenomedullin expression and improves trophoblast invasion via the adrenomedullin pathway Daniel Kraus1, Liping Feng1, R. Phillips Heine1, Haywood Brown1, Amy Murtha1, Chad Grotegut1 1 Duke University, Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Durham, NC
OBJECTIVE: Smoking reduces preeclampsia risk, but the mechanism of this effect is unknown. Adrenomedullin (ADM) is a vasoactive peptide highly expressed in the placenta. Placentas of preeclamptics exhibit decreased ADM expression. Furthermore, placentas of mice lacking ADM exhibit a preeclampsia phenotype of decreased villi branching and fewer decidual natural killer cells. Our aim was to determine the impact of cigarette smoke extract (CSE) on trophoblast invasion and if the effect was mediated by ADM. We also sought to determine whether placentas from smokers demonstrated enhanced ADM expression. STUDY DESIGN: HTR-8/SVneo trophoblast cells were incubated for 24 hours in Matrigel-invasion chambers with six treatment groups: nonstimulated (NS), ADM, ADM inhibitor (Inhibitor), 1% CSE, ADM ⫹ Inhibitor, and 1% CSE ⫹ Inhibitor. Cells that penetrated through the chambers were quantified, invasion indices were calculated, and compared using a one-way ANOVA with Bonferroni corrections. Next, term placental tissue was obtained from eleven smokers and eleven non-smokers and processed for immunohistochemistry. The intensity of ADM staining in the syncytiotrophoblasts was evaluated by three blinded observers using a four-point intensity scale and compared between the groups. RESULTS: Trophoblast cells treated with either ADM or CSE exhibited increased invasion compared to NS [1.5-fold (Figure, *p⬍0.01) and 1.45-fold (Figure, *p⬍0.01), respectively]. Co-treatment with the ADM inhibitor significantly attenuated the increased invasion seen with both ADM or CSE (Figure, ^p⬍0.01). Placentas from smokers demonstrated more intense ADM staining compared to placentas from non-smokers (p⬍0.004). CONCLUSION: CSE increases trophoblast cell invasion via an ADM-mediated process, and placental ADM expression is increased among term smokers. These findings provide evidence that the ADM pathway may play a role in the protection from preeclampsia seen in smokers.
Matrigel invasion assay
American Journal of Obstetrics & Gynecology Supplement to JANUARY 2013