125: Identification of cervical-vaginal biomarkers of recurrent preterm birth by proteomic analysis
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124 METABOLISM OF 17-HYDROXYPROGESTERONE CAPROATE BY HUMAN HEPATIC AND PLACENTAL MICROSOMES RU YAN 1 , TATIANA NANOVSKAYA 1 , DONALD MATTISON 2 , GARY D.V. HANKINS 1 , MAHMOUD AHMED 1 , 1 University of Texas Medical Branch, Ob- stetrics and Gynecology, Galveston, Texas, 2 Obstetric-Fetal Pharmacology Re- search Units (OPRU) Network, Center for Research For Mother and Children NICHD, Bethesda, MD, Bethesda, Maryland OBJECTIVE: Administration of 17-hydroxyprogesteronecaproate (17-P) is as- sociated with prevention of preterm deliveries in 30% of the patients. However, the mechanism of action of 17-P and whether it is a drug or prodrug remains unclear. Recently, we demonstrated that 17-P is not hydrolyzed by human hepatic and placental homogenates. The aim of this investigation is to determine whether 17-P is metabolized by human hepatic and placental microsomes by a pathway that does not involve its hydrolysis by esterases. STUDY DESIGN: Crude microsomal fractions from human livers and term pla- centas were ued. A dual radioactive labeled 17-[3H]-hydroxy progestrone [14 C]-caproate was utilized. The metabolites formed were separated and identified by HPLC/MS and their amounts determined by scintillation spectrometry. RESULTS: Hepatic and placental microsomes catalyzed the formation of several hydroxylated derivatives of 17-P that retained the tritium and 14C atoms. In human liver, the metabolites formed were divided into the following three groups accord- ing to the extent of their hydroxylation as revealed by their mass: mono-, di- and tri-hydroxylated 17-P which accounted for 63, 37 and 1% of the total formed, respectively. The major mono-hydroxylated compound formed by the liver ac- counted for 38% of the total metabolites formed. In human placenta, only mono- hydroxylated metabolites were formed. Two of these metabolites accounted for 16% of the total and were not formed by hepatic microsomes. The rates of metab- olites formation by the placenta were approximately 7% of that by the liver. CONCLUSION: Human liver CYP isozymes metabolize 17-P to mono-, di- and tri-hydroxylated compounds while in the placenta only mono-hydroxylated com- pounds are formed. Two of the metabolites formed by the placenta are not formed by the liver. At this time, it is unclear whether one or more of 17-P metabolites could contribute to its reported physiological effect. Supported by the Obstetric Pharma- cology Research Network (OPRU/NICHD). 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.135 125 IDENTIFICATION OF CERVICAL-VAGINAL BIOMARKERS OF RECURRENT PRETERM BIRTH BY PROTEOMIC ANALYSIS LEONARDO PEREIRA 1 , JANE HITTI 2 , JODI LAPIDUS 3 , DAVID ESCHENBACH 4 , MICHAEL GRAVETT 4 , SRINIVASA NAGALLA 5 , 1 Oregon Health & Science University, Materna Fetal Medicine, Portland, Oregon, 2 University of Washington, Obstetrics & Gynecology, Seattle, Washington, 3 Oregon Health & Science University, Portland, Oregon, 4 University of Washington, Seattle, Wash- ington, 5 PROTEOGENIX, INC, Beaverton, Oregon OBJECTIVE: To identify cervical-vaginal fluid (CVF) protein biomarkers asso- ciated with recurrent preterm birth (RPTB) compared to non-recurrent preterm birth (PTB) among symptomatic women in preterm labor. STUDY DESIGN: Gestational age (GA) matched women with RPTB and non- recurrent preterm birth (N5 in each group) were chosen from a prospective observational cohort of women in spontaneous preterm labor at 20-34 weeks= gestation, after excluding intra-amniotic infection. RPTB was defined a multiparity with PTB in the index pregnancy and a history of PTB in all previous deliveries. Non-recurrent PTB was defined as multiparity with PTB in the index pregnancy, but no previous PTB. CVF proteome analysis was performed using fluorescence 2 D gel analysis, multidimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and label-free quantification (spectral counting). RESULTS: There were no differences between the 2 groups in terms of GA and cervical dilation at sample collection, maternal age, parity, or other demographic variables. Mean GA at delivery was earlier in the RPTB group compared to the non-recurrent PTB group as expected (30.5 vs. 40.0 weeks respectively, p.02). Both individual patient-level data and pooled sample comparisons suggested a distinct protein expression profile associated with RPTB that differs from non- recurrent PTB. CVF proteome analysis demonstrated increased relative abundance of cytoskeletal and cell-cell adhesion proteins (alpha-actin) and decreased relative abundance of inflammatory proteins (calgranulin A, cystatin A, leukocyte elastase inhibitor). CONCLUSION: Changes in cell-cell adhesion and inflammatory proteins pro- vide insights into the mechanism involved in RPTB and could potentially lead to the development of a rapid test to distinguish RPTB from non-recurrent PTB. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.136 126 PREGNANCY ASSOCIATED ALTERATIONS OF THE INFLAMMATORY MEDIATORS AND PROTEIN ZMEDIATORS AND PROTEIN Z TREVOR MILLER 1 , DEBRA HOPPENSTEADT 2 , JOSEPHINE CUNANAN 3 , JOHN GIANOPOULOS 2 , JAWED FAREED 3 , 1 Society of Perinatal Obstetricians, chicago, Illinois, 2 Loyola University Chicago, Maywood, Illinois, 3 Loyola University Chicago, , Illinois OBJECTIVE: Pregnancy associated hypercoagulable state is reported to result in the generation of several markers of thrombotic activation, however the role of inflammatory mediators is not completely understood. Pregnancy related condi- tions such as pre-eclampsia and preterm labor appear to occur due to a strong stimulus or maternal response. This abnormal inflammation process is non-adap- tive and includes the activation of cells, complement and the soluble components of the coagulation system. There is also an increase in endothelial cell markers that assist in leukocyte adhesion and are activated by thrombin, interleukins and tumor necrosis factor. The objective of this study was to determine the inflammatory and hemostatic responses that cause complications in pregnancy in order to determine the risk stratification in pregnant women. STUDY DESIGN: Blood samples from pregnant women (n50) were collected and citrated plasma samples were profiled for CD 40 Ligand (CD 40L), Tumor necrosis factor alpha (TNF) Interleukin-6 (IL-6), Interleukin-8 (IL-8) (R&D Sys- tems, Minneapolis, MN) and protein Z (P-Z) (Stago, Parsipanny, NJ) by using commercially available sandwich ELISA methods. The results were compiled and compared to aged match normal healthy non-pregnant female controls (n40). The t-test was used to determine statistical differences between groups. RESULTS: Statistically significant differences were found between non-preg- nancy women and pregnant women with regards to plasma levels of TNF and CD40 L (P0.05). No significant differences were observed between these two groups for levels of both IL-6 or IL-8. CONCLUSION: These results suggest that some inflammatory mediators are up- regulated in normal pregnancy as compared to the non-pregnant state. New re- search suggests a relationship between specific pregnancy complications (i.e. pre- eclampsia and preterm labor) and certain inflammatory mediators which may serve as potential disease markers. Understanding their patterns in normal pregnancy may help to elucidate their potential role gestational pathologies. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.137 127 EFFECT OF MATERNAL N-ACETYLCYSTEINE (NAC) ON CERVICAL INCOMPETENCE (CI) WITH RESCUE CERCLAGE KEUN-YOUNG LEE 1 , JI-EUN SONG 1 , HYUN-AH JUN 1 , CARL WEINER 2 , 1 Hallym University, Obstetrics and Gynecology, Seoul, South Korea, 2 University of Kansas, Obstetrics and Gynecology, Kansas City, Kansas OBJECTIVE: NAC is a potent antioxidant that in animal models negates the impact of LPS. We tested the hypothesis to evaluate that the combination of anti- biotics with NAC would improve the outcome of rescue cerclage for CI. STUDY DESIGN: Case-control study. 86 women with advanced cervical dilata- tion (2cm) and bulging membranes were treated by rescue cerclage. All received antibiotics. 37 women received NAC and 49 did not. Amniocentesis was performed pre-cerclage to reduce membrane tension. MMP-8 PTD Check TM was used as a rapid bedside test to identify intra-amniotic inflammation. We assessed pregnancy outcome using NAC and MMP-8 as independent variables. RESULTS: NAC treated and non-treated women were demographically similar. 54% of NAC treated women were MMP-8 positive compared to 59 % of those untreated. There was no significant difference in the mean prolongation of gesta- tion by MMP-8 status. The mean prolongation of pregnancy was similar regardless of NAC administration (NAC-treated, 38.5d vs. NAC non-treated, 37.0d, pNS). MMP-8 status did not alter the efficacy of NAC. Both the mean prolongation days of pregnancy and birth weights were similar in MMP-8 positive women whether or not NAC was given. CONCLUSION: This case control study does not support the administration of NAC to improve the result of rescue cerclage. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.138 SMFM Abstracts www.AJOG.org S48 American Journal of Obstetrics & Gynecology Supplement to DECEMBER 2007
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Transcript of 125: Identification of cervical-vaginal biomarkers of recurrent preterm birth by proteomic analysis
124 METABOLISM OF 17-HYDROXYPROGESTERONE CAPROATE BY HUMAN HEPATIC ANDPLACENTAL MICROSOMES RU YAN1, TATIANA NANOVSKAYA1, DONALD MATTISON2,GARY D.V. HANKINS1, MAHMOUD AHMED1, 1University of Texas Medical Branch, Ob-stetrics and Gynecology, Galveston, Texas, 2Obstetric-Fetal Pharmacology Re-search Units (OPRU) Network, Center for Research For Mother and ChildrenNICHD, Bethesda, MD, Bethesda, Maryland
OBJECTIVE: Administration of 17�-hydroxyprogesteronecaproate (17-P) is as-sociated with prevention of preterm deliveries in 30% of the patients. However, themechanism of action of 17-P and whether it is a drug or prodrug remains unclear.Recently, we demonstrated that 17-P is not hydrolyzed by human hepatic andplacental homogenates. The aim of this investigation is to determine whether 17-Pis metabolized by human hepatic and placental microsomes by a pathway that doesnot involve its hydrolysis by esterases.
STUDY DESIGN: Crude microsomal fractions from human livers and term pla-centas were ued. A dual radioactive labeled 17�-[3H]-hydroxy progestrone [14C]-caproate was utilized. The metabolites formed were separated and identified byHPLC/MS and their amounts determined by scintillation spectrometry.
RESULTS: Hepatic and placental microsomes catalyzed the formation of severalhydroxylated derivatives of 17-P that retained the tritium and 14C atoms. In humanliver, the metabolites formed were divided into the following three groups accord-ing to the extent of their hydroxylation as revealed by their mass: mono-, di- andtri-hydroxylated 17-P which accounted for 63, 37 and �1% of the total formed,respectively. The major mono-hydroxylated compound formed by the liver ac-counted for 38% of the total metabolites formed. In human placenta, only mono-hydroxylated metabolites were formed. Two of these metabolites accounted for16% of the total and were not formed by hepatic microsomes. The rates of metab-olites formation by the placenta were approximately 7% of that by the liver.
CONCLUSION: Human liver CYP isozymes metabolize 17-P to mono-, di- andtri-hydroxylated compounds while in the placenta only mono-hydroxylated com-pounds are formed. Two of the metabolites formed by the placenta are not formedby the liver. At this time, it is unclear whether one or more of 17-P metabolites couldcontribute to its reported physiological effect. Supported by the Obstetric Pharma-cology Research Network (OPRU/NICHD).
0002-9378/$ - see front matterdoi:10.1016/j.ajog.2007.10.135
125 IDENTIFICATION OF CERVICAL-VAGINAL BIOMARKERS OF RECURRENT PRETERMBIRTH BY PROTEOMIC ANALYSIS LEONARDO PEREIRA1, JANE HITTI2, JODI LAPIDUS3,DAVID ESCHENBACH4, MICHAEL GRAVETT4, SRINIVASA NAGALLA5, 1Oregon Health &Science University, Materna Fetal Medicine, Portland, Oregon, 2University ofWashington, Obstetrics & Gynecology, Seattle, Washington, 3Oregon Health &Science University, Portland, Oregon, 4University of Washington, Seattle, Wash-ington, 5PROTEOGENIX, INC, Beaverton, Oregon
OBJECTIVE: To identify cervical-vaginal fluid (CVF) protein biomarkers asso-ciated with recurrent preterm birth (RPTB) compared to non-recurrent pretermbirth (PTB) among symptomatic women in preterm labor.
STUDY DESIGN: Gestational age (GA) matched women with RPTB and non-recurrent preterm birth (N�5 in each group) were chosen from a prospectiveobservational cohort of women in spontaneous preterm labor at 20-34 weeks=gestation, after excluding intra-amniotic infection. RPTB was defined a multiparitywith PTB in the index pregnancy and a history of PTB in all previous deliveries.Non-recurrent PTB was defined as multiparity with PTB in the index pregnancy,but no previous PTB. CVF proteome analysis was performed using fluorescence 2 Dgel analysis, multidimensional liquid chromatography tandem mass spectrometry(2D LC-MS/MS) and label-free quantification (spectral counting).
RESULTS: There were no differences between the 2 groups in terms of GA andcervical dilation at sample collection, maternal age, parity, or other demographicvariables. Mean GA at delivery was earlier in the RPTB group compared to thenon-recurrent PTB group as expected (30.5 vs. 40.0 weeks respectively, p�.02).Both individual patient-level data and pooled sample comparisons suggested adistinct protein expression profile associated with RPTB that differs from non-recurrent PTB. CVF proteome analysis demonstrated increased relative abundanceof cytoskeletal and cell-cell adhesion proteins (alpha-actin) and decreased relativeabundance of inflammatory proteins (calgranulin A, cystatin A, leukocyte elastaseinhibitor).
CONCLUSION: Changes in cell-cell adhesion and inflammatory proteins pro-vide insights into the mechanism involved in RPTB and could potentially lead to thedevelopment of a rapid test to distinguish RPTB from non-recurrent PTB.
0002-9378/$ - see front matterdoi:10.1016/j.ajog.2007.10.136
126 PREGNANCY ASSOCIATED ALTERATIONS OF THE INFLAMMATORY MEDIATORS ANDPROTEIN ZMEDIATORS AND PROTEIN Z TREVOR MILLER1, DEBRA HOPPENSTEADT2,JOSEPHINE CUNANAN3, JOHN GIANOPOULOS2, JAWED FAREED3, 1Society of PerinatalObstetricians, chicago, Illinois, 2Loyola University Chicago, Maywood, Illinois,3Loyola University Chicago, , Illinois
OBJECTIVE: Pregnancy associated hypercoagulable state is reported to result inthe generation of several markers of thrombotic activation, however the role ofinflammatory mediators is not completely understood. Pregnancy related condi-tions such as pre-eclampsia and preterm labor appear to occur due to a strongstimulus or maternal response. This abnormal inflammation process is non-adap-tive and includes the activation of cells, complement and the soluble components ofthe coagulation system. There is also an increase in endothelial cell markers thatassist in leukocyte adhesion and are activated by thrombin, interleukins and tumornecrosis factor. The objective of this study was to determine the inflammatory andhemostatic responses that cause complications in pregnancy in order to determinethe risk stratification in pregnant women.
STUDY DESIGN: Blood samples from pregnant women (n�50) were collectedand citrated plasma samples were profiled for CD 40 Ligand (CD 40L), Tumornecrosis factor alpha (TNF) Interleukin-6 (IL-6), Interleukin-8 (IL-8) (R&D Sys-tems, Minneapolis, MN) and protein Z (P-Z) (Stago, Parsipanny, NJ) by usingcommercially available sandwich ELISA methods. The results were compiled andcompared to aged match normal healthy non-pregnant female controls (n�40).The t-test was used to determine statistical differences between groups.
RESULTS: Statistically significant differences were found between non-preg-nancy women and pregnant women with regards to plasma levels of TNF and CD40L (P�0.05). No significant differences were observed between these two groups forlevels of both IL-6 or IL-8.
CONCLUSION: These results suggest that some inflammatory mediators are up-regulated in normal pregnancy as compared to the non-pregnant state. New re-search suggests a relationship between specific pregnancy complications (i.e. pre-eclampsia and preterm labor) and certain inflammatory mediators which may serveas potential disease markers. Understanding their patterns in normal pregnancymay help to elucidate their potential role gestational pathologies.
0002-9378/$ - see front matterdoi:10.1016/j.ajog.2007.10.137
127 EFFECT OF MATERNAL N-ACETYLCYSTEINE (NAC) ON CERVICAL INCOMPETENCE (CI)WITH RESCUE CERCLAGE KEUN-YOUNG LEE1, JI-EUN SONG1, HYUN-AH JUN1, CARLWEINER2, 1Hallym University, Obstetrics and Gynecology, Seoul, South Korea,2University of Kansas, Obstetrics and Gynecology, Kansas City, Kansas
OBJECTIVE: NAC is a potent antioxidant that in animal models negates theimpact of LPS. We tested the hypothesis to evaluate that the combination of anti-biotics with NAC would improve the outcome of rescue cerclage for CI.
STUDY DESIGN: Case-control study. 86 women with advanced cervical dilata-tion (�2cm) and bulging membranes were treated by rescue cerclage. All receivedantibiotics. 37 women received NAC and 49 did not. Amniocentesis was performedpre-cerclage to reduce membrane tension. MMP-8 PTD Check TM was used as arapid bedside test to identify intra-amniotic inflammation. We assessed pregnancyoutcome using NAC and MMP-8 as independent variables.
RESULTS: NAC treated and non-treated women were demographically similar.54% of NAC treated women were MMP-8 positive compared to 59 % of thoseuntreated. There was no significant difference in the mean prolongation of gesta-tion by MMP-8 status. The mean prolongation of pregnancy was similar regardlessof NAC administration (NAC-treated, 38.5d vs. NAC non-treated, 37.0d, p�NS).MMP-8 status did not alter the efficacy of NAC. Both the mean prolongation daysof pregnancy and birth weights were similar in MMP-8 positive women whether ornot NAC was given.
CONCLUSION: This case control study does not support the administration ofNAC to improve the result of rescue cerclage.
0002-9378/$ - see front matterdoi:10.1016/j.ajog.2007.10.138
SMFM Abstracts www.AJOG.org
S48 American Journal of Obstetrics & Gynecology Supplement to DECEMBER 2007
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