- Email: [email protected]
Chronic hypoxia increases IL-6, TNF-, and matrix metalloproteinases (MMP2 and MMP9) expression of fetal guinea pig hearts
SMFM Abstracts S9 Continental Ballroom 5, Hilton San Francisco 18 CHRONIC HYPOXIA INCREASES IL-6, TNF-, AND MATRIX METALLOPROTEINASES (MMP2 AND MMP9) EXPRESSION OF FETAL GUINEA PIG HEARTS CHIEN OH1, YAFENG DONG2, LOREN THOMPSON1, 1University of Maryland at Baltimore, Obstetrics and Gynecology, Baltimore, Maryland, 2University of Kansas, Obstetrics and Gynecology, Kansas City, Kansas OBJECTIVE: Maternal hypoxia has been demonstrated to affect vascular and structural changes in the fetal heart. Recent research on adult hearts has demonstrated ischemia remodels the matrix of the heart through upregulation of inflammatory cytokines and subsequent upregulation of matrix metalloproteinases (MMPs). The hypothesis of this study is that fetuses exposed to chronic maternal hypoxia will demonstrate similar changes in inflammatory cytokines and MMPs which may affect function of the heart as an adult. STUDY DESIGN: To test the hypothesis, timed pregnant Duncan-Hartley guinea pigs (term gestation O 60d) were exposed to either hypoxia (10.5% O2, 14d, HPX, n=6) or normoxia (room air, NMX, n=6). At near term, agematched fetuses were excised from anesthetized mothers and fetal hearts removed for analysis. The apex of the left ventricle was frozen in liquid N2 until ready for study. Total RNA was isolated and quantified, then reverse transcribed by Omniscript RT Kit (Qiagen). Inflammatory cytokines and enzymes associated with cardic remodeling in fetal heart (IL-1b, IL-4, IL-6, TNF-a, MMP-2, and MMP-9) were quantified by Real-Time PCR. Melting curve was identified for product specification. 18S subunit was used as input control. RESULTS: Chronic hypoxia significantly (P!0.05) upregulated fetal cardiac IL-6 (4.1 fold), TNF-a (8.1 fold), MMP-2 (6.1 fold) and MMP-9 (5.5 fold) mRNA levels. Expression levels of other cytokines and substrates tested, such as IL-1b, Procollagen -a1 and -a2, were similar between normoxic and hypoxic fetal hearts. CONCLUSION: Chronic hypoxia induces fetal cardiac expression of several inflammatory cytokines associated with upregulation of MMP-2 and -9, shown to be important in cardiac remodeling. Further, this study identifies selected gene-related changes that may be associated with important adaptations in the hypoxic fetal heart. The clinical implications of these findings suggest that remodeling of the fetal heart in response to intrauterine stress may have an impact on cardiac function, both in utero and after birth.
20
0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.023
19
PROTEOMIC ANALYSIS OF THE HUMAN AMNIOTIC FLUID (AF) TO PREDICT HISTOLOGICAL CHORIOAMNIONITIS (HCA) IN UTERO IRINA A. BUHIMSCHI1, LISSA K. MAGLOIRE1, STEPHEN F. THUNG1, CHRISTIAN M. PETTKER1, GUOMAO ZHAO1, CAROLYN M. SALAFIA2, CATALIN S. BUHIMSCHI1, 1Yale University, Ob./Gyn.& Reprod.Sci, New Haven, Connecticut, 2Columbia University, Department of Epidemiology, Larchmont, New York OBJECTIVE: Subclinical HCA is regarded as a risk factor for morbidity and poor long term neurodevelopmental outcome of the neonate. Proteomic analysis of the AF provides an opportunity for early recognition of HCA and targeted treatment of the fetus, in utero. Our purpose was to determine the relationship between HCA and presence in the AF of biomarkers characteristic of inflammation (defensins 2 and 1, calgranulins C and A). STUDY DESIGN: 139 consecutive women (GA=27.0G0.4w) with singleton pregnancies admitted with PTL/PPROM were enrolled prospectively. All had indicated amniocentesis to rule out inflammation/infection. A proteomic fingerprint (MR score) from fresh AF was generated using SELDI-TOF. The MR score ranges from 0-4 (none to all biomarkers present). Presence or absence of the biomarkers was interpreted in relationship to placental pathology at delivery. Criteria for HCA were 3 stages (st I: intervillositis, st II: chorionic inflammation, st III: inflammation of both chorion and amnion) and 4 grades of inflammation of the amnion, chorion-decidua and chorionic plate. RESULTS: 1) The prevalence of HCA was 66% (91/138) with the following distribution: st I: 10%, st II: 16%, st: III 40%; 2) The severity of AF inflammation (determined by the MR score) significantly correlated with stages of HCA (0.468, p!0.001), grades of amnionitis (0.498, p!0.001) and choriodeciduitis (r=0.474, p!0.001); 3) The MR score correlated with the presence of clinically significant inflammation (grades 2-4) in the chorionic plate (r=0.475, p!0.001), amnion (0.482, p!0.001) and chorioadeciuda (p=0.451, p!0.001); 4) Calgranulin C (EN-RAGE, ligand for the RAGE receptor) had the strongest predictive value for amnionitis (OR: 4.8 [95%CI: 1.5-15.2] and choriodeciduitis [OR: 8.3 [95%CI: 1.5-44.4], independent of amniocentesis-to-delivery interval or GA at amniocentesis or delivery. CONCLUSION: Proteomic analysis of the AF has the ability to predict HCA at amniocentesis. This methodology may identify the fetuses that might benefit most from rapid interventions to prevent damage in utero. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.024
FETAL CORD BLOOD MONONUCLEAR CELLS COLLECTED AT TERM FROM HIV1 INFECTED WOMEN HARBOR TRANSCRIPTIONALLY ACTIVE INTEGRATED HIV GREG HAIR2, MICHAEL LINDSAY1, PROVIRAL DNA JANE ELLIS1, HARRIET WILLIAMS1, BRUCE SUNDSTROM2, 1Emory University, Department of 2 Gynecology and Obstetrics, Atlanta, Georgia, Emory University, Department of Pathology and Laboratory Medicine, Atlanta, Georgia OBJECTIVE: Strategies to limit perinatal transmission of human immunodeficiency virus (HIV) include antepartum/intrapartum antiretroviral therapy and pre-labor cesarean section with resulting reduction in the vertical transmission rate from 25% to 2%. In the current study we examined fetal cord blood cells for the presence of integrated provirus which is generally not detectable by current methods used to screen neonatal blood. STUDY DESIGN: Placental tissue and cord blood were obtained from 21 HIV infected women who received prenatal care and delivered at Grady Memorial Hospital in Atlanta, Georgia. Prenatal care was provided in accordance with current guidelines for care of HIV+ parturients. All specimens were obtained under Emory University IRB approved protocols. RNA and DNA were prepared from isolated placental or cord blood mononuclear cells (CBMCs) by standard methods. Integrated provirus or HIV cDNA standard concentrations were then determined by a two step modified absolute concentration Nested Real-Time qPCR assay. Realtime PCR measurements of multiply spliced (MS) or unspliced (US) HIV mRNA transcripts were conducted to determine active transcription. Quantitative measurements of Y chromosomal DNA by real-time PCR were performed for select maternal/ fetal blood pairs using DYS14 primer pairs to confirm the absence of maternal contamination of fetal cord blood. RESULTS: Placental cells isolated from these tissues all contained detectable levels of integrated HIV proviral DNA. Five (24%) of the corresponding samples of fetal CBMCs also had detectable levels of HIV proviral DNA, possibly suggesting a higher rate of intrauterine transmission than has been reported. Furthermore, all HIV+ CBMCs had detectable levels of MS and US proviral mRNA transcripts indicating that integrated HIV proviral DNA in all infected CBMCs was transcriptionally active. CONCLUSION: Findings suggest that even with use of antiretroviral therapy in the ante-and intrapartum periods some fetuses harbor integrated HIV proviral DNA that may retain the potential to become transcriptionally active. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.025
21
TLRS, THE LOWER GENITAL TRACT AND PREGNANCY JUAN GONZALEZ1, JINGHUA CHAIR2, MICHAL ELOVITZ2, 1Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, 2University of Pennsylvania, Philadelphia, Pennsylvania OBJECTIVE: Pregnancy is considered a state of immunosuppression. Expression of the Toll-like receptors, the backbone of the innate immune system, in the lower genital tract during pregnancy has not been explored. Pathogens activate inflammatory pathways through TLRs. However, the absence of these receptors, as observed in knock-out mice, can produce overwhelming infection and host death. We sought to investigate whether pregnancy altered expression of TLRs in the lower genital tract. STUDY DESIGN: MRNA expression of TLR-2, TLR-3, TLR-4 and TLR-9 were investigated in non-pregnant (NP) and pregnant CD-1 mice. Five different groups were utilized with 3-6 animals per treatment group: nonpregnant mice, timed-pregnant mice on day 12, day 16, day 18, day 19 and post-partum day 0. Uterine, cervical and placental tissues were harvested and RNA extracted. Quantitative PCR was performed using an ABI 7900. Statistical analysis utilized One-way ANOVA and SNK method for pairwise comparisons. RESULTS: TLR-2, TLR-3, TLR-4 and TLR-9 mRNA expression were all significantly increased in pregnant uterine tissue compared to NP. Maximal up-regulation occurred on D18 and 19(term) with TLR-2 being 6.7!, TLR-3 being 2.6!, TLR-4 being 7! and TLR-9 being 4.2! increased compared to NP. In the cervix, TLR-2, TLR-3 and TLR-9 were all significantly increased in pregnant vs NP. In the cervix, TLRs were maximally increased in midgestation (D16) with TLR-2 being 70!, TLR-3 being 74!, TLR-4 being 17! and TLR-9 being 76! increased compared to NP. In the placenta, TLR mRNA decreased throughout gestation but only TLR-4 was significantly decreased at term. CONCLUSION: The down-regulation of TLRs in the placenta supports the concept of immunosuppression at the maternal-fetal interface. However, in the lower genital tract, pregnancy results in a potent up-regulation of TLR, suggesting an enhancement of the innate immune response. Future studies are required to determine if this change in TLR expression results in an overexaggerated immune response or serves to protect the pregnancy from invading organisms. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.026
20
0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.023
19
PROTEOMIC ANALYSIS OF THE HUMAN AMNIOTIC FLUID (AF) TO PREDICT HISTOLOGICAL CHORIOAMNIONITIS (HCA) IN UTERO IRINA A. BUHIMSCHI1, LISSA K. MAGLOIRE1, STEPHEN F. THUNG1, CHRISTIAN M. PETTKER1, GUOMAO ZHAO1, CAROLYN M. SALAFIA2, CATALIN S. BUHIMSCHI1, 1Yale University, Ob./Gyn.& Reprod.Sci, New Haven, Connecticut, 2Columbia University, Department of Epidemiology, Larchmont, New York OBJECTIVE: Subclinical HCA is regarded as a risk factor for morbidity and poor long term neurodevelopmental outcome of the neonate. Proteomic analysis of the AF provides an opportunity for early recognition of HCA and targeted treatment of the fetus, in utero. Our purpose was to determine the relationship between HCA and presence in the AF of biomarkers characteristic of inflammation (defensins 2 and 1, calgranulins C and A). STUDY DESIGN: 139 consecutive women (GA=27.0G0.4w) with singleton pregnancies admitted with PTL/PPROM were enrolled prospectively. All had indicated amniocentesis to rule out inflammation/infection. A proteomic fingerprint (MR score) from fresh AF was generated using SELDI-TOF. The MR score ranges from 0-4 (none to all biomarkers present). Presence or absence of the biomarkers was interpreted in relationship to placental pathology at delivery. Criteria for HCA were 3 stages (st I: intervillositis, st II: chorionic inflammation, st III: inflammation of both chorion and amnion) and 4 grades of inflammation of the amnion, chorion-decidua and chorionic plate. RESULTS: 1) The prevalence of HCA was 66% (91/138) with the following distribution: st I: 10%, st II: 16%, st: III 40%; 2) The severity of AF inflammation (determined by the MR score) significantly correlated with stages of HCA (0.468, p!0.001), grades of amnionitis (0.498, p!0.001) and choriodeciduitis (r=0.474, p!0.001); 3) The MR score correlated with the presence of clinically significant inflammation (grades 2-4) in the chorionic plate (r=0.475, p!0.001), amnion (0.482, p!0.001) and chorioadeciuda (p=0.451, p!0.001); 4) Calgranulin C (EN-RAGE, ligand for the RAGE receptor) had the strongest predictive value for amnionitis (OR: 4.8 [95%CI: 1.5-15.2] and choriodeciduitis [OR: 8.3 [95%CI: 1.5-44.4], independent of amniocentesis-to-delivery interval or GA at amniocentesis or delivery. CONCLUSION: Proteomic analysis of the AF has the ability to predict HCA at amniocentesis. This methodology may identify the fetuses that might benefit most from rapid interventions to prevent damage in utero. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.024
FETAL CORD BLOOD MONONUCLEAR CELLS COLLECTED AT TERM FROM HIV1 INFECTED WOMEN HARBOR TRANSCRIPTIONALLY ACTIVE INTEGRATED HIV GREG HAIR2, MICHAEL LINDSAY1, PROVIRAL DNA JANE ELLIS1, HARRIET WILLIAMS1, BRUCE SUNDSTROM2, 1Emory University, Department of 2 Gynecology and Obstetrics, Atlanta, Georgia, Emory University, Department of Pathology and Laboratory Medicine, Atlanta, Georgia OBJECTIVE: Strategies to limit perinatal transmission of human immunodeficiency virus (HIV) include antepartum/intrapartum antiretroviral therapy and pre-labor cesarean section with resulting reduction in the vertical transmission rate from 25% to 2%. In the current study we examined fetal cord blood cells for the presence of integrated provirus which is generally not detectable by current methods used to screen neonatal blood. STUDY DESIGN: Placental tissue and cord blood were obtained from 21 HIV infected women who received prenatal care and delivered at Grady Memorial Hospital in Atlanta, Georgia. Prenatal care was provided in accordance with current guidelines for care of HIV+ parturients. All specimens were obtained under Emory University IRB approved protocols. RNA and DNA were prepared from isolated placental or cord blood mononuclear cells (CBMCs) by standard methods. Integrated provirus or HIV cDNA standard concentrations were then determined by a two step modified absolute concentration Nested Real-Time qPCR assay. Realtime PCR measurements of multiply spliced (MS) or unspliced (US) HIV mRNA transcripts were conducted to determine active transcription. Quantitative measurements of Y chromosomal DNA by real-time PCR were performed for select maternal/ fetal blood pairs using DYS14 primer pairs to confirm the absence of maternal contamination of fetal cord blood. RESULTS: Placental cells isolated from these tissues all contained detectable levels of integrated HIV proviral DNA. Five (24%) of the corresponding samples of fetal CBMCs also had detectable levels of HIV proviral DNA, possibly suggesting a higher rate of intrauterine transmission than has been reported. Furthermore, all HIV+ CBMCs had detectable levels of MS and US proviral mRNA transcripts indicating that integrated HIV proviral DNA in all infected CBMCs was transcriptionally active. CONCLUSION: Findings suggest that even with use of antiretroviral therapy in the ante-and intrapartum periods some fetuses harbor integrated HIV proviral DNA that may retain the potential to become transcriptionally active. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.025
21
TLRS, THE LOWER GENITAL TRACT AND PREGNANCY JUAN GONZALEZ1, JINGHUA CHAIR2, MICHAL ELOVITZ2, 1Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, 2University of Pennsylvania, Philadelphia, Pennsylvania OBJECTIVE: Pregnancy is considered a state of immunosuppression. Expression of the Toll-like receptors, the backbone of the innate immune system, in the lower genital tract during pregnancy has not been explored. Pathogens activate inflammatory pathways through TLRs. However, the absence of these receptors, as observed in knock-out mice, can produce overwhelming infection and host death. We sought to investigate whether pregnancy altered expression of TLRs in the lower genital tract. STUDY DESIGN: MRNA expression of TLR-2, TLR-3, TLR-4 and TLR-9 were investigated in non-pregnant (NP) and pregnant CD-1 mice. Five different groups were utilized with 3-6 animals per treatment group: nonpregnant mice, timed-pregnant mice on day 12, day 16, day 18, day 19 and post-partum day 0. Uterine, cervical and placental tissues were harvested and RNA extracted. Quantitative PCR was performed using an ABI 7900. Statistical analysis utilized One-way ANOVA and SNK method for pairwise comparisons. RESULTS: TLR-2, TLR-3, TLR-4 and TLR-9 mRNA expression were all significantly increased in pregnant uterine tissue compared to NP. Maximal up-regulation occurred on D18 and 19(term) with TLR-2 being 6.7!, TLR-3 being 2.6!, TLR-4 being 7! and TLR-9 being 4.2! increased compared to NP. In the cervix, TLR-2, TLR-3 and TLR-9 were all significantly increased in pregnant vs NP. In the cervix, TLRs were maximally increased in midgestation (D16) with TLR-2 being 70!, TLR-3 being 74!, TLR-4 being 17! and TLR-9 being 76! increased compared to NP. In the placenta, TLR mRNA decreased throughout gestation but only TLR-4 was significantly decreased at term. CONCLUSION: The down-regulation of TLRs in the placenta supports the concept of immunosuppression at the maternal-fetal interface. However, in the lower genital tract, pregnancy results in a potent up-regulation of TLR, suggesting an enhancement of the innate immune response. Future studies are required to determine if this change in TLR expression results in an overexaggerated immune response or serves to protect the pregnancy from invading organisms. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.026