Title: Oxidative Stress, Gene Expression, and Protein Changes Induced in the Human Placenta during Labor
Abstract: Malperfusion of the placenta has been implicated as a cause of oxidative stress in complications of human pregnancy, leading to release of proinflammatory cytokines and anti-angiogenic factors into the maternal circulation. Uterine contractions during labor are known to be associated with intermittent utero-placental perfusion. We therefore tested whether oxidative stress, proinflammatory cytokines, and angiogenic regulators were increased in placentas subjected to short (<5 hours) and long (>15 hours) labor compared with nonlabored controls delivered by cesarean section. In addition, broader changes in gene transcripts were assessed by microarray analysis. Oxidative stress, activation of the nuclear factor-κB pathway, tumor necrosis factor-α and interleukin 1β all increased in placental tissues after labor. Stabilization of hypoxia-inducible factor-1α and increased vascular endothelial growth factor soluble receptor-1 were also observed. By contrast, tissue levels of placenta growth factor decreased. Apoptosis was also activated in labored placentas. The magnitude of these changes related to the duration of labor. After labor, 55 gene transcripts were up-regulated and 35 down-regulated, and many of these changes were reflected at the protein level. In conclusion, labor is a powerful inducer of placental oxidative stress, inflammatory cytokines, and angiogenic regulators. Our findings are consistent with intermittent perfusion being the initiating cause. Placentas subjected to labor do not reflect the normal in vivo state at the molecular level. Malperfusion of the placenta has been implicated as a cause of oxidative stress in complications of human pregnancy, leading to release of proinflammatory cytokines and anti-angiogenic factors into the maternal circulation. Uterine contractions during labor are known to be associated with intermittent utero-placental perfusion. We therefore tested whether oxidative stress, proinflammatory cytokines, and angiogenic regulators were increased in placentas subjected to short (<5 hours) and long (>15 hours) labor compared with nonlabored controls delivered by cesarean section. In addition, broader changes in gene transcripts were assessed by microarray analysis. Oxidative stress, activation of the nuclear factor-κB pathway, tumor necrosis factor-α and interleukin 1β all increased in placental tissues after labor. Stabilization of hypoxia-inducible factor-1α and increased vascular endothelial growth factor soluble receptor-1 were also observed. By contrast, tissue levels of placenta growth factor decreased. Apoptosis was also activated in labored placentas. The magnitude of these changes related to the duration of labor. After labor, 55 gene transcripts were up-regulated and 35 down-regulated, and many of these changes were reflected at the protein level. In conclusion, labor is a powerful inducer of placental oxidative stress, inflammatory cytokines, and angiogenic regulators. Our findings are consistent with intermittent perfusion being the initiating cause. Placentas subjected to labor do not reflect the normal in vivo state at the molecular level. There is currently much interest in the role that placental oxidative stress plays in the pathophysiology of complications of human pregnancy.1Hubel CA Oxidative stress in the pathogenesis of preeclampsia.Proc Soc Exp Biol Med. 1999; 222: 222-235Crossref PubMed Scopus (537) Google Scholar, 2Myatt L Cui X Oxidative stress in the placenta.Histochem Cell Biol. 2004; 122: 369-382Crossref PubMed Scopus (626) Google Scholar, 3Burton GJ Jauniaux E Placental oxidative stress: from miscarriage to preeclampsia.J Soc Gynecol Invest. 2004; 11: 342-352Crossref PubMed Scopus (534) Google Scholar The cause of the oxidative stress is not certain, but the major complications of pregnancy have long been associated with deficient conversion of the uterine spiral arteries, suggesting that impaired perfusion of the placenta is the initiating insult.4Brosens IA Morphological changes in the utero-placental bed in pregnancy hypertension.Clin Obstet Gynaecol. 1977; 4: 573-593PubMed Google Scholar, 5Kim YM Bujold E Chaiworapongsa T Gomez R Yoon BH Thaler HT Rotmensch S Romero R Failure of physiologic transformation of the spiral arteries in patients with preterm labor and intact membranes.Am J Obstet Gynecol. 2003; 189: 1063-1069Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 6Kim YM Chaiworapongsa T Gomez R Bujold E Yoon BH Rotmensch S Thaler HT Romero R Failure of physiologic transformation of the spiral arteries in the placental bed in preterm premature rupture of membranes.Am J Obstet Gynecol. 2002; 187: 1137-1142Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 7Brosens IA Robertson WB Dixon HG The role of the spiral arteries in the pathogenesis of preeclampsia.Obstet Gynecol Annu. 1972; 1: 177-191PubMed Google Scholar Deficient conversion results in the spiral arteries retaining more smooth muscle cells in their walls than normal, and we recently proposed that this leads to exaggerated intermittent perfusion of the placenta.8Hung T-H Skepper JN Burton GJ In vitro ischemia-reperfusion injury in term human placenta as a model for oxidative stress in pathological pregnancies.Am J Pathol. 2001; 159: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar Experiments involving hypoxia-reoxygenation of villous explants in vitro have supported this hypothesis, demonstrating that placental oxidative stress can be induced rapidly by an ischemia-reperfusion-type insult. Generation of oxidative stress is associated with activation of the p38 and SAPK MAPK, and the nuclear factor (NF)-κB signaling pathways; increased secretion and production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β); and apoptotic changes localized principally to the syncytiotrophoblast.9Cindrova-Davies T Spasic-Boskovic O Jauniaux E Charnock-Jones DS Burton GJ NF-κB, p38 and stress activated protein kinase mitogen-activated protein kinase signaling pathways regulate proinflammatory cytokines and apoptosis in human placental explants in response to oxidative stress.Am J Pathol. 2007; 170: 1511-1520Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 10Hung T-H Skepper JN Charnock-Jones DS Burton GJ Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia.Circ Res. 2002; 90: 1274-1281Crossref PubMed Scopus (330) Google Scholar, 11Hung T-H Charnock-Jones DS Skepper JN Burton GJ Secretion of tumor necrosis factor-α from human placental tissues induced by hypoxia-reoxygenation causes endothelial cell activation in vitro: a potent mediator of the inflammatory response in preeclampsia.Am J Pathol. 2004; 164: 1049-1061Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 12Tjoa M-L Cindrova-Davies T Spasic-Boskovic O Bianchi DW Burton GJ Trophoblastic oxidative stress and the release of cell-free feto-placental DNA.Am J Pathol. 2006; 169: 400-404Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 13Bainbridge SA Belkacemi L Dickinson M Graham CH Smith GN Carbon monoxide inhibits hypoxia/reoxygenation-induced apoptosis and secondary necrosis in syncytiotrophoblast.Am J Pathol. 2006; 169: 774-783Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar The finding that these changes can be blocked by antioxidants in vitro9Cindrova-Davies T Spasic-Boskovic O Jauniaux E Charnock-Jones DS Burton GJ NF-κB, p38 and stress activated protein kinase mitogen-activated protein kinase signaling pathways regulate proinflammatory cytokines and apoptosis in human placental explants in response to oxidative stress.Am J Pathol. 2007; 170: 1511-1520Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar strongly suggests they are a response to increased production of reactive oxygen species. However, the cellular events occurring during hypoxia-reoxygenation are complex because of the rapid rate of reaction of reactive oxygen species with scavengers and other biomolecules. Consequently, in vitro manipulations may not accurately reflect in vivo responses. It is not possible experimentally to induce changes in utero-placental perfusion in pregnant women for obvious ethical reasons. We therefore took advantage of the experiment-of-nature that occurs when an otherwise healthy placenta is subjected to intermittent blood supply during labor to assess the impact of ischemia-reperfusion in vivo on placental gene expression and cytokine profile. Uterine contractions cause compression of the uterine vasculature and, hence, transient reductions in uteroplacental blood flow.14Brar HS Platt LD DeVore GR Horenstein J Medearis AL Qualitative assessment of maternal uterine and fetal umbilical artery blood flow and resistance in laboring patients by Doppler velocimetry.Am J Obstet Gynecol. 1988; 158: 952-956Abstract Full Text PDF PubMed Google Scholar, 15Fleischer A Anyaegbunam AA Schulman H Farmakides G Randolph G Uterine and umbilical artery velocimetry during normal labor.Am J Obstet Gynecol. 1987; 157: 40-43Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 16Ramsey E Donner M Placental vasculature and circulation.Anatomy, Physiology, Radiology, Clinical Aspects Atlas and Textbook. Georg Thieme Publishers Stuttgart, Stuttgart1980Google Scholar Recent Doppler ultrasound studies have demonstrated a linear inverse relationship between uterine artery resistance and the intensity of the uterine contractions during labor.14Brar HS Platt LD DeVore GR Horenstein J Medearis AL Qualitative assessment of maternal uterine and fetal umbilical artery blood flow and resistance in laboring patients by Doppler velocimetry.Am J Obstet Gynecol. 1988; 158: 952-956Abstract Full Text PDF PubMed Google Scholar The absence of end-diastolic flow is observed during contractions when the intrauterine pressure exceeds 35 mmHg.15Fleischer A Anyaegbunam AA Schulman H Farmakides G Randolph G Uterine and umbilical artery velocimetry during normal labor.Am J Obstet Gynecol. 1987; 157: 40-43Abstract Full Text PDF PubMed Scopus (92) Google Scholar This intermittent perfusion can be expected to provide the basis for ischemia-reperfusion type injury of the placenta. As a result, conversion of xanthine dehydrogenase to xanthine oxidase (XD/O), a hallmark of ischemia-reperfusion injury, is enhanced in placental tissues after vaginal delivery compared with nonlabored cesarean controls.17Many A Roberts JM Increased xanthine oxidase during labour—implications for oxidative stress.Placenta. 1997; 18: 725-726Abstract Full Text PDF PubMed Scopus (39) Google Scholar Analysis of placental energy metabolism also confirms significant ischemia during labor because there is a marked reduction in the tissue concentration of ATP, and of the ATP/ADP ratio, in placentas delivered vaginally compared with cesarean controls.18Bloxam DL Bobinski PM Energy metabolism and glycolysis in the human placenta during ischaemia and in normal labour.Placenta. 1984; 5: 381-394Abstract Full Text PDF PubMed Scopus (26) Google Scholar In addition, labor is associated with a significant depletion of vitamin C in maternal plasma, amniotic fluid, and fetal plasma, indicating increased utilization of antioxidant pathways.19Woods JR Cavanaugh JL Normkus EP Plessinger MA Miller RK The effect of labor on maternal and fetal vitamins C and E.Am J Obstet Gynecol. 2002; 187: 1179-1183Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Finally, umbilical venous and arterial samples of neonates born by vaginal delivery contain higher glutathione levels compared with those delivered by cesarean section (CS),20Raijmakers MT Roes EM Steegers EA van der Wildt B Peters WH Umbilical glutathione levels are higher after vaginal birth than after cesarean section.J Perinatal Med. 2003; 31: 520-522Crossref PubMed Scopus (21) Google Scholar and placental generation of lipid peroxide and superoxide are also enhanced. The hemodynamic consequences of labor for the placenta are, however, potentially confounded by the endocrine stimuli that initiate the labor process. In most models of parturition, prostaglandins (PGs) are identified as the effector molecules for uterine contractions and cervical ripening. PG formation is known to be stimulated by proinflammatory cytokines that are secreted locally by intrauterine tissues. In preparation for labor, there is a switch in the myometrium from the production of prostacyclin, which suppresses uterine activity along with progesterone, to increased synthesis of PGE2 and PGF2α, which are uterotonic. These PGs arise via the action of cyclooxygenase (COX) enzymes, primarily via transcriptional up-regulation of the inducible isoform COX-2.21Kniss DA Cyclooxygenases in reproductive medicine and biology.J Soc Gynecol Invest. 1999; 6: 285-292Crossref PubMed Scopus (108) Google Scholar Although these PGs are primarily uterotonic, they could also have an effect on placental gene expression. To separate the hemodynamic effects of labor from the stimuli initiating labor, we determined whether the responses were related to the duration of labor by investigating placentas subjected to short (<5 hours) and long labor (>15 hours). The hypothesis tested in this study was that ischemia-reperfusion during labor stimulates oxidative stress, increased production of proinflammatory and anti-angiogenic cytokines, and apoptosis in placentas from vaginal deliveries compared with nonlabored cesarean controls. A panel of antibodies reflecting these protein changes has been chosen, based primarily on our previous in vitro findings.9Cindrova-Davies T Spasic-Boskovic O Jauniaux E Charnock-Jones DS Burton GJ NF-κB, p38 and stress activated protein kinase mitogen-activated protein kinase signaling pathways regulate proinflammatory cytokines and apoptosis in human placental explants in response to oxidative stress.Am J Pathol. 2007; 170: 1511-1520Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar We also assessed the impact of the labor process on the broader pattern of placental gene expression using microarray analysis in the long labor samples. Antibodies to the phosphorylated and total forms of IκB, Hsp27, TNF-α, cleaved caspase-3, and cleaved caspase-9 were from Cell Signaling Technology (Beverly, MA). Anti-COX-2 antibody was from Cayman Chemical (Ann Arbor, MI), anti-HNE from Axxora (Nottingham, UK), anti-Hsp90 from Stressgen Bioreagents Corp. (York, UK), anti-VEGF-R1 and PlGF from Abcam (Cambridge, UK), and anti-VEGF-A from Santa Cruz Biotechnology (Santa Cruz, CA). The horseradish peroxidase-conjugated secondary antibodies were from Amersham Biosciences (Bucks, UK). Alexa 488 and Alexa 568 fluorescently labeled antibodies were from Molecular Probes Invitrogen Detection Technologies (Leiden, The Netherlands), and biotinylated secondary antibodies were from Vector Laboratories (Peterborough, UK). The study was approved by the local research ethics committees in Cambridge and London. Human term (38 to 41 weeks) placentas were obtained from normal uncomplicated pregnancies, with written informed patient consent, immediately after either elective nonlabored CS or vaginal delivery. In patients delivering vaginally the start of labor was defined as the onset of regular uterine activity leading to dilatation of the cervix. Progress of labor was recorded using a partogram,22Philpott RH Castle WM Cervicographs in the management of labour in primigravidae. I. The alert line for detecting abnormal labour.J Obstet Gynaecol Br Commonw. 1972; 79: 592-598Crossref PubMed Scopus (35) Google Scholar, 23Philpott RH Castle WM Cervicographs in the management of labour in primigravidae. II. The action line and treatment of abnormal labour.J Obstet Gynaecol Br Commonw. 1972; 79: 599-602Crossref PubMed Scopus (109) Google Scholar and all patients progressed satisfactorily to a noninstrumented vaginal delivery. Placentas were collected from women delivering after <5 hours of total labor (short labor) and >15 hours (long labor). There was no evidence of ascending infection in the vaginal deliveries, and clinical details of the placentas used for protein analysis and immunohistochemistry (IHC) are presented in Table 1.Table 1Clinical Details of the PlacentasGroupMA (years)GA (weeks)Mode of deliveryReason for CSLabor stages (minutes)Baby's weight (g)CS2639LSCSPrevious LSCS3800CS2338LSCSTwo previous LSCS4000CS3039LSCSPrevious LSCS3250CS3239LSCSMaternal request3450CS2838LSCSTwo previous LSCS3900SL3240SVD150, 90, 43880SL2140SVD248, 20, 53820SL2840SVD70, 9, 92750SL3138SVD160, 3, 63080SL3840SVD240, 40, 23820LL3239SVD950, 85, 53830LL3641SVD980, 35, 53940LL3441SVD930, 35, 53640LL3841SVD960, 30, 123460LL3641SVD840, 115, 53680CS, cesarean section; SL, short labor; LL, long labor; MA, maternal age; GA, gestational age; LSCS, low segment cesarean section; SVD, standard vaginal delivery. Open table in a new tab CS, cesarean section; SL, short labor; LL, long labor; MA, maternal age; GA, gestational age; LSCS, low segment cesarean section; SVD, standard vaginal delivery. Samples (each ∼40 to 50 mg wet weight) were taken from two standardized sites midway between the chorionic and basal plates from lobules free of visible infarction, calcification, hematoma, or tears, and processed within 5 to 10 minutes of delivery. After a brief rinse in ice-cold phosphate-buffered saline, samples were either snap frozen in liquid nitrogen for mRNA and protein analysis (stored at −80°C until use) or fixed in 4% paraformaldehyde and embedded in wax for IHC. Tissue homogenization to obtain protein lysates and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed as previously described.12Tjoa M-L Cindrova-Davies T Spasic-Boskovic O Bianchi DW Burton GJ Trophoblastic oxidative stress and the release of cell-free feto-placental DNA.Am J Pathol. 2006; 169: 400-404Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar Proteins were revealed and quantified using Image J software (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/). Membranes were reprobed with antibody recognizing β-actin to control for protein loading. The values are expressed as a percentage of the control lysate (100%) for each experiment. Sample number was restricted to five per group (CS, short labor, long labor) because it is only possible to compare between groups if the lysates are run on the same gel. Paraformaldehyde-fixed tissues embedded in paraffin wax were sectioned at 7 μm. After dewaxing and blocking of endogenous peroxidases by incubation with 3% H2O2 for 30 minutes, the sections were incubated with nonimmune serum for 20 minutes. Primary antibodies were applied overnight at 4°C, and binding was detected using Vectastain Elite ABC kits (Vector Laboratories) and SigmaFast DAB (Sigma, Poole, Dorset, UK), according to the manufacturers' instructions. Sections were lightly counterstained with hematoxylin. When necessary, antigen retrieval was performed before blocking using 0.01 mol/L sodium citrate buffer at pH 6.0 in a pressure cooker for 2 minutes. Negative controls were performed by replacement with equal concentrations of nonimmune or isotype-matched irrelevant control. Sections were incubated with primary antibodies overnight as in the colorimetric protocol, washed, and incubated for 1 hour at room temperature with species-specific Alexa 488 or Alexa 568 secondary fluorescent antibodies (used at 1/200; Molecular Probes) in blocking buffer. Sections were washed in Tris-buffered saline and subsequently mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories). Images were captured using a Leica confocal microscope (LeicaTCS-NT; Leica Instruments GmbH, Wetzlar, Germany). Total RNA was isolated either from snap-frozen placental tissue or from tissue collected in RNA Later (Ambion, Inc., Austin, TX) using TRI reagent (Sigma). The RNA was quantified by spectrophotometry (Nanodrop Technologies, Wilmington, DE) and RNA integrity assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies UK Ltd., Stockport, Cheshire, UK). In brief, 20 μg of total RNA from each sample and the common reference RNA (a pool of equal masses of RNAs from 20 placentas) were reverse-transcribed using a master mix containing SuperScript II reverse transcriptase in the first strand buffer with 0.1 mol/L dithiothreitol (Invitrogen, Paisley, UK), 5 μg/μl oligo (dT)23 (Sigma), 50× aminoallyl dUTP/low dTTP dNTP mix (25 mmol/L dATP, 25 mmol/L dCTP, 25 mmol/L dGTP, 10 mmol/L dTTP, and 15 mmol/L aa-dUTP), and 20 U/μl RNaseOUT (Invitrogen). The cDNA was purified using a Microcon-30 filter (Millipore, Bedford, MA) and fluorescently labeled with either Cy-3 or Cy-5 monofunctional reactive dye (GE Health Care UK Ltd., Little Chalfont, Buckinghamshire, UK). Dye-labeled cDNAs were purified using a polymerase chain reaction (PCR) product purification kit (Qiagen Ltd., Crawley, West Sussex, UK) and dried before hybridization. Equal amounts of the labeled cDNAs were dissolved in hybridization buffer (40% deionized formamide, 5× Denhardt's solution, 5× standard saline citrate, 50 mmol/L Tris-HCl, 0.1% sodium dodecyl sulfate, 1 mol/L sodium pyrophosphate, 4 mg/ml yeast tRNA, and 8 mg/ml Poly dA), boiled, and hybridized at 42°C for a minimum of 18 hours to custom human cDNA arrays (HMN1 and HMN2) representing ∼15,000 genes in total (http://www.path.cam.ac.uk/resources/microarray/microarrays/). After hybridization and washing, the arrays were scanned using an Axon 4100A scanner and the images analyzed using BlueFuse software (BlueGnome, Cambridge, UK). The raw data were normalized both within arrays and between arrays using the LIMMA software package (linear models of microarray data, http://bioinf.wehi.edu.au/limma/). Transcript abundance data were compared between cesarean control and prolonged labor samples using the popular Cyber-T algorithm.24Hung SP Baldi P Hatfield GW Global gene expression profiling in Escherichia coli K12: effects of leucine-responsive regulatory protein.J Biol Chem. 2002; 277: 40309-40323Crossref PubMed Scopus (116) Google Scholar This algorithm is an unpaired t-test, modified by the inclusion of a Bayesian prior based on the variance of other transcripts in the data set25Baldi P Long AD A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes.Bioinformatics. 2001; 17: 509-519Crossref PubMed Scopus (1313) Google Scholar and is widely used. For further statistical analysis, GeneSpring Expression Analysis software (Silicon Genetics, Redwood City, CA) was used. Gene ontologies were identified using Fatigo26Al-Shahrour F Diaz-Uriarte R Dopazo J FatiGO: a web tool for finding significant associations of Gene Ontology terms with groups of genes.Bioinformatics. 2004; 20: 578-580Crossref PubMed Scopus (882) Google Scholar (http://www.fatigo.org). The transcripts identified as having significantly different abundance were analyzed using the Ingenuity Pathway Analysis package (Redwood, CA). The MIAME for both HMN1 and HMN2 arrays have been submitted and accepted in European Bioinformatics Institutes (http://www.ebi.ac.uk), and their accession numbers are A-MEXP-361 and A-MEXP-362, respectively. The ABI Prism 7700 sequence detection system (TaqMan; Applied Biosystems, Warrington, Cheshire, UK) was used to perform real-time polymerase chain reactions according to the manufacturer's protocols. Ct values for each transcript were compared with those for 18S ribosomal RNA, which according to the gene array results remained relatively constant in abundance. All primers and probes were obtained from Applied Biosystems, Assays-on-Demand, and used a 5′-FAM reporter and 3′-nonfluorescent minor groove binder. The thermocycler parameters were 2 minutes at 50°C, followed by 40 cycles of 15 seconds 95°C and 1 minute at 60°C for PCR amplification. All data are presented as means ± SEM. Statistical analysis was performed using Statview (SAS Institute Inc., Cary, NC). Western blot measurements were analyzed using analysis of variance, and significant differences between groups were identified using the protected least significant differences (PLSD) post hoc test at P < 0.05. Differences between two groups were evaluated using Student's unpaired t-test. In all cases, results were considered significant at P < 0.05. Expression of the heat shock proteins (Hsp27 and Hsp90) and peroxidation of lipids (anti-HNE) were used as markers of oxidative stress. The relative amount of Hsp90 was significantly increased in the long labor samples, whereas that of Hsp27 peaked in the short labor samples and then fell to nonlabored levels (Figure 1). IHC localized Hsp27 principally to the syncytiotrophoblast (see below, Figure 6A). The relative amount of HNE between the three groups was not significantly different when analyzed by analysis of variance because of the small sample number and large variability. However, there was an obvious trend indicating increased lipid peroxidation in labored samples, and the difference was significant when the cesarean controls were compared with all of the labor samples combined (P = 0.023 analyzed by Student's t-test).Figure 6Immunostaining for Hsp27 (A), CuZn-SOD (B), COX-2 (C), TNF-α (D), sVEGF-R1 (E), PlGF (F), PAI-2 (G), and M30 (H) localized these markers principally to the syncytiotrophoblast.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mn-SOD increased in the short labor samples but returned to control levels in long labor placentas (Figure 2). Catalase increased with labor duration, and there was also a significant difference in catalase expression between the two labor groups (Figure 2). The expression in Cu/Zn-SOD also seemed to increase with labor duration, but this trend was not statistically significant by analysis of variance (Figure 2, Figure 6). There was no effect on glutathione peroxidase (Figure 2). The NF-κB pathway has been implicated in responses to oxidative stress. Phosphorylation and subsequent degradation of the IκB protein allows translocation of NF-κB to the nucleus, where it regulates gene expression. The ratio of phosphorylated IκB to total protein increased markedly with the duration of labor, reflecting activation of the NF-κB pathway. There was a significant difference between the short and long labor samples (Figure 3). Activation of NF-κB suggests an increase of proinflammatory cytokines and COX-2 after labor. Western blots confirmed that TNF-α, IL-1β, leptin, and COX-2 were all significantly increased in labored placentas compared with cesarean controls. Of these, only COX-2 was significantly different between short and long labor (Figure 3). IHC demonstrated localization of COX-2 and TNF-α principally to the trophoblast (see Figure 6, C and D). We also examined the effect of labor on placental vascular endothelial growth factor (VEGF-A), its soluble receptor (sVEGF-R1), and placental growth factor (PlGF). VEGF-A increased during labor in a similar manner, and there was a significant difference between the two labor groups (Figure 4). Placental sVEGF-R1 was significantly increased in both the short and long labor samples, the increase being 2.5-fold in long labor samples compared with cesarean controls (Figure 4). HIF-1α has been implicated in the regulation of both these proteins,16Ramsey E Donner M Placental vasculature and circulation.Anatomy, Physiology, Radiology, Clinical Aspects Atlas and Textbook. Georg Thieme Publishers Stuttgart, Stuttgart1980Google Scholar and we found that HIF-1α increased with the duration of labor, reaching statistical significance in the long labor samples (Figure 4). In contrast, PlGF decreased with labor duration, the difference reaching statistical significance in the long labor samples. HIF-1α and PlGF expression was significantly different between long labor and short labor. Plasminogen activator inhibitor type 2 (PAI-2) showed a trend toward a decrease in the long labor samples, but the differences failed to reach statistical significance (Figure 4). IHC localized the immunoreactivity of sVEGF-R1 (Figure 6E), PlGF (Figure 6F), and PAI-2 (Figure 6G) principally to the syncytiotrophoblast. Both TNF-α and IL-1β are known to be proapoptotic, and their elevation may lead to an increase of apoptosis in placental tissues subjected to labor. Consistent with this hypothesis, there was a significant increase in levels of cleaved caspase-3 and cleaved caspase-9 in the labored samples, with no significant difference between the two labor groups (Figure 5). Cleaved PARP also showed a strong trend toward an increase in the long labor samples, but analysis of variance was not significant (P = 0.078) because of large sample variation. It is important to note that IHC staining for M30 confirmed trophoblast apoptosis after labor (Figure 6H). To summarize, the above results clearly show an increase in oxidative stress, production of inflammatory cytokines and soluble VEGF-R1, and trophoblastic apoptosis in the placenta during labor. cDNA microarray analysis was performed using custom-made human cDNA arrays containing ∼15,000 probes in total. Placentas from uncomplicated normal pregnancies were used: seven from women undergoing labor longer than 15 hours and 10 from women undergoing elective CS. After normalization of the raw data from all 17 with LIMMA, the data were analyzed using Cyber-T. The conditions were tuned by repeated random sampling of the dat