REVIEWARTICLEpublished: 12 November 2014doi: 10.3389/mmu.2014.00567Oxidative stress damage as a detrimental factor in pretermbirth pathologyRamkumar Menon*Department of Obstetrics and Gynecology, School of Medicine, The University of Texas Medical Branch, Galveston, TX, USAEdited by:Jeffrey A. Keelan, University ofWestern Australia, AustraliaReviewed by:Gaurav K. Gupta, Harvard MedicalSchool, USAWalter Balduini, Universit degli studidi Urbino Carlo Bo, Italy*Correspondence:Ramkumar Menon, Department ofObstetrics and Gynecology, School ofMedicine, The University of TexasMedical Branch, 301 University Blvd.,Galveston, TX 77555-1062, USAe-mail: ram.menon@utmb.eduNormal termandspontaneouspretermbirths(PTB) aredocumentedtobeassociatedwith oxidative stress (OS), and imbalances in the redox system (balance between pro- andantioxidant) have been reported in the maternalfetal intrauterine compartments. The exactmechanism of labor initiation either at term or preterm by OS is still unclear, and this lackof understanding can partially be blamed for failure of antioxidant supplementation trials inPTB prevention. Based on recent ndings from our laboratory, we postulate heterogeneityin host OS response. The physiologic (at term) and pathophysiologic (preterm) pathwaysof labor are not mediated by OS alone but by OS-induced damage to intrauterine tissues,especially fetal membranes of the placenta. OS damage affects all major cellular elementsin the fetal cells, and this damage promotes fetal cell senescence (aging). The aging of thefetal cells is predominated by p38 mitogen activated kinase (p38MAPK) pathways. Senesc-ing cells generate biomolecular signals that are uterotonic, triggering labor process. Theaging of fetal cells is normal at term. However, aging is premature in PTB, especially inthosePTBscomplicatedbypretermprematureruptureofthemembranes, whereele-ments of redox imbalances and OS damage are more dominant. We postulate that fetalcell senescence signals generated by OS damage are likely triggers for labor. This reviewhighlights the mechanisms involved in senescence development at term and preterm byOS damage and provides insight into novel fetal signals of labor initiation pathways.Keywords: oxidative stress, pretermbirth, premature rupture of fetal membranes, inammation, oxidative damage,senescence, senescence-associated secretory phenotypeTheWorldHealthOrganizationrecentlyestimatedthe globalpreterm birth (PTB) rate for singleton gestation at 9.6% (1). ThePTB rate has increased in the United States by as much as 30%duringthelast25 yearsdespiteadvancesinmedicalcare(1, 2).Twenty-eight percent of all neonatal deaths (deaths within the rst7 days of life) that are not related to congenital malformations aredue to PTB (1, 2). The most common phenotype of PTB is spon-taneousPTB ofunknown etiology. Approximately 60% ofPTBsare spontaneous, and 3040% ofthese are preceded by pretermpremature rupture of the fetal membranes (pPROM) (310). Thecurrent management of pretermlabor andpPROMis basedlargelyon inhibiting uterine contractions (7, 1125). This approach hasnot been successful, as such interventions are usually performedtoo late in the process to succeed. A second problem with the cur-rent management of preterm labor is that only women who haveclear risk factors (abnormally short cervixes) or a history of PTBsare targeted for interventions designed to prevent PTB (2630).The vast majority ofPTBs occur in women who are consideredlow-risk because they are either on their rst pregnancies or haveonly had termbirths previously (3137). Although the rate of PTBis lower in these women (35%), they make up the largest volumeof clinical practice. Simple interventions that can be applied to thisgroup are likely to have the largest impact on PTB rates. Knowl-edge gaps in current literature about causality and causally linkedpathways make it difcult to provide appropriate or personalizeinterventions based on the specic risk prole of an individual (6).Riskfactorsof PTBandpPROMcanbeclassiedintotwomajorcategories, staticanddynamic. AsshowninFigure 1, allthe risk factors outlined in the outermost layer can be called staticriskfactorsastheyareunlikelytochangeduringthecourseofpregnancy. Independently or in combination, these static risk fac-tors can either predispose or cause the dynamic risk factors thatarecommonlydiagnosedasclinicalrisksorpathologiesassoci-atedwithadversepregnancyoutcomes.Epigeneticchangesthatareindependentof DNAbasevariationsgeneratedbycomplexinteractionsbetweenvariousriskfactorsduringpregnancycanalsocontributetodynamicclinical risksbyalteringexpressionof certain genes. These changes can transition between static anddynamic risks. Static and dynamic risk factors produce pathwaysand pathophysiologies depicted in the inner circle with a uniquebiomarker prole contributing to labor-inducing changes, result-ing in PTB or pPROM. The nal effector pathways culminatinginlaboranddeliveryincludeinammationandoxidativestress(OS). In normal pregnancies, these are generated by various fetaland maternal factors that signal the end of pregnancy. In PTB, thematernalfetal signals and their causal origins are still unclear asthey arise from complex etiologies and redundant pathways.Inammation is a well-studied pathophysiology ofboth PTBandpPROM(3840). Thisreviewisanattempt toshedsomelight on one of the under-studied mechanistic pathways: OS dam-agetointrauterinetissues andhowit mayimpact pregnancyoutcomes.www.frontiersin.org November 2014 | Volume 5 | Article 567 | 1 Menon Oxidative stress damage in preterm birthFIGURE 1 | As depicted in this gure, preterm labor (the innermostcircle) is an end result of multitudes of complex interacting pathologiesand pathophysiologic pathways. The external layer (the outermost circle)shows static risk factors, including epidemiologic and genetic risk factors,that can lead to multiple disease processes as depicted in the middle circle.Epigenetic markers can be dynamic, and that complex interaction betweenthe host environment and risk factors can produce epigenetic changes,which can lead to diseases contributing to nal effecter pathways (the blueshaded area). Various diseases may also cause epigenetic changes in thegenes of preterm labor pathways. Spontaneous preterm labor leading topreterm birth is a complex syndrome comprising multiple diseases, each ofwhich can be independent initiators of labor-inducing pathways. All thesedisease processes can trigger inammation and oxidative stress (OS).However, the extent and biomolecular characteristics of inammation andOS are dependent on the type of risk that initiated the process, theircomplex interactions with the geneticepigenetic system, and the diseasethat they cause. The nal terminal pathways that involve inammation andOS trigger labor-inducing signals from fetal membranes and decidua andcause cervical ripening, myometrial contractions, and membrane rupture,resulting in preterm labor and delivery. *Any exogenous factor that canimpact pregnancy outcomes. Many of the static risk factors in the outercircles are also called environments.INFLAMMATION, A WELL-DOCUMENTED FEATURE OFLABOR AND DELIVERYLabor anddelivery at termandpretermhave anunderlyingpathophysiology markedby inammatory mediators. Pretermlabor is hypothesizedtobe drivenby overwhelming inam-mation that eclipses fetal uterotonic signals of organ matu-rity. Approximately 50%of PTBs and 70%of pPROMs areassociatedwithintra-amniotic infection(IAI) andinamma-tion. Histological and microbiological ndings indicate that focalinfectionandinammationmayplayasignicant roleinthepathogenesisof PTBandpPROM. InammatorychangesthatprecedePTBsuchas leukocyteactivation, increasedinam-matory cytokines and chemokines, and collagenolysis of theextracellular matrix metalloproteinases (MMPs), resulting in lossof membrane structural integrity, myometrial activation, andcervical ripening are well documented by various experi-mental and clinical studies (9, 38, 41). Recent studies havereportedthat the heterogeneityinthe inammatoryresponseFrontiers in Immunology | Inammation November 2014 | Volume 5 | Article 567 | 2 Menon Oxidative stress damage in preterm birth(cytokines/chemokines, toll-like-receptors, and their interactions)isassociatedwithIAI andPTBriskfactors(4245). PTBandpPROMarewell-documentedhost responsediseasesinwhichoverwhelming immune activationcantrigger labor-associatedchanges. The biomolecular markers that trigger labor-associatedchanges aredifferent, andthedifferenceis attributedtobothepidemiologic and clinical risk factors.SIGNIFICANCE OF OS AND OXIDATIVE DAMAGEOftheseriskfactors, IAI, behavioralfactors(cigarettesmoking,alcohol intake, anddrug use), obesity, malnutritionor antioxidant-decientdiets, physiologicandpsycho-socialstressors, environ-mental pollutants, genotoxic agents, and geographic location aresome of the most common and well-studied risk factors of bothPTB and pPROM (3, 10). The role ofinammation induced bythese risk factors in PTB pathophysiology is well studied and mul-tiple biomarkers are reportedtobe associatedwiththe adverse out-come. OS, characterized by generation of reactive oxygen species(ROS), isaninseparablecomponentof inammation. Genera-tionof ROSasapartof theaerobicenergybuildingprocessisinevitable and is well balanced (redox status) by an array of enzy-maticandnon-enzymaticantioxidant systems (4650). Redoxbalanceismaintainedthroughtheproductionandsubsequentelimination ofROS. Cells are able to protect themselves againstOS by the nely tuned regulation of redox status through endoge-nousenzymes,antioxidants,andothercellularmechanisms.Atlowlevels, ROS, oftengeneratedinbiologicalsystems, isessen-tial for cell division and survival, cell signaling, inammation andimmune functions, autophagy, and stress response (5052). How-ever, an overwhelming redox imbalance compromises a biologicalsystemsabilitytodetoxifythesehighlyreactivemoleculesortorepair any damage caused by them (53). Therefore, the former istermedOSand the latter oxidative damage(see Glossary) (54).Oxidative damage due to ROS generation has been linked to thedevelopmentofadultdiseases, includingcardiovasculardisease,cancer, chronic inammation, and neurologic disorders. The twomain sources ofROS in human cells are: (1) mitochondrial and(2) non-mitochondrial. Mitochondria generate ROS as a byprod-uct of the electron transport chain during respiration, and the rateofROS production is proportional to the rate ofmitochondrialrespiration. The ROS production rate is thus higher when meta-bolic rates are high (55, 56). A vast majority of ROS in the humanbody is produced by mitochondria, and mitochondria are the pri-mary sources of superoxide productionother thanphagocytic cellsduring innate immune defense.Figure 2 depicts the mechanismof ROS generation, antioxidantsystem, and potential damage by ROS that can cause damage dur-ing pregnancy complications. The gure also models the potentialofROSingeneratinginammationanddamagetocellularele-ments that can generate pathways leading to pPROMor PTB. Thisgure represents only a few key mediators of ROS generation anddoes not include all known endogenous antioxidants.FIGURE 2 | A model of reactive oxygen production, its inactivation byantioxidant systems, and potential consequences of redox imbalances.Superoxide radicals are generated during normal cellular respiration(mitochondrial) and also during infection following phagocytosis.Non-enzymatic and enzymatic mechanisms generate super oxide radicals(O2 from O2) that are converted into H2O2 by superoxide-dismutase (SOD).H2O2, a reactive oxygen species (ROS), is reduced to water by severaldifferent enzymes, mainly by catalase. Hydroxyl and/or superoxide radicals arecreated from H2O2 by a Fenton reaction that includes Fe3+. H2O2 is a potentialtransactivator and can activate a master transcription factor NF-B, resulting inactivation of several proinammatory genes including many uterotonic genes.The Hydroxyl and/or superoxide radicals can cause DNA, protein, and lipiddamage. DNA damage as detailed in this review can lead to telomerereduction and senescence. DNA damage of the cell can also cause apoptosis.One of the transactivator proteins is a nuclear factor of erythroid 2-relatedfactor 2 (Nrf2). When activated in response to ROS, this protein cantransactivate Hemeoxygenase-1 that can also neutralize H2O2, providing aregulatory mechanism. Our studies have shown that Nrf2 can causeanti-inammatory PPAR- activation. Depending on the dose and type ofROS, inammatory and/or anti-inammatory/antioxidant pathways may beinitiated. This model shows that OS-associated pathologic events result inspontaneous preterm birth and pPROM. This is not a universal model for allOS-related pathologies. Damage to cellular elements has been welldocumented in cancers and other diseases.www.frontiersin.org November 2014 | Volume 5 | Article 567 | 3 Menon Oxidative stress damage in preterm birthHETEROGENEITY IN ANTIOXIDANT FUNCTIONSGeneration ofROS is tightly regulated by an array ofenzymaticand non-enzymatic ways to keep redox balance. This balance inhumans is maintained by a well-balanced antioxidant system thatis responsiblefor homeostaticbalance, whichmaintains phys-iologicfunctionsbut preventsoxidativedamage. Antioxidants,ingeneral, aresubstances that decreasetheseverityof OSbyforminglessactiveradicalsorbyquenchingdamagecreatedbyfree-radical chain reactions. They can slow down or prevent dam-agetobodycellsandlowertheriskof infectionbyimprovingimmune function. Antioxidants can be classied into three majorcategories: (1)lowmolecularantioxidants[glutathione(GSH),vitaminsCandE,bilirubin,andurate],(2)enzymesthatneu-tralizefreeradicals[superoxide-dismutase,catalase,glutathioneperoxidase(GPx), DTdiaphorase, andperoxiredoxin], and(3)non-enzymatic proteins (thioredoxin, glutaredoxin, andmetl-lothionines). Antioxidantscanalsobeclassiedasprimaryandsecondary. Primaryantioxidantsareendogenousenzymes[e.g.,superoxidedismutase(SOD),catalase,andGPx]actingatthesitewherefreeradicalsareformed. Secondaryantioxidantsareexogenousmoleculesobtainedthroughfood(e.g.,VitaminsA,C, E, carotenoids). As detailed in Table 1, regardless ofprimaryor secondary, endogenous or exogenous status, antioxidants havespecic functions.WHY ANTIOXIDANT SUPPLEMENTATION FAILS INPREVENTING ADVERSE PREGNANCY OUTCOMES?A healthy pregnancy is characterized by a stable balance betweenROS and antioxidants (46, 47, 49). Redox imbalance is an under-lyingpathologicfeatureof manypregnancycomplications. Allof the PTB and pROM risk factors detailed above are capable ofcausing redox imbalance, leading to the production ofsuperox-ide, hydrogen peroxide, hydroxyl ions, and nitric oxide that canTable 1 | Antioxidants and their potential roles associated withreactive oxygen species (ROS) mediated damage.Antioxidants Potential roleSuper oxide dismutase (SOD) Direct protection against ROSGlutathione peroxidase (GPx)Catalase (Cat)PeroxiredoxinGlutathione Non-specic reductionVitamin CVitamin E Protection against lipid peroxidation-CaroteneTransferrin Metal sequestrationLactoferrinFerritinMetallothionein (MT)DNA repair enzymes Repair enzymesMacroxyproteinasesGlutathione transferase (GST)Thioredoxins (Trx)damage collagen matrix and consume antioxidant defenses. Theseeventscantriggeruterinecontractions(labor),leadingtoPTB.Basedonthisimbalancetheory, antioxidantclinical trialshavebeen primarily carried out to compensate for deciencies and tomaintain redox balances. However, these trials expected to reduceOS and improve pregnancy outcomes have met with minimal suc-cess (36, 5761). Numerous reasons can be cited for the failure ofthese trials, but the following primary factors are highlighted:1. OS-inducedtissueinjuryordamageisthelikelypathologyandtrigger of several downstreameffects likeinitiationoflaborormembranerupture. OSdamagegeneratesaviciouscycleof events(enhancedinammation, cell death, andtis-sue destruction), and antioxidants do not regulate these eventsand are unlikely to impact OS damage. Therefore, it is unlikelythat reversing OS through antioxidant supplementation wouldreduce the risk of adverse pregnancy outcomes.2. Clinical trials are conducted based on the generalized theorythat OS is an underlying pathology. This theory is unlikely; thismay be the case for a limited subset ofsubjects with specicexposures with ROS as an initiator of downstream events.3. Biomarker screenings for ROS are not done prior to interven-tiontodeterminethetypeof ROSortoassesstheamountofROS-associated damage. Even the type ofbiomarker mea-sures may not be reective of risk exposure or the extent of ROSbecause these biomarkers may represent an end stage indicativeof tissue damage and not necessarily risk.4. Like inammation, ROS is not a homogeneous phenomenon.Different tissues andcells, cytoplasmic andnuclear membranes,and the contents of different organelles (inner and outer mem-branes of mitochondria) may react differently to various stres-sors, producing distinct patterns of ROS. The antioxidants usedin trials may curtail a particular ROS pathway or production ofa specic free radical, but depending on the type of risk factor(e.g., cigarette smoke vs. poor nutrition), the ROS pathway andantioxidant requirement may be different.5. Reactive oxygen species-associated damage can be any ofthemajorfunctionalelementsmentionedearlier, andthedegreeof damage is likely dependent on the site, risk factors, and typesof triggers that initiate an ROS-mediated response.AMBIGUITY IN ANTIOXIDANT AND OS DAMAGE MARKERANALYSISMany studies have reportedantioxidant deciencies as a causal fac-tor associated with adverse pregnancy outcomes. In the wake of afailed antioxidant supplementationtrial, my laboratory conducteda cross-sectional study examining twoantioxidant enzymes (SOD1and catalase) and two OS damage markers (F2-Isoprostane lipidperoxidationproductand8-OxodGDNA damage).Bankedmaternal plasma samples were collected from subjects at the timeof deliveryadmissionforeitheratermlabor(n =19), pretermlabor (n =18), or pPROM (n =13). An ELISA-based analysis wasperformed using these samples. If each of the markers is analyzedindependently, multitudes ofinterpretations can be derived. Forexample, if SODwas the only analyte tested, as showninFigure 3A,SODwashigherattermandreducedsignicantlyinPTBandpPROM. These data are normally interpreted as heightened OS atFrontiers in Immunology | Inammation November 2014 | Volume 5 | Article 567 | 4 Menon Oxidative stress damage in preterm birthFIGURE 3 | Maternal plasma levels of two antioxidants super oxidedismutase (SOD) and catalase and two OS markers, 8-oxodG andF2-Isoprostanes. (A) Antioxidant enzyme Super oxide dismutaseconcentrations. (B) Antioxidant enzyme catalase concentrations.(C) Lipid peroxidation product F2-Isoprostane concentration. (D) bDNAoxidation product 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG)concentration. *P