Similarly, the recentlyobserved autoxidation and subsequent HOM formation in the oxidationof saturated hydrocarbons cannot be initiated by O3 andrequire different mechanistic steps for initiating and propagatingthe autoxidation sequence. This Perspective reflects on these recentfindings in the context of the direct aerosol precursor formationin urban atmospheres.Gas-phase hydrocarbonautoxidation is a rapid pathway for the productionof in situ aerosol precursor compounds. It is a highway to moleculargrowth and lowering of vapor pressure, and it produces hydrogen-bondingfunctional groups that allow a molecule to bind into a substrate.It is the crucial process in the formation and growth of atmosphericsecondary organic aerosol (SOA). Recently, the rapid gas-phase autoxidationof several volatile organic compounds (VOC) has been shown to yieldhighly oxygenated organic molecules (HOM). Most of the details onHOM formation have been obtained from biogenic monoterpenes and theirsurrogates, with cyclic structures and double bonds both found tostrongly facilitate HOM formation, especially in ozonolysis reactions.Similar structural features in common aromatic compounds have beenobserved to facilitate high HOM formation yields, despite the lackof appreciable O Part ofthe inability to hinder the problem raises from our incomplete understandingof volatile organic compound (VOC) oxidation in the gas phase. Perhapsmost alarmingly, the formation of particulate air pollution from gaseoussources is particularly poorly understood.Airpollution shortens the lifespan and decreases the well-beingof an increasing number of lives.12 HOM were originallyfound in the Finnish Boreal forest14 and were promptly connected to the “missingorganic compounds” expected to explain organic aerosol measurements.17 Consequently, they were shown to be the key compound class involvedin the formation and growth of atmospheric secondary organic aerosol(SOA) mass.18 The current understanding ofHOM was recently reviewed by Bianchi and co-workers19 in order to synthesize the decade of observations intoa more meaningful format. They defined HOM as species that are (i)formed via a sequential autoxidation of peroxy radicals (RO2) (ii) under atmospherically relevant conditions and (iii) containsix or more oxygen atoms. This definition contains the gas-phase aerosolprecursors formed by a rapid sequential oxidation through consecutiveH-shift reactions, while also excluding common plant-synthesized,highly oxygenated carbohydrates . The HOM contain severalhydrogen-bond-donating functional groups such as alcohols and hydroperoxidesand aggregate by a peroxide “dimer” formation, and theirgeneral stability on surfaces and in condensed phase media is stilllargely in doubt. This strict definition currently leaves out certainimportant aerosol forming molecules with similar properties in clusteringand condensing reactions, such as multicarboxylic acids , which donot have known fast gas-phase formation routes. Also, the oxygenatedcompounds left out from the HOM definition  are considerably more stable in condensedphase, and while they have a similar propensity for hydrogen bondingand clustering reactions, it is possible that they do not react significantlyin the condensed phase.Recently the rapidgas-phase autoxidation of endocyclic alkenesinitiated by a reaction with ozone  was shown toyield highly oxygenated organic molecules (HOM).22 In practice,AVOCs cover all hydrocarbon subclasses containing all functional groupsfrom aromatic to aliphatic compounds. In fact, the division to AVOCand BVOC  is chemically completely arbitrary;often, the same compounds are emitted by both anthropogenic and biogenicprocesses =CH2] and ethylene CH2=CH225). Generally, AVOCs arethought to consist mainly of aromatic compounds and alkanes, and commonfor these species is that they do not react appreciably with O3. This more chemically rigorous classification of AVOC isadopted here too to allow for an easier discussion, notwithstandingthe shortcoming that several common anthropogenic compounds do notfit in . The ozonolysis of double bonds is an important initiation stepin the formation of atmospheric particulate matter, and thus a divisionof compounds that either react or do not react with O3 isuseful. Moreover, most of the information we currently have on HOMformation has been gained through ozonolysis oxidation experiments.Anthropogenic volatile organic compounds(AVOC) are hydrocarbonsreleased by human activities. They are emitted from countless activitiesof the society, such as traffic, energy production, industry, fromsolvents, adhesives, paints, lubricants, and wear-reducing products,from cosmetics and personal care-products, and so on, and so on 1.20−22 I27 Aromatic systems have doublebonds coupled to cyclic structures , and the pathways up to five oxygen-containingbicyclic peroxy radicals (BPR) are well-established . Sub37 have inspected the first steps of alkylbenzene-derivedRO2 H-shifts by detailed quantum chemical computations,whereas others have mainly reported product compositions and overallHOM yields.41 It seems that AVOC HOM from both aromatic and alkane sources haveescaped observations due to the same reasons HOM were not found inthe first place: (i) The yields of HOM generally fit into common measurementuncertainties , (ii) the sampling from experimental setups has not beenoptimized for free radicals and sticky aerosol precursors , and (iii) oxidation studies and mass spectrometricdetection are performed at reduced pressure and low O2,decreasing, or even preventing, the formation of HOM.The detailed pathwaysto HOM in aromatic compounds remain far fromunderstood, notwithstanding the relatively many publications alreadydevoted to the topic. Currently only Wang et al.This perspectiveconcerns AVOC HOM formation by autoxidation witha focus on the recent advances in our understanding and on the remainingchallenges in uncovering the underlying mechanistic steps leadingto these in situ aerosol precursors. The text is structured as follows.First the AVOC HOM reported in recent literature is presented, followedby the related challenges in instrumental detection methodologies,field observations, and theoretical computations. Then the mere lackof chemical intuition in finding the correct reaction sequences isdetailed and discussed, with the last chapters dedicated to an examinationof the burning questions that are difficult, or even impossible, toanswer with the current research methodologies.37 a collection of compounds including polyaromaticsby Molteni et al.,38 the sequential andauto-oxidation of benzene and HOM formation in several other systemsby Garmash et al.,39 toluene and 1,2,4-methylbenzeneby Zaytsev et al.,40 and the photo-oxidationof toluene and naphthalene by Wang et al.41 Numerous earlier works have investigated the formation of bicyclicperoxy radicals that are the starting material for HOM formation fromaromatics  yet 39  and was very recently inspected by Garmash etal.39  and 46)39 (46)).47). Most of the studieshave applied selective adduct-forming negative polarity reagent ionsfor a sensitive detection of a wide variety of HOM. Especially theutilization of nitrate ion (NO3–)-basedcharging has been instrumental for the original discovery and subsequentinsights . Recent52) or a collection of reagent ions  are produced in a controlled manner atatmospheric pressure in a separate ion production unit, after whichthe reagent ions and the targeted sample are mixed with the help ofelectric fields in an ion–molecule reaction region. Subsequentlythe charged sample is drawn into the mass spectrometer through anorifice or a capillary, with practically no dilution, as the charginghappens before the entrance of the vacuum chambers. The careful controlover the ion pool that meets the sample improves the sensitivity andreduces the secondary chemistry tremendously. Also other reagent ionintroduction designs have been implemented; for example, Sipiläet al.54 used sulfuric acid injection directlyinto the sample flow, to induce HSO4– formation in reactions with NO3–, followedby its clustering with the target molecules. The controlled mannerby which well-chosen ions can be produced together with the extremeselectivity of the clustering reaction make the APcCI currently anunrivaled method for gas-phase HOM detection.In APcCI methods either a single reagent ion  dueto low-pressure ionization sources  and due to nonselective ionization schemes,making charge availability problematic. Nevertheless, with the noveltype of ion transfer optics, they have been shown useful in recentHOM research and, with further optimization, could become of greatimportance. Especially with several carefully chosen proton-transferschemes and the proton transfer being performed at atmospheric pressurecould allow for a selective detection of both the reagents and a widearray of products with a single instrument. Yet, this type of an approachremains to be demonstrated in atmospheric research.Novel proton-transfer-reactionmass spectrometers (PTRMS) workwith largely the same principles as the APcCI-MS, and certain instrumentalconfigurations have shown promising developments in the detectionof HOM.56 Yet, the growing number of studies probing HOMand subsequent new-particle and SOA formation in a laboratory andin the field, and the increasing variety of reagent ion chemistriesused in HOM detection, are slowly unifying the perception of HOM importancein gas-phase particulate matter formation.Withouta doubt, the community would benefit from more instrumentationcapable of detecting these crucial, low-volatile, and reactive aerosolprecursors in situ. Arguably, the HOM research community suffers fromthis scarcity of detection methods, and some have even criticizedHOM to be merely an artifact of the used instrumentation.In contrast to theapplied mass spectrometric detection methods,spectroscopic and chromatographic techniques could supply direct informationon the product structure and would be highly beneficial for inferringthe presently uncertain HOM structures. However, their inherent experimentallimitations prevent a utilization of their benefits. In spectroscopictechniques, the spectral overlaps of similar functional groups withinsimilar chemical neighborhoods constitute a tremendous problem forspeciation. On the one hand, the less-oxidized products and intermediatespresent at higher concentrations than HOM will contain the exact samefunctional groups in the exact same molecular positions ,maximizing the spectral overlaps. In chromatographic techniques, onthe other hand, the transfer limitations of the elution columns generallyprevent the highly functionalized and polar HOM to pass through. Additionally,the high surface activities and reactivity of polyperoxide HOM meanthat, if they can travel through the chromatographic column, theyare unlikely to do it without a change in their molecular structure.Moreover, HOM completely lack authentic standards, and, as a currentlyfully gas-phase species, their stability in condensed phases is amystery. The HOM standards would have to be formed in situ with acontrolled gas synthesis, a chore that is in practice close to impossibleto accomplish without a multitude of secondary unwanted reaction products.57 If the technical limitationscan be overcome, then a combination of chromatographic separationand spectroscopic detection would provide a currently unrivaled structuralcharacterization for the HOM, yet nothing like this seems availablein the foreseen future. Another fundamental technical obstacle comesfrom the detection limit. Individual HOM compounds are present frombelow 105 cm–3 to up to ∼108 cm–3 gas-phase concentrations even in optimallaboratory settings. Such minute quantities present challenges forany detection methods notwithstanding the nature of the target material.Thus perhaps a more fruitful approach is to develop chemical ionizationmass spectrometry toward a structural analysis methodology.While the chromatographic and spectroscopic techniques would beideal in solving detailed HOM structures, their successful applicationrequires a surmounting of these technical challenges first. Recently,gas chromatography has been used for the quantification of severalmultifunctional aromatic oxidation products, which are the likelyprestages of HOM formation through a sequential oxidation.2O-mediatedH to D exchange,11 ion-mobility spectrometry(IMS),58 and MS-MS fragmentation experiments59 have all been used to verify the assumptionthat several HOM isomers are formed from the oxidation sequences.If single-oxidation pathways and their subsequent products could beisolated, then a threshold photoionization  and potentially other compound-specific characteristicscould be used for a molecular identification. However, currently itseems that only in very specific cases a single oxidation pathwaycould be responsible for the HOM formation, and generally isomersare formed. For an understanding of the chemistry the most importantisomers to distinguish would be those that are susceptible to a verydifferent subsequent chemistry, for example, double bonds versus cyclicstructures and bridged oxygens  versuscarbonyls, which hinder and enhance the oxidation propagation in veryspecific ways.While mass spectrometers are generally far from ideal at extractingthree-dimensional molecular information, they are able to resolvefeatures of the molecular structure. Several methods can be used toinfer a deeper insight into the HOM construction. D64 For example, distinguishingbetween carbonyl and alcohol groups with a reference to molecularcomposition from the MS measurement. In such a technology, a rapidapplication of several ionization schemes would be highly beneficial.The recently developed multischeme chemical ionization inlet (MION50) type of approach would allow this, enablingthe rapid switching between multiple reagent ion schemes in both ionpolarities. Even the utilization of several concomitant proton-transferschemes could be envisioned, enabling an overcoming of the chargebalance problem mentioned above.As yet unexplored, structural information onoxidation productscould also be inferred indirectly by utilizing several well-chosenreagent ion schemes. Only a certain type of reagent ions bind to certainfunctional groups, and thus, a chemical filtering of the product distributionbecomes possible.65 The a-pinene product distribution considerably changes from subsecondto tens of seconds reaction time. The endoperoxide bridge reportedlyimportant for the rapid early oxidation propagation seems to be absentalready at 30 s reaction time according to the MS-MS analysis. Similarchanges in peroxy radical distributions as a function of reactiontime in isoprene oxidation have been discussed by Wennberg et al.66 While the long reaction times are rather trivialto obtain when larger reactor setups are utilized, the short reactiontimes pose considerable challenges for the CIMS research methodology.CIMS is poorly suited for the study of rapid chemical changes dueto the finite time needed to charge the sample by ion–moleculereactions. Especially the inlets optimized for a sticky and reactivereaction product detection  have sheathflows and characteristic long charging times. The faster CI-inlets,the MION working by ion injection,50 andthe “cluster CIMS” with a transverse ionization geometryby Zhao et al.67 are more suited for thistype of research . Thus, often it is likely enoughto merely estimate the sum of HOM from the mass spectrometer signalfrom products with HOM compositions. This carries some extra uncertaintydue to the varying detection sensitivities for molecules with differentfunctional groups, yet the difference in charging efficiency is likelyrelatively small for such a highly oxygenated reaction products.51 Furthermore, it is rather easy to propose HOMcompositions based on simple mechanistic rules concerning their formationpathways. For example, if an OH H-abstraction reaction started theoxidation sequence and the only bimolecular steps were O2 additions, then likely a succession of products with varying, highoxygen content, the same amount of C atoms, and two fewer H atomsare seen, the second H being lost during an −OH ejection afteran oxidation-terminating H-shift from a hydroperoxide C. However,in reality every branching point, especially C–C bond scissionsand H-atom scrambling, complicate this task, leading to a wider arrayof possible product molecular structures. However, currently mostof the observed HOM products pertain to certain oxidation propagationrules 18 and co20 in considering the vast amountof different petrochemically derived volatile chemical products (VCP)emitted from the daily routines of the society.Most likely places for AVOC HOM observationsare polluted urbanatmospheres and places in close proximity to industrial activitiesand combustion sources. These environments are characterized by avast number of different emitted chemicals and airborne particulatesurfaces where HOM can deposit on. Generally urban atmospheres canhave high AVOC HOM concentrations as exemplified recently by McDonaldet al.19 The inherent complication in this research isa result of the rapidly growing number of potentially relevant uniquemolecular structures that contribute to the important reaction steps.Often it is not a simple task to decide which pathway to follow computationally,and the previously generated rules of thumb slowly lose their meaningas a function of molecular complexity. The substituent effects offunctional groups that affect the H-transfer rates are also not necessarilyadditive. This rapidly increases the number of species to consider,and an even larger number results from their mutual reactions—oftenbeing referred to as the combinatorial problem of organic chemistry.For example, isomers  are almost certainly also reacting differently,yet even the conformation of the molecule  can have a strong influenceon its subsequent chemistry.42Most of the information on HOM formation mechanisms and molecularstructures have been obtained by quantum-chemical computations.72 which havespecial importance in growing atmospheric SOA,74 was resolved only by an application of farmore sophisticated computational methodology . Thus, parts of the potential energysurfaces (PES) must be described very accurately, which consumes alot of effort and, consequently, time. Very recently, the rapid formationof HOM from α-pinene, the most studied HOM-forming system, wasuncovered by a more detailed look on the same ozonolysis-derived intermediatesthat had been already considered several times.75 Specifically it was the improved descriptionof the excess energy partitioned into the postozonolysis vinoxy radicalintermediate that was required for uncovering this important oxidationpathway.65Acoupled technical hurdle comes from the fact that several ofthe key reactions require state-of-the-art computational tools, whichconsume a vast amount of computational resources. For example, recentlythe decades old mystery of gas-phase “dimerization”of peroxy radicals into oxygen-bridged peroxides,64 and the use of these with controlled fragmentation to obtain experimentalconstraints.76 This also amply highlightsthe general need for joint theoretical-experimental approaches, asparts of the problem are easier to address computationally, whereasother areas are easier to resolve in laboratory settings. The synergisticapplication of both is likely to be the most fruitful one, in whichtheory is used to guide the experimental design, and experimentalresults are used to constrain the further theoretical argumentation.For the time being, computations will likely remain as the most reliablewindow we have on the structures of these highly functionalized gas-phaseaerosol precursors.As the experimental speciationand quantification of individualHOM compounds will remain extremely challenging, the role of computationswill likely only increase in the future. Especially of importancefor the applied CIMS methodologies are the studies that associatereagent ion and target binding energies with instrument sensitivity77 The gained oxidized functionalities help the subsequent hydrogenabstraction reactions  and thus enable a propagationof the oxidation chain. With each added oxygen, the several sitesof potential H abstractions become more equal, increase the possibilityfor isomeric reaction channels, and directly relate to the mutualexperimental-theoretical challenge of determining the HOM-formingpathways and subsequent product structures. The fast radical rearrangementsthat do not transform the product chemical composition but only tweakthe spatial structure occur very rapidly, and they are not easilytracked by the available research methodologies.The biggestobstacle in describing HOM formation in any VOC systemlies in the rapidly growing amount of potential branching pathways.These happen both in the (pseudo-) unimolecular oxidation propagationand in the cross combination of practically all the radical intermediatesinvolved  abstraction andsubsequent ketohydroperoxide formation. Importantly, in autoignitionthe loosening of the C–H bonds and subsequent H-abstractionhappens due to heat, whereas in an atmospheric autoxidation the looseningis brought about by nearby functional groups. Very recently Wang etal. showed how these effects can be additive and, thus, how the heatcan be used to initiate autoxidation in naked alkanes too, providinga tangible segue between these two phenomenological VOC oxidationregimes.33 While the previous low-temperaturecombustion research could not observe the HOM formation due to pertaininginstrumental limitations, it is likely that considerable insight couldstill be gained from a revisit to the extensive ignition delay andcool flame literature, potentially providing important experimentaland theoretical constraints.80More details on HOM compositions and a solid hypothesis for thechemical nature and formation mechanism of these compositions wassoon reported by Ehn and co-workers.36 reported how even the naked alkanes have thepotential for H-shift reactions, but they are too slow to providea significant competition for other RO2 loss processes.Thus, bimolecular RO2 reactions yielding RO  seem pivotal for the initiation of alkane autoxidationand formation of HOM VOCs. Its involvementis clear already from the first principles in considering the radicalintermediates propagating the sequence, but unfortunately it is aknown complexity we have not afforded to dwell into yet. The sophisticatedtheoretical computations have simply been too resource-demanding,and the mass spectrometric detection methodologies have not been ableto follow distinct reaction pathways. Thus instead the HOM researchhas focused on finding a single, theoretically sound oxidation pathwayable to explain a certain prominent HOM formation. Nevertheless, ithas been clear from the beginning that other pathways must contributeand lead into isomeric HOM products. Recently, Noziere and Vereeckenn of HOM 5.2 and RO radicals areat theheart of AVOC autoxidation  and carbon-centered radical (C*) rearrangementsare inaccessible, alkoxy radicals are needed to propagate the oxidationsequence. RO undergo much more rapid H-shift reactions than RO2, and their more energetic nature makes for more varied branchingpathways.83 Alkoxy radicals are also proneto decompose, and their special role in the breaking of carbon ringsthat hinder HOM formation has been noted previously.75 Especially in the naked, straight-chain, unbranched alkanes therole of RO is heightened, as the parent compounds do not have feasibleRO2 hydrogen-shift reactions available.36 Alkoxy radicals are even indirectly related to the molecular mechanismof peroxide “dimer” formation by RO2 + RO2 reactions, in which two ROs will stay entangled before theO2 breaks apart and the peroxide −C-O-O-C–bridge is formed.72 Very recently alkoxyradicals were also shown to undergo bimolecular reactions even withanother (stabile) radical species such as RO2,84 which highlights the poor understanding we haveof their importance for the atmospheric particulate matter formationand on atmospheric chemistry in general.The conversions between ROxidation 5. When R85 compared thisto the point in which ozone-pollution research was some decades ago,that is, the details do matter. Yet this common practice of lumpingin atmospheric and combustion chemistry modeling is likely still usefulfor several analysis, but for ambient pollutant formation and removal,especially relating to particulate matter formation, a number of moleculesand oxidation processes must be considered individually. With relativelycomplicated atmospheric molecules such as mono- and sesquiterpenesthis constitutes a considerable task in considering the wide varietyof possible reaction pathways. Fortunately, the number of specieslikely involved in the formation of most of the atmospheric SOA isfar more limited than the whole VOC pool, and a thorough knowledgeof some tens of atmospheric systems is probably enough to adequatelyestimate the aerosol-forming potential of most gas-phase environments.Likewise, the pool of small atmospheric radicals that react with theseaerosol prestages is dominated by a handful of species, decreasingthe amount of interactions that must be explicitly accounted for.Yet, even this reduced number of chemical systems results in a largenumber of reactions and even larger number of products to consider,and thus sophisticated machine-learning methodologies or other similarapproaches can hopefully soon provide order to this apparent chaos.The HOM and linkedSOA research are rapidly approaching a statuswhere lumped rate coefficients and simplified mechanistic rules donot suffice to explain the phenomena at the required detail. Veryrecently McFiggans et al.2 reactivity, and (v) role of apparent minor channels. This listingis not meant to be exhaustive and surely excludes several other burningissues that remain to be resolved.Several mechanistic aspectscan be envisioned to influence thelikelihood of AVOC HOM formation, yet their relevance remains unclearand prevents a determination of their ultimate importance. Among themost significant uncertainties are (i) competition for oxidation initiationand propagation, (ii) role of hydrogen scrambling, (iii) role of catalyticand photolytic reaction steps, (iv) role of differing RO2 propagation ratescan vary significantly. However, under atmospheric conditions nearlyall O2 reactions are fast due to oxygen being in a verylarge excess, which is commonly exploited in theoretical computationsby omitting their calculation. Nevertheless, even the O2 addition rates can vary by several orders of magnitude , and in certain cases this additionalcomplexity could be important to account for. This competition forthe oxidation propagation continues throughout the whole HOM formationsequence and is of special importance for each radical intermediate.The competition between different RO2 H-shift sites islikely the most determining factor in HOM formation and currentlyvery poorly constrained, as only a few RO2 H-shift rateshave been inspected experimentally . The OH abstraction rate depends on the chemicalneighborhood, and practically every C–H abstraction in a moleculewill occur at a different rate, with only certain abstractions leadingto subsequent HOM formation. The following steps are likewise pronefor a similar competition, and even with the same primary radicalproduction rate the following R + Og., refs  and 43)2 propaga89) can severely complicate the assessment of correct pathways. Itleaves no marks to the transformed product chemical composition andis generally a very fast reaction step. Previously it was noted toyield peroxy acids at the expense of hydroperoxides, yet in the cyclohexeneozonolysis system an NO2 addition to the reaction mixtureapparently leads to the formation of highly oxygenated acylperoxynitrates.81 Mechanistically the H-scrambling reaction isvery intriguing, as it can in a sense reverse the course of the reactionand return the radical site to its previous location.The H-scramblingreactions in the oxidized radicals , enabling a reinitiation of the oxidation sequence. Similarlythe role of photolysis in inducing HOM formation has not been assessed.The photolysis of several oxidation products,91 even RO2 radicals,92 couldprovide an alternative route to the HOM formation. The aromatics andtheir derivatives provide an exceptionally good example, as the absorptionspectra of the first- and second-generation oxidation products shiftto progressively longer wavelengths and higher absorption cross sections,facilitating their photolysis under a solar illumination.91 However, the concentration of every new productgeneration is likely to be lower than that of the parent, and thusthe influence is likely to stay minor. Nonetheless, this could proveto be a significant source, for example, during early mornings whensolar irradiance quickly increases and the accumulated processed materialfrom the previous night photolyzes.93The roleof oxidation propagation, or even initiation, by catalyticpathways is currently poorly understood. Recently Monge-Palacios etal.2 leading to oxidation propagation and termination willhave a special importance, with the rates of common RO2 + R′O2 reactions spanning many orders of magnitude.94 Especially for the AVOC HOM formation the ROinvolvement seems critical  and will remain difficult to account for. Theseare problematic, especially in cases where the rest of the potentialenergy surface of the minor channel would be far more attractive foran HOM formation but is ruled out during an early inspection of themechanism due to computational limitations, as generally to find evenone pathway with rigorous treatments is a laborious task.In any real-life VOC oxidation system the cross reactions of differentROcritical 4 and 5. Theoverarching realization is that HOM with a usual 0.1–10%yield generally fit into uncertainties of the previous studies. Thus,finding out the small differences in the pathways that enable HOMformation will remain very challenging but is likely to be the keyin finding the HOM from the representative AVOCs. Furthermore, withoutsignificant leaps in detection and computational methodologies, itwill remain very difficult to ascertain the importance of these less-clearoxidation sequences. Nevertheless, with the recent surge on the computationalmethod development that exploits machine- and deep-learning strategiesenabled by ever-increasing computing power, the needed theoreticaladvancements do not seem that science fiction anymore. Nonetheless,the experimental techniques have more real-life physical limitationsto overcome and, thus, are likely to lag behind. Time will show.33 and Garmash et al.39While the decade of work on HOM formation by atmospheric autoxidationhas increased our understanding of complex organic oxidation in thegas phase, several dark spots still exist and prevent us from describingthe formation of HOM from the diversity of AVOC. The lessons learnedabout the likelihood of H-shift reactions are directly transferrablebetween BVOC and AVOC, yet far more experimental-theoretical studiesare required to solidly anchor the underlying chemistry. As a topicalexample, also the autoxidation of the first- and second-generationreaction products must be studied , not the least by remembering that the H-shiftreactions are faster in the oxidized intermediates than in the nakedparent hydrocarbons. Currently this has been only briefly inspectedby Wang et al.2 environments in VOC oxidation research havea high potential to skew the results by limiting the collision frequencyand, in considering the real atmosphere, collisions with the correctcoreactants. The theoretical RO structure activity relationships (SAR)by Vereecken et al.95 are a good starting point inestimating the relevance of alkoxy radicals, whereas apparently nothingsimilar exists for the C*. While SAR are a common and practical toolin atmospheric chemistry research, such simplifications are also abig part of the reason why it took such a long time to find the pathwaysto HOM in the first place. In considering ambient HOM formation, weare still far away from any useful SAR development, and thus system-specifictreatments are a must. Ultimately, this research will evolve enoughto allow for an SAR development, but when, and how, is still too earlyto say.The rapid reaction sequence propagationby alkoxy radicals andcarbon-centered radical (C*) rearrangements are likely of high importancefor an accurate description of the reaction mechanisms but lack methodologyfor their experimental inspection. The commonly applied low-pressureand low-O


Dengue control approaches are best informed by granular spatial epidemiology of these viruses, yet reconstruction of inter- and intra-household transmissions is limited when analyzing case count, serologic, or genomic consensus sequence data. To determine viral spread on a finer spatial scale, we extended phylogenomic discrete trait analyses to reconstructions of house-to-house transmissions within a prospective cluster study in Kamphaeng Phet, Thailand. For additional resolution and transmission confirmation, we mapped dengue intra-host single nucleotide variants on the taxa of these time-scaled phylogenies. This approach confirmed 19 household transmissions and revealed that dengue disperses an average of 70 m per day between households in these communities. We describe an evolutionary biology framework for the resolution of dengue transmissions that cannot be differentiated based on epidemiologic and consensus genome data alone. This framework can be used as a public health tool to inform control approaches and enable precise tracing of dengue transmissions. Dengue virus (DENV) causes an estimated 390 million infections each year, 96 million of which manifest as clinical disease  information, along with temporal and spatial epidemiologic data, enabled fine-scale reconstructions of transmission chains and house-to-house spread, as well as genomic confirmation of within-household transmission clusters. This approach could be used in public health to reconstruct fine-scale DENV transmissions and support dengue control and prevention efforts.The samples used in this study were virus isolates, which originated from samples collected in a study described in Thomas et al. , the Illumina MiSeq next-generation sequencing system , and, for gap filling, the Applied Biosystems 3130 Sanger sequencing platform . We generated consensus genomes by using the in-house developed ngs_mapper reference mapping pipeline and manually curated to ensure consensus accuracy (>1% (minimum Phred of 30) called by Lofreq, a base caller previously shown to confidently call DENV iSNVs at this frequency  to determine model fit and the coefficient point estimate and 95% CI to estimate the average rate of DENV spread between households. We performed all regression analyses by using Stata 15.1 .We performed crude estimates of the dispersal rate of DENV at an inter-household scale by a univariate linear regression model, which fit the difference in sampling days between confirmed transmission pairs as a single predictor, and distance between confirmed transmission pairs as the outcome variable. We fit this model by using all transmission pairs confirmed by the Bayesian consensus analysis, the iSNV analyses, or both. We used the coefficient of determination   Table 3.>2 shared iSNVs  were on average 19 days apart  and a regression coefficient indicating that DENV disperses an average of 70 m/day (95% CI 54–86 m/day) within subdistricts in this study population , these coefficient estimates are nevertheless preliminary and are subject to potential nonindependence of transmission pair distance measurements and would benefit from confirmation in greater sample sizes in other study populations. This distance dependent dispersal between households was not noted in a prior Singapore study, which plotted uncorrected consensus genetic distance against geographic distance and sampling times . Even though our regression analysis indicated a good model fit (RAlthough our findings were derived from an intensive, 4-year active cluster investigation across a very well sampled geographic area, ascertainment bias was a potential weakness in our study. All sequences were either derived from index case-patients accessing care for febrile illness or their contacts who were only sampled in households within 100–200 m of the index case-patient. However, our dense sampling of index cases over a 4-year study period ensured a large number of sequenced specimens across the Kamphaeng Phet region across multiple cluster investigations. Two of the 9 genetically confirmed transmission pairs were detected between persons who were not sampled in the same index case–contact cluster, highlighting the incremental value of genomic data over epidemiologic data alone in DENV transmission tracing. Future improvements in deep sequencing methods (In conclusion, we have provided proof-of-concept evidence to support a new evolutionary epidemiologic framework that incorporates both within-host and between-host viral variation to determine fine-scale DENV transmissions. Our framework offers precision tracing of DENV at inter- and intra-household scales and leverages recent similar applications used for study of influenza and Ebola viruses (Additional information about precision tracing of household dengue spread using inter- and intra-host viral variation data, Kamphaeng Phet, Thailand.


The uterus is the organ for embryo implantation and fetal development. Most current models of the uterus are centred around capturing its function during later stages of pregnancy to increase the survival in pre-term births. However, in vitro models focusing on the uterine tissue itself would allow modelling of pathologies including endometriosis and uterine cancers, and open new avenues to investigate embryo implantation and human development. Motivated by these key questions, we discuss how stem cell-based uteri may be engineered from constituent cell parts, either as advanced self-organising cultures, or by controlled assembly through microfluidic and print-based technologies. Bergmann et al. discuss the construction of synthetic uteri to model the earliest stages of human embryogenesis and associated pathologies. They highlight the constituent components from which a synthetic uterus may be engineered, propose a modular approach to assembling synthetic uteri and discuss how these technologies can shed light on implantation failure and uterine pathologies. The goal of such stem cell-based uterus models is to engineer a defined, flexible, and scalable system to address fundamental questions of reproductive biology. Major topics include mechanisms and pathologies of embryo implantation, embryogenesis, crosstalk between the developing embryo and the mother, and pathologies of the female reproductive tract. The first trimester of pregnancy is a particularly dynamic and critical stage when the embryo organises the body plan before the foetal growth phase in the second and third trimesters. Although complex in vitro models capable of complete recapitulation of the structure and function of the uterus may be necessary for a clinical setting to increase survival chances of premature fetuses5, such models would lack the scalability and experimental flexibility required for drug discovery and genetic screening.The primary function of the uterus is to provide a suitable environment for the embryo to implant and gestate to full term. Recent single-cell transcriptional atlases of the uterus during the menstrual cycleIn this review, we discuss the construction of stem cell-based uterus models to illuminate the earliest stages of human embryogenesis and associated pathologies. We outline the developmental origin of the uterus and consider its anatomy, function, and diseases. Building on recent breakthroughs in organoid culture, we highlight the constituent components from which a stem cell-based uterus may be engineered and propose a modular approach to assembling uterine models. We discuss approaches based on self-organisation as well as controlled assembly, either through microfluidic or print-based methodologies and conclude how these technologies can be used to tackle questions about implantation failure and key pathologies of the uterus.6  ducts.In mammals, the uterus originates from the intermediate mesoderm which is positioned between the paraxial and lateral plate mesoderm after gastrulation6 Fig. , week 3.. 7 Fig. , week 5.12. Intermediate mesoderm‑derived coelomic epithelial cells invaginate to form the Müllerian (paramesonephric) ducts at CS14–17, which elongate caudally along the Wolffian ducts at CS18–23. In mouse, the Müllerian and Wolffian ducts express Lhx1 and Wnt signalling is required for Wolffian duct elongation14. Histological studies of human embryos suggest that this morphogenetic process is conserved15. At CS23, Wolffian and Müllerian ducts together form the bipotential genital tract16. In males, the Müllerian ducts degenerate and the Wolffian ducts persist to form male reproductive organs. In females, it is the Müllerian ducts that develop into the female reproductive tract while the Wolffian ducts degenerate6. In either case, sex determination is controlled by gene expression from the X and Y chromosomes18.Morphogenetic rearrangements at CS12–16 lead to the insertion of the Wolffian ducts into the cloaca, the precursor of the bladder16. At week 16, the developing human uterus starts to form glands in the endometrium. Endometrial glands gradually increase complexity and develop branches within the stroma until birth and will continue to develop postnatally until puberty11. This contrasts with mouse development where glands are formed exclusively after birth20. Nevertheless, both human and mouse endometrial gland formation requires WNT signalling22. Prenatal uterine development concludes with the formation of myometrium at week 22 and the uterus assumes its adult shape23. The final steps of human uterus development occur during puberty when the uterus further matures under the influence of sex steroid stimulation and initiates the menstrual cycle.Development of the uterus at week 8–9 commences with the fusion of the Müllerian ducts, which will undergo morphogenesis to form the uterus, fallopian tubes, cervix, and upper vaginal tract. Müllerian duct fusion in humans results in one central uterine cavity, in contrast to rodents, where Müllerian duct fusion is less extensive to allow the formation of two separated uterine hornsThe uterus is located within the pelvic area, between the bladder and the rectum. In humans, it is on average about 7 cm long and weighs around 60 g in the non-pregnant state (non-gravid), extending to up to five-fold in size during pregnancy (gravid). The uterine body contains a triangular uterine cavity with the isthmus leading to the cervix, which connects to the vaginal opening Fig. . The ute24. Most other mammalian species experience an oestrous cycle, in which the uterus undergoes remodelling throughout the cycle without shedding25. During human menstruation, only the endometrial functionalis layer facing the uterine cavity is shed while the basalis layer situated towards the myometrium remains unaffected by hormonal changes , musculature (uterine prolapse), and genetic malformations . There are around 9400 cases of endometrial cancers each year in the UK, with a 92% 5-year survival rate when diagnosed at the earliest stage compared to 15% when diagnosed at stage four55 which are likely to be compromised due to their carcinogenic origin. Consequently, we suggest that the construction of stem cell-based uteri should be based on either pluripotent stem cells (PSCs) or patient-specific primary cultures, depending on the research objective.The assembly of a stem cell-based uterus requires developmentally authentic cell lines as constituent parts Fig. . Previou57. However, these conditions were poorly defined, and the resulting cells were not fully characterised. Miyazaki et al. addressed this problem by differentiating human-induced PSCs (iPSCs) into endometrial stromal fibroblast-like cells in a 14-day embryoid-body-based approach in defined conditions58. The protocol closely follows in vivo endometrium development: iPSCs progress from primitive streak through intermediate mesoderm, coelomic epithelium and Müllerian duct-like stages. The first part of the differentiation is based on a protocol used to generate kidney progenitor cells59. Intermediate mesoderm-like cells express markers including LHX1 and PAX2. The fact that the intermediate mesoderm-like cells derived by this protocol can be used to make both kidney and endometrial-like cells, lends credibility to the developmental authenticity of the system. BMP inhibition prevents the acquisition of renal identity, while WNT activation is required for Müllerian duct and uterine gland development in vivo61. The resulting endometrial stromal fibroblast-like cells were hormone-responsive and resembled primary endometrial stromal cells as determined by RNA sequencing (RNA-seq). This protocol provides an encouraging starting point for iPSC-derived models of human endometrium.The differentiation of PSCs into the endometrial epithelium and stromal-like cells presents a promising avenue Fig. . The fir63. Purified endometrial cell cultures have been used to understand physiological and pathological processes, including trophoblast invasion64. However, endometrium-derived primary culture offers limited potential for use in in vitro models since they cannot be propagated in vitro for many passages and tend to differentiate63. Recent advances in organoid culture demonstrate that when epithelial cells from endometrial primary cultures are dissociated, separated from the stromal compartment, and seeded into a 3D-extracellular matrix scaffold, they are capable self-organisation into hollow spheres formed by a single layer of columnar epithelium69. These spheres are called endometrial organoids as they recapitulate several physiological characteristics of the endometrium, and RNA-seq shows organoids share many similarities with in vivo endometrial glands70. In contrast to two-dimensional primary cultures, endometrial organoids can be readily propagated, stay genetically and phenotypically stable, and retain the capacity to respond to hormonal stimuli71. Strikingly, when exposed to sex hormones, such as progesterone or oestrogen, organoids develop characteristics of early pregnancy and are able to recapitulate the menstrual cycle70. Single-cell profiling identified several sub-populations in endometrial organoids, including proliferating, secretory, ciliated, and putative stem cell populations70. A subset of cells harbours the potential to generate either secretory or ciliated epithelial cells, which is consistent with the proposition of a progenitor population in vivo66. Overall, endometrial organoids represent the first step towards 3D-modelling of the endometrium and offer a sustainable source of endometrial epithelial cells74.Primary cultures of human adult tissue represent an alternative approach for building a stem cell-based uterus. Endometrium biopsy-derived epithelial and stromal cultures can be maintained in vitro and respond to hormonal stimuli75. Such modularity is advantageous due to its flexibility, whilst the use of minimal component sets avoids unnecessary confounding factors that may obscure interpretation and confers a degree of scalability. Here, we focus on three key approaches: (i) self-organising cultures to model early implantation; (ii) controlled assembly approaches to model trophoblast invasion, placentation, and embryo maintenance, or pathologies of the uterus; (iii) scaffold-based approaches that may be used to model pathologies of the uterus, with potential clinical implications.Due to the broad range of topics relating to uterine pathologies and maternal–embryo interactions, a single endometrial culture system is unlikely to address all biological questions of interest. Instead, a modular approach is required, with stem cell-based uteri variously assembled from the minimal components necessary to answer key questions84. Similarly, the culture of embryos with endometrial stromal cells provides opportunities for investigating aspects of trophoblast invasion at the earliest stages89. Multi-layered co-cultures combining epithelial and stromal cells offer a more complete model of early implantation92 but are limited by the availability of uterine tissue and embryo samples. Whilst issues with the availability of maternal tissues may be mitigated by appropriate community resources, such as biobanks, embryos are likely to remain the key bottleneck to these applications.The co-culture of embryos alongside endometrial epithelial monolayers has long been suggested as a model for implantation and to gauge mechanisms of apposition and competence of blastocysts for maternal–embryo interactions93 represent a suitable advancement over monolayer cultures, allowing for more complete modelling of endometrial tissues including the characteristic glandular structure. Since changes in endometrial vasculature represent a phenotypic readout of implantation, the incorporation of vasculature within organoid models would provide an important improvement . It remains to be seen if such protocols can be adapted to allow survival and self-organisation of vascularised endometrial organoids.Endometrial organoidsent Fig. . Blood v102. Trophoblast stem cells are capable of self-renewal and differentiation into syncytiotrophoblast and trophoblastic vesicles104. The latter may provide suitable surrogate models for human blastocysts to overcome the ethical and practical limitations associated with human embryo research. Indeed, the culture of trophoblast spheroids alongside endometrial epithelial cells116 or stromal cells119 has already been demonstrated. A key step forward would therefore be the co-culture of vascularised endometrial organoids with trophoblast organoids . A combination of embryonic and extraembryonic tissues is likely to be required to faithfully recapitulate maternal–embryo interactions, rather than extraembryonic trophoblast tissues alone . The inclusion of immune components into organoid models would be an asset to delineate mechanisms of immune tolerance, albeit at the expense of increased complexity. Careful consideration should be given as to which components of the immune system to include in order to retain tractability of the organoid approaches. The abundance of uterine natural killer cells within the uterus during pregnancy may present a natural starting point. Due to the plasticity of components of the immune system care must be taken to minimise undesired transdifferentiation of immune components in culture. Fluorescence labelling and live-cell imaging will allow tracking of immune cell movements and, combined with single-cell profiling endpoint analysis, will provide candidate regulators for immunomodulatory functions of the uterus.Selective incorporation of components of the immune systemDecidualisation and placentation represent protracted biological processes that extend over large spatial scales. Trophoblast invasion, which is an important part of placentation, has been quantified using invasion assays. Traditional approaches include the culture of trophoblast cells in Matrigel-coated trans-well inserts with their invasiveness being monitored. These techniques are limited in scope and incapable of capturing the complex interplay between trophoblast and maternal tissues. Full modelling of trophoblast invasion, placentation, and pathologies such as placenta accreta, requires tissue constructs more akin to the maternal interface.141 including muscle142 and neural tissues143. These approaches are based on the layered deposition of appropriate gel-embedded cells or bio-inks by 3D printing technologies144. The increased spatial scale of these tissue constructs over organoids may require the integration of functional vasculature for oxygen and nutrient supply. Blood vasculature has already been induced in Matrigel and hyaluronic acid (HA)-based hydrogels146 and the deposition of epithelial-cell-laden hydrogel channels within printed constructs effectively allows the formation of vasculature150. Another exciting approach entails sacrificial networks, which are cell matrices with embedded dissolvable filaments. Upon the breakdown of these filaments, the created space can be seeded with endothelial cells to generate defined vasculature155. In the future, sacrificial networks may be routinely combined with print-based approaches156 (reviewed in refs. 157). A limitation of explicit engineering of vasculature is the decreased complexity of branching compared to natural systems. Alternative approaches include seeding of vascularised cell aggregates97 onto a basal scaffolding on which subsequent layers of stromal and epithelial cell-laden gels or organoid-containing gels are deposited , heart184, liver185, oesophagus186, trachea187, bladder188, liver189, and vagina190. Decellularisation of rat and pig uteri has been shown to preserve vasculature192, and allow for preliminary recellularisation using neonatal rat uterine cells and rat mesenchymal stem cells192 or mixtures of stromal and epithelial stem cells from human endometrial origin191. Studies in sheep have systematically compared different decellularisation approaches of uteri for their ability to support recellularisation of sheep PSCs189. The possibility of using animal scaffolds presents an opportunity for clinical settings (Fig. 193. Finally, the ability of donor uteri from deceased patients to sustain live births174 may open up the possibility of donor scaffolds. Together, these early studies suggest that repair or reconstruction of uteri using pathogen-free, patient-specific cells may represent a long-term treatment solution to some of the most severe uterine pathologies.Partial reconstruction of the uterus has been achieved using tissue grafts168 Fig. . This apngs Fig. . However194. Stem cell-based approaches to mimic the maternal environment synergise with recent advances in modelling embryogenesis124, and together will illuminate the critical stages of human periimplantation development. Modelling primate implantation with stem cell-based uteri is charged with biomedical potential and provides a unique opportunity for basic science. The development of multi-organoid systems and controlled structural assembly of uterus models should facilitate large-scale genetic and drug screening, enable cross-species comparative studies, and shed new light in the field of human embryogenesis that has previously been obscured. Stem cell-based uterus and embryo research will raise important ethical questions that will require careful consideration by the research community and beyond. Ultimately, advances in basic science go hand-in-hand with progress in clinical applications, and the next few years promise to see striking steps forward in the advancement of women’s reproductive health.Investigation of the earliest stages of implantation and maternal–embryo interactions with human embryos has long been limited by the 14-day rulePeer Review File


Immunotherapy has emerged as a promising strategy for cancer treatment, in which durable immune responses were generated in patients with malignant tumors. In the past decade, biomaterials have played vital roles as smart drug delivery systems for cancer immunotherapy to achieve both enhanced therapeutic benefits and reduced side effects. Hydrogels as one of the most biocompatible and versatile biomaterials have been widely applied in localized drug delivery systems due to their unique properties, such as loadable, implantable, injectable, degradable and stimulus responsible. Herein, we have briefly summarized the recent advances on hydrogel-by-design delivery systems including the design of hydrogels and their applications for delivering of immunomodulatory molecules , immune cells and environmental regulatory substances in cancer immunotherapy. We have also discussed the challenges and future perspectives of hydrogels in the development of cancer immunotherapy for precision medicine at the end. In the past decade, cancer immunotherapy has shifted the paradigm for the treatment of cancers that it activates patients’ own immune systems to attack and kill cancers. Till now, cancer immunotherapy includes five main classes, immune checkpoint blockade (ICB) therapy, lymphocyte promoting cytokine therapy, chimeric antigen receptor T-cell (CAR-T) therapy, agonistic antibodies and cancer vaccines . The sucin situ chemical polymerization render them implantable in the body with minimal surgical wounds and the in situ entrapment of therapeutic molecules or cells by simple syringe injection at target sites  to the backbone polymer. With facilitainge the interactions with cellular and extracellular biomolecules, the hydrogel can also carry immune cells for therapeutic purposes (Injectable hydrogels formed otherapy . Injectaotherapy . Such maotherapy . Firstly(GM-CSF) . Thirdlyin situ , whilst degradation can occur in the polymer backbone or at the crosslinks, and is typically mediated by hydrolysis or enzyme activity  which is grafted onto the platelet. When the hydrogel is injected into the tumor site, inflammation will initiate platelet activation and release aPD-L1, which achieves a controlled release  to construct a localized drug delivery system for combining chemotherapy and immunotherapy -hairpin DNA into dendrimer-dextran hydrogels to silence multidrug resistance protein-1 (MRP1). When the hairpin is opened by hybridizing with a complementary target (MRP1 mRNA), this interaction becomes weaker, allowing simultaneous release of the drug and knockout of the gene encoding the protein responsible for drug resistance  nanogel. After the mice were subcutaneously injected with murine IL-12-loaded CHP nanogel (CHP/rmIL-12), the concentration of serum rmIL-12 stays relatively high for 12–24 h ester-functionalized hyaluronic acid (HA-PCLA) that loaded with OVA expressing plasmid (pOVA) and GM-CSF to boost the maturation of dendritic cells (DCs). By the controlled release of pOVA and GM-CSF through a single subcutaneous injection, the hydrogel system effectively eliminated the B16/OVA melanoma tumors -specific aptamer. Once intratumoral injection, this hydrogel gelled in situ and released the CpG oligonucleotide, achieving remarkable synergistic tumor elimination as well as generating long-lasting immune memory to prevent recurrences (Chemotherapy and radiotherapy can induce immunogenic cell death (ICD), such antitumor immune response would be further amplified if there are concurrent immune adjuvants inside the tumor. Adjuvants are nonspecific immunostimulants, which enhance the body’s immune response when combined with antigens. For example, Hou function . Liu et urrences .et al. developed an anti-inflammatory nanofiber hydrogel self-assembled by steroid drugs for local delivery of (αPD-L1). Effective therapeutic effects were observed with inhibition both local tumors and abscopal tumors once injected with αPDL1-loaded hydrogel.Immunological checkpoints are a class of immunosuppressive molecules that can regulate the intensity and duration of immune responses, thereby avoiding damage of normal tissues. During the occurrence and development of tumors, immune checkpoints become one of the main regulators for immune tolerance . Immune et al. prepared a new type of injectable adhesive hydrogel based on thermosensitive nanogels containing catechol groups and loaded with MnO2 nanoparticles. After intratumoral injection, the concentrated nanogel dispersion transformed into an adhesive hydrogel in situ. The photothermal effect of the loaded MnO2 nanoparticles induces immunogenic cell death and releases a large amount of autologous tumor-derived protein antigens under near-infrared radiation. These antigens captured by the hydrogel are ideal immunostimulatory substances to avoid the consequences of tumor heterogeneity. They also recruit more dendritic cells to stimulate a strong and durable anti-tumor immune response mediated by CD8+ T cells  as antigens, Toll-like receptors 3 (TLR3) agonist, polyinosinic:polycytidylic acid [poly(I:C)] to construct the vaccines, which robustly recruit, activate and mature DCs in vitro and in vivo by sustained release of TCL and poly (I:C). The antigen persistence at the injection site and antigen drainage to lymph nodes were significantly improved by hydrogel  have been widely applied for the treatment of metastatic melanoma. Meanwhile remarkable research efforts have been focusd on adopative cell therapy (ACT), which heavily relies on the administration of allogeneic T cells . Furtheret al. used Pluronic F-127 as a NaHCO3 releasing carrier to focally alleviate extracellular tumor acidity  multiple imaging-guided injection techniques should be developed for deep-sited tumors administration; 2) The dose of immune drugs is also a vital parameter. For in vitro experiments, a drug dose is usually chosen based on the effectiveness of the drug. In clinical practice, however, a strict dose screening should be performed according to the drug release curve of the hydrogel. High concentration of anticancer agents can also cause severe acute injury to the healthy parenchyma due to the burst release. If the drug release from the hydrogel does not proceed at a uniform rate, it can also lead to increase the risk of drug resistance; 3) It will be feasible to construct the final system on the basis of approved clinical materials as it is also more likely to get clinical approval.In spite of the successful exploration of hydrogel drug delivery systems for cancer immunotherapy, several challenges still need to be considered for the future development and potential applications. Firstly, the controlled release of drug from hydrogels still remains as an issue since immunological drug are released by diffusion and hydrogel degradation, both of which are uncontrollable processes. Those hydrogels are responsive to stimulation can release immunological drugs, but the research in this area is only limited to via immunotherapy as well as other aspects for improving human health.In summary, the delivery systems based on hydrogels have exhibited excellent promises for cancer immunotherapy as the unique characteristics of hydrogels ensure locally and sustainably delivering immunotherapeutic agents to target tumor sites with reduced drug dose and side effects. However, challenges still exist, which requires experts from multidisciplinary fields work more closely. We believe this smart delivery system will continue contributing in tomour threatment


Different mechanisms have been proposed in lymphomatous involvement of the optic nerve. They include isolated optic nerve lymphoma, optic nerve lymphoma associated with primary central nervous system (CNS) lymphoma, or with systemic lymphoma. We present one case of non-Hodgkin lymphoma of the optic nerve in a Human Immunodeficiency Virus (HIV) patient and discuss the mechanism of metastasis, classification of optic nerve involvement with clinical and radiologic features as well as treatment options. Despite the uncommon nature of optic nerve infiltration by lymphoma, prompt evaluation should be considered in patients with a history of lymphoma and visual symptoms as delays in treatment can result in permanent vision loss. The recommended initial workup includes neuroimaging and cerebrospinal fluid evaluation. Treatment options are not standardized but include intravenous and intrathecal chemotherapy, corticosteroids, and radiation. Non-Hodgkin lymphoma (NHL) is the seventh most common cancer in both men and women. It is more common in the presence of advanced HIV infection and is almost always of B-cell origin. AIDS-related lymphomas include Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), AIDS-related primary central nervous system lymphoma (PCNSL), primary effusion lymphoma, plasmablastic lymphoma, Kaposi's sarcoma-associated herpesvirus (KSHV)-associated multicentric Castleman’s disease, and classic Hodgkin’s lymphoma. In the combination antiretroviral therapy (ART) era, the incidence of AIDS-related lymphoma has decreased; however, the incidence is still higher compared to patients without HIV/AIDS. HIV-associated DLBCL and PCNSL tend to present with severe immunosuppression and are often linked to Epstein-Barr virus (EBV) infection . DLBCL iA 51-year-old Hispanic male presented with left facial pain and edema and was ultimately diagnosed with Hodgkin lymphoma, nodular sclerosis type, by anterior and posterior left buccal mucosa biopsy. PET scan showed extensive fluorodeoxyglucose (FDG)-avid lymphadenopathy within the neck, chest, abdomen, and pelvis consistent with the known diagnosis. He completed one cycle of ABVD in December 2019. One month later, he presented with rapid enlargement of the left testicle. Due to the high standardized uptake values (SUV) of the solid hypoechoic left testicular mass, left-sided orchiectomy was performed with pathology revealing high grade diffuse large B-cell lymphoma germinal center type, c-Myc positive with positive fluorescence in situ hybridization (FISH) result for Myc rearrangement; suggestive of 14q or IGH rearrangement, negative for BCL6 rearrangement and IGH/BCL2 fusion, t, for which he completed six cycles of DA-EPOCH-R with intrathecal methotrexate for central nervous system (CNS) prophylaxis. At that time, a bone marrow biopsy showed a normocellular marrow with no abnormal lymphoid aggregates or granulomas. Immunostains for CD3, CD20, and CD138 as well as kappa and lambda in situ hybridization (ISH) and Epstein-Barr encoding region (EBER) ISH were performed and interpreted as negative for involvement by a large B-cell lymphoma or Hodgkin lymphoma. Verbal and written consent were obtained from the patient to discuss this case report.One month after completing six cycles of DA-EPOCH-R with intrathecal methotrexate for CNS prophylaxis, he presented to the emergency department with worsening painless right blurry vision which progressed to complete vision loss associated with periorbital headache. He had no previous ophthalmologic history. On initial examination, he had visual acuity to hand motion in the right eye and a relative afferent pupillary defect (RAPD). Dilated fundoscopic examination of the right eye showed optic disk edema and retinal pigment epithelium atrophy without hemorrhage, microaneurysms, and anterior vitreous detachment (AVD) in the right eye. Magnetic resonance imaging (MRI) of the orbits was suspicious for lymphomatous involvement of the right optic nerve including slight asymmetric thickening of the canalicular and orbital segments of the right optic nerve as well as asymmetric hyperintense short tau inversion recovery (STIR) signal and hyperenhancement involving the pre-chiasmatic and canalicular segments of the right optic nerve Figures , 2.MRI of the brain showed abnormal enhancement of the left temporal leptomeninges, pituitary infundibulum, and the dura along the left tentorium suggestive of meningeal carcinomatosis as well as abnormal enhancements in the ependyma of the lateral and third ventricles. Lumbar puncture was performed and yielded cerebrospinal fluid (CSF) with an abnormal population of B-cells with CD19, CD20, uniform CD10 expression with monoclonal kappa surface light chain immunoglobulin expression, consistent with CSF involvement by the patient’s known diffuse large B-cell lymphoma. CSF was negative for bacteria and fungi. A systemic infectious disease evaluation was negative.After MRI and CSF evaluation, the patient was started on corticosteroids and high-dose IV methotrexate with improvement in his right eye symptoms.Optic nerve involvement can be further classified into primary intraocular lymphoma, PCNSL, and optic nerve involvement with metastatic CNS disease.Primary intraocular lymphoma implies malignant lymphoid cells invading the retina, vitreous or optic nerve head with or without CNS involvement . It commPrimary CNS lymphoma (PCNSL) is an uncommon entity accounting for roughly <0.7% of all malignant lymphomas and <1% of intracranial tumors , howeverPatients with PCNSL develop neurologic signs over weeks and are not limited to focal neurological deficits, mental status changes, and behavioral changes but also intracranial hypertension syndrome with vomiting and papilledema on ophthalmoscopy . On imagOptic nerve involvement with CNS disease is a rare and aggressive disease. It presents with secondary CNS involvement in patients with systemic lymphoma. Malignant lymphoma cells may reach the CNS by hematogenous spread, direct invasion from contiguous structures, or extension along nerves. MRI with gadolinium contrast shows leptomeningeal, dural, subependymal, and cranial nerve involvement with thickening and T2 hyperintense enhancing portions which can be smooth, irregular, or nodular.Role of neuroimagingMRI is the preferred modality as it best characterizes the meningeal, cerebrospinal fluid, and axonal portions of the optic nerve. Typical findings include enlargement of the optic nerve, enhancement of the nerve sheath (MRI with gadolinium contrast), or tram-track sign  . These fLumbar puncture, CSF analysis, and optic nerve biopsyCSF analysis is pivotal in the diagnosis of lymphomatous involvement of the optic nerve. It is unclear how many lumbar punctures are necessary in patients who are suspected to have developed CNS lymphoma; however, it is often enough for diagnosis and precludes the need for an optic nerve biopsy. Routine CSF studies include cell counts, protein, glucose, and lactate dehydrogenase (LDH). Glucose is often very low in patients with CNS lymphoma. Cytology, when combined with flow cytometry, is the gold standard for diagnosis. Flow cytometry is also important as it assists with the prognosis and identification of patients who might develop drug resistance . Flow cyOptic nerve biopsy is reserved for patients who have progressive and significant vision loss despite appropriate empiric treatment or when lumbar puncture and CSF studies are indeterminate for diagnosis of CNS lymphoma and no etiology is identified for the optic neuropathy. Routine optic nerve biopsy is not necessary in most cases and it should be done by a specialist as it carries significant risk to patients who maintain visual acuity regardless of lymphomatous involvement of the optic nerve . MethodsTreatmentAlthough there is no consensus in treatment, most patients are treated with high-dose IV methotrexate, intrathecal methotrexate, intravenous corticosteroids, and radiation therapy. In patients with AIDS-related lymphoma, combination ART is warranted as higher CD4 and lower international prognosis index (IPI) have been independently associated with improved overall survival. Rituximab use is controversial in HIV-DLBCL patients. Even though rituximab has not been associated with a high toxic-death rate, it has also not been associated with improved overall survival  10], ex, ex10], Patients with history of NHL presenting with optic neuritis should have evaluation for lymphomatous infiltration of the optic nerve. When signs and symptoms are concerning for optic nerve lymphoma, CSF evaluation with cytology and flow cytometry and neuroimaging including MRI with contrast are warranted. HIV is associated with increased risk of developing DLBCL. Our case supports hematogenous dissemination in patients with systemic NHL. Optic nerve biopsy is not routinely indicated except in patients who have failed empiric therapy or those with high index of suspicion for optic nerve lymphoma in the presence of negative diagnostic studies.


The ‘Developmental Origins of Health and Diseases’ hypothesis posits that prenatal maternal diet influences offspring growth and later life health outcomes. Dietary assessment has focused on selected nutrients. However, this approach does not consider the complex interactions between foods and nutrients. To provide a more comprehensive approach to public health, dietary indices have been developed to assess dietary quality, dietary inflammation and risk factors for non-communicable diseases. Thus far, their use in the context of placental development is limited and associations with offspring outcomes have been inconsistent. Although epidemiological studies have focused on the role of maternal diet on foetal programming, the underlying mechanisms are still poorly understood. Some evidence suggests these associations may be driven by placental and epigenetic changes. In this narrative review, we examine the current literature regarding relationships between key validated diet quality scores  in pregnancy and birth and long-term offspring outcomes. We summarise findings, discuss potential underlying placental and epigenetic mechanisms, in particular DNA methylation, and highlight the need for further research and public health strategies that incorporate diet quality and epigenetics. The importance of adequate nutrition during foetal life for long-term health is well documented . The ‘DeAdequate maternal nutritional intake is an essential factor for a healthy pregnancy and optimal child development. However, dietary assessment is difficult to elucidate due to its complex interactions between diverse foods and nutrients . To provAlthough epidemiological studies have focused on the role of maternal diet on foetal programming, underlying mechanisms are still poorly understood. Perturbations in the maternal environment may be transmitted across the placenta and thereby affect the development of the foetus. The placenta plays a fundamental role in maternal-foetal exchanges during pregnancy and an adequate maternal nutritional status is necessary to support placental homeostasis and development . AlteredSome evidence has shown that these associations may be also driven by epigenetic changes . EpigeneThus, the aim of this narrative review is to examine and synthesize the literature regarding associations between maternal dietary quality, evaluated by several dietary scores, and dietary inflammation state with birth outcomes and long-term offspring health. We examine the underlying mechanisms including the placental dysfunction, which in turns impacts foetal growth, and the epigenetic mechanisms, more specifically DNA methylation, and conclude with future perspectives in this area of nutritional researchThis is a narrative review of the current literature on the relationships between maternal dietary quality and dietary inflammation status and, birth and child outcomes, placental development and epigenetics. The literature search was performed in Pubmed, Web of Science, Scopus and the Cochrane database by using the following keywords separately or in combination: Maternal diet, nutrition, dietary inflammatory index, dietary quality scores , macronutrients, micronutrients, pregnancy, periconception period, foetal programming, child development, birth anthropometry, obesity, body mass index, placenta, epigenetic mechanisms, DNA methylation and cord blood. Relevant free-access abstracts were identified and peer-reviewed human studies, meta-analyses, systematic reviews and interventional studies were included. Other inclusion criteria were: (1) English language; (2) recording of the diet before or during pregnancy; (3) assessment of offspring measurements and prematurity; and (4) published papers within the last 15 years. Additionally, we included animal studies in our literature search in relation to maternal diet associations with placental outcomes and epigenetics. The websites of key government departments and agencies were consulted to identify important recommendations and reports such as the “Dietary Guidelines for Americans 2010”.®), was developed to characterise the inflammatory potential of the diet [Optimisation of maternal diet during critical windows of development may positively impact on childhood weight status and future health trajectory ,20. Howethe diet ,29,30,31Macronutrients are broadly defined as nutrients that provide calories or energy and are required in large amounts to maintain body functions and carry out the activities of daily life ,33. Duri12 and iodine were below national guidelines, thus highlighting that multiple micronutrient deficiencies can still be prevalent in developed countries and targeted supplementation may be required in certain groups [Micronutrients are vitamins and minerals needed by the body in very small amounts to enable production of enzymes, hormones and other substances needed for normal growth and development ,42. Globn groups ,45. For n groups .Folate is a key micronutrient for early stages of placental development  and the 12, B2, and B6; betaine; choline; and methionine implicated in one-carbon metabolism (OCM) have been found to change DNA methylation in foetal tissues and in the placenta [Additionally, certain micronutrients are thought to play a role in epigenetic modification pathways including DNA methylation, thus establishing links between early nutrition programming and long-term health trajectories and disease risk ,48. Folaplacenta ,48,52. Fplacenta ,22. Thusplacenta ,22,41,53An overview of dietary indices commonly used in the general population is presented in First validated in 2009 (Generation 1)  and lateSeveral dietary metrics based on the MD have been created including the Mediterranean Diet Scale  developed by Trichopoulou et al., Mediterranean Food Pattern (MFP), the MD Score (MDS), Mediterranean Lifestyle (MEDLIFE) index and the MedDiet score ,55. The Originally developed in 1995, the HEI evaluated adherence to key dietary recommendations from the Dietary Guidelines for Americans . The oriCompared to the HEI, the AHEI-2010 incorporates foods, nutrients, dietary recommendations predictive of chronic disease risk  and mortality . It consThe DASH score was developed for the therapeutic reduction in blood pressure based on dietary patterns and food/nutrient components from the DASH Trial . It is pMany of the aforementioned dietary scores have been modified and adapted for characterising maternal diet quality during pregnancy. Mediterranean diet scores such as Trichopolou’s MDS and Khoury’s score have often been modified in observational studies for pregnancy to provide the most useful information in terms of conceptual suitability and applicability . For exaIn summary, these specific dietary metrics all share certain characteristics such as a diet high in vegetables, legumes and fruits, indicative of better diet quality and sustained habits, and were associated with one or more important maternal health outcomes such as pre-eclampsia, GDM, altered blood glucose metabolism and postpartum obesity ,81,82,83As mentioned above, studies investigating maternal diet have mainly focused on adequate intake of selected macronutrients and micronutrients during pregnancy. In recent years, there has been growing interest in examining associations between overall maternal diet quality (by using the previously described scores) and child development. Here we summarise the literature regarding the relationship between maternal dietary quality and dietary inflammatory potential with birth outcomes related to growth and prematurity.n = 1078 mother–neonate pairs) reported that higher DII scores in obese mothers during pregnancy were associated with increased neonatal adiposity [n = 1057 mother–child pairs) did not report associations with birth anthropometry but an increased risk of prematurity among female offspring was observed [z score in infants born to obese mothers [Chronic inflammation during pregnancy that is observed in obese women may alter physiological mechanisms of the developing foetus. Obesity-induced inflammation is characterised by abnormal cytokine and adipokine levels . Some stdiposity , while tobserved . Conversobserved . Additio mothers . There a mothers . A higheSome studies have examined the potential benefits of the Mediterranean diet on foetal development, considering the MD as a whole rather than focusing on the effect of its individual components. Foetal growth restriction (FGR) and SGA were the main outcomes examined. In a prospective observational study of 3207 Caucasian pregnant mothers in Rotterdam, low adherence to a MD in early pregnancy was associated with decreased intra-uterine size with a lower placental and a lower birth weight . InverseOther studies reported associations between maternal MD and prematurity. In a study from the French West Indies, where dietary patterns present several characteristics of the MD diet, no overall associations were found between MD adherence and birth weight. However, there was a decreased risk of prematurity associated with greater MD in overweight and obese women . In the To conclude, two recent systematic reviews of literature reported growing evidence of the potential benefits of the MD diet for child development, but other randomized trials are needed to confirm these results ,98. MoreSeveral studies have examined birth outcome associations with these dietary indices see . In a SpA systematic review and meta-analysis of RCTs provides evidence that the DASH diet could play a role in glycaemic control and improve gestational outcomes in women with cardiometabolic disorders such as GDM, hypertension or obesity . Among tA recent meta-analysis of 11 randomized trials including 1985 women has shown that compared with control diets, a low GI diet significantly reduced fasting and 2-h postprandial blood glucose levels and proportion of LGA infants . HoweverIn line with the DOHaD hypothesis, maternal diet associations with offspring health may not be restricted to birth and may have longer term implications. Here we summarise the literature regarding the relationship between maternal dietary quality metrics and dietary inflammatory potential with offspring outcomes related to childhood growth and obesity. A summary of the studies and their key findings is presented in Data regarding long-term associations of maternal dietary inflammation with offspring anthropometric outcomes are scarce. Sen et al. reported positive associations between maternal DII (measured during the first and second trimesters) and offspring anthropometric measures in mid-childhood (between ages 6 and 10), which included BMI z-score and waist circumference as well as more direct measures of adiposity such as fat mass index, fat-free mass index (FFMI), trunk fat mass index and fasting insulin . The E-DChatzi et al. studied anthropometric outcomes in 997 mother–child pairs in the American Project Viva study in conjunction with 569 mother–child pairs from the Greek Rhea study. They found that maternal adherence to the MD during pregnancy was associated with lower child adiposity, leptin and blood pressure levels . It is iThe analysis of results from the Growing Up Today Study II across follow-up ages of 12–23 years indicate that greater maternal adherence to the Alternate Mediterranean Diet (aMED) score (adapted to the US population) showed similar findings with a lower risk of offspring overweight or obesity . HoweverTahir et al. report that higher HEI-2015 scores during the third trimester of pregnancy were associated with lower infant body fat percentage at six months . Howeverz-score >85th percentile) as well as a lower late-childhood fat mass index (FMI) [early- and mid-childhood OWOB (mean age range 2.8–6.1 years) [Despite its importance in controlling blood pressure and placental vascular flow, research regarding maternal DASH score associations with offspring adiposity and childhood obesity are scarce. Strohmaier et al. report an inverse association between peripregnancy DASH scores and offspring overweight risk at 12–23 years; however, this association was no longer observed after adjusting for maternal BMI and lifestyle factors before pregnancy . Recent ex (FMI) . Interes1 years) .z-score in the unadjusted analysis. However, this association was no longer present after adjusting for previously mentioned confounders, particularly maternal pre-pregnancy BMI [Results from the Southampton Women’s Survey of 907 mother–child dyads identified associations between higher maternal GI and GL with greater offspring adiposity (body-fat mass) at 4 and 6 years . These rancy BMI . Similarancy BMI . LimitedThe placenta plays a fundamental role in the various maternal-foetal exchanges during pregnancy and has endocrine functions that are essential for the maintenance of the pregnancy as well as for the development of the foetus . TogetheDifferent markers of altered placental function reflected by weight, morphology, vascular development and transport function have been described. Markers of modifications in placental transport capacity include alterations of exchange surface area, barrier thickness and cell components of the different gestational ages of the placenta. These histopathological changes predispose to reduced nutrient transfer to the foetus. Additionally, vasculogenesis and angiogenesis are both critical processes to the maternal-foetal exchange. Vasculogenesis is de novo formation of blood vessels in the developing embryo while angiogenesis corresponds to new vessels from pre-existing vessels . The foeAlterations in maternal nutritional status, such as undernutrition, have been described as impairing placental development and function, including placental weight, its function in transporting various nutrients, as well as its vascular development (de novo vessel formation) and the process of angiogenesis . The patStarting with animal studies, some researchers have looked at the associations between high-fat diet or micronutrient and placental development . Feeding2, B6 and B12) also have antioxidant activity, thus playing an important role in early stages of placental development, including trophoblast invasion and angiogenesis [12 deficiency in rats altered placental phospholipid ratios, underscoring the potential of micronutrients in influencing placental development and composition [Other studies also evaluated the effect of specific micronutrients on placental development and inflammation. Reduced placental weight has been reported in severe iron deficiency in rats . This isogenesis ,133. Morogenesis . Folate ogenesis ,47. Thisogenesis . Furtherposition .Maternal nutritional status during pregnancy plays a major role in foetal development, and accumulating evidence suggests that maternal hyperglycaemia may have adverse effects on embryonic and placental development . ObservaExcessive omega-6 fatty acid intake and insufficient omega-3 fatty acid intake are two key components that adversely affect foetal programming . An Icel12 in in vitro/in vivo models of deficiency have also all demonstrated pro-inflammatory effects in the placenta including upregulation of pro-inflammatory cytokines [Other studies have evaluated the effect of micronutrients on placental development. In a prospective study of 1650 pregnancies, low iron status in early pregnancy was inversely related to placental size . The synytokines .However, research on maternal dietary score associations with placental development is scarce. In the Generation R cohort, women with low adherence to a Mediterranean diet pattern during early pregnancy had smaller placentas and also tended towards higher uteroplacental vascular resistance compared to those with high adherence to the MD pattern. However, it is important to note that an established, validated MD score was not used in this study . Recent In 1942, Conrad H Waddington defined epigenetics as “the branch of biology that studies the causal interactions between genes and their products which bring the phenotype into being” . EpigeneEpigenetic modifications have been proposed as mechanisms by which a suboptimal nutrition state could contribute to the “developmental plasticity of the individual”. Early-life exposures have been described to be recorded in the “cellular memory” and having the potential to influence cellular functions with long-term effects ,156. The12, choline and betaine) was associated with hypermethylation of the agouti gene [The first evidence is based on findings from animal studies. Murine models have provided evidence about how early nutritional exposure is involved in epigenetic mechanisms, programming of the individual and its phenotype . A classuti gene .Recent human data are becoming available regarding the associations between maternal diet and DNA methylation. It has been described that some micronutrients are implicated in the one-carbon metabolism pathway, which supports multiple physiological processes, essential for human development including DNA methylation ,161. The2, B6, and B12) [12 and methylation of the imprinted genes insulin-like growth factor 2 (IGF2) in maternal and cord blood. IGF2 is a key factor in human growth and development. The methylation patterns of IGF2 in promoter 3 of cord blood samples were negatively associated with serum levels of vitamin B12 only in maternal blood [IGF2) and transposable elements such as long interspersed nuclear element 1 (LINE-1) [The Pune Maternal Nutrition Study cohort in India have provided evidence of the importance of the one-carbon metabolism in foetal programming . OCM nutand B12) . Some stand B12) . In Gamband B12) ,164. Metand B12) . Modificand B12) ,165. Anoal blood . Other r(LINE-1) ,167. One(LINE-1) . However(LINE-1) .In recent years, some interventional studies have focused on the impact of GI dietary intervention in mothers during pregnancy and evaluated their relation to DNA methylation in the progeny. Exposure to adverse conditions during pregnancy like GDM is increasing and has been largely shown to affect foetal development . New eviPOMC) promoter. The POMC gene is described as a key factor in the control of energy balance [Several animal studies have reported that maternal overnutrition or dietary restriction were associated with direct consequences on foetal development. Some investigations conducted in murine models reported that maternal overnutrition programs long-term epigenetic modifications ,175,176. balance .IGF2 in the adulthood [TAPBP) gene were associated with BMI z-score and systolic blood pressure (BP) percentile in female and systolic and diastolic BP percentile in male offspring at 5 years [Epidemiological studies conducted in humans have also linked the impact of maternal under- and over-nutrition during critical periods of development with the child development . One of dulthood . These rdulthood . It has  5 years . Recent  5 years . However 5 years . A meta- 5 years .While more research concerning maternal obesity and individual micronutrient intake and offspring epigenetics has been conducted, far fewer studies have examined dietary patterns or specific types of maternal diets, which yet reflects dietary quality and food interactions.MEG3-IG DMR in cord blood leukocyte among girls, in response to lower maternal adherence to a MD pattern in early pregnancy [MEG3, IGF2, PLAGL1 andSGCE/PEG10 in cord blood samples [The MD pattern has also been associated with higher intake of specific nutrients such as folates that have been shown to influence DNA methylation . Howeverregnancy . One oth samples . Genomic samples . Imprint samples . There a samples . Furthern = 338) and circulating cytokines (n = 105) [MEG3 imprinted gene [Inflammation has recently been proposed as another pathway by which maternal diet could affect the epigenome . The horn = 105) . Althougted gene . DespiteIn summary, it has been largely shown that specific micronutrients are involved in the DNA methylation process; however, further investigations regarding the potential role of maternal dietary quality or inflammation are necessary.In this review, we examined and summarised findings from a growing body of research investigating associations between maternal dietary inflammatory potential and dietary quality, determined by various scores and foetal development, childhood adiposity, placental measures and epigenetics. The main results suggest that diets of the highest quality, as assessed by the HEI, AHEI, MD, DASH and GI/GL scores, during pregnancy are beneficial for a wide range of maternal and offspring outcomes. However, findings in human studies remain inconsistent.The evidence regarding DII and E-DII scores highlight the importance of having a less pro-inflammatory diet in pregnant women for improving birth outcomes and reducing the risk of prematurity. The findings in relation to dietary quality associations with long-term child development and placenta development are either conflicting or limited. Finally, few studies investigating relationships between dietary scores and DNA methylation exist. Thus, future research investigating the potential influence of dietary inflammatory status and dietary quality on these outcomes is warranted.The observed heterogeneity of study populations, ethnicity, differences in dietary assessment methods, timing of dietary assessment and outcome measures may contribute to the observed inconsistent results. Most studies are observational in nature, thus causality cannot be established. However, it has been shown that low-GI dietary intervention may lead to decreased risk of adverse neonatal outcomes . These oMost of the research to date regarding early life nutritional programming has focused on the mother but emerging evidence suggests that paternal nutritional status may also have an effect on child development, independently of maternal diet ,104. In In conclusion, improving our understanding of parental nutritional programming may help inform the development of more effective public health messages, diet and lifestyle interventions and pregnancy guidelines to promote intergenerational health and longevity. Several studies have shown that dietary quality and dietary inflammation state during pregnancy play a role in child development, even if results from some studies remain inconsistent or limited. Strategies aiming to promote adherence to a less pro-inflammatory and better-quality diet across all stages of maternal and childhood life may be of considerable importance to public health.