Filamentous fungi are highly productive cell factories, many of which are industrial producers of enzymes, organic acids, and secondary metabolites. The increasing number of sequenced fungal genomes revealed a vast and unexplored biosynthetic potential in the form of transcriptionally silent secondary metabolite biosynthetic gene clusters (BGCs). Various strategies have been carried out to explore and mine this untapped source of bioactive molecules, and with the advent of synthetic biology, novel applications, and tools have been developed for filamentous fungi. Here we summarize approaches aiming for the expression of endogenous or exogenous natural product BGCs, including synthetic transcription factors, assembly of artificial transcription units, gene cluster refactoring, fungal shuttle vectors, and platform strains. Aspergillus, Penicillium, Trichoderma, Fusarium, and Neurospora species) are highly efficient cell factories, often used industrially for the production of a diverse range of products such as proteins, enzymes, organic acids, and SMs , cholesterol-lowering (lovastatin), immunosuppressant (cyclosporine), anticancer (paclitaxel), and food additive (carmine) compounds (Secondary metabolites (SM), commonly referred to as natural products, are chemical substances that are produced by living organisms, often bearing distinctive pharmacological effects. The exploitation of microorganisms for generating these valuable products for our societies in an economical manner has a great history. Notably, the use of filamentous fungi in industrial biotechnology is well established. With the introduction of synthetic biology, new tools and alternative methods are provided to further aid the metabolic engineering and exploitation of fungal workhorses. Filamentous fungi  and in the protection against harsh environments (pigments and iron-chelating siderophores), but SMs are also used for chemical signaling . AlthougThe core skeleton of fungal SMs is produced by dedicated biosynthetic enzymes that belong to a few distinct families: nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), terpene synthases (TPS), dimethylallyltryptophan synthases (DMATS), or combinations thereof . NRPS anvia a transesterification reaction. The activated substrates/intermediates are then transported to the C domain, which is responsible for the formation of the peptide bond. Eventually, the final product is released by a terminal thioesterase domain , a β-ketoacyl synthase (KS), and an acyltransferase domain (AT). The AT domain selects and loads both starter and extender monomers, while the KS domain catalyzes C-C bond formation between two adjacent substrates/intermediates. The ACP domain is responsible for storing and shuttling monomer substrates and synthesized intermediates during the elongation process. PKS enzymes are extremely diverse, many contain optional domains that introduce further chemical modifications, generating an incredible variety of products .Molecules can be also constructed from hybrid NRPS-PKS assembly lines leading to mixed NRP-PK products, such as the bacterial bleomycin, rapamycin, epothilones, or the fungal fusarin C, pseurotin A, tenellin, and cytochalasin E. . NRP-PK de novo detection algorithm for SM BGCs) aim to combine genomic data and transcriptomic data to predict coordinately regulated genes including fungal BGCs   or the “gal BGCs . As homogal BGCs .SM production is often regulated by a stimulus, and without it, the product of the BGC is not synthesized. When no known metabolite is connected to a BGC, the cluster is called “cryptic” or “orphan.” In most cases, BGCs react to various environmental stimuli, but often the connection between regulators and the stimuli is unknown. Under laboratory conditions, native environmental signals may not be present, rendering BGCs transcriptionally silent. Since cryptic BGCs appear to be silent under laboratory conditions , alternaAspergillus ochraceus is capable of producing 15 compounds in addition to the previously known aspinonene  in A. nidulans resulted in increased penicillin and sterigmatocystin production  caused upregulation of the in-cluster inpA and inpB NRPS genes, as well as the asperfuranone BGC, located on a different chromosome    results in transcriptional suppression, while deletion of sorR2 results in early-stage transcriptional activation of the sorbicillin BGC, but with hardly any sorbicillin production  . Similarar/GaaX) . It is bar/GaaX) . The sor pathway . Overexpoduction . AlthougAspergillus and Penicillium species (0.1%–5.0%) (ku70/ku80 genes encode a protein complex functioning in the non-homologous end-joining (NHEJ) DNA repair pathway, which favors random integration of the transformed donor DNA. Deletion of either of these genes is highly advantageous for fungal strains that are employed for precise genome editing and HR-mediated DNA delivery. Inactivation of either of the fungal homolog of the human ku70/ku80 genes drastically decreases or eliminates the functionality of the NHEJ DNA repair pathway and highly increases the efficiency of targeted DNA delivery through HR  and high rate of random integration of the transformed DNA. Targeting efficiency to the desired location is relatively low in 1%–5.0%) , and it rough HR . With through HR .Agrobacterium-mediated transformation, particularly employed when little or no genetic tools are available for that host. A. tumefacians is a gram-negative plant pathogen that has been shown to be capable of transferring its T-DNA  into the genome of several filamentous fungi. This made it possible to achieve successful DNA delivery in fungal hosts that cannot be transformed with traditional methods (via homologous recombination using long homologous flanking sequences (>1000 bp)  .Xanthomonas. The DBD builds up from 33 to 34 amino acid long tandem repeats, which determines the targeted DNA sequence. These repeats can be altered to recognize one specific nucleotide and by combining these repeats in sequential order, the protein can be targeted to any DNA sequence (preceded by a thymine or cytosine base). Direct fusion of the TALE DBD and with restriction endonuclease (FokI) domain created guidable TALENs, meanwhile fusions to transcription activation domains (ADs) to created STFs for targeted transcriptional regulation (TALE-TFs)  and transcription activator-like effector (TALE) nucleases were established for locus-specific genome engineering applications. Engineering the DNA binding domain (DBD) of these nucleases allows targeting specific genomic loci. ZFNs are artificial restriction enzymes and are typically generated by fusing DBSs with the FokI nuclease domain. DNA targeting is provided by fusing together three to six DNA-binding zinc-finger proteins, each of which is capable to recognize a specific 3 bp DNA sequence. Although ZFNs are relatively small proteins, which are easy to deliver to the host, their targeting efficiency is rather weak and the relatively high levels of off-target effects may lead to cytotoxicity . The nexALE-TFs) .The CRISPR systems and their CRISPR-associated (Cas) proteins have recently been repurposed for transcriptional gene regulation in eukaryotes , where tStreptococcus pyogenes Cas9 (SpCas9) is 5′-NGG-3′  located downstream the protospacer, meanwhile the Cas12a nuclease recognizes 5′-TTTV-3′  sequences located upstream of a typically 20–24 nucleotide long protospacer , that must be followed by a protospacer adjacent motif (PAM). This PAM sequence is recognized at the DNA level by the protein and is unique for different Cas proteins. These unique PAM sequences limit the number of sequences that one can target since they show minimal flexibility for different nucleotides: For example, the PAM sequence recognized by the commonly used tospacer . Carefultospacer , which rA. fumigatus, A. oryzae, Neurospora crassa, Pyricularia oryzae, Trichoderma reesei, Ustilago maydis, and P. rubens  sequence from A. nidulans supports autonomous vector replication in several filamentous fungal species  can be used to enable fine-tuning of gene expression or controlling entire pathways. Synthetic biology-based tools have been established in several model bacterial and eukaryotic systems, and recently also in filamentous fungi where they are still relatively underdeveloped compared to more common hosts.The Design-Build-Test-Learn (DBTL) cycle of synthetic biology represents a systematic and efficient workflow for the optimization of biological systems for speciffic functionalities . Complex genetic systems can be constructed in a modular manner with desired features–synthetic regulatory tools and rewired expression of biosynthetic pathways–enabling an affordable genetic engineering of biological systems. Synthetic transcription units can be rapidly assembled by cloning methods supporting the assembly of multiple DNA fragments . Inducible promoters are found in a similar manner, but employing a well-defined chemical  as a potential inducer that can be added in various amounts to repress or enhance gene expression levels  in various filamentous fungi like A. nidulans  from Psilocybe cubensis in A. nidulans  organisms, and therefore they are suitable for the industrial production of compounds destined to be used in humans. The most commonly employed hosts for the characterization of heterologous BGCs are A. nidulans and A. oryzae  that renders NHEJ DNA repair less favorable, facilitating precise genomic integration of heterologous genes via HR. A1145 has been successfully used to elucidate a diverse range of biosynthetic pathways belonging to all major classes of SMs  and three constitutive cassettes. There are four versions of these vectors, each carrying a different selection marker, that can be co-transformed to allow the overexpression of up to 16 heterologous genes in the recipient NSAR1 strain . NSlPD1 was successfully engineered to generate a strain that produces higher titers of kojic acid and uses cellulose as starting material  and is therefore suitable for HR-mediated genetic engineering. These results highlight the P. rubens 4xKO strain as a valuable option for SM research.Another important fungal workhorse for industrial applications is industry . The CSIntations . This idntations . As prooB, and C . This stT. reesei. For decades, this organism has been used in industry for its astonishing ability to produce cellulolytic enzymes such as cellulases and hemicellulases (tmus53 (ligD homolog) and pyrG was engineered , which encodes a reporter gene for white/blue selection placed under the control of a constitutive promoter. Additionally, the COSI contains two 500 bp sequences flanking the reporter cassette that can be used for HR-mediated target gene replacement, whereby GOIs can be easily inserted and overexpressed. Since the COSI is equal in each recipient strain, only one integration cassette has to be designed and built. As a proof of concept, the DIVERSIFY platform has been successfully used to overexpress a fluorescent reporter (mRFP) and cellobiohydrolase, and for the production of 6-MSA, a model polyketide (Undoubtedly, the fungal platforms discussed above provide a rich choice for researchers who want to investigate unknown biosynthetic pathways or produce industrially relevant metabolites. Nevertheless, despite the broad availability of hosts, not all species might be capable to produce the desired natural product, and even when they are, it is very likely that one species performs better than another in terms of yield. This is difficult to predict and engineering more species at once to optimize production can be extremely time-consuming as well as costly. To reduce the workload and facilitate simultaneous cloning and screening of more hosts, a group of researchers recently developed the first multispecies fungal platform for heterologous gene expression . The firlyketide . BecauseClearly, filamentous fungi are powerful instruments for the elucidation of biosynthetic pathways and the production of SMs. In many cases, though, the desired compounds are produced at very low yields which are unsuitable for commercial applications. The development of a great array of hosts, each with specific benefits, as well as multispecies platforms that allow fast and simultaneous screening of several fungi with reduced workloads, will offer researchers the tools to readily optimize the production yield of metabolites of interest. Ultimately, this is necessary to engineer efficient fungal cell factories that are ready to be employed at an industrial scale.Thanks to the rapidly expanding number of filamentous fungal genome sequences and the advanced bioinformatics tools that are now available, it has become obvious that filamentous fungi represent an untapped reservoir of natural products. Each genome contains a high number of BGCs for which the product has not been identified, and many of these BGCs are transcriptionally silent under laboratory conditions. The products of these clusters can be unearthed with the combined efforts of bioinformatics, chemistry, and synthetic biology, to reveal new chemistries and biological activities of interest. A major challenge, however, remains prioritizing these BGCs for their potential value since the bioinformatics tools available at the moment cannot reliably predict the resulting products. Hence, much relies on laborious empirical testing, whereby many BGCs have to be expressed to screen for bioactive compounds. Here, we have discussed conventional tools and the development of new synthetic biology tools that aid in the transcriptional activation of silent BGCs in filamentous fungi, therefore offering new approaches for compound discovery .via homologous recombination or advanced synthetic cloning methods. The only approach that ensures transcriptional activation of an entire BGC is promoter replacement of all its genes, but this is generally a laborious and cumbersome task. Furthermore, the number of established strong fungal promoters and fungal selection markers is still limited, and this represents a bottleneck towards rapid or consecutive genomic modifications. An alternative to serial replacement of promoters could be the utilization of promoters from BGCs which are transcriptionally active in the selected host, or synthetic promoters containing regulatory elements for orthogonal regulatory systems such as STFs. The more recent CRISPRa systems further increase the number of available activator tools adding a new layer of control, since such systems do not require BGC refactoring anymore.Modulating global regulatory systems does not require prior knowledge regarding the cluster-specific regulation mechanism of a given BGCs, and although numerous genes can be affected when using this approach, transcriptional activation of BGCs of interest and subsequent production of new metabolites are not guaranteed. Instead, overriding the native regulatory system of the cluster can result in a more direct transcriptional activation. Such approaches involve partial or complete BGC refactoring, and require a deep level of understanding about the number of genes in the BGC, and/or its cluster specific regulators. Refactoring approaches can take place within a host which supports the assembly of numerous DNA fragments, often achieved Since genetic manipulation and precise engineering of a non-model or wild type fungal strains is challenging—mainly due to the low rate of HR and difficulties to grow these organisms in laboratory conditions—the most versatile method is to express BGCs in an established heterologous host. Ideal host strains are those in which it is easy to perform genetic manipulation, that are convenient to cultivate at different scales, for which compatible genetic tools are available, and convenient downstream processing steps have been established such as rapid compound screening and a clean metabolite spectrum. A combination of advanced transcriptional activation tools and established expression hosts can ensure reliable, targeted transcriptional activation and efficient methods for compound identification.Although new host strains and tools are continuously being developed for the characterization of cryptic BGCs in filamentous fungi, high-throughput BGC screening remains a major challenge. Automatized engineering of protoplasted filamentous fungi using microtiter plates and robotic liquid handling robots have been successfully established , as wellIn the future, the combination of emerging genetic tools, tailored heterologous hosts with high metabolic capacity, and automated systems, will facilitate the development of highly efficient, targeted, multiplexing-compatible transcriptional activation applications for novel natural product discovery. This, coupled with the development of bioinformatics tools that are able to prioritize the most valuable BGCs within genomic sequences, will revolutionize the field and eliminate time-consuming and costly wet lab procedures, while starkly increasing the chances of identifying novel and potent bioactive compounds.


Posttraumatic headache (PTH) attributed to traumatic brain injury (TBI) is a secondary headache developed within 7 days after head injury, and in a substantial number of patients PTH becomes chronic and lasts for more than 3 months. Current medications are almost entirely relied on the treatment of primary headache such as migraine, due to its migraine-like phenotype and the limited understanding on the PTH pathogenic mechanisms. To this end, increasing preclinical studies have been conducted in the last decade. We focus in this review on the trigeminovascular system from the animal studies since it provides the primary nociceptive sensory afferents innervating the head and face region, and the pathological changes in the trigeminal pathway are thought to play a key role in the development of PTH. In addition to the pathologies, PTH-like behaviors induced by TBI and further exacerbated by nitroglycerin, a general headache inducer through vasodilation are reviewed. We will overview the current pharmacotherapies including calcitonin gene-related peptide (CGRP) monoclonal antibody and sumatriptan in the PTH animal models. Given that modulation of the endocannabinoid (eCB) system has been well-documented in the treatment of migraine and TBI, the therapeutic potential of eCB in PTH will also be discussed. Traumatic brain injury (TBI) is caused by external mechanical forces such as compressive, tensile, and shear forces to the head resulting in mortality and long-term disability. More than 60 million people worldwide are affected by TBI each year and about half of the world’s population have one or more TBI over their lifetime ,2. HumanIt is known that brain tissue damage by TBI occurs in two-step processes: Primary and secondary injuries. During the primary injury, brain tissue deformation occurs and neurons can undergo disruptive morphological changes and cell death in the impact areas by mechanical force or penetration. From minutes to months after primary injury, secondary injury evolves as the results of consequent metabolic, cellular and molecular events which spread the injury to the surrounding and distant areas. There are several TBI models that aimed to replicate the pathological and symptomatic characteristics of human patients. TBI models are basically classified into two major types either with a focal or a more diffuse injury. In the focal injury models, impact is directly impacted to the brain such as the sensorimotor cortex, where the most pathological damages are observed. Accordingly, brain damage is relatively limited to the regions of impact. The typical focal injury model is induced by the controlled cortical impact, which utilizes a pneumatic piston to impact on the desired brain areas . In the As animals cannot verbally express pain, the pain-related behavioral changes are commonly used as headache indicators. Allodynia is one of the characteristics of headache sufferers including patients with head trauma ,22, whicA number of studies have shown cephalic hyperalgesia in TBI animal models, though the duration is varied ranging from days to months post-injury depending on the severity and type of injury. For instance, Wattiez et al. examined allodynic behavior in two different TBI models; severe compressive impulse induced variable shock front and acceleration/deceleration insult caused hypersensitivity in both cephalic and extracephalic regions for at least 63 days, whereas the repeated weight drop model resulted in cephalic allodynia for 2 weeks without altering plantar tactile pain threshold . The susPhotophobia, a sensory disturbance provoked by light, is the main symptom of migraine sufferers as well as the PTH patients . ConsistIn addition to photophobia, PTH patients often experience phonophobia, the hypersensitivity to sounds. This symptom has been less addressed in the animal model, though Fanselow’s group reported phonophobia-like behavior using the fluid percussion model . AnimalsTo gain the mechanistic insight of PTH, several studies have been conducted on the use of headache-inducing agents in the PTH models. The commonly used headache triggers are nitric oxide donors which induce vascular dilation, such as nitroglycerin (NTG) or sodium nitroprusside (SNP). NTG administration generally causes transient hyperalgesia in animals  and humaCalcitonin gene-related peptide (CGRP) is known to be a vasodilating neuropeptide and plays a pivotal role in the migraine pathogenic mechanism. CGRP levels in plasma were found to increase during migraine attack , and intThe trigeminovascular system is critically involved in the initiation and propagation of PTH, although impairment of other brain regions after TBI also contributes to the PTH development. The trigeminovascular system innervates the cerebral arteries, dural and pial blood vessels and sinuses, where nociceptive sensitization is originated . This sySatellite glial cells (SGCs) are functionally similar to astrocytes, however, SGCs have a unique anatomical structure that surrounds the TG neurons. The close contact between SGCs and TG neurons implies an intimate cross-talk to modulate neuronal excitability and pain sensitivity . SeveralThe meninges are three protective membrane layers encompassing the brain and spinal cord. They are composed of pia, arachnoid, and dura matters that contain blood vessels and enclose the cerebrospinal fluid. Studies from mild TBI patients and TBI animals demonstrated that brain trauma could cause meningeal vascular damage and cell death, as well as the breach of glial limitans . Of noteMeningeal Mast cells (MCs) are localized in the vascularized tissues including skin and dura mater, especially near the blood vessels and in close apposition to the terminals of the dural afferents . MeningeCortical spreading depression (CSD) is a pathophysiologic phenomenon characterized by transient cortical gray matter depolarization and subsequent hyperpolarization that propagate to the superficial cortical regions at a rate of 2 to 5 mm/min, resulting in suppression of spontaneous electrical activity . CSD hasTG neurons innervate centrally to the TNC in the brainstem and C1 and C2 dorsal horn where the nociceptive signal is relayed to the second-order neurons, and then projects to thalamus and sensory cortex for processing the nociceptive signal. In turn, TNC neurons receive descending signals from several brainstem regions such as periaqueductal gray (PAG), locus coeruleus and rostral ventromedial medulla (RVM) as well as hypothalamus . These mTrigeminal nociceptive inputs from cranial vessels are relayed by the second-order TNC to the ventroposterior medial nucleus of thalamus (VPM), sensory cortex, and several subcortical areas such as reticular formation, cerebellum, and midbrain. Consistent with the results observed in TNC in the weight drop model, both serotonin and NK1R were significantly increased in the secondary somatosensory cortex and ventroposterior thalamus . ConversClinicalTrials.gov, substantial number of PTH clinical trials have been planned, enrolled, or completed that include the use of CGRP monoclonal antibody (mAb), botulinum toxin, triptan, metoclopramide, prazosin, and others ). Current intervention for PTH is mostly based on the treatment strategy for primary headache because of its similar clinical presentations to migraine or tension-type headache. However, considering that the pathogenic mechanisms of PTH are likely to be distinct from that of a primary headache, development of the evidence-based PTH-specific treatment is desirable. Several therapeutic agents have been tested using the PTH models. The chief among them are CGRP related medication and sumatriptan, although other therapeutic substances including IL-2 [According to the ing IL-2 ,91, 48/8ing IL-2 , oxytociing IL-2 , SNC80, ing IL-2 , and onaing IL-2  were alsing IL-2 , althougAs mentioned above, the anti-CGRP treatment such as anti-CGRP mAb and CGRP receptor antagonists has been utilized in clinical interventions to migraine patients . The theSumatriptan is currently used as a prescribed medication for acute migraine attack. Sumatriptan belongs to a chemical compound called triptan, the agonist of serotonin receptors especially 5-HT1B/5HT1D . In addiThe endocannabinoid (eCB) system is composed of amine or glycerol derivatives of N-acyl fatty acids such as 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamide (AEA), the degrading and biosynthetic enzymes, and their binding receptors CB1, CB2, GRP55, and others . It is w2+ concentration in which adenylate cyclase and cGMP receptor were involved [It was reported that AEA administration to the TG culture increased miniature excitatory post-synaptic currents, and the increase was blocked by the TRP receptor antagonist capsazepine, but not the CB1 receptor antagonist AM251 , suggestinvolved . Recentlinvolved .These results are consistent with the “clinical endocannabinoid deficiency” theory proposed by Russo based on the findings that eCBs tone was lower in painful conditions such as fibromyalgia, bowel syndrome, and migraine . PreviouAEA administration prior to NTG injection reduced nociceptive behavior and attenuated neuronal activity in TNC based on the expression of c-fos ,124. TheIt was reported that eCB levels have been altered in several TBI animal models; in our previous study, AEA was showed to be increased in the ipsilateral cortex , which iIt is still speculated that the eCB signaling is effective for the treatment of PTH, although accumulating evidence indicates that exogenous cannabinoids and the inhibitors of the eCB degrading enzymes have potent therapeutic efficacy on TBI. Since eCBs interact not only with the CB receptors but also with the nociceptive receptors such as TRPV1 ,146, theThus, it is promising that enhancement of eCBs by inhibition of the eCB catabolic enzymes or direct administration of eCB ligands can ameliorate PTH, because augmentation of eCB signaling has been demonstrated to mitigate migraine-like pathology and TBI induced neuronal damage. However, it is unknown how the eCB system can modulate the ascending and descending pain pathways in which eCB receptors and enzymes are substantially expressed. Based on previous findings including TBI, migraine, and other inflammatory and neuropathic pain models, we hypothesize that eCBs can modulate PTH in various regions . As mentA recent study demonstrated that intravenous injection of CGRP in PTH patients exacerbated the migraine-like symptoms , suggest


Childhood vaccination is among the most effective public health interventions available for the prevention of communicable disease, but coverage in many humanitarian settings is sub-optimal. This systematic review critically evaluated peer-review and grey literature evidence on the effectiveness of system-level interventions for improving vaccination coverage in protracted crises, focusing on how they work, and for whom, to better inform preparedness and response for future crises.Realist-informed systematic review of peer-reviewed and grey literature. Keyword-structured searches were performed in MEDLINE, EMBASE and Global Health, CINAHL, the Cochrane Collaboration and WHOLIS, and grey literature searches performed through the websites of UNICEF, the Global Polio Eradication Initiative (GPEI) and Technical Network for Strengthening Immunization Services. Results were independently double-screened for inclusion on title and abstract, and full text. Data were extracted using a pre-developed template, capturing information on the operating contexts in which interventions were implemented, intervention mechanisms, and vaccination-related outcomes. Study quality was assessed using the MMAT tool. Findings were narratively synthesised.n = 17). Almost all campaigns operated using multi-modal approaches combining service delivery through multiple pathways (fixed and roving), health worker recruitment and training and community engagement to address both vaccination supply and demand. Creation of collaterals through service integration showed generally positive evidence of impact on routine vaccination uptake by bringing services closer to target populations and leveraging trust that had already been built with communities. Robust community engagement emerged as a key unifying mechanism for outcome improvement across almost all of the intervention classes, in building awareness and trust among crisis-affected populations. Some potentially transformative mechanisms for strengthening resilience in vaccination delivery were identified, but evidence for these remains limited.50 studies were included, most describing interventions applied in conflict or near-post conflict settings in sub-Saharan Africa, and complex humanitarian emergencies. Vaccination campaigns were the most commonly addressed adaptive mechanism  apparently irrespective of context, but evidence remains piecemeal. Adapting these approaches for local system resilience-building remains a key challenge.The online version contains supplementary material available at 10.1186/s12913-022-08653-4. Childhood vaccination one of the most effective interventions in the armoury available to public health policymakers and practitioners –6, but tThe challenge of low coverage in humanitarian settings is compounded by the chronic nature of many contemporary crises, and a long-term shift in displacement patterns in humanitarian settings. Data from the UN show a steady upward trend in the average length of humanitarian crises worldwide to at least 9 years , suggesthow they work, and contextual factors shaping this  there needed to be sufficient programmatic detail in the article to form a clear view of intervention design and how it had been implemented, and [ii] interventions had to operate at meso- or macro-level. Meso-level interventions included supply-side measures such as area-based interventions , health sector interventions addressing system resilience directly, or alternatively specifying activities under one or more of the WHO’s six health system building blocks; or interventions targeting specific tranches of the vaccination delivery pathway e.g. cold-chain maintenance; or demand-side interventions focused on refugee or otherwise displaced populations in crises with demonstrated effects on population health outcomes (e.g. cash transfer programmes), with the overall objective of increasing population demand for, and uptake of, vaccination. Macro-level interventions, on the other hand, addressed system resilience at national level directly, or concurrently addressed a number of the WHO’s six health system building blocks at national level; or described interventions targeting specific tranches of the vaccination delivery pathway at national level. Micro-level interventions such as the use of tailored text-messaging or other forms of individualised outreach were excluded from the review. Shorter-term activities such as vaccination campaigns were included provided there was demonstrable evidence that these contributed in some way to longer-term resilience-promotion .A full outline of inclusion and exclusion criteria is given in Table The primary outcomes of interest in this study were population level vaccination coverage for the vaccines outlined in Table https://www.technet-21.org/en/) websites, because of the role these organisations and initiatives have played in producing and collating evidence and technical guidance to support RI delivery including in humanitarian contexts. All literature searches were performed between 3rd and 9th September 2021. A sample search strategy is provided in Additional file Keyword-structured searches were performed in MEDLINE, EMBASE and Global Health , CINAHL (via EBSCOHost), the Cochrane Collaboration and WHOLIS. These were accompanied by targeted searches for grey literature through the United Nations Children’s Fund (UNICEF), GPEI and Technical Network for Strengthening Immunization Services , a validated tool developed which has been developed to help facilitate appraisal of public health research studies with multiple study designs and interventions within a single framework . Resultsn = 50 studies focused on childhood vaccination delivery were included after screening , followed by South Sudan and Afghanistan , and Cameroon, Haiti and Somalia . Four studies (8%) addressed interventions applied across multiple settings. There was considerable diversity in health system and wider crisis contexts in which interventions were being implemented. Considering crisis settings, over half of the included studies described interventions in settings of active conflict or that were immediately post-conflict ; a third were implemented in complex humanitarian emergencies including large-scale population displacement ; and two studies (4%) focused on interventions implemented in the aftermath of natural disasters . Eight studies (16%) addressed multi-dimensional interventions, some of which included campaigns as one aspect of what was delivered but might also incorporate community mobilisation activities and governance or surveillance system strengthening among other activities. Health financing interventions were addressed in 7 studies (14%), including an evaluation of the effects of aid on immunization-related outcomes, and 2 studies on the application of national performance incentive policies implemented at the level of facilities or health workers. Community engagement activities were also considered in 4 studies (8%). Other papers addressed more narrowly focused interventions such as service integration , those geared towards improving governance coordination in vaccination delivery , health information  and health workforce . Finally, most studies addressed either interventions targeting multiple antigens from the routine schedule  or those aimed at improving coverage of different classes of poliomyelitis immunisation . A smaller number considered cholera or measles vaccination specifically.We identified eight broad intervention classes Fig. , of whicn = 29, 58%) were mixed-methods program evaluations, but methodological approaches across the complete set were diverse. We did not identify any systematic reviews that explicitly addressed vaccination delivery in humanitarian settings and met the inclusion criteria for the study – specifically with regard to intervention design and delivery. This partly reflects a primary focus of this study on gathering information on intervention mechanisms, as well as measured effects. Significant methodological limitations were identified across almost all of the included studies  describing campaigns delivered in complex emergency settings, and a large proportion (n = 7) focused on cholera prevention or control, with measles (n = 4) and polio (n = 3) also commonly targeted pathogens. Most studies were either post-hoc campaign evaluations (n = 9) or cross-sectional surveys assessing campaign impact (n = 8). These studies were distinguished from material on supplementary immunisation activities (SIAs) by the duration of engagement during the intervention, with an understanding that campaigns were short – up to a maximum of a few months in length for each individual round. Included articles described campaigns delivered in a broad range of country contexts, with the best-represented country again being Nigeria (n = 4), and two studies from Cameroon.A total of 17 studies in this category met the inclusion criteria, most  domestically and from international donors and other non-governmental actors. Those studies reporting the largest effects all concerned multi-component campaigns involving vaccination delivery through multiple service delivery modes , but accompanied by community mobilisation activities, health worker recruitment and training, and support to cold chain improvement, among other interventions. For example, two linked studies addressing different aspects of the same cholera vaccination campaign among displaced persons in Borno, in northern Nigeria, showed 90% (95% CI 88–92%) first dose, and 73% complete (68–77%) oral cholera vaccine (OCV) coverage in the target population following a multi-dimensional intervention involving door-to-door and fixed-site delivery modes, multiple information dissemination and communication routes  and health worker capacity building. However, the two studies also note important contextual factors contributing to success: the campaign benefited from a long-standing partnership governance model in which the Ministry of Health led but agencies and non-government organisations (NGOs) supported on the ground delivery, and preparedness in two key areas before the outbreak: training in cholera preparedness that had incidentally been run by the Nigeria Centre for Disease Control earlier the same year, and the licensing of OCV for use in Nigeria in the months prior to the outbreak , 34.These studies also emphasised the importance of prior networks built through polio eradication work in Northern Nigeria, and the ability of campaigns to capitalise upon this. Two further evaluation studies from Nigeria, considering polio and measles campaigns respectively, documented the use of polio eradication infrastructure to support short-term activities, including the use of existing GPEI governance structures, outreach capabilities especially in less secure areas that had been developed previously in partnership with the Nigerian military, and the integration of measles surveillance within case-based information gathering on acute flaccid paralysis (AFP). In these cases, near-term, adaptive responses built on long-term capacities in the vaccination delivery system in Nigeria intended to bolster resilience against the spread of poliomyelitis, although the results reported by the first study should be treated with caution because administrative coverage estimates exceed 100% , 36.Two studies from Cameroon considered campaign rollout for cholera and meningococcal vaccines against the backdrop of COVID-19 spread nationally. These studies emphasised the importance of central coordination through the Ministry of Health, the recruitment and training of cadres of vaccination workers (including community mobilisers to enhance outreach into remote and insecure communities) and the use of multiple communication modes with target populations – including through community leaders – to enhance uptake. Both studies reported large improvements in administrative vaccination coverage following the respective campaigns, but noted large regional variations. Neither study included a formal cross-sectional survey or time-series analysis to statistically evaluate campaign impact, so results should be treated with caution , 38.Drawing transferable lessons from other campaign-focused studies in this review is limited by the diversity in contexts in which they were implemented, ranging from multi-vaccine delivery in low-income settings in the midst of active conflict , 40, to Seven studies considered health financing interventions that ranged in focus from adaptive capacity building, to potentially transformative activities. These included two that looked at macro-level financing – specifically the value or otherwise of development aid in promoting improved health outcomes, including for vaccination, and system-strengthening in Gavi supported countries , 56; oneStudies on P4P and disbursement mechanisms involved changes of a potentially transformative nature for system resilience by changing structures and accountability systems governing financing for vaccination delivery. Of the three studies that looked at P4P, two were from time-series analyses from Burundi , 51 in tThe study on disbursement mechanisms did not consider any of our primary outcomes, but did address intermediate effects (secondary review outcomes) including successful completion of health worker payments, widely understood to be a key operational challenge for programme delivery in LMICs in general, and humanitarian settings in particular . In thisOne study considered the integration of private providers into RI delivery through a public-private partnership (PPP) in Uruzgan province in central Afghanistan, which had historically suffered from both profound insecurity and chronic underinvestment in primary health care services . This crp < 0.001), third-dose DTP  and all-vaccination coverage  over 5 years from 2011 to 2015, albeit from low levels  the provision of multiple service delivery pathways  and [ii] the use of intensive community mobilisation activities that were delivered in a culturally sensitive way . The secThree studies considered governance and coordination activities, two emphasising adaptive capacity through civil-military engagement, and the final study describing transformative capacity change through inter-governmental cooperation.The two studies on civil-military engagement both addressed the use of military and/or security personnel to improve access and polio vaccination uptake in security compromised areas of Angola  and NigeA final study considered a cross-border governance and coordination initiative between the Kenyan, Somalian, Ethiopian and South Sudan governments to reduce the risk of polio transmission in the context of ongoing population movement. This was essentially a preparedness and planning intervention in which, with the support of WHO, representatives from respective country Ministries of Health met on a regular basis to identify important formal and informal population crossing points and transit hubs, agree on selection and recruitment of immunisation staff, and develop materials for workforce training and surveillance strengthening, among other activities. Identified improvements in vaccination coverage, strengthened cross-border situational awareness were reported . This coTwo studies focused on the contributions of workforce interventions to improving vaccination outcomes, operating at different levels: one on the value-added of technical surge capacity to support immunisation delivery at local level, and the second on the contribution of community volunteers in mobilising service users to take up vaccination . Both studies were from Nigeria and both focused on polio vaccination delivery , 67. TheThe second study looked at volunteer community mobilisers in Nigeria to address persistently low uptake in security compromised areas, vaccine scepticism, and distrust in government and officials. In this study, volunteer community mobilisers had an integrative role including supporting RI, but also water, sanitation and hygiene and other intervention promotion. These were financed through stipends and other  incentives but the main livelihood source is elsewhere. This intervention showed evidence of improvements in coverage but also a sharp decline in MOVs (from 4.5% in 2014 to 0.8% in 2018) which the study attributes to high levels of trust in volunteer community mobilisers because of their social position, cultural sensitivity of their approach, engagement modes including house-to-house visits, and their ability to update programme coordinators with critical soft information on pockets of low uptake .Three studies focused solely or primarily on situational awareness through health intelligence and surveillance activities – although as noted elsewhere in the results section, many of the interventions in other categories involved surveillance/health intelligence-focused components. All three studies were concerned with mechanisms for strengthening situational awareness in contexts featuring population movement and/or ongoing insecurity that could undermine the efficacy of more conventional approaches.Two of the studies considered systems for strengthening awareness for campaign delivery and evaluation. One examined the use of geographical information system (GIS) technology to support microplanning processes for a measles campaign in Nigeria in 2017–18, focusing particularly on applications in conflict-affected northern states where tracking of mobile populations was much more challenging than in stable areas. Specifically, GIS mapping was used to geo-locate population centres to inform the positioning of fixed-sites for the campaign, and resulted in a reduction in the number of wards with zero vaccination coverage by comparison with states where standard population estimation approaches were used. The mechanisms by which this intervention were thought to have worked included [i] more accurate enumeration of target populations especially in the context of ongoing population movement, and [ii] a clearer view of ward boundaries than conventional (hand-drawn) approaches – both of which improved microplanning accuracy . The secA final, descriptive, study from Iraq considered the use of outreach surveillance activities linked to AFP case ascertainment for polio, to help assess uptake and effectiveness of immunisation in Iraq. This intervention involved use of AFP outreach workers to gather soft intelligence on RI delivery to pinpoint areas where microplanning for delivery needed to improve , and to help build awareness of services available among affected populations . The autAlthough – as the summary table in Additional file Study outcomes focused predominantly on measures of administrative coverage and missed opportunities for vaccination. Three of the studies relied on narrative assessments of impact without formal statistical testing –73; the Eight studies addressed sustained, multi-dimensional interventions. These interventions also tended to act at multiple levels, spanning macro-level governance and coordination changes, through to health worker training, and community outreach activities. Three of these studies looked at integrated, strategic interventions in response to poliomyelitis outbreaks, all in conflict-affected settings –77 and aOf these three studies, two considered regional activities in response to polio outbreaks in conflict-affected countries in the MENA, both involving multi-phase response plans not just to interrupt initial transmission, but then to focus on high-risk areas for importation. These evaluation reports indicate that through intensive community engagement it is possible to raise vaccine coverage even in areas of profound insecurity and constant population movement, but also that focused surveillance is really important in allocating resources appropriately and that multiple data points were triangulated for this . The firA further four studies looked at integrated, preventive responses, three focused on SIAs –81 and oThis review identifies a series of interventions that may support vaccination system resilience capacities for improved routine childhood vaccination coverage  in settings of protracted humanitarian crisis. Most of these interventions reinforced adaptive resilience capacities but some  had transformative potential even if study results did not necessarily indicate radical change had been achieved within the term of the study. Although the diversity of settings described in this review precludes easy generalisation regarding important contextual factors, more successful interventions relied on leadership from domestic ministries of health, funding and – importantly – flexibility from agency and donor partners, and an ability to negotiate safe access for vaccinators and outreach workers. However, findings drew heavily on evidence from polio programming, and to a lesser extent cholera and measles vaccination delivery, potentially limiting generalisability to delivery of other antigens in these settings.Considering adaptive capacity, vaccination campaigns were the most extensively evidenced interventions and while their effects may appear to be short-lived, many of the examples considered here both built on existing capacities and contributed to development of new ones in a range of areas, including through introduction of novel governance structures and workforce capacities developed during previous activities. The most successful campaigns were multi-dimensional interventions that incorporated a mix of service delivery approaches , intensive community mobilisation efforts, health worker training, and supply chain strengthening work. They were also often multi-phased to help both break chains of transmission (where the primary strategic objective was outbreak control) and prevent future outbreaks. The long-term sustainability of capacity strengthening through campaigns necessarily depends, however, on mobilisation of funding and other resources beyond the intervention lifecycle – some of which can be addressed by sustaining intervention through SIAs. None of the studies exclusively focused on campaigns offered detail on sustainability planning post-intervention.Other adaptively-focused interventions included service delivery changes such as service integration and the use of mobile health teams. All included studies on service integration demonstrated improvements in vaccination coverage and course completion rates albeit with varying effect sizes and in studies of generally low quality. Mobile health teams seemed to improve vaccination coverage by enabling outreach especially into poorer and more marginalised communities, by improving trust through regular interactions and the supporting activities community mobilisers drawn from the communities they served.Some common mechanisms emerged across the higher-impact interventions identified in the review. For example, strengthening trust and increasing the range of access points were key themes especially among communities living in security-compromised zones, and many interventions directly addressed this through outreach models including the use of community volunteers, messaging through community (including religious) leaders, but also more controversially through cooperation with security personnel. Political and ethical challenges revolving around the deployment of security personnel in support of routine vaccination are considerable and this route is unlikely to be feasible or desirable in all settings. On the other hand, community volunteers were frequently identified as critical in tackling scepticism towards government and officials, but also contributed to bolstering situational awareness through improved case ascertainment for VPDs .Finally, a large proportion of the papers related directly or indirectly to lesson learning from polio eradication, and the resilience-building contributions GPEI infrastructure could make with respect to routine immunization more generally. Particular areas of learning included the use of multi-modal approaches to vaccination and surveillance in security-compromised areas, the value and use of existing outreach networks into communities, and messaging strategies. Objectives for polio control  are very different to those for many other VPDs where primary aims are more likely to be interruption chains of transmission in the context of outbreaks or to keep the burden of mortality and morbidity low in the face of endemicity (for diseases such as measles for example). This should not, however, preclude the use of infrastructure developed for polio control to support wider RI objectives, especially given calls elsewhere to consider how GPEI activities can better support routine immunisation delivery overall and lessons learnt through these regarding effective community engagement strategies in security-compromised areas , 84.Evidence in some areas was notable by its scarcity. For example, material on governance and financing was both limited and showed conflicting evidence on vaccination-related outcomes, although some promising interventions were identified in these domains, including the use of direct disbursement mechanisms for health worker payment, and a model of cross-border cooperation in the Horn of Africa to support preparedness and planning to reduce poliomyelitis risk. In addition, while some studies touched on leadership and oversight capacity development, we identified no studies that explicitly focused on leadership models contributing to resilience capacities for RI. Similarly, evidence on governance reforms such as decentralisation, which have been implemented in some eligible countries (e.g. Kenya and Tanzania) was not forthcoming, although one study briefly considered the importance of decentralised decision-making as a contextual contributor to the success of a nutrition-immunisation integration intervention . FinallyA number of systematic reviews have been published in recent years addressing the effectiveness of interventions in humanitarian settings, some including data on vaccination delivery , 85, andNevertheless, many of the central messages from this review – including the value of recruiting local staff to improve trust and vaccination uptake, flexibility in service delivery modes (including the use of mobile services), and use of electronic systems to strengthen supply chain management and health intelligence – support findings from reviews and guidance elsewhere , 88, 89,Limitations to the findings reported here relate to both the nature of the underlying evidence base, and the way in which the review was conducted. As the quality appraisal results show  coincided fairly precisely with the duration of the crises of interest. This is often not the case: there may be delays of up to several years before RPs are formulated and agreed – as in the case of the Syria Crisis response, for example. Secondly, an important goal of this review was to capture data from a wider range of sources than the peer-reviewed literature alone, to better capture emergent best practice. Structured searching of grey literature sources remains challenging and it is likely that some relevant material was missed.Some of the criteria applied to searches in this study may also have excluded relevant material. The restriction to three focus languages  may have preventing us from accessing material published in other languages although preliminary searches did not suggest sizeable, relevant literatures elsewhere. Secondly, the focus on material published since 2001 will have excluded earlier material on vaccination delivery in other settings in the 1980s and 1990s that may have informed our findings.As with all systematic reviews, our findings also cannot account for unpublished or negative results. This may explain the lack of data relating to absorption as a resilience mechanism – that they were not reported simply because they involved services performing in much the same way but more intensively . Because of the diversity of intervention types, study designs and study contexts included, it was neither possible nor desirable to produce summary statistics of intervention effects beyond those reported in the results section.Finally, we noted imbalances in the geographical representativeness of studies included. On one hand, the balance of included evidence was skewed strongly towards unstable, low income settings. This is unsurprising given that most countries in protracted crisis today fall into this category, but it reduces the potential transferability to middle income countries hosting large displaced populations today. Evidence from countries such as Jordan, Iran and Turkey, for example – all of which host large populations displaced by conflict – was notably absent in this review. On the other, a large proportion of included studies were conducted in Nigeria – reflecting historical challenges with vaccine delivery and particularly polio control in the north of the country.Findings from this review suggest that no “silver bullet” solution exists to promoting resilience in vaccination delivery systems in protracted humanitarian crises, and adaptations are likely to be needed across a range of fronts to address significant access barriers (many of which stem directly from population displacement and chronic insecurity), low trust in service providers, and limitations to effective VPD prevention and control imposed by national borders. Periodic intensification of vaccination delivery via campaigns and SIAs is likely to be a mainstay of adaptive responses to crises whatever the context, to account for shortfalls in routine delivery. However, there is a strong steer from this work for recruitment of non-traditional workforce cadres from within affected communities – including community mobilisers – to help enhance uptake over the long-term, especially in security compromised areas where trust in government and agency representatives may be low.Resilience in vaccination coverage is also likely to be enhanced through the concurrent use of multiple service channels to reach affected populations, including mobile health teams and integration with in-demand services such as nutritional support. However, there will inevitably be trade-offs in cost terms to expanded service availability in this way given resource constraints in many humanitarian settings, and the success of any of these interventions will ultimately depend on the willingness of domestic and international actors (including donors) to ensure stability in funding flows to crisis-affected countries.Strengthening the resilience of vaccination delivery systems in protracted humanitarian crises depends on system adaptation across a range of areas, including bolstering access through strengthened outreach, multiple service pathways and better integration with other essential services, as well as demand-generation activities. Future work should consider evidence not just on adaptive and transformative measures to support improvements in vaccination coverage in these settings, but also economic analyses given the significant resource constraints under which decision-makers in humanitarian contexts have to operate.Additional file 1: Appendix 1. Developing a working analytical framework for the review.Additional file 2 Appendix 2. List of eligible settings and their eligibility periods.Additional file 3: Appendix 3. Sample search strategy.Additional file 4: Appendix 4. Summary table of included studies.Additional file 5: Appendix 5. Overall summary of quality appraisal results.


The current literature demonstrates that not only is exercise during pregnancy safe, but it has substantial maternal and infant benefits and appears to influence infant growth/size throughout pregnancy and at birth. However, many existing studies have investigated only the effects of prenatal exercise on birth weight. The purpose of this review was to determine the impact or association of maternal physical activity during pregnancy on neonatal body composition assessed between birth and two weeks of age. Electronic database searches were conducted on 29 July 2019 for randomized control trials and cohort studies, with an updated search completed on 8 January 2021. A total of 32 articles that met eligibility criteria were selected for review. Overall, prenatal exercise was not associated with infant body composition at birth. Yet, five of the studies identified suggest that infant body composition could be influenced by higher volumes of mid-to-late term prenatal physical activity. This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and registered in PROSPERO (Registration No. CRD42020160138). Scientific evidence has demonstrated the safety and efficacy of exercise during the perinatal period ,2,3,4,5.Birth weight has been used historically to indicate a healthy intrauterine growth environment; however, birthweight is not a strong predictor of important future health outcomes , particuTaken together, understanding the relationship between physical activity during pregnancy and infant body composition is essential. Exercise during pregnancy appears to influence infant growth/size ,13; howeThis systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The review protocol was pre-registered with the International Prospective Registrar of Systematic Reviews (PROSPERO) registration number CRD42020160138. We followed the PRISMA reporting guidelines see .Eligible study designs included randomized controlled clinical trials (RCTs), prospective cohort studies, and retrospective cohort studies that assessed physical activity levels at any point during pregnancy and assessed infant body composition within two weeks of delivery. Studies were included regardless of intensity, duration, or mode of physical activity utilized. Only studies that were peer-reviewed with scientific credibility were included. The decision to include non-RCTs was based on several factors, including lack of existing RCTs assessing infant body composition as an outcome; poor adherence to interventions, making it difficult to truly evaluate the relationship between physical activity levels and infant outcomes; and the idea that other study designs may better allow assessment of physical activity as a continuous variable versus as a grouping variable .Women of all pre-pregnancy body mass indices (BMIs) were included. Studies including women with gestational diabetes and preeclampsia were excluded. These specific conditions have known and particular effects on infant body size/composition and would make it difficult to discern the impact of physical activity on infant outcomes. Studies including multiple gestation pregnancies were also excluded.For the mode of exercise, studies including all modes of physical activity were included . The physical activity assessment could be through self-reporting , compliance to an intervention, or objective assessments . Exercise data could be collected during any timepoint of pregnancy . For the outcome of infant body composition, the study had to include an assessment or estimate of infant adiposity (and not just birth weight). These included ponderal index, BMI, abdominal circumference, or body fat percentage from bioelectrical impedance, dual X-ray absorptiometry (DXA), skinfold anthropometry, or air-displacement plethysmography. Any studies including an initial assessment of body composition on infants >2 weeks of age were excluded.Electronic searching of PubMed/MEDLINE, EMBASE (Elsevier), Web of Science Core Collection (Thomson Reuters), and CINAHL (EBSCO) databases took place on 29 July 2019. Phrases and controlled vocabulary headings for each component of the population and outcome framework (PICO) were combined using OR and then using AND . The search strategy was first created in PubMed See  and thenThe risk of bias (RoB) was assessed on all studies selected for inclusion. Randomized control trials (RCTs) were evaluated using Version 2 of the Cochrane risk-of-bias tool (RoB 2) . Each RCRoB in case-control and single-arm cohort studies was evaluated using a modified Newcastle-Ottawa Scale (NOS)  See App. Again, All articles retrieved from the electronic databases were imported to EndNote, and duplicates were removed. After deduplication, titles and abstracts were uploaded to Rayyan, a web and mobile app for systematic reviews, for screening . In stagExtracted data included year published, study design, sample size, ethnicity, pre-pregnancy weight status, time point of pregnancy, physical activity assessment , infant body composition assessment, timepoint for infant body composition assessment, and main results found. Data from each study were extracted by one reviewer (BM) and checked by study team members . All extracted data were organized by design type and risk of bias score, as shown in n = 26) , wrong study design (n = 7), and wrong outcome (n = 23) ; 32 articles were included for evaluation. Of these, 13 were randomized controlled trials [The PRISMA flow chart  shows thcome n =   were rated as having “some concerns” for the overall potential risk of bias ,27,28,29For case-control and cohort studies, the risk of bias was generally low to moderate. We did not detect strong evidence of publication bias in these studies See .Overall, this systematic review suggests maternal PA during pregnancy has minimal implications on neonatal adiposity, except for extremely high volumes of PA. However, due to the variability in numerous elements of the study design , it remained a challenge to synthesize findings. These data suggest that maternal physical activity does not appear to have a negative or detrimental impact on the body composition of the neonate, which is an important conclusion given fear of harm to the unborn child is a factor that prevents many women from getting or staying active during pregnancy . In thisOnly one of thirteen randomized clinical trials had findings indicating that maternal PA has an impact on neonatal anthropometrics . SpecifiOut of the 16 single-arm cohort studies that were identified, three studies found that maternal physical activity levels were associated with smaller amounts of neonatal adiposity ,34,36. IThe second cohort study by Dahly et al., 2018, determined that the prevalence of babies born with very high fat mass was lowest among women that engaged in frequent moderate-intensity exercise early in pregnancy . While nThe third cohort study, by Harrod et al., 2014, found that increasing levels of maternal physical activity, particularly during late pregnancy, are negatively correlated to neonatal adiposity . In thisOut of three case-control cohort studies included, one study found a relationship between maternal physical activity and neonatal body composition . Clapp aAlthough a cohort design allows for free-living PA to be reported and/or recorded in addition to sports and exercise leisure-time PA, a randomized control trial that implements an intervention may be able to pinpoint specific PA modifying factors  and determine a direct causal relationship between exercise and infant body composition. It is plausible that specific modes, intensities, and/or volumes can have differential effects on infant body composition. Once again, this poses significant challenges to drawing broad conclusions about the relationship between general physical activity and infant adiposity.While the Pregnancy Physical Activity Questionnaire (PPAQ) is commonly used across the literature, it has been found to overestimate leisure-time PA across all activity categories . The PPAAnother factor that warrants consideration is the timepoint in pregnancy at which maternal physical activity assessments are taken. Some studies focus on early pregnancy, others on late pregnancy, and some assess throughout See . Upon caAnother aspect of study design that may play a role in interpreting the relationship between maternal PA and infant anthropometrics is the accuracy of the infant assessment. The majority of included studies utilized skinfolds and/or ponderal indices, both of which are considered poor and imprecise measures of assessing infant body composition . They arEvidence suggests that only extreme levels of physical inactivity/activity seem to influence neonatal adiposity. Specifically, higher levels of maternal physical activity and vigorous maternal physical activity, particularly when performed throughout late pregnancy, are consistently associated with decreased neonatal fat mass. There may be a “threshold” point where moderate exercise is beneficial but heavy exercise is detrimental, at least with respect to birth weight . It is uIt is also possible that a minimal level of physical activity exists to prevent overgrowth. In fact, moderate maternal physical activity promotes appropriate fetal growth. For example, work from Barakat et al. suggests that light intensity resistance exercise training performed during the second and third trimester might attenuate the adverse consequences of maternal body weight before pregnancy on neonatal anthropometrics . SpecifiDespite the inherent limitations discussed above, which limit the ability to draw conclusions across studies, some important findings were noted. Collectively, the studies suggest that continuing a regular physical activity regimen during pregnancy does not compromise fetoplacental growth. This is critically important, as many women do not engage in appropriate levels of PA due to fear of harm to the unborn child . PregnanGiven the ongoing obesity epidemic, there is a need to identify children at risk of developing obesity as early in life as possible . It is a


Hearing loss is the most common neurosensory disorder, and with the constant increase in etiological factors, combined with early detection protocols, numbers will continue to rise. Cochlear implantation has become the gold standard for patients with severe hearing loss, and interest has shifted from implantation principles to the preservation of residual hearing following the procedure itself. As the audiological criteria for cochlear implant eligibility have expanded to include patients with good residual hearing, more attention is focused on complementary development of otoprotective agents, electrode design, and surgical approaches. The focus of this review is current aspects of preserving residual hearing through a summary of recent trends regarding surgical and pharmacological fundamentals. Subsequently, the assessment of new pharmacological options, novel bioactive molecules , nanoparticles, stem cells, and gene therapy are discussed. Hearing loss is the most common neurosensory disorder when considering all sensory deficits and carries a tremendous economic and social burden, considerably impacting patients’ quality of life.In recent decades, hearing loss has become an increasingly concerning health issue. According to the World Health Organization, around 466 million people worldwide have disabling hearing loss  has become the gold standard of treatment and is constantly being improved and adapted to patients’ actual needs. Given the results obtained since the initial implementation in 1971 [As we witness a steady increase in knowledge regarding the inner ear’s physiology and with the development of targeted therapies, many lines of research regarding CI and ways to increase its functionality are being explored. However, few of these results will actually reach mass adoption and clinical usage. Many of the novel treatment options are in accordance with the Gartner Hype Cycle for Emerging Technologies, but only a few will prove their actual benefit. Thus, we believe that a review of the current knowledge is necessary to sum up the principles that are being considered for future implementation. Through this review, we outline the surgical principles of CI, with emphasis on recent technical improvements, and discuss emerging therapies focused on improving the outcomes of cochlear implantation.As selection criteria for CI are continuously evolving and more patients are eligible for implantation, the preservation of residual hearing is becoming increasingly studied. Sustained trauma to the cochlea during the advancement of the electrode array was identified as a critical factor that can deteriorate residual hearing; therefore, in recent years, increasing attention has been focused on the surgical procedure.The aim of preserving residual hearing led to an emphasis on avoiding mechanical trauma to the cochlea, leading to the use of the term ‘‘soft’’ surgical technique. After the first mention of the soft-surgery concept by Lehnhardt in 1993 , severalCochlear implantation most commonly begins with cortical mastoidectomy followed by posterior tympanotomy and preparation of the device well. Non-mastoidectomy approaches, such as the pericanal approach, the suprameatal approach, and the transcanal approach, still require drilling to create the groove that shelters the electrode . One priNoise and vibration during surgical drilling has become a concern for the potential reduction in residual hearing. Pau et al. determined that drilling at the level of the promontory exceeds 100 dB SPL, with further increased SPL when the endosteal layer is exposed, and exceeding 130 dB SPL when the round window membrane is touched by the drill . VibratiThe use of hyaluronic acid (HA) in cochlear implantation is not a new concept, as Donnelly demonstrated it in 1995 by using HA to facilitate electrode insertion into fresh human temporal bone specimens . Other aAnother emerging surgical concept is that of partial electrode insertion in patients with residual hearing who are candidates for electric–acoustic stimulation. Lenarz et al. hypothesize that partial electrode insertion could offer protection for the lower frequencies, and if hearing loss progresses postoperatively, the electrode can be further inserted .Intraoperative electrocochleography (ECochG) has been utilized during CI surgery and can provide the surgeon objective feedback during electrode insertion. Although it is not a method used to reduce inflammatory reaction by itself, whenever ECochG amplitude or phase responses are modified, the surgeon can adjust the course of the electrode in order to obtain normal intracochlear potentials and limit potential physical trauma to the cochlea .In addition to the soft surgery principles, it has been hypothesized that applying hypothermia to the surgical site has the potential to reduce immediate and delay secondary hearing loss. In preclinical studies, local application of controlled hypothermia rendered a protective effect by decreasing the auditory function loss associated with electrode insertion ,19. TakiThe cochlear implant electrode array is positioned in the scala tympani when normal anatomy is present. The round window membrane or a basal turn cochleostomy are the two most common ways to access the scala tympani in order to insert the electrode array . Each haThe round window membrane (RWM) is a natural boundary of the scala tympani. It has become an increasingly popular route for electrode insertion in cochlear implantation, particularly for hearing preservation. Preliminary comparisons revealed that the round window approach causes much less traumatic than insertion through a cochleostomy . DrillinRobotics in neurotology is a relatively recent subject is intended to help with various aspects of cochlear implant surgery, such as drilling a keyhole route to the middle ear for implants, inner ear access, and electrode insertion into the cochlea . InsertiEarly experimental studies on cadaveric cochleae revealed that robotic-assisted insertion systems reduced cochlear trauma associated with CI electrode insertions compared to manual insertions ,31. CompFollowing the first reports of cochlear implantations using robot-assisted electrode insertion, the results obtained after a radiological and audiological evaluation showed that this technique is less traumatic ,33,34. ATorres et al. also described their experience using straight and pre-curved electrodes but did not correlate the electrode type results, instead emphasizing the electrode array’s scalar translocation and functional outcomes. They observed no differences in speech perception in a sound-isolated room between manual electrode array insertion and robot-assisted techniques. However, they did observe that robot-assisted insertion reduced the number of translocated electrodes compared with manual insertion  but lackAs this new technique is promising, studies are still needed to determine safety, utility recommendations, and cost effectiveness. Further developments in robotic technologies and more clinical evidence may convert this surgical approach into a feasible, affordable option. Meanwhile, using intraoperative ECochG during CI surgery can offer the surgeon objective feedback during electrode insertion.Current surgical principles are outlined in The pharmacological substances with the best results in the preservation of post-implantation hearing are yet to be determined. The same also applies to the dose, delivery, and the timing of administration for currently available pharmacological substances.As the passing of the electrode through the scala tympani initiates a foreign body inflammatory reaction, pharmacological compounds with anti-inflammatory properties are among the most studied. The most well-known and scrutinized pharmacological compounds for this purpose are still corticosteroids. Their efficacy inspired their use in various hearing preservation protocols in an attempt to decrease any incidental loss of hearing . AlthougThe authors of one study recommend using intravenous (i.v.) dexamethasone one hour before surgery in order to reduce possible surgery-induced hearing loss ; others Although topical administration of steroids hypothetically implies higher intracochlear concentrations , animal To obtain the best functionality from a cochlear implant, preservation of the spiral ganglion neurons (SGN) is also of utmost importance, along with the cochlear components, as they are the target of electrical stimulation . Rejali Glial-cell-line-derived neurotrophic factor (GDNF) and BDNF attached to the cochlear implant electrode via biodegradable calcium phosphate hollow nanospheres also induced spiral ganglion nerve axons to sprout and grow towards the electrode to form direct contact and close the anatomical gap between them .Neurotrophin-3 (NT-3) was studied alongside BDNF or as a standalone substance. Through an experimental animal study using guinea pigs, Pfings et al. delivered intracochlear NT-3 via a viral vector, followed by cochlear implantation. Results were compared to empty viral vector inoculation and cochlear implantation, as well as cochlear implantation without inoculation. Overall results show that NT-3 cochlear inoculation was effective in maintaining a higher number of SGN compared to implanted cochleae with empty virus inoculation and implanted cochleae with no inoculation. Unfortunately, in the subjects with NT-3 inoculation, SGN preservation in the vicinity of the cochlear implant variated from 4% to 49% in animals with no residual hearing and between 4% and 67% in animals with residual hearing. These inconsistencies in SNG density could be an effect of NT-3 or could be derived from electrical stimulation of the implant, which is known to sustain nerve function; therefore, further studies with no electrical stimulation are needed .Various studies have been conducted on the mechanism of insulin growth factor 1 (IGF-1) and its role as a neurotrophic agent with anti-inflammatory properties capable of inhibiting apoptosis and promoting cell regeneration, making it a potential inner ear protector ,52,53,54Inhibitors of the c-Jun N-terminal kinase-like CEP-1347, SP 600125, and D-JNKI-1 have been investigated as antiapoptotic substances in preclinical and clinical studies since 1900 ,60,61. DD-JNKI-1was investigated in a phase III clinical trial, HEALOS , for acute sensorineural hearing loss (ASNHL). The intratympanic AM-111 administration was well tolerated and safe but failed to meet the study’s primary endpoint. Analysis of pure-tone average (PTA) scores revealed significant hearing recovery in the profound idiopathic sudden sensorineural hearing loss (ISSNHL) subpopulation but not in the severe ISSNHL subpopulation . To datePioglitazone is an antidiabetic drug of the thiazolidinediones (TZDs) class used to treat type 2 diabetes mellitus. TZDs act by activating peroxisome proliferator-activated receptors (PPARs), a group of nuclear receptors exerting an anti-inflammatory and antiproliferative effect, as well as regulating the cellular redox balance. When used with gentamicin in in vitro studies conducted by Sekulic-Jablanovic et al., pioglitazone demonstrated a protective cochlear effect against oxidative stress and prevented gentamicin toxicity ,68. As PIn 2018, pioglitazone was included as a 1.2% gel injection compound in a phase III multicentric, double-blind, placebo-controlled clinical trial  including adults with sudden sensorineural hearing loss. Participants were randomly allocated to one of three groups: intratympanic pioglitazone gel injection (STR001-IT) followed by 12 weeks of 5mg sustained-release oral pioglitazone tablets (STR001-ER), intratympanic pioglitazone followed by 12 weeks of oral placebo treatment, and intratympanic placebo injections followed by 12 weeks of oral placebo treatment. Results have yet to be published . No precOther PPAR activators, such as fenofibrate, rosiglitazone, and other antidiabetic drugs, including metformin, are mentioned in the literature as having an otoprotective effect, but none of them have been studied in conjunction with cochlear implants ,73,74,75N-acetyl cysteine (N-ACC) is a precursor and the substrate for glutathione synthesis, an endogenous antioxidant. When studied with noise-induced cochlear damage, N-ACC reduced reactive oxygen species (ROS), protected mitochondria, and inhibited inflammation and necrosis . When stAn in vivo animal study conducted by Eastwood et al. using a pledget loaded with N-ACC applied on the round window for 30 min before cochleostomy and electrode insertion brought up some detailed information. Whereas local N-ACC exposure led to a non-significant change in threshold shift in the low and mid frequencies, the high frequencies exhibited an increased threshold shift in the first week after surgery, with complete resolution by the fourth week compared to saline. Although there were no signs of active inflammatory reaction, the experimental group did display signs of ossification .The results of a 2021 in vivo study contradicted the favorable effects of N-ACC in vitro. Jaudoin et al. prepared an N-ACC liposomal gel, which was applied transtympanically one week before and during cochlear implantation. After seven days, the hearing thresholds increased in all frequency ranges compared with the group with no N-ACC. When tested on normal hearing guinea pigs, the liposomal gel induced a hearing loss that remained constant until the following two weeks of the study . These oTaurodeoxycholic acid, a bile acid taurine conjugate of deoxycholic acid, was used by Shah et al. in an in vitro study mimicking EIT. By analyzing apoptosis, levels of ROS, nitric oxide, and hair cell (HC) density, the authors observed that TDCA protected against HC loss in a dose-dependent manner .A summary of all the pharmaceutical agents described above is presented in When opting for a cochlear implant as a therapeutic solution for hearing loss, access to the inner ear is created through the round window or a cochleostomy; therefore, the electrode can also support local drug delivery to reduce EIT in order to avoid high systemic drug concentrations with possible adverse effects, blood labyrinthine barrier (BLB) limitation, or the “first-pass” effect . The desGlucocorticosteroids were intensively studied to reduce EIT and DEX-eluting electrodes, resulting in a considerable amount of research data ,88,89,90Chen et al. used an electrode coated with polycaprolactone (PCL) loaded with 5%, 10%, and 20% DEX to obtain prolonged release. In an artificial perilymph, the electrode released a stable DEX concentration for up to 270 days with no toxicity for the HEI-OC1 cell line. At five weeks after CI, they measured a minor ABR threshold shift in favor of the PCL-DEX-coated electrode when compared to the uncoated electrode. They also observed reduced cochlear fibrosis and better preservation of the SGNs .The first human study with a dexamethasone-eluting cochlear implant electrode elaborated by MED-EL (CIDEXEL) is an ongoing clinical trial that began in June 2020 , with no results available yet.As SGNs seem to be protected by brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT3), implanting electrode arrays coated with fibroblasts overexpressing these neurotrophins appears to be a therapeutic alternative for EIT and residual hearing preservation ,50.PLGA-coated electrode arrays treated with DEX, aracytine (Ara-C), Ara-C+DEX, and nicotinamide adenine dinucleotide (NAD+) were used recently in an animal study . The ABRHydrogel-coated electrodes filled with recombinant human IGF1, recombinant human growth factor (HGF), a mixture of both were implanted into guinea pig cochleae by Kikkawa et al. . The coaAs researchers noted that systemic drug delivery into the cochlea was hindered by the limitations mentioned earlier and that local RW application limited permeation into the inner ear, new methods of drug deliveries emerged. One of the main focuses is on loading drugs into/onto nanoparticles (NPs). The use of these nanovectors has been investigated due to their promise for improved drug stability, increasing drug storage capacity, and improving target specificity .BDNF is a macromolecule whose delivery to the inner ear can improve the electrode–neural interface after a cochlear implantation. Nanoporous silica and nanoporous poly-L-glutamic acid (PGA) nanoparticles were studied for this task . Wang etPoly-L-glutamic acid can also be used as a building block for BDNF carriers. Nanoporous PGA particles encapsulating BDNF demonstrated a protective effect on the spiral ganglion neurons after intracochlear administration . No studOne study was intended to improve the functionality of cochlear implantation using rolipram delivered into the cochlea through a lipidic nanocapsule. An interesting observation of this study was that rolipram alone did not to improve ABR results, but the lipid nanocapsule containing rolipram increased the spiral ganglion survival rate .The aim of regenerative medicine applied to hearing loss is to restore hearing without the need for a hearing aid. Currently, regenerative therapies are combined with cochlear implants to improve their functionality. When used as monotherapy, stem cells offer limited benefits and have reduced capacity to restore cochlear function .As repopulation of the cochlea with hair cells with the help of stem cells is technically complex, increasing the population of spiral ganglion neurons appears more feasible in the initial stages. Following this principle of SGN repopulation, stem cells can be associated with CI to achieve better outcomes. BDNF-overexpressing mesenchymal stem cells (MSCs) introduced into a guinea pig cochlea as a CI electrode coated with ultra-highly viscous alginate were more effective in protecting SGNs from degeneration compared to BDNF injected before CI insertion .Roemer et al. described a protocol for elaborating biohybrid cochlear electrodes using autologous mononuclear cells derived from bone marrow (BM-MNC). The authors chose this cellular line for its immunomodulation, neuroprotection, neurorestorative, and neurogenesis properties. Before clinical use, they evaluated cytotoxicity in an in vitro model and studied the insertion forces in a 3D human cochlear model. Three candidates for contralateral cochlear implantation were included in the clinical trial. The authors compared the performance of the biohybrid electrode to that of the contralateral standard non-coated cochlear implant. Whereas none of the patients manifested adverse reactions and the electrode impedances were identical between the ears, speech perception was similar in both ears in just one patient. A second patient’s speech perception in the biohybrid electrode-implanted ear exceeded that in the contralateral ear, whereas speech perception was reduced in the third patient . AlthougWith the advancement of gene sequencing methods, we can now identify a consistent number of genes linked to inherited hearing loss . As hearIn order to deliver in vivo cochlear gene therapy, a vector is needed. Two main platforms can be differentiated: viral vectors  and non-viral vectors  . The uniAs some of these vectors can be administered transtympanically for an indirect diffusion into the inner ear, a direct access into the cochlea is desirable through the round RWM, the oval window (OW), cochleostomy, or through a semicircular canal.Gene therapy was combined with a cochlear implant by Pinyon et al. in 2014, using a guinea pig model . They usNeurotrophin-3 and BDNF have both been proven to be important for SGN growth, survival, and regrowth ,106,107.Simultaneous hearing loss restoration strategies are being developed as we witness rapid technological and biological progress. All of these efforts combined and implemented could greatly benefit patients in the future.Improved electrode designs, surgical advancements, and microsurgical and robotic instruments will make otologic surgical procedures less traumatic, more accurate, and tailored to each patient’s unique anatomy and needs. Surgical principles are clearly outlined based on current concepts. In the case of robotic surgery utility, guidelines need to be further presented, and cost effectiveness has to be taken into consideration. Locally applied hypothermia is a feasible option due its current use in other surgical areas and due to the fact that is does not present a side-effect profile.As new drugs with otoprotective properties are studied and new implant materials are developed, cochlear implant drug delivery opens new possibilities for the immediate short-term future. To date, many of the researched substances have not been tested in clinical trials. The only pharmaceutical agent being tested in a clinical trial is MAPK/JNK pathway inhibitor, but the study is not associated with cochlear implantation. Corticosteroids are the only compounds that are frequently used as a potential agent for the preservation of residual hearing, but no clear evidence suggests that one administration route or dose is more favorable than the others.When improved understanding of nanotechnologies, gene treatments, and stem cells is achieved, construction of a bionic ear could become more compelling. At present, most of these therapies are experimental, as there are still ethical issues (stem cell therapy), as well as toxicity, stability (nanoparticles), and efficiency issues to be addressed.