In nursing students, high stress levels can lead to burnout, anxiety, and depression. Our objective is to characterize the epidemiology of perceived stress, stressors, and coping strategies among nursing students in the Middle East and North Africa region.We conducted an overview of systematic reviews. We systematically searched PubMed, Embase, PsycInfo, and grey literature sources between January 2008 and June 2020 with no language restrictions. We included any systematic review reporting measurable stress-related outcomes including stress prevalence, stressors, and stress coping strategies in nursing students residing in any of the 20 Middle East and North Africa countries. We also included additional primary studies identified through a hand search of the reference lists of relevant primary studies and systematic reviews.Seven systematic reviews and 42 primary studies with data from Bahrain, Egypt, Iraq, Jordan, Oman, Pakistan, Palestine, Saudi Arabia, and Sudan were identified. Most studies included nursing students undergoing clinical training. The prevalence range of low, moderate, and high perceived stress among nursing students was 0.8–65%, 5.9–84.5%, and 6.7–99.2%, respectively. Differences related to gender, training period, or the type of tool used to measure stress remain unclear given the wide variability in the reported prevalence measures across all stress levels. Common clinical training stressors were assignments, workload, and patient care. Academic training-related stressors included lack of break/leisure time, low grades, exams, and course load. Nursing students utilized problem focused , emotion focused (regulating the emotion), and dysfunctional (venting the emotions) stress coping mechanisms to alleviate their stress.Available data does not allow the exploration of links between stress levels, stressors, and coping strategies. Limited country-specific prevalence data prevents comparability between countries. Reducing the number or intensity of stressors through curriculum revision and improving students’ coping response could contribute to the reduction of stress levels among students. Mentorship, counseling, and an environment conducive to clinical training are essential to minimize perceived stress, enhance learning, and productivity, and prevent burnout among nursing students.The online version contains supplementary material available at 10.1186/s13643-021-01691-9. Mental health-related conditions are becoming increasingly prevalent among healthcare professionals worldwide . ProfessPsychological stress can impact nursing students’ academic and clinical performance  as well The Middle East, as with many regions worldwide, has a shortage of professional nurses , 23. PubSeveral studies –46 and sWe conducted a systematic overview of published systematic reviews on stress, stressors, and coping strategies among nursing students in the MENA region. Our systematic overview is part of a series of research and publications aimed to improve the quality of evidence generated in the MENA region by synthesizing available literature on population health issues in the region –50. ThisTwo independent reviewers (AA and SC1) systematically searched PubMed, Embase, and PsycInfo for any type of review on stress, stressors, and coping strategies on any country in the MENA region published between January 2008 and June 2020. Search terms related to stress, coping strategies/behaviors, and countries’ names were used. The full-search strategy is available in In this review, we include countries in the MENA region where Arabic, English, French, and/or Urdu are the primary official languages and/or the medium of instruction in the colleges/universities . The 20 A systematic review was defined as a literature review that had explicitly used a systematic literature search of at least one electronic database to identify all studies that met pre-defined eligibility criteria along with a study selection process . ReviewsUsing Rayyan software, duplicates were removed . Two indThe methodological quality of the included systematic reviews and primary studies was assessed by two independent reviewers . The AMSTAR measurement tool  was usedA customized tool was used to assess the quality of the included primary studies to accommodate the specific issues related to the methodology and the assessed outcomes. A quality assessment checklist was based on the Cochrane approach for risk of bias (ROB) assessment  using anThe characteristics of the included systematic reviews and primary studies were synthesized narratively. To quantify the stress levels among nursing students in the MENA region, available data on the prevalence of perceived stress was summarized using prevalence ranges according to three stress levels: low, moderate, and high, as defined by the different tools utilized in the studies. Prevalence measure variations according to gender, nature of ongoing training, and the type of measurement tools were explored. A measurement tool was considered validated if a validation record in the specific language was retrievable from published literature.Reported stressors among nursing students are categorized according to the training period: clinical, academic, and stressors external to training periods. For our review, clinical training stressors are classified into six domains as per the perceived stress scale for stressors . The totReported stress coping strategies among nursing students in the MENA countries are categorized according to three mechanisms as per the theory of psychological stress and coping : problemRecommendations for stress management are synthesized based on the available evidence into three main categories: nursing students, the nursing institutions, and nursing faculty and educators.In our overview, we include 7 systematic reviews and 42 primary studies on the epidemiology of perceived stress among nursing students containing data for at least 1 MENA country , Stress Assessment Scale (SAS), Physio-Psychosocial Stress Scale (PPSS)), stressors , Student Stress Survey (SSS), Students Stress and Coping Inventory (SSCI), Student Clinical Stressor Scale (SCSS), Student Nurse Stress Index (SNSI)), or stress coping strategies ((The Coping Behaviors Inventory (CBI), abbreviated version of the full COPE Inventory (Brief COPE), Coping Orientation to Problems Experienced (COPE), Adolescent Coping Orientation for Problem Experiences (ACOPE), Revised Ways of Coping Strategies Questionnaire (RWCSQ), and Students Stress and Coping Inventory (SSCI)). Only 28 out of 42 primary studies (67.5%) had a sample size of 100 or above and 20 out of 42 primary studies (47.6%) used a probability-based sampling.Quality assessment of the included primary studies is summarized in Table Table Some studies reported significantly higher stress levels in nursing students living in rural areas , having Table Based on the total number of primary studies, stressors related to clinical training are most commonly reported among nursing students followed by academic stressors. The predominantly reported specific stressors during clinical training are associated with assignments and workload  and patient care . Lack of break/leisure time, getting lower grades than anticipated, and examination and course load are the main specific sources of academic stressors. External stressors related to the physical environment include lack of recreation facilities; absence of a calm, safe, and secure environment; and congested classrooms. External stressors related to intrapersonal reasons reported by the nursing students include uncertainty about their future career, change in sleep pattern, and financial problems.No significant differences are observed in the type and level of stressors between students in private nursing schools and those in public schools  or accorTable The reported relationship between stress levels and the used coping strategies is inconsistent. Two included studies , 91 suggSome included studies suggested differences in the type of specific coping strategies used according to gender , 85, acaRecommendations reported in the included systematic reviews on how nursing students, nursing faculty and educators, and nursing institutions can aim to reduce stress levels, and manage stressors to maximize knowledge gain and productivity among nursing students are synthesized Table .Table 4Our overview synthesizes the evidence on nursing students in the MENA countries about perceived stress, stressors, and the stress coping strategies utilized by them to manage stress. We reviewed 7 systematic reviews and 42 primary studies that include data from nine the MENA countries namely, Bahrain, Egypt, Iraq, Jordan, Oman, Pakistan, Palestine, Saudi Arabia, and Sudan. Prevalence data from the majority of studies suggest that moderate and high stress levels predominate among nursing students in the region. Differences related to gender, training period, or the type of tool used to measure stress remain unclear given the wide variability in the reported prevalence measures across all stress levels. Commonly reported stressors among nursing students are related to clinical training , and academic training . Studies report the utilization of three predominant stress coping mechanisms: problem-focused , emotion-focused (regulating the emotion), and dysfunctional coping (venting the emotions). The most commonly utilized strategies within the problem-focused mechanism include active coping which in turn includes, specific strategies namely “problem understanding and solving” and “seeking social support.” Similarly, within emotion-focused mechanism, positive reinforcement and growth strategy, which includes “staying optimistic” and “wishful thinking” were more common. In the case of dysfunctional coping mechanism, behavioral, and mental disengagement, “avoidance” and “transference” were the most commonly used strategies and specific strategies respectively.While moderate stress levels are reported in studies conducted in China , Hong KoSimilar to our findings, studies conducted in non-MENA countries have reported clinical training stressors, particularly clinical assignments and workload, as the most common stressors among nursing students –135. SpeA global systematic review excluding the MENA concluded that the most common stressors among nursing students are academic stressors (workload and problems associated with studying) followed by clinical stressors  . This caMost of the published literature focuses on assessing academic and clinical stressors. The importance and potential impact of stressors external to training periods, such as the physical environment and intrapersonal stressors have been less studied. Likewise, the lack of a standardized approach to categorize stressors in these studies makes it difficult to compare results between studies. The grouping of stressors in this overview may be useful for future research on this topic. Researchers may choose to assess stressors based on academic, clinical, or factors external to training.The wide variation found in coping strategies utilized by nursing students in the MENA countries and worldwide –135, 142The relationship between socio-economic factors and stress levels or stressors could not be confirmed, as most of the included primary studies are cross-sectional and not designed to assess causal associations. A possible link between stress levels and factors, such as gender  and liviPublished literature indicates a potential link between stressors and the coping strategies utilized by nursing students , 85. A rTo manage stress among nursing students, it is highly recommended by several published studies that nursing institutions must recognize their role in improving stress management , 115–121To our knowledge, this is the most comprehensive, up-to-date, systematic overview synthesizing several dimensions of stress and coping behavior in a key population of the health care system for the MENA region. We searched multiple grey and non-grey literature sources for systematic reviews and primary studies to provide comprehensive evidence on the epidemiology of perceived stress among nursing students in the region. This compilation of evidence will serve as a benchmark for nursing students, nursing faculty and educators, and nursing institutions to help direct future interventions to optimize learning and prepare nursing students to manage stress effectively. Moreover, most of the included primary studies (12 out of 13) with prevalence data utilized a validated tool to measure the prevalence of perceived stress which minimizes the bias from the measurement tool. Most of the validated tools utilized in the studies are designed to assess clinical stressors in nursing students , 144–146Some included systematic reviews had specific inclusion criteria, such as, nursing students undergoing clinical training only or with a certain standard of methodological quality, which could explain the limited number of included primary studies in the systematic reviews. Moreover, none of the included systematic reviews have included studies published in a language other than English which could have led to an incomplete selection of primary studies relevant for the MENA countries. Out of the seven included systematic reviews, five had global geographical coverage and did not search grey literature or specific sources relevant for countries of the MENA region. Despite our best efforts to include all available data, data on the topic in other country-specific grey literature sources could exist. The included systematic reviews have searched data up to August 2018. In the absence of recent systematic reviews and to complete the collected data with recent studies published in the past 2 years, we conducted a hand search of primary studies in grey and non-grey literature sources. Despite this updated search, other recent primary studies could have been missed. Out of the seven included systematic reviews, five , 68, 69 Our findings suggest that the stress level among nursing students in the MENA region ranges from moderate to high. The limited data on stress prevalence among nursing students in all the MENA countries prevents the estimation of its magnitude with certainty; hence, comparability of stress prevalence between countries and other regions is also not possible. Differences due to gender, clinical training period, or type of tool used remain unclear given the wide variability in the reported prevalence measures across all stress levels. Nursing students commonly report stressors related to both clinical and academic training components of the nursing curriculum. Studies report an equal utilization of three predominant stress coping strategies by the nursing students: problem focused , emotion focused (regulating the emotion), and dysfunctional (venting the emotions). The link between stressors, and coping strategies and stress levels remains unclear. Although the significance of using the problem-solving approach to manage stress is well-established, there is a need to identify effective strategies to reduce excessive stress and increase the utility of positive coping strategies. Nursing institutions should establish a strong support system for students and educators to equip them with effective coping strategies. Reducing the number or intensity of stressors through curriculum revision and improving students’ coping response could contribute to the reduction of stress levels among students. Nursing faculty and educators are encouraged to mentor students to develop and strengthen problem-based, rather than emotion-based behavior to cope with stress and to provide a supportive clinical learning environment. While stress may not be preventable, it appears coping with stressors especially during the clinical training of the nursing curriculum is essential to maximize knowledge gain and productivity and prevent burnout among nursing students.Additional file 1: Table S1. The 2009 PRISMA checklist for reporting a systematic review. Table S2. PRIO-harms checklist for reporting an overview of systematic reviews (OoSRs). Table S3. Characteristics of the included systematic reviews. Table S4. Prevalence of stress, stressors and coping strategies among nursing students in the MENA countries with available data. Table S5. Quality assessment of included systematic reviews. Table S6. Quality assessment of included studies.


The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties. Extracellular vesicles (EVs) are generated by most cells as part of their normal physiology and released into the extracellular environment to maintain tissue homeostasis. Although they are delimited by a lipid membrane bilayer and share some surface features , the conBroadly, EVs can be categorized into large EVs (<5000 nm) which originate from the plasma membrane (apoptotic bodies and microvesicles/ectosomes) ,10 and sWhile it is conventionally accepted that EVs are secreted into biological fluids which enables their free shuttling between cells, in tissues EVs inevitably encounter the ECM and therefore must be able to traverse it. This is manifested in either an active manner, such as that exhibited by cancer derived EVs, or in a more passive manner that seems to depend on the combined properties of EVs and ECM . The ECMThere are three general mechanisms that govern EV formation: apoptosis, ectosomal shedding, and exosome biogenesis via endosomal pathways . ApoptotBudding entails plasma membrane protrusion from the cytosol. Additionally, newly-formed or inherent cellular protrusions can undergo a pearling process, followed by subsequent scission and release. These processes refer to different mechanisms of ectosomal shedding, however, they usually imply the same outcome; the formation of ectosomes/microvesicles. All these mechanisms involve the lipid bilayer and cytoskeletal structures such as actin; however, on rare occasions actin is not present, particularly in vesicles shedding from the distal tips of actin-based projections ,24. PearExosomes differ from apoptotic bodies and microvesicles in that they originate from endosomal pathways that are traditionally involved in endocytic recycling or lysosomal degradation A,B. EndoWhile the mechanisms governing EV biogenesis differ, as described above, the range of initial interactions with downstream target cells are similar. When EVs are released into the extracellular space, they either flow freely  or interAlthough therole of EVs in intercellular communication has been appreciated, it has also been proposed that they can act as independent metabolic entities that influence their surrounding microenvironment. This has been demonstrated by taking EVs that were isolated from media conditioned by neural stem cells, and incubating these in fresh media while monitoring metabolite consumption and release. Aspartate and glutamate were among the most abundantly released metabolites, whereas asparagine was the most consumed metabolite, suggesting high levels of asparaginase activity . AdditioIn addition to these biochemical properties, EVs play an active role in matrix remodeling through surface molecular interactions or the downstream effector roles of their cargo ,36. The Transmembrane proteoglycans with heparan sulfate chains, such as syndecan-1, associate with and can be attacked by heparanase. EV biogenesis pathways are known to retain heparanase on the surface of released EVs, and upon exposure to ECM, heparanase degrades their heparan sulfate components and liberates associated signaling molecules, as demonstrated in exosomes derived from myeloma, lymphoblastoid and breast cancer cell lines ,37,38. GUnlike EVs from cancer sources in which these surface enzymes were identified and explored, stem-cell-derived EVs have not been characterized by surface functionality. It is not clear whether enzymes identified in cancer correlate with those under normal physiological conditions. Interrogation of Exocarta  (exocarta.org) indicates that >100 matrix-related proteins are linked to mesenchymal stem-cell-derived exosomes, contributing to 12% of the total protein entries for mesenchymal stem cell exosomes. However, neither clear description nor further validation of these entries has been reported in the literature. Nevertheless, there is emerging evidence demonstrating the downstream functional and structural changes in the ECM spurred by stem-cell-derived-EVs ,44,45,46EVs can passively navigate the ECM. While the porosity of the ECM (<50 nm) is usually not large enough to allow for passive EV transport and diffusion, a recent study demonstrated a comprehensive mechanism in which matrix mechanics, in conjunction with the fluctuating deformation of EVs, contribute to EV transport. EVs in stiff stress-relaxing systems escape confinement more rapidly than EVs in soft stress-relaxing systems. Stress-relaxation refers to a gradual decrease in stress over time upon the application of constant strain, and mostly applies to viscoelastic materials . For exaAdditionally, matrix composition and consequent changes in its mechanical properties affect EV release and content. For example, an early study revealed that stress-relaxation of fibroblasts triggered an ectocytic process of transient membrane budding that resulted in the release of small plasma membrane derived vesicles into the collagen matrix in which they were cultured . In anotCOL1A1 expression in normal fibroblasts, which is indicative of scarring. Furthermore, higher levels of miR-21-5p, which is associated with fibrosis, have been noted within EVs from fibrocytes that were cultured on rigid plates and on decellularized fibrotic lung scaffolds, compared to those cultured on soft plates and non-fibrotic lung scaffolds, respectively [Whether these features alter the content of EVs is unclear. A study into the effect of fibrosis on EV content and bioactivity showed that lung stiffness due to fibrosis increases pro-fibrotic miRNA (miR-21) content within the EVs, which further accentuates fibrosis by lung-derived fibroblasts. EVs derived from fibrotic rat lung fibrocytes, which are bone-marrow-derived cells expressing hematopoietic and stromal cell markers , signifiectively . These fAlthough EVs play a role in ECM modulation by regulating matrix degradation/synthesis by target cells, it is largely unknown whether they continuously and actively regulate the ECM as intrinsic cues. Functional roles of EVs released into native ECM have not been reported in this context; however, matrix-bound vesicles (MBVs) have been seen in decellularized tissue from multiple sources and in decellularized scaffolds ,18,19. ITo expand on these findings, a 2020 study aimed to distinguish between the two putative EV populations: EVs in biological fluids and matrix-bound vesicles. Two systems were used to corroborate previous observations of matrix-specific vesicles . DecelluBone marrow mesenchymal stem cells (BM-MSCs), adipose derived stem cells (ADSCs) and umbilical cord stem cells were used in a manner similar to that described above for fibroblasts, to establish that miRNA content is unique to cellular origin. Separate clustering of the generated miRNA libraries demonstrated clear differences in the miRNA profiles of EVs from the three different cell types . In addiEVs isolated from liquids such as blood plasma and cell culture media are not specific to the ECM but have the capacity to mediate repair by activating signaling pathways that contribute to matrix modulation ,43,44,45The secretomes of ADSCs and BM-MSCs have been widely studied for their regenerative potential, which can be powerful because they approximate the regenerative action of their parent cells. Some of the regenerative effects that are imparted by the secretome have been attributed to purified EV fractions from these cells . FurtherELN), FBLN4, FBLN5, LOX, LOXL, and LTBP-4, as only fibrillin-1 expression increased after 30 days; the authors concluded that over 30 days, cells might have adapted to the stimulatory effect of EVs [ELN, FBLN-5 and LOX gene expression increased in the EV-treated cells, compared to conditioned media controls  and this was coupled with dense elastic matrix deposition, which served to highlight the anti-proteolytic roles of such stem cell-derived EVs [ADSC EVs play a potent role in the induction of matrix protein synthesis. This is underpinned by the finding that smooth muscle cells (SMCs) increased elastin and collagen deposition after 30 days of culture on 3D fibrin gel constructs when stimulated with EVs, compared to non-conditioned/control media and EV-depleted conditioned media. However, this was not strongly reflected in transcripts of genes typically involved in elastin fiber formation and maturation, such as tropoelastin , and TGF-β [More recently, ADSC EVs were found to display therapeutic potential for attenuating osteoarthritis, partly through maintaining the chondrocyte-populated matrix and protecting cartilage from degeneration. These EVs decreased MMP-1, MMP-13 and ADAMTS-5 expression in human chondrocytes in vitro, and osteoarthritic cells in the presence of IL-1β ,57. Furtnd TGF-β . CollectA mouse in vivo model of wound healing and repair revealed reduced scar size in full-thickness dorsal wounds treated with ADSC EVs. Collagen III levels were elevated at the wound site while collagen I levels were decreased . CutaneoPTEN and SPRY-1. These effects were reversed upon inhibiting miR-21-3p, which implicates miR 21-3p in a targeting, functional or regulatory role. Studies on EVs obtained from umbilical cord blood plasma, while not widely investigated, show promise in wound healing similar to stem-cell-derived EVs (Vascularization of injured tissue is crucial in maintaining tissue survival and reducing scarring. EVs isolated from umbilical cord blood plasma improve wound closure, re-epithelialization, and endothelialization, contemporaneous with enhanced vascularization . Fibroblived EVs .EVs from diverse sources share some similarities but ultimately can elicit different responses, primarily because they reflect the functionality of their source cells. The existence of ECM-specific EVs leads to the proposal that they play an active role in tissue homeostasis. This concept is further supported by the presence of EVs in explanted decellularized tissues, where they can contribute to regenerative effects, presumably by recapitulating aspects of missing cell–cell signaling in these scaffolds. The wound healing benefits of EVs can be attributed to their modulation of proteolysis in matrix remodeling but the effectors for this modulation are unknown. Stem cell EVs are of substantial interest due to their angiogenic and matrix-modulatory roles, which collectively contribute to improved healing. Although numerous studies have investigated matrix-modulatory and angiogenic roles separately, an understanding of the interactions between these two important aspects of the healing response will deliver improved repair by EVs. Stem cell derived EVs will benefit from more characterization as this knowledge will allow for the selection of matched EVs for specific outcomes, and should overcome current translational limitations in ECM repair.


Drosophila), a mode of AP patterning has evolved that seems not to overtly rely on oscillations or sequential gene activities, providing an opportunity to study the evolution of pattern formation mechanisms.Oscillatory and sequential processes have been implicated in the spatial patterning of many embryonic tissues. For example, molecular clocks delimit segmental boundaries in vertebrates and insects and mediate lateral root formation in plants, whereas sequential gene activities are involved in the specification of regional identities of insect neuroblasts, vertebrate neural tube, vertebrate limb, and insect and vertebrate body axes. These processes take place in various tissues and organisms, and, hence, raise the question of what common themes and strategies they share. In this article, we review 2 processes that rely on the spatial regulation of periodic and sequential gene activities: segmentation and regionalization of the anterior–posterior (AP) axis of animal body plans. We study these processes in species that belong to 2 different phyla: vertebrates and insects. By contrasting 2 different processes  in species that belong to 2 distantly related phyla (arthropods and vertebrates), we elucidate the deep logic of patterning by oscillatory and sequential gene activities. Furthermore, in some of these organisms (e.g., the fruit fly  Segmentation, also known as metamerism, refers to the organization of the body into repeating units of similar structure along the anterior–posterior (AP) axis. Among bilaterian animals, several clades feature segmented body plans, including annelids, arthropods, tardigrades, cephalochordates, and vertebrates, among others . SegmentInsects could be classified into 2 broad categories: simultaneously segmenting (or long-germ) insects and sequentially segmenting (or short-germ) insects . RecentlTribolium castaneum as a representative of short-germ insects and the fruit fly Drosophila melanogaster as a representative of long-germ insects, since they are the most studied species of their respective categories. We should bear in mind, however, that segmentation and regionalization in insects show remarkable evolutionary flexibility .,174.Tribavefront , whereasavefront . Interesastoderm .Tribolium [opa and dichaete [Tribolium blastoderm, where other maternal and zygotic gradients exists  that could be directly involved in regulating pair-rule gene regulation. Moreover, it is unclear if the same model is applicable to later stages of segmentation in the germ-band. However, in axn RNAi embryos, the cad gradient was shown not to regress in the Tribolium germ-band. Concomitantly, gap gene waves , which are nonperiodic version of pair-rule waves, were observed to continue propagating and shrinking without being arrested into stable expression domains in this genetic background, as predicted by speed regulation model [Hence, a single model can explain both blastoderm and germ-band segmentation in short-germ insects . The Wntribolium ,8. Experribolium ,8. Howevdichaete ) or whaton model . This suLfng reporter LuVeLu in mouse PSM explants [In vertebrates, it has recently been reported that pharmacological inhibition of FGF leads to a dose-dependent lengthening of the oscillatory period for the explants . Furtherexplants . These sexplants .In line with the role of Wnt/FGF signaling in frequency modulation in amniotes, sustained oscillations were observed in mouse PSM explants cultured under experimental conditions that maintained a uniform level of Wnt and FGF activities, without any sign of variability in frequency . On the Tribolium [Tribolium (replacing the clock with a genetic cascade). In line with this model, reactivating the gap gene hb all over the embryo resulted in 2 distinct responses [Kr in Drosophila is regulated by 2 enhancers, one initiates its expression and the other maintains it [Drosophila embryo has been shown to be mediated by the pioneer factor Zelda and at late-acting enhancers by the pioneer factor Opa [Tribolium to be mediate by opposing gradients of opa [zelda [opa and/or zelda are regulated by (or interact with) the Wnt/Cad gradient. Interestingly, in vertebrates, it was shown that some enhancers and/or genetic programs mediate the initiation of segmentation clock waves posteriorly, and others mediate their anterior expressions [But if the frequency of the segmentation clock is modulated along the AP axis via a speed regulator gradient, how could this be achieved at the molecular level? In a recently devised model of AP patterning in ribolium ,200, segesponses . Within esponses  Fig 4H)Triboliumesponses ,10. Thistains it . Interesctor Opa ,144. Ones of opa  and zelda [zelda , where tressions –204.Lfng mutant mice, the coupling delay is shortened, and oscillatory period is concomitantly decreased in intact PSM tissue but not isolated PSM cells [Lfng mutant mice [In an alternative hypothesis, the period of the segmentation clock could be spatially modulated by a protein production time delay that increases along AP axis . In yet SM cells . ConsistSM cells ,206. Theant mice ,206. TheMonodelphis domestica, which displays a lengthening in somitogenesis period from 1 hour at cervical levels to 4.5 hours at caudal levels [Tribolium, where the rate of segment addition varies along the AP axis [Clock-based segmentation programs enable the conversion of periodic temporal patterns (oscillations) into spatial patterns (segments). However, the pacemaker mechanisms that control the periodicity of the clock itself remain poorly understood. Strikingly, the oscillatory period of cyclic genes displays great plasticity in both vertebrates and arthropods. First, traveling waves slow down and become narrower as they reach the anterior part of the PSM or SAZ as described above. Second, the somitogenesis period in vertebrates is not constant throughout axis formation but rather increases gradually as the caudal most somites are laid down . An extrl levels . Similar AP axis ,210.Like the slowdown of traveling waves along the PSM/SAZ, and in line with the speed regulation model , the graTribolium [In addition to clock frequency variation along the AP axis, the period of the segmentation clock varies significantly across species. The segmentation clock oscillates every 30 minutes in zebrafish , 1.5 houribolium , and 5 hribolium ,184. Thiribolium ,212,213.ribolium .Hes/Her time-delayed negative feedback loops that are thought to underlie cyclic gene oscillations [Hes/Her genes are referred to as the transcriptional delay and represent the total time delay before feedback inhibition can take place [Hes/Her orthologs are longer in vertebrate species with slower clock period, and removing introns from the mouse Hes7 gene to accelerate splicing results in a moderate acceleration of oscillations [Hes/Her gene expression kinetics are most likely the result of larger differences in the global rates of biochemical reaction speeds between species. Recent studies have shown that the production and degradation of many proteins, not only Hes/Her proteins, is accelerated in mouse PSM cells compared to human cells [Currently, the predominant hypothesis concerning the segmentation clock pacemaker in vertebrates is based upon the llations . The cumke place . These tllations ,216. Thean cells . Similaran cells . It thusHes7 still exhibit residual segmentation, and the period of Wnt oscillations remains unchanged [Hes7 locus has been swapped for its human ortholog differentiate into PSM cells that oscillate with the characteristic mouse-specific period, thus confirming that differences in Hes7 genomic sequence cannot explain the interspecies differences in clock period [Hes7 is thus unlikely to be the fundamental pacemaker for the mammalian segmentation clock. Alternatively, other Hes genes may work redundantly with Hes7 as pacemakers, thus complicating the interpretation of the single mutant phenotype.These studies also suggest that Hes/Her genes may not represent the core segmentation clock pacemaker, at least in mouse. Rather, the clock period may be an emergent property of system-wide kinetics. In line with this hypothesis, mice carrying null alleles of nchanged ,219. Mouk period . Hes7 isDrosophila is based on a French Flag mechanism, in which the maternal Hb gradient acts as a master regulator. The maternal Hb gradient, however, plays less prominent role in Tribolium: Flattening the maternal Hb gradient in Tribolium  only slows down the onset of the sequential activation of gap genes [Tribolium [cad by RNAi completely abolishes gap gene expressions in Tribolium, and manipulating the Wnt/Cad gradient in several genetic backgrounds leads to stereotypical changes in the spatiotemporal dynamics of gap gene expression waves similar to those observed for pair-rule genes in the same genetic backgrounds [cad gradient is nonregressing and induces sequential waves of gap genes according the nonperiodic and gradient-based version of the speed regulation model . The moTribolium are the main regulator of downstream Hox genes. Little experiments, however, have been done to study Hox gene regulation in Tribolium other than those showing their disruption upon gap gene knockdowns , anHoxA-D) [Hox4 genes control patterning of the cervical somites that form the neck [Hox5, Hox6, and Hox9 paralogous groups are involved in the development of thoracic segments [Hox10 paralogs are required for the specification of lumbar identity and are capable of suppressing rib formation when overexpressed in the thoracic region [Hox11 genes are essential for sacrum specification and Hox13 genes correspond to the caudal vertebrae [In most vertebrates, genome duplications have resulted in 4 separate Hox clusters (HoxA-D) . MammaliHoxA-D) . There iHoxA-D) ,224. Desthe neck , whereassegments . Moreovec region . Similarertebrae ,228.Hox13 paralogs displaying the strongest effect and also triggering axis termination [The Hox gene expression domains that determine axial identity are thought to be established by a “Hox clock” that drives the sequential activation of Hox genes in the progenitor domain . This semination . Thus, tmination ,230. ThiWhat drives the sequential activation of Hox genes is highly debated. Several lines of evidence argue for a functional relevance of the fact that the 3′ to 5′ organization of the Hox cluster matches the temporal sequence of Hox gene activation. All genes in a Hox cluster are initially in a compact chromatin state that prevents transcription . OpeningTribolium. Early Hox genes (which are anteriorly expressed and located at 3′ end of the cluster) are activated by Wnt signaling through asymmetric distribution of Wnt-responsive enhancer sequences on the 3′ side of the cluster [However, it is so far unclear whether chromatin regulation is the main driver of Hox colinearity or just a safeguard to prevent the accidental premature activation of more posterior Hox genes, which exhibit dominant negative effects over anterior ones. It has been recently shown that a relay mechanism (or a genetic cascade) composed of cross-regulatory interactions between Wnt, Cdx, and Gdf11/TGFβ acts upstream of Hox genes and (partly) mediates their collnear activation ,236–238, cluster . Later o cluster ,239. Hen cluster .cad concentration is reduced in Tribolium  and regionalization (by regulating Hox genes). Pair-rule genes also coregulate Hox genes, providing an additional mechanism for coupling segmentation and regionalization. In Tribolium, although gap genes and pair-rule gene regulation represent 2 separate processes running in parallel, limited coupling between segmentation and regionalization has been documented [Segments and fates along the AP axis of insects and vertebrates have species-specific registry. Any misalignment between regionalization and segmentation leads to incorrect segmental fates (homeotic transformation) that potentially leads to severe consequences on the organism fitness. Hence, the processes of segmentation and regionalization are usually coupled. How this coupling is carried out is well understood in cumented ,113.Hoxb6 leads to somite formation and segmentation clock defects in tail bud-stage mouse embryos [In vertebrates, segmentation and regionalization are linked together through their common coupling to axial elongation. As described above, axial elongation takes place in the posterior progenitor domain, which corresponds to the regressing primitive streak at early stages and to the tail bud later in development. This posterior progenitor domain is not only the source of PSM cells but is also responsible for setting up the Wnt/FGF gradients that control segmentation and the point of origin for segmentation clock oscillations. At the same time, Hox gene expression patterns are also set up in this posterior progenitor domain, where they control the timing of mesodermal cell ingression . This me embryos . In cont embryos , has chaDrosophila and the short-germ insect Tribolium utilize the same set of genes to mediate segmentation and regionalization of their AP axis, neither the gene expression dynamics nor the underlying patterning mechanisms seem to be similar in the 2 species. Specifically, both gap and pair-rule genes arise as sequential waves in Tribolium, whereas they arise more or less simultaneously in Drosophila  leads to the down-regulation of the gap gene domain immediately posterior to it and overexpression of the gap gene domain immediately anterior to it . Based  cascade . Howevertterning , right [Tribolium and Drosophila seem to be harder to reconcile. In Drosophila, pair-rule gene regulation heavily depends on instructive cues from upstream gap genes, whose expression patterns form significantly earlier than those of pair-rule genes. On the other hand, gap and pair-rule genes are expressed in parallel in Tribolium with limited interaction between the 2 gene classes, although the nature and extent of this interaction is currently unclear. A possible bridge between segmentation in the 2 species is the zebra enhancers of pair-rule genes in Drosophila. As discussed earlier, while each one or pair of pair-rule stripes in Drosophila is initially mediated by a stripe-specific enhancer, there exist late-acting “zebra enhancers” that drive the full 7-striped pattern of each pair-rule gene. While zebra enhancers were hypothesized to mediate the stabilization of the pattern already formed by the stripe-specific enhancers, they could have evolutionarily originated as a “clock enhancer” to mediate pair-rule oscillations in more ancestral sequentially segmenting insects (like Tribolium) [Tribolium) could act later on to stabilize clock-oscillations into stripes  . The strribolium , and theribolium ), and inosophila .As highlighted in this review, vertebrates and short-germ insects share an impressive number of features related to segmentation and regionalization mechanisms ? Several models for short- to long-germ evolution have been suggested in this and other articles [There remain many open questions in the field of segmentation and regionalization in both insects and vertebrates. What is the molecular mechanism underlying the speed regulation of the segmentation clock and regionalization genetic cascade? How is the segmentation clock wired? The amniotes segmentation clock seems to be composed of several constituents . Are these constituents independent, coupled (as indeed shown for Wnt and Notch), or all part of the same clock? Is the insect pair-rule clock composed of multiple coupled clocks? Is the wiring of this clock evolutionary flexible? –31. How articles ,12,51 thTribolium continues to grow. A tissue culture assay, RNAi screen, and a growing number of live imaging and genomic editing techniques have been developed for Tribolium [Tribolium has been implemented [cis-regulatory level. When combined with the wealth of cis-regulatory data already available for Drosophila segmentation, this opens an avenue for understanding the evolution of patterning mechanisms at the molecular level.In vertebrates, answering these questions has been hindered in part by the complexities of culturing and manipulating somitogenesis stage embryos, especially in the case of mammalian species. As an alternative, in vitro models of the mammalian segmentation clock have now been established ,184,257.ribolium –267. Reclemented , promisiThese novel systems and techniques hold great promise to enable the detailed dissection of mechanisms controlling segmentation and regionalization in vertebrates and insects, and more generally, embryonic patterning mechanisms that rely on the spatial regulation of oscillatory and sequential gene activities.


Biomarkers can contribute to clinical cancer therapeutics at multiple points along the patient’s diagnostic and treatment course. Diagnostic biomarkers can screen or classify patients, while prognostic biomarkers predict their survival. Biomarkers can also predict treatment efficacy or toxicity and are increasingly important in development of novel cancer therapeutics. Strategies for biomarker identification have involved large-scale genomic and proteomic analyses. Pathway-specific biomarkers are already in use to assess the potential efficacy of immunotherapy and targeted cancer therapies. Judicious application of machine learning techniques can identify disease-relevant features from large data sets and improve predictive models. The future of biomarkers likely involves increasing utilization of liquid biopsy and multiple samplings to better understand tumor heterogeneity and identify drug resistance. A biomarker is a measurable indicator that predicts disease presence, severity, or response to treatment. Levels of biomarkers can be clinically useful by guiding disease diagnosis, or by revealing the pharmacodynamics of drug treatment. Approved and experimental biomarkers can be classified based on their clinical uses. These clinical uses parallel the progressive utilization of biomarkers during the development of cancer therapeutics. Companion diagnostics is the development of predictive biomarkers in conjunction with novel therapeutics. It identifies patients who are likely to respond to the treatment or to experience severe toxicity. An early example is estrogen receptor assays which are implemented in the prescription of the estrogen receptor modulator tamoxifen. Since then, others have been developed including measurement of HER2 levels prior to treatment of breast cancer with the anti-HER2 antibody pertuzumab, and measurement of PD-L1 levels prior to treatment with the anti-PD-L1 antibody pembrolizumab . CompaniWhile companion diagnostics focuses on predictive biomarkers, all types are utilized in both patient care and the phases of drug development. Diagnostic biomarkers suggest the presence of a disease or can classify patients into subtypes. Elevated levels of these diagnostic biomarkers may suggest the presence of cancer, and thus can be used as a screening tool in healthy individuals or can support other diagnostic measures such as imaging and biopsy. Several long-used cancer diagnostic biomarkers include prostate-specific antigen (PSA), used for diagnosis of prostate cancer ; cancer Prognostic biomarkers predict the patient’s overall survival, independent of therapy. Examples of diagnostic biomarkers with prognostic value include CA 19-9 and CA 125, which can predict overall survival in PDAC and ovarian cancer, respectively . Carcinovia measurement of pERK) in non-small-cell lung cancer (NSCLC) patients receiving BRAF inhibitors can indicate direct drug-target interaction (via fludeoxyglucose (18F) measured by PET/CT] to determine first if the drug is hitting its primary target and second if the drug is mediating tumor suppression  status are used to differentiate molecular subtypes of breast cancer. HR status was found to be associated with survival, making it also a prognostic biomarker . PrognosCancer-specific mutations are an appealing source of potential biomarkers. Genomic analyses have been applied to biomarker identification in both inherited and sporadic cancers. Early applications of gene mutations involved the identification of germline mutations that serve as prognostic biomarkers of elevated cancer risk, such as p53 mutations in Li-Fraumeni syndrome or BRCA mutations in hereditary breast cancer . These tThe convoluted mutational profile of most sporadic cancers increases the complexity of genomic analysis. Advances in sequencing technologies including single-cell sequencing have further underscored tumor heterogeneity and provided insight into the variation in patient responses to therapy. Predictive biomarkers when combined with targeted therapies have shifted treatment decisions from a focus on tumor type to gene-directed individualized treatment plans. For instance, FDA-approved biomarkers such as activating mutations in EGFR predict effectiveness of EGFR inhibitors like gefitinib . PatientProteomics attempts to directly analyze the main mediators of cellular function by quantifying protein activity and location . The dynMany genomic and proteomic datasets are large, nonlinear, and multidimensional. The high number of variables measured for each clinical sample can require sophisticated data analysis strategies to differentiate signal from noise and adjust for multiple comparisons. Machine learning can be used to make predictions that incorporate and simplify multivariate information and can determine which variables  are relevant biomarkers. The methods to select disease-relevant features while eliminating redundancy and noise range from linear models to neural networks . In seleThere are many existing web servers and bioinformatic analysis tools. Among pan-cancer human—omics datasets, web servers aggregating patient data are available for DNA mutation, methylation, mRNA, micro-RNA, long non-coding RNA, and protein information. DNA mutation servers include cBioPortal , GSCALite and CaPSSA. DNA methylation is available at MEXPRESS, GSCALite, and MethSurv. There are many databases of mRNA data including GENT2, PROGgeneV2, LOGpc, SurvExpress, PRECOG, and Oncomine which all have more than 15,000 patient samples. OncoLnc combines mRNA micro-RNA and long-noncoding RNA data. Proteomics patient datasets are available through CPTAC, TCPAv3.0, and TRGAted .Model development typically involves separate training and testing data. The quality of a model is determined by how much more effectively it classifies test data than would be expected by other available means. Validation can be achieved through independent datasets, but ideally also involves animal experiments or clinical trials . ClinicaBiomarkers can be derived from tumor tissue, blood, other biologic fluids, and even imaging. Blood-based biomarkers, or liquid biopsies, have become increasingly attractive in patient care . Liquid CTCs are a rare, migratory cell population shed from a tumor and believed to play a role in metastasis. CTCs have both diagnostic and prognostic value across several tumor types including PDAC , breast ctDNA is tumor-released single- or double-stranded DNA that enters the bloodstream and can be detected for diagnosis, guidance of treatment, and monitoring of disease progression. Recently, ctDNA has been used to guide clinical decision-making in several tumor types including CRC , NSCLC , and metExosomes are small cell-derived vesicles that are shed from myriad cell types into biological fluids under normal and pathological conditions. They carry molecular constituents of their host cells, including proteins, lipids, mRNAs, and miRNAS . They haBiomarkers are particularly important in immunotherapy as immune checkpoint inhibitors have demonstrated impressive responses across multiple tumor types. However, most patients do not benefit from this therapy and there is a growing need for biomarkers that can predict patient response.Some patients with PD-L1 positive tumors have improved clinical outcomes. However, the utility of this biomarker is inconsistent across tumor types . IncreasImmune status of the TME, including tumor-infiltrating immune cells and cytokine profiles, can be predictive biomarkers for immunotherapy. In melanoma, tumor-infiltrating immune cell subsets such as CD4+, CD8+, and FOXP3+ T cells correlate with improved treatment efficacy and disease outcome . An incrGenomic biomarkers such as tumor mutational burden (TMB), neoantigen load, and microsatellite status are all clinically relevant biomarkers of immunotherapy. Tumors with high TMB are thought to have increased neoantigen burden making them immunogenic and more responsive to immunotherapy. High TMB is correlated with response to immunotherapy in several cancer types including NSCLC , melanomAs the use of biomarkers in clinical cancer therapeutics advances, more frequent screening to evaluate drug efficacy and the development of resistance will be desirable. For patients undergoing these repeated screenings, the noninvasive nature of liquid biopsy is advantageous. ctDNA has been applied in detecting resistance to EGFR tyrosine kinase inhibitors and can replace a tumor biopsy in the decision to transition to third-generation EGFR tyrosine kinase inhibitors . While sThe complexities of tumor biology, its evolution over time and heterogeneity both between patients, and within a single tumor all add challenge to the development of biomarkers that meaningfully impact clinical cancer therapeutics. Biomarkers have the most clinical relevance when they are reproducibly and accurately measurable, clinically feasible, and prospectively validated in randomized clinical trials. Biomarkers are increasingly available at all phases of patient care, from screening and prevention to evaluations of drug efficacy and tumor response. Some biomarkers, such as those related to the EGFR pathway have already become approved decision-making tools for selecting cancer therapeutics. More are under development and may add insights to important areas of research such as tumor immune modulators and the tumor microenvironment. Blood-based biomarkers promise to reduce the potential for biopsy complications and make it easier to test repeatedly for tumor evolution. Many new trials are utilizing companion diagnostics where biomarker assays guide the use of targeted cancer drugs . As the


Efficient cancer therapy has been the research focus in biomedicine field for decades. Available therapeutic modalities for cancer treatment in clinic include surgery, chemotherapy, and radiotherapy. Surgery is difficult to completely remove tumors from normal tissues, and residual tumor cells are readily to relapse and result in treatment failure. In this regard, chemotherapy and radiotherapy are usually required to eradicate residual tumor cells after tumor resection by surgery. However, both chemotherapy and radiotherapy are challenged by serious side effects because of their off-target damage to normal tissues. Moreover, tumor cells are likely to activate anti-apoptotic signaling pathways to resist treatment. Very recently, immunotherapy has been demonstrated an promising way in fabricating long-term antitumor immunity to fight against cancer recurrence and metastasis, but only present efficacy to a subset of patients. In light of the above mentioned issues, it is imperative to develop more efficient and safe strategies for cancer treatment.The interdisciplinary research of material chemistry and biomedicine provides more opportunities to overcome these challenges. Enormous endeavors have been devoted in the past decades from the following aspects.(1) To improve the efficacy of chemotherapy or radiotherapy, intelligent drug nanocarriers have been designed and constructed to increase drug retention in tumors. Drugs are released from nanocarriers in response to specific tumor microenvironment, such as low pH or high glutathione level. Moreover, external stimuli  have also been employed for drug controlled release.(2) Phototherapeutic platforms have been developed based on some semiconducting materials or photosensitive agents, which can generate local hyperthermia or reactive oxygen species (ROS) under light irradiation. Since visible light hardly penetrates human tissues, near infrared (NIR) light with much deeper tissue penetrating depth has been widely investigated for phototherapy in recent years. In addition to light, ultrasound with extraordinary tumor penetrability has also been employed for generating ROS in tumors, which is termed as sonodynamic therapy.(3) Tumor therapy drugs have been combined with imaging agents, such as upconversion nanoparticles, quantum dots, or fluorescence dyes. Under the guidance of imaging agents, both tumor location and drug delivery process can be facilely monitored, providing possibilities to achieve personal medicine and tumor theranostics.(4) Besides, some nanomaterials themselves can be designed with therapeutic functions, such as radioprotection, anti-inflammation, or immune activation for tumor vaccines.Wang et al. reported a covalent organic framework nanomaterials as carriers for DOX loading, showing high drug loading capacity and pH-/redox-sensitive release in tumor cells. Howaili et al. synthesized a plasmonic nanogel composed of Au nanoparticles and polymer-based hydrogel nanoparticles. Such plasmonic nanogel was employed as drug carrier for curcumin delivery and dual pH-/photo-responsive release. In addition, Zhao et al. investigated the potential of black phosphorus quantum dots as carriers of Chinese herbal medicine for lymphoma therapy. Lin et al. synthesized a new kind of polymer nanocarriers with minimal cytotoxicity and high stability. The polymer nanocarriers improved antioxidant activity of curcumin, showing great potential as protection agent in caner radiotherapy. Gupta et al. developed solid lipid nanoparticles as carriers to improve the solubility, stability, and bioavailability of curcumin.In this topic, we present original research and review articles with focus on functional nanomaterials for cancer diagnostics and therapy. In the direction of functional nanocarriers, Feng et al. developed a multifunctional nanosystem with bovine serum albumin as “mother ships” material. The nanosystem was demonstrated to be selectively accumulated within tumors, disrupt extracellular matrix for tumor penetration, and generate ROS to induce tumor cell apoptosis. This work offer a new avenue for improving photodynamic therapy via reprogramming tumor microenvironment. To improve tumor hypoxic environment, Huang et al. designed an oxygen-sufficient nanoplatform with polymer-perfluorocarbon nanoparticles as oxygen carriers. The nanoplatform not only improved the ROS generating efficiency of sonodynamic process, but also overcame tumor drug resistance to enhance therapeutic outcomes. As a Food and Drug Administration (FDA)-approved NIR photosensitizer, indocyanine green (ICG) has been widely used in oncology. Sevieri et al. summarized the applications and perspectives of ICG nanoparticles, offering insights for its further biomedical applications. Zhang et al. reviewed the progress of photodynamic therapy with internal light sources from the aspects of chemiluminescence, bioluminescence, and Cerenkov radiation, all of which circumvent the limitation of light penetration in body. Jia et al. coated Fe3O4 with mesoporous silica and polydopamine for drug delivery and synergistic photothermal/chemodynamic therapy.In the direction of phototherapy/sonodynamic therapy, Yu et al. reviewed the recent progress of rare-earth-doped nanoparticles for tumor fluorescence imaging and theranostics in the second near infrared window . Hong et al. reported theranostic platform based on upconversion nanoparticles, which can accumulated in the mitochondria of tumor cells. Under NIR light irradiation, such nanoplatform emitted fluorescence signal for imaging-guidance and produced ROS for tumor killing. Tang et al. reviewed the advances of noble metal nanoclusters (Au and Ag) in biosensing, bioimaging, and cancer therapy.In the direction of cancer diagnostics and theranostics, Shang et al. studied antitumor activity and tumor-killing mechanism of zirconia nanoparticles (ZrO2), providing a promising alternative to traditional chemotherapy drugs for tumor treatment. When camouflaged with platelet membrane (PLT), the obtained PLT@ZrO2 nanoparticles successfully escaped from immune recognition and targeted tumor cells, thereby achieving prolonged blood circulation time and tumor targeting delivery Shang et al.. Seré et al. reported a nanovaccine based on ovalbumin-conjugated mesoporous silica nanoparticles, which was attractive as a replacement of dendritic cell-based vaccines.Besides, We hope this Research Topic will provide researchers information to understand the advanced strategies of functional nanoplatforms in cancer diagnostics and therapy, inspiring new ideas for future research directions and research activities.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Proper development of the placenta is vital for pregnancy success. The placenta regulates exchange of nutrients and gases between maternal and fetal blood and produces hormones essential to maintain pregnancy. The placental cell lineage primarily responsible for performing these functions is a multinucleated entity called syncytiotrophoblast. Syncytiotrophoblast is continuously replenished throughout pregnancy by fusion of underlying progenitor cells called cytotrophoblasts. Dysregulated syncytiotrophoblast formation disrupts the integrity of the placental exchange surface, which can be detrimental to maternal and fetal health. Moreover, various factors produced by syncytiotrophoblast enter into maternal circulation, where they profoundly impact maternal physiology and are promising diagnostic indicators of pregnancy health. Despite the multifunctional importance of syncytiotrophoblast for pregnancy success, there is still much to learn about how its formation is regulated in normal and diseased states. ‘Omics’ approaches are gaining traction in many fields to provide a more holistic perspective of cell, tissue, and organ function. Herein, we review human syncytiotrophoblast development and current model systems used for its study, discuss how ‘omics’ strategies have been used to provide multidimensional insights into its formation and function, and highlight limitations of current platforms as well as consider future avenues for exploration. The placenta is a temporary organ that forms during pregnancy. It serves crucial functions to sustain pregnancy, promote fetal growth and development, and stimulate adaptive changes in maternal physiology and metabolism. These functions include (but are not limited to) hormone production and metabolism, hemodynamic adaptations, and serving as a physical barrier separating maternal and fetal circulations. The placental barrier enables nutrients, gases, and wastes to diffuse between maternal and fetal blood, yet protects the fetus from potentially harmful factors including toxins, pathogens, and maternal immune reactivity. Structural adaptations have evolved to enable the placental barrier to execute its versatile requirements as both protector and nurturer, culminating in the formation of a unique multinucleated syncytium consisting of millions of nuclei connected by a continuous cytoplasm, called syncytiotrophoblast (STB). In humans, STB facilitates implantation and ultimately lines chorionic villi where it bathes in maternal blood. STB secretions and debris are deposited into the maternal circulation, where they have important roles in modulating maternal physiology as well as diagnostic potential for fetal-placental aberrations and pregnancy disease. Despite the importance of STB formation and function for fetal development and pregnancy outcome, limitations of cell and animal models have left much to be discovered. In the first part of this review, we will briefly highlight the ontogeny and diverse functions of human STB as well as models commonly used for its study. Then, we will discuss how various omics technologies have provided unprecedented insights into understanding STB formation and function, including current limitations, challenges, and opportunities for future investigation.There are two types of STB that arise during different stages of human embryogenesis: a primitive STB that mediates implantation and decidual erosion during the second week after fertilization, and a definitive STB that lines chorionic villi from the third week and beyond. Whether these two STB subtypes are distinct entities or the gradual evolution of the same lineage as gestation progresses is unclear. The primitive STB first appears around the time of implantation as the blastocyst breaches the uterine surface epithelium, likely through intercellular fusion of underlying cytotrophoblasts (CTBs) at the embryonic pole of the blastocyst. The primitive STB rapidly expands into the decidua and erodes uterine stroma, glands, and capillaries. The cavities generated within the primitive STB, called lacunae, become filled with blood and glandular secretions from eroded decidual tissue, providing a source of early nutrition for the conceptus. The primitive STB provides the groundwork in which pillars of CTBs proliferate, forming primary villi. These primary villi traverse the entirety of the primitive STB and ultimately connect together to encircle the conceptus as the CTB shell, which serves to anchor the conceptus to the decidua basalis. Villi also branch extensively to create smaller floating villi that remain within spaces between villi (intervillous spaces), increasing the surface area of the villus tree. Chorionic villi are formed when extraembryonic mesoderm and blood vessels emanating from the allantois infiltrate the proximal cores of the primary villi during the third week of development. Thus, the villus core includes an inner meshwork of mesoderm-derived stroma consisting of fibroblasts and immune cells (notably macrophages) as well as blood vessels that are contiguous with the fetal circulation via the umbilical vessels. The core is lined by a trophoblast bilayer containing an outer STB layer and an inner CTB layer, which are physically separated from the stroma by a laminin-rich basement membrane. Blood vessels known as spiral arteries course through the decidua basalis and supply maternal blood to the intervillous spaces. Since STB lines the outer surface of the villi, it directly bathes in maternal blood and forms a key site of interaction between maternal and fetal tissue. A schematic illustrating the primitive and definitive STB is presented in ELF5, TP63, ID2, and TEAD4. Simultaneously, factors implicated in nutrient transport, immunomodulation, and hormone biosynthesis and metabolism are induced. The integration of multiple signaling pathways and transcription factors (TFs) are implicated in the process of CTB differentiation, including suppression of WNT and activin/transforming growth factor beta (TGF-β) signaling as well as activation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) pathways. CTB differentiation also involves the activity of many TFs and epigenetic regulators including PPARG, DLX3, GCM1, TFAP2A, OVOL1, and many others. A detailed characterization of TFs implicated in trophoblast differentiation is discussed in several comprehensive reviews  and ERVFRD-1 (encodes syncytin-2). Syncytin-1, which is expressed in STB, binds to the neutral amino acid transporter ASCT-2 expressed mainly by CTBs. Syncytin-2, on the other hand, is expressed in small clusters of CTBs and binds to MFSD2A, which is expressed by STB . Preeclampsia is a serious disease characterized by vascular damage and hypertension in the mother during the latter half of pregnancy that can result in further organ deficiency and damage. Currently, the only definitive treatment is to remove the placenta and therefore deliver the baby, which can lead to complications associated with prematurity if performed prior to 37 weeks. FGR is the failure of a fetus to achieve its growth potential as predetermined by genetic and epigenetic factors . Low birModels commonly used to study human STB are listed in ex vivo analyses that are biopsied or removed only when diseased, it is possible to collect placental tissue from pregnancies deemed healthy. To study STB biology, villus explants can be cultured for defined time periods, which is advantageous to study STB function while preserving tissue integrity  and is often considered clinical waste, it is possible to conduct experiments using placental tissue. Moreover, unlike many other tissues used for ntegrity . CTBs cantegrity . IsolateChoriocarcinoma cell-lines are a valuable tool to study STB biology due to their resiliency and extended lifespan in culture. BeWo cells were derived from a brain metastasis, serially cultivated, and are adapted to cell culture . DiffereIn 2018, culture conditions to maintain trophoblast stem cells (TSCs) from human embryos or first-trimester placentas were determined . These cHuman embryonic stem cells (hESCs) and pluripotent stem cells (hPSCs) cultured under defined culture conditions differentiate into cells with features consistent with trophoblasts, including STB . In part‘Omics’ technologies provide a holistic and integrative approach toward the study of biological systems. To obtain a systems level understanding of biology and disease, large-scale data on DNA, RNA, protein, and/or metabolites are produced, which are then organized by computational tools to provide a framework for the hierarchical contribution of integrated cellular pathways . The terGenomics is the study of the genome and the genetic basis underlying disease. The emergence of high-throughput methods, such as genotype arrays and next-generation sequencing (NGS), have enabled large-scale analyses of DNA sequences to identify copy number variations, small insertions and deletions, as well as single nucleotide variations between individuals, tissues, or cells . EpigenoTranscriptomics is the study of the transcriptome that comprises the entire set of transcripts present in a cell or organism. Transcriptomics provides insight into particular types and levels of RNA molecules, including mRNA and non-coding RNA , and is often used to evaluate changes in gene expression . TranscrThe proteome is the set of all expressed proteins in a cell, tissue, or organism. Proteomics research encompasses protein expression profiles, post-translational modifications, and protein networks related to cellular function, and it endeavors to understand the biological functions of proteins, which holds promise in biomarker discovery . ProteomData obtained from omics experiments require detailed bioinformatic analysis and statistics to organize and interpret the information. Methods used for analysis of omics datasets include enrichment and network analyses, or other statistical tools. The integration of multiple omics by interpreting data from several sources has enabled a more detailed understanding of biological processes . TherefoOCT4) were downregulated , and cell fusion , whereas downregulated genes included those associated with mitosis (GABRP) were highly expressed in >70 μm hESC-derived STB, but not in primary CTB-derived STB, whereas the reverse was true for others   were identified as putative markers of STB in early pregnancy. Proteins encoded by these transcripts (which promote cell invasion in some cancers) showed reduced expression as gestation advanced, indicating that hESC-derived STB might represent the invasive STB population that forms soon after implantation  . To deteantation .OCT4 . The subset of these sites containing TF motifs was associated with genes controlling trophoblast invasion and placental development, and protein–protein interaction data were incorporated to construct a network highlighting candidate TFs that may be important in these processes  cultured in defined conditions (such as with BMP4 or in micromesh culture) provide evidence that these cells possess STB-like gene expression profiles . Tsuchid in situ . Li et antiation . The useethylome .Notably, the aforementioned studies applied hESC/hPSC differentiation paradigms that typically resulted in a heterogenous mixture of trophoblast-like cells rather than STB exclusively, with limited temporal control over differentiation events. Additionally, cells arising from these differentiation paradigms do not fulfill all criteria for trophoblast identity, suggesting either incomplete reprogramming to trophoblast or the possibility that cells other than trophoblast are formed, namely mesoderm or amnion . Neverthin vivo environment when studying early developmental events. Separate cell clusters were enriched in STB, CTB, or extravillous trophoblast (EVT) marker genes. Moreover, three time-dependent genetic networks were characterized between days 6 and 10, including an early (possibly pre-implantation) stage associated with epithelial development, a middle (peri-implantation) stage featuring expression of fusion-related genes, and a late (post-implantation) stage featuring genes associated with cell migration. The authors then determined when STB segregated from cells expressing CTB and EVT markers, demonstrating that STB first appeared in co-cultures between days 7 and 8. Putative upstream regulators were screened to identify TBX3 as a novel TF required for STB formation, which was validated through knockdown experiments using JEG-3 cells  could lead to variability in gene expression levels. -3 cells . CollectVarious omics approaches have also been applied to develop and profile hTSC, naïve hPSC, and organoid models that have been utilized to study STB development. For instance, PEG3, ZFAT, and PROSER2-AS1) in transdifferentiated cells that was not apparent in placenta-derived hTSCs  but not from conventional primed hPSCs (post-implantation epiblast) . These ned hTSCs . Io et aed hTSCs . Guo et ed hTSCs . Collectin vitro, they also spontaneously generate functionally active, hCG-secreting STB toward the luminal surface of the organoid. RNA-seq analysis comparing CTB organoids with primary villus CTBs (freshly isolated or differentiated for 72 h into STB) was conducted, demonstrating similarities in gene expression between organoids, villus CTBs, and in vitro-generated STB in monolayer culture . The majority of these studies take advantage of transcriptomics, such as DNA microarray, to better understand the molecular underpinnings governing CTB differentiation and fusion into STB. SIK1, which was rapidly upregulated in BeWo cells exposed to forskolin and whose silencing via CRISPR/Cas9 strongly abrogated cell fusion and, to a lesser extent, biochemical differentiation  and biochemical differentiation (including production of hormones such as hCG and hPL) during syncytialization through independent mechanisms , 2008. Tntiation . To addrntiation .in situ to STB, suggesting that they may play roles in STB development or function. For example, one such gene was DLG5, which regulates apical polarity complexes and epithelial-to-mesenchymal transition, and thus may be important for maintaining STB integrity  cultured under differentiating conditions. Transcripts decreased in OVOL1-deficient cells included those vital for STB endocrine function. Furthermore, knockdown of OVOL1 in BeWo cells, primary CTBs, and BMP4-treated hESCs reduced expression of several ERV genes including ERVW-1 and ERVFRD-1. By examining the microarray dataset for potential intermediary targets of OVOL1, it was discovered that genes encoding key factors that maintain CTBs in a progenitor state such as MYC, ID1, and TP63 were downregulated following induced differentiation and were subsequently shown to be direct targets of OVOL1 regulation. Therefore, OVOL1 facilitates CTB differentiation and induction of STB-associated transcripts by repressing genes that maintain progenitor traits  in both models following STB formation, and whose protein expression was confirmed via western blotting and immunohistochemistry    . Vento-T ADHFE1) . Liu et ication) .CSH2, CSHL1, GH2, and CGA), PSGs, and genes associated with immunity (HPGD and class II human leukocyte antigens). Interactome analysis of receptor-ligand pairs revealed abundant potential for communication between STB and decidual cells. For example, STB and decidual cells express corresponding ligand-receptor pairs for WNT family members, prostaglandins, hormones, cytokines, and growth factors that may help modulate tissue-specific functions during pregnancy  showed altered methylation of numerous CpGs within and near genes linked to cell differentiation and commitment, with upregulated and downregulated genes showing decreased and increased methylation, respectively. Furthermore, integration of the RNA-seq dataset with genome-wide localization of key histone marks using ChIP-seq indicated that syncytialization was associated with a gain in transcriptionally active marks  among genes that were either expressed constitutively or upregulated following forskolin treatment, with no change in repressive histone modifications  compared to other placental lineages, consistent with the notion that DNA methylation serves as a key epigenetic mechanism for gene regulation in the placenta (Epigenomics techniques have been used to provide more detailed insights into the dynamic epigenetic networks orchestrating villus STB development. ications . DNA metormation . Recentlplacenta .TEAD4 and OVOL1) as well as genes with novel regulatory roles in this process . These findings prompted subsequent investigations into the functional roles of specific HDAC enzymes  during STB formation, which identified HDAC1 and HDAC2 as critical mediators driving CTB differentiation , and other genes associated with immunomodulatory functions . Genes downregulated in STB included negative regulators of differentiation  and genes encoding proinflammatory TFs . Moreover, while promoter enrichment of repressive histone markers remained low in STB, increased and decreased polymerase II binding to promoters of a subset of genes during differentiation was closely correlated with increased and decreased active histone marks, respectively  were detected in samples collected from the basal plate, whereas higher levels of formate and very low-density lipoproteins were identified in samples collected closer to the chorionic plate  cellular heterogeneity of placental tissue, researchers have analyzed endogenous and secreted metabolites from placental explants and CTBs cultured for defined periods of time. For example, o 20% O2 . Horgan tabolism . Functiotabolism .Sub-proteomics analyses have been used to characterize components of the STB microvillus membrane and mitochondria. 2 species generation, and the tricarboxylic acid cycle. Although this analysis was conducted using whole placental tissue, immunostaining subsequently localized selected mitochondrial proteins to STB, including TFRC, PRDX3, and HSPE1  blood. Omics technologies have provided deeper insight into the plethora of substances produced and released by STB. STBEVs are released by STB into maternal circulation and have the potential to provide important diagnostic information about placental health. Therefore, studies have used omics strategies to characterize the composition of STBEVs. 2 tensions  on exosome release and composition in first-trimester CTBs. In total, over 160 exosomal proteins were detected using LC-MS/MS. Lower O2 tensions were associated with increased levels of EV-associated proteins, particularly those associated with hypoxia and IL-8 signaling. Other exosomal proteins were related to cellular movement  (nd VEGF) .One limitation in STBEV omics research has been the variable extraction protocols resulting in differing findings about the number and identity of proteins detected in STBEVs. In order to identify consistencies among the various studies, de novo mutations and common fetal aneuploidies like trisomy 21 , rigorous analysis, validation with multiple techniques or models using independent samples, comprehensive discussion of limitations, and objective interpretation of results. The possibility of using a multi-omics approach is gaining traction to circumvent some of the limitations associated with a “one omics” strategy. For example, in vivo milieu is not reflected in culture models. For instance, stark differences in trophoblast gene expression profiles are apparent when co-cultured with decidualized stromal cells compared to culture without these cells, highlighting the importance of modeling the in vivo environment as closely as possible (Another challenge in understanding STB biology is the limited availability of cell and tissue models. Placental biopsies following delivery are frequently used to evaluate changes in placental function in normal and diseased states, but in many cases the specific role of STB is uncertain because biopsies contain many cell-types in addition to STB. Moreover, samples may vary even if collected from different regions of the same placenta . This vapossible .ex vivo analyses. It is not yet possible to isolate and culture intact, purified STB from placentas, explants, or organoids to study STB in isolation or for use in functional studies. Although the recent upsurge in single cell omics has revolutionized the capacity to study specific cell lineages in complex tissue, STB poses a unique challenge for preparing single cells for analyses because of its enlarged, multinucleated nature. Future exploration incorporating single nucleus RNA-seq may enable a more robust characterization of STB at various stages of its life cycle, whereas spatial transcriptomics offers a promising avenue to study STB biology while preserving its native architectural integrity.Another limitation in the study of STB is its characteristic morphology and fragility that complicates cell isolation for Omics technologies are advancing at a startling rate and are poised to continue providing unparalleled insights into STB development and function. Integration of multiple omics strategies in combination with hypothesis-driven mechanistic approaches and improvements in available cell models will enable a better understanding of how this cell lineage functions in normal and diseased states. Given the importance of STB for pregnancy health and its unique position contiguous with maternal blood, studies of STB biology that incorporate omics approaches will be instrumental for driving discovery of novel biomarkers of placental stress or pregnancy disease.AJ, MJ, and SR wrote and edited the manuscript. GJB edited the manuscript and contributed to preparation of the tables. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.