Probiotics are live microorganisms that confer health benefits on the host. However, in recent years, several concerns on their use have been raised. In particular, industrial processing and storage of probiotic products are still technological challenges as these could severely impair cell viability. On the other hand, safety of live microorganisms should be taken into account, especially when administered to vulnerable people, such as the elderly and immunodeficient individuals. These drawbacks have enhanced the interest toward new products based on non-viable probiotics such as paraprobiotics and postbiotics. In particular, paraprobiotics, defined as “inactivated microbial cells (non-viable) that confer a health benefit to the consumer,” hold the ability to regulate the adaptive and innate immune systems, exhibit anti-inflammatory, antiproliferative and antioxidant properties and exert antagonistic effect against pathogens. Moreover, paraprobiotics can exhibit enhanced safety, assure technological and practical benefits and can also be used in products suitable for people with weak immunity and the elderly. These features offer an important opportunity to prompt the market with novel functional foods or nutraceuticals that are safer and more stable. This review provides an overview of central issues on paraprobiotics and highlights the urgent need for further studies aimed at assessing safety and efficacy of these products and their mechanisms of action in order to support decisions of regulatory authorities. Finally, a definition is proposed that unambiguously distinguishes paraprobiotics from postbiotics. Since the first observation by Metchnikoff more than 100 years ago, the popularity of probiotics boosted substantially. In the last decades, particularly in the last five years, a large body of experimental and clinical evidence on the health benefits of probiotics has appeared . The ongLactobacillus rhamnosus sepsis associated with probiotic therapy. A study regarding 89 patients with Lactobacillus bacteremia reported the mortality rate of 26% within 1 month and 48% within 1 year following infection onset. Even if rare, endocarditis due to Lactobacillus infection results in a high mortality rate, averaging 30%. A randomized controlled trial Probiotic prophylaxis in patients with predicted severe acute pancreatitis (PROPATRIA) highlighted a significantly increased mortality (16% vs. 6%) due to bowel ischemia (9 vs. 0) among severe acute pancreatitis patients subjected to probiotic administration [Saccharomyces was the most frequent genus with 47 cases, followed by Lactobacillus (26 cases), Bifidobacterium (12 cases), Bacillus (5 cases), Pediococcus (2 cases) and Escherichia (1 cases), respectively [Akkermansia muciniphila, Faecalibacterium and Bacteroides species) will keep being launched more and more often [According to the earlier revised definition by the International Scientific Association for Probiotics and Prebiotics (ISAPP), probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” . Thus, pectively ,11,12. Oectively . Viabilire often .All these drawbacks related to the administration of viable microorganisms led to the interest in non-viable probiotic preparations. Since 2004, increasing evidence has been suggesting that some health benefits of physiologically active bacteria are not strictly associated with their viability. In fact, probiotic products also contain dead cells, which can produce a biological response as effectively as their live equivalents, highlighting the fact that probiotic products may be further used beyond their expiry. This is called the “probiotic paradox” , i.e., both live and dead cells can produce a biological response. Though there may be a potential benefit from the consumption of dead microorganisms, they cannot be classified as probiotic . Hence, Although the molecular mechanisms underlying paraprobiotic action still need a thorough investigation, scientific evidence has shown that, similarly to probiotics, molecules present on the cell surface  could constitute the first line of interaction between paraprobiotics and the host, thus mediating the beneficial effects .Paraprobiotics have been proven to modulate anti-inflammatory and positive immune responses in animals and humans, with some advantages if compared to probiotics. Non-viable microbial cells may exhibit enhanced safety, i.e., reduced risk of sepsis and antibiotic resistance, as well as technological and practical benefits, i.e., longer shelf life, since the cold chain is not required for microorganism viability and stability. These features also enable their application in underdeveloped regions ,18.Lactobacillus and Bifidobacteria that have been awarded the GRAS  and QPS  status for intentional addition to food and feed by the FDA and EFSA respectively [Escherichia coli, Bacillus, Saccharomyces) and next-generation probiotics that need to be studied for their safety profile  are emerging [Another great advantage is no loss of bioactivity when administered in combination with antibiotics or antifungal agents . Killed ectively , other pemerging ,20. Theremerging .An overview of the current state of the scientific literature setting “paraprobiotics” or “inactivated probiotics” as search terms, which revealed a hike in the number of articles published in the last ten years in different research areas, helps us to realize the growing interest in the inactivated microbial cells .Industrial processing and storage of probiotic products still represent technological challenges as these could severely impair probiotic cell viability, putatively a key requisite for the probiotic effects. As a matter of fact, probiotic products actually contain viable and non-viable cells that could both contribute to the beneficial effects on human health ,23,24. OThe use of paraprobiotics allows overcoming several of these drawbacks and opens up new perspectives in the design of novel functional foods, significantly simplifying industrial handling and marketing. In fact, paraprobiotics could be added to several foods that, due to their chemical or physical properties, do not offer a suitable environment for the survival of probiotics (such as fruit juice). Furthermore, paraprobiotics do not directly interact with food matrices and do not modify their organoleptic features . In addiOn the other hand, it should be taken into consideration that inactivated cells should be unable to produce secreted metabolites  and enzymes which could have a relevant role in the probiotic health effects. Due to these features, novel foods containing paraprobiotics are less affected by storage and transport conditions, thus guaranteeing a prolonged shelf life and assuring economic advantages. These points could offer an important opportunity to prompt the functional food market with safer and more stable products. Inactivation of probiotic cells can be achieved using physical or chemical treatments capable of modifying microbial cell components  and physiological functions (enzyme activities and membrane selectivity) without completely destroying the cell structure. Conventional and emerging technologies for the production of paraprobiotics comprehensively reviewed by de Almada et al.  include Lactobacillus acidophilus, Lacticaseibacillus casei, and Bifidobacterium animalis [In particular, thermal treatments that are still the most widely used processes for producing paraprobiotics at laboratory and industrial levels  damage tanimalis .Similarly, high hydrostatic pressure and high-pressure homogenization treatments also used in combination with thermal processes can cause membrane rupture due to shear stress, as well as alteration of ribosomes and irreversible protein denaturation and coagulation leading to the inactivation of biological functions mediated by enzymes, extensive loss of solute and reduction of intracellular pH ,38.More recently, the application of high-intensity ultrasound (HIUS) in the inactivation of probiotics has been reviewed . The effOn the contrary, other inactivation methods more specifically target particular cell components. Microbial inactivation by ionizing radiation (gamma rays or X-rays) is mainly due to the damage of nucleic acids caused by oxidative radicals originating from the radiolysis of water . SimilarA sound body of scientific evidence shows the beneficial effects of paraprobiotics on human and animal health, thus definitively assessing that cell vitality is not an absolute prerequisite for the health effects . HoweverL. rhamnosus GG cells were equally effective in decreasing IL-8 production in the intestinal epithelium cells (Caco-2 cells), but their mechanisms of action involved different pathways [The ability to modulate the adaptive and innate immune systems represents the key feature of the paraprobiotic action ,22. Intrpathways . L. acidophilus (treated at 60 °C for 30 min) were able to inhibit the attachment of pathogenic bacteria  to Caco-2 cells [L. rhamnosus CNCM I-3698 and Lactobacillus farciminis CNCM I-3699 (autoclaved at 120 °C for 15 min) exhibited co-aggregative abilities toward Campylobacter jejuni and higher exclusion potential against binding of this pathogen to mucin compared to the live counterparts. The ability of inactivated cells to adhere to different intestinal matrix models  was also reported [Aggregation and adhesion are also important properties of probiotics, being involved in gut colonization and antagonistic effect against pathogens. These features could be affected by experimental conditions used for cell inactivation. Ostad et al. demonstrated that both live and heat-inactivated forms of fecal -2 cells . Similarreported .Lactobacillus reuteri strains  toward select pathogens. The reported data highlighted that these properties were strictly strain-specific. The inactivated cells (80 °C for 10 min) adhered to Caco-2 cells, although to a slightly lesser extent compared to the live counterparts, while the pathogen inhibition abilities were significantly reduced. The adhesion and antagonistic potential of the probiotic strains were lost upon exposure to 5 M LiCl, thus indicating the involvement of surface proteins [More recently, Singh et al. investigated the adhesion and antagonistic activity of several probiotic proteins .As a matter of fact, morphological changes on several heat-treated lactic acid bacteria strains were observed by field-emission scanning electron microscope (FE-SEM). In particular, resulting cell surfaces of all the heat-treated bacteria were rougher and more uneven than those of viable untreated cells. This finding was accompanied by a decrease of the adhesive ability of heat-killed bacteria with increasing temperature. Interestingly, heat treatment decreased the adhesion ability but did not affect the immunostimulatory activity .However, further studies are required to definitively assess whether paraprobiotics retain their adhesion and pathogen exclusion abilities and clarify how these features are affected by the inactivation processes, applying the most up-to-date methodologies. It should also be kept in mind that models used to assess cell adhesion in vitro only represent simplifications of in vivo conditions and the counting of truly adherent paraprobiotic cells could still present several experimental difficulties .Bifidobacterium longum SPM1207 isolated from healthy adults and orally administrated to rats retained the ability of lowering cholesterol, blocking the body weight increase and relieving or eliminating constipation in rats, as also shown for the viable probiotic cells [Another interesting feature of probiotics is their ability to remove cholesterol from media via several possible mechanisms including assimilation during growth and incorporation into the cell membrane ,50. It hic cells . Howeveric cells .Detailed studies aimed at investigating the molecular mechanisms at the basis of the exhibited health effects of paraprobiotics and performing comparisons with viable counterparts are still required and crucial to set up inactivation protocols that preserve their beneficial action. Paraprobiotics are defined as “inactivated microbial cells (non-viable) that confer a health benefit to the consumer” . ConsideFlow cytometry is a potential analytical technique that holds the potential to quantify non-viable cells in a matrix. Over the last 20 years, flow cytometry has gained increased popularity in microbiological research since it allows the determination of viable bacteria but also the enumeration of damaged/dead cells . This teThe flow cytometry principle is based on dual nucleic acid staining with a cell-permeant dye  and a cell-impermeant dye (propidium iodide). Thiazole orange or equivalents permeates membranes of total cells and stains the nucleic acids with green fluorescence. Propidium iodide penetrates only bacteria with damaged membranes, causing a reduction in thiazole orange fluorescence when both dyes are present. Thus, live cells with intact cell membranes fluoresce bright green, bacteria with slightly damaged membranes exhibit both green and red fluorescence, whereas bacteria with broken membranes fluoresce red ,53. The However, it has to be considered that by using flow cytometry, it is possible to assess the content of cells and their viability, but no taxonomical information on the microorganisms that are present can be obtained. Since the concept of “probiotic” also requires the identification of probiotic microorganisms in the final product, flow cytometry should be used as a complementary method along with PCR methods to allow paraprobiotics detection, count and identification. In addition, a study of Klein et al. showed the suitability of a proteomic approach based on capillary electrophoresis coupled to mass spectrometry (CE/MS) for quality control of inactivated probiotics preparations .Soejima et al. have recently launched digital PCR as a robust tool for the routine analysis of heat-killed lactobacilli-supplemented foods . This asDespite favorable perspective on the use of paraprobiotics, several aspects of this concept are not fully understood yet. The term “paraprobiotic” is misleading by itself, for it suggests, if literally taken, that it is effective only if administered in the presence  of a probiotic. The proliferation of overlapping terminology and the absence of a universally recognized definition induce vagueness, which makes challenging communication both between researchers and of the concept to the consumer. The current situation requires a consensus panel in order to draw attention to the confusion that reigns within the probiotic glossary and to address the emerging terms in the “biotics” field with the objective of establishing a generally agreed terminology. Some confusion also derives from the definition of paraprobiotics that includes non-viable intact or broken cells  or the cellular extract. This entails a partial overlap with the term “postbiotics”, defined as extracts of non-viable probiotics and comprising cell membrane components such as surface proteins, lipopolysaccharides, teichoic acids, etc. ,57.We propose defining the term “paraprobiotic” as “inactivated microbial cells (non-viable), specifically, cells as a whole, including both structural components and synthesized or excreted metabolites that confer a health benefit to the consumer.” In addition, we recommend defining the term “postbiotics” as “compounds derived from microbial metabolism synthesized by cells or produced in the matrix by enzymatic action.” Postbiotics can be single metabolites or even very complex mixtures. A detailed definition of the different terms and the cell components involved in the biological activities are reported in There are several key points to be clarified in order to support regulatory authorities for defining the requirements for the registration and approval of foods and dietary supplements containing paraprobiotics. In addition to a punctual and unequivocal definition recognized at the international level, specific attention is required with regard to paraprobiotics production methods, quality control criteria, how to detect and quantify their presence and how to assess their safety and efficacy.Means of inactivation may affect the physiological activity of the resulting dead cells and the stability of their beneficial effects during shelf life anyway. This is another aspect to deepen and clarify to make the best possible use of paraprobiotic opportunities .Given that the viability of probiotics in food is a critical factor, some food matrices are more suitable to deliver probiotics. The food carrier also affects bacterial susceptibility to harsh gastrointestinal conditions , ability to adhere to intestinal epithelial cells and immunomodulatory properties. Since the buffering capacity of milk and milk fat assures probiotic survival through processing and storage, dairy products, including yoghurt and other fermented milks, cheese and frozen fermented dairy desserts, represent an ideal and marketable carrier of probiotic bacteria to consumers ,59.Lactobacillus and Bifidobacterium, have been actually found in milk culture, yoghurts and cheese [Particularly, yoghurt is a very efficient probiotic vehicle compared with other food products, but also ice cream, which is rich in milk fat, is effective in enhancing microbial survivability and acid tolerance ,61. Neved cheese .Paraprobiotics could be of particular interest for the dairy sector, since they remain stable in a wide range of pH and temperature, allowing the incorporation into foods with high acidity and before thermal processing without loss of functionality. Their addition does not change the sensory properties of the product, thus avoiding detrimental modifications like high acidification after fermentation in yoghurt . ActuallUp to now, only a few works have addressed the potential application in the dairy industry of paraprobiotics added or generated during processing, and clinical studies have been performed to investigate the efficacy of the products containing paraprobiotics and their health effect at the physiological level .Lactobacillus gasseri CP2305  has been proven to improve sleep quality and normalize bowel habits in subjects under stressful conditions [Some studies reported that paraprobiotics affect behavior under chronic or prolonged stress situations. The daily consumption of paraprobiotic nditions . The samnditions  and it wnditions .Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lb. acidophilus has also been recognized to prevent impaired barrier function in gut epithelium in human intestinal Caco-2 cells [A yoghurt containing paraprobiotic cells derived from -2 cells . Lactobacillus paracasei CBA L74 showed an increase in butyrate producers , Bacteroides and different Roseburia and Blautia oligotypes [Non-viable bacteria in fermented milk are also useful to modulate gut microbiota. A clinical study conducted on young children fed with a cow’s milk fermented product containing the heat-killed probiotic strain igotypes .A clinical study by Liu et al. provided evidence that pasteurized yoghurt with no viable lactic acid bacteria consumed twice daily for seven weeks was effective in improving constipation symptoms and intestinal health .Lactococcus lactis inactivated upon pasteurization applied to fermented milk have been reported to possess antihypertensive potential and to remove cholesterol, probably via cholesterol attachment to their cell surface thanks to the chemical and structural properties of the cell wall peptidoglycans [Nongrowing and dead cells of oglycans .Finally, although the high-intensity ultrasound (HIUS) technology showed the potential to generate paraprobiotics while improving the fermented dairy product processing , there are no studies focusing on the contribution of paraprobiotics in sonicated dairy products .Although dairy products are currently the most common food carrier to deliver probiotics and paraprobiotics, an increasing number of non-dairy food matrices is attempted as potential for delivery of these microbial components. In fact, in recent years, the increasing health concerns regarding dairy products resulted in a shift towards non-dairy foods such as cereal-based products, fruit and vegetable drinks and ice cream ,71.There are several reasons why people might be looking for substitutes of probiotic and paraprobiotic dairy products, such as milk protein allergy, as 2–3% of children under three have a milk allergy ; lactoseThese concepts are even more important for the elderly people that may have multimorbidity and diverse risk factors. Considering that life expectancy has increased rapidly in the recent years as well as the cost of medical care, the development of new non-dairy functional foods becomes essential to promote healthy diets and improve life quality. Therefore, both probiotics and paraprobiotics could be extensively used in functional non-dairy foods, medicine, supplements and fodder ,77.Nowadays, functional foods containing probiotics represent an industrial sector with growing market shares and large commercial interest . HoweverTo overcome these disadvantages, paraprobiotics can be added as ingredients in non-dairy products. Their application in foods and beverages could comprise an important alternative for specific cases in which probiotics are damaged and do not survive during processing and/or shelf life, although these aspects are poorly explored and deserve thorough investigation.Helicobacter therapy [Saccharomyces or Bifidobacterium probiotic strains, that provide health benefits when administered with diet were recorded by de Almada et al. [At the clinical level, there is currently an increasing interest in the use of heat-killed preparations of different probiotic strains in the management of intestinal, allergic respiratory and topical diseases and also as support in  therapy . So far,a et al. .10 heat-killed L. paracasei MCC1849 in healthy young adults [Bifidobacterium longum on obesity and blood glucose levels in an obese diabetes murine model were evaluated by administering inactivated Bifidobacterium cells orally for five weeks. The treated mice showed a significant decrease of body weight gain, adipose tissue mass and blood glucose levels, as well as reduced levels of cholesterol and triglycerides [Murata et al. confirmed the safety of continuous oral intake of a powder containing about 1×10g adults . In addiycerides .Helicobacter pylori infection [Currently, some paraprobiotic products with proven effects in the prevention or treatment of some diseases are commercially available as supplements . Many ofnfection ,84 or innfection ,86.L. gasseri CP2305 strain inactivated by heat. The daily consumption for 12 weeks of sports drinks containing heat-inactivated L. gasseri CP2305 contributed to recovery from fatigue and alleviation of anxiety and depressive moods in young athletes facing stressful situations [Lacticaseibacillus casei 01 inactivated by ohmic heating. Interestingly, this drink exhibited hypoglycemic activity in vitro (through the inhibition of α-glucosidase and α-amylase) and was effective in reducing postprandial glycemia in healthy individuals [To date, applications of paraprobiotics added or generated during the processing of non-dairy foods are very scarce . One of tuations . Furthertuations . The contuations . Very reividuals .Moreover, for the widespread application of paraprobiotics in foods, a few conditions should be addressed, such as ease of production, good solubility in foods, lack of or reduced interaction with foods and stability during processing and storage. Last but not least, the elucidation of the mechanisms of action of paraprobiotics, including the determination of specific active compounds responsible for the beneficial effects, comprise key information to enable not only the application of paraprobiotics in foods, but also their regulation by health authorities . Modulation of the host’s adaptive and innate immune system is a key mechanism underlying the beneficial effects exerted by probiotics on animal and human health, in particular in the prevention and treatment of pathologies such as atopic dermatitis, gastrointestinal and respiratory diseases, virally induced infectious diseases, infections and allergies.Innate immune cells, including neutrophils, monocytes, macrophages and natural killer cells, represent the first defense system activated by the host to face off with infection by pathogens, even if these cells are only slightly specific in the recognition of their targets. However, they provide an interface between innate and adaptive immune response as they act as professional antigen-presenting cells (APCs), thus promoting the specific immune response that involves different subsets of T helper cells , regulatory T cells and B cells. As innate and adaptive immunity are deeply interconnected, probiotics have a multifaceted role in immunomodulation. The reported effects exerted by probiotics on host immune response include, among others, modulation of the profile of secreted cytokines, both proinflammatory (interleukin 12 (IL-12), IL-3, tumor necrosis factor alpha (TFN-α), interferon gamma (IFN-γ), etc.) and anti-inflammatory ; stimulation of the production of immunoglobulins  by B cells; inhibition of the signaling pathways such as the nuclear factor-κB pathway ,94,95,96The innate immune response depends on transmembrane pattern recognition receptors (PPRs) such as Toll-like receptors (TLRs) of host epithelial and immune cells  that link microbial-associated molecular patterns (MAMPs) present on the microorganisms and intracellular nucleotide-binding oligomerization domain (NOD) proteins that sense MAMPs in the cytoplasm and recognize pathogens that replicate intracellularly ,98. As aLactobacillus strains on mouse splenocyte proliferation and the activation of mouse dendritic cells, demonstrating their efficacy in modulating interleukin secretion [Lactobacillus rhamnosus GG cells both reduced the production of IL-8 upon flagellin induction in human epithelial colorectal adenocarcinoma Caco-2 cell line [L. rhamnosus ATCC 7469 were both able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6) or regulatory (IL-10) functions in mouse macrophages [L. brevis KCCM 12203P cells in comparison with their viable counterpart have been assessed [B. animalis subsp. lactis, BB-12 and L. rhamnosus GG on cultured Caco-2 cells mainly on barrier integrity and production of inflammatory mediators have been investigated [Several studies investigated the impact of paraprobiotics on the immune system response in comparison with their probiotic counterpart mainly in different cell lines. For instance, Chuang et al. examined the effects of three heat-killed ecretion . Lopez eell line . It has rophages . More reassessed  and the stigated .In many cases, mechanisms and health effects of paraprobiotics are very similar to those exerted by probiotics in terms of stimulation of cytokine expression and activation of the cell signaling system, indicating that the loss of viability due to cell inactivation does not hamper beneficial functions. Reduction in the expression of proinflammatory cytokines  and enhanced synthesis of anti-inflammatory cytokines (IL-10) is a key mechanism associated with beneficial effects on anti-inflammatory diseases. A switch in Th1/Th2 cell differentiation that involves the increased expression of IL-12 and IFN-γ and the decreased expression of IL4 and IL13 thus enhancing Th1 cell population is the basis of positive effects on allergies and atopic dermatitis treatment ,22,108. In the last decade, evidence of beneficial effects of paraprobiotics in prophylaxis and treatment of several pathologies including diarrhea, colitis, respiratory, intestinal and liver diseases induced by alcohol, inflammation and allergies have been corroborated by in vivo studies in murine models and human clinical trials ,27,108. Lactobacillus gasseri TMC0356 cells to increase the expression of IL-12 and interferon-α and -β receptor 1 and the activities of splenic natural killer cells demonstrated that oral administration of the paraprobiotic could modulate cell-mediated immunity [Lactococcus lactis G50 enhanced intestinal immunity and inhibited the growth of enteric H2S-producing bacteria [Lactobacillus pentosus b240 induced an enhanced secretion of the salivary immunoglobulin A (sIgA) that plays a key role in mediating humoral mucosal immune response [L. pentosus b240 exerted protective effects against the common cold in elderly adults (reducing its incidence rate) related to the ability of this paraprobiotic to improve mucosal immunity through enhanced secretion of sIgA. The elderly group following the supplemented diet also experienced an increased general well-being independently from common cold, thus suggesting other beneficial effects due to paraprobiotic consumption [Lactobacillus paracasei MCC1849 cells caused similar effects  on a pre-specified subgroup of subjects of young healthy adults susceptible to common cold [Short-lived senescence-accelerated mouse prone 1 (SAMP1) has been used as an animal model to investigate paraprobiotic effects on age-related immunosenescence. The ability of heat-killed immunity . In a sibacteria . The decbacteria . In a raresponse . Moreovesumption . Interesmon cold .Lactobacillus plantarum L-137 cells has been previously shown in healthy adults including a limited subgroup of elderly subjects [Lactobacillus fermentum and Lactobacillus delbrueckii subjected to an extensive high-temperature post-production treatment) has proved to positively affect social behavior and reduce baseline corticosterone levels (stress hormone) in healthy mice, suggesting potential anxiolytic and antidepressant effects of paraprobiotics [A strict relationship between the enhancement of immune functions and the improvement of health-related quality of life (QOL) prompted by heat-killed subjects . More reobiotics .As a matter of fact, anxiety, stress and depression are frequently encountered in the elderly and constitute additional risk factors for their well-being. In this frame, the possibility to use paraprobiotics as a natural aid to alleviate anxiety, stress and depression symptoms could be particularly important for the elderly, as they are more susceptible to drug-induced side effects.In the last years, evidence of the beneficial impact that probiotics (prebiotics and synbiotics) could have on cognitive functions and neurological diseases via the microbiota–gut–brain axis are gaining increasing attention .Although knowledge in this area remains limited and further studies are needed to definitively assess the health-promoting effects of paraprobiotics, these findings reinforce the idea of the potential impact of paraprobiotics for ameliorating general well-being, a perspective that could be particularly important for frail subjects.Paraprobiotics represent an important opportunity for the development of innovative functional foods suitable for people with weak immunity. These products are also characterized by greater stability and can be stored without the cold chain, thereby facilitating industrial handling and wide commercialization.However, there are several aspects that need to be defined and others on which the scientific community must do substantial work.Probiotic cell inactivation may be achieved by physical or chemical procedures, which may alter cell structure and function, making bacteria lose their ability to grow and reproduce while preserving (or not) the beneficial effects exhibited by their viable counterparts. Thus, a further challenge is the use of appropriate methodologies to evaluate their biological activity and to identify the components responsible for the health effect. In addition, some challenges exist when considering food as a delivery vehicle for paraprobiotics, similarly to what happens for probiotics. Easy production, i.e., fast, controlled and affordable inactivation methods, good solubility into foods and limited interaction with food components are other aspects to be taken into account for practical application of paraprobiotics in foods. Their application must then necessarily go through the development of an approach able to correlate the biological response with the physical state and vitality of the microbial strain and to allow quality control of these products and regulatory interventions .


Bos indicus) with improved (Bos taurus) breeds gained momentum and economic relevance in several countries to increase milk production. While production performance of the crossbred offspring is high due to hybrid vigor, they suffer from a high incidence of reproductive problems. Specifically, the crossbred males suffer from serious forms of subfertility/infertility, which can have a significant effect because semen from a single male is used to breed several thousand females. During the last two decades, attempts have been made to understand the probable reasons for infertility in crossbred bulls. Published evidence indicates that testicular cytology indices, hormonal concentrations, sperm phenotypic characteristics and seminal plasma composition were altered in crossbred compared to purebred males. A few recent studies compared crossbred bull semen with purebred bull semen using genomics, transcriptomics, proteomics and metabolomics; molecules potentially associated with subfertility/infertility in crossbred bulls were identified. Nevertheless, the precise reason behind the poor quality of semen and high incidence of sub-fertility/infertility in crossbred bulls are not yet well defined. To identify the underlying etiology for infertility in crossbred bulls, a thorough understanding of the magnitude of the problem and an overview of the prior art is needed; however, such systematically reviewed information is not available. Therefore, the primary focus of this review is to compile and analyze earlier findings on crossbred bull fertility/infertility. In addition, the differences between purebred and crossbred males in terms of testicular composition, sperm phenotypic characteristics, molecular composition, environmental influence and other details are described; future prospects for research on crossbred males are also outlined.Crossbreeding of indigenous cattle ( Bos taurus and Bos indicus is being practiced in several countries  around the world are engaged in milk production, mainly in developing countries . The daiountries  resultinountries . Crossbrountries . Since tountries . Even thountries . Female ountries . Globallountries .Infertility in a bull is defined as the inability to achieve pregnancies, whereas sub-fertile bulls  delay conception, prolong the calving season, reduce calf weaning weights, and increase the numbers of females culled, thereby resulting in economic losses and threatening the sustainability of a livestock operation . Breed vBos taurus male with Bos indicus female, although not sterile, show increased incidences of sub-fertility and/or infertility compared to their parents (Bos indicus) and European taurine cattle (Bos taurus) exhibit several morphological and physiological differences. At chromosome level, the Y-chromosome in Bos taurus is submetacentric while that in Bos indicus is acrocentric. Therefore, it was proposed that the lower fertility in zebu crosses with European cattle could be due to small deletions or position changes between the synapse region of the X and Y chromosomes, or to alterations in genes participating in the regulation of reproduction  are always sterile. The male progeny born via crossbreeding of a  parents . Despiteoduction . With thoduction . While toduction  indicateDespite these fertility problems, crossbreeding is commonly adopted to improve the milk productivity of indicine and non-descript cattle; the reason for the higher incidence of sub-fertility or poor semen quality in crossbred bulls compared to purebred bulls is not fully understood. Understanding the reason behind infertility/subfertility in crossbred bull will assist development of strategies to improve crossbred bull reproduction. Therefore, in this review, we compiled and analyzed earlier findings on crossbred bull fertility/infertility. In addition, the differences between purebred and crossbred males in terms of testicular composition, sperm phenotypic characteristics, molecular composition, environmental influence, and other details are described and future prospects for research on crossbred males are also outlined.B. indicus × B. taurus (22%) bulls than purebred Bos indicus (13%) and Bos taurus (15%) bulls  bulls . More th%) bulls . Among t%) bulls . Ejacula%) bulls . EjaculaEjaculate quality in crossbred bulls is also influenced by the level of exotic inheritance , althougThe incidence of sub-fertility/infertility in crossbred bulls is high compared to purebred bulls. Sire conception rate was higher in indigenous bulls , compared to Holstein Friesian crossbred  and Jersey crossbred  bulls . AnotherHigher culling rates (40–70%) due to poor semen quality, poor sperm cryotolerance and sub-fertility/infertility were reported in crossbred bulls . In a stBos indicus males (Bos taurus (40%) and Bos indicus (29%)  . A signius (29%) . Singh eus (29%) .In an effort to understand the reasons behind the higher incidence of infertility/sub-fertility in crossbred males, several researchers studied the physiological and molecular differences between crossbred and purebred males. Some studies proposed axiomatic evidences at genetic , hormonaDuring spermiogenesis, chromatin compaction occurs by replacing histones with protamines; little or no cytoplasm remains in spermatozoa . TherefoPRM1 is an abundant transcript found in spermatozoa of crossbred bulls  based kits were developed  in purebred bulls compared to crossbred bulls  were upregulated, whereas genes involved in WNT pathway (involved in sperm motility initiation and inhibition of ubiquitination) were downregulated in crossbred testes. The genes involved in steroidogenesis, including CYP17A1 gene  were downregulated in crossbred bull testis. Genes associated with steroidogenesis are downregulated in crossbred bull testis compared to purebred testis. Downregulated steroidogenesis-related genes and their involvement as a group in different biological process, molecular function and pathway are shown in An appropriate molecular environment in the testis is required for proper spermatogenesis. In a recent study, the testicular transcripts related to sperm function and fertilizing potential were altered in crossbred bull testis compared to purebred bull testis . This stIn a study by Successful reproduction depends upon an optimal endocrine milieu. Development of testis, puberty, sexual maturity and spermatogenesis require certain levels of reproductive hormones, mainly follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone . AlthougBos taurus × Bos indicus) cattle  cattle . The X a) cattle . This PA) cattle . Synapse) cattle .Inter-species breeding can cause structural and/or molecular/genetic discrepancy between the PAR of X and Y chromosome in the offspring and interferes in the usual synapse and recombination during male meiosis, resulting in infertility . For insBos taurus with Bos indicus, because the Y chromosome of Bos taurus is submetacentric whereas in Bos indicus it is acrocentric with visible p-arms. The Y chromosomal arms are of different sizes in Bos indicus and Bos taurus. Furthermore, the location of PAR on the Y chromosome differs as it is located on the short arm of Bos taurus Y chromosome but on the long arm of Bos indicus Y chromosome. The difference between the Y chromosome of Bos taurus × Bos indicus is due to pericentric inversion or centromere transposition. The Crossbred (Bos taurus × Bos indicus) bulls have a submetacentric Y chromosome (Bos taurus and Bos indicus (which have different Y chromosome morphology and a different location of PAR in the Y chromosome) can result in structural and/or molecular differences between the PAR of X and Y chromosome in the male offspring. Thus, normal pairing and recombination between the PAR of X and Y chromosome during male meiosis would be disrupted. This may eventually lead to improper spermatogenesis and infertility in crossbred bulls. Although the location of PAR on the short or long arm does not completely block the pairing between X and Y chromosomes, it interrupts the success of X-Y pairing by having genetic consequences in structural chromosomal reorganization. For instance, isochromosome formation results in duplication or complete deletion in one of the sex chromosomal regions (The same reasoning can be speculated for the progeny born out of the crossbreeding romosome . As ment regions .SRY), DEAD box polypeptide 3-Y chromosome (DDX3Y) and Testis-specific protein on Y chromosome (TSPY)] of Holstein Friesian crossbred bulls compared to Sahiwal (Bos indicus) bulls. Furthermore, PRAMEY , TSPY , ZNF280BY , and HSFY  in the Y chromosome of F1 hybrid cattle-yak bulls than in cattle bulls. However, in-depth studies involving a large number of different purebred and crossbred males are required to understand the effect of a discrepancy between the PAR of X and Y chromosome on male fertility.Disparity between sex chromosomes due to species hybridization or crossbreeding may also lead to deletions and duplication of genes in the offspring. This is supported by the findings of Bos taurus with Bos indicus might eventually lead to improper spermatogenesis and infertility in crossbred bulls because of differences in the Y chromosome. The Y chromosome of Bos taurus is submetacentric, whereas in Bos indicus it is acrocentric. Differences in location of PAR in the Y chromosome might disrupt the process of normal pairing and recombination between the PAR of X and Y chromosome during male meiosis. Information about the influence of the level of exotic blood on fertility in crossbred males is very limited. Schematic representation of the possible cellular and molecular alterations in crossbred testis and spermatozoa that might culminate in subfertility/infertility is given in Undoubtfully, crossbreeding of low-producing zebu cows with exotic bulls of high genetic merit has resulted in the production of superior genotypes with hybrid vigor and enhanced milk production efficiency. On the other hand, it is evident from the foregoing information that the magnitude of reproductive problems is higher in crossbred bulls compared to purebred bulls. Until recently, little research had been done to identify the underlying etiological factors for this high incidence of infertility/sub-fertility in crossbred bulls. During the last decade, considerable research has been conducted; published information suggests alterations at the level of testis, spermatozoa, seminal plasma and male reproductive hormones, in crossbred bulls compared to purebred bulls. It is also evident that crossbreeding of Bos taurus, Bos indicus, and their crossbred male offspring should be studied to understand the origin of these reproductive problems in crossbred males. Expression of genes such as DDX3Y, RINGO, and SPATA7 and metabolites such as hypotaurine, L-malic acid, selenocysteine, D-cysteine, and chondroitin 4-sulfate are to be studied further to determine their role in crossbred male reproduction. Testicular transcriptomic study indicated an inability of the crossbred testis to maintain protein stability and steroidogenesis, which could be the pressure point for reproductive problems in crossbred bulls. Therefore, research to understand and improve protein stability and steroidogenesis in crossbred bulls will be important in the future. In addition, tailored freezing techniques and specific extenders need to be tested because poor freezability of spermatozoa is an important problem in crossbred bulls. Although sperm mRNAs (messenger RNA) were studied to some extent in crossbred bulls, the other RNAs such as transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), mitochondrial RNA (mt-RNA), small non-coding RNA (sncRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA) need to be investigated to broaden our understanding about crossbred bull fertility. Studies involving microRNA, small interfering RNA, their intracellular delivery and target genes in crossbred bulls are particularly warranted. Furthermore, use of technologies such as gene transfer, editing, slicing and knockout may be useful in future to understand and improve the fertility in crossbred bulls.Detailed and large-scale studies involving crossbred males with different levels of exotic inheritance are required to have a clear understanding about infertility/sub-fertility in crossbred bulls. Genes in the pseudo autosomal regions of X and Y chromosome of AK, KE, and TD conceptualized the review. All authors were involved in literature review and development of the manuscript, and approved the manuscript 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.


Abnormalities in programmed cell death (PCD) signaling cascades can be observed in the development and progression of various cardiovascular diseases, such as apoptosis, necrosis, pyroptosis, ferroptosis, and cell death associated with autophagy. Aberrant activation of PCD pathways is a common feature leading to excessive cardiac remodeling and heart failure, involved in the pathogenesis of various cardiovascular diseases. Conversely, timely activation of PCD remodels cardiac structure and function after injury in a spatially or temporally restricted manner and corrects cardiac development similarly. As many cardiovascular diseases exhibit abnormalities in PCD pathways, drugs that can inhibit or modulate PCD may be critical in future therapeutic strategies. In this review, we briefly describe the process of various types of PCD and their roles in the occurrence and development of cardiovascular diseases. We also discuss the interplay between different cell death signaling cascades and summarize pharmaceutical agents targeting key players in cell death signaling pathways that have progressed to clinical trials. Ultimately a better understanding of PCD involved in cardiovascular diseases may lead to new avenues for therapy. The development and homeostasis of multicellular organisms depend not only on the regulation of cell survival and renewal, but also on the processing of those cells that are no longer needed or pose a potential danger to the organism. This includes the removal of cells at risk of neoplastic transformation or those infected by pathogens . ProgramAt present, increasing studies suggest that PCD is a significant cause of cardiovascular diseases such as ischemia/reperfusion (I/R) injury, myocardial infarction (MI) and cardiomyopathy . Each PCMoreover, many clinical trials have found that the application of appropriate drugs could target cell death signaling pathways in cardiovascular diseases. A clinical phase 2 trial of simvastatin found that targeting inhibition of apoptosis through miR-15a-5p could protect the myocardium in non-coronary surgery, and another trial of berberine found that it could regulate myocardial autophagy through AMPK/mTOR pathway to promote myocardial protection in postoperative patients . HoweverHere, we discuss the role of PCD in cardiovascular disease, the interactions between different PCD signaling cascades, and describe the application of drugs targeting cell death signaling pathways in cardiovascular pathology. Moreover, we summarize the latest progress on the relevant studies that have progressed to clinical trials and put forward a novel understanding for the better transformation of PCD pathways in cardiovascular medicine.Apoptosis is usually induced by intrinsic and extrinsic  triggers. The intrinsic pathway, also called the mitochondrial or Bcl-2-mediated pathway, is activated by Bh3 proteins  to initiate apoptosis when facing intracellular stress caused by various physicochemical factors . ActivatIt has been found that myocardial apoptosis can simultaneously exert detrimental effects on cardiac function. For instance, Wu et al. found that bradykinin (BK) could reduce myocardial apoptosis by reducing caspase-3 expression in a human cardiac c-Kit + progenitor cell (hCPCs) myocardial infarction model, thereby improving ischemic heart disease , Uberti Necrosis is a type of PCD characterized by lytic necrosis and could drive inflammation, in which the RIPK1/RIPK3/MLKL axis may play an essential role . ReceptoFerroptosis, characterized by mitochondrial atrophy, increased membrane density, and reduced mitochondrial cristae, is a new form of PCD discovered recently, which proceeds with cell morphology and function different from the various modes of death described above . FerroptCurrent studies have found that cardiovascular diseases caused by high iron levels are associated with ferroptosis . Baba etAutophagy is a highly conserved catalytic process that leads to the autolysosomal degradation of primary cytoplasmic contents, removing abnormally aggregated proteins and excess organelles. Generally, the activation of autophagy is triggered by undernutrition and oxidative stress . It is aIn the normal physiological state of the heart, cardiomyocytes, vascular epithelial cells, and smooth muscle cells, autophagy plays an essential role in organelle function and cell renewal. Autophagy is also activated in response to cardiovascular stress, including I/R and heart failure. Sciarretta S et al. found that in ischemic stress, Rheb, as a GTP-binding protein, could inhibit autophagy by activating mTORC1, thereby aggravating post-ischemic infarct size . During Pyroptosis is an inflammatory PCD process that rapidly initiates innate immune responses through pattern recognition receptors (PRRs), and different inflammatory bodies can sense different pathogen-associated molecular patterns and damage-associated molecular patterns (PAMPs and DAMPs) that induce pyroptosis . ClassicAn increasing number of studies have shown that cardiovascular risk factors can activate nod-like receptor protein 3 (NLRP3) inflammasomes in cells. In addition, NLRP3 inflammasomes mediated pyroptosis was observed in various cardiovascular diseases. Studies suggested that NLRP3 inflammasomes and related pyrogenic signaling molecules played an essential role in the progression of cardiovascular diseases  Liu et . For exaThere is now increasing evidence that different PCD pathways in cardiovascular diseases are interconnected and affect each other on multiple levels. Studies have found that the PI3K/AKT pathway can regulate autophagy by targeting mTOR, and can also regulate apoptosis through the Bad pathway . mTOR kiCurrently, interventions targeting apoptosis may have a significant impact on the treatment of cardiovascular diseases. Bcl-2 family, p53 gene, IEG family, ICE gene family, and Fasl-Fas system play essential roles in initiating and developing apoptosis . AccumulNecrosis is a class of cell death initiated by death receptor ligands, mediated through death receptors, occurs in the inhibition of apoptotic pathways. The death receptor-receptor interaction protein-mitochondria-oxygen free radical pathway is proved to be an essential regulator of cardiac necrosis, and current clinical trials have found that drugs targeting this pathway can reduce cellular necrosis and improve cardiac function . Verma SUnlike classical apoptosis, there is no hallmark of apoptosis such as chromatin condensation during ferroptosis, but it is accompanied by mitochondrial shrinkage and accumulation of lipid oxides. Studies have revealed that overloaded iron would cause irreparable damage to multiple organs. The use of inhibitors of apoptosis in clinical trials does not also inhibit ferroptosis, while the use of iron chelators can inhibit the ferroptosis process of cells. Wongjaikam S et al. demonstrated that a combination of lamotrigine and desferrioxamine as chelation therapy could reduce the level of myocardial iron by restoring sarco/endoplasmic reticulum calcium ATPase (SERCA) levels . FarmakiAutophagy plays a broad role in developing cardiovascular pathology and can act as both a suppressor and a promoting factor of cardiovascular disease. Although some drugs and regimens can modulate autophagy, few clinical trials have assessed their role in the cardiovascular system. Among them, Qing et al. conducted a phase 2 clinical trial of berberine and found that it could reduce myocardial injury by regulating the AMPK/mTOR pathway; Hua et al. elucidated that simvastatin could reduce myocardial injury by regulating the expression of LC3-II/LC3-I, Beclin- 1, and AMPK phosphorylation, which in turn improved cardiac function  Hua et . AnotherPyroptosis is a new programmed inflammatory death mainly regulated by classical and non-classical pathways, the classical pathway of which is mediated by caspase-1 dependence, and the non-classical inflammasome pathway is mediated by caspase-4/5/11 . NumerouWhile research into cardiovascular disease has focused on the molecular mechanisms of individual PCD types, it is now clear that different PCD pathways do not operate in isolation. Indeed, a more likely explanation basing on current knowledge about the various possibilities of triggering and reconnecting PCD signaling cascades suggests that autophagy, apoptosis, ferroptosis, pyroptosis, and necrosis constitute a pluralistic, coordinated cell death system in which one pathway can flexibly compensate for the other . For exaUp to now, while the increasingly important role of different PCD is unveiled, few clinical trial studies are conducted to treat cardiovascular diseases by pharmacological intervention to PCD . TherefoApart from cardiovascular disorders, PCD is also involved in other diseases, including respiratory and digestive tumors . HoweverWhile the underlying biological mechanisms regarding the PCD remain uncovered, therapeutic manipulation of cell death pathways holds great promise. At present, among various types of diseases, the most studied is the intervention of drugs in various tumors: recent clinical trials reported that the combination of deferoxamine (DFO) and deferiprone (DFP) effectively improves hematologic malignancies . The comTherefore, our review may pave the way for further novel research in the future. On the one hand, we hope to uncover complex signaling pathways between different PCD and clarify their functions in cardiovascular pathologies. On the other hand, acceleration in clinical transformation is warranted to enable PCD-targeted agents to be applied to detect, prevent, and treat cardiovascular diseases.


Dendritic cells (DCs) are immune specialized cells playing a critical role in promoting immune response against antigens, and may represent important targets for therapeutic interventions in cancer. DCs can be stimulated ex vivo with pro-inflammatory molecules and loaded with tumor-specific antigen(s). Protocols describing the specific details of DCs vaccination manufacturing vary widely, but regardless of the employed protocol, the DCs vaccination safety and its ability to induce antitumor responses is clearly established. Many years of studies have focused on the ability of DCs to provide overall survival benefits at least for a selection of cancer patients. Lessons learned from early trials lead to the hypothesis that, to improve the efficacy of DCs-based immunotherapy, this should be combined with other treatments. Thus, the vaccine’s ultimate role may lie in the combinatorial approaches of DCs-based immunotherapy with chemotherapy and radiotherapy, more than in monotherapy. In this review, we address some key questions regarding the integration of DCs vaccination with multimodality therapy approaches for cancer treatment paradigms. Malignant growth depends on the failure of the immune system to recognize cancer cells, due to the ability of tumor cells to effectively silence the “danger signal” required for immunological activation. When the immune surveillance process fails, one of the protective mechanisms against tumor growth is lost. Strategies are being developed to correct this vaccination failure .Since their discovery in 1973, Dendritic Cells (DCs) were considered the crucial Antigen-Presenting Cells (APCs) for the activation of the adaptive immune system and may represent important targets for therapeutic cancer interventions .In mouse models, it was extensively demonstrated that DCs prepared for immunotherapeutic use can capture tumor antigens, which are released from tumor cells, and present them to T cells in tumor-draining lymph nodes. This DCs capability results in the generation of tumor-specific Cytotoxic T Lymphocytes (CTLs) that contribute to tumor rejection ,4. To acSince the 1990s, DCs were used in clinical trials for therapeutic vaccination of cancer patients. As natural DCs constitute only about 1% of peripheral blood mononuclear cells (PBMCs), several ways to generate DCs from precursors have been investigated for DCs’ vaccination purposes.Natural circulating DCs or monocytes are isolated from autologous PBMCs obtained by apheresis. In case of monocytes, ex vivo differentiation into DCs are required. Both natural circulating DCs and monocyte-derived DCs are matured cells, as this is essential for effective T-cell activation . DCs matDuring the vaccine manufacturing process, DCs are loaded with relevant tumor antigen(s) to induce a tumor-specific immune response in patients. Several methods to load DCs with antigen have been reported .Protocols describing the specific details of DCs vaccination manufacturing vary widely. Differences cover all aspects of DCs vaccination, including culture methods, usage of DCs subsets, maturation strategies, antigen types and loading techniques. In addition, to obtain a highly effective product, the route of administration must also be considered.Regardless of the employed protocol, side effects analyzed in the majority of DCs vaccination protocols resulted minimal, consisting mainly in flu-like symptoms, fever and local reactions in accordance with the injection site. Toxicity is extremely uncommon when DCs vaccination is given as monotherapy . These dA functional immune response is a complex and multi-step process. At least four components of the immune response are necessary for a positive result: the presence of appropriate APCs, the quality of induced CD4+ T helper cells, the control of regulatory T cells (Tregs) and the breakdown of the immunosuppressive tumor microenvironment. Thus, the ultimate role for vaccines may lie in the combination with other treatments. Clinical studies combining DCs vaccination with chemotherapy, radiotherapy (RT) and/or other targeted therapy have been performed, confirming, in Phase I, the safety of DCs ,13,14,15Immunotherapy is a fast-moving field, and it is a major pathway of developmental therapeutics for metastatic patients. Lessons learned from early trials move to the hypothesis that combinatorial approaches of immunotherapy with DCs, chemotherapy, and RT rather than monotherapies can improve the efficacy of the cancer treatment option . The aimNatural DCs present in the human blood represents only about 1% of PBMCs. Despite the limited number, some authors have described the possibility of using natural DCs in clinical trials without an extensive culture period. Recently, it has been demonstrated that is possible to obtain more than 10 million plasmacitoid DCs, and an even higher number of BDCA-1 myeloid DCs, after a single leukapheresis, despite their low frequency among PBMCs . Some clA major advantage of natural circulating DCs is their rapid isolation procedure with antibody-coated magnetic beads. Natural DCs subsets are postulated to be more powerful compared with monocyte derived DCs due to their unique functional properties and crosstalk capacity .To overcome the limit of natural DCs low number in the peripheral blood, starting from 1994 investigators have sought a way to generate, ex vivo, large amounts of DCs, starting from the CD14+ cell subset from apheresis, with the aim of using them as “second generation” vaccines for patient treatment ,21. MonoAs mentioned above, ex vivo generation of a large amount of DCs can occur using different culture protocols. After isolation, CD14+ cells are cultured with cytokines, typically granulocyte-macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for four to five days, after which DCs display an immature phenotype.Historically, in the first clinical studies involving the use of DCs for patient treatment, the authors planned to use immature or semi-immature DCs-derived monocytes. Later trials have demonstrated that mature DCs use is better in terms of enhanced migratory capacity, immunogenicity, expression of HLA-DR and costimulatory molecules. These findings are strongly related to an improved clinical outcome ,22. TakeThe choice to use mature DCs in clinical trials is closely related to the optimization of the ex vivo DCs culture protocol, mainly in terms of the maturation cocktail. In vivo, the contact with the pathogens or tissue injury starts the DCs maturation process; the outcome depends on the type of signals that the DCs receive. Ex vivo DCs maturation depends on a variety of stimuli to which DCs are submitted during the culture process. The best DCs preparation protocol should give origin to DCs with a high migration capacity towards lymph nodes, DCs able to present antigens and co-stimulation to T cells, and finally DCs able of surviving to T-cell activation.The most widely used DCs maturation cocktail includes cytokines GM-CSF and IL-4 and tumor necrosis factor-α (TNFα) in combination with IL-1β, IL-6, prostaglandin E2 (PGE2) or monocyte-conditioned medium ,25. ThisSome authors also theorized the addition of Interferon γ (INFγ), Poli-IC and Toll Like Receptors (TLR) ligands or costimulatory pathways (CD40-CD40L) to the maturation cocktail, thus resulting in the production of high levels of IL-12, which directs a Th1-type T cell response ,29,30,31Some authors showed that DCs may also be activated by electroporation with mRNA encoding constitutively active TLR4 and CD40 ligand, obtaining DCs able to suppress Tregs functions and to reprogram Tregs to Th1 cells under certain circumstances ,34.An important aspect in terms of DCs preparation for clinical use is the time requested for cell manufacturing. Traditional protocols have a duration of seven to eight days and foresee medium and repeated cytokines addition. In a good manufacturing practice (GMP)-compliant context, this promotes increased costs in terms of consumables, as well as operator activity in a clean room. Some groups have demonstrated that it is possible to obtain DCs with the safety and efficacy requested by regulatory agencies using short culture protocols in three to five days ,36.Together to the shortening of the culture, in the last years manufacturing challenges also concerned the transfer from culture flasks to bags and, finally, bioreactors. This constitutes a great advantage in terms of GMP requirements and allows an easier and safer passage from research DCs preparation protocols to automatic DCs preparation of commercial drugs. Manufacturing cells to be used as drugs for patient treatment were traditionally prepared in “open” polystyrene-based vessels both for adherent and suspension cell cultures. Open cell culture systems require the opening of flasks or plates for media changes and other cell culture manipulations ,37. To dAntigen processing and presentation is a biological mechanism used by APCs to capture, process and expose the antigen on the cell surface in association with the histocompatibility complex. DCs are specialized APCs effective for carrying out this role, since they are able, once the antigen has been captured, to move into the lymph nodes and to present it to T lymphocytes activating them. For this capability, the practical use of DCs as delivery vehicles for antigen presenting finds extensive application in the immunotherapeutic field for a wide range of tumor types.To date, several methods have been evaluated for loading DCs with tumor antigen in antitumor immunity induction. However, the best approach for loading DCs to induce the strongest CTLs responses has not yet been identified .Tumor cell lysates are the most used source to load DCs. The lysis procedure mostly assures that tumor cells die before antigen loading, thus guaranteeing the safety of these cells. Freeze-thaw processing, UVB irradiation to induce cellular necrosis and apoptosis, manual and/or mechanical breakdown of the tumor, and whole tumor Hypoclorous acid Oxidation (HOCl) are the most common procedures to prepare tumor lysates ,41,42,43Fusion of DCs with tumor cells is another strategy to deliver all tumor cell antigens onto DCs to generate a specific CTL immune response .DCs loading with peptide(s) is an alternative modality to induce antitumor responses. Peptides with defined epitopes have the advantage of inducing epitope-specific immune response limiting possible autoimmune reaction. However, this approach can limit the studies only to those tumors for which specific antigens have been identified. Furthermore, the same epitope can induce different individual reactivity and limit the application to MHC-matched patients only. In addition, the possibility of tumor escape from immune recognition is higher than using a tumor antigens broad spectrum. To overcome this limitation, loading DCs with whole protein may be preferred.Liposome-mediated fusion, electroporation, and osmotic loading are the most popular methods for loading DCs with protein . The useThe described methods for loading tumor antigens are generally valid; but due to the different nature of tumor cells and their variety, a method may be more suitable for some types of tumors but not for others, in this way provoking tumor immune responses of different efficiency ,45. IdenThe therapeutic use of cancer vaccines has recently been reviewed as a consequence of clinical trials series that have yielded encouraging clinical outcomes. First, treatment of metastatic prostate cancer with sipuleucel-T , a cell product based on enriched blood APCs that are briefly cultured with a fusion protein of prostatic acid phosphatase (PAP) and GM-CSF with the result of an approximately a four month longer median survival in Phase III trials ,48. SipuMultiple clinical trials have investigated the role of immunotherapy combined with radio/chemotherapy in cancers . Mixed aThe combination of DCs with chemotherapy seems unusual, as chemotherapy is known to have an immunosuppressive effect: cytotoxic chemotherapy induced depletion of leukocytes due to non-specific cell death of proliferating cells ,62,63 isRecent studies have indeed shown that chemotherapy also has out of target immunological effects, like immunogenic cell death (ICD) of tumor cells, which cause enhancement of anti-tumor immunity ,65, and Chemotherapy creates a cytokine milieu for optimal expansion of antitumor effector cells; for this reason, chemotherapy has been applied in combination with adoptive T-cell transfer ,67, withMany chemotherapeutic drugs, such as cyclophosphamide, temozolomide (TMZ), and gemcitabine seem to be the cause of tumor cell death by anti-tumor immunity .Administration of cyclophosphamide prior to DCs therapy was found to prolong survival in mouse models of mesothelioma, melanoma and colon carcinoma compared to monotherapy. This appears associated with a decrease in Tregs and a subsequent increase in T cells.TMZ is an alkylating agent mainly used to treat glioblastoma (GBM) and melanoma due to its ability to cross the blood brain barrier. TMZ at high doses induces lympho-ablation while at low doses it mainly targets Tregs. Administration of TMZ prior to DCs therapy in patients with melanoma reduced circulating Tregs: studies indicate that administration of TMZ before or during DCs therapy could strengthen the efficacy of DCs therapy, while DCs therapy followed by TMZ may adversely affect DCs-induced antitumor immunity . Some daIn 2008, Heimberger et al. suggested that chemotherapy is not necessarily counterproductive to immunotherapy as long as it is administered outside the therapeutic window, during which vaccine induced CTLs are more reactive and active against tumors . OutsideAdministration of gemcitabine improves antitumor immunity by depleting MDSCs and Tregs. In mouse models of colon and pancreatic cancer, gemcitabine treatment before and after DCs therapy prolonged survival compared to untreated mice at greater extent than monotherapies. In a mouse pancreatic model, simultaneous treatment of DCs therapy and gemcitabine delayed tumor growth and prolonged survival in comparison with both monotherapies . DCs-basSo far, no clinical studies have been reported that exclusively tested the combination of chemotherapy and DCs vaccination. In melanoma, a phase III study shows encouraging data following combined treatment with chemotherapy and DCs vaccination with the addition of a Cyclooxygenase-2 (COX-2) inhibitor, and in lung cancer with the addition of autologous T cells. The two randomized trials show a longer overall survival than the one deriving from chemotherapy alone ,83,84.RT is commonly considered an immunosuppressive agent that non-selectively affects rapidly dividing cells ,86. In pHowever, several studies have suggested that the immune system has an important role with regard to radiation’s therapeutic effects, promoting tumor cell death in the radiation area; for all these reasons, combining radiotherapy with DCs therapy may give origin to synergistic effects and extend clinical reactions. Stone and colleagues were among the first to demonstrate the involvement of the immune system in radiation’s therapeutic effects . In a chOne of the best-studied mechanisms through which radiation can enhance immune responses or immunotherapy efficacy is the upregulation of class I MHC ,95,96,97Radiation also appears to activate DCs and enhance tumor cross-presentation by causing the release of tumor antigens via inflammatory cell death, DCs activation, migration and cross-presentation of tumor antigens resulting in tumor-specific T cells activation and proliferation . It has As radiation therapy induces the death of immunogenic cells, it causes the release of damage-associated molecular patterns and tumor-derived antigens, leading to initiation and migration of DCs towards lymph nodes and induction of systemic antitumor immune responses ,105,106.Many other immunostimulatory mechanisms have emerged from studies on the effect of RT. Among these, the upregulation of FAS represents an interaction mechanism between radiation and the immune system ,115,116 Other mechanisms concern changes in the vascular endothelium that enhance extravasation of immune cells  and incrAmong therapies, cryoablation should also be mentioned.Cryoablative therapy is a process that uses extreme cold to destroy tissue. Ablation occurs in tissue that has been frozen by at least three mechanisms: (a) the formation of ice crystals which disrupt cell membranes; (b) the coagulation of blood, thereby interrupting blood flow; and (c) the induction of apoptosis. Cryoablation is mostly indicated for tumors at an early stage or those not eligible for surgery. The synergies of local ablative techniques together with systemic treatments are currently under investigation in interventional oncology. Given that cryoablation provides a pool of antigens visible for the immune system to induce an immune-specific activation directed against the tumor cells, it could be a very useful adjuvant anticancer therapy .DCs vaccination may be more efficient in combination with therapies that break the suppressive tumor microenvironment. Tumor escape mechanisms are different and include: the secretion of immunosuppressive cytokines and activation of negative regulatory pathways, the loss of tumor antigen expression or downregulation of class I MHC expression with impaired immune recognition, and the expansion and recruitment of Treg and MDSCs . FurtherIt is well known that tumor cells are able to upregulate the expression of checkpoint molecules , leading to anergy of cytotoxic T cells in the tumor microenvironment . Immune Despite promising results, ICI also showed limitations: PD-1 inhibitors have an objective response rate ranging from 50% to 80% in melanoma, Merkel cell carcinoma and carcinoma squamous cells; an average of 15–30% in most other cancers; while virtually no improvements have been seen in cancers such as pancreatic cancer . CombiniHowever, contrary to preclinical data, clinical data on combined treatment with ICI and DCs vaccination in humans are still limited. In 2009, Ribas et al. reported the safety of the combination of tremelimumab (CTLA-4 mAb) and DCs vaccination in patients with melanoma  in whichIn recent years, the rapid development of cancer immunotherapy, such as TILs, NK, CTLs, cytokines, induced killer cells (CIKs) and other immune cells, has provided a novel approach to a cancer treatment considered as a fourth-line cancer therapy . Among tStudies have shown that CIKs/DC-CIKs combined with different chemotherapy regimens for the treatment of gastric cancer shows better efficacy than that shown by treatment with chemotherapy alone. However, clinical trials of CIKs/DC-CIKs cellular immunotherapy are still in the initial phase of study.With the help of DCs, CIK cells could eradicate the malignant cells in three ways: direct disintegration of malignant cells; induction of malignant cells apoptosis by secreting several cytokines; direct regulation of immune response by killing malignant cells. Interestingly, DC-CIKs immunization also generates long-lived CD8+ T cells, suggesting DC-CIKs immunotherapy may be effective to prevent cancer relapse .Also NKT cells exhibit several desirable anticancer properties due to their ability to directly lysate cancer cells. These cells secrete both TH1 and TH2 cytokines which can modulate T cell activity and promote DCs maturation through CD40L/CD40 mediated interactions . The synDCs vaccines have so far shown limited efficacy in tumor immunotherapy. Factors that could contribute to their ineffectiveness are not well understood even if numerous attempts have been made to improve their efficiency. Previous studies involving DCs vaccine injections have shown that only a small fraction of DCs actually reach lymph nodes, possibly explaining why many patients do not respond well to therapy.Among studies, Mitchell et al. found that priming the vaccine site with a booster antigen improves DCs vaccines efficacy in patients with GBM .The authors conducted a randomized, blinded study in which 12 patients received vaccine site preconditioning through Tetanus/diphtheria (Td) Toxoid, a potent booster antigen. Patients who received TdT had significantly more antigen specific DCs in the drainage lymph nodes of the vaccine site than patients who received DCs alone. Patients who received TdT also had a significant increase in progression-free and overall survival, suggesting that DCs increased migration to vaccine site-draining lymph nodes is correlated to improved progression-free and overall survival.To investigate the mechanism of this behavior, the authors observed that serum levels of chemokine (motif C-C) ligand 3 (CCL3) were increased in the patients. Since virtually all people have been vaccinated against tetanus, the booster antigen can locally attract CD4 + T cells that release the CCL3 chemokine, upregulating CCL21 expression.These assumptions have recently been confirmed by Eoli and colleagues . In a phTaken together, these data suggest that DCs vaccine responses in GBM patients could be improved through TdT preconditioning, and further proposing that DCs migration to VDLN could serve as a biomarker to predict patient response. Mitchell and colleagues speculated that TdT injection would work by inducing local inflammation at the vaccine site, and were surprised to see a systemic effect . IncreasSurgical resection with curative intent aims at removing, in the oncological patient, all the tumor burden, although often residual occult disease remains and can eventually lead to relapses .The application of adjuvant treatment such as DCs-based immunotherapy with the aim of killing cancer cells might reduce relapse chances.It is becoming increasingly clear that higher tumor burden is associated to higher tumor-induced immune suppression: several soluble elements secreted by tumor cells are known to suppress infiltrated effector T cells ,157,158.Adjuvant DCs vaccination has been extensively studied in GBM, melanoma and other tumors. GBM is commonly treated with maximally safe surgery and adjuvant temozolomide (TMZ) combined with radiotherapy, which is the so-called Stupp protocol . In contIn addition to a direct clinical benefit for patients, adjuvant DCs vaccination could also prove useful in improving subsequent treatment response in the event of relapse. These effects were retrospectively observed with administration of ipilimumab in patients with relapse after adjuvant DCs vaccination for stage III melanoma .The full nature of the estimated beneficial effects of DCs vaccination is much more complex than any immune monitoring or molecular biology tool at this stage . EarlierThe hypothesis of cancer immunotherapy was first demonstrated by Coley and colleagues. The key concept of cancer immunotherapy is that the immune system can be manipulated to achieve cancer control and, ideally, therapy. Coley demonstrates this statement by using a blend of bacterial toxins to treat patients with inoperable sarcomas . The antThe over 200 clinical trials testing DCs vaccines have shown that DCs are highly immunogenic and safe, often able to activate an antitumor immune response. Some trials have shown that DCs can induce a durable tumor regression and clinical response in treated patients. However, despite several positive data, Sipuleucel-T immunotherapy remains the only therapy to obtain FDA approval to date .DCs research remains critical and DCs will continue to be tested as cancer vaccines in new stages of cancer (earlier stages), new tumors and new combinations.The synergy between cancer vaccines and conventional therapy (chemotherapy and radiation) has demonstrated a potential role for immunotherapy in multi-modal treatment paradigms. With an improved understanding of tumor biology, checkpoint biology, and immune evasion, we will be able to time and deliver therapy to maximize tumor reactions, promote in situ antitumor immune responses, and enhance patient outcomes.Combination agents, useful for improving DCs activity, should ideally (1) reduce tumor mass, as high disease burdens harness the clinical efficacy of DCs-schedules; (2) not impair the immune system of the host; (3) release the brakes of ongoing immune response; and (4) synergize directly or indirectly with DCs functions.The timing of these combinatorial approaches should be carefully considered, as it could affect the efficacy of combination therapies. In addition, determining the optimal combination of therapies likely depends on multiple factors including the patient’s condition, tumor type, stage and composition of the tumor microenvironment. Therefore, a punctual characterization of individual patients will help to select immunotherapies that most likely will work synergistically with DCs therapy.Further research on interactions between immunotherapy and other therapies is needed to incorporate cancer vaccines into current standard of care to maximize the antitumor potential of each treatment modality.In the near future, surgery, cytotoxic therapies and immunotherapy will form a “triple alliance”, which already demonstrates great potential in the treatment of cancer patients. While the potential impact of such regimens is recognized, an optimal combination treatment still has to be established in order to improve clinical outcomes and provide a “true personalized therapy”.


Although these cells have not undergone trans-differentiation into cardiomyocytes after transplantation, but could improve cardiac function through their high secretory profile and mostly by immunomodulatory and proangiogenic effects.2 This can explain their promising impacts, despite extremely low engraftment degree 3 or other possible issues such as ageing and host tissue response. Thus, these sorts of cell therapy could have just been an adjuvant to the common reperfusion or coronary bypass graft procedures.1 In the context of regeneration as the second category, however, scientific community hoped to have the privilege of remuscularizing damaged myocardium. This attempt has started by using cKIT+ cells as the resident progenitor cells of the heart as well as cardiosphere-derived cells.2 However, lineage tracing studies showed that cKIT+ cells mainly differentiated into non-myocytes cells of the heart,4 disproving their remuscularization capacity. The introduction of in vitro generated human cardiomyocytes from directed differentiation of pluripotent stem cells (hPSC-CM) brought some new hope in the context of remuscularization. While the main goal of this approach was regeneration, concerns on the possible arrhythmogenic events were hindered its application. Bottom-line, this hPSC-CMs are now being tested in a doctor-initiated clinical trial (jRCT2053190081). Clinical trials which were performed for cell therapy of cardiovascular diseases, in general, nor induced considerable cardiac repair, neither regeneration, but just neutral or marginal improvement. Two decades of preclinical experiments and clinical trials, however, taught us to first resolve the following issues before taking any further actions:Two decades of hopes and failures in search for next generation therapeutics of cardiovascular diseases, is coming to an end by approaching 2021. The beginning of 21st century was coincided with a burst of hopes toward introduction of novel cell-based therapies for damaged myocardium. Therapeutics which could surpass the intermittent satisfactory reflow and/or revascularization therapies. By far, one can subdivide such preclinical attempts and clinical trials into two categories; those attempted repair and those aimed regeneration.Clearly identifying the desired therapeutic outcome; cardioprotection or regenerationChoosing optimal cell type preclinicallySelecting the best route of administration and developing tractable delivery systemsIdentifying long-term safety and efficacy in preclinical large animal modelsEstablishing general regulations for more consistent and rigorously designed clinical trials with randomized, adequately powered, placebo-controlled, blinded arrangements and evaluations based on clinically relevant markersSelecting ideal patient subsetsResearch and development toward good manufacturing practice (GMP) of advanced therapeutic medicinal products (ATMP) in order to have scalable clinical grade products and reduce batch-to-batch differences.2. While they lack direct remuscularization capacity, they might deliver regenerative signals to myocardial resident cells. As another potential candidate, gene therapy can be pursued for cardiovascular therapeutics. Three decades of introducing gene therapy products, did not include a successful candidate for heart. However, there is a big opportunity here and that is exploring candidate genes which either participate in cardioprotection or regeneration.5 For cardioprotection, genes underlying important proteins of immunomodulation, anti-apoptosis, anti-fibrosis and pro-angiogenesis can be targeted. On the other hand, genes involved in cell cycle reentry or redox regulation can be targeted for regeneration. Last but not least, replacement of RNA therapeutics for gene therapy, as a more direct modulator of gene expression by means of microRNAs or long non-coding RNAs, and most recently modRNAs, opened a new avenue in the research toward biotechnology-related therapeutics of cardiovascular diseases.6 Although, the development of next generation therapies for heart has been fairly slow, future would definitely celebrate the emergence of such products for a healthy heart life.It is worth noting that apart from cell therapy, previous experiences led into other biotechnology-related products (i.e. everything related to the biology of the cells that can be harvested or synthesized by advanced technology) as candidates for cardioprotection or regeneration. Extracellular vesicles which are membrane-enclosed entities secreted from cells, with a cargo of specific proteins, small RNAs and other signaling molecules, have emerged as promising therapeutics for cardiovascular diseases. Depending on their cells of origin, they can induce immunomodulatory, anti-apoptotic, anti-fibrotic and proangiogenic effects


Women with polycystic ovary syndrome (PCOS) are generally considered to be central obese and at higher risks of metabolic disturbances. Imaging methods are the golden standards for detecting body fat distribution. However, evidence based on magnetic resonance imaging (MRI) and computed tomography (CT) is conflicting. This study systematically reviewed the imaging-based body fat distribution in PCOS patients and quantitatively evaluated the difference in body fat distribution between PCOS and BMI-matched controls.PUBMED, EMBASE, and Web of Science were searched up to December 2019, and studies quantitatively compared body fat distribution by MRI, CT, ultrasound, or X-ray absorptiometry (DXA) between women with PCOS and their BMI-matched controls were included. Two researchers independently reviewed the articles, extract data and evaluated the study quality based on Newcastle-Ottawa Scale (NOS).47 studies were included in systematic review and 39 were eligible for meta-analysis. Compared to BMI-matched controls, higher accumulations of visceral fat , abdominal subcutaneous fat , total body fat , trunk fat , and android fat  were identified in PCOS group. However, no significant difference was identified in all the above outcomes in subgroups only including studies using golden standards MRI or CT to evaluate body fat distribution . Moreover, meta-regression and subgroup analyses showed that young and non-obese patients were more likely to accumulate android fat.PCOS women seem to have abdominal fat accumulation when compared with BMI-matched controls. However, MRI- and CT- assessed fat distribution was similar between PCOS and controls, suggesting central obesity may be independent of PCOS. These findings will help us reappraise the relationship between PCOS and abnormal fat deposition and develop specialized lifestyle interventions for PCOS patients.PROSPERO, identifier CRD42018102983. Polycystic ovary syndrome (PCOS) is an endocrine disease associated with obesity and multiple metabolic complications, including insulin resistance, diabetes, and cardiovascular diseases , 2. AccoAccording to the World Health Organization, body fat distribution is another factor that determines the metabolic risks associated with obesity . VisceraTherefore, it is essential to quantitatively study body fat deposition of PCOS through imaging methods to help us get in-depth knowledge of the fat distribution of women with PCOS. This study systematically reviewed the imaging-based body fat distribution in PCOS patients and quantitatively evaluated the difference between PCOS and BMI-matched controls from 8 aspects: visceral fat, abdominal subcutaneous fat, total body fat, trunk fat, android fat, and gynoid fat. Our findings provide new insights into the fat distribution patterns in PCOS patients, which is of great significance for understanding the etiology of PCOS and guiding lifestyle interventions in clinical practice.Systematic database searches were performed in PUBMED, EMBASE, and Web of Science updated in Dec 2019. The declarations of Preferred Reporting Item for Systematic Reviews and Meta-analyses (PRISMA) were followed. The protocol of this systematic review and meta-analysis was previously registered on PROSPERO (CRD42018102983). We developed the “full text” search strategy based on the combination of the following keywords (subject item plus free items): (polycystic ovary syndrome OR PCOS) AND  AND (magnetic resonance OR ultrasound OR computerized tomography OR X-ray). Detailed search strategies were listed in The populations being studied in this review were women diagnosed with PCOS and the populations of comparator were BMI-matched control women without PCOS. This review was based on observational studies, therefore interventions were not applicable. The main outcomes in this study were imaging-based body fat distribution including visceral fat, abdominal subcutaneous fat, total body fat, trunk fat, android fat, and gynoid fat in quantity. Moreover, we also included total body fat, trunk fat, android fat, and gynoid fat in percentage as secondary outcomes. Studies that satisfied the following criteria were included in the present meta-analysis: (1) studies that investigated the distribution of body fat including visceral fat, abdominal subcutaneous fat, total body fat (both in quantity and in percentage), android fat (both in quantity and in percentage), gynoid fat (both in quantity and in percentage), and trunk fat (both in quantity and in percentage) between women with PCOS and controls; (2) body fat distribution was measured by standard imaging methods including MRI, CT, ultrasound, and X-ray absorptiometry (DXA); (3) When duplication of same subject population occurred, the most recent study or study with the largest sample was included. Exclusion criteria were: (1) studies that employed testing technologies other than the standard imaging methods such as bioelectrical impedance; (2) studies without BMI adjustment; (3) studies that lack sufficient data to perform quantitative or qualitative analysis. Articles in languages other than English were excluded.Two researchers (Zhu SQ and Hu CP) reviewed retrieved studies independently. Baseline characteristics relating to study and its participant  were extracted. The definition of PCOS was defined by Rotterdam criteria, National Institutes of Health (NIH) criteria, the Androgen Excess and PCOS (AE-PCOS) Society criteria, or based on the original articles –15. DefiNewcastle-Ottawa Scale (NOS) was employed to assess the qualities of the included studies . Two resWe evaluated the variation of fat distribution between women with PCOS and controls. The between-study standardized mean difference (SMD) and 95% confidence intervals (CIs) were deployed. SMD>0 represents higher fat distribution in PCOS as compared to that of controls, while SMD<0 indicated the opposite. Between-study heterogeneity was assessed by I2-statistics and Q-test. Random-effects model was used when significant heterogeneity was observed. Subgroup analyses were performed to explore the source of heterogeneity. The impact of a study on the overall effect was assessed through sensitivity analysis. Besides, meta-regression was performed on outcomes that have more than 10 included studies. Publication bias was examined using Egger’s regression test. Two-tailed P < 0.05 was considered as significant. Trim and Filled Analysis was used to further evaluate the variation when significant bias was tested in Egger’s regression test. Statistical analyses were performed using STATA 12.0 .A total of 1284 articles were yielded through electronic search strategy and hand search. After excluding duplicates and screening abstracts based on selection criteria, 122 full-text articles were further assessed for eligibility. After excluding 15 for duplicated datasets, 13 for not using standard imaging methods, 29 for insufficient data, 18 for undesirable controls or outcomes, 47 articles finally remained for qualitative synthesis and 39 were found eligible for meta-analysis . Increased accumulation of trunk fat  and android fat  was identified in women with PCOS. PCOS women also had higher absolute value of gynoid fat , whereas the percentage of gynoid fat in women with PCOS was comparable to that in healthy controls . Interestingly, there was no statistically significant difference in all outcomes in MRI or CT subgroup.  , upper body fat (including trunk fat and android fat) and lower body fat (gynoid fat). Previous researches have shown that visceral fat and upper body fat are related to higher risks of metabolic disorders such as hypertension and type 2 diabetes, and lower body fat is associated with reduced metabolic risks –63. In PContrary to previous studies which reported an elevated prevalence of central obesity estimated with WC in women with PCOS , in thisFurthermore, imaging methods may also affect the results. When studies using DXA and ultrasound as measurements were also included in the meta-analysis, the results showed a higher accumulation of total body fat and upper body fat  in women with PCOS compared to BMI-matched healthy women, which was consistent with previous knowledge. The conflicting results between the different imaging methods may be related to bias from methods. Despite that DXA has been widely used for estimating regional body fat, the results of DXA analysis could be confounded by hydration of lean soft tissue . It has In meta-regression and subgroup analyses, we found that age was inversely associated with increased visceral fat accumulation in women with PCOS. Similarly, prospective cohort studies have demonstrated that the risks of central obesity and metabolic diseases increased in young PCOS patients but were attenuated in later life , 71. HowThis study systematically reviewed the articles that investigated the difference of image-assessed fat distribution between women with PCOS and BMI-matched controls and the robustness of results was verified by sensitivity analyses and tests of publication bias. Through comprehensive subgroup analyses of possible confounders, we found that different imaging methods may be the dominant source of heterogeneity. Given the lack of convenience and efficiency of golden standards MRI and CT, the small sample size is a common problem in studies using MRI or CT to detect fat distribution. This meta-analysis facilitated the integration of these data and found similar abdominal fat distribution between PCOS and BMI-matched healthy controls in MRI or CT subgroup. Despite the above advantages, there are some limitations in this study. Firstly, the main limitation of such an extensive review is that the selection bias in comparator populations. The optimal source of controls should be from the community, but less than half of the included studies met the criteria. In studies that do not use community-based controls, they were more inclined to recruit controls from schools, hospital employees, or medical examiners. This population may be healthier and lead to an overestimation of the difference between PCOS and control groups. Secondly, although details of these possible confounders were extensively extracted from the original studies and displayed in In conclusion, this systematic review and meta-analysis summarized the current evidence focused on imaging-based body fat distribution in women with PCOS and found similar fat distribution patterns assessed by golden standards MRI or CT between women with PCOS and BMI-matched controls, indicating central obesity may be independent of PCOS and exacerbate metabolic dysregulation in PCOS patients. Moreover, younger patients and non-obese patients were more inclined to accumulate android fat. These results facilitated the understanding of the relationship between PCOS and ectopic fat deposition, and will support the establishment of specialized lifestyle interventions for PCOS patients.The original contributions presented in the study are included in the article/YL and HY designed the study and evaluated the data. SZ, CH, and ZL collected the information and analyzed the data. SZ, ZL, and FS wrote the manuscript. CW and YL critically revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by grants from the Natural Science Foundation of Jiangsu Province (BK20200223), the Natural Science Foundation of Shandong Province (ZR2020QH051), the Young Scholars Program as well as Fundamental Research Funds (21520079614029) of Shandong University to YL.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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