Brain pathologies are characterized by microscopic changes in neurons and synapses that reverberate into large scale networks altering brain dynamics and functional states. An important yet unresolved issue concerns the impact of patients’ excitation/inhibition profiles on neurodegenerative diseases including Alzheimer’s Disease, Frontotemporal Dementia, and Amyotrophic Lateral Sclerosis. In this work, we used The Virtual Brain (TVB) simulation platform to simulate brain dynamics in healthy and neurodegenerative conditions and to extract information about the excitatory/inhibitory balance in single subjects. The brain structural and functional connectomes were extracted from 3T-MRI (Magnetic Resonance Imaging) scans and TVB nodes were represented by a Wong-Wang neural mass model endowing an explicit representation of the excitatory/inhibitory balance. Simulations were performed including both cerebral and cerebellar nodes and their structural connections to explore cerebellar impact on brain dynamics generation. The potential for clinical translation of TVB derived biophysical parameters was assessed by exploring their association with patients’ cognitive performance and testing their discriminative power between clinical conditions. Our results showed that TVB biophysical parameters differed between clinical phenotypes, predicting higher global coupling and inhibition in Alzheimer’s Disease and stronger N-methyl-D-aspartate (NMDA) receptor-dependent excitation in Amyotrophic Lateral Sclerosis. These physio-pathological parameters allowed us to perform an advanced analysis of patients’ conditions. In backward regressions, TVB-derived parameters significantly contributed to explain the variation of neuropsychological scores and, in discriminant analysis, the combination of TVB parameters and neuropsychological scores significantly improved the discriminative power between clinical conditions. Moreover, cluster analysis provided a unique description of the excitatory/inhibitory balance in individual patients. Importantly, the integration of cerebro-cerebellar loops in simulations improved TVB predictive power, i.e., the correlation between experimental and simulated functional connectivity in all pathological conditions supporting the cerebellar role in brain function disrupted by neurodegeneration. Overall, TVB simulations reveal differences in the excitatory/inhibitory balance of individual patients that, combined with cognitive assessment, can promote the personalized diagnosis and therapy of neurodegenerative diseases. Neuroscience is showing a growing interest in merging results at different scales of complexity to achieve a global and comprehensive knowledge of the brain and its mechanisms. In this context, brain modeling can be used to bridge the gap between cellular phenomena and whole-brain dynamics, both in physiological  and pathological conditions . The VirNeurodegenerative pathologies ranging from Alzheimer’s Disease, Frontotemporal Dementia, and Amyotrophic Lateral Sclerosis are reportedly characterized by a disrupted balance between excitation and inhibition.Glutamate and GABA concentrations are relevant to the excitatory/inhibitory balance. An increase or decrease in their concentrations can lead to hyper/hypo excitation or inhibition, possibly contributing to neurodegeneration. Indeed, hyperexcitation is thought to play a pivotal role in Alzheimer Disease, Frontotemporal Dementia, and Amyotrophic Lateral Sclerosis pathogenesis , but mulDespite the importance of excitatory/inhibitory balance disruption in pathologies, excitation/inhibition experimental determination, e.g., evaluating GABA and glutamate concentrations, in single subjects is yet to reach clinical adoption because of longer acquisition times, the need for spectral editing techniques not implemented on clinical scanners and the lack of community guidelines and protocols standardization. Since TVB is designed to extract information about connectivity and network parameters including those linked to inhibition/excitation pathways in single human subjects, starting from data that can be acquired with clinical scanners, it has a true potential to foster personalized and precision medicine, especially in those neurodegenerative conditions mentioned above.It is important also to highlight that TVB analysis should include not only cerebral nodes and their structural connections to one another, but also the cerebellum. Recently, it was shown that integrating cerebro-cerebellar connections can improve TVB predictive capability in healthy subjects . This isIn this work, we exploited TVB capabilities to (i) characterize each group of subjects by providing personalized excitation/inhibition profiles and (ii) assess the cerebellar impact on brain dynamics generation in Healthy Controls and in Alzheimer’s Disease, Frontotemporal Dementia, and Amyotrophic Lateral Sclerosis. TVB simulations were performed using the Wong-Wang model , which aIn this work individual’s subject analysis was conducted as described in Sixty patients affected by neurodegenerative diseases were recruited at the IRCCS Mondino Foundation, as part of a study on cognitive impairment published in All subjects underwent a neuropsychological examination based on a standardized battery of tests to assess their global cognitive status . For anatomical reference, a whole brain high-resolution 3D sagittal T1-weighted scan  was also acquired.All subjects underwent MRI examination using a 3T Siemens Skyra scanner with a 32-channel head coil. The protocol included resting-state fMRI (T0 image was obtained averaging the 10 volumes acquired with no diffusion weighting. DW data were denoised and corrected for Gibbs artifact (0 image using the eddy tool [FMRIB Software Library (FSL)1; 0 image using the brain extraction tool  denoising  and 31 cerebellar  labels  labels  in MNI15) labels .TVB workflow includes several steps: (1) incorporation of subject SC matrices; (2) selection of a mean field/neural mass mathematical model; (3) simulation of the rs-fMRI time-course per node and creation of the simulated FC matrix (simFC); (4) model parameters tuning to achieve the best matching between simFC and expFC matrices; (5) final simulation of brain dynamics with the optimal model parameters as described in detail by The Wong-Wang model  implemeni denotes the firing rate of the excitatory (E) and inhibitory (I) populations, Si identifies the average excitatory or inhibitory synaptic gating variables at local area, i, and Ii is the input current to the excitatory and inhibitory populations at local area, i. All parameters described in i), the strength of excitatory (NMDA) synapses (JNMDA) and the strength of local excitatory recurrence (w+). Thus, this model retains information on both global brain dynamics and local excitatory/inhibitory balance and is particularly interesting for the investigation of pathological conditions.where rFor each set of parameters combination, resting-state BOLD fMRI time-courses were simulated over 6 min length using a Balloon-Windkessel hemodynamic neurovascular coupling model  and the To investigate the impact of the cerebellum on brain dynamics generation, simulations were performed using three different combinations of connections and nodes :•Whole-brain network: whole-brain nodes and connections.•Cortical subnetwork: cerebral cortex nodes and connections (excluding cerebro-cerebellar connections).•Embedded cerebro-cerebellar subnetwork: cerebral cortex nodes but also considering cerebellar nodes and hence the cerebro-cerebellar connections.For each of these three networks predictive power was evaluated as the mean PCC between expFC and simFC matrices in different clinical conditions .Statistical tests were performed using SPSS software version 21 .To assess whether biophysical parameters derived from TVB differ according to the clinical condition, optimal model parameters were tested for normality (Shapiro-Wilk test) and differences between groups were assessed with non-parametric tests  when they did not present a Gaussian distribution.p < 0.05) explained neuropsychological scores variance in all subjects together.A multiple regression analysis was performed to investigate the relationship between individual scores of the 5 cognitive domains  and the optimal model parameters. Neuropsychological scores in each cognitive domain were considered as dependent variables while model parameters combined with age, gender, and group category were used as predictors in a backward approach. The regression algorithm automatically removed one or more predictors to identify which of them significantly  model parameters alone, (ii) neuropsychological scores alone, and (iii) a combination of both. To visualize and assess the sensitivity and specificity of the best discriminative variables, receiving operating characteristics (ROC) curves and corresponding areas under the curve (AUC) were calculated.Finally, a k-mean cluster analysis was performed to reconstruct subjects-specific excitation/inhibition profiles. The number of clusters was an input parameter, arbitrarily set equal to 4, matching the number of variables considered . A frequency analysis of the clusters in terms of clinical groups enabled us to reach a deeper understanding of their respective excitatory/inhibitory balance profiles. This percentage of subjects belonging to one of the clusters in each clinical group was used to qualitatively describe the occurrence of specific patterns of excitatory/inhibitory balance in the population.PCC obtained with the three networks were normally distributed (Shapiro-Wilk test), thus parametric tests were used to compare them between different conditions. First, to assess TVB predictive power for each clinical condition, a one-way ANOVA was performed between the PCC of each network across groups . Then, to assess the impact of each specific network on TVB predictive power, a multivariate general linear model (GLM) with Bonferroni correction was chosen to compare PCC values of the three networks within each group.https://github.com/BrainModes/fast_tvb. Dataset will be available at 10.5281/zenodo.5796063.All codes used for this study are freely available. The optimized TVB C code can be found at i, JNMDA, w+) parameters were adjusted iteratively to optimize the model fit to empirical data. Optimal model parameters were found across the whole-brain network of each subject.Both global (G) and local ; Amyotrophic Lateral Sclerosis patients showed higher JNMDA than Healthy Controls (p < 0.05); no statistically significant differences were found when comparing Healthy Controls and Frontotemporal Dementia with other groups.The biophysical parameters derived from TVB were compared between groups to assess whether, at group level, their value could differ according to the clinical condition. Both global and local biophysical parameters showed significant differences between groups : Alzheimp < 0.05) explain a percentage of the variance  in Each of the four clusters identified with the k-means analysis was characterized by a different combination of values of TVB-derived biophysical parameters, as reported in •NMDA).Cluster 1 is mainly characterized by medium to strong overexcitation , is characterized by a high global coupling strength  and medium to strong overinhibition .Cluster 2, in addition to showing strong overexcitation .Cluster 3 is the only one characterized by medium or low values of G, J•i) and high global coupling strength between nodes .Cluster 4 is mostly characterized by overinhibition  Whole-brain network, (ii) Cortical subnetwork, (iii) Embedded cerebro-cerebellar subnetwork as in p < 0.01) than those obtained with the whole-brain network, while PCCs of the cortical subnetwork were significantly higher (p < 0.003) than those of the whole-brain network in Alzheimer’s Disease and Frontotemporal Dementia  conditions integrating cerebro-cerebellar connections in TVB. Importantly, by adopting the Wong-Wang model to model brain dynamics, we gained information on local excitatory/inhibitory balance at the single subject level.NMDA represents the strength of excitatory synapses in the network, Ji denotes the strength of inhibitory synapses and w+ the strength of recurrent excitation.Parameters derived from TVB simulations using the Wong-Wang model yield information on global brain dynamics and on the local excitatory/inhibitory balance. In particular, the global scaling factor G denotes the strength of long-range connections, and higher global coupling means a greater weighting of the global over the local connectivity. The remaining three parameters define the balance between excitation and inhibition in the simulated network: JOur results revealed that different clinical groups were characterized by specific TVB parameters providing new clues for the interpretation of the dysfunctional mechanisms in local microcircuits.i were higher in Alzheimer’s Disease patients compared to Healthy Controls suggesting that Alzheimer’s Disease subjects were characterized by increased global coupling and overinhibition. This increased G-value in our Alzheimer’s Disease group could be interpreted as a compensatory mechanism counteracting altered cerebral connectivity, but it might also underly the hypersynchrony typically characterizing disrupted networks in patients  , althougpatients . Furtherpatients .i also compared to Frontotemporal Dementia patients, strengthening the tenet that the pathophysiological mechanisms underlying the two diseases are different. Indeed, Frontotemporal Dementia showed G and Ji values similar to Healthy Controls, consistent with the similarity of cortical neural synchronization in Frontotemporal Dementia and Healthy Controls  or used to establish cluster membership in a data-driven approach.G-value could be due to an increased strength of long-range connectivity or increased synchrony between nodes. It is worth noting that cluster 2 was more common in pathological conditions than in Healthy Controls, and this is in line with the frequent observation of hyperexcitation and hypersynchrony in Alzheimer’s Disease (While at group level there were noticeable differences in excitatory/inhibitory parameters, when considering single-subjects’ profiles, K-means clusters were not group specific; this suggests an heterogeneous excitatory and inhibitory balance across subjects that could be exploited for future personalized interventions. Considering the biophysical meaning of TVB parameters, cluster 1 was mainly characterized by overexcitation and was more frequent in Healthy Controls and Amyotrophic Lateral Sclerosis. As well as being consistent with the fact that hyperexcitability is a well-known feature of Amyotrophic Lateral Sclerosis patients , the pre Disease , Frontot Disease , and Amy Disease . Cluster Disease , in line Disease . Unfortu Disease , GABAergIt is important to point out that Frontotemporal Dementia patients appeared to be the most distributed between clusters, without a main cluster membership, and this evidence reflects the heterogeneity of our Frontotemporal Dementia cohort, which is in line with the wide spectrum of neurotransmitters deficits which has been already observed in Frontotemporal Dementia .Cerebellar impairment has been consistently observed in neurodegenerative diseases, although the cerebellum has been rarely considered in neurodegenerative conditions. Disease-specific clusters of cerebellar atrophy have been found in Alzheimer’s Disease, Frontotemporal Dementia, and Amyotrophic Lateral Sclerosis . FunctioIn our work, the integration of cerebro-cerebellar connections in the structural matrix improved the predictive power of TVB simulations in pathological conditions, supporting the concept of a cerebellar involvement in neurodegenerative conditions and confirming the sizeable contribution of cerebro-cerebellar connectivity to simulated brain dynamics . This imOne potential concern about the present study is the small sample size. However, it should be noted that one of the most interesting aspects of TVB is to uncover subject-specific characteristics in clinical groups. In this perspective, the use of a small sample does not detract relevance from this study, also considering that other TVB applications on small cohorts can be found in literature . An inteA technical aspect to consider is also that, in this study, whole brain parcellation was performed combining the AAL atlas with SUIT in order to account for the cerebellum. The different parcel sizes may influence structural/functional connectivity and brain dynamics . In thisTVB-derived biophysical parameters provided a unique description of the excitatory/inhibitory balance both at group and single subject levels. An extremely high performance was achieved in patients’ discrimination combining TVB parameters and neuropsychological scores. Moreover, the integration of cerebro-cerebellar connections in TVB improved the predictive power of the model in neurodegeneration. Overall, this work opens new perspectives for the use of TVB to explore neurodegenerative mechanisms, supports the involvement of the cerebellum in determining brain dynamics in neurodegenerative diseases, and suggests a novel approach to obtain physiological information relevant to future personalized diagnosis and therapy.This manuscript has been released as a Pre-Print at BioRxiv .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the IRCCS Mondino Foundation. The patients/participants provided their written informed consent to participate in this study.AM performed research, analyzed data, and wrote the manuscript. FP, ED’A, and CG designed research, coordinated the work, and provided critical revision. PV and AP performed and coordinated brain MRI examinations and provided critical revision. AC, MC, and SB were involved in clinical data acquisition. VJ provided critical revision. All authors contributed to the article and approved the submitted version.


Polycystic ovary syndrome (PCOS) is an endocrine systemic disorder mainly characterized by a hormonal and metabolic disbalance that leads to oligo/anovulation, hyperandrogenism and the formation of ovarian cysts. Despite the progress that has been reached in its diagnosis and management, little is known about the molecular mechanisms and signaling pathways underlying the pathogenic mechanisms. In this sense, recent research has suggested that the influence of multiple factors, including age, environment, lifestyle and the disease state environment can change the clinical presentation of PCOS via epigenetic modifications. Variants in the genes encoding for proteins involved in steroidogenesis and glucose homeostasis play a crucial role in the development of the disease. Other genes involved in inflammation and cell proliferation seem to undergo an epigenetic control. Moreover, lifestyle factors influence the PCOS course and prognosis, including diet and physical activity, which are fundamental in reducing oxidative stress, inflammation and in improving metabolic and hormonal parameters. In the present review, literature evidence on molecular and epigenetic mechanisms related to PCOS etiology will be discussed, with a particular attention on the positive influence of diet and physical activity as nonpharmacological ways of intervention in the management of the disease. Polycystic ovary syndrome (PCOS) is an endocrine, systemic disorder that affects 5–20% of women in reproductive age and is mainly characterized by a sex hormone imbalance causing the formation of water-retained cysts in the ovarian antral follicles . The horIt has to be considered that about 75% of PCOS women suffers from anovulatory infertility , and tho-oligo/anovulation + hyperandrogenism + polycystic ovaries;-oligo/anovulation + hyperandrogenism;-hyperandrogenism + polycystic ovaries;-oligo/anovulation + polycystic ovaries .Currently, the most used diagnostic criteria are the Rotterdam criteria (2003). According to them, PCOS is present if two of the following three criteria are manifested: oligo/anovulation, hyperandrogenism and the presence of ovarian cysts. Following the same criteria, four phenotypes could be determined :-oligo/aDespite these well-characterized phenotypes, the pathogenesis of PCOS remains unclear; it is a multifactorial disorder that involves genetic, epigenetic and environmental variables , as wellMoreover, lifestyle factors influence the PCOS course and prognosis. Despite the progress that has been reached in its diagnosis and management, little is known about the molecular mechanisms and signaling pathways underlying the pathogenic mechanisms. In this sense, recent research has suggested that the influence of multiple factors, including age, environment, lifestyle and the disease state environment, can change the clinical presentation of PCOS via epigenetic modifications. In the present review, the molecular and epigenetic mechanisms of diet and physical activity are discussed, with a particular attention on their positive influence in the management of the disease and their efficacy as nonpharmacological ways of intervention .Dysfunctional granulosa cells (GCs), characterized by a poor apoptosis and increased proliferation, have proved to be typical of PCOS and are usually associated with impaired ovulation and folliculogenesis . ElevateDysfunctional GCs also present increased mitochondrial autophagy that leads to mitochondrial injuries with consequent decreased membrane potential and mtDNA content . More geThe excess of androgens is directly related to the arrest in the antral follicle development and anovulation: in murine models, androgens have proved to enhance the activity of the C-type natriuretic peptide (CNP) and natriuretic peptide receptor 2 (CNP/NPR2) pathway that in turn stops oocyte meiosis impairing ovulation .Androgens also correlate with GCs dysfunction, favoring in them the accumulation of advanced glycation end products (AGEs): male hormones, in fact, cause the overexpression of the AGE receptor in GCs through the activation of the endoplasmic reticulum stress . In theiMoreover, GCs undergo the epithelial mesenchymal transition (EMT) during the ovulation period, and this process is regulated by the integrin-interacting protein kindlin 2; it has been demonstrated that an excess of testosterone in PCOS women significantly increased the expression of kindlin 2, thus affecting EMT and ovulation . In addiIn this kind of patient, a strict correlation between hyperandrogenism and abdominal adiposity has been highlighted as well. An in vitro study showed that in normal-weight patients, subcutaneous abdominal stem cells developed too early to adipocyte: these new adipose cells were characterized by a high content of lipids and an overexpression of PPARγ and CEBPα, which are two genes involved in adipogenesis processes . In addiThe genetic predisposition in PCOS is unquestionable and several genes are demonstrated to be involved in the etiology of the disease . Indeed,Considering that most PCOS women are at an increased risk of developing glucose intolerance and type 2 diabetes mellitus (T2DM), the gene Calpain 10 (CAPN10), located on the 2q chromosome and usually associated with insulin resistance, has been investigated . In partCritical genes that encode for steroidogenic enzymes, including cytochrome P450 enzymes (CYP), have also been investigated. In this regard, polymorphisms in CYP11A, CYP17, CYP19 and CYP21 genes are related to the alteration in the steroidogenesis with an overproduction of androgen hormones ,47,48.Since androgen effects are mediated by their receptors, it has been assumed that the androgen receptor (AR) gene is involved in hormonal disbalance. The AR, located on the X-chromosome at Xq11–12, presents a genetic polymorphism in exon 1, defined by a poly-glutamine (CAG) repeat region (8–35 repeats in most cases) : a shortMoreover, polymorphisms that occur in the sex hormone–binding globulin (SHBG) gene (chromosome 17p12—p13) seem to be crucial: it has been shown that a shorter (TAAAA) pentanucleotide repeat is responsible for an altered gene transcription . As a coIn addition, reproductive anomalies in PCOS suggest the presence of mutations in folliculogenesis-related genes: for example, a recent study demonstrated that luteinizing hormone (LH) and luteinizing hormone receptor (LHR) genes show variants strongly related to PCOS phenotypes . The FSHDespite the difficulties to offer a complete report about the genetic basis of PCOS, common genetic risk factors can be identified and linked to metabolic and endocrine dysfunctions associated to PCOS. Next generation genetic analysis will allow incorporating huge, genotyped datasets, thereby detecting a genetic architecture across PCOS diagnostic categories.Epigenetic mechanisms have been recently supposed to participate in PCOS pathogenesis: in particular, a huge amount of data is demonstrating that DNA methylation and microRNAs (miRNAs) have an altered pattern in the blood, serum, adipose tissue, granulose cells and theca of women with PCOS. Thus, PCOS women can have a different epigenetic regulation that seems to trigger and support the progression of the disease , possiblDNA methylation consists in an enzymatic reaction that adds a methyl group generally at the carbon in the 5′ position of the pyrimidine ring of a cytosine followed by a guanine called CpG dinucleotides . A huge Furthermore, recent studies have highlighted that the mechanisms of DNA methylation possess a high plasticity across age and DNA methylation, and gene expression and the corresponding phenotype can be modified through therapeutic intervention in animal models, thus opening the possibility of therapeutic interventions in PCOS women as well ,59,60.A general DNA methylome profiling of GCs recently has revealed an altered methylation in genes regulating pivotal ovarian functions in PCOS : in partSeveral studies investigated the genome-wide DNA methylation levels in the peripheral blood of women with PCOS . Among The molecular and morphological changes in the ovarian tissue that are typical of PCOS can be related to epigenetic alterations, as demonstrated in animal models of PCOS.TET1), axon guidance (ROBO-1), inhibition of cell proliferation (CDKN1A), inflammation (HDC) and insulin signaling , are found to be hypomethylated in PCOS women as well, and it has been suggested that this epigenetic process could be inheritable, since three of them  resulted in being hypomethylated in daughters suffering from PCOS [For example, the fetal programming induced by prenatal testosterone has been proven to be related with defects in the expression of ovarian steroidogenic genes correlated with the insulin pathway in the ovaries of fetal ewes . Similarrom PCOS .A few studies have stated significant differences in the methylated genes of the ovarian tissue between PCOS women and controls, which correlate with hormone activity (such as AMH), transcriptional regulation, inflammation, glucose metabolism and insulin signaling . FurtherTNIK) usually involved in cell proliferation: the hypermethylation of the corresponding gene upregulates TNIK in PCOS ovarian tissue; moreover, the hyperexpression of TNIK seems to be related to Wnt signaling pathways, finally altering women metabolic profile [The epigenetic regulation affects also the Traf2- and Nck-interacting kinase ( profile .TNF) and the hypomethylation of aldo-keto reductase 1 C3 (AKR1C3), calcium-sensing receptor (CASR), growth hormone-releasing hormone receptor (GHRHR), resistin (RETN) and mastermind-like domain 1 (MAMLD1) lead to hyperandrogenism, causing the hormone disbalance that is crucial for the pathogenesis of PCOS [(LHR) is overexpressed in PCOS nonobese women; in contrast, the insulin receptor gene (INSR) in under expressed in PCOS obese subjects [In addition, epigenetic mechanisms correlate with the androgen excess. The hypermethylation of tumor necrosis factor  and autophagy (Becn1) [Moreover, considering that ovarian autophagy is critical for follicular development , the gen (Becn1) . Interes (Becn1) . [Together with the DNA methylation, microRNAs (miRNAs), which are small noncoding RNAs, are involved in epigenetic processes regulating gene expression at the post-transcriptional level . The int SLC7A5) . Actuall SLC7A5) .Altered miRNA expressions are closely associated with the occurrence of diseases, including endocrine and metabolic disorders. Recently, the roles of miRNAs in PCOS pathology have attracted considerable attention . For exaIn addition, different miRNAs have been linked to PCO pathogenesis and different studies have enlightened the importance of miRNAs in cumulus GCs for ovarian follicle development. For example, Cai and collaborators  have proAnother miRNA involved in GC proliferation is miR-93, whose expression has been proven to be upregulated in PCOS ovarian samples. This miRNA seems to regulate folliculogenesis and GC proliferation via targeting CDKN1A and promoting G1/S transition . An elevSimilarly, miR-221/miR-222 has been evidenced as downregulated in GCs of PCOS samples and to be related to GC decreased proliferation, probably as a consequence of an elevated androgen level and overexpression of p27/kip1 .A few members of the miR-17-92 cluster  have been found to be downexpressed in the ovarian tissues of PCOS women . These mAdditionally, other miRNAs have been related to the serum concentration of key hormones or of their receptors: for example, Song and collaborators have evidenced a downregulation of serum miR-592, whose targets are represented by luteinizing hormone/chorionic gonadotropin receptor (LHCGR), which regulates follicle development, and by IGF-1 receptor (IGF1R), which has been linked to insulin resistance [A few studies have studied the putative differences in the chromatin accessibility between PCOS women and controls, examining ovary samples but also samples derived from adipose tissue. For example, Hosseini and colleagues have investigated histone acetylation and methylation levels of cumulus cells (CCs) in infertile PCOS patients, and their correlation with the expression of the ovarian aromatase gene . This stA recent interesting study has evidenced that adipose stem cells (ASCs) of normal-weight PCOS women present an augmented lipid accumulation accompanied by an overexpression of the key master regulator genes of adipogenesis required for adipocyte maintenance/differentiation and specific changes in chromatin accessibility and transcriptional regulation compared to control stem cells . InteresThe data presented here below allow concluding that methylation in DNA and miRNAs (and in part modifications in the chromatin status) is altered in PCOS women in the blood, serum, adipose tissue, granulose and theca cells. Thus, women with PCOS present a changed epigenetic regulation, which might be triggered by an adverse intrauterine environment or by postnatal environmental elements, including diet and obesity. These epigenetic alterations can be considered as biomarkers for the diagnosis and treatment of PCOS patients.An autoimmune predisposition could be involved in PCOS pathogenesis . The freIt has been demonstrated that, in women affected by PCOS, CD4(+), CD25(+) and CD127(−/low) T regulatory cells present a decreased activity with a consequent impaired immunosuppression . MoreoveAntiendometrial antibodies were also found in PCOS women and they have been demonstrated to associate with the presence of oxidized proteins, suggesting a link between the pathogenesis of the disease and the oxidative stress in the uterine microenvironment . InteresRecent studies showed the presence of activating autoantibodies (AAb) to the gonadotropin-releasing hormone receptor (GnRHR) ,103 thatLow levels of LH and FSRH autoantibodies were also found, but their role in enhancing hyperandrogenism in PCOS women is still doubtful .Improving nonpharmacological interventions is fundamental in the management of PCOS. Lifestyle changes aim to achieve better anthropometric parameters that usually reflect better metabolic and hormonal profiles affecting positively ovarian functions . ObesityFirst of all, identifying and correcting abnormal eating disorders is a successful strategy in providing weight loss . In factIn order to improve the metabolic profile, it has to be considered that sleep duration is crucial in developing worse PCOS phenotypes: a recent study demonstrated that poor sleep is associated with sleep apnea disorders, which in turn are responsible for increased levels of triglycerides, a higher percentage of liver fat and increased waist circumferences in adolescents affected by PCOS . The resIn this perspective, it is clear that diet and physical activity are keystones to achieve lifestyle changes which are effective in obtaining the improvement both in the metabolic and in hormonal profile .Quantitative and qualitative changes in dietary patterns are needed to have an impact on the management of PCOS . GeneralThe effect of a ketogenic diet on PCOS outcomes has also been investigated. A ketogenic diet is usually characterized by high fat, moderate protein and very low (40–50 g/die) CHO intake . EvidencIn this regard, in PCOS women, oral supplementation with natural molecules has been surprisingly useful in reducing inflammation. It has been shown that a magnesium–zinc–calcium–vitamin D cosupplementation reduces oxidative stress and inflammation in women affected by PCOS, increasing antioxidant capacity . This isAn enhanced glucose and lipid metabolism has been observed in PCOS women also during vitamin D and calcium supplementation that positively affects triglyceride, cholesterol, testosterone levels, dehydroepiandrosterone sulfate (DHEAS), SHBG and hs-CRP . In addiThe inositol supplementation seems to be particularly effective in PCOS women. Inositol is a hexahydroxy cyclohexane (six-carbon ring compound) whose two isoforms, myo-inositol (MI) and D-chiro-inositol (DCI), respectively mediate insulin uptake in the ovaries and insulin-dependent androgen production . In partThe ovarian microenvironment is also influenced by the oral supplementation with L-carnitine; in a murine model, acetyl and propionyl L-carnitine have proved to enhance ovarian functions though the amelioration of the ovarian oxidative stress status, preventing the upregulation of SIRT1, SIRT3 and SOD2. Moreover, the same study demonstrated that L-carnitine acts against glycative molecular pathways . In PCOSIn addition, a nonpharmacological intervention with flavonoids should be considered in women suffering from PCOS because of their large anti-inflammatory and antioxidant activities . In muriEscherichia and Shigella leads to an altered production of short-chain fatty acids, while a lower abundance of Lactobacillus and Bifidobacterium species impairs the release of anti-inflammatory metabolites and reduces the immune response [Lactobacillus and Bifidobacterium abundance with a positive effect on LH/FSH ratio, testosterone, homeostasis model assessment-insulin resistance (HOMA-IR), α-1-acid glycoprotein (α-AGP) and leptin [Finally, it must be considered that a balanced diet modulates intestinal biodiversity that, in turn, has an impact on the PCOS phenotype. The gut microbiota refers to a group of nonpathogenic microorganisms with anti-inflammatory, antioxidant, immune and metabolic properties for which a relationship with several diseases, including PCOS, has been established . In muriresponse . In thisd leptin . In murid leptin .Therefore, gut microbiota plays a crucial role in PCOS pathogenesis and the use of probiotics as modulator of intestinal microorganisms should be considered as an alternative therapeutic strategy.In order to obtain significant lifestyle changes, and in addition to a balanced diet, physical activity is fundamental for improving both metabolic and hormonal parameters as well as reducing risk factors for cardiovascular diseases . As showBesides improving BMI and cardiorespiratory functions, aerobic exercise seems able to affect catecholamine-induced lipolysis in PCOS women. A 6-week aerobic training has proved effective in increasing in vitro atrial natriuretic peptide (ANP)/isoproterenol-lipolysis, enhancing the sensitivity to ANP and isoproterenol and the postreceptor response via cAMP-dependent protein kinases . This coMoreover, an aerobic protocol seems capable to reduce inflammation markers in women affected by PCOS: especially in obese women, the inflammatory status is predominant  and oftePhysical activity also affects mitochondria that have been shown to be involved in PCOS pathogenesis . In PCOSCertainly, physical activity is effective in improving antioxidant defenses. In PCOS women, a 12-week aerobic training has proved to increase superoxide dismutase (SOD) and the total antioxidant capacity, and reduce MDA and AMH .FNDC5 with the consequent release of irisin, which is also produced in response to ATP depletion after exercise [UCP1 gene and increase the expression of GLUT4 in the brown adipose tissue, leading to an enhanced glucose uptake; it is also involved in lipolysis mechanisms triggering the AMP (cAMP)–protein kinase A (PKA)–perilipin–hormone-sensitive lipase (HSL) pathway [Finally, the role of exercise-induced irisin should be considered. Irisin is a hormone-like myokine whose production in the skeletal muscle is mediated by physical activity and seems to be crucial in improving biochemical aspects of several metabolic diseases, including PCOS . Exercisexercise . The impexercise . Indeed, pathway .Because of its effect on inflammation, metabolism, endocrine and fertility parameters, physical activity should be always considered primary in the management of PCOS .PCOS is a multifactorial disorder whose real pathogenesis is still unclear. Continuous interaction between genetic and environmental factors seems to influence different phenotypes of the disease through epigenetic and molecular mechanisms.Differently from previous literature reviews in the field, the present work does not focus on a single topic, but it offers an overview on PCOS pathogenesis and nonpharmacological treatments, focusing on epigenetic mechanisms related to PCOS pathogenesis and on the reversal effects of diet and physical exercise. These approaches have a predominant role in triggering several pathways that affect PCOS clinical manifestations in a positive way. Next generation genetic analysis will allow incorporating huge, genotyped datasets, possibly generating genetic risk scores enhanced with clinical information, environment and lifestyle data for a precision medicine approach to PCOS diagnosis and treatment. Considering the above, more attention should be focused on these aspects to better improve the nonpharmacological strategies that appear fundamental in the management of PCOS.


Infertility is a global health concern affecting 48 million couples and 186 million individuals worldwide. Infertility creates a significant economic and social burden for couples who wish to conceive and has been associated with suboptimal lifestyle factors, including poor diet and physical inactivity. Modifying preconception nutrition to better adhere with Food-Based Dietary Guidelines (FBDGs) is a non-invasive and potentially effective means for improving fertility outcomes. While several dietary patterns have been associated with fertility outcomes, the mechanistic links between diet and infertility remain unclear. A key mechanism outlined in the literature relates to the adverse effects of inflammation on fertility, potentially contributing to irregular menstrual cyclicity, implantation failure, and other negative reproductive sequelae. Therefore, dietary interventions which act to reduce inflammation may improve fertility outcomes. This review consistently shows that adherence to anti-inflammatory diets such as the Mediterranean diet  improves fertility, assisted reproductive technology (ART) success, and sperm quality in men. Therefore, integration of anti-inflammatory dietary patterns as low-risk adjunctive fertility treatments may improve fertility partially or fully and reduce the need for prolonged or intensive pharmacological or surgical interventions. Infertility is defined as a failure to conceive after more than one year of unprotected intercourse in the absence of other reproductive pathologies . InfertiA key modifiable risk factor is preconception diet, which has been the focus of a range of studies to date. Substantial evidence suggests that modifying preconception dietary patterns to conform with Food-based Dietary Guidelines (FBDG) is beneficial to fertility outcomes ,9. TheseDietary interventions which reduce inflammation, such as anti-inflammatory diets, in men and women during the preconception period may therefore improve fertility outcomes. However, the role of these dietary patterns in promoting fertility has not yet been established. The aim of this review is to collate the available evidence regarding the role of anti-inflammatory dietary patterns in female and male fertility, focusing on inflammation as the primary mechanism underpinning the efficacy of these interventions. We also highlight the key gaps in the literature and outline recommendations for future research in this field.While previous research has focused on individual nutrients or single food groups, the importance of assessing dietary patterns as a determinant of overall health and disease risk has been recognised for some time ,13. DietTwo main methods are typically used in the research literature to quantify dietary patterns. The first method uses a priori defined numerical indices intended to capture adherence to specific dietary patterns that have been pre-defined on the basis of previous scientific evidence . A prior®) is an example of an a priori diet quality index developed to estimate the inflammatory potential of a diet .,90.89,90It has also been suggested that high intakes of soluble fibre can modulate inflammatory processes in response to the production of short-chain fatty acids, in particular butyrate, which is involved in the activation of transcription factors that regulate the expression of genes encoding proinflammatory cytokines ,97,98. LThe specific mechanisms whereby anti-inflammatory components may directly influence fertility outcomes remain unclear. Findings from observational studies indicate  that these anti-inflammatory dietary patterns attenuate pro-inflammatory markers during pregnancy and the preconception period. Some have highlighted that adherence to the MedDiet facilitates weight loss and reductions in central adiposity, potentially resulting in improved fertility, which is consistent with other studies on hypocaloric diets reporting improvements in inflammation and fertility in the short-term ,106. AddWestern dietary patterns  are typically associated with higher levels of inflammation  14,110],11014,11Further, the introduction of Western-style dietary patterns to East Asian regions (such as Hong Kong and South Korea) is thought to be a key driver of obesity and cardiovascular disease in the region . In tandAlthough several mechanisms are postulated, adherence to Western dietary patterns is thought to be involved in the upregulation of several genes (Interleukin (IL)-6, IL-1β, tumour necrosis factor [TNF]) involved in pro-inflammatory pathways ,118,119.Preconception nutrition has been linked to fertility, and in particular to ART success , with liRegular menstrual cyclicity is a core component of fertility, and irregular menstrual cycles  may indicate anovulation, substantially decreasing the ability to conceive . Diet haA cross-sectional study by Onieva-Zafra  comparedPutative mechanisms have been described linking diet with menstruation via inflammation-related pathways. Prostaglandins (such as PGF2-α and PGE2), which are associated with inflammation, are responsible for mediating blood flow to endometrial tissue, thereby controlling local hypoxia and smooth muscle contraction and in turn supporting menstrual bleeding . Anti-inAdhering to anti-inflammatory dietary components appears to have some benefits in relation to menstrual parameters, but it remains unclear whether improved menstrual cyclicity translates to improved fertility outcomes in this context. Since most of the studies in this area are observational, firm conclusions are precluded. This highlights the need for further well-designed, adequately powered, and appropriately controlled studies to establish the key links between diet and menstruation as a proxy measure of fertility. Endometriosis is one of the most common gynaecological conditions, affecting six to twelve percent of reproductive-aged women. In this condition, endometrial tissue that normally lines the uterus is found ectopically. Endometriosis is described as an estrogen-dependent state of chronic inflammation and has implications for fertility, affecting 35 to 45% of women with infertility . InflammIn a recent systematic review  of nine Based on these preliminary findings, it appears that interventions incorporating anti-inflammatory diet components may improve inflammation, oxidative stress, and ultimately alleviate pain and the overall symptoms and severity of endometriosis; however, their impact on pregnancy rates among women with endometriosis remains unclear . ImportaPCOS is a common endocrine condition presenting with reproductive, metabolic, and psychological features . InfertiLow-grade inflammation is common in women with PCOS, with studies reporting that C-reactive protein (CRP) levels are typically elevated . This heA recent RCT investigated the effects of an anti-inflammatory dietary intervention on fertility parameters in 150 adult overweight women with PCOS. In this sample, participants were either presented with an anti-inflammatory dietary combination with physical activity alone for 12 weeks or with the addition of metformin. Improvements in menstrual cyclicity and spontaneous pregnancy were reported in the diet and physical activity group, with concomitant seven percent weight-loss, and these effects were not inferior to those observed in the metformin group . This haA Cochrane systematic review by Lim et al.  in 2019 2. An observational study [Several studies have examined preconception diet in women undergoing ART, including intracytoplasmic sperm injection (ICSI) and IVF. In women undergoing ICSI, studies have produced inconsistent results. Hoek et al.  conducteal study  of 2659 al study . The PREal study  is one oal study .Few studies have also investigated the relationship between DII scores as an indicator of the inflammatory potential of a diet and outcomes related to IVF treatment in subfertile and infertile women. In a cross-sectional study of 144 infertile women from Iran, Diba-Bagtash et al.  reportedFindings in relation to the MedDiet specifically have been mixed. In a prospective cohort study  of 244 nAbnormal sperm characteristics contribute to failed reproductive attempts and underpin 30–40% of male infertility and ~30% of subfertility cases requiring ART . OxidatiA Western-style diet is thought to increase oxidative stress through promoting weight gain and insulin resistance, which are linked with infertility and poor sperm quality . FurtherIn a systematic review and meta-analysis of RCTs, Salas-Huetos et al.  reportedConversely, Western dietary patterns which include processed meat, potatoes, full-fat dairy products, coffee, alcohol, and sugar-sweetened beverages have been consistently associated with poor sperm quality and fecundability. A recent cross-sectional study by Nassan et al.  examinedFew studies have investigated the explicit relationship between DII scores and semen quality in males. In a cross-sectional analysis of 209 healthy male university students (aged 18–23 years) in Spain, a pro-inflammatory dietary pattern (as indicated by a positive DII score) was positively associated with total and progressive sperm motility, but had no relationship with total sperm count or morphology . In contIn light of the existing data, adopting a healthy diet incorporating anti-inflammatory components may have potentially beneficial effects in both women and men trying to conceive (subfertile couples). However, well-designed prospective studies and clinical trials are warranted to provide more definitive evidence in this context.Whilst current evidence for the potential utility of anti-inflammatory diets in fertility outcomes is promising, there are several limitations that must be acknowledged. First, nutritional intervention studies exhibit a vast diversity in treatment regimens, comparators, frequencies, and formulations. This heterogeneity precludes appropriate comparisons from being made between studies and makes the reported outcomes difficult to interpret.Second, the MedDiet is a dietary pattern based on the traditional cuisines from the 1960s of Greece  and other countries that border the Mediterranean Sea. As such, a singular MedDiet does not exist. Some individuals following the MedDiet, or any other anti-inflammatory diet, may be consuming relatively more or less fruits, vegetables, or dairy, than others. This is further complicated by disparities in the diet quality indices that were used to quantify adherence to a given diet, given that the scoring systems used to quantify adherence are not homogeneous and do not always produce comparable results. As such, many of the aforementioned studies included in this review have assessed adherence to a MedDiet using the Mediterranean Diet Score (or an adaptation of this), developed by Trichopoulou et al. [Third, evidence regarding female dietary patterns and female fertility outcomes, particularly in relation to IVF, relies mostly on observational study designs and early outcomes related to clinical pregnancy, with largely inconsistent results . InherenLastly, whilst dietary indices are useful for identifying and improving anti-inflammatory diet adherence for a specific population, at present there is a lack of consensus related to the application of specific MedDiet tools across different countries and regions . This isFuture research should focus on addressing the above limitations in order to develop high-quality nutrition intervention studies that are able to properly address these important research questions. The use of controlled studies  with appropriately powered sample sizes is critically important to ensure the reliability of the results, while epidemiological and population-based studies are important for ensuring that findings have some degree of external validity.TM trends indicating that searches for ‘diet and inflammation’ and ‘anti-inflammatory diet’ have steadily increased over the past 10 years, rising by 44% and 67%, respectively (from December 2010 to November 2020). In comparison, searches for ‘national dietary guidelines’ or country-specific resources such as the ‘Dietary Guidelines for Americans’ or the ‘Australian Guide to Healthy Eating’ peaked in 2004–2006 and then steadily declined over the subsequent five-year period. As the effectiveness of nutrition education depends upon relevance to the individual [Novel dietary advice such as introducing anti-inflammatory diets in a clinical context can engage curiosity, increase motivation, elicit exploratory behaviour, and promote learning ,185. Condividual , and gendividual ,193. Diedividual , but ratIn this review of the literature, we have highlighted that, despite some inconsistencies in the literature, adherence to an anti-inflammatory dietary pattern is generally associated with improved female  and male  fertility-related outcomes, which are thought to occur through mediation of anti-inflammatory pathways. Whilst the current evidence is not sufficiently designed to allow for controlled analysis, following a healthy dietary pattern has no risk involved with a possible plethora of perceived benefits. Further, integration of these dietary patterns as low-risk adjunctive therapies for fertility could improve the efficacy of concomitant interventions or reduce the need for more invasive or unwarranted surgical or pharmacological interventions. Whilst diet is unlikely to remove the need for ART, it offers an easily implementable and low-risk option to assist men and women toward achieving their desired fertility outcomes.


Despite promising breakthroughs in diagnosing and treating acute coronary syndromes, cardiovascular disease’s high global mortality rate remains indisputable. Nearly half of these patients died of ischemic heart disease. Primary percutaneous coronary intervention (PCI) and coronary artery bypass grafting can rapidly restore interrupted blood flow and become the most effective method for salvaging viable myocardium. However, restoring blood flow could increase the risk of other complications and myocardial cell death attributed to myocardial ischemia-reperfusion injury (IRI). How to reduce the damage of blood reperfusion to ischemic myocardium has become an urgent problem to be solved. In preclinical experiments, many treatments have substantial cardioprotective effects against myocardial IRI. However, the transition from these cardioprotective therapies to clinically beneficial therapies for patients with acute myocardial infarction remains elusive. The reasons for the failure of the clinical translation may be multi-faceted, and three points are summarized here: (1) Our understanding of the complex pathophysiological mechanisms of myocardial IRI is far from enough, and the classification of specific therapeutic targets is not rigorous, and not clear enough; (2) Most of the clinical patients have comorbidities, and single cardioprotective strategies including ischemia regulation strategies cannot exert their due cardioprotective effects under conditions of hyperglycemia, hypertension, hyperlipidemia, and aging; (3) Most preclinical experimental results are based on adult, healthy animal models. However, most clinical patients had comorbidities and received multiple drug treatments before reperfusion therapy. In 2019, COST Action proposed a multi-target drug combination initiative for prospective myocardial IRI; the optimal cardioprotective strategy may be a combination of additive or synergistic multi-target therapy, which we support. By establishing more reasonable preclinical models, screening multi-target drug combinations more in line with clinical practice will benefit the translation of clinical treatment strategies. Translating single, mechanistic basic experimental research to clinically beneficial outcomes is not a parallel, idealized process. Predicting clinically meaningful outcome endpoints is difficult, even from a well-recognized, robust preclinical indicator. The contradiction between successful animal research and ineffective translation of clinical effects has become an urgent problem to be solved . Due to In 1960, Jennings and colleagues  proposedCardioprotective strategies are classified according to the mode of action, that is, ischemic conditioning (episodes of brief ischemia and reperfusion), pharmacological protection , and physical methods .The components of ischemic conditioning include ischemic preconditioning (IPC), ischemic postconditioning (IPostC), and remote ischemic preconditioning (RIC) . IPC redThe discovery of specific therapeutic targets in basic research provides a theoretical basis for drug research, and the cardioprotective properties of new drugs offer more therapeutic methods for clinical application .Acute myocardial IRI can lead to autonomic dysfunction and is positively associated with mortality in patients with MI, making vagus nerve stimulation a promising therapeutic target , 24. TheCardioprotective strategies are classified according to the duration of action, including before ischemia, during ischemia and reperfusion, and after reperfusion. In a clinical setting, ischemia time is difficult to predict, and the myocardium is ischemia before symptoms appear, so we only discuss cardioprotective strategies after ischemia. Interventions . NecrosMicrocirculatory perfusion cannot be restored after recanalizing ischemic coronary vessels, mainly manifested as prominent low perfusion area and severe inhibition of blood flow , 74, 75.Multitarget cardioprotective therapy is defined as multiple cardioprotective agents or interventions targeting different targets acting together to exert additive or synergistic cardioprotective effects. A single intervention strategy can also be considered a multi-target strategy if it protects against multiple targets . To solvDiabetic patients have increased adverse cardiovascular events and worse clinical outcomes after acute MI. Despite the lack of clinical evidence, animal studies suggest diabetic hearts are resistant to cardioprotective strategies . A revieDiabetes increases myocardial susceptibility to IRI and alters the myocardial response to ischemic regulatory strategies by disrupting normal intracellular signaling . DiabeteMyocardial ischemia from cardiac surgery may also lead to IRI. Brief administration of a volatile anesthetic before initiation of reperfusion, known as anesthetic postconditioning, reduces MI size and improves cardiac function. The study confirms that sevoflurane postconditioning (SPC) protects normal rat hearts from IRI, and its mechanism depends on NO-mediated recovery of autophagic flux . HoweverIncreased expression of autophagy activation-related proteins in isolated rat hearts exerts cardioprotection. However, no increase in autophagy activation-related protein expression was found in left ventricular biopsies from CABG patients undergoing IPC . This di2S) attenuates hyperglycemia-induced oxidative stress and mitochondrial dysfunction induced during myocardial IR in hyperglycemic rats by enhancing the SIRT1/NrF2 signaling pathway and restores the cardioprotective effect of SPC. This experiment is the first to explore the relative roles of STAT1\/Nrf2 signaling in regulating mitochondrial dysfunction and oxidative stress in H2S, restoring SPC-mediated cardioprotection in diabetic rats. Diabetes also leads to impairment of the JAK-STAT3 pathway. The antioxidant N-acetylcysteine restores the cardioprotective results of SPC in diabetic rats by enhancing STAT3 activity and adiponectin and reducing Fox1 and CD36  can reduce MI size in normal rats , 109. HoAn effective cardioprotective strategy is to reduce angiotensin II production (using angiotensin-converting enzyme inhibitors or angiotensin receptor blockers) , 113. HoRenal denervation therapy, a novel therapeutic modality for refractory hypotension, has been proven to protect hypertensive rat myocardium from IRI by improving NO bioavailability, inhibiting oxidative stress and GRK2 signaling . Renal dStudies found that hyperlipidemia interferes with cardioprotective signaling and directly damages the myocardium , 115. HyIschemic regulatory strategies have a strong protective effect on myocardial IRI. However, the cardioprotective effects of IPC, IPostC, RIC, and SPC were inhibited in hyperlipidemia –126. Osi+ channel activator diazoxide is also inhibited under hyperlipidemic conditions  plays a protective role in hyperlipidemic hearts . The carAging can block the protective effects of ischemic regulation strategies against myocardial IRI , 137. A 2S can upregulate autophagy by activating the AMPK/mTOR pathway in aging hearts, thereby restoring the cardioprotective effect of IPostC  criteria were proposed . The IMPThe research on preclinical multi-target strategies focuses on discovering cardioprotective phenomena, and the specific protective mechanisms of different cardioprotective strategies cannot be determined , 131. MeOne of the reasons for the failure of the clinical translation may be the significant difference in comorbidity status between preclinical animals and clinical patients. Therefore, the development of animal models that are closer to clinical practice has become an urgent issue.in vitro aging myocardial tissue model based on human-induced pluripotent stem cell-derived cardiomyocytes, providing a promising new platform for studying cardiovascular disease and other age-related diseases  thickness allows nutrients to diffuse to the innermost cells, maintaining viability, and preventing ischemic damage cultures , 158.Human atrial preparation technology has also received extensive attention. The study by Kleinbongard et al.  demonstrTransgenic mouse hearts are widely used for human heart disease research. However, the standard mouse heart’s fast beating (400–600 bpm) limits its ability to evaluate its kinetics. In a recent experiment, isolated mouse hearts were stabilized at 120–130 bpm at 37°C by applying 300 μM lidocaine . The mouMost experiments in large mammals anatomically and physiologically closer to humans than mice and other rodents have been performed in young, healthy animals that lack the confounding factors specific to patients with clinical MI. To date, there are no reliable data from large animal models addressing confounding factors in cardioprotection . SimilarIn addition to the lack of reliable preclinical data to support translational strategies for cardioprotection, issues of rigor and reproducibility of preclinical studies are more general concerns. Lack of blinding and randomization principles, small data volume, and statistical testing methods with free significance thresholds lead to high false-positive results that cannot be replicated in preclinical experiments.Multicenter tests that consider the robustness of interventions to numerous confounders and unknown variables are an exception in studies of cardioprotective strategies , 165. ThTo this end, the National Heart, Lung, and Blood Institute-sponsored and formed a clinical trials network as a model for developing a shared collaborative infrastructure for research, named CAESAR . The consortium will study promising heart-protective therapy in mouse, rabbit, and pig models of myocardial IRI in a manner similar to a multicenter clinical trial . The overall goal of CAESAR is to screen for truly effective cardioprotective strategies in limiting infarct size through rigorous preclinical evaluation and to provide recommendations for clinical trials to test these cardioprotective strategies , 168. InIn animal models of IRI, various cardioprotective therapies can effectively reduce infarct size. However, conventional animal models of IRI cannot adequately reproduce the phenomenon of IRI in patients. Here, we envision that multi-target cardioprotection is necessary to achieve therapeutic effects in these animal models and effectively apply cardioprotection to patients. Interventions combined with drugs with robust mechanisms of action, efficacy, and safety tested in preclinical experiments, combined with appropriate routes of application, are good candidates for translational clinical trials. At the beginning of such experimental designs, a factorial method could demonstrate the additive effect of combination therapy, but this approach would increase the number of patients required. Therefore, it may be better to test the combination in patients and controls by confirming the additive effect in an animal model.Another factor that must be considered is that most clinical patients receive drug therapy before reperfusion therapy and have different comorbidities. There are no reliable data to fully demonstrate the cardioprotective effect of a multitarget strategy in large animals with IRI presence of confounding factors. Therefore, preclinical animal models should be as close to clinical reality as possible to confirm the practical protective effect of multi-target strategies.Animal experiments on cardioprotection need to improve rigor and reproducibility, and establishing a multi-center experimental network similar to the standard of clinical trials would be beneficial. Reliable preclinical data must be available to support tests of cardioprotective strategies in humans. The transition from single-center animal experiments to initial clinical proof-of-concept trials, and even directly to clinical outcome trials, is somewhat unreasonable and disorganized. Such an approach is undoubtedly burying heart protection strategies in the grave. In the era of personalized medicine, clinical outcome studies should be conducted in carefully selected patient cohorts to prove or deny efficacy. While specific cardiac assist interventions are protective in some patients, they are not a problem as long as they are safe for most patients. Modern oncology research can develop highly effective treatments for selected small cohorts, as can current cardiovascular medicine research.Finally, based on the examples we discussed in previous sections, some potential combination therapies for multi-target cardioprotective strategies include:(1)RIC combined with a drug with a different mechanism of action.(2)A drug that inhibits the cell death pathway and a drug that activates an endogenous cardioprotective pathway.(3)A single drug acts on different protection targets, such as cyclosporin A.(4)Background drug combination therapy .Establishing an animal model with the coexistence of multiple confounding factors and confirming that the multi-target strategy has a cardioprotective function in this model will have guiding significance for the translation of clinical trials.All authors reviewed the literature and contributed to the preparation of this manuscript.


Drought stress has severely hampered maize production, affecting the livelihood and economics of millions of people worldwide. In the future, as a result of climate change, unpredictable weather events will become more frequent hence the implementation of adaptive strategies will be inevitable. Through utilizing different genetic and breeding approaches, efforts are in progress to develop the drought tolerance in maize. The recent approaches of genomics-assisted breeding, transcriptomics, proteomics, transgenics, and genome editing have fast-tracked enhancement for drought stress tolerance under laboratory and field conditions. Drought stress tolerance in maize could be considerably improved by combining omics technologies with novel breeding methods and high-throughput phenotyping (HTP). This review focuses on maize responses against drought, as well as novel breeding and system biology approaches applied to better understand drought tolerance mechanisms and the development of drought-tolerant maize cultivars. Researchers must disentangle the molecular and physiological bases of drought tolerance features in order to increase maize yield. Therefore, the integrated investments in field-based HTP, system biology, and sophisticated breeding methodologies are expected to help increase and stabilize maize production in the face of climate change. Zea mays L.) has been anticipated to become the most important crop as its importance goes beyond food and feed , and ear stages (grain filling) of maize are the most susceptible to drought stress, with yield losses of up to 25, 50, and 21%, respectively . It inte closure .2 absorption and subsequently leads to a decrease in photosynthetic activity, is one of the first reactions to drought stress  analysis and transcriptome-wide association studies (TWAS) to evaluate expression-trait associations and identify candidate genes linked to a trait based on gene expression variation .via this approach. Additionally, for QTL fine mapping and cloning of the genes present within it, generally large populations are required to get high map resolution  approach has been practiced to identify favorable alleles for drought tolerance in maize. Unlike linkage mapping, it utilizes diverse populations for QTL fine mapping by harnessing the historical recombination . GWAS waZmTPA  for post-transcriptional gene silencing, DNA methylation, and histone changes affect gene activity and play an important role in gene expression to cope with environmental stresses , ND2005 and ND2006, TZE-W Pop DT STR C4, and TZE-Y Pop DT STR C4 have been successfully developed using conventional breeding methods such as recurrent selection, inbreeding, pedigree breeding, backcrossing, and hybridization . The sucThe application of molecular markers has improved the efficiency of breeding drought tolerant crops significantly . Unlike Advanced genomics-assisted breeding techniques including MAS, marker-assisted back crossing (MABC), marker-assisted recurrent selection (MARS), and genomic selection (GS) have opened up new possibilities for drought tolerance enhancement. Under WS circumstances, Arabidopsis thaliana, which is a DNA sequence that is beyond a QTL for transpiration efficiency. However, there is no report in the literature on the cloning of genes underlying QTLs in any crop species, or determining a suitable QTL for gene cloning is still difficult  to protect photosynthetic machinery during drought stress conditions. E. coli enzyme encoding choline dehydrogenase, which has been linked to maize abiotic stress. At the germination and young seedling stage, transgenic maize plants accumulated more glycine betaine and exhibited improved GY and drought resistance than non-transgenic plants. ZmPYL3, ZmPYL9, ZmPYL10, and ZmPYL13 genes played important role to impart drought resistance in transgenic plants by enhancing ABA signaling, proline, and other drought related marker genes. A transgenic gene-silencing approach was used to modulate the ethylene biosynthesis in maize and determine its effect on GY under drought stress. The results showed that there was significantly higher GY, decreased and an increase in kernel number/ear in transgene-positive events compared to nulls  and obtained 6% increased GY under drought conditions , transcription activator-like effector nucleases (TALEN), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease protein 9 (Cas9) system, have provided targeted gene modification in plants . The CRIbidopsis , rice , water efficient maize for Africa (WEMA), and affordable, accessible Asian (AAA) drought tolerance maize project. Under DTMA project nearly 160 drought tolerant maize varieties superior to the commercial maize varieties have been released between 2007 and 2013. These cultivars have been tested in experimental trails at farmers’ fields, and disseminated among farmers in 13 African countries through different national agricultural research systems and private seed companies . As per Another project, WEMA was in pipeline from 2008 to 2018 to develop drought-tolerant maize for smallholder farmers in Asia. Similarly, a project is ongoing at Indian Council of Agricultural Research (ICAR)-Indian Institute of Maize Research (IIMR), India for genetic enhancement of maize for the development of high yielding and climate resilient hybrids with the objective to develop drought tolerance new productive inbreds and hybrids . Variousviz., transgenics and genome editing have widened the range to improve desired traits in shortest period of time. Among crop plants, Arabidopsis, Brachypodium distachyon, and rice are the excellent model plants to study the drought stress responses. It provides a useful heterologous system to test new genes discovered in cereal crops like maize and their relative molecular responses. As a wild grass, Brachypodium has never been subjected to human selection, which is why it has a very promising framework for tracking drought-tolerance mechanisms in grasses that may have been lost during temperate cereal crop’s domestication. Hence, the research work done in these model plants should be transferred to maize by using an accurate experimental design, including high throughput phenotyping platforms to thoroughly investigate tissue- and cell type-specific drought responses. It is crucial to strengthen the coordination among different research groups and institutions working to develop drought tolerance in maize. In addition, partnership among public and private sector could not only prove instrumental to provide promising results, but also help in designing potential drought-tolerant strategies that might have a huge impact on global maize production.Currently, the agriculture sector is facing the biggest challenge of climatic change with frequent spells of drought. In addition, drought being a complex trait further complicates the speedily development of drought tolerant maize cultivars. A number of advanced QTL mapping, genomic-assisted breeding methods, genetic engineering, and genome editing tools have been implemented successfully to harness the commercial stress-tolerant maize varieties. With the deployment of recent advanced technologies, researchers have provided some insight on the genetic architecture and regulatory pathways of drought tolerance mechanism in maize. The rapid trait mining has detected several genomic regions regulating drought-associated traits that can be utilized for direct selection. But still the candidate genes involved to create genetic variants for these traits are unexplored. Hence, it is highly important to execute the multi-omics studies, genetic designs, and pertinent analytical methods in integration to clearly understand the molecular regulatory mechanisms of drought response. The other approaches SeS and YK wrote the manuscript. SK and ShS contributed in structuring the manuscript. RK conceptualized the idea. RK and SR edited the manuscript thoroughly. All authors read and finalized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Between 2020 and 2021, the COVID-19 pandemic severely strained health systems across countries, leaving millions without access to essential healthcare services. Immunization programs experienced a ‘double burden’ of challenges: initial pandemic-related lockdowns disrupted access to routine immunization services, while subsequent COVID-19 vaccination efforts shifted often limited resources away from routine services. The latest World Health Organization (WHO) and United Nations Children’s Fund (UNICEF) estimates suggest that 25 million children did not receive routine vaccinations in 2021, six million more than in 2019 and the highest number witnessed in nearly two decades. Recovering from this sobering setback requires a united push on several fronts. Intensifying the catch-up of routine immunization services is critical to reach children left behind during the pandemic and bridge large immunity gaps in countries. At the same time, we must strengthen the resilience of immunization systems to withstand future pandemics if we hope to achieve the goals of Immunization Agenda 2030 to ensure vaccinations are available for everyone, everywhere by 2030. In this article, leveraging the key actions for sustainable global immunization progress as a framework, we spotlight examples of strategies used by five countries—Cambodia, Cameroon, Kenya, Nigeria, and Uganda—who have exhibited exemplar performance in strengthening routine immunization programs and restored lost coverage levels in the last two years of the COVID-19 pandemic. The contents of this article will be helpful for countries seeking to maintain, restore, and strengthen their immunization services and catch up missed children in the context of pandemic recovery and to direct their focus toward building back a better resilience of their immunization systems to respond more rapidly and effectively, despite new and emerging challenges. Between 2020 and 2021, the COVID-19 pandemic severely strained health systems across countries, leaving millions without access to essential healthcare services. Routine Immunization programs experienced a ‘double burden’ of challenges due to the COVID-19 pandemic and associated disruptions and to the subsequent COVID-19 vaccination efforts shifting often limited resources away from routine services. The latest World Health Organization (WHO) and United Nations Children’s Fund (UNICEF) estimates suggest that 25 million children missed routine vaccinations in 2021, six million more than in 2019 and the highest number witnessed in nearly two decades.Recovering from this sobering setback requires a united push on several fronts. Intensifying the catch-up of routine immunization services is critical to reach children left behind during the pandemic and bridge large immunity gaps in countries. At the same time, we must strengthen the resilience of immunization systems to withstand future pandemics if we hope to achieve the goals of Immunization Agenda 2030 to ensure vaccinations are available for everyone, everywhere by 2030.This article spotlights examples of strategies used by five countries—Cambodia, Cameroon, Kenya, Nigeria, and Uganda—that have exhibited exemplar performance in strengthening routine immunization programs and that have restored lost coverage levels back to pre-pandemic levels to identify country-level learnings and inform and support other countries to adopt similar practices. No personal or patient-level health information was gathered for this work, and this work is not considered human subjects research. Coverage data was obtained from WHO-UNICEF and reported at the country-level. Across the world, the COVID-19 pandemic has disrupted essential health services, adversely impacting significant gains in health outcomes achieved in recent decades. This is particularly so for routine immunizations, with recently published WHO-UNICEF estimates showing historic reductions in immunization coverage in 2021, with 25 million children missing out on life-saving vaccines, the highest number since 2006 [As a result of this monumental drop in coverage, a disproportionate number of children in low- and middle-income countries are left most vulnerable to vaccine-preventable diseases. With rising immunity gaps, the risk of large outbreaks is imminent, with cases of measles already reported in Africa and Eastern Mediterranean, and wild polio virus 1 (WPV1) detected outside the endemic countries in Asia . This thRoutine immunization programs were impacted by a ‘double burden’ of managing the disruptive effect of the COVID-19 pandemic as well as the subsequent historic COVID-19 vaccination efforts to improve population immunity against the virus. Nearly all countries reported some form of disruption to routine immunization (RI) services in 2020 and early 2021, with primary and community care among the most affected service delivery settings . In manyDespite these sobering trends, some countries improved vaccination coverage during the pandemic—39 countries recovered or almost recovered to pre-pandemic levels in 2021. But over two years, only 24 countries achieved higher coverage in 2021 than in 2019 . This noThe vulnerability of global immunization systems demonstrates the urgent need to maintain, restore, and strengthen routine immunization systems in order to address the widening immunity gap in the context of the pandemic and build resilience for future shocks. Despite the complexities of managing immunization programs during a continually evolving pandemic, several countries have shown that improving immunization coverage is possible. Documenting and spotlighting learnings from these countries will help other countries to adopt similar practices to increase vaccination equity.This article presents examples from five selected countries—Cambodia, Cameroon, Kenya, Nigeria, and Uganda—that have either maintained or increased their Diphtheria, Tetanus, and Pertussis–containing vaccine (DTP3) coverage rates in 2021 compared to 2019/20 see . ThroughClosing immunity gaps and reaching missed communities require intensified efforts that are well planned and informed by evidence. In Cambodia, Kenya, Nigeria, Cameroon, and Uganda, multiple rounds of catch-up activities were conducted to service high-risk communities throughout the pandemic to address coverage gaps and inequities. These included a mix of intensified and targeted outreaches and campaign style periodic intensification of routine immunization (PIRIs) activities e.g., integrated child health days (ICHDs) and local immunization days (LIDs). In Cameroon, districts in conflict-affected regions were serviced through three rounds of periodic intensifications of routine immunizations (PIRI), which resulted in 28% improvement in DTP3 coverage in the South West Region in 2020/2021. To support with planning these outreach efforts, Clinton Health Access Initiative (CHAI) Cameroon helped track on a quarterly basis district level service delivery and immunization coverage indicators. We used an Excel/power BI-based dashboard, which enabled the rapid identification and prioritization of districts that required intensified catch-up activities. Similarly, in Cambodia, CHAI supported with data review and field assessments to assess and update the Expanded Programme for Immunization (EPI) list of high-risk communities with recent pockets of missed children, while in Kenya, CHAI supported the roll-out of tracking tools to monitor PIRI microplanning progress and review the accuracy and completion of immunization micro plans. In Nigeria, CHAI supported program planning for a catch-up campaign and identifying zero-dose children in 145 districts across the country, including redesigning microplanning and other data tools to accommodate infection prevention and control (IPC) needs in the context of the pandemic.Despite challenges brought on by the pandemic, these countries continued to strengthen health information system capacities to capture and use routine immunization data for planning and implementation. CHAI provided technical support to EPI across all levels in Cambodia, Uganda, and Kenya to strengthen data management and review capacities to promote immunization data use for planning and decision making. By promoting a systemic approach that leverages existing data, underserved communities can be identified and necessary resources allocated for rapid course correction. In Cambodia, CHAI supported the development of a new visualization dashboard that provided easy access and review of coverage gaps at all levels  to enable prompt follow-up and action. The dashboard was made available in English and the local language, Khmer, for ease of access and user-friendliness. In Kenya, CHAI was instrumental in supporting the Health Management Information System (HMIS) team in separating PIRI and RI indicators within the DHIS2 platform to enable clear performance monitoring for supplementary immunization activities. In Cameroon, CHAI supported the identification and characterization of zero-dose communities using triangulation of demographic, geographic, and immunization data. Through this effort, health areas with the highest risk or probability of zero-dose children were prioritized for targeted action. CHAI also supported mentoring activities to improve data completeness, timeliness, and quality into DHIS2, resulting in an increase of 18% in timeliness, 5% in data quality, and 5% in completeness in the Adamawa region in Cameroon. In Nigeria, to inform decision making, CHAI was instrumental in the roll out of the PowerBI tool for the visualization of real-time campaign immunization coverage and reach. In Uganda, CHAI strengthened identification of underserved areas within health facility catchment areas through monthly reviews of health facility immunization registration data, resulting in in ~50% increase in the number of children from underserved villages vaccinated against DTP3 and MR1 .Across the spectrum of activities undertaken to plan, implement, and monitor the COVID-19 vaccine roll-out, many of the countries who demonstrated resilience in the last two years capitalized on these activities for the mutual benefit of routine immunizations. ○The design and implementation of an integrated NCD screening and COVID-19 vaccination pilot in two provinces, which was initially implemented at mass vaccination sites and later shifted to health center fixed sites after the acute emergency phase (as booster dose delivery picked up pace). In the first five months during implementation at mass vaccination sites, the pilot successfully resulted in the referral or linkage to care for approximately ~1600 adults with previously undiagnosed diabetes or hypertension. By the end of the second phase of the pilot (February 2022), which has now been transitioned to government implementation, a total of ~2700 adults had been referred for appropriate follow-up care.○To optimize healthcare worker capacity and national health budgets to provide hard-to-reach communities with immunization services, CHAI supported with analyzing COVID-19 and routine immunization coverage data at the provincial, district, and village levels to inform the implementation of such integrated outreach in previously underserved communities.To improve integrated delivery of services and promote a life course approach to vaccination, the Cambodian Ministry of Health, with support from CHAI and other partners, developed policies to integrate routine immunization into the COVID-19 outreach strategy in 2021, with a focus on prioritizing hard-to-reach communities. CHAI’s support included the following:The Ugandan Ministry of Health, with support from partners, developed and disseminated operational guidelines to support the symbiotic delivery of health services alongside COVID-19 vaccination, care, and treatment. This included models that encourage task shifting among healthcare workers and hybrid offsite and onsite approaches to supportive supervision to better balance workload and delivery of health services at the district level.○TEACH, which combines traditional microplanning methods with appropriate technology.○A family-centered integrated PHC approach that translates into the national strategy of improving access to basic health services.○SCALES 3.0 , a strategy that incorporates integration of services, performance-based incentives, data use for action, and decentralized demand generation.Data reporting helped countries recognize and react to the cannibalistic effect COVID-19 vaccination was having on healthcare workers’ ability to deliver other primary healthcare (PHC) services, including routine immunization. The government of Nigeria decided to separate the service delivery and data collection functions of COVID-19 vaccination to support front line healthcare workers to prioritize the dual duty of COVID vaccinations and other PHC services. CHAI provided guidance in developing strategies and adopting technical guidelines for the integration of COVID-19 vaccination with other PHC services, including immunization, Vitamin-A, and ante-natal care (ANC) services. Three main strategies were promoted, which combined to boost coverage by 70% by October 2022. The strategies included the following:In Cameroon, Kenya, and Nigeria, COVID-19 vaccination training was also used to refresh frontline healthcare worker knowledge on routine immunization and promote broader immunization best practices.In Kenya, through support from CHAI, several county-level EPIs leveraged COVID-19 vaccine outreach services, targeting teachers in schools to co-deliver HPV vaccinations to eligible adolescent girls enrolled in the schools.Early intervention by governments to provide clear directives to health facilities was instrumental in minimizing service delivery disruptions. For example, the mid-2020 nationwide directive in Nigeria to continue routine outreach sessions resulted in an 11.6% increase  in serviLearning from the largest disruption to immunization service delivery in three decades and the largest vaccination rollout in history, a few countries are prioritizing investments in systems that enable multisectoral collaboration with strong community participation for agile decision making. For example, the Cambodian government is in the process of developing a PHC booster strategy to ensure all individuals can access a quality package of care in the public sector—from prevention to early diagnosis and management across the life course, encompassing maternal and child health, communicable diseases, selected non-communicable diseases, mental health, and other ageing-related illnesses. This strategy aims to emphasize stronger community engagement and new models of service delivery that could strengthen the resilience of health systems. CHAI is supporting by updating the community participation policy, which will redefine the governance, roles, and responsibilities for community heath workers within existing community structures. In Uganda, the government is adapting routine immunization for the use of Smart Paper Technology, which enables individual-level tracking of COVID-19 vaccine recipients and includes a reminder function for subsequent doses. CHAI is supporting the country to develop systems and processes that reduce missed opportunities for vaccination at every encounter with the health system, which has had a demonstrable impact on all antigens of note, including sustained increase in the number of vaccinated  in the supported districts in 2021 [The importance of integrating routine immunization into primary healthcare systems has never been clearer. As countries continue to recover and adopt lessons from the last few years, to not only mitigate the effects of backsliding but to reach those who were previously unreached, integrated and holistic PHC systems offer the best way forward for supporting resilient and sustainable routine immunization programs. Investing in broader health system strengthening and improving linkages with communities will help advance immunization goals. With the future of the COVID-19 pandemic remaining uncertain, it is vital to focus on building back better the resilience of our immunization systems to respond more rapidly and effectively to challenges, and ensure all children continue to have access to lifesaving vaccinations despite new and emerging concerns. Learnings from the exemplary countries presented in this article provide insights into the various possibilities that can be unlocked with the right commitment and support.


Idiopathic ventricular fibrillation is a rare cause of sudden cardiac arrest and a diagnosis by exclusion. Unraveling the mechanism of ventricular fibrillation is important for targeted management, and potentially for initiating family screening. Sudden cardiac arrest survivors undergo extensive clinical testing, with a growing role for multimodality imaging, before diagnosing “idiopathic” ventricular fibrillation. Multimodality imaging, considered as using multiple imaging modalities as diagnostics, is important for revealing structural myocardial abnormalities in patients with cardiac arrest. This review focuses on combining imaging modalities  and the electrocardiographic characterization of sudden cardiac arrest survivors and discusses the surplus value of multimodality imaging in the diagnostic routing of these patients. We focus on novel insights obtained through electrostructural and/or electromechanical imaging in apparently idiopathic ventricular fibrillation patients, with special attention to non-invasive electrocardiographic imaging. Sudden cardiac arrest (SCA) continues to be an international health problem and can be a fatal outcome of several cardiac diseases . SCA is When we re-evaluate the first described cases of idiopathic ventricular fibrillation, possibly dating back to 1929, it is questionable if they would have received the same diagnosis today . Many caThe 2013 Heart Rhythm Society (HRS)/European Heart Rhythm Association/Asia Pacific Heart Rhythm Society (APHRS) expert consensus statement defines IVF as “resuscitated cardiac arrest, preferably with documentation of VF, for which known cardiac, respiratory, metabolic, and toxicological etiologies have been excluded through clinical evaluation”. Or as stated in 1997 “the terminology that best acknowledges our current inability to identify a causal relationship between the clinical circumstance and the arrhythmia” ,11. To gNon-invasive imaging is a well-implemented part of the evaluation of SCA survivors and enables the detection of several structural diagnoses. The following sections of this review provide an overview of the yield of different imaging modalities during the evaluation of SCA survivors.The diagnostic use of echocardiography already starts during the acute phase of cardiac arrest, as it is able to identify signs of several acute and potentially reversible causes. Echocardiography can detect cardiac tamponade, CAD  and secondary signs of acute pulmonary embolism or hypovolemia . After tConcealed cardiomyopathies contribute to a part of the underlying etiology of IVF patients, as shown both on genetic and clinical basis ,43. One After echocardiography, CMR is indicated in all UCA patients . CMR serRe-evaluating the diagnosis of IVF in patients based on the expanded medical knowledge and advanced diagnostic techniques will eventually lead to the discovery of unidentified pro-arrhythmic factors. Hence, Groeneveld et al. re-evaluated CMR images to study the prevalence and morphology of mitral annulus disjunction (MAD) and MVP in a multicenter IVF cohort. Mitral valve abnormalities have been previously associated with UCA ,68. MAD Cardiac computed tomography (CT) can accurately quantify ventricular volume, mass and function, showing a good correlation with CMR. It is, however, less frequently performed in SCA survivors and not recommended in consensus statements for the cardiac evaluation of SCA survivors or for diagnosing cardiomyopathies due to radiation exposure ,37,38,39When CMR reveals abnormalities consistent with sarcoidosis ; PET-CT imaging is indicated and is a competent imaging modality to help diagnose sarcoidosis. PET-CT can visualize a specific pattern for cardiac sarcoidosis with patchy uptake ,33,34. FCompared to echocardiography and CMR, cardiac CT is less frequently performed in diagnosed IVF patients ,5,21. WiThe 12-lead ECG has sufficiently improved our understanding of the electrical activity in the heart by measuring an attenuated, dispersed reflection of the electrical signals from the heart on the body surface. The possible role of an invasive electrophysiological study in the diagnostic work-up of SCA patients, when other imaging modalities reveal no abnormalities, is described in a review by Haïssaguerre et al. . They diECGI facilitates the non-invasive measurement of the electrical activity at the level of the heart muscle by mathematically reconstructing these signals from an array of body-surface ECGs and a patient-specific heart-torso geometry  81]. Th. Th81]. ECGI investigations for validation included computer models, torso-tank experiments and eventually in vivo validation ,85. PossConduction and repolarization abnormalities, and abnormal focal excitation, can serve as a substrate for arrhythmias without proven structural abnormalities . A basisThis review describes different imaging modalities proven or potentially useful for the identification of a substrate for VF. Revealing the underlying etiology in SCA remains a challenge. The importance of a complete workup is vital for discovering a disease and improving management. Echocardiography and CMR should be performed on all UCA survivors. Coronary, cardiac, or PET-CT are indicated in specific cases. Promising new techniques are deformation imaging and ECGI, which might be implemented in the standard diagnostic assessment of IVF patients after further investigation.