Articular cartilage is a connective tissue consisting of two phases: a solid composed of collagen, proteoglycans, proteins, and chondrocytes, and a liquid made up of water and electrolytes. It can be divided into four zones , each presenting a different cellular organization, collagen fiber architecture and depth . These sIt is known that articular cartilage is an avascular tissue with little cellularity, making it difficult to heal. As a consequence, cartilage injuries can generate several complications for the individual, such as loss of mobility, degeneration and osteoarthritis (OA), directly affecting the quality of life. Not to mention that it represents an economic burden .Current procedures that seek to repair chondral tissue are microfracture stimulation, autograft and osteochondral allograft, and cellular implant therapies based on tissue engineering principles. Cell therapies and regenerative techniques are important and there are two main examples: autologous chondrocyte implantation (ACI) and mesenchymal stromal cells (MSCs). MSCs have received considerable research attention, due to ease of collection, ability to proliferate and differentiate cells, non-rejection by the patient, and paracrine effect on local cellular machinery .This Research Topic aimed to widening the knowledge on the strategies being used for articular cartilage regeneration, describing the state-of-the-art and exploring the innovative approaches in cartilage restoration field. This issue currently includes 14 articles related to different innovative propositions associated with cartilage injuries.In Dai et al. investigated the function of LncRNA H19 (H19) in BMP2, finding out that H19 regulates BMP2-mediated hypertrophic differentiation of MSCs by promoting the phosphorylation of Runx2, that maturates chondrocytes. H19 may play a role in cartilage differentiation, cartilage phenotype maintaining, and cartilage hypertrophic differentiation. They evaluated nine types of porcine tissue through a simple standard decellularization protocol.Stem-cell-based and gene-enhanced tissue engineered cartilage is promising in the treatment of cartilaginous pathologies, especially traumatic cartilage defects . Bone moHanai et al. found that a soluble decellularized ECM (dECM) for each of the nine tissues harvested exhibited variations in their biochemical characteristics and growth factor distribution. On cell culture, it appeared to promote cell differentiation toward the specified used ECM tissue phenotype and decellularization was successful with reducing cellular components in every tissue. The present results are important in the field of musculoskeletal regeneration therapy, since tissue derived soluble ECMs could be employed to regenerate tissues combined with some appropriate scaffolds seeded with stem cells.At present, tissue engineering and regenerative medicine have focused on extracellular matrices (ECMs) to function as a natural scaffold . Hanai eWang et al. attempted to analyze the cartilage reconstruction effect of bone marrow MSC-derived small extracellular vesicles (BMSC-sEVs) and showed an increase of the tissue in the cartilage lacuna and hypertrophic cartilage cells in the deep area of the bone under the cartilage, besides higher rates of cell proliferation and migratory activity and alleviated G1 stagnation of the cell cycle of OAsEV which may provide guidance regarding their therapeutic applications as early and minimally invasive therapies for TMJOA.Researching less invasive treatments for Temporomandibular joint osteoarthritis (TMJOA), Tejedor et al. demonstrated that the enhanced regenerative potential of MRL mice is attributed, in part, to their MSCs that exhibit PYCR1-dependent higher glycolytic potential, differentiation capacities, chondroprotective abilities, and regenerative properties than BL6 MSCs, what could be a promising tool in the treatment of OA. Comparing the MSCs of these two mouse species, Tejedor et al. showed that the slow-proliferating MRL MSCs display a higher migration potential than the fast-proliferating BL6 MSC and found that mesencephalic astrocyte derived neurotrophic factor (MANF) was specifically highly produced by MRL MSC as compared to MSC derived from other mouse strains, and that MANF highly produced by MRL MSC contributes to their capacity to tend to reduce the OA score.Comparing Murphy Roths Large (MRL), who possess remarkable capacity to regenerate several musculoskeletal tissues, and BL6 mices regeneration to a ear punch, Da Cruz et al. demonstrated that post-adipose-derived stem cells (ADSC) stimulation by type V collagen (Col V) treatment induced a significant regeneration of cartilage in an osteoarthritic rabbit articular cartilage model suggesting that surgical-induced OA treated with ADSCs stimulated by Col V may prevent the progression of cartilage injury and indicating that ADSCs/Col V may be a therapeutic target for the treatment of osteoarthritis. Also using a defect produced in the articular cartilage of rabbits, Yang et al. developed a staged regeneration strategy that combines endogenous cell recruitment and pro-chondrogenesis approaches for in situ articular cartilage regeneration with growth factor (GF)-loaded scaffold who facilitated cell homing, migration, and chondrogenic differentiation and promoted the reconstructive effects of in vivo cartilage formation.To delay the development of OA and promote joint cartilage regeneration, Li et al. used extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel) to evaluate the therapeutic effect of this natural biomaterial on this repair, comparing the control group treated with microfractures to the group with microfractures associated with ECM/SVF-gel, finding that autologous ECM/SVF-gel displays a curative effect on articular cartilage regeneration in a rabbit model, besides being a simple, time-sparing, cost-effective, enzyme-free and minimally invasive preparation process.Studying the regeneration of defects in articular cartilage, Mustapich et al. studied a strategy for enhanced microfracture treatment with stromal cell-derived factor-1 (SDF-1), demonstrating a simple cost-effective one-step process for improving the quality of cartilage defect repair in a rat model of microfracture. In this perspective, Lu et al. found that this approach played a unique role in cartilage resurfacing of adult rabbits despite the fact that self-healing dominates cartilage repair in young rabbits less than 9 months old.Microfracture is one of the most widely used techniques for the repair of articular cartilage. However, microfracture often results in filling of the chondral defect with fibrocartilage, which exhibits poor durability and sub-optimal mechanical properties. Jacob et al. showed emerging techniques, exploring tissue engineering with new methods of cultures and their results, and stated that for improved chondrogenesis the cells require both growth factors and mechanical forces to bring about more physiological cellular responses. The author demonstrated satisfactory results in the medium-term follow-up with the MaioRegen presenting faster return to sports and a low rate of complication and failure, while the Agili-C showed encouraging results through significant improvement in the assessments.After categorizing treatment options for osteochondral lesions into repair and regenerative techniques, Li et al. presented the cells in the joint tissues and their role in OA pathogenesis, the numerous ways these cells communicate, and concluded that application of stem cell-derived EVs in OA treatment is an emerging field in regenerative medicine.OA is a multifactorial disease that presupposes local and systemic factors and has multiple pathogenetic mechanisms, which must be considered when exploring new treatment options. Najar et al. This is an essential understanding of the immunological profile and functions of MSCs as a graft and understanding of the mechanisms involved in the effects of MSCs for better therapeutic targeting.Cartilage tissue engineering has potential for the treatment of cartilage pathologies, providing biomaterials that can be adjuvants to already established treatments to improve the clinical outcome, providing a higher quality regeneration and in the shortest possible time. There are clearly several cellular regulatory pathways involved in the therapeutic effect of MSCs, and the broad cellular and molecular changes that accompany MSC apoptosis, autophagy, and senescence may be essential for their therapeutic effects according to Pachito et al. reviewed thirty-nine studies focusing on the comparison of PRP to a related product, types of anticoagulants, centrifugation protocols, commercial kits, processing time, methods for activation, and application concomitantly to other substances, finding a great variability embed in each step necessary for the preparation of PRP which may justify the variability of clinical effects of PRP across different clinical trials.A treatment widely used in different clinical situations is Platelet-rich plasma (PRP), but universal standardization of procedures for its preparation is still lacking. In this editorial, it was possible to discover a large number of innovative aspects, with extremely promising research fields that could change the course of the treatment of OA and other pathologies that affect the articular cartilage. Despite that, the articles have some methodological limitations and further research should be carried out focusing on the difficulties raised, in order to increase the level of evidence, safety and efficacy of the new therapies.Innovative technologies and discoveries take time to be translated from an idea to clinical practice . Moreove


Molecular biomarkers play a marginal role in clinical practice for hepatocellular carcinoma (HCC) diagnosis, surveillance and treatment monitoring. Radiological biomarker: alpha-fetoprotein is still a lone protagonist in this field. The potential role of molecular biomarkers in the assessment of prognosis and treatment results could reduce the health costs faced by standard radiology. The majority of efforts are oriented towards early HCC detection, but the field faces an important challenge to find adequate biomarkers for advanced HCC management.Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and the fourth cause of tumor-related death. Imaging biomarkers are based on computed tomography, magnetic resonance, and contrast-enhanced ultrasound, and are widely applied in HCC diagnosis and treatment monitoring. Unfortunately, in the field of molecular biomarkers, alpha-fetoprotein (AFP) is still the only recognized tool for HCC surveillance in both diagnostic and follow-up purposes. Other molecular biomarkers have little roles in clinical practice regarding HCC, mainly for the detection of early-stage HCC, monitoring the response to treatments and analyzing tumor prognosis. In the last decades no important improvements have been achieved in this field and imaging biomarkers maintain the primacy in HCC diagnosis and follow-up. Despite the still inconsistent role of molecular biomarkers in surveillance and early HCC detection, they could play an outstanding role in prognosis estimation and treatment monitoring with a potential reduction in health costs faced by standard radiology. An important challenge resides in identifying sufficiently sensitive and specific biomarkers for advanced HCC for prognostic evaluation and detection of tumor progression, overcoming imaging biomarker sensitivity. The aim of this review is to analyze the current molecular and imaging biomarkers in advanced HCC. Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and, in terms of mortality, represents the fourth cause of tumor-related death  . In the It represents the main primary malignancy of the liver, followed by intrahepatic cholangiocarcinoma (CCC) .The 5-year HCC survival rate is about 18%, appointing it as the second most lethal tumor after pancreatic cancer , with a Systemic therapy is the only therapeutic approach for advanced HCC, with atezolizumab-bevacizumab as the current first-line therapeutic choice, along with Lenvatinib or Sorafenib if the principal option is not feasible .Despite the recent noteworthy improvements in the development of new therapies for advanced HCC, there is still a lack of biomarkers able to predict the treatments’ efficacy or identify subgroups of patientswhich would benefit from a certain drug. Alpha-fetoprotein (AFP) confirmation is the only molecular biomarker with recognized prognostic value .This review focuses on the potential role of molecular and radiological biomarkers in predicting the therapeutic efficacy and survival in advanced HCC patients, with a role in identifying specific subgroups of patients who could benefit from definite medical treatments or predicting early therapeutic failure to optimize the standard of care in these patients.A biomarker is a biochemical or clinical factor that provides reliable clues about disease burden and outcome, especially in response to specific treatments with relevant prognostic and predictive implications. The histotype, the prevalence of a tumor and the existence of efficacious treatments are important elements affecting the potential value of a biomarker .There are several parameters that can be considered valuable prognostic parameters for HCC: serum proteins such as AFP and its glycosylated form (Lens culinaris agglutinin-reactive AFP or AFP-L3), des-γ-carboxy prothrombin , tumor number and dimension, status of surgical margins, neoplastic stage, vascular invasion, portal hypertension, and Child-Pugh score .The traditional and well-proven imaging biomarkers are widely recognized as the gold standard in HCC management (diagnosis and treatment monitoring), they are represented by ultrasound (US), contrast-enhanced ultrasound (CEUS), computed-tomography (CT), and magnetic resonance imaging (MRI). The last two methods in particular are the gold standard for the assessment of neoplastic burden after treatment based on the standardized rules applied to imaging techniques: the Response evaluation criteria in solid tumors (RECIST) and their modified versions (modified-RECIST and RECIST 1.1) .Even adverse events represent potential response biomarkers during systemic treatment . It has been reported that hypertension, diarrhea and hand-foot skin reaction are correlated to sorafenib clinical efficacy .Due to the presence of chronic liver disease at different stages  in patients developing HCC, the relevant clinical biomarkers used as prognostic factors at the basis of the Child-Pugh score are liver function  and portal hypertension with its possible complications  .Regarding molecular biomarkers, it is important to underline that the wide heterogeneity of HCC’s molecular features has potentially relevant therapeutic and prognostic implications. In fact, HCC represents a complex environment involving both neoplastic and non-tumor cells (mainly belonging to the immune system). The reciprocal interplay between these elements is crucial to enhance the success of the different therapeutic options available.Despite the advances in terms of therapeutic options, serum AFP is still the main molecular biomarker used for diagnostic and prognostic purposes, and treatment response monitoring. Nevertheless, its role is still debated, especially in HCC surveillance and diagnosis , due to In the last decades no significant improvements have been achieved in the field of molecular biomarkers for HCC that still have weak roles in clinical practice. Among them the fucosylated fraction of AFP, proteoglycans such as glypican 3 and versican, DCP and PIVKA II have been principally investigated for early-stage HCC detection, treatment response monitoring and tumor prognosis evaluation ,17,18.Biochemical biomarkers have also been gradually included in some clinical staging algorithms; however, these still remain at the periphery of both the diagnostic and prognostic processes.The routinely applied therapeutic/prognostic decisional algorithm Barcelona clinic liver cancer (BCLC), traditionally based on tumor burden, liver function , and albumin-bilirubin “ALBI” score), and patient’s physical status (ECOG), in its most recent version introduced for the first time AFP as a criterium for liver transplant evaluation and to assess the eligibility to systemic treatment with ramucirumab . Other rNowadays, the main unmet need in HCC management remains the discovery of sensitive and specific biomarkers providing precocious information about the progression and prognosis of advanced HCC compared to traditional radiological biomarkers.The recently developed the BALAD score, based on the combination of biochemical values  and a panel of three biomarkers  have been proposed as a method to detect the worse prognosis, reflecting neoplastic progression and metastasis . HoweverThe serum is the most accessible source of biomarkers and a simple and non-invasive method to monitoring HCC treatment. Serum biomarkers can be categorized as protein markers, growth factors and circulating nucleic acid .AFP is still the principal and most reliable serum biomarker for HCC diagnosis, treatment management, and prognosis evaluation despite numerous attempts to investigate other possible molecules . The proIt has been demonstrated that an AFP reduction of more than 20% after 4 weeks of antiangiogenetic treatment is an important predictor of a patient’s response to therapy and survival ,31.p = 0.001) compared to the results granted by the RECIST and mRECIST criteria . It. It81]. The NLR showed a positive correlation with HCC recurrence after liver transplantation , and itsIn a retrospective analysis performed in patients with advanced HCC (BCLC stage C) treated with glass microsphere radioembolization, the post-treatment increase of NLR was associated with a reduced OS, although pre-treatment values did not show a significant association .p < 0.0001) . H. H108]. An accurate evaluation of tumor response after therapy has become an essential part in the management of advanced HCC not only for determining treatment efficacy, but also for subsequent therapeutic planning and as a marker for survival. Traditionally, the European Association for the Study of the Liver (EASL) guidelines recommend that the evaluation criteria for tumor progression or remission should incorporate measurements of viable enhancing areas of the tumor . The modIn a phase II study of brivanib in advanced HCC, mRECIST was able to demonstrate a higher response and disease control rate and a longer time to progression than the classical volume-based criteria . SimilarHowever, mRECIST criteria rarely reflect the effect of therapies that result in decreased enhancement without necrosis, typical of molecular targeted agents. In this clinical setting, it would be beneficial to measure tumor perfusion, because vascular changes can happen long before there is any evidence of tumor volume variation on conventional imaging. As a result, perfusion imaging techniques such as dynamic contrast-enhanced (DCE) ultrasound (US), CT or MRI begin to play a critical role in the evaluation of antiangiogenetic therapies . All theOn perfusion CT, HCC has been reported to show substantially higher perfusion  compared to normal liver tissue . Jiang ep = 0.01) was found between percentage variation of perfusion parameters, from baseline to follow-up, and overall survival rate [After antiangiogenic drugs or HCC directed therapies, a decrease in BF and BV has been shown within days of initial treatment ,115. In val rate .Similarly, Liang et al. reported that signal parameters of DCE-MRI over tumor and liver parenchyma correlated with tumor response and survival in HCC patients receiving a combination of radiotherapy with an anti-angiogenic agent . In a rep = 0.002, <0.001 and 0.003 respectively). Moreover, a decrease of AUC and an increased/unchanged value of time to peak (TP) and MTT were associated with longer survival  [Among different contrast enhanced imaging techniques, dynamic contrast-enhanced ultrasonography (DCE-US) has emerged as a versatile tool for monitoring anti-angiogenetic treatments since it is a non-invasive method that easily allows repeated examinations and provides both morphological and functional data. Several studies demonstrated that DCE-US can be used to predict tumor responses and patient survival in HCC patients treated with antiangiogenetic agents ,120. In A variety of imaging protocols, other than perfusion techniques have been proposed as biomarkers in patients with advanced HCC.A recent study demonstrated that baseline imaging features showing aggressive tumor biology and in particular, satellite lesions, atypical HCC, peritumoral arterial enhancement, and larger lesion size, can serve as imaging biomarkers for OS and liver decompensation in patients receiving both sorafenib and selective internal radiation therapy for HCC .Tumor stiffness and fibrosis could be another factor in predicting the therapeutic response in patients with advanced HCC since the abnormal cellular microenvironment of neoplastic conditions can be associated with increased stiffness .p < 0.001) and significant liver injury in advanced HCC patients treated with sorafenib  [Magnetic resonance elastography (MRE) is a relatively novel technique that has been shown to be superior to US-based elastography for the assessment of liver stiffness (LS) . In a st= 0.001) .p < 0.01). Moreover, stiffness was significantly correlated with intratumoral T lymphocytes in tumor biopsies [Similar results were obtained by Qayyum et al. in 9 HCC patients treated with pembrolizumab. Early changes in tumor stiffness evaluated by MRE were correlated significantly with OS (R = 0.81) and time to progression  is a standardized diagnostic system for the analysis of CT and MR imaging features in patients at risk of HCC development . Recent A precise association between imaging biomarkers and prognostic factors is still difficult, but useful for a rudimentary definition of predictive imaging features. It could be useful to include these characteristics into an algorithm alongside morphologic criteria. Nevertheless, further validation studies are required in order to increase its potential effectiveness in HCC management, patients’ outcome improvement, and shaping individualized treatment plans and follow-up.It is known that tumor heterogeneity is closely related to tumor prognosis, most notably in HCC lesions. The recent developed radiomics technique can obtain intratumoral heterogeneity in a non-invasive way that is relevant to patient prognosis. This is an emerging field in which high dimensional features are mathematically extracted from radiological images with subsequent conversion of images into mineable data . RecentlA multi-feature-based radiomic signature was identified to be an independent biomarker for OS and TTP in patients with advanced HCC treated with apatinib plus TACE. The combined use of a radiomic signature and AFP in the clinical-radiomic nomograms performed better than radiomic nomograms alone .p = 0.011), for predicting β-arrestin1 phosphorylation-positive HCC and OS [In a recent retrospective study, a radiomic-based model for predicting β-arrestin1 phosphorylation in HCC was developed using visual imaging features on preoperative CT images . ImagingC and OS .Although promising, none of these functional imaging biomarkers have undergone enough standardization and validation to be used in clinical practice. Further studies are warranted to determine unclear aspects such as optimal timing and best quantitative dynamic parameter to assess responses to HCC treatment.A large number of prognostic biomarkers for advanced HCC have been reported; however, their clinical applications are still far away in comparison to other cancers . Serum bDue to the high heterogeneity of HCC, further research is necessary to validate the potential of non-invasive biomarkers in the management of advanced tumors.


Many previous studies indicate that heart failure (HF) increases the risk of cognitive dysfunction and stroke, showing the logic that several physiological factors associated with cardiac dysfunctions affect homeostasis in the cerebral circulation. In the chronic process of HF patients, it is suggested that reduced cerebral blood flow (CBF) and abnormal auto-regulation might result in impaired perfusion, metabolic insufficiency, and regional or global structural deteriorations in the brain. However, the mechanism underlying HF-induced brain disease remains unclear. Cardiac dysfunction in patients with HF or HF-induced several physiological abnormalities may cause brain dysfunction. Possible physiological factors should be considered for future studies to prevent brain disease as well as cardiovascular dysfunction in patients with HF. It has been reported that coronary heart disease almost triples the risk of a stroke, and cardiac failure is associated with a more than fivefold increased risk . In the HF, one of the leading causes of morbidity and mortality, is a systemic disease but affects the other organ system, especially the brain . For exaIn severe chronic HF patients, global CBF is reduced by 14–30% . Kim et On the other hand, Choi et al.  reportedPrevious studies demonstrated some compensatory mechanisms  against changes in arterial pressure and cardiac output to maintain adequate CBF . Thus, iCerebral autoregulation, which maintains steady-state cerebral blood flow in a relatively stable manner over a range of perfusion pressures from 60 to 150 mmHg ,33, is aArterial baroreflex function is the compensatory mechanism for acute changes in arterial pressure to maintain an adequate perfusion pressure. Ogoh et al.  demonstrIn addition to arterial baroreflex, the cardiopulmonary baroreflex is also essential for CBF regulation. An increase or decrease in central blood volume (CBV) possibly elevates or reduces cardiac output and consequently causes over- and under-cerebral perfusion . OrthostMoreover, the vascular bed of the external carotid artery is essential to regulate intracranial blood flow. For example, resistance-exercise-induced large arterial blood pressure increases common carotid artery blood flow but internal carotid artery blood flow that supplies blood to the cerebral cortical region is well maintained, while external carotid artery blood flow largely increased accordingly as a large increase in arterial blood pressure . This fiIt is known that sympathetic nerve activity and the renin–angiotensin-aldosterone system are enhanced in HF patients . The comAs well as the CBF compensatory mechanism, we may need to consider possible HF-induced physiological factors that decrease CBF rather than cardiac dysfunction in HF patients. One of them is, for example, greater arterial stiffness that is associated with left ventricular (LV) remodeling and reduced LV function ,56. HF pBased on the anatomy, cardiac function should directly affect CBF regulation because of the cerebral artery connected to the aortic arch. Indeed, changes in arterial blood pressure and cardiac output are directly associated with CBF regulation. However, cardiac dysfunction in patients with HF directly does not cause impairment in CBF regulation. The compensatory mechanism of CBF regulation; cerebral autoregulation, baroreflexes, and external carotid artery vascular bed, against cardiac dysfunction, are also modified in patients with HF. Thus, these alterations may affect CBF regulation. However, since sympathetic nerve activity and the renin–angiotensin–aldosterone system that play a role in blood volume regulation are enhanced in HF patients, the effect of these physiological factors on the compensatory mechanism of CBF regulation needs to be considered. Additionally, one of the possible important physiological factors of cerebral hypoperfusion in patients with HF is arterial stiffness. Our previous study demonstrated that arterial stiffening modified dynamic pulsatile hemodynamic transmission from the aorta to the brain. Cardiac dysfunction and cerebral unfavorable circulatory homeostasis may be linked by a common factor: central arterial stiffening rather than direct cardiac dysfunction.


Personalized medicine is care that is tailored to an individual patient. In contrast, randomized trials are designed to report evidence of benefits and harms “on average”. The average trial participant though, is often healthier than older patients seen in clinical practice. A variety of methods have been proposed which offer improvements to traditional (but questionable) practices of one-subgroup-at-a-time examinations of treatment effect heterogeneity, but translating evidence from these advancements has received less attention. Data Science initiatives have allowed broader data sharing, data harmonization and data synthesizing approaches, as well as an enormous maturing of interactive visualization techniques. Motivated by the SPRINT study and using advanced approaches of marginalized standardization and the Predictive Approaches to Treatment effect Heterogeneity (PATH) statement, we show how researchers, regulators, clinicians and patients can "find themselves" on the treatment effect continuum and be better informed of potential individualized evidence through interactive visualizations.


Despite the implementation of specific dose constraints on healthy tissues to maintain the theoretical risk of late toxicity below 5% five years after radiotherapy (RT), many patients experience “unusual” toxicity during their oncological follow-up. This narrative review describes the methods of individual radiation sensitivity (iRS) diagnosis, their impact on the RT workflow as well as initiatives to support clinical decision-making initiatives.(1) Background: radiotherapy is a cornerstone of cancer treatment. When delivering a tumoricidal dose, the risk of severe late toxicities is usually kept below 5% using dose-volume constraints. However, individual radiation sensitivity (iRS) is responsible  for unexpected toxicities after exposure to a dose that induces no toxicity in the general population. Diagnosing iRS before radiotherapy could avoid unnecessary toxicities in patients with a grossly normal phenotype. Thus, we reviewed iRS diagnostic data and their impact on decision-making processes and the RT workflow; (2) Methods: following a description of radiation toxicities, we conducted a critical review of the current state of the knowledge on individual determinants of cellular/tissue radiation; (3) Results: tremendous advances in technology now allow minimally-invasive genomic, epigenetic and functional testing and a better understanding of iRS. Ongoing large translational studies implement various tests and enriched NTCP models designed to improve the prediction of toxicities. iRS testing could better support informed radiotherapy decisions for individuals with a normal phenotype who experience unusual toxicities. Ethics of medical decisions with an accurate prediction of personalized radiotherapy’s risk/benefits and its health economics impact are at stake; (4) Conclusions: iRS testing represents a critical unmet need to design personalized radiotherapy protocols relying on extended NTCP models integrating iRS.  More than 19 million new cancer cases are diagnosed worldwide each year . RT aims at deactivating tumor cells by using high-energy ionizing radiation to induce critical DNA breaks, particularly double-strand breaks (DSBs). Due to their physical properties, photons, which are commonly used in most RT facilities, can cause unwanted irradiation of the healthy tissues located within the RT field and surrounding the target, leading to specific toxicity. In clinical practice, we differentiate early expected toxicity transitorily, which has rapid effects such as dividing epithelial cells within 3 months from RT; and late toxicity, which can occur months to years after the completion of RT—an estimated 18% of adult survivors cope with permanent consequences . The staIndividual radiation sensitivity (iRS) has a substantial impact on the determinants of late RT toxicity, among other factors. iRS characterizes an individual’s tissue or cellular reaction to exposure to ionizing radiation. It is here used in the context of radiation doses that would normally induce no toxicity in the majority of the population classified as normal-responding individuals . As withHere, we aim to review the iRS diagnosis methods and estimate the impact of an a priori knowledge of a patient’s iRS in the RT workflow, using extended normal tissue complication probability (NTCP) models and initiatives to support decision-making. Recently, the clinical aspects of RT late toxicity have been thoroughly described—as simplistically summarized in Among RT-induced effects, one of the most frequent is RT-induced fibrosis. It involves complex molecular, cellular, and tissue mechanisms currently investigated to generate hypotheses for tissue response biomarkers, predictive assays, or mitigating treatments.The effects observed after RT may result from direct, intervention-related  and/or systemic mechanisms  integrated at the temporal-spatial level. The dose delivered to a given structure, the tissue organization, the degree of cellular differentiation, and their proliferative and regenerative capacity impact the variability of tissue damage and clinical expression. In addition, inter-individual variability occurs in fibrosis severity after a given RT plan in a specific anatomic area. The nature of the radiation-induced phenomena depends on the time elapsed since the irradiation: the phenomena are generally physical, chemical, and then biological, evolving from the microscopic scale  to the macroscopic scale . The most critical consequences at the cellular level involve DNA . The maiFinally, the irradiated cells either survive with accurate genetic information, survive with unrepaired lesions in more or less critical genes or die in their first generations. RT-induced death results from the various lethal contributions that may coexist, such as mitotic death, senescence, and apoptosis . Ionizing radiations can indirectly create cellular stress through the production of reactive oxygen species (ROS) ,16,17 thROS may be generated in the extracellular compartments or within the mitochondria . ROS areLow ROS levels in the cellular compartments promote detoxification by the upregulation of reducing factors  and the transcription of antioxidant enzymes  ,23. As tThe post-actinic inflammation begets a tissue remodeling process through a cross-talk between the repair and fibrogenesis pathways ,29,30,31The following conditions may expose the patient to a higher probability/severity of toxicity for comparable dose/volume constraints as in the overall population not experiencing toxicities. Technical factors should be carefully examined when investigating the mechanisms behind putative radiation-induced toxicities. Several therapeutic parameters may increase the risk of RT toxicity. There is a correlation between the probability of occurrence and the severity of tissue reactions on the one hand and the RT dose delivered in a specific body volume and in a certain time interval on the other. The total dose delivered is a major determinant of the outcome. Fraction size also has a relevant impact on late toxicities, with hypofractionation being more at risk of causing late damage than the conventional (1.8–2.2 Gy/day) fractionation schedules considering the same endpoint and the same volume of irradiated tissue . SimilarAll of these aspects should be modeled and integrated when planning RT, considering the specificity of dose-response for the different irradiated organs and tissues. Healthy organs at risk (OARs) surrounding tumor volumes are thus defined as structures to be spared more or less radically by the treatment when planning RT. Recommended doses for OARs are mainly derived from retrospective clinical experience accumulated over more than a century of practice ,37 and cThe effects of the total dose, daily dose, dose rate, and treatment timing can be included in NTCP models. These models are predictive tools used in RT to estimate the risk of treatment toxicities. They convert relevant characteristics of the dose distribution in OARs into a predicted probability of achieving the outcome of interest. They can merge the combination of the dose distribution with patient, disease, and treatment characteristics . NTCP moNTCP models can also include dose-modifying factors explicitly describing individual patients’ radiosensitivity, such as polygenic risk scores or results from biomarker assays ,44,45,46It is acknowledged that a concomitant treatment during RT influences the RT’s effects . HormonaTissue radiation sensitivity is a function of the developmental, self-renewal, and senescence dynamics of the organ . DysfuncA few infrequent hereditary diseases are characterized by a high iRS . Such paA large number of autosomal recessive genetic syndromes associated with radiosensitivity are associated with DNA repair dysfunction, strongly suggesting that DSBs are the cause of radiation-induced cell death . HomozygIt has been estimated that more than 5–10% of patients suffering from high/moderate—but still “unusual”- toxicity during/after RT have none of the phenotypic alterations, risk factors, and pathogenic mutations involved in radiosensitive syndromes . DecipheIn the field of genome sequencing techniques and tools, radiogenomic studies have improved the knowledge of common genetic alterations associated with RT-induced toxicities. These studies have identified biomarkers through candidate gene studies, prediction models based on SNPs, or Genome-Wide Association Studies (GWAS)  see Tab below.Applied to the individual risk of RT-induced skin fibrosis, clinically significant gene expression patterns and regulators were associated with iRS. Several single nucleotide polymorphisms (SNP) were identified, most of them belonging to the DNA damage response (DDR) pathway -including ATM, a central kinase in the initiation of DDR . Ho et aMitochondrial haplogroups are also associated with iRS, the haplogroup H being radioprotective, while haplogroups J and U are associated with RT-induced skin fibrosis .The use of skin transcriptome emerged in the early 2000s . QuarmbyForrester et al. identified a basal transcriptomic signature to distinguish patients prone to develop RT-induced skin fibrosis using fibroblasts from healthy skin. ,96. OverPhenotype-specific splicing events have also been highlighted: 152 genes were alternatively spliced between radiosensitive and radioresistant fibroblasts. They were associated with gene ontology terms linked to fibrosis’ pathogenesis, such as the integrin-mediated signaling pathway  and extrEpigenetic modifications include histone modifications, such as acetylations and methylations, DNA methylation, particularly on the CpG island, non-coding RNAs, and three-dimensional chromatin organization . Few stuAt the molecular level, oxidative stress, DNA and biomolecule damage, DNA repair, cell death, and local and systemic reactions are involved in the occurrence of RT toxicities. The association of genes or their products or regulators in these different pathways was studied as a potential biomarker for toxicity prediction .The challenge is to implement a reproducible assay with high sensibility and sensitivity . AlternaPredictive biomarkers of iRS  can be pIn the radiomic approach, normal tissue imaging in response to RT or dose distribution  may predNumerous studies tried to identify mutations and polymorphisms in patients with different types of cancers. Single nucleotide polymorphism (SNP) studies are limited to a candidate gene approach, with a tissue specificity for each genetic determinant and “linkage disequilibrium” for which some SNPs can catch most of a regional genetic variation. Even if most studies have produced statistically significant results, the models were often not applied to validation cohorts . The mosSensitivity to RT can be associated with rare cancer-prone syndromes . ATM proApoptosis is the most crucial pathway of programmed cell death. Ionizing radiation can induce cell apoptosis phenomena linked to DDR with activation in a p53-dependent manner, the release of cytochrome C of mitochondria, and apoptosome formation ,124,125.The oxidative stress response has been described for a long time as being involved in determining the response to irradiation. Some assays to predict radiation toxicities have focused on the quantification of enzymes involved in DNA repair mechanisms of the base, nucleotide, single DNA, or double DNA strand breaks: one such first study on 38 patients with head and neck cancer showed that the dysfunction of these pathways might be associated with some risk of toxicity depending on the treatment’s outcome .The last 20 years offered exponential advances in genomic technology, as nowadays, SNP or GWAS studies can be performed directly from a blood sample. This technology is on the edge of offering new tools for targeted screening in high-risk individuals, but more research is needed if GWAS is to pay off the investment required see : nowadaySystematic screening of radiosensitivity in patients scheduled to undergo radiotherapy is undoubtedly a way to further reduce the morbidity of radiotherapy in a personalized fashion. The development of high-performance iRS diagnostic tests should make this strategy feasible in routine practice. A prerequisite for this is that such testing has to be integrated into treatment costs. Pre-therapeutic radiosensitivity testing would then be used to adapt cancer treatment a priori.Currently, iRS screening tests have ~90% accuracy. Therefore, there is no clinical obstacle to implementing iRS screening in practice. Therapeutic modifications could then mainly consist of treatment de-escalation with a reduction in the administered doses or the irradiation of more limited, selected volumes. Strategies based on iRS testing have to rely on evidence-based medicine, such as non-inferiority clinical trials, to ensure that tumor control is at least equivalent to a personalized treatment as with standard treatment and that its associated toxicity rates are indeed lower.Another prerequisite for the implementation in routine practice is that a standard technique for iRS screening should be recognized internationally as there are currently multiple and non-consensual iRS approaches.The recommendations of the American Society for Radiation Oncology  are currTherefore, the question of patient management according to their iRS remains open, as “unusual” toxicity events may occur even in patients without any identified genetic mutation. Although there is no international guidelines, physicians facing toxicities that are not explainable by technical factors or comorbidities in a given patient may be willing to investigate their iRS to adapt anticancer and supportive care management. It is estimated that about 90% (unpublished data) of pATM testing in France falls in this category. Given that the probability of iRS in a specific patient is high, physicians could propose customized monitoring of toxicities during further radiotherapy courses based on an accurate testing method.Knowledge of the prediction of adverse events after RT has improved in the last years ,135,136,In another field, mobile applications can be worth the remote monitoring of outpatients to improve the collection of toxicity data and the patients’ compliance and adherence to supportive treatments, which may help to avoid higher rates or the increased severity of acute and late toxicities ,143.Various physical, technological and biological approaches may be proposed to improve the profile of tolerance to RT in patients with normal phenotypes identified as radiosensitive . It may When RT avoidance represents suboptimal therapy, technical RT adaptations should be proposed to minimize toxicity.Reducing the tissue volume irradiated at intermediate/high doses may help to reduce the incidence of toxicities—in 2022, over 90% of routine RT treatments rely on photon-based RT.Photon-based techniquesConformal irradiation, i.e., “conventional” tridimensional radiotherapy, using a linear electron accelerator producing “high energy” megavoltage-photons has gradually been replaced by Intensity-Modulated Radiation Therapy (IMRT) in many tumor sites treated with curative intent. Stereotactic Body Radiation Therapy (SBRT) has also been massively implemented in routine practice since the development of extracranial SBRT equipment with in-room imaging and the versatility of linear accelerators. SBRT is used in curative indications and increasingly also for metastases, sometimes with repeated courses. A shared trait of both IMRT and SBRT is the use of photons. The “unfavorable” dose deposit of photons, i.e., with a significant amount of dose under the skin continuing their course in tissues behind the tumor, has somewhat been compensated by the technological advances, i.e., with the use of many (coplanar or non-coplanar) beams and sophisticated photon fluence modulation using dynamic multileaf collimators. Manipulation of photon beams have resulted in the ability to deliver very steep dose gradients with better conformality. Clinically, these advances have translated into reduced volumes of organs at risk of receiving a high dose and, subsequently, into lower rates of clinically relevant toxicities. Control for setup uncertainties and tumor or organ tissue volumes (inter-fractions and intra-fractions) is exploited to reduce radiotherapy margins; this is the field of image-guided (IGRT) and adaptive radiotherapy (ART).However, this better conformation has not fully eliminated the issue of low to intermediate doses to healthy tissues , and parTissue volume-sparing with other forms of radiotherapy than IMRT or SBRTAs with -MV photon-based IMRT and SBRT, brachytherapy and hadrontherapy can be used to reduce the irradiated volumes . HadrontConcerning protons, major technological advances have been made in the last 15 years, which have favored the expansion of protontherapy (PT) centers worldwide. Production of secondary neutrons has been reduced with active (pencil beam scanning) delivery (compared to passive PT).It is also important to note that intriguing PT-induced toxicity profiles were recently reported , suggestThe physical properties of carbon ion beams and protons show a dosimetric advantage due to their characteristic dose deposit patterns. As linear energy transfer increases with the range in proton beams, RBE values can rise from about 1 in the entry channel to about 1.7 in the distal falloff region. In contrast, with carbon ions, the number and complexity of ionizations increase along with an increase in LET up to 100 keV per nucleon, which is the overkill area where death mechanisms are “saturated.” The resulting complex clustered DNA damage is more difficult to repair and can have a better therapeutic effect by killing tumor cells. However, low carbon ion doses could also induce complex unrepaired chromatid anomalies , which mFinally, proton therapy and ion therapy might be considered to reduce the volumes subjected to irradiation volumes—radiobiological and radiosensitivity investigations are ongoing.Brachytherapy is also an effective way to reduce irradiated volumes using sources placed in the patient body. Currently, this is most often performed using a high dose rate due to radioprotection issues; it can also be performed as intracavitary or can be interstitial.HyperfractionationHyperfractionation is one modality of altered fractionation that has been historically used to reduce long-term toxicities. It delivers fraction doses of less than 1.8 Gy (often 1.2 Gy) more than once daily   or five Hyper-radiosensitive patients are usually denied radiotherapy. Due to a lack of evidence-based data in moderately radiosensitive patients (group 2 in ATM studies) with molecularly detected iRS, no specific fractionation or technique recommendation has yet been proposed in radiosensitive patients . While tRadiation effects including cell cycle arrest, DNA repair, and cell death) are well documented. However, specific gene expression data after different modalities of radiotherapy  are lacking implications for iRS testing and treatment adaptations could be important and clinically relevant.New Frontiers: temporal fractionation (such as FLASH) and spatial fractionationAnother area of investigation in radiosensitive patients could be FLASH radiotherapy due to its dramatic normal tissue-sparing effects ,160. ThiOther normal tissue-sparing technologies under development include high-dose spatial fractionation (GRID), allowing valleys of low doses along the beam paths . Data specific to iRS are lacking. Toxicities should be managed already from the early phase using appropriate supportive care to reduce the risk of them transforming into consequential late effects in some instances. Steroids or non-steroidal anti-inflammatory treatments—topical or systemic—are frequently used. RT may be continued, interrupted momentarily, or even permanently, depending on the severity of the effects. In addition to symptom-oriented supportive care, pre-rehabilitation and rehabilitation may improve long-term tolerance ,166,167.(1)Anti-inflammatory drugs or angiotensin II receptor antagonists ; cortico(2)Long-term use of antioxidant drugs such as superoxide dismutase or tocopherol (vitamin E), preferably combined with pentoxifylline, to mitigate fibrosis, as evidenced in randomized trials .(3)Bevacizumab, is an effective treatment of radionecrosis due to its intrinsic anti-edematous properties and preventive activity against anarchic angiogenesis, especially in the brain ,171.(4)Endoscopic or minimally invasive procedures, may help to relief stenoses , adhesions, and strictures (bowel). Argon plasma electrocoagulation of telangiectasia is an effective approach for skin or bleeding mucosae . Several(5)Hyperbaric oxygen (HBO) is effective in better oxygenating fibrotic/hypoxic tissues, especially lymphedema, jaw osteoradionecrosis, and proctitis . Clinica(6)In situ injection of autologous mesenchymal stem cells, recently advocated in the frame of accidental irradiation .Late toxicity is generally considered irreversible, characterized by fibrosis and/or necrosis. When identified during follow-up, countermeasures can be proposed to mitigate these effects, such as:The understanding of increased intrinsic radiosensitivity in patients has substantially improved in the last decade. Despite possible morbidity and sequelae of “unusual” toxicities, systematic detection in patients with a normal phenotype has not yet been implemented in routine practice, mainly because proofs of performance of individual radiosensitivity testing have been available only recently. Other more complex reasons are the lack of financial support for this kind of monitoring and the lack of a proper assessment of the benefits of avoiding sequelae for the numerous patients who undergo radiotherapy at some point in their cancer history. Yet, it is challenging to estimate the risk/benefit ratio of radiotherapy in a given patient with a normal phenotype but with unusual toxicity for which iRS is suspected but not tested. iRS testing could contribute to a more clinically relevant decision. These gaps in knowledge in iRS testing should be addressed in order to design truly adapted radiotherapy protocols  and set up more frequent monitoring and more systematic supportive care.


The cerebral cortex forms early in development according to a series of heritable neurodevelopmental instructions. Despite deep evolutionary conservation of the cerebral cortex and its foundational six-layered architecture, significant variations in cortical size and folding can be found across mammals, including a disproportionate expansion of the prefrontal cortex in humans. Yet our mechanistic understanding of neurodevelopmental processes is derived overwhelmingly from rodent models, which fail to capture many human-enriched features of cortical development. With the advent of pluripotent stem cells and technologies for differentiating three-dimensional cultures of neural tissue in vitro, cerebral organoids have emerged as an experimental platform that recapitulates several hallmarks of human brain development. In this review, we discuss the merits and limitations of cerebral organoids as experimental models of the developing human brain. We highlight innovations in technology development that seek to increase its fidelity to brain development in vivo and discuss recent efforts to use cerebral organoids to study regeneration and brain evolution as well as to develop neurological and neuropsychiatric disease models. Human tissues are frequently inaccessible to experimentation. In particular, long-term perturbational studies involving human cells resort to cell-based models. This is especially challenging for organs such as the brain, where limited or no stem cell populations can be robustly isolated and cultured in vitro. The isolation of mouse embryonic stem (ES) cells ,2, the dIn parallel with the advances in stem cell differentiation into neuroectoderm in serum-free conditions using defined combinations of small molecules, a protocol for differentiating organoids as models of cortical development was introduced . By cultThe advent of human ES and iPS cells enabled the adoption of these three-dimensional culture protocols for human cells ,13,14 Fb. In parProtocols for differentiating cortical organoids have been further refined, and cell types that emerge within brain organoids have been further characterized. For example, the emergence of cortical astrocytes has been demonstrated ,15,16, aMore recently, by using multi-electrode arrays to measure spontaneous and evoked activity, organoids have been shown to be able to respond to light stimulation , and to Together, these studies have demonstrated a remarkable capacity of neural differentiations to self-organize, recapitulating many complex features of the developing brain, including cell type heterogeneity, cellular organization, and even cell-cell interactions necessary to enable complex patterns of neural activity to emerge b,c.Transcriptomic and epigenomic datasets generated from primary human tissue serve as an invaluable resource for benchmarking in vitro models. For example, brain organoids have been profiled using single cell transcriptomics and compared against microdissected bulk tissue transcriptomic data from developing human cerebral cortex, revealing that the molecular programs underlying human cortical neurogenesis are broadly expressed in cortical organoids . This stSimilarly to transcriptomic analyses, epigenomic and epitranscriptomic (mRNA modifications) profiling have been applied to brain organoid and primary human tissue samples, which also revealed that broad neurodevelopmental trajectories and transitions are recapitulated in organoids sampled at different stages of differentiation ,37,38,39While transcriptomic and epigenomic data are tremendously valuable in that they enable genome-wide comparisons, they are unable to capture the dynamic neurodevelopmental processes that give rise to the complex cell types of the developing brain. Mechanistic studies of cortical development have been overwhelmingly conducted in mice. These studies have underscored the role of radial glia as the neural stem cells of the brain ,43,44. RIn many species outside mice, such as ferrets, macaques, and human developing cerebral cortex, radial glia extensively diversify into outer radial glia  in the outer subventricular zone (OSVZ) ,57,58,59Importantly, oRG cells have been shown to give rise to oligodendrocyte progenitor cells (OPC) , althougGlutamatergic neurons of the cerebral cortex are generated by radial glia and IPCs during human development ,56,57,58GABAergic neurons of the cerebral cortex originate in the ganglionic eminences ,79,80,81A deeper understanding of radial glia heterogeneity and differentiation trajectories in the human cerebral cortex will provide critical resources for benchmarking cerebral organoids as models of human neural development and may provide important insights into the cell of origin of brain tumors ,88,89. WTimelapse microscopy studies conducted in cerebral organoids have demonstrated that the characteristic behaviors of radial glia subtypes are recapitulated in organoids ,96, as wCerebral organoid protocols are being widely adopted to model human neurodevelopment, the consequences of genetic mutations, and in the context of evolutionary comparisons. In parallel, advanced methods are being developed to apply next-generation technologies to brain organoid culture. Publications describing detailed protocols as well as hands-on training workshops represent important steps towards developing protocol commons, as well as quality control standards that could be agreed upon by the scientific community to support the generation of interpretable data. One difference in experimental design that could lead to different experimental outcomes involves the choice of neural induction protocol. Some studies have relied on protocols that are more directed and involve dual smad inhibition ,13,14, wAnother important issue that is rarely discussed is the fact that lines from very few donors have thus far been utilized by most organoid studies. There is an unmet need to establish lines from donors that represent a more complete spectrum of human genetic diversity. Validation of their differentiation capacity using newly developed protocols that increase the reproducibility of cerebral organoid differentiations ,102 willA pervasive cell culture-associated cell stress response has been identified as a potential confounding factor in organoid experiments ,33. SomeSimilarly, most cell types that form the cerebrovasculature  do not aOther cell types that are involved in cerebrovascular interactions, such as pericytes, smooth muscle cells, fibroblasts, fibromyocytes, or perivascular macrophages , remain During development, neurons form local and long-range interactions with cells located in ontogenetically distinct regions e. While Physical co-culture of two or more brain region-specific organoids enables interactions between cells that emerge from different sets of progenitors to be studied in vitro. This approach is sometimes referred to as an ‘assembloid’ assay f, and alHowever, organoid fusions require parallel organoids to be differentiated into distinct regions, and in most cases, organoid fusion is not performed until these regional identities are fully acquired. As an alternative strategy, a growing number of studies are employing methodologies that deliver localized sources of developmental morphogens into parts of an organoid, such that they can mimic the effects of organizer regions that normally pattern the developing neuroepithelium. As a result, multiple brain regions can be simultaneously induced within the same organoid ,137.Another experimental approach that enables studies of neural interactions involves the separate induction of brain organoids that are cultured separately while allowing for neurons to form reciprocal axonal connections. These “connectoid” cultures have been shown to develop more complex neural activity profiles  than indCortical organoid differentiation protocols have been convincingly shown to generate cortical lineage neurons. However, the brain contains dozens of neuronal subtypes that ultimately give rise to its complex function. In this section, we will discuss broadly how the diversity of neurons in the mammalian forebrain is specified and how these are modeled using organoids.Cortical neurons are specified along two major axes. First, their position in the six-layered cortical plate defines their morphology and connectivity. For example, “deep layer” neurons located in layers five and six send axons to subcortical areas, including the spinal cord and thalamus, respectively, whereas “upper layer” neurons of layers two, three, and four project to other cortical layers, to the contralateral hemisphere, or to the striatum . The speThe second axis follows the surface of the cerebral cortex, which can be divided into dozens of anatomically and functionally distinct areas, called aerial or regional identities. During early development, developmental morphogens secreted by organizer regions induce the expression of patterning transcription factors in radial glial cells, which subsequently give rise to glutamatergic neurons (reviewed by ). DifferAs an early application of the organoid technology, Sasai et al. demonstrated that cortical neuron differentiation can be further refined by harnessing lessons from developmental biology to increase regional specificity. In their experiments, inhibiting FGF signaling favored the specification of caudal cortical identities (NR2F2+ ve neurons), whereas activating Wnt and BMP signaling favored the specification of medial structures such as the cortical hem and choroid plexus . SubsequRegulatory programs underlying cortical neuron specification and arealization have been extensively studied in mice . TargeteFinally, epigenomic datasets generated from distinct areas of the cerebral cortex are beginning to emerge ,145,146,One of the key applications of stem cell-based technologies is regenerative medicine. Pluripotent stem cell derived neurons have been successfully transplanted into neonatal immunocompromised mice and shown to develop projection patterns consistent with their molecular identities ,148. MorSeveral pioneering studies have successfully transplanted intact organoids into adult mouse brains ,152,153.In one study, organoids were transplanted into 3-year-old cynomolgus monkeys . By 12 wWhile chimeric animal studies are necessary to understand the fidelity and function of in vitro derived human brain tissue and are a necessary first step for regenerative medicine approaches, they are not without controversy. Biomedical researchers, ethicists, and the public have questioned whether and when transplanting human neural tissue into experimental animals leads to human-like perception or cognition. Would chimeric animals modeling neuropsychiatric diseases potentially experience human disease symptoms in a distressing manner? The National Academies of Science, Engineering, and Medicine published a report on neural chimeric tissues in 2021  that calThe discovery of reprogramming factors has allowed for the generation of pluripotent stem cells from patient cells, allowing for in vitro studies in a patient-specific genetic background. As a result, one of the most common applications of iPS technology today is disease modeling. Two early studies differentiated glutamatergic neurons from iPS lines derived from idiopathic schizophrenia  and TimoIn addition to patient-derived cells, genome engineering technologies using CRISPR have also enabled the generation of ‘isogenic’ iPS lines where a putative disease-causing variant can be introduced into control iPS lines or corrected in a patient iPS line. This approach offers tremendous potential for screening large numbers of candidate mutations as well as for assessing the role of genetic background in specific disease-associated variants. However, rigorous validation of isogenic iPS lines will be necessary because non-specific ‘passenger’ mutations are frequently introduced during clonal selection of isogenic lines .Cerebral organoids generated from patient-derived iPS lines have also been applied to studies of disease mechanisms. For example, organoids derived from an iPS line derived from a patient with compound heterozygous truncating mutations in CDK5RAP2 have been shown to exhibit slower growth kinetics compared to control iPS lines . This stThe advent of new technologies, such as single cell barcoding and CRISPR perturbation screening, has enabled parallel and multiplexed perturbations of genes in cerebral organoids. Esk et al. applied this approach to study the effects of loss of function of 173 candidate microcephaly genes . This stPAFAH1B1) encodes one of the five genes implicated in primary lissencephaly in humans [Mechanisms underlying microcephaly have been successfully studied using mouse models thanks to the conservation of many biochemical processes of cell division. By contrast, lissencephaly is a condition inherently difficult to model in mice due to the lissencephalic nature of the mouse cerebral cortex. LIS1 , which involve the contribution of many genetic variants. Polygenic risk architecture may be difficult to capture in animal models.Starting from syndromic disorders that include nervous system alterations, iPS cells derived from patients with Timothy syndrome have been used to identify neural deficits associated with the condition , includiSeveral studies utilized pluripotent stem cells derived from patients with DiGeorge syndrome, which is caused by a microdeletion at the 22q11.2 locus. Cortical differentiations of these lines have been shown to result in precocious gliogenesis  and a deMECP2 have been shown to exhibit abnormal patterns of oscillatory network activity that can be partially rescued by Pifithrin-α, a TP53 inhibitor [MECP2 encodes a CpG binding protein and is mutated in patients with Rett syndrome ,182. Retnhibitor , consistnhibitor . These sStudies of ASD pathobiology have attracted substantial interest in the scientific community due to the complex nature of this early-onset condition. Human genetics studies have implicated hundreds of genetic variants that might underlie ASD ,189,190.Copy number variants in the 16p11.2 locus are strongly associated with ASD diagnosis ,192. StuSYNGAP1 mRNA is enriched in glutamatergic neurons, but its expression can also be detected in radial glia [High-confidence ASD risk genes discovered through whole exome sequencing studies coalesce into several categories, including gene expression regulation and synaptic transmission. Understanding the points of convergence between them that might underlie the highly stereotypical nature of ASD symptoms remains challenging. However, early analyses have uncovered brain regions and developmental time points that are enriched for ASD risk gene expression. Specifically, by intersecting ASD candidate risk genes with gene expression information from the developing human brain , two stuial glia . Emerginial glia . This fiIn an effort to systematically screen high-confidence ASD risk genes for their function in neurodevelopment, several studies have begun to interrogate neurodevelopmental phenotypes for multiple genes in parallel, using iPS-derived neurons and cerebral organoids.Cederquist et al. generated isogenic pluripotent stem cell lines carrying loss of function mutations for 27 high confidence ASD risk genes and differentiated the cells in a pooled assay to glutamatergic neurons of the prefrontal cortex . Loss ofLalli et al. investigated progenitor cell proliferation and neuronal differentiation phenotypes across 13 high-confidence ASD risk genes using a pooled CRISPR screen approach in neural progenitor cells . This stSUV420H1 , ARID1B, and CHD8 loss of function using comprehensive single cell RNA sequencing analysis. In this study, heterochrony of neuronal maturation and excessive production of GABAergic versus glutamatergic neurons were observed [An organoid-based study investigated the neurodevelopmental consequences of observed . Transcrobserved .Together, these studies suggest that loss of function mutations of at least a subset of high-confidence ASD risk genes disrupt radial glia proliferation or neuronal differentiation. This is consistent with clinical reports suggesting that patients with ASD can have either increased or decreased head circumference relative to the general population . HoweverComplementing the studies of rare risk variants, organoids derived from idiopathic ASD patients have identified phenotypes of increased abundance of cortical interneurons ,206 in aAdditional studies are needed to investigate the consequences of ASD risk gene mutations on additional neurodevelopmental phenotypes, including cell behavior, epigenetics, neurophysiology, synaptogenesis, cell dynamics, gliogenesis, and connectivity. Progress in addressing these questions will require advances in culture methods that promote neuronal maturation ,209, scaOne of the general themes that has emerged thus far from organoid-based modeling of neurodevelopmental phenotypes associated with psychiatric variants is that such mutations frequently lead to aberrant timing of neurodevelopmental events. Such heterochronicity could lead to disorganized development of neural circuits, likely leading to functional abnormalities. Given the inherent variability of cerebral organoid differentiations as well as the current inability to precisely control many neurodevelopmental processes, such as the positional identity of radial glia, insights gained from organoids should be carefully validated to determine their predictive value.However, validating early neurodevelopmental phenotypes in human tissue can be challenging due to the limited availability of suitable research material. Still, insights gained from brain organoids can and should lead to predicted consequences in brain structure or function. Functional imaging studies that have been performed in patients with psychiatric conditions can serve as a point of reference . SimilarAnother area of growing interest is the application of organoid technology to interrogate mechanisms underlying neurodegenerative phenotypes. Neurodegenerative disorders involve many genetic and non-genetic risk factors and are thought to involve dysfunction of homeostatic mechanisms involving not only neurons but also non-neuronal cells. Four major disease areas are being actively studied: Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and Down syndrome.Multiple studies have leveraged patient derived iPS cells to recapitulate deficits in neuronal survival as well as abnormalities in biochemical processes relevant to these conditions ,225,226.Cerebral organoids have also been applied to the study of neuronal phenotypes associated with neurodegeneration. Organoids derived from patients with familial Alzheimer’s disease as well as Down syndrome have been shown to recapitulate toxic accumulation of the Ab fragment of the amyloid precursor protein ,229. DisELAVL4, a splicing factor involved in neuronal maturation, was identified as a core master-regulatory gene upregulated in mutant cells, leading to transcriptome-wide changes in splicing and changes to synaptic recycling rates. This could be reversed by inhibition of PIKFYVE, a lipid kinase that regulates endolysosomal trafficking, consistent with findings in a mouse model of C9ORF72 hexanucleotide repeat expansion that underlies both frontotemporal dementia and amyotrophic lateral sclerosis [In an excellent demonstration of organoid technology for disease modeling, a recent study modeled neuronal phenotypes using iPS lines carrying MAPT mutations that underlie frontotemporal dementia . Extensiclerosis .The mammalian neocortex is a highly evolved structure, but even across mammals it shows substantial variation in size, cellular organization, and folding pattern . Many hyThree prominent hypotheses have been promoted to account for the expansion of the neocortex. One, changes to the proliferative expansion of the neuroepithelial stem cells, which serve as the founder population of cortical radial glia . Two, a Cerebral organoids have the potential to serve as an experimental platform for establishing causality between genetic changes between species and specific neuro-developmental or neurophysiological phenotypes. Two main approaches have emerged so far in the field.The first approach has been to use organoids to study the function of genetic variants that have been identified as derived from the human lineage ,251,252.The second approach has been to derive brain organoids from multiple species. Thanks to the discovery of somatic reprogramming factors, it is possible to derive brain organoids from many species, including great apes ,258,259.cis- and trans-regulatory programs for transcriptional divergence between species [Organoids derived from tetrapoid iPS cells generated by fusing cells from different species provide another avenue for interrogating  species . As an eIn the last two decades, pluripotent stem cell-derived brain organoids have advanced from a novel finding to the premier research tool for studying human brain development and disease. These models are utilized by hundreds of researchers. Many challenges remain to improve the fidelity of organoids relative to in vivo tissues and to expand the features of brain development and function that can be effectively studied in these cell culture models. However, there is tremendous enthusiasm to tackle these issues, as outlined in the research reviewed here. In vitro differentiation of human pluripotent stem cells to cerebral organoids offers a tremendous opportunity to understand human neurodevelopment, identify the consequences of disease-relevant mutations, and begin to develop strategies to advance regenerative medicine applications.


Depression in pregnancy affects nearly one in five women in low- and middle-income countries and is associated with adverse obstetric and neonatal outcome. Burden of morbidity is high, but specialized mental health resources are meager. Effective low intensity psychosocial interventions hold promise to fill the treatment gap for maternal depression. In this paper, we aim to describe the process of development of a stepped care model incorporating screening, providing brief intervention, and referral pathways developed for managing depression in pregnancy in antenatal care health facilities in India.Using complex intervention development and evaluation method of Medical Research Council, United Kingdom, we searched evidence-based strategies from preexisting manuals, conducted formative research for need assessment and stakeholder engagement, and developed the intervention following an expert review panel. We conducted pilot testing to assess the feasibility and acceptability of intervention supplemented by three focused group discussions.Manual review identified psychoeducation, empathetic listening, behavior activation, and supportive counseling as important elements. Need assessment revealed huge gap in perinatal mental health knowledge. Nearly 92% of total 272 perinatal women had poor awareness and 35%–70% of total 62 health-care providers had poor knowledge. In qualitative interview, women reported depressive symptoms as a normal part of pregnancy and had poor help seeking, behavior symptoms of depression were more prominent. A stepped care algorithm was developed for screening all expectant mothers in each trimester for depression using Patient Health Questionnaire-9 (PHQ-9). Women with PHQ-9 score >19 or reporting self-harm ideation were urgently referred to psychiatrist. Women with PHQ-9 score 5–19 were given brief intervention for depression in pregnancy intervention by antenatal nurse. The intervention developed consists of three sessions of psychoeducation, relaxation exercise, and mental health promotion, each lasting 20 min and at gap of 2 weeks each. Service providers and mothers reported good acceptability of psychosocial intervention and reported satisfaction with content and delivery of intervention.Low intensity brief psychosocial interventions can be adapted for implementation if relevant stakeholders are engaged at each step right from development of such as screening, intervention pathway to delivery, and effectiveness study. Perinatal period due to inherent biological, psychological, and social transitions make women more vulnerable to develop depressive and anxiety disorders. Depression is the second most common perinatal mental health disorder affecting up to one in ten women in high-income countries and nearly one in five women in low- and middle-income countries (LAMIC). Research indicates that untreated maternal depression is associated with detrimental effects on maternal physical health during pregnancy, obstetric outcome, infant outcome, and early child development.Globally, depression is the leading cause of mental health disability, and the magnitude is increasing with successive year. Demand for addressing maternal depression has increased considerably, but mental health professionals available to screen and provide effective intervention remain meager especially in LAMIC. Literature has emerged in favor of effective low-intensity psychosocial intervention models for addressing maternal depression and has shown improvement in perinatal depression, health-care seeking, breastfeeding as well as physical growth and risk of infection in infants.,9 Specifically, stepped care models utilizing existing antenatal health-care staff or peers for screening maternal depression and providing brief intervention have shown promising results across South Asian countries.,9Prevalence rates of maternal depression in the past decade have increased not only due to increased risk factors such as loss of social rubric of the joint family system for childcare support, more mothers being employed with variable maternity leave benefits, rise in domestic violence cases, and increased global conflictual situation but also due to positive changes such as increased awareness, advocacy movement, and isolated research or program-based active screening for maternal mental health in a few countries. For effectiveness in LAMIC, such models need to be simple enough for lay health-care workers to use and adapt in different health care and cultural settings.With the promises shown in research setting for these programs, challenges have also emerged concerning feasibility, scalability, sustainability, and acceptance for real-time integration of these models in existing maternal child health programs. Medical Research Council, United Kingdom  proposes four stages for implementation of complex psychosocial interventions: Development, feasibility, evaluation, and implementation. Index study will focus only on development and feasibility aspects only.The purpose of this study is to describe the process of developing and implementing a stepped care model incorporating screening, providing brief intervention and referral pathways developed for managing depression in pregnancy in antenatal care health facilities in India. This model was developed as a part of multicentric randomized controlled trial (RCT) for developing a brief intervention for depression in pregnancy (BIND-P) in four centers across different parts of India.The study received ethical approval from the Institutional Ethical Committee of all four study sites. Participant recruitment was started only after trial registration in Clinical trial registry of India (CTRI/2018/07/014836). Written informed consent was taken from all participants after explaining them nature and purpose of study. Anonymity of participants was ensured, and due standard treatment was provided to all screen-positive mothers with depression including urgent referral for severe cases. Contact detail of one resource person from each study site was provided to approach to in case of need.The study was conducted in the antenatal outpatient department of the Department of Obstetrics attached to medical colleges across India: Lady Hardinge Medical College (LHMC), Delhi (Northern India); BKL Walwalkar Rural Medical College (BKLWRMC), Ratnagiri, Maharashtra state (Western India), Yenepoya Medical College (YMC) Mangalore and Dharwad Institute of Mental health and Neuroscience (DIMHANS) Karnataka (Southern India). All three sites added diversity in terms of culture and level of healthcare setting. The first three sites were tertiary care centers, and fourth one was a secondary level health-care facility. Study centers also offer a mix of rural (Ratnagiri) as well as urban settings and public health facility (LHMC and DIMHANS) as well as private health facility .A mixed-method study was used; triangulation of methods was done to extract information from multiple sources. Based on guidelines of developing complex interventions, following five steps were followed for intervention development and feasibility assessment:To select evidence-based approaches from literature with proven effectiveness for developing screening, providing brief intervention, and referral pathways for maternal depression.Knowledge attitude survey and qualitative interviews with all stakeholders, service users, as well as service providers.Consultation review with subject experts involving psychiatrist, psychologist, obstetrician, pediatrician, and public health experts to decide structure and content of intervention.To determine feasibility and acceptability of stepped care model delivery using qualitative interview with stakeholders.Finalizing the model based on feedback received after expert consultation review. search was done for preexisting evidence available regarding screening referral and brief intervention pathways for maternal depression. The aim was to examine individual elements of such interventions or strategies for adaptation in our setting. Electronic databases such as EMBASE, PsycINFO, MEDLINE, CINAHL, and the Cochrane Central Register were searched using keywords “maternal depression, perinatal depression, postnatal or postpartum depression” AND “stepped care model, stratified model, RCT, low-intensity psychosocial intervention, effectiveness trial and clinical trial” from 2007 to 2018. Search was restricted to individual-based psychosocial interventions with structured manuals with proven effectiveness in a large-scale RCT, simple enough to be delivered by nonspecialized health care worker in LAMIC suiting to our resources and cultural context.In India, screening for mental disorders in perinatal period is not incorporated in routine maternal health-care pathways. Thus, utilizing distillation-matching-method principles, Perinatal Mental Health Project (PMHP) manual on basic counseling skills: A guide for health-care workers in maternal care, A Guide to Maternal Mental Health by White Swan Foundation (WSF) for expectant mothers and families, Lifeline basic counseling skills participant manual, Africa Focus on Intervention Research for Mental health, (AFFIRM) perinatal counseling manual. Of theses, WSF manual was a resource guide for family and patient only; PMHP, AFFIRM manuals focused more on training aspect of health-care workers rather than clinical care implementation pathways. THP intervention had shown good effective size but had a long duration of therapy. Hence, elements of relevance for our manuals such as psychoeducation, cognitive restructuring, mother–child relationship, mental health promotion strategies, and involvement of significant family members were distilled from preexisting manuals. Results are tabulated in Evidence review showed most effective interventions utilized components of cognitive behavior therapy, problem-solving therapy, interpersonal therapy, empathetic listening, supportive counseling. We found five programs which either focused on training of healthcare workers or self-help skills available in the public domain, namely; Thinking Healthy Program (THP) manual,A cross-sectional survey was carried out for assessing knowledge about maternal depression using a semi-structured proforma based on Perinatal Depression Monitor among consecutively selected 270 perinatal women attending routine antenatal clinic (ANC) or postnatal clinic (PNC) and 62 health-care providers  working in ANC/PNC clinics or wards of two study centers Delhi and Ratnagiri. Details regarding survey methodology have been reported earlier.It showed only 8.51% of perinatal women had awareness regarding maternal depression. Among nursing professional surveyed, most (71%) regarded it as normal part of pregnancy, only 45% considered it as biological and only 23% regarded antidepressant medications as a part of treatment for same. Among medical professionals, up to 90% recognized it as a medical problem, 70% considered it as having biological cause needing antidepressant medications. Sociodemographic details have been reported earlier.Purposive sampling was done on the following two groups of voluntary participants  from two centers Delhi and Ratnagiri.Participants understanding about maternal depression and expectations from screening and intervention strategies: Focused group discussion (FGD) were carried out in the successive women of 18–40 years of age group visiting antenatal or PNC diagnosed as having depressive disorder by a psychiatrist as per ICD 10, Diagnostic Criteria for Research  willing to voluntarily participate. Interview was conducted using a prestructured guide regarding their understanding of psychological, social, and cultural aspects of depression, explore thinking-styles of problem-solving, expected determinants in any interventionNursing professionals understanding about maternal depression, need of screening, self-perceived ability and willingness to deliver intervention: In-depth interviews were conducted with purposively sampled volunteer nurses working in the antenatal and postnatal OPD. Staff of varying age and experience was recruited to understand the issues involved in delivering a psychological intervention in a hospital setting. We explored the nurse’s daily work profile, their views about psychosocial intervention delivery, feasibility, challenges of working with peripartum women and families, health beliefs, help-seeking behavior and presence of existing resources for dealing with perinatal mental health problems. Interviews allowed free discussion while permitting maximum elaboration of participant response. Issues raised in one interview were used as probes for the next interviewAll interviews followed an iterative process till themes were saturated with no prior fixed sample size. All interviews were recorded, transcribed verbatim, and analyzed as per framework analysis. Two researchers familiarized themselves with the data, codes were generated, and subthemes were generated manually.Overall, 35 mothers with depressive disorders  and 12 Nursing professionals were interviewed. Key findings regarding mother’s views are highlighted in Most nursing professionals welcomed brief screening as an essential thing to be incorporated in perinatal care, but most had a concern about intervention is either time consuming or too difficult and expressed the need of training for counseling skills and continued supervision and assistance. Key findings regarding nursing professional’s views are highlighted in To develop an intervention grounded in evidence-based interventions yet culturally well-adapted to and feasible for the Indian population, we established a multidisciplinary team with expertise in several domains. The teams included two clinical psychologist, eight psychiatrists, three obstetricians, one pediatrician, two public health experts, and one psychiatry nursing faculty with considerable experience in mental health conditions and puerperal disorders.A stepped care model was developed taking into account extensive review of literature done for low-intensity psychosocial interventions for peripartum disorders, expert panel and author’s own clinical experience and formative research done in service providers and users in perinatal clinics. The main input for the intervention method was based on the outcome of semi-structured interviews, survey results, and WHO mhGAP Manual, “Thinking Healthy” that included techniques of active listening, collaboration with family, and homework activity added to the routine maternal child care service model.A model was developed incorporating the following steps: Provision of screening of all mothers visiting antenatal OPD by asking questions of short 2 item Patient health questionnaires (PHQ 2). Those who score ≥3, were subjected for further evaluation with PHQ-9.Screen positive (PHQ-2 score >3) mothers were assessed for severity of depression by using 9-item PHQ 9.Based on PHQ-9 rating, participants can be divided into two groups.PHQ-9 Score >19 (severe depression) or yes to Question 9 of PHQ-9 assessing self-harm ideation→ Urgent referral to psychiatrist.Low to Medium Risk-→ PHQ-9 score <19 mild to moderately severe case of depression, antenatal nurse to provide brief intervention, BIND-P→ referral to a psychiatrist if no improvement after 6 weeks of BIND-P intervention or progression to severe symptoms during the course of intervention.anybody with past history of psychiatry illness, irrespective of screening tool score should be sent for perinatal care planning to psychiatrist, even if currently in remission).,Developing skills such as counseling-based psychoeducation, empathic listening, and befriending specific to the intervention moduleDevelop appropriate referral practices for moderate to severe cases.continued training. Reorientation program with feedback sessions conducted for the trained nurses on a quarterly basis besides the on-site assistance in daily dealing of cases.An interactive case-based approach and role play were incorporated in training module for skill building during the training with the provision of Pre and Post-Test assessments were carried out to examine the change in knowledge with training and assessing competence by demonstration of skills learned on healthy volunteer actors from research team.Feedback from service users and providers.The fourth step was a qualitative assessment of draft stepped care model and intervention through a pilot study in Delhi. Ten antenatal and ten postnatal women with depression having PHQ-9 <19 were purposively picked and given intervention after informed consent. After 6 weeks of start of pilot trial, FGDs were conducted in service users and providers to ascertain the feasibility, acceptability, and adaptability of intervention content or frequency or modality. Three FGDs were held with a group of 5–6 people each, consisting of nursing professionals, antenatal women, and postnatal women each. An interview guide was created to ask question regarding usefulness of intervention, views about content, any suggestions to improve or any facilitation strategy suggested, any difficulty encountered. All FGDs were recorded, transcribed, and key thematic areas coded and analyzed manually.All mothers found it acceptable, 83% reported it to be useful and report having learned new perspective about importance of their mental health for maternal–child relationship and infant growth. They appreciated the self-help skills taught as a part of mental health promotion. Sixty-six percent of mothers did not find the last session about domestic violence useful and reported it to be removed from intervention or to be kept only for targeted population reporting it. Mothers also expressed difficulty in coming for fortnightly sessions when their ANC visit is monthly.All nursing professional reported no difficulty in using PHQ for screening after training. Nearly 50% suggested to keep intervention sessions limited to two or three to reduce attrition. Nearly 25% expressed concern about extra workload and suggested monetary incentivization as a suggestion to keep staff motivated. Nearly half expressed concern about long-term sustainability of these practices in the absence of its incorporation in programmatic approach and suggested if translation of research can be converted into implementation by making it a mandatory top-down order for inclusion in routine functioning and involving hospital administration.,22No changes were suggested in stepped care model. Based on result of pilot study, BIND-P intervention was modified, and last session concerning domestic violence was removed, elements of mother–infant interaction were shifted to the first session of the intervention, and sessions were reduced to three focusing on psychoeducation, relaxation exercise, and mental health promotion. Final result of stepped care model remains same as described in  Furthermore, there have been multiple disjointed efforts of assessing and intervening either in antenatal period or only in postpartum period; however, there is an incumbent need of developing systematic protocols for assessing mothers right from conception for preemptively identifying high-risk mothers in need and continuing the momentum of care postnatally also, preferably up to 6 months to 1 year after delivery.Despite years of research, most focus still remains on “postpartum depression” neglecting the fact that of all the disorders surrounding peripartum period more than half have onset antedating the delivery. The model development process is based on a well-established theory of behavior change utilizing the principle of assess, build, create, and deliver. The use of mix methods allowed for need assessment as well as contextualizing the strategy from stakeholder’s perspective and building an intervention with strong theoretical foundation.This paper describes the formative phase of developing one such stepped care model based on MRC, UK framework for development, and evaluation for complex interventions.,18The qualitative interview showed mothers are not able to recognize depressive symptoms, and behavioral symptoms were more prominent than cognitive symptoms. It led to the inclusion of behavior activation strategies in BIND-P intervention, similar to THP, and AFFIRM interventions.–18 It demonstrates that such low-intensity interventions can be implemented to reduce the treatment gap.Preliminary service provider’s feedback showed it is possible to incorporate brief screening elements in busy antenatal OPDs, and if session lengths are kept short and number of sessions limited, nursing professionals may incorporate it in their schedule as in BIND-P intervention. Most other manual-based programs have 8–14 sessions of 40to 60 min each.per SE is a simple psychosocial intervention of three sessions of 20–30 min each to be given at a gap of fortnight and needs a formal training and needs competence assessment posttraining.BIND-P not only is an intervention but also is a strategic pathway for stepping up care by incorporating universal screening in maternal–child health service delivery, risk stratification and based on risk assessment, referral, or brief intervention delivery within obstetric setting. It is simple enough for any Accredited Social Health Activist or Auxiliary Nurse Midwife to deliver following due training. BIND-P intervention Concern for this or any such strategic program is need of continued supervision and regular retraining and handholding sessions for health-care provider to ensure fidelity of service delivery and scalability of such interventions. Interventions based on THP manual and AFFIRM manual have also shown similar promises and challenges.If implemented well, any nursing professional with basic training can be equipped with skills to screen for depression during antenatal visits of mother, manage the mild cases at their own level by psychological intervention and refer moderate or severe cases to mental health expert. It could transform a routine antenatal setting into a one-stop clinic providing holistic services for physical as well as mental health needs.For increasing the generalizability, effectiveness analysis research is being undertaken at four sites representative of culturally diverse settings of country  and simultaneously incorporating all possible health settings of antenatal care, that is, secondary as well as tertiary health care facilities.Literature review was based on structured review not a systematic review. We restricted our search to LAMIC countries individual-based interventions provided by healthcare workers, we may have missed group based or self-help or peer led innovative models and their inherent elements. Effectiveness trial is running at four centers but index study shared data about pilot testing at Delhi and Ratnagiri centers only. Sample size for postintervention development assessment was small and might have yielded limited response, but it was appropriate for a qualitative formative phase of study. Qualitative analysis was done manually due to nonavailability of access to paid qualitative software. It is also likely speculated that service users and service providers interviewed for the study may not have been able to provide in depth inputs about stepped care pathway or intervention because of the absence of any such screening program in India in routine health-care settings.BIND-P stepped care pathway is a systematic path for integration of mental health principles in general health-care settings. Results from the formative phase of the study show engagement with stakeholders during pathway development can give good insights regarding implantation challenges and barriers to effect adaptability. The preliminary result suggests this model is feasible, acceptable and has the potential for scaling it up for integration in reproductive child health programs once effectiveness analysis results of the model implementation are available