The bilateral effects of deep brain stimulation (DBS) on motor and non-motor symptoms of Parkinson’s disease (PD) have been extensively studied and reviewed. However, the unilateral effects—in particular, the potential lateralized effects of left- versus right-sided DBS—have not been adequately recognized or studied. Here we summarized the current evidence and controversies in the literature regarding the lateralized effects of DBS on motor and non-motor outcomes in PD patients. Publications in English language before February 2021 were obtained from the PubMed database and included if they directly compared the effects of unilateral versus contralateral side DBS on motor or non-motor outcomes in PD. The current literature is overall of low-quality and is biased by various confounders. Researchers have investigated mainly PD patients receiving subthalamic nucleus (STN) DBS while the potential lateralized effects of globus pallidus interna (GPi) DBS have not been adequately studied. Evidence suggests potential lateralized effects of STN DBS on axial motor symptoms and deleterious effects of left-sided DBS on language-related functions, in particular, the verbal fluency, in PD. The lateralized DBS effects on appendicular motor symptoms as well as other neurocognitive and neuropsychiatric domains remain inconclusive. Future studies should control for varying methodological approaches as well as clinical and DBS management heterogeneities, including symptom laterality, stimulation parameters, location of active contacts, and lead trajectories. This would contribute to improved treatment strategies such as personalized target selection, surgical planning, and postoperative management that ultimately benefit patients. For instance, the left cerebral cortex is dominant for motor control and verbal processing, whereas the right cerebral cortex is dominant for spatial cognition, body schema, proprioceptive control, and inhibition control2. The basal ganglia—a group of subcortical nuclei—is critical for information integration and processing of the cortex input for motor and cognitive functions3. Thus, it is postulated that the functions of basal ganglia are hemisphere-specific as well. Indeed, both neurobiological5 and structural6 basis as well as the electrophysiological pattern asymmetry7 suggest the functional laterality of basal ganglia. Therefore, interventions to left versus right basal ganglia may demonstrate distinctive effects on motor and cognitive features.Cerebral lateralization refers to the functional specialization of the two cerebral hemispheres8. Deep brain stimulation (DBS) is a well-established neurosurgical treatment for controlling motor symptoms and reducing levodopa-induced complications in advanced PD10. Currently, two main structures of the basal ganglia, the subthalamic nucleus (STN) and the globus pallidus interna (GPi), are primarily targeted in DBS surgery11. Bilateral and symmetric placement of DBS is the strategy adopted in the majority of cases as patients with advanced PD often show bilateral disabling motor symptoms12. Thus, bilateral effects of DBS on motor and non-motor symptoms have been extensively investigated13. In contrast, the unilateral effects—in particular, the potential lateralized effects of left- versus right-sided DBS, given the postulated functional lateralization of basal ganglia—have not been adequately recognized or studied. Moreover, to the best of our knowledge, no systematic review has been composed regarding this issue. In this review, therefore, we focus on the evidence and controversies regarding the potential lateralized effects of DBS on motor and non-motor symptoms in PD. We also highlight limitations of the current literature and potential factors that may influence the interpretation of the evidence.Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by a selective and progressive loss of dopaminergic neurons in the substantia nigra, resulting in a dopamine deficiency in the basal ganglia16, several studies have suggested that there exists a superiority of unilateral STN DBS in improving motor functions18. Schulz et al. enrolled 12 right-handed PD patients with stable bilateral STN DBS and reported that, in the off-medication condition, unilateral left-sided and bilateral STN stimulation resulted in better improvement in non-lateralized motor endpoints and overall motor scores than unilateral right-sided stimulation17. However, by studying 45 PD patients treated with bilateral STN DBS, Tabbal et al. found no differences between unilateral left- and right-sided STN stimulations in the improvement of gait speed in the off-medication state14. The differences between unilateral left- and right-sided stimulations on the improvement of rigidity and bradykinesia were not reported in this study, because authors focused mainly on discrepancies in the effects of ipsilateral and contralateral stimulation on these appendicular motor features14. Similarly, a comparable amelioration of the motor score and the working memory performance (see “neurocognition and speech” section) was described by Hershey et al. between unilateral left- and right-sided STN stimulation conditions in 49 PD patients with bilateral STN DBS in the off-medication state15. Instead, they suggested a potential causal role of the disease asymmetry to explain the different effects of unilateral STN DBS between the more and less affected side on motor functions and working memory performance15. Interestingly, although Lizarraga et al. reported no differences in motor and gait scores in the off-medication state between unilateral left- and right-sided stimulation conditions in 22 PD patients with the presence of the gait dysfunction after receiving bilateral STN DBS, they showed that unilateral right-sided STN stimulation might produce slightly greater improvement in gait kinematics  than left-sided stimulation18. They later postulated that the right (non-dominant) hemisphere may be dominant for axial motor control2. Castrioto et al. also reported no significant difference between unilateral left- and right-sided stimulation conditions on motor scores in 22 PD patients with bilateral STN DBS in the off-medication condition. However, they proposed the presence of a ‘dominant-STN’, the stimulation of which may lead to improvements of motor symptoms, similar to bilateral stimulation16. In this study, 11 (50%) participants presented a ‘dominant-STN’, in 8 of whom the dominant-STN was contralateral to the most affected side. Interestingly, the ‘dominant-STN’ phenomenon was associated with longer disease duration and tremor predominance16. A subsequent study further demonstrated that the unilateral stimulation of the ‘dominant-STN’ could offer improvement in gait parameters, which were comparable to those after bilateral stimulation. Whereas in patients without the ‘dominant-STN’, only bilateral stimulation could significantly improve gait parameters (n = 10)19. However, it is currently difficult to preoperatively select patients with underlying ‘dominant-STN’.Whether the unilateral left- and right-sided DBS have different effects on motor symptoms in patients with PD remains debated. Though the lateralization effect is not observed in all patients14. However, the difference between unilateral left- and right-sided DBSs on ipsilateral motor effects was not investigated in this work. In fact, few studies in the current literature focused on this issue. Shemisa et al. investigated 73 right-handed PD patients treated with either unilateral GPi (n = 26) or STN (n = 47) DBS, and this study provided limited evidence of a similar ipsilateral motor improvement in the off-medication state after either unilateral left- or right-sided DBS stimulation, regardless of target20. This study, however, should be interpreted in caution as the target selection was not randomized and could be biased by several baseline factors.As mentioned above, Tabbal et al. reported that unilateral STN DBS reduced rigidity and bradykinesia both ipsilaterally and contralaterally23. Compared with patients with right-sided lesions, PD patients who underwent left globus pallidotomy are more likely to experience postoperative declines in verbal fluency23. However, in the era of DBS surgeries, the lateralized effects of DBS on cognitive functions of PD have not been as adequately investigated as the effects of bilateral stimulation.Lateralized deficits in cognitive performance, especially in language-mediated functions, have been recognized in the era of lesional surgeries for PDn = 13) or right-sided (n = 7) GPi DBS in the on-medication condition24. Six patients who underwent the unilateral left-sided GPi DBS showed a tendency for cognitive decline (as measured by cognitive impairment index), which was not observed in patients who had received right-sided GPi DBS24. In another study, 6 PD patients were consecutively recruited to investigate the changes in cognitive performance after staged bilateral GPi DBS in the on-medication condition25. Among the 5 patients who received left-sided stimulation first, 4 showed a decline in semantic fluency. In contrast, only 1 of the 5 patients showed a further decline after the second, right-sided surgery25. Rothlind et al. included both staged bilateral GPi (n = 23) and STN (n = 19) DBS cases to evaluate cognitive outcomes in the on-medication condition. Participants were randomized to receive either staged bilateral GPi or STN DBS to avoid the selection bias. Authors reported that the deleterious effect of unilateral DBS on semantic fluency were largely due to the left-sided treatment26. Patients who initially received right-sided DBS displayed a significant decline in semantic fluency only after left-sided surgery26. Moreover, Zahodne et al. compared the cognitive performance of 22 PD patients with unilateral GPi (n = 12) or STN (n = 10) DBS to that of 19 PD controls27. They found that the unilateral DBS surgery was associated with declines in letter and semantic verbal fluency in the on-medication condition and suggested that such changes may be more common after left-sided DBS27. In contrast, Tröster et al. stated that in PD patients who received unilateral pallidal surgery, the decline in on-medication semantic verbal fluency was not associated with the side of surgery28. However, this finding should be interpreted prudently, as both pallidotomy  and DBS  surgeries were combined in the final analysis to increase the statistical power28.For GPi DBS, Vingerhoets et al. compared the cognitive outcomes at the 3-month follow-up to that at the preoperative baseline in a cohort of PD patients who received unilateral left-  and the side of STN stimulation in 49 PD patients with bilateral STN DBS in the off-medication condition15. Instead, the authors demonstrated that stimulation on the more affected side of the brain aggravated the SDR, whereas stimulation on the less affected side did not, suggesting that clinical asymmetry might interact with STN DBS to impact behavioral responses15. David et al. used a memory-guided sequential reaching task to examine the role of STN DBS in the intensive as well as integrative and coordinative aspects of motor control in 10 right-handed PD patients treated with bilateral STN DBS in the off-medication condition. They reported that unilateral right-sided STN stimulation significantly increased finger latency and reduced finger velocity  when compared to left-sided stimulation, but the endpoint error  was similar between the two stimulation conditions30. One study that investigated cognitive-motor functions in prosaccade and antisaccade tasks in 10 right-handed PD patients with bilateral STN DBS in the off-medication state also found no difference in the spatiotemporal or the cognitive aspects of oculomotor control between unilateral left- and right-sided STN stimulations31.Similarly, whether STN DBS has a potential lateralized effect on cognitive functions for PD remains controversial. In a preliminary study, Lueken et al. systematically evaluated the performance in the Wisconsin Card Sorting Test of 8 right-handed PD patients with bilateral STN DBS in the on-medication condition, and left- and right-sided stimulation was given in a counterbalanced manner. Compared to right-sided stimulation, selected measures of executive functions were more compromised under left-sided stimulation35. Concordant with the findings from Rothlind et al. and Zahodne et al. as mentioned above, Schulz et al. also suggested that the declines in syntactic and lexical language performance, speech rate, and laryngeal–articulatory coordination for PD patients with bilateral STN DBS may be principally due to the left-sided stimulation17. Sjöberg et al. compared the verbal fluency of 6 PD patients receiving unilateral left-sided STN DBS to that of 10 PD patients receiving bilateral STN DBS in the on-medication condition. At approximately 1.5-year follow-up, the bilateral STN DBS showed a more deleterious effect on both letter and category verbal fluency. A reasonable explanation is that the group receiving unilateral DBS may represent patients who were at an earlier stage of the disease, which may influence their neurocognitive performance32. More recently, Yilmaz et al. reported no significant discrepancy in verbal fluency or visual orientation between unilateral left- and right-sided stimulations in 29 right-handed PD patients with bilateral STN DBS in the on-medication condition33. In contrast, both Witt et al. (n = 12) and Schmalbach et al. (n = 13) reported a mild but significant shift of visuospatial attention towards the right side and a neglect of left-sided visual stimuli under unilateral stimulation of the left STN, similar to the neglect syndrome after right hemispheric lesions. Such declines in visuospatial attention could be corrected by stimulating the right STN35.The lateralized effects of STN DBS on changes in verbal fluency (dominant hemisphere) and visuospatial attention (non-dominant hemisphere) are also a subject of debate38. Ray et al. reported that the performance in stop-signal task declined after left-sided STN stimulation compared to that after right-sided treatment in 16 right-handed PD patients with bilateral STN DBS in the on-medication condition36. In contrast, Mirabella et al. did not find any difference in the performance in stop-signal task between unilateral left- and right-sided STN stimulations by evaluating 10 right-handed PD patients with bilateral STN DBS in the off-medication condition37. More recently, Mancini et al. recruited 20 right-handed PD patients with unilateral STN DBS  and also reported no main effect of stimulating lateralization on the on-medication performance in countermanding task38. However, the discrepancies in findings between these studies should be carefully interpreted, because the PD cohort selection  and the medication status  during the task were different across studies, which might limit the interpolation of the results.The difference in the inhibition control performance between unilateral left- and right-sided STN stimulations has also been investigated39. Similar results have also been reported by other groups41. Wang et al. examined the speaking rate and articulatory accuracy of syllable repetitions in 20 right-handed PD patients with unilateral STN DBS  before the surgery and at 3- to 6-month follow-up after withdrawal of anti-parkinsonian medication. They concluded that a decrease in articulatory accuracy and speaking rate was associated with unilateral left-sided STN DBS40. They also reported a significant decline in vocal intensity and vowel duration baseline in 3 right-handed PD patients with unilateral left-sided STN DBS at the 3-month follow-up. Such a decline was not observed in patients with unilateral right-sided STN DBS41.Regarding other speech-related functions, Santens et al. reported negative effects of unilateral left-sided STN DBS on prosody and articulation (and hence intelligibility) by separately stimulating the left and right STN in 7 PD patients with bilateral STN DBS in the off-medication state42. They measured the mood response with a computerized version of the Visual Analog Scale in 24 PD patients with bilateral STN DBS. Left-sided stimulation resulted in significantly greater amelioration in valence than right-sided stimulation, and trend-level significance was seen for apathy ratings. No significant differences were observed for anxiety ratings or emotional arousal between unilateral left- and right-sided stimulations42. Birchall et al. reported a similar improvement in depressive symptoms at the 6-month follow-up compared to the preoperative baseline between PD patients with unilateral left-sided (n = 26) STN stimulation and those with unilateral right-sided (n = 24) STN stimulation43. In another study, the authors prospectively assessed the apathy score change preoperatively and postoperatively at the 6-month follow-up in PD patients with either unilateral GPi  or unilateral STN  DBS, and concluded that the incidence of postoperative apathy was not associated with the laterality of GPi or STN DBS44.To date, few studies have assessed the lateralized effects of DBS on mood in PD patients, and findings were not conclusive. Campbell et al. reported that left-sided STN DBS may produce greater mood improvement than right-sided STN DBS45. They reported that the improvement in subjective sleep quality at the 6-month follow-up was greater in right-sided stimulation group than in the left-sided group. However, the preoperative baseline sleep quality score was also worse in the right-sided stimulation group. Therefore, it is possible that the reported results were due to more easily detectable improvements in patients with worse baseline sleep dysfunction rather than a direct lateralized stimulating effect of the treatment45. To the best of our knowledge, no other studies have been published to support or oppose this finding.Interestingly, Amara et al. measured the short-term effect of unilateral STN DBS on sleep quality in 53 PD patients 45 have analyzed two separate cohorts with respective unilateral left- and right-sided DBS implantation. One study also compared the group with unilateral left-sided STN DBS with that with bilateral treatment32. Consequently, differences in baseline characteristics between the two groups may limit the interpretation of the results. Other studies42 recruited PD patients with simultaneous or staged bilateral DBS and collected the outcome data under unilateral on-stimulation conditions in a randomized or counterbalanced manner. Among these, some studies applied randomized stimulation in consecutive days to ensure a sufficient ‘washout’ interval. However, some required patients to complete tests under different stimulating conditions on the same day with a shorter ‘washout’ period. In addition, the medication conditions  also differed across studies. For example, the majority of the studies that evaluated the potential lateralized stimulating effects on motor outcomes were conducted in the off-medication condition to exclude the interference of drug effects. In contrast, several studies investigating the cognitive outcomes reported their results in the on-medication state to reduce the possibility for motor symptoms to limit the neurocognitive performance. Actually, current literature suggests both beneficial and detrimental cognitive effects of levodopa in PD46. Moreover, if the analysis included a comparison between the pre- and post-operative status in the on-medication condition, the different management of postoperative medication could be another confounding factor. Last but not least, the follow-up periods in different studies ranged from months to years, and several studies only assessed the clinical outcomes in a subacute setting. Therefore, the discrepancies in the study design may considerably limit the interpretation and comparison of the literature.To compare the differential effects of unilateral left- versus right-sided DBS in PD patients, several studies47. The asymmetrical loss of dopaminergic innervation in the striatum has been suggested as the root of the symptom laterality in PD. Because of the hemispherical lateralization of several cognitive and affective functions and the potential role of impaired dopaminergic transmission in non-motor symptoms in PD, several studies have been focused on the correlation between the symptom laterality and the prevalence and severity of several aspects of cognitive and neuropsychiatric domains in PD50. For instance, one literature review concluded that right-sided motor symptom predominance  was associated with deficits in language-related functions and verbal memory in PD patients. In contrast, patients with left-sided motor symptom predominance  showed worse performance in tasks of spatial attention, visuospatial orienting, and memory and mental imagery48. However, several studies, failed to identify such a relationship in patients with early-stage unmedicated PD50. These studies suggest a negative effect of dopamine-replacement therapy on the development of lateralized cognitive deficits in relation to the symptom laterality. Nevertheless, symptom laterality serves as a non-negligible factor for the interpretation of the putative lateralization of DBS efficacy in PD.One of the prominent clinical characteristics in PD is an asymmetrical distribution of motor symptoms at the onset and during the disease course53—was not systematically and objectively analyzed in the articles included in this review. We suggest that the precise and individualized determination of cerebral dominance would be beneficial for delineating the potential lateralized effects of DBS in PD.Furthermore, right- or left-handedness—a critical phenotype for hemispheric language dominance58. For example, York et al. reported that declines of several aspects of neuropsychological outcomes after STN DBS in PD patients may be related to the trajectory as well as electrode location in a hemisphere-specific manner54. Witt et al. also indicated an association between surgical trajectory through the head of the caudate nuclei and declines in global cognitive performance in PD patients with STN DBS55. Specifically, Tripoliti et al. found that the medially located electrodes in the left STN and the high stimulating intensity of the left electrode were significantly associated with the poor outcome of the speech intelligibility after bilateral STN DBS surgery57. More recently, Petry-Schmelzer et al. reported that the inter-individual variability of non-motor outcomes  after STN DBS in PD patients may depend on the location of neurostimulation58.The surgical trajectory and electrode placement in relation to the STN topography are critical sources of variation in postoperative outcome in PD59. Limited evidence suggests that the microlesional effect could contribute to the early transient verbal fluency decline following DBS surgery, while the long-term fluency impairment might be related to the direct effect of STN DBS60. Later, two retrospective studies concordantly reported that the postoperative cognitive change at approximately 1-year follow-up was not correlated with the number of MER trajectories61. Prospective studies are warranted to confirm these findings in the future.Moreover, the number of microelectrode recording (MER) trajectories has been hypothesized to contribute to cognitive declines following DBS surgery, because the degree of local injury along the trajectory or at the target site would increase as the number of MER passes increase63. As mentioned above, Tripoliti et al. presented a correlation between the high stimulating intensity of the left electrode and the result of poor speech intelligibility after bilateral STN DBS surgery57. Moreover, severe gait disturbances and freezing episodes often appear in patients with long-term high-frequency DBS64. Evidence suggested an amelioration of gait disorders with low-frequency (60 Hz) STN stimulation in PD65. In addition, several studies showed that a lateralized STN neuromodulation  can also affect the gait disturbance and posture in PD67. For example, Fasano et al. showed that reducing the STN stimulation voltage on the contralateral side of the leg with a longer step length could improve the frequency and duration of gait freezing66. Twenty-two participants with bilateral STN DBS were assessed in four stimulation conditions  in a subacute setting, while the chronic effect of stimulating intensity reduction on axial and appendicular motor functions was not investigated. More recently, a PD case with Pisa syndrome following chronic bilateral STN DBS successfully treated with left-sided stimulating intensity reduction was reported by Lizarraga et al.67. The lateral trunk flexion angle reduced from 12° to 4° by reducing the left-STN stimulation voltage from 4.8 to 3.8 V67. Although these findings are preliminary and should be interpreted with caution given the methodological drawbacks and limited sample size, the potential feasibility of the lateralized STN neuromodulation strategy suggests the presence of a functional lateralization of the basal ganglia.Varying stimulating parameters  may also considerably affect the treatment efficacy and introduce adverse effects in PDThe main limitation of this comprehensive review relies on the overall low-quality of the current literature investigating the potential lateralized effects of DBS in PD. The majority of the studies were conducted on small samples, possibly due in part to the substantial burden of clinical evaluation under various stimulus conditions. Most of the studies assessed clinical outcomes only in a subacute setting or at a short-term follow-up. Few studies used or specified a randomized and blinded evaluation paradigm to minimize biases. Furthermore, potential confounding factors discussed above were not well addressed in the current literature, which considerably limited the interpolation of the findings. For instance, effects of location of active contacts and trajectories on neurocognitive outcomes were poorly discussed in the literature involved in this review. Furthermore, authors paid little attention to the correlation between changes in motor and non-motor symptoms when investigating the presumed lateralized DBS effects. This could be interesting because a lack of correlation might suggest different underlying mechanisms of the potential lateralized DBS effects on motor and non-motor domains of PD.There is insufficient evidence in current literature to draw solid conclusions about the lateralized effects of DBS on motor and non-motor outcomes in PD. Several factors, such as the inherent bias in experimental design, symptom laterality, and location of active contacts with respect to the putative topography of the nuclei, surgical trajectory, and DBS programming strategies should be considered for the accurate interpretation of results. Nevertheless, limited evidence suggests potential lateralized effects of STN DBS on both axial motor symptoms and language-related functions in PD.68), surgical planning, and postoperative management that ultimately benefits patients.To date, most of the studies have focused on STN DBS, and the potential efficacy of lateralization of GPi DBS in PD has not been adequately investigated. In addition, whether STN and GPi DBS exert differential lateralization effects on motor and non-motor outcomes in PD also warrants further investigation. Ongoing research focusing on delineating the lateralized effects of DBS can lead to a better understanding of the therapeutic mechanisms of DBS in PD. This would potentially contribute to improving treatment strategies such as personalized target selection  OR (right[Title/Abstract])) OR (sided[Title/Abstract])) OR (left-sided[Title/Abstract])) OR (right-sided[Title/Abstract])) OR ) AND (deep brain stimulation[MeSH Terms])) AND (parkinson’s disease[MeSH Terms])) AND (english[Language]). The search results were verified manually. Of the 243 publications, 225 articles were excluded after title and abstract screening. Subsequently, the full texts of potentially relevant publications were analyzed. The references, citations, and similar articles suggested in PubMed for the included articles were also screened for additional eligible studies. The literature search was conducted by Z. Lin and C. Zhang. Finally, after duplicate removal, 27 publications were included in this review (Table


We hypothesized that a deep-learning algorithm using HE images might be capable of predicting the benefits of adjuvant chemotherapy in cancer patients. HE slides were retrospectively collected from 1343 de-identified breast cancer patients at the Samsung Medical Center and used to develop the Lunit SCOPE algorithm. Lunit SCOPE was trained to predict the recurrence using the 21-gene assay (Oncotype DX) and histological parameters. The risk prediction model predicted the Oncotype DX score > 25 and the recurrence survival of the prognosis validation cohort and TCGA cohorts. The most important predictive variable was the mitotic cells in the cancer epithelium. Of the 363 patients who did not receive adjuvant therapy, 104 predicted high risk had a significantly lower survival rate. The top-300 genes highly correlated with the predicted risk were enriched for cell cycle, nuclear division, and cell division. From the Oncotype DX genes, the predicted risk was positively correlated with proliferation-associated genes and negatively correlated with prognostic genes from the estrogen category. An integrative analysis using Lunit SCOPE predicted the risk of cancer recurrence and the early-stage hormone receptor-positive breast cancer patients who would benefit from adjuvant chemotherapy. As excellent prognosis in many of these patients have been known, many efforts to identify those patients with high risk of recurrence, who would benefit from adjuvant chemotherapy (ACTx), were made using gene expression profiling6. Currently, several multigene assays, such as the 21-gene assay (Oncotype DX), PAM50, and Mammaprint, are used to stratify patients and guide ACTx according to the recurrence risk in HR-positive, and lymph node- negative breast cancer after extensive clinical validation8.Breast cancer is the most common cancer in women worldwide, and hormone-receptor (HR)-positive, lymph node-negative diseases account for nearly half of all breast cancer cases10. Moreover, the instability of RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue in real-world practice might compromise its accuracy and interfere with the appropriate translation of the RS results11. Therefore, the development of a simpler and more efficient method for assessing recurrence risk using permanent tissue is necessary. As the RS from the 21-gene assay is mainly characterized by the proliferation genes group score  and the mitotic count is associated with the RS7, a comprehensive pathological examination of mitosis and other cell–cell interactions features, consistently reflects the RS.Despite the proven clinical utility of RS for the 21-gene assay, its effectiveness in patients with HR-positive, lymph node-negative, early stage breast cancer remains controversial, along with its financial burden in countries outside of the US12. Based on The Cancer Genome Atlas (TCGA) pan-cancer analysis, Lunit SCOPE was able to predict an abundance of cancer-associated stroma in pancreatic adenocarcinoma and a consensus of molecular subtype 4 of colon cancer13, as well as tumor-infiltrating lymphocytes in immunogenic tumors such as renal cell carcinoma, melanoma, and urothelial cancer14.Thus, we developed a deep learning (DL)-based HE image analyzer called Lunit SCOPE that identifies and quantifies various histological parameters from HE-stained whole slide images (WSIs). Previously, the Lunit SCOPE was shown to accurately detect tumor cells as well as other cells in a microenvironment, and it clearly predicted mitosis in each cell in breast cancerAs Lunit SCOPE accurately identifies the comprehensive features of HE slides, especially regarding mitotic count and the infiltration of immune cells or stromal cells, we hypothesized that histological parameters analyzed using Lunit SCOPE would predict the RS from the 21-gene assay, revealing potential prognostic and predictive biomarkers of ACTx in early stage hormone receptor-positive breast cancer.The Lunit SCOPE divides the HE slide image into histological parameters through three panels, including the tissue, structure, and cell panel. The process used to develop the Lunit SCOPE and workflow of this study are illustrated in Fig. The study included a total of 1875 patients with HE-stained WSIs and clinical information, including cancer recurrence and survival Table a. The opThe top 10 important histological parameters for predicting the RS > 25 based on the 21-gene assays are listed in the variable importance plot  , with a 3.128 coefficient followed by the T-stage, N-stage, age, and adjuvant chemotherapy. The details of the multivariate and univariate Cox proportional hazard models for disease-free survival (DFS) in the prognosis validation cohort are shown in Supplementary Table The RS values of the 898 SMC prognosis validation cohort and 532 TCGA cohort were used to validate the Lunit SCOPE model. The mean value for the output of the SMC model development cohort and validation cohort were 0.040 and 0.090, respectively   and multivariate analysis with age, T-stage, and N-stage (p = 0.117)  worse prognosis in high-risk patients without ACTx. ACTx status in 583 low-risk predicted patients was no significant difference in cancer recurrence and survival (p = 0.092). The clinical characteristics of the four groups divided by the predicted risk and adjuvant treatment are summarized in Supplementary Table To confirm the utility of our model, DFS of each risk group was compared according to whether ACTx was done or not. From the 363 patients who did not receive ACTx, the 104 high-risk patients had a lower survival rate than the low-risk patients (p < 0.001), while the median output of the former cohort was higher than that of the latter. Based on Lunit SCOPE predictions, among the 532 HR- positive breast cancer, high risk group showed significantly worse prognoses in cox proportional hazard model (p = 0.023) with the more advanced stages of cancer .532 TCGA breast cancer cohort was used as the external validation set. The survival rate of TCGA cohort was worse than that of the prognosis validation cohort (p < 0.001). Age was not significantly correlated in both cohorts using Kendal's method, but age was a variable that was not significant in survival in both cohorts. The distribution of predicted risk by cancer stage and age was shown in Supplementary Figure Predicted risk increased significantly with increasing stage, in both the prognosis validation cohort and the TCGA cohort (p < 0.05), 228 significant Gene Ontology and KEGG pathway terms were identified. The top-5 gene ontology functional terms and pathways are shown in Fig. p-values. Mitotic cell cycle, cell cycle process, cell cycle, nuclear division, and cell division were the enriched biological processes observed following Gene Ontology analysis of the top 300 genes. Among the various cellular parameters, spindle and chromosome, which play an important role in the cell cycle, were significantly enriched. Furthermore, protein binding was significantly enriched. The cell cycle was identified as another significant term in the KEGG analysis. The details of the functional terms, genes, and significance of the top 100 functions are available in Supplementary Table We analyzed TCGA cohort gene expression data associated with the predicted risk using 532 diagnostic slide images. The top 300 genes that had the highest correlation coefficient with the predicted risk were used for the functional enrichment analysis of the BP, CC, and MF for the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Based on the Bonferroni-corrected significance threshold .Of the 21 genes assessed during the Oncotype DX test, the correlations of 16 genes with the predicted risk, excluding the reference gene, were measured and ordered by correlation coefficient , BCL2 Apoptosis Regulator and SCUBE2  are known to be a favorable prognostic marker on breast cancer recurrence22. The directionality of the correlations between the expression of recurrence-related genes and the predicted risk indicates that the pathology-based predictions of this model were consistent with those obtained using the 21-gene expression assay.The 21-gene expression assay test included proliferation, estrogen, HER2, invasion, and other cancer-related gene categories. Based on the Lunit SCOPE predictions using pathology images, the five genes associated with cancer proliferation had a positive correlation with the predicted risk. This suggests that the expression of proliferation, cell cycle, and progression genes ultimately affected the components of the pathology image, which were associated with cancer recurrence. Excluding ESR1, which was not significant, three genes in the estrogen category were negatively correlated with the predicted risk. The 8. The cutoff of 21 gene-assay changes based on age 50, but this model predicted based on pathology image does not reflect age. Therefore, this model can underestimate the risk of young patients. Another limitation was represented by the selection bias present in the retrospective analysis, as patients who did not receive chemotherapy were associated with other clinical factors, such as poor performance status or poor compliance. Moreover, physicians would choose patients who are clinically high-risk to receive ACTx. This factor could contribute to worse clinical outcomes in patients with ACTx compared to those without ACTx. To overcome this limitation, a well-designed prospective clinical trial is required.There are several limitations to the current study. First, the RS of the model development cohort did not have a range that was sufficient to predict RS. Recent clinical trials have shown that endocrine treatment alone is not inferior to endocrine treatment plus chemotherapy in patients with an RS of 11–25, and a more well-validated RS cutoff for the decision to add chemotherapy to the standard treatment would be 25In conclusion, the Lunit SCOPE predicted the early stage HR-positive breast cancer patients with a high risk of recurrence, as well as those who would benefit from adjuvant chemotherapy.23. The remaining 898 images without RS were used as a prognosis validation cohort to confirm the prognostic and predictive values of the predicted risk.The protocol for this retrospective study was approved by the Ethics Committee of the Institutional Review Board (IRB 2018-03-038-002) of Samsung Medical Center (SMC). Informed consent was also waived by Ethics Committee of the Institutional Review Board. All experiments were performed in accordance with relevant guidelines and regulations and all experimental protocols were approved by SMC. A total of 1343 pathology slide images, derived from anonymized HE-stained tissue samples from breast cancer patients with histologically confirmed hormone receptor-positive tumors, were acquired using a WSI scanner  at a magnification of 40 ×. Of the total of 445 images from patients with a 21-gene assay RS obtained from Oncotype DX , 255 images with clinical information were used to develop the model predicting the high risk of recurrence (RS > 25), and the 190 images with RS were used as a validation cohort to estimate the predictive performance using AUROC. We have used the HE images from the same block that were used for Oncotype DX test to minimize possible problems due to intratumoral heterogeneity24.A total of 532 samples with both digital pathology images and image-matched RNA sequencing data from primary tumor tissues from the TCGA BRCA cohort were also included in the data analysis. Data from the HR-positive and human epidermal growth factor receptor-2 (HER2) negative cases (excluding advanced stage patients) were used for the external validation of the prognostic significance assessment25. The WSIs were tiled into 50% overlapping 4096 × 4096 patches to analyze and quantify the histologic parameters. The performance of these models was evaluated by measuring the distance between the outputs of two images using the validation set with accuracy, intersection over union (IoU), and mean average precision (mAP).For training, anonymized HE-stained tissue slides were reviewed by expert pathologists . The informative regions from these slides were manually selected and annotated by expert pathologists. Next, we trained convolutional neural networks (CNNs) to decipher various types of histologic parameters26.The histological parameters that were quantified using Lunit SCOPE had a count distribution based on tissue, structure, and cell type. We applied the Trimmed Mean of M-values (TMM) count normalization for the histological parameters count to make accurate data proportions comparisons between samples without missing the data composition27. To filter out the genes with low expression levels, the genes with counts per million (cpm) values < 1 in at least half of the samples were excluded28. The raw read counts were normalized using TMM and logCPM transformation with limma voom. Finally, the expression levels of 17,649 genes were used for this analysis29.RNA-seq data for breast cancers were obtained from TCGA Broad Institute GDAC Firehose. The RNA sequencing raw count samples, quantified using RNA-seq expectation maximization32.To determine the biological functions associated with the predicted risk based on the 21-gene assay, we performed a Pearson correlation analysis. The top 300 highly correlated genes were selected as related genes, and an enrichment analysis was performed for the BP, CC, and MF terms in the Gene Ontology and KEGG pathway database using the RDAVIDWebService tool in Bioconductor33. The RF model was trained with the out-of-bag (OOB) training data from 255 images with binarized 21-gene assay (RS > 25). The method provides the importance index of the input variable for classification with the reprioritization component of RS assessments. The model was developed using bootstrap samples with RS, and the OOB samples were used as test samples. A variable’s importance was defined as the mean decrease in the tree’s performance for the randomly permuted OOB samples. The loss of function for minimizing the gini was used for the model assessment metrics in the classification problem to assess the goodness-of-fit and predictive performance of the RS from the 21-gene assay.Fast unified RFs for survival, regression, and classification (RF-SRC), a non-parametric statistical estimation was used to predict the RS from the 21-gene assay based on Lunit SCOPESupplementary Information 1.Supplementary Information 2.


Inherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs. Inherited retinal diseases (IRDs) comprise a genetically and clinically heterogeneous subgroup of vision disorders with over 270 different causative genes identified so far . Many IRRPE65-associated IRD, recent success in the field of gene augmentation therapies is largely driven by preclinical studies performed in animal models .–17.10–17Three differentiation stages of a retinal organoid can be described Fig. . At stagImportant advances have been made in the development of protocols that are used to differentiate stem cells into retinal cells, improving the formation of well-defined retinal cell types and structures, as well as the efficiency and reproducibility Table .Table 1BOne of the earliest successful differentiation methods used adherent (2D) cultures to direct embryonic stem cells into an anterior neural fate. Adding Wnt/BMP signalling inhibitors alongside IGF-1 to the media, induced the formation of photoreceptor-marker positive cells, but these were not the main cell types in the adherent culture . InhibitA key step to obtain stratified neural retinas was the transition to non-adherent (3D) protocols. Mouse embryonic stem cell (ESC) aggregates cultured in suspension under low-growth factor conditions together with Matrigel matrix improved the formation of optic cups mimicking the embryonic optic cup with apical-basal polarities . The addA combination of 3D and 2D protocols that did not require the addition of small molecules differentiated iPSCs to mature and light-responsive photoreceptor cells with rudimentary OS. This was achieved by reducing the RA concentration between D50 and D70, and prolonging the culturing times . AlternaMost of these protocols share common media components, but a switch in the timing and addition of certain molecules helped to improve the yield of neuroretinal vesicles obtained. Bone morphogenetic proteins play a role in establishing dorsal/ventral patterning of the retina , and speRetinal organoid technology offers the possibility to obtain retinal tissue for a wide range of applications and research questions. To illustrate, here we highlight the potential of retinal organoids for therapeutic transplantation and as a model to assess therapeutic strategies.Photoreceptors are often the first cell type lost in many retinal diseases. When that occurs and the adjacent retinal layers remain intact, transplantation of healthy photoreceptors could be a potential treatment option. Photoreceptors derived from human stem cells are an exceptional and unlimited source of human cells for transplantation. Transplanted photoreceptors were first thought to integrate into the ONL of degenerating retina and improve vision in mouse models , 37. HowCrx−/− mice retinas were able to produce ERG responses [Gnat−/− mice displayed features of mature photoreceptors, such as inner and outer segments. When transplanted to Rho−/− and Prph2−/− mutants, the donor cells showed rhodopsin and PRPH2 positivity that were lacking in the endogenous retinas, although they did not contain OS [Nrl−/− mice, a model where rod cells take a different fate and become S-cones [Aipl1−/− mice that lack photoreceptors transplanted L/M cones expressed the presynaptic protein ribeye and photopigments. However, the functionality of these cells was not assessed [Transplanted embryonic stem cell-derived photoreceptors from 2D cultures into adult esponses ; howeveresponses , 23, 24 ntain OS . Transpl S-cones  was repo S-cones . Donor Lassessed .+/SSEA4− cells possess the characteristics of retinal progenitors and in rd1 mice, organoid-derived C-kit+/SSEA4−-transplanted cells protected the ONL and improved visual function. The photoreceptor-specific cell surface markers CD24 and CD73 can also be used to isolate photoreceptors [The source and developmental stage of transplanted cells, as well as their enrichment and delivery methods, are key to successful transplantation. The body of photoreceptor precursor cell isolation and transplantation work suggests that fully mature photoreceptors may be more fragile and unfit for transplantation than retinal progenitor cells. Fluorescence-activated cell sorting (FACS) of the progenitor cell marker C-kit, a type III receptor tyrosine kinase, and the pluripotency marker SSEA4, can be used to isolate retinal progenitor cells without tumorigenic components , 47. C-Keceptors , 49. Labeceptors . Photoreeceptors . Microfleceptors . Dependieceptors . Integraeceptors , 54, 55.rd1 mice and form synaptic connections with the host retina [S334ter−3 rats, a model of severe retinal degeneration [Transplantation of retinal sheets is an alternative to single-cell suspension transplants. Transplanted mouse iPSCs-derived retinal sheets can survive in the subretinal space of t retina . The hESt retina . The samt retina . Transplneration . hESC-deneration . Transplneration .Other physical factors may play an important role in transplantation. The OLM is made of cadherin–catenin complexes that mediate cell–cell adhesion and serves as a barrier for the integration of transplanted photoreceptor precursor cells . DisruptDespite these advances and the availability of well-characterised GMP-compliant retinal cells via stem cell differentiation, there are still many challenges to be overcome to achieve the level of successful transplantation that is needed to restore high acuity vision.Therapies based on adeno-associated virus (AAV) vectors are gaining momentum as a potential treatment for retinal diseases. One major reason is the accessibility of the eye, which makes it suitable for intravitreal or subretinal injection surgery. AAVs can infect human cells, allowing long-term expression of the transgene after a single dose.There are several known AAV serotypes , with diDelivery of AAV by intravitreal injections is a safe method, but it often leads to low transduction efficiencies of photoreceptors. A novel injection system using peripapillary intravitreal injection promises to be a safe and efficient alternative to standard intravitreal injections . This, tCEP290- (NCT03913130 and NCT03913143), USH2A- (NCT03780257), and RHO- (NCT04123626) associated IRD. Retinal organoids have been instrumental to evaluate the efficacy and safety of several AON molecules that modulate splicing to either correct defects introduced by deep-intronic variants in CEP290 [ABCA4 [USH2A [Another type of therapeutic strategies are RNA-based therapies, such as antisense oligonucleotides (AONs), which are becoming popular to treat IRD , 77. AON2A- NCT030257, andn CEP290  and ABCAA- NCT037257, and 4 [USH2A . This imThe advent of new analytical methods that include and integrate multiple next-generation “omics” technologies is on par with the progress of organoid technology Fig. . This faGDPD1 in the RP17 locus of patients with autosomal dominant retinitis pigmentosa (RP) [The epigenetic landscape and intragenic/intergenic variants play an important role in regulating gene expression. One such epigenetic feature is a 3D organization of chromatin in the nucleus. The chromatin organization can be examined at a high spatial resolution, using methods such as chromosome conformation capture (3C) assays. In these assays, first the chromatin-deoxyribonucleic acid (DNA) complexes in the nucleus are crosslinked with formaldehyde. Then, the tissue is fragmented, and the DNA is extracted, ligated, and digested with restriction enzymes. Interacting loci are amplified with polymerase chain reaction (PCR) and quantified by sequencing methods , 83. A hosa (RP) . The lowosa (RP) , suggestAnother promising application of retinal organoids for epigenetic analysis might be chromatin accessibility assay via the transposase-accessible chromatin using sequencing (ATACseq). ATACseq can identify regions of open chromatin and cell-specific transcription factors binding sites via the action of hyperactive Tn5 transposase and high-throughput deoxyribonucleic acid (DNA) sequencing . It was The small size and heterogeneity of cell types in retinal organoids pose significant technical challenges for techniques with higher material demand, lower sensitivity, or difficulty to distinguish between the signal coming from different cell types; this is also true for epigenetic analyses. However, for a disease where a primary tissue is difficult to obtain, such as in IRDs, organoids provide a unique opportunity to study the epigenetic landscape of a specific patient genome in the correct cellular context.The retinal organoid system can also be used to quantify the changes in expression levels of retina-specific genes between a patient, or gene-edited line, and control, or following a therapeutic intervention. For this, ribonucleic acid (RNA) is converted into single-strand complementary DNA (cDNA) using a retroviral enzyme, reverse transcriptase with either oligo-dT for priming from the poly-A tails or random hexamers. The cDNA is used as a template for amplification of the gene of interest with PCR . The resA snapshot of all RNA transcripts can be identified with microarrays or RNA-sequencing (RNA-seq). Microarray analysis utilises a collection of specifically designed short DNA oligos, which gives a quantifiable fluorescent signal upon hybridization with a cDNA from retinal organoids. With microarray analysis, the rhodopsin-positive cells of D26 and D34 ESC-derived organoids were compared to P12 early postnatal mouse photoreceptors, revealing that D34 were more mature and closer resembling P12 photoreceptors . These aThe array technique allows for transcriptome-wide gene expression profiling, including the ability to detect alternative splicing events, whereas RNA-seq allows for the identification of a larger number of genes than microarrays and is also applicable to unknown transcripts . Here, tThe averaged gene expression profiles resulting from the bulk RNA-seq analysis limit its applicability to the mixed cell populations that comprise a retinal organoid. The gene expression profiles of individual cells yielded by single-cell RNA-seq (scRNA-seq), on the other hand, allow expression data to be refined to specific cell types . ScRNA-sAIPL1, underlying severe inherited retinal dystrophy Leber congenital amaurosis type 4 (LCA4), unexpectedly showed no difference in gene expression of AIPL1 itself or that of other retinal specific genes, but it did of the cortical development gene NEUROD6 at D25, D60, D88, and D123; furthermore, in the patient-derived organoids, photoreceptors developed normally [Several studies have compared the expression of a gene of interest in patient-derived organoids versus controls. Organoids derived from a patient with splicing factor-related RP showed differential expression of a number of genes related to ciliary structure and photoreceptor morphology . RNA-seqnormally . In contnormally .RS1, the expression of RS1 alongside retinopathy-associated genes IQCB1 and OPA1 was reduced compared to isogenic control organoids [RP2 gene encodes for a GTPase-activating protein linked to a severe form of X-linked RP. Isogenic disruption of RP2 in retinal organoids shared expression profile similarities to patient-derived organoids, including upregulation of a number of pro-apoptotic genes compared to isogenic controls that correlated with photoreceptor cell loss [A gene expression profile of patient-derived retinal organoids can be compared with that of an isogenic control generated by CRISPR/Cas9-mediated correction, thereby reducing inter-individual variation and more precise control of phenotypes associated with a disease-causing mutation. For example, retinal organoids bearing an RP-causative mutation in RPGR showed upregulation of necrosis and inflammation receptors when compared to both isogenic and unrelated controls . Similarrganoids . Alternarganoids . The RP2ell loss .Crx and it is driven, amongst others, by the protein Musashi-1, which promotes the inclusion of photoreceptor-specific exons [CEP290, which causes LCA10, were higher in patient-derived retinal organoids compared to RPE or other cell types [In the retinal organoid system, RT-PCR and RNA-seq can also enable mapping of developmental switches in splicing and give an insight into the retina-specific alternative splicing events, where several distinct mRNAs are produced from the same gene, leading to different isoforms. Photoreceptors have a high level of alternative splicing with many unique or enriched isoforms. The alternative splicing programme in photoreceptors is independent of ic exons , 105. Alll types . Retinalll types , 79 and ll types .REEP6.1, which harboured a mutation in a family with autosomal recessive RP, correlated with the time course of photoreceptor development [DYNC2H1 increased in maturing retinal organoids over time to become the dominant transcript at D200, correlating with the non-syndromic RP in patients with mutations affecting the retinal isoform only [ABCA4-associated progressive disorder of the retina that is initially characterised by a loss of central vision, retinal organoids exhibited enhanced splicing defects, such as pseudoexon insertion caused by deep-intronic variants, compared to transfection of gene constructs in non-retinal cells, and were used to show the therapeutic potential of an AON for splice correction [PRPF31 patient organoids revealed changes in alternative splicing of key components involved in the splicing process, as well as part of the microtubule and ciliary networks [PRPF31 mutation in retinal organoids and RPE rescued these molecular and cellular defects [Mutations can also occur in retinal specific exons; for example, RT-PCR of control retinal organoids showed an increase in expression of the alternatively spliced isoform elopment . Similarorm only . In Starrrection . RNA-seqnetworks . CRISPR/ defects .To conclude, transcriptome analysis via the RNA-seq method has become a pivotal tool in the analysis of retinal organoid development and disease mechanisms. The disadvantage of RNA-seq is the difficulty in reconstructing full-length transcripts from the assembly of reads, particularly when alternative splicing into highly similar isoforms occurs. Long-read sequencing provides amplification-free, single-molecule sequencing of cDNAs and eliminates the need for the library assembly step, and thus can be more sensitive for the alternative splicing events in distinct cell populations of the retinal organoid , 110. OtAn increase in RNA transcript numbers does not necessarily lead to an increase in the amount of the encoded protein. RNA or protein stability and post-translational modifications also contribute to protein levels , 112. WeDue to the small size of the laminated retinal organoid, it is challenging to obtain sufficient material for WB analysis. Although one study reports pooling together at least 16 stage-1 (D18-D35) organoids per condition , this apA more exploratory technique, mass spectrometry (MS) analysis can identify and quantify multiple proteins in complex mixtures by measuring the mass-to-charge ratio of peptides that are labelled with isotopes , 119. FoMetabolomics is another -omics approach that allows sensitive and semi-quantitative detection of low molecular weight molecules at a given time. The “metabolome” can rapidly change and reflect the dynamics of a biological system . While mImaging techniques, such as hyperspectral imaging (HSpec), can be used to assess the metabolic activity of the organoids. HSpec showed that retinol and RA signal intensity increased following an increase in CRX expression. The metabolic state can also be imaged with fluorescence lifetime imaging microscopy (FLIM) that shows glycolytic activity by measuring free and bound NADH distribution. In D150 organoids, the glycolytic region appeared in the laminated outer edge that corresponds to the emergence of a highly metabolically active photoreceptor layer . RecentlInterestingly, for retinal organoids, an easy and renewable source of material is the culture medium. Many factors are released to the medium, and they could provide valuable information about the stage of the organoids and the active functions. Furthermore, the used medium is usually discarded and thus the metabolomic studies throughout the complete retinal organoid differentiation could be established. For example, high-performance liquid chromatography (HPLC) system coupled to MS allowed the analysis of the media content of retinal organoids treated with retinotoxic antibiotic moxifloxacin . AnotherELOVL4 and CERKL. In addition, sphingolipidosis, such as Gaucher disease, or complex lipid disorders like Sjögren-Larsson syndrome, also present a retinal phenotype. Thus, lipidomics is a very appealing approach to study the mechanism of disease in cellular models. Retinal organoids can be used to study how changes in lipid metabolism affect retinal health; for example, deoxysphinganine-induced photoreceptor apoptosis in retinal organoids, supporting the potential association of serine and lipid metabolism disruption in macular telangiectasia type 2 [Lipidomics is another -omics strategy based on measuring the different types of lipids by combining extraction protocols with detection methods such as liquid chromatography-mass spectroscopy. In the past, lipids in the retina of multiple species have been exhaustively studied –141. Depa type 2 .Most studies of retinal organoids rely on immunohistochemistry (IHC) as a gold standard for characterisation of differentiation and identification of protein and cellular changes . This miRP2 and CRISPR-mediated RP2 knock-out. The cell death also coincided with an increase in apoptotic cells visualised by a TUNEL assay where the 3′ ends of the fragmented DNA are labelled with fluorescently tagged dUTP nucleotides. Significantly more TUNEL-positive cells were present in the ONL at D150 in the patient and knock-out organoids compared to the control which pinpointed a peak in photoreceptor cell death, with subsequent thinning of the ONL, by D180 [IHC can also be adapted for quantitative analysis; for example, the survival of cells in ONL in retinal organoids can be measured, either as cell counts or ONL thickness. This method was used to show photoreceptor degeneration in the ONL of retinal organoids derived from a patient with a mutation in  by D180 .While severe photoreceptor layer abnormalities or large cysts are visible under bright field microscopy , this teA technique called optical coherence tomography (OCT) allows non-invasive imaging in the eye in vivo and can also be applied to organoids to reveal the internal organisation via the difference in light wave reflectance between nuclear and plexiform layers. An appearance of a layer with high reflectance was reported at D150 near the outer laminated edge of the organoid  while anThe depth of imaging with the multiphoton technique is limited by the penetration of the excitation. Clearing is a chemical treatment that equalises the reflection indexes of the cell membranes and cytoplasm, thus minimizing light scattering, and allows light to travel through a thicker tissue while preserving its excitation and emission efficiency. PACT (passive clarity technique) clearing requires organoids to be fixed with paraformaldehyde, and embedded into hydrogel monomer, after which the resulted hydrogel-organoid hybrid is cleared from the membrane’s lipids , 148. AlSome potential future applications, such as high throughput testing of drugs or gene therapies, demand an increase in the analysis speed and screening capacity. With an estimated capacity of 200,000 samples per day, a 3D automated reporter quantification (3D-ARQ) platform combines a microplate reader and an excitation/emission detection for the quantitative screening of retinal organoids stained with fluorescent dyes, for example Calcein AM dye that accumulates in the cytoplasm of live cells and is used in viability assays, or JC-1 that allows visualization of the mitochondria rich inner segments of photoreceptors .Often, the subcellular structures of the organoids are of interest, for example, the organization of the membranes inside the OS of photoreceptors, or the structure of the cilium. Light microscopy cannot resolve subcellular structures due to the limited resolving power, whereas transmission electron microscopy (TEM) utilises electrons instead of light and can achieve resolution well below 1 nm. However, it requires elaborate sample preparation with the organoid section undergoing fixation, dehydration, and resin embedding. Imaging with TEM revealed photoreceptor-characteristic organisation and structures, such as the mitochondria rich inner segment, connecting cilium, basal bodies, adherens junctions at the OLM, and microvilli along with membranous disks inside immature OS , 152. TEcis retinal, a chromophore essential for rhodopsin activity, which also serves as a control for the response being opsin-mediated [Light responsiveness and synaptic transmission are two crucial features of the functional maturation of photoreceptors and the retina. The light response of a cell can be accessed by measuring a change in its ionic currents in response to light using a patch-clamp. With the patch-clamp, the glass micropipette filled with an electrolytic solution serves as a recording electrode. The electrode touches the membrane of the cell and the suction is applied to establish a tight contact between the pipette and the membrane. This forces the flow of ionic current across the sealed patch of the cell membrane to enter the pipette , 156. Phmediated . The OS mediated . Althoug2+ indicative of the inward dark current that is characteristic of photoreceptors. The fluorescent calcium dye Fura-2 allows for visualization of the increase Ca2+ influx in response to cGMP-analogue in cells of D45 and D90 [2+ influx in response to light stimulation was recorded in photoreceptors and in OFF bipolar cells but very few ON bipolar cells [Another method to investigate the functionality of retinal organoids utlises confocal or multi-photon microscopy to measure changes in intracellular Ca and D90 , D175 [1 and D90 , and in  and D90 . In cellar cells .Two-photon microscopy has been combined with patch-clamp electrophysiological recordings to test membrane localization and functionality of optogenetic constructs in retinal organoids. Several types of light-sensitive microbial opsins were expressed in cells of the retinal organoids and excitatory photocurrents were observed for the depolarizing opsins alongside inhibitory photocurrents for the hyperpolarizing opsins .PRPF31, causing the degeneration of the mid-peripheral retina, implied a significantly reduced response to GABA, but no changes in response to cGMP-analogue [2+ imaging or patch clamping where the origin of the response is more clearly identified.Micro-electrode arrays (MEAs) contain multiple electrodes embedded into a ceramic or glass carrier . Retinalanalogue . The advAn important potential addition to testing photoreceptor-driven responses would be to generate a disruption of the elements of the phototransduction pathway, such as by CRISPR/Cas9-mediated knock-out, to confirm the source of the responses. While testing the light responsiveness of retinal organoids in vitro is a “holy grail” of organoid characterisation, there is still a lot of work to be done before it could be used as a routine and reproducible measure of photoreceptor function in disease or following rescue.cis-retinal. A major challenge in retinal organoid technology to study photoreceptor disease has been to produce properly stacked OS. Mature retinal organoids contain OS-like structures containing membranous disks that are loaded with opsins [In the retina, the OS of cone and rod photoreceptors are specialised primary cilia that contain stacks of deep membrane folds of the plasma membrane, or double-membrane disks enclosed by the plasma membrane, respectively . The strh opsins , but lach opsins , 163. Noh opsins .-trans-retinal into 11-cis-retinal [OS membrane disks are renewed and shed daily , 165. Wh-retinal , 169. Du-retinal , 170–172-retinal .Another structure yet to be replicated is the fovea, a substructure in the centre of the human retina that is required for high-acuity colour vision. The absence of rod photoreceptors and other retinal cell types in the foveal pit allows light to directly stimulate densely packed cones. The fovea is specific to human and non-human primates and is selectively affected by diseases such as macular degeneration. The fovea develops postnatally in the area of the retina where the development of rods from late retinal progenitor cells is inhibited, and the location of this area is likely to be determined by transcription factors involved in the definition of the axis of the retina . While tBlood vessel and immune-related cells are also missing from retinal organoids; these are derived from the mesoderm and during development migrate into the developing optic vesicles. The addition of these cells to retinal organoids would likely require assembloid type technology to bring together cells from different developmental lineages, which was pioneered in brain organoids . RecentlThe small size and inherent heterogeneity of cell types in retinal organoids pose significant technical challenges for the types of analysis with higher material demand, lower sensitivity, or difficulty to distinguish between the signal coming from different cell types. It is, however, important to realise the potential of the retinal organoids to reveal neuroretina-specific gene regulation, pathological processes, and therapeutic manipulation.It would be desirable to increase the efficiency and reproducibility of organoid production and decrease the labour required for their production. To do so, first, it is important to understand the reasons for the innate heterogeneity between iPSC lines and why some lines generate organoids more efficiently than others. More reproducible organoid generation from a particular cell line can be achieved by a better understanding of epigenetic control of iPSC pluripotency , as wellIn conclusion, we now have a number of state-of-the-art techniques that can facilitate analysis of the retinal organoids at different levels of complexity to understand disease mechanisms and test potential therapies. An expansion of the analytical toolbox will increase the benefit of the retinal organoid model for teasing apart the molecular mechanism of genetic blinding diseases.


Patients recovering from COVID-19 commonly report persistence of dyspnea, exertional fatigue, and difficulties in carrying out their daily activities. However, the nature of these symptoms is still unknown. The purpose of the study was to identify limiting causes of cardiopulmonary origin for the performance of physical exercise in post-COVID-19 condition that could explain the symptomatic persistence of dyspnea or fatigue-related symptoms. Thirty-two non-hospitalized patients with post-COVID-19 condition  completed a clinical examination including echocardiography, submaximal and maximal cardiorespiratory fitness tests (Ekblom-Bak and Bruce’s protocols), and a battery of validated questionnaires about fatigue and exercise intolerance. Four participants (12.5%) reported an abnormal cardiac response to exercise during the submaximal test, which aroused suspicion of the presence of chronotropic incompetence. All of them were confirmed with a positive diagnosis maximal exercise test after cardiology screening, even with a comprehensive clinical examination, resting ECG, and echocardiogram, without other findings. No statistical differences were found in any physiological variables or questionnaire values, between patients with positive and negative diagnoses. Chronotropic incompetence and other autonomic disorders may appear in patients with mild forms of COVID-19 presentation and may persist in the long term, being responsible for exercise intolerance after resolution of acute infection. Clinicians should be aware that chronotropic incompetence and other autonomic disorders may be a complication of COVID-19 and should consider appropriate diagnostic and therapeutic interventions in these patients, especially when early exercise-related fatigability is reported. After a year of the coronavirus disease COVID-19) pandemic, it has become evident how SARS-CoV2 can be responsible for damage in the central nervous system (CNS) 9 pandemi), is affDamage in the ANS and CNS can lead to important dysfunctions in terms of heart rate (HR), blood pressure (BP), and systemic inflammatory response ,7. In a Chronotropic incompetence is defined as a limitation to increase HR in response to the metabolic demands proposed by exercise and may be responsible for the appearance of fatigue and exercise intolerance in patients with post-COVID-19 condition . During The purpose of the study was to identify limiting causes of cardiopulmonary origin for the performance of physical exercise in post-COVID-19 condition that could explain the symptomatic persistence of dyspnea or fatigue-related symptoms commonly referred by this population.This cross-sectional study examined the RECOVE cohort including non-hospitalized post-COVID-19 patients (NCT04718506) . After cParticipants were recruited for the study after they expressed interest on the registration website . ParticiA complete clinical examination, including electrocardiogram and echocardiography, was performed to rule out cardiovascular diseases. A resting echocardiogram was performed following standard procedures  by a teaThe cardiorespiratory fitness assessment included a submaximal cycle ergometer test with HR and rate of perceived exertion (RPE). Heart rate variability (HRV) was collected for 1 week by means of the root mean square of successive differences (RMSSD) using the Welltory app  as a marWhen chronotropic incompetence was suspected, to confirm this diagnosis, patients underwent a maximal graded exercise test according to Bruce’s treadmill protocol . To diagParticipants were provided with a battery of self-rating questionnaires  to evaluate dyspnea, fatigue, and exercise intolerance in daily living activities: Chalder Scale  , Fatiguet-test for independent samples. Calculations were performed using IBM SPSS v. 20.0 .Descriptive data analysis included means and standard deviations. Crosstabs and chi-squared analysis were used to examine the distribution of symptoms between people with positive and negative chronotropic incompetence diagnoses. Mean differences were identified by Student’s From 62 patients who expressed interest in the study from 1 February to 15 April, 32 complied with inclusion/exclusion criteria and were enrolled in the study . These tBaseline characteristics of participants are shown in Four participants (12.5%) reported an abnormal cardiac response to exercise during the submaximal test, i.e., HRmax ≤ 70% of estimated HRmax with an elevated RPE > 16  which aroused suspicion of the presence of chronotropic incompetence. To confirm this diagnosis, patients underwent a maximal graded exercise test according to Bruce’s treadmill protocol. Not one of them was able to reach 80% of the age-estimated HRmax (M ± SD = 69.6% ± 5.0%); therefore, chronotropic incompetence was diagnosed. Comprehensive clinical examination and resting ECG and echocardiography parameters were withing normal limits . LikewisIn this study, we examined a cohort of non-hospitalized patients with post-COVID-19 condition to identify a possible explanation for the exercise intolerance and fatigue, commonly referred by this population. The results showed the presence of chronotropic incompetence in 12.5% of ambulatory patients as a possible central cause for these symptoms and not only due to skeletal muscle dysfunction as other have suggested . To our The presence of autonomic dysfunction, understood as increased sympathetic activity and loss of parasympathetic counter-regulation that characterizes other cardiovascular diseases and the hyperacute phase of COVID-19 ,30, may A new contribution of the present study is the use of a submaximal, safe, short, reproducible, and noninvasive test (Ekblom-Bak protocol) for the identification of patients with exercise limitations and may assist in the identification of cardiac complications such as chronotropic incompetence. As a practical application, we found that people achieving HRmax ≤ 70% of estimated HRmax with an elevated RPE > 16  were likely to get a positive diagnosis. In fact, this finding is clinically relevant in individuals with post-COVID-19 condition complaining of dyspnea and post-exercise malaise or fatigue, because basing intensities of exercise on predicted maximal HR could be nefarious. This would lead to an overestimation of the target heart rate during exercise, whereby the subsequent inability to achieve it will cause discomfort, as well as a sensation of shortness of breath, and, in the short term, compromise adherence to the training program. Given that the Ekblom-Bak protocol is submaximal in nature, we can also conclude that the limitation of exercise appears not only at high intensity, but also at moderate intensities, which clearly implies the commitment of the activities of daily life. In addition, the use of adapted exercise tests as a screening tool in people with post-COVID-19 condition seems important considering that spirometry may fail in identifying exertional intolerance and dyspnea in this population . The ongThe persistent clinical manifestations of post-COVID-19 condition are extraordinarily varied and multisystemic in nature . FatigueHRV has previously been used in patients with acute COVID-19 as a noninvasive measure of autonomic function, finding that, after an early increase in parasympathetic activity, patients with elevations of poor prognostic biomarkers, such as CRP (C-reactive protein), exhibited a prior loss of HRV ,35. ThisIt must be noted that the current findings are limited by the reduced sample size, the lack of a control group, and the absence of previous stress tests. We cannot rule out the possibility that failure to achieved estimated HR is justified by COVID-19 alone. Nonetheless, this study is strengthened by the novelty of the findings and its practical implications  for clinicians dealing with patients with post-COVID-19 conditions or other conditions characterized by unexplained fatigue and exercise intolerant symptoms.Chronotropic incompetence and other autonomic disorders may appear in patients with mild forms of COVID-19 presentation and persist in the long term, being responsible for exercise intolerance after resolution of the acute infection. Clinicians should be aware that chronotropic incompetence and other autonomic disorders may be a frequent complication of COVID-19 and should consider an appropriate diagnostic test, especially when early exercise-related fatigability is reported. Our research group supports that, in those patients in whom structural damage caused by SARS-CoV2 infection has been ruled out, a stress test should be performed to complete the diagnosis. Carrying out an 8 min Ekblom-Bak test is a simple, accurate, reproducible, non-time-consuming, and low-risk tool, in any clinical setting, including primary care. The presence of simple criteria ) can indicate the diagnosis of chronotropic incompetence. Once confirmed by ergospirometry, the decision on the prescription of physical exercise must be individualized. The direct effects of training on the reversal of chronotropic incompetence are not well known; thus, new studies in people with post-COVID-19 condition must be carried out.


Residents of Long-Term Care Facilities (LTCFs) represent a major share of COVID-19 deaths worldwide. Information on vaccine effectiveness in these settings is essential to improve mitigation strategies, but evidence remains limited. To evaluate the early effect of the administration of BNT162b2 mRNA vaccines in LTCFs, we monitored subsequent SARS-CoV-2 documented infections and deaths in Catalonia, a region of Spain, and compared them to counterfactual model predictions from February 6th to March 28th, 2021, the subsequent time period after which 70% of residents were fully vaccinated. We calculated the reduction in SARS-CoV-2 documented infections and deaths as well as the detected county-level transmission. We estimated that once more than 70% of the LTCFs population were fully vaccinated, 74%  of COVID-19 deaths and 75% (36%−86%) of all documented infections were prevented. Further, detectable transmission was reduced up to 90% (76–93% 90%CI). Our findings provide evidence that high-coverage vaccination is the most effective intervention to prevent SARS-CoV-2 transmission and death. Widespread vaccination could be a feasible avenue to control the COVID-19 pandemic. Widespread vaccination has the potential to significantly reduce SARS_CoV-2 infections and deaths, and subsequently improve social and economic conditions . AvailabAmong all populations, residents of Long-Term Care Facilities (LTCFs) represent a major share of COVID-19 deaths, with a 7-fold higher incidence of death compared to the general population in the US ,7. As a In this study, we aimed to quantify the early effect of the administration of the BNT162b2 mRNA COVID-19 vaccine on reducing the risk of SARS-CoV-2 transmission and COVID-19 death in LTCFs in Catalonia (Spain).Documented COVID-19 infections, identified with a molecular test (PCR or antigen) independently of symptoms, were assumed to capture most infections. Documented deaths attributable to COVID-19 included those with laboratory confirmation and those only meeting clinical and epidemiologic criteria. Detected county-level transmission was used as an indicator of transmission and defined as at least one documented infection in any facility within a county per unit of time. LTCFs residents were defined as those living in a LTCFs and older than 64y. The general population, or “community”, was defined as all people in a county not living in LTCFs. All vaccinated individuals received the two doses of the BNT162b2 mRNA vaccine. For further details see We generated multiple time series of daily confirmed infections, deaths, and vaccinations in LTCFs, aggregated by healthcare area level n=9), and in the broader Catalonia region. Similarly, we collected daily confirmed infections in the general population, at the healthcare-area level and regional level. For each of these time series, we took a moving weekly average around each day to smooth some of the daily variation of reporting. Further, we generated a time series of the detected county-level transmission  by week. The pre-vaccination period was from July 6 to December 27 2020, when vaccination in LTCFs began. We evaluated the impact of vaccines during an evaluation time period from February 6 to March 28, 2021, the subsequent time period after which 70% of residents were vaccinated with two doses. The 70% threshold was chosen to represent the estimated herd immunity - the estimated level of immunity in a population that prevents uncontrolled spread of infections [, and in We built regression models to predict the number of infections and deaths using community infections as inputs. These models were calibrated during the pre-vaccination period and then used to generate predictions in the absence of vaccines during the evaluation period. We compared the models’ counterfactual predictions with observations; discrepancies were used to quantify the effects of the vaccine. Further, for each county with at least one pre-vaccination week with a transmission event and at least one week without one (n=36), we built logistic regressions to estimate the probability that a documented transmission would be observed, using community infections as input. These models were trained on the pre-vaccination period and then used to predict transmission events in the evaluation period; the aggregated county-level predicted probabilities and the aggregated observed transmissions were compared to quantify vaccine effectiveness. See We quantified the effect of administering the BNT162b2 vaccine (a) on reducing deaths and documented infections among residents older than 64y, and (b) on reducing detected transmission caused by SARS-CoV-2 in LTCFs in Catalonia.In this study we show that high vaccination coverage (over 70%) prevented around 3 out of 4 expected COVID-19 deaths in subsequent weeks, which is consistent with the vaccine effect estimated in clinical trials  and obseFurthermore, LTCFs represent enclosed populations, where transmission is caused both by external introduction of the virus, mostly by the staff , and intOur methodological assumptions may lead to underestimation of the true vaccine effect. First, our definition of transmission events at the county-level does not capture the facility-level transmission; thus, our results may fail to capture more dramatic facility-level reductions in transmission. Second, the counterfactual estimates of deaths produced by our models may be underestimations of mortality during the evaluation period. Indeed, the documented deaths were generally higher than those estimated by our models in December and January . It is pIn addition, we acknowledge that our investigation has limitations. We assume that the rigorous screening standards in LTCFs in Catalonia led to infection ascertainment close to 100% and also that the ascertainment of community infections does not change dramatically over time. While this may appear unrealistic, we suspect it is not far from the truth, as public health authorities in Catalonia substantially increased the infection screening efforts in LTCFs before the vaccination campaign .Further, regression models were not designed for accurate infection (or deaths) forecasting and as such, they may not fully capture the epidemiological dynamics. However, our efforts were focused on proper inference of the expected epidemic trajectory in the absence of vaccination. This goal is achieved as our models appear to reasonably capture the overall dynamics during the pre-vaccination time periods, even at high spatial granularity –S2.Finally, there could be unmeasured confounders, such as behavior or policy changes, not captured by our models, that may have changed the dynamics of transmission between the community and LTCFs over time --this may be the case in the health area Alt Pirineu i Aran –S2. In mIn spite of these limitations, our analyses provide evidence that vaccination may be the most effective intervention in controlling SARS-CoV-2 spread and subsequent risk of death available to date. If our findings continue to be confirmed by future studies, widespread vaccination could be shown to be a feasible avenue to control the COVID-19 pandemic.1