The essential goal of neoadjuvant chemotherapy (NACT) is to downstage the primary tumor making it amenable for breast conservation surgery (BCS). However, since the safety of this surgery is paramount, post-NACT breast conservation rates remain low. As per the recommendation of the 2018 Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) overview of long-term post-NACT follow-up, we have devised a protocol for imaging, localization, rad-path analysis, and documentation of radiotherapy techniques to ensure the safety of post-NACT breast conservation.This is a retrospective cohort of 180 breast cancer patients who received NACT and were operated on by a single surgical oncologist from 2015 to 2020. After selection based on published guidelines, patients were treated with neoadjuvant systemic (chemo or hormone) therapy. In cases where primary tumors responded and reduced to 1–2 cm in size mid-NACT, the residual tumors were localized by clips under ultrasound guidance and calcification was wire localized. All patients were treated using appropriate surgical and oncoplastic techniques where indicated. Negative margins were ensured by intra-operative rad-path analysis. Adjuvant chemotherapy and radiotherapy were given as per protocol.In 81 cases that required mastectomy at presentation, we were able to achieve a 72.8% post-NACT BCS rate with the help of oncoplasty. Overall, 142 of 180 (80%) patients were treated with breast conserving surgery of which 80% (121 of 142) were oncoplasty. Margins were assessed on intra-operative frozen and re-excised in the same setting. No positive margins were reported in final histopath of 142 breast conservation procedures. Post-operative complication rates after breast conservation in the first year were at 17% (24 of 142 including two major complications). Patient reported outcomes were satisfactory with increased satisfaction for breast conservation compared with immediate breast reconstruction.Employing oncoplastic breast surgery (OBS) techniques following stringent protocols for accurate localization of the residual tumor, intra-operative rad-path analysis, and adjuvant treatments, we show successful breast conservation in 72.8% of our mastectomy-qualified patients after downstaging by NACT. We also report satisfactory outcomes for post-NACT surgery, patient-reported satisfaction, and survival. Breast cancer is the most diagnosed cancer and the leading cause of cancer-related death in India and globally, 2. PopuConventional BCS is limited to selected patients where it has been shown to be oncologically safe and demonstrable esthetically superior outcomes . A hurdlOncoplastic techniques have been demonstrated to expand the indications for breast conservation in a variety of situations –22. MultHere, we present an audit of our cohort of 180 breast cancer patients who were treated with post-NACT surgery, assessing breast conservation rates, oncological outcomes, and 1 year patient reported outcomes measures (PROMs). We present a series of precautions and procedures that we carried out to ensure safe breast conservation to address the lacunae/issues raised by the EBCTCG overview. At our center, we routinely perform OBS whenever needed and possible for all breast tumors, including LABC and LOBC downstaged with NACT. In recent times, the scope of patients for whom guidelines recommend neoadjuvant therapy has been broadened . In thisThis is a single institutional study involving retrospective analysis of prospectively collected data. The following pathological criteria were used for patient selection for neoadjuvant systemic treatment: (1) LABC (not including node positive T1 and T2 LABC cases), (2) LOBC, (3) luminal HER2-positive and HER-positive  tumors greater than 2 cm; (4) Triple Negative Breast Cancer (TNBC) greater than 3 cm, (5) luminal HER2 negative large T2/T3 tumors, and (6) any patient with clinically or histologically proven positive axillary nodes. Patients with demonstratable metastatic disease were excluded. All patients underwent definitive breast surgery post-NACT in the period between January 2015 and December 2020. Patients were deemed qualified for upfront mastectomy based on the following criteria: (1) LABC; (2) LOBC; that is, Stage IIB and above (T3N0 but not T2N1) as described by Simos et al. ; (3) smaData included demography, medical history, clinical findings, pathological reports , details of NACT, surgical intervention, pre- and post-operative images of patients, post-surgery complications, follow-up details, and PROMs.Triple assessment based on clinical examination, imaging and image-guided core needle biopsy was routinely used to establish a diagnosis. Systemic staging was assessed based on Positron Emission Tomography (PET) imaging. Patients were selected for neoadjuvant treatment based on decisions made by the multidisciplinary team (MDT). After clinical staging, NACT was administered based on NCCN guidelines.After every other cycle of chemotherapy and at the completion of NACT, the patient was monitored clinically and radiologically. Clinical response of the primary tumor to therapy was calculated according to the RECIST Ver 1.1 criteria . The patFor all cases, the residual tumor was pre-operatively localized by ultrasound (USG) imaging. In cases where the tumor responded to NACT, the residual tumor was clipped mid-NACT at ~1cm size by USG guided insertion of liga clips. At least four clips were used to facilitate identification by intra-operative USG Figure 1In this report, paraffin slides of margin tissue were accessed from the pathology laboratory to verify the negative margin outcome. Slides of tissue assessed at frozen were quality checked for tissue integrity on Mantra (PerkinElmer). Frozen and pathology slides were imaged on an OS-15  bright field digital scanner. These digital images were blinded and shared from our server with the second pathologist (SB) to assess concordance in margin assessment.In every case, wide local excision of the residual tumor was identified by palpation as well as USG of the residual tumor and the marker clips used for localization. Post-excision margin adequacy was assessed on the table by USG or by specimen mammography. In addition, all specimens were sent for frozen section margin assessment to the pathologist. Any close margin (1–2 mm) seen on specimen mammogram or reported on frozen section  was revised in the same sitting before restoration of the breast form. Margin guidelines based on the consensus on margin safety by the American Society of Clinical Oncology, Society of Surgical Oncology, and American Society for Radiation Oncology Consensus Guideline  were folThe surgical plan was determined based on assessment at diagnosis, post-therapy clinical-radiological assessment of residual disease, residual tumor location, and extent of calcification, breast size, and ptosis. Patients were counseled for safety and esthetic outcomes. Final decisions were based on patient choice. Volume displacement and volume replacement techniques were employed in all cases where conventional breast conservation was deemed unsuitable. In cases where breast conservation was not feasible mastectomy with immediate whole breast reconstruction was performed. Very few patients did not undergo reconstruction. The surgical procedures used are described here.BCS with primary breast closure was performed in patients with small residual tumors with adequately sized breasts or tumors in favorable locations.Oncoplastic surgeries have been classified based on the recent recommendations of the American Society of Breast Surgeons  and as eIn certain situations, a complete mastectomy was performed. Seventy percent cases of mastectomy were followed by immediate breast reconstruction by implant or LD flaps. Dermal sling was employed for larger ptotic breasts, which has been shown to be a safe procedure . For casPost-surgical oncological management decisions regarding chemoradiation protocols were undertaken by a multidisciplinary clinical team in accordance with guidelines.Surgical outcome data were recorded as minor or major based on the presence or absence of surgical complications such as seroma, delayed wound healing, fat necrosis, lymphedema, and infection and the interventions required to treat them. Major complications were those that required surgical intervention, whereas minor complications could be treated by therapy in the clinic.Margin status was assessed by histopathology of frozen sections intra-operatively and paraffin section post-operatively. Patients were carefully monitored for local and distant recurrences, quarterly for the first 2 years post-surgery and then every 6 months. Suspicious symptoms or signs were assessed by appropriate imaging and histological confirmation wherever feasible.Patients were evaluated for patient reported outcome measures (PROMs) using the BREAST-Q questionnaire  at 1-yeaData were analyzed using Fishers Exact Count Statistic (when less than five cases), chi-square test, Wilcoxon Rank Test, and Student’s t-test using the stat and BSDA  package One hundred eighty breast cancer patients treated with neoadjuvant therapy and operated between January 2015 and December 2020 were included in the study. A majority of cases (86%) were diagnosed at biopsy as IDC (155 of 180), and the rest were IDC with DCIS  and ILC . Most tumors were T2 at presentation , and 85% of patients were node positive (153 of 180). Most of these cases were N2 on imaging . A significant number of cases were clinically stage IIIA at presentation . The three cases that were treated with neoadjuvant therapy post an excisional biopsy performed at other centers. Excision biopsy is performed on lumpectomy specimens. These are the results of surgical procedures performed for diagnosis, immediately after a suspicious imaging report or clinical examination, without any initial biopsy. This is a common practice in many centers. Such patients often come to our center for reconstructive surgery to regain breast shape, remove scars, and so forth. This is a common practice in many centers. Such patients often come to our center for reconstructive surgery to regain breast shape, remove scars, and so forth. Since margin positivity is often not assessed after such procedures, patients are always assessed for residual tumor by imaging or a histopathological review of specimen tissue when available. The three cases included here came to us post such a procedure from other centers with reported positive margins. Two of these cases also had positive nodes that had not been treated. The cases included in this cohort were young age (< 40 years) and offered NACT prior to surgery.p = 6.39e−07), and all except 1 were post-menopausal. Seventy-seven percent (38 of 49) of HER2-positive  patients received trastuzumab, a very high rate in an Indian setting for the use of HER2-directed therapy due to the prohibitive cost involved  received hormonal therapy and 51% (38 of 76) were given Anthracycline- or Anthracycline- and Paclitaxel-based chemotherapy. Patients on hormone therapy were significantly older  than the chemotherapy group , involved . Fifty-eOBS dominated as the choice of surgery in our cohort Figure 2The clinical response to treatment was determined by RECIST criteria Ver 1.1. . The patPatients were classified as qualified for upfront BCS (99 of 180) or upfront mastectomy (81 of 180). The basis for qualification for mastectomy is summarized in Out of 81 cases that initially qualified for mastectomy, 59 showed a good response to NACT and hence were converted to BCS/OBS with the conversion rate from mastectomy to BCS/OBS of 72.8%. The rest underwent mastectomy with reconstruction (15) or mastectomy alone (7)  breast-conserving surgeries were performed in the cohort. A substantial proportion (79%) of the breast conservative operations is oncoplastic procedures (OBS) (121 of 142). The types of OBS carried out in the cohort and for the subset that qualified for upfront mastectomy are shown in Patients who were selected for volume displacement Level 2 surgeries were also counseled for contralateral symmetrization. Only patients who consented for symmetrization were treated with this oncoplastic technique (40 cases). In addition, three cases of perforator flap volume replacement and 11 cases of immediate breast reconstruction received simultaneous symmetrization.p = 0.0012)  as compared with average resection volume in OBS procedures  received adjuvant chemotherapy. A majority of HER2-positive  patients received adjuvant trastuzumab (57%), while only 45% (25 of 56) TNBC patients and 49% (36 of 74) luminal HER2-negative patients received adjuvant chemo- or hormonal therapy. Most patients received adjuvant radiotherapy (RT) (92%—167 of 180). Of the patients who did not receive RT, seven were lost to follow up, and six patients refused RT. Of these six patients, none had recurred at the time of last follow-up (average follow-up for these patients = 39 months).The frequency and type of post-surgical complications observed in the NACT cohort in the first year post-surgery are shown in We assessed margin positivity rates and the minimum tumor margin size in BCS and OBS surgeries. Due to the extreme care during the surgical procedure with stringent radiological and pathological analysis and revision of margins in the same setting (see Methods), none of our patients needed a second surgery. To independently verify intra-operative frozen assessment of negative margins, pathology margin slides were imaged, blinded, and shared with an independent US-board certified pathologist (SB) for assessment of margin involvement. One hundred sixty-two slides  from 23 patients were re-examined. All the examined margins were found to be negative. In patients with scattered tumor in excised tissue, much wider than 2-mm margins were taken to ensure not to miss any scattered residual disease. The average margin in breast conservation surgeries was 12.2 ± 13.4 mm, with larger average margins for OBS compared with BCS (12.8 ± 14.3 mm compared with 8.5 ± 5.3 mm). The median follow-up for this cohort is 38 months . Sixteen cases (9%) have less than 1-year follow-up. At median follow-up (38 months), the entire cohort had an overall survival (OS) rate of 90.5%, distant disease–free survival of 84%, and loco-regional recurrence free survival at 93% Table 6.P < 0.001) by Mann–Whitney Test.The PROM scores from the BREAST-Q questionnaire  QuestioTable 8 Breast conservation gives the best surgical outcome for breast cancer in appropriately selected patients . RecentlThe long-term EBCTCG overview of neoadjuvant compared with adjuvant therapy  did not Oncoplastic techniques have previously been shown to drastically increase the scope of breast conservation and reduce the rate of mastectomy with better margin positivity and re-excision rates , 56–58. Here, we report post-NACT outcomes for a series of 180 patients. All cases were operated by a single surgical oncologist after extensive patient counseling and with the aim of providing the best result to the patient in terms of oncological safety, long-term outcomes, cosmetic outcome, and patient satisfaction. Mastectomy has been shown to have an adverse effect on quality of life end points for most patients . In our The flaws brought out by the EBCTCG overview about the inconsistency of the protocol of imaging, tumor localization, and rad-path analysis were mitigated by adherence to a strict protocol as discussed. In case of tumors that responded well to NACT, the tumor center was localized mid-NACT at ~1–2 cm by USG-guided insertion of at least four liga clips see Figure 1A recent meta-analysis on outcomes of post-NACT BCS , reporteThe relatively short median follow-up of 38 months (~3 years) may have biased some of the oncological outcome presented here. This cohort covers 6 years of retrospective data from 2015 to 2020. The first 3 years (2015–2018) have a median follow-up of 50 months and account for 60% of the cases. Cases from 2019/2020 have shorter follow-up of 29–20 months. However, many of the patients in this cohort are in active follow-up and the outcomes of this cohort will be subsequently updated. In addition, in the time covered by this retrospective cohort, the practice was to offer the PROM questionnaire only once at the time of the 1 year follow-up. We are therefore unable to present long-term patient outcomes.With a range of oncoplastic techniques, we were able to achieve a rate of 80% breast conservation in our cohort despite having 61% (110/180) LABC cases. Oncoplasty conferred the ability to excise a variety of volumes as was required in each case with adequate margins to make the surgery oncologically safe. These included cases where the residual tumor was large or the excised specimen sent for the frozen section showed scattered tumor foci. The esthetic outcomes were deemed acceptable based on the satisfactory PROM scores. Carefully carried out breast oncoplasty has the potential to increase breast conservation rates and patient satisfaction in the post-NACT setting. The incorporation of oncoplastic techniques helps treat larger tumors, achieves better cosmetic outcomes, and maintains comparable survival rates as that of mastectomy.Our experience shows that meticulous protocol for imaging and targeting the tumor with mid-cycle clipping, intra-operative evaluation of margins, stringent RAD-PATH analysis, and application of appropriate oncoplastic and RT techniques confers a major benefit in terms of surgical, oncological, and patient-reported outcomes. Using these protocols, we were successful in avoiding re-excisions by a second surgery and providing breast conservation in our socio-economic conditions. In fact, avoiding second surgeries should be an aim even in the developed world as it would help save on resources. The inclusion of oncoplastic surgery in the armamentarium of surgical techniques will improve breast care for patients presenting with larger tumors and more advanced disease, which is found to a larger extent in developing countries.dr.koppiker@prashanticancercare.org.The datasets presented in this article are not readily available because The dataset is composed of clinical data with follow up of individual patients treated by us and is therefore not shareable with others outside our institutions. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by PCCM-CTCR Independent Ethics Committee. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CK was involved in the conception and design, financial support, administrative support, manuscript writing, and final approval of the manuscript and was generally accountable for all aspects of the work; DK was involved in the collection, analysis, visualization and interpretation of data, manuscript writing, final approval of the manuscript; MK was involved in the collection, analysis and visualization and interpretation of data, manuscript writing; SK was involved in the collection, analysis and visualization of data; MP was involved in the collection and analysis of data; UD, CD, BV, NJ: Assembly of data; VZ in data interpretation and manuscript writing; NG, AJ in data collection and analysis; RU, RB, NN in data collection; PV in data analysis; LB Administrative support; GT and SN data assembly and collection; JP in data interpretation; SB in data analysis and interpretation. All authors contributed to the article and approved the submitted version.


Receiver operating characteristic curve analysis showed that miR-486-5p expression could predict atrium LVA . Also, miR-486-5p plasma levels were associated with AF-type . In addition, miR-486-5p expression was positively correlated with LVA percentage, left atrial (LA) area, and LA volume . These findings suggest that miR-486-5p expression might have prognostic significance in LVA extent in patients with AF.Atrial fibrillation (AF) is the most common arrhythmia worldwide, affecting 1% of the population over 60 years old. The incidence and prevalence of AF are increasing globally, representing a relevant health problem, suggesting that more advanced strategies for predicting risk stage are highly needed. miRNAs mediate several processes involved in AF. Our aim was to identify miRNAs with a prognostic value as biomarkers in patients referred for AF ablation and its association with LVA extent, based on low-voltage area (LVA) maps. In this study, we recruited 44 AF patients referred for catheter ablation. We measured the expression of 84 miRNAs in plasma from peripheral blood in 3 different groups based on LVA extent. Expression analysis showed that miR-486-5p was significantly increased in patients with broader LVA (4-fold,  Atrial fibrillation (AF) is the most common heart rhythm disorder. It affects approximately 1% of the population over 60 years of age and can affect 10% of the population over 75 years of age; thus, AF incidence increases with age. The incidence and prevalence of AF are increasing globally, representing a relevant health problem since it highly increases the risk of death in those who suffer AF; in addition, AF contributes to a significant increase in health costs . In persIn this study, we aimed to identify miRNAs with a prognostic value as biomarkers in consecutive patients referred for AF ablation. Additionally, we evaluated if miRNA expression is also associated with LVA extent , based on LVA maps, and the possible mechanisms that can contribute to AF pathophysiology.The demographic, clinical, and treatment characteristics of the participants are shown in Plasma expression profiles for a panel of 84 miRNAs showed that miRNAs levels were differentially regulated according to LVA percentage groups in peripheral blood. The expression levels of miRNAs—hsa-let-7b-5p, hsa-miR-320a, and hsa-miR-486-5p—were increased in patients with higher LVA percentages a.The area under curve data showed that only hsa-miR-486-5p may be a good predictor of LVA percentage in patients in Stages 1 and 3 in plasma from peripheral blood .Regarding miRNA profile based on AF type, first, we found an association between LVA percentage and AF type a,b, resuOur data also showed that hsa-let-7b-5p and hsa-miR-486-5p were differently expressed depending on AF type a. The arCorrelation analysis revealed that miR-486-5p expression was positively correlated with LVA percentage a, LA areRegulated miR-486-5p was predicted to target multiple genes involved in several pathways related to cardiovascular diseases. The KEGG analysis included pathways that are related to the AGE-RAGE signaling pathway in diabetic complications; cellular senescence; valine, leucine, and isoleucine degradation; propanoate metabolism; FoxO; p53; TGF-beta; AMPK; Insulin; signaling pathways regulating the pluripotency of stem cells; mTOR; and focal adhesion signaling pathways .Atrial fibrosis is a common pathological feature of AF, characterized by the excessive deposition of extracellular matrix proteins, such as collagen and fibronectin, within the atrial tissue; this fibrosis can contribute to the initiation and maintenance of AF by promoting conduction abnormalities, electrical remodeling, and structural changes in the atrial tissue . Our stuIt has been previously reported that miR-486-5p also plays a role in regulating the expression of genes that are involved in electrical conduction and structural heart remodeling . Several2+ release, impairment in the sarcoplasmic reticulum Ca2+ load, and decreased Ca2+ re-uptake via SERCA2a. These abnormalities are common in atrial cardiomyocytes of AF patients. In addition, it was reported that mutations in the PCCA gene were related to long QT syndrome, which is often associated with FA [Targeting analysis showed that dysregulated genes, targeted by miR-486-5p, could be involved in AF maintenance and fibrosis, supporting our results . miR-486 with FA . The CAD with FA .In summary, miR-486-5p and its targeted genes have been implicated in the pathophysiology of AF and the formation of scar tissue in the heart. Higher levels of miR-486-5p are associated with a greater risk of developing AF and a greater degree of cardiac fibrosis, which can contribute to impaired cardiac function.This study has some limitations that need to be taken into account at the time of data interpretation. Firstly, and very importantly, this study was conducted with a small sample size in a single center. Thus, the expression of miR-486-5p needs to be validated in a larger sample size. One of the limitations of our study is that it is a non-randomized retrospective study. In addition, 16% of our patients had already undergone pulmonary vein ablation. Patients with radiofrequency lesions beyond the pulmonary veins were not included in the analysis because they may interfere with the level of myopathy determined via electroanatomic voltage mapping. Thus, the quantification of the LVA percentage was performed outside the pulmonary veins, so this does not influence the conclusions of this study. Spontaneous low-voltage regions are a surrogate marker for atrial fibrosis, but histological validation is missing. The spatial distribution and extent of LVZ depend largely on spontaneous rhythm and the site and frequency of atrial pacing, as well as the mapping catheter and interelectrode distance. However, all our patients were mapped in spontaneous rhythm without pacing, using catheters with the same electrode size, interelectrode distance, and automatic acquisition setting validated in our previous studies. Multipolar catheters may be prone to suboptimal contact in several LA regions. The definition of the total LA surface area may not be in line with previous studies since, for the present analysis, it was defined as the LA body area without the PV antrum regions, LA appendage orifice, and mitral valve. Although it is our belief that it should not alter the conclusions of this study (regarding the amount of LVA), our conclusions should be interpreted under this definition. To conclude, the temporal pattern and intermittent ECG monitoring determined the AF burden, although this did not really correspond to long-term ECG monitoring.We included consecutive AF patients referred for pulmonary vein ablation despite having optimal pharmacological therapy at the University Clinical Hospital of Santiago de Compostela. The exclusion criteria were age under 18 years, any latent infectious condition, and pregnancy, and there was no history of malignant chronic kidney disease or osteoarthritis present. Patients with radiofrequency lesions beyond the pulmonary veins were not included in the analysis because they might interfere with the level of myopathy determined via electroanatomic voltage mapping. Due to the fact that previous radiofrequency ablation beyond the pulmonary veins generates areas of low voltage at the time of the EAM, patients with previous AF ablation outside the pulmonary veins were excluded from the analysis. Also, patients with paroxysmal, persistent, and long-standing persistent were included. Antiarrhythmic drug therapy (ADT) was continued during the blanking period (defined as 3 months after ablation), and after this period, only ADT was restarted in case of recurrence. This study complies with the Declaration of Helsinki and was approved by the Clinical Research Ethics Committee of Galicia (MRM-miRAF-2017-01). All of the patients signed informed consent.AF was classified, according to the American Heart Association and American College of Cardiology, as paroxysmal ; persistent (failure to terminate in 7 days); long-standing persistent (lasting for more than 12 months) .In all patients, AF was recorded using a 12-lead electrocardiogram (ECG) within the 6-month period before ablation. Computed tomography (CT) or magnetic resonance imaging (MRI) was routinely performed and used to guide the manipulation of the catheter at the time of the procedure.As previously described by López-Canoa et al. , patientAll procedures were performed under general anesthesia or conscious sedation with blood pressure monitoring. Trans-esophageal echocardiography or CT-angiography was performed in all patients to rule out the presence of left atrial thrombus before ablation. If already present, OACs were not interrupted before the procedure. Vitamin K antagonists were continued with a target INR between 2 and 3. Direct oral anticoagulants (DOACs) were discontinued on the day of the procedure and resumed the same day. After groin puncture, intravenous heparin was administered to maintain an activated clotting time between 300 and 350 s throughout the whole procedure. Concerning the ablation, irrigated tip ablation catheter with contact force sensing technology was systematically employed. RF lesions were placed in temperature-limited power control mode . The ablation was guided via automatic ablation annotation and minimum force–time integral and, later, minimum ablation index values, local electrogram attenuation, and impedance changes. Antral or wide-antral PVI was performed at operator’s discretion, but ostial ablation was avoided. Entry and exit blocks of the PVs were assessed with or without intravenous adenosine, and if needed, touch-up applications were applied at the gap sites to achieve block. Point-by-point pulmonary vein isolation was performed in all patients using contact force sensing technology  following high-density bipolar voltage mapping. The LVA percentage was defined via the size of the LA fibrotic area, derived from a bipolar voltage map created simultaneously with LA surface reconstruction, guided by an EAM system  using a multipolar mapping catheter . Patients in AF rhythm at the start of the procedure systematically underwent electrical cardioversion in the electrophysiology laboratory. For those patients who relapsed after at least two cardioversions, EAM was performed in AF, adjusting the cut-off . AdequatC. elegans as an exogenous normalizer (spike-in cel-miR-39) [Sequences of 84 different predesigned mature miRNAs (listed in -miR-39) . All cDNhttp://mirwalk.umm.uni-heidelberg.de/ (accessed 20 February 2023)). EnrichR was used for GO terms and KEGG pathway enrichment analyses (https://maayanlab.cloud/Enrichr/ (accessed on 20 February 2023)). MiRNA–genes–pathways networks were visualized with Cytoscape software 3.9.1 (http://cytoscape.org/ (accessed on 28 February 2023)). In silico analyses were performed to fully understand the functional role of differentially regulated miRNAs.miRNA-targeted genes were retrieved from the miRWalk 3.0 database  analysis, were performed using GraphPad Prism 9 . Numerical data were presented as mean and standard deviation (SD), interquartile range (IQR), or standard error of the mean (SEM). In all analyses, a two-tailed Thus, miR-486-5p may be involved in the pathogenesis of AF and LVA extent, and its dysregulation could contribute to the development and progression of these conditions. Further research is needed to fully elucidate the role of miR-486-5p in the pathogenesis of AF and its potential as a biomarker or therapeutic target for the treatment of AF and associated LVA extent.


This cohort study evaluates the association of neighborhood income mobility and risk of neonatal and maternal morbidity among patients in Ontario, Canada. Do adverse birth outcomes differ among women who move from a low- to higher-income neighborhood between births?This population-based cohort study included 95 617 women residing in a low-income neighborhood at their first birth, and all had universal health care. Women who moved to a higher-income neighborhood between births had lower rates of severe maternal morbidity/mortality than those who remained in a low-income neighborhood between births (12.0 vs 13.3 per 1000 births), and their newborns experienced lower rates of severe neonatal morbidity/mortality (48.0 vs 50.9 per 1000 live births).These findings suggest that women who move from a low-income to a higher-income neighborhood between births experience less morbidity and death, as do their newborns. Residing in a low-income neighborhood is generally associated with worse pregnancy outcomes. It is not known if moving from a low- to higher-income area between 2 pregnancies alters the risk of adverse birth outcomes in the subsequent birth compared with women who remain in low-income areas for both births.To compare the risk of adverse maternal and newborn outcomes among women who achieved upward area-level income mobility vs those who did not.This population-based cohort study was completed in Ontario, Canada, from 2002 to 2019, where there is universal health care. Included were all nulliparous women with a first-time singleton birth at 20 to 42 weeks’ gestation, each residing in a low-income urban neighborhood at the time of the first birth. All women were then assessed at their second birth. Statistical analysis was conducted from August 2022 to April 2023.Movement from a lowest-income quintile (Q1) neighborhood to any higher-income quintile neighborhood (Q2-Q5) between the first and second birth.The maternal outcome was severe maternal morbidity or mortality (SMM-M) at the second birth hospitalization or up to 42 days post partum. The primary perinatal outcome was severe neonatal morbidity or mortality (SNM-M) within 27 days of the second birth. Relative risks (aRR) and absolute risk differences (aARD) were estimated by adjusting for maternal and infant characteristics.A total of 42 208 (44.1%) women  experienced upward area-level income mobility, and 53 409 (55.9%) women  remained in income Q1 between births. Relative to women who remained in income Q1 between births, those with upward mobility had a lower associated risk of SMM-M (12.0 vs 13.3 per 1000 births), with an aRR of 0.86  and aARD of −2.09 per 1000 . Likewise, their newborns experienced lower respective rates of SNM-M (48.0 vs 50.9 per 1000 live births), with an aRR of 0.91  and aARD of −4.7 per 1000 .In this cohort study of nulliparous women living in low-income areas, those who moved to a higher-income area between births experienced less morbidity and death in their second pregnancy, as did their newborns, compared with those who remained in low-income areas between births. Research is needed to determine whether financial incentives or enhancement of neighborhood factors can reduce adverse maternal and perinatal outcomes. Moreover, they tended to compare women of low- and high-income groups,16 rather than being confined to women living in low-income areas, namely, those at highest risk for maternal and neonatal morbidity and death. Existing research also largely focused on income mobility between a woman’s early life and that measured in adulthood,19 rather than a change in her income position exclusively within her reproductive years, which may provide insight on the mutability of risk between pregnancies, while offering a key period for prevention and intervention. Identifying women and newborns at higher risk of morbidity cannot only impact their health trajectories, including their risk of disability and death, but has well-defined economic, emotional, and social consequences for their families and the local health care system.20Neighborhood income characterizes the economic profile of a local geographic area. Prior research on neighborhood income and adverse pregnancy outcomes generally focused on indirect proxies of newborn risk, such as preterm birth, small or large-for-gestational-age birth weight, and a low Apgar score,21 Findings from our other studies suggest that immigrant women and their newborns have an overall lower risk of severe maternal morbidity or maternal mortality and severe neonatal morbidity and neonatal mortality, respectively, compared with Canadian-born women and their newborns, all of whom were residing in low-income neighborhoods.23 In the current study, comprising mothers living in low-income urban neighborhoods, we evaluated their subsequent risk of adverse maternal and newborn outcomes, while comparing those who achieved upward area-level income mobility between 2 consecutive births vs those who did not experience any upward income mobility.The current study builds on previous research in which we found that women residing in the lowest-income neighborhoods are at highest risk of having a newborn with concomitant preterm and severe small-for-gestational-age birth weight.This retrospective population-based cohort study was conducted in Ontario, Canada’s most populous and ethnically diverse province, with universal health care insurance available to all permanent residents. Included were all nulliparous women who had 2 consecutive singleton hospital-based live births or stillbirths at 20 to 42 weeks’ gestation. The study was restricted to those residing in an urban low-income quintile (Q1) neighborhood at the time of the first birth, aged 15 to 50 years, and who had a valid Ontario Health Insurance Plan number at each birth hospitalizations  reporting guideline. Ethics approval was received from the Health Sciences Research Ethics Board at the University of Toronto.This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  urban area at the second birth, which was compared with remaining in an urban income Q1 neighborhood at both births  a Q1 to Q2 or Q3 neighborhood, (2) a Q1 to Q4 or Q5 neighborhood, each relative to (3) remaining in a Q1 neighborhood at both births.35 The SMM measure was developed and validated by the Canadian Perinatal Health Surveillance System,36 and includes 40 unique indicators using the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, Canada (ICD-10-CA) diagnostic codes37 and the Canadian Classification of Health Interventions (CCI)38 procedural codes. Examples of SMM indicators are maternal ICU admission, severe preeclampsia, and use of assisted ventilation  or all-cause maternal mortality (SMM-M) in the second pregnancy. SMM is a near-miss proxy that is associated with maternal death and prolonged hospital length of stay.39 SNM-M was based on the English-version Neonatal Adverse Outcomes Indicator, a validated measure consisting of 23 components, 16 neonatal diagnoses and 7 procedures that relate to different body systems,39 and recorded using ICD-10-CA diagnostic codes and CCI procedural codes. Examples of SNM-M indicators include neonatal death, newborn seizure, newborn resuscitation, and ventilatory support  live birth or stillbirth arising before 37 weeks’ gestation, again, occurring in the second pregnancy.The primary newborn outcome was a composite of severe neonatal morbidity (SNM) or all-cause neonatal mortality (SNM-M) in the second pregnancy. SNM is a near-miss proxy that is associated with neonatal death and hospital readmission among infants in their first year of life.40 The top 20 most common SMM and SNM indicators in the second pregnancy were also presented, irrespective of neighborhood income mobility between pregnancies.Means and proportions were assessed using standardized differences, contrasting women with any upward mobility vs no upward mobility. A standardized difference value of >0.10 was considered to be clinically important.41 to estimate relative risks (RR) and absolute risk differences (ARD). This approach is based on marginal probabilities of the outcome of interest, called population-average (mean) probabilities of outcomes for exposed and unexposed study participants. The 95% CIs of RR and ARD were estimated by bootstrap method with resampling 1500 replicates.41 The same modeling approach was also used to assess each outcome by the degree of upward mobility, namely, from Q1 to Q2 or Q3, or Q1 to Q4 or Q5, each relative to no upward mobility between pregnancies. All RRs and ARDs were adjusted for maternal age at the second birth hospitalization ; birth interval ; maternal world region of origin 43; prepregnancy hypertension within 1 to 365 days before the second birth hospitalization; prepregnancy diabetes within 1 to 365 days before the second birth hospitalization and gestational diabetes identified at the second birth hospitalization. For the outcome of SNM-M, RRs and ARDs were further adjusted for any structural congenital anomaly diagnosed in the second infant’s birth hospitalization.For each binary study outcome, women with any upward mobility were compared with those with no upward mobility, using an adapted approach to logistic regression analysis by Austin45 Accordingly, an additional analysis was completed for the corresponding main model of any upward income mobility and SMM-M or SNM-M. Therein, the interaction term of “neighborhood income mobility between births and immigrant status” was added to each respective model. Even so, it was decided, a priori, that adjusted RRs (aRRs) and adjusted ARDs (aARD) would be shown for these 2 groups, irrespective of the statistical significance of the interaction terms.Immigrants may have a different health trajectory and pregnancy outcomes than nonimmigrants.P < .05, and all tests were 2-tailed.Statistical analysis was conducted from August 2022 to April 2023. All analyses were performed using SAS version 9.4 (SAS Institute Inc). Statistical significance was set at There were 1 403 386 births during the study period. Of these, the final cohort comprised 95 617 nulliparous women residing in an urban low-income Q1 neighborhood at their first birth, and who had a subsequent birth during the study period , had a longer time interval between births , and were more likely to be nonimmigrants (28 299 participants [67%] vs 33 283 participants 62.3%) . AdditioAmong the top 20 most prevalent SMM indicators in the second pregnancy, postpartum hemorrhage with red cell transfusion or procedures to the uterus or hysterectomy was most the common (3.2 per 1000 births)  (eTable 3 in The rate of PTB in the second pregnancy was lower among women with any upward income mobility between births (57.2 per 1000 births) than those who remained in an income Q1 area (61.0 per 1000 births), with a corresponding aRR of 0.95  and aARD of −2.9 cases per 1000 births  (eTable 4 in There was no significant interaction detected between “neighborhood income mobility between births and immigrant status” and the risk of SMM-M (footnote of eTable 6 in For the outcome of SNM-M, there was no significant interaction between “neighborhood income mobility between births and immigrant status” (footnote of eTable 7 in In this large, population-based cohort study of nulliparous Canadian women initially residing in low-income urban areas, changing to any higher residential income neighborhood between first and second births was associated with a lower risk of SMM-M and SNM-M. For SMM-M, this apparent inverse association was consistent for upward movement to any higher-income neighborhood, while for SNM-M, it was most pronounced by a greater degree of upward mobility.48 The current findings align with prior US research on PTB.49 A matched-sibling design in California found that women with upward mobility from a very disadvantaged neighborhood between 2 consecutive live births had a reduced odds of PTB, but not small-for-gestational-age birth weight.49 Another study from California observed a lower risk of PTB among White and Latina women with upward income mobility between early life and adulthood, but not in Black women.19 African American women who achieved upward mobility from early-life residence in poor Chicago neighborhoods did experience a significant reduction in their risk of PTB, however,17 along with a lower risk of small-for-gestational-age birth weight.18 As in the current study, the greatest benefit was seen in newborns whose mother had moved to the highest income neighborhoods.18Few studies have examined neighborhood income mobility in relation to pregnancy outcomes and there is no prior research on SMM-M and SNM-M, which are important public health indicators of maternal and neonatal health and quality of health care.51 Aside from neighborhood factors that may alter the risk of SMM-M, SNM-M, or PTB are those of the woman herself, namely, educational attainment, health literacy, assertiveness, and both mental and physical resilience. Women who tend to move to higher-income areas may possess such attributes, compared with those who remain within a low-income area, and thus, achieve a better health state at the time of their second pregnancy.52Moving from a low- to higher-income neighborhood may be positively associated with maternal health. More affluent neighborhoods tend to have lower crime rates, more safe green spaces, reliable housing, and better access to healthier foods, education, and health services. Such neighborhoods may also introduce fewer chronic stressors, and generally have higher air and water quality, with reduced vehicular traffic.54 However, upward social mobility may lessen the accumulation of such adverse exposures,55 and potentially introduce protective exposures for the mother-to-be. Likewise, a fetus starts off with a 42-week maximum gestational window during which it, too, may be positively influenced by its mother’s place of residence, and a more optimal environment for in utero development.56 Future research might evaluate whether downward neighborhood income mobility between pregnancies, contrasted with continued residence in a higher-income area, is associated with adverse birth outcomes.58A consistent and pronounced association with upward mobility was seen for the outcomes of SMM-M and SNM-M. A cumulative pathway life course model suggests that the longer a person is exposed to socioeconomic challenges, the greater their accumulation of poor physical, psychological and cognitive attributes, which may further affect reproductive health.59 A future study might also investigate whether improving the infrastructure and available services within low-income areas, akin to those found in higher-income neighborhoods, is associated with better pregnancy outcomes for women and infants.32 Such a study could include community benefits agreements that aim to prevent mere gentrification, while ensuring that neighborhood improvements are beneficial to the community as a whole, as well as to adjacent areas.60Certainly, greater understanding is needed about the causal pathways that might explain why upward neighborhood income mobility appears to be associated with a lower risk of SMM-M, SNM-M, and PTB. Details should include neighborhood characteristics, particularly local access to high-quality perinatal health care and follow-up care, primary reason for moving, as well as maternal characteristics, behaviors, and experiences that promote or constrain achieving a healthy pregnancy. To overcome some limitations arising from observational study designs, an interventional study using neighborhood-level randomization may be warranted; this can reduce selection bias and unmeasured confounding. Such an approach has been successful in prior research aimed at reducing the risk of obesity and diabetes.61 and SMM and SNM outcomes were based on validated measures routinely captured within hospital administrative data. The SMM and SNM composites consist of a broad range of conditions and procedures, which minimizes the risk of under-ascertainment of each outcome.39 We also accounted for several important individual-level factors that may confound the relation between neighborhood income mobility and each study outcome, including the interpregnancy birth interval.This large population-based cohort study was conducted in a province with a high degree of ethnic diversity and immigration, and a universal health care system. Validated data sets captured nearly all hospital births during the study period,62; however, details of individual-level income were unavailable herein. Women lacking an up-to-date residential address may have been incorrectly assigned a neighborhood income Q; yet, this was unlikely to be the case, as Ontario Health Insurance Plan activity is updated at every antenatal and hospital delivery episode of care. Also, we did not capture if a woman moved more than once between pregnancies in our study, since her residential postal code at the index hospitalization for the first and second deliveries births were used to determine neighborhood income quintile at each birth.Lack of information on early maternal life exposures, race, employment, cigarette or substance use, body mass index, miscarriage, or quality of care could potentially lead to residual confounding. If a woman remained in neighborhood income Q1 during both of her pregnancies, it was not known if she remained within the same domicile. Neighborhood income quartile offers a measure of area- and individual-level income status63 Cohort entry also required a woman to have 2 births, which likely introduced selection bias, in that anyone who died after her first birth, underwent a hysterectomy, or who simply did not achieve a second pregnancy would have had a different trajectory after her first index delivery. As only women initially living in low-income urban areas were studied, these findings may not be generalizable to those living in low-income rural areas, where there may be less access to health care services.65As only singleton births were included herein, the current findings may not apply to women with a multifetal pregnancy, whose risk of SMM and SNM is higher.In this cohort of universally insured nulliparous Canadian women residing in low-income urban neighborhoods, mobility to a higher-income area between births was associated with a lower risk of SMM-M and SNM-M. Among women living in low-income areas, research is needed to determine whether financial incentives can improve pregnancy outcomes, or whether enhancement of neighborhood factors may reduce adverse maternal and perinatal outcomes.


The study of mild cognitive impairment (MCI) is critical to understand the underlying processes of cognitive decline in Parkinson’s disease (PD). Functional connectivity (FC) disruptions in PD-MCI patients have been observed in several networks. However, the functional and cognitive changes associated with the disruptions observed in these networks are still unclear. Using a data-driven methodology based on independent component analysis, we examined differences in FC RSNs among PD-MCI, PD cognitively normal patients (PD-CN) and healthy controls (HC) and studied their associations with cognitive and motor variables. A significant difference was found between PD-MCI vs PD-CN and HC in a FC-trait comprising sensorimotor (SMN), dorsal attention (DAN), ventral attention (VAN) and frontoparietal (FPN) networks. This FC-trait was associated with working memory, memory and the UPDRS motor scale. SMN involvement in verbal memory recall may be related with the FC-trait correlation with memory deficits. Meanwhile, working memory impairment may be reflected in the DAN, VAN and FPN interconnectivity disruptions with the SMN. Furthermore, interactions between the SMN and the DAN, VAN and FPN network reflect the intertwined decline of motor and cognitive abilities in PD-MCI. Our findings suggest that the memory impairments observed in PD-MCI are associated with reduced FC within the SMN and between SMN and attention networks. Mild cognitive impairment (MCI) is highly prevalent in PD (PD-MCI) 2 and is known to be a risk factor for PD with dementia3. Yet, PD-MCI is a rather heterogeneous condition, encompassing several subtypes of cognitive decline, thought to confer different risks of progression to dementia4. This heterogeneity reflects the diverse neuropathological processes of PD-MCI5 which have been most consistently associated with the progressive accumulation of α-synuclein, amyloid-β and pathologic tau species, particularly in frontal, temporal and cingulate areas6.Parkinson’s disease (PD) is currently viewed as a complex neurodegenerative disease that involves several cerebral areas and neurotransmitter systems. Although motor symptoms are the hallmark of clinical diagnosis, non-motor symptoms play a prominent role in the course of the disease. For many patients, the most disruptive non-motor manifestations of PD are cognitive impairment and dementia, which reach a long-term prevalence of up to 80%7. However, these two profiles do not always capture the heterogeneous cognitive deterioration of PD-MCI. Instead, a range of cognitive impairments arises from the progressive and heterogeneous involvement of distinct neural networks, modulated by dopaminergic and cholinergic neurotransmitter systems8.PD research has led to the characterization of two cognitive deterioration profiles: a fronto-executive dysfunction mainly driven by dopaminergic loss and manifesting as deficits in flexibility, planning, working memory and reinforcement learning; and a cholinergic cortical dysfunction leading to memory deficits and dementia 9. Here, we use the seven major RSNs identified in Yeo et al.10 namely the visual (VN), sensorimotor (SMN), dorsal attention (DAN), ventral attention (VAN), limbic (LN), fronto-parietal (FPN) and default mode (DMN) networks, plus the striatum. As described below, four of these RSNs  have been implicated in the pathology seen in PD-MCI patients.A wide body of evidence supports the existence of a hierarchically organized resting state network (RSN) system in which association networks serve higher-level cognitive functions11, and its main hubs suffer from dopamine denervation in PD12. Meanwhile, attentional orienting is driven by both the ventral attention network (VAN)13 and the dorsal attention network (DAN)14, which have also been associated with PD-MCI17. The orienting attentional system is associated with the cholinergic neurons of the basal forebrain, and the cholinergic neurotransmitter loss present in PD-MCI subjects has been linked to attention deficits in this system18. Finally, the SMN mainly serves primary motor functions, although it also coordinates with other cognitive networks. Although the role of the SMN in PD-MCI is an ongoing discussion. On one side, SMN disruption could be a reflection of motor impairments in PD-MCI patients, which is reflected in their scores in the "movement disorders society unified Parkinson’s disease scale” (MDS-UPDRS)19. On the other side, SMN disruptions have been related to impaired sensory integration for motor function in PD20. The SMN has also been associated with the fronto-executive dysfunction cognitive profile observed in PD, mainly impacting the FPN. For example, the SMN plays a crucial role in verbal short-term memory, coordinating with the fronto-temporal areas21. More evidence of the SMN cognitive role arises from disruptions in this network in association with cognitive impairment in PD and even in Alzheimer´s disease (AD)24.The FPN is a critical component in executive function and working memory25. Methodological approaches to the analysis of RSFC have employed RS functional magnetic resonance imaging (fMRI). Studies of RSFC of the diseased brain have adopted a variety of methods, however, most attempt to determine whether there is an increase  or a decrease  within or between RSNs. Such studies are improving our understanding of the functional brain changes underlying PD-MCI. Conventional methods for analyzing data in RSFC fMRI studies cannot effectively capture and differentiate the shared underlying factors or conflicting processes that arise from distinct functional patterns observed in the brains of healthy and diseased individuals, using a data-driven approach. In the present work, we sought to use a recently implemented methodology that is ideal for examining the interaction between multiple brain systems or networks at the FC level. These FC patterns  represent different functional mechanisms between networks. Afterwards, we associate those between-network FC-trait features with the cognitive performance tests used for proper diagnosis and cognitive subtype classification in PD-MCI in line with Movements Disorders Society (MDS) criteria level II26. Although few studies have used MDS criteria level II27, it is thought that these criteria give better diagnostic sensitivity and specificity than criteria level I26. This is why in the present work we use criteria level I to infer which MCI cognitive domain is associated with the FC-traits. The data-driven methodology that we used here is termed ConnICA, which implements Independent Component Analysis (ICA) to extract robust independent FC-traits from a set of individual FC matrices29. An FC-trait represents a FC pattern between resting state networks in our FC matrices with a possible role in cognitive, motor, or clinical impairments.Here, we focus on alterations in the RS functional connectivity (FC) between these RSNs in PD-MCI. Resting state functional connectivity (RSFC) can be defined as a significant temporal correlation between functionally related brain regions in the absence of any stimulus or taskconnICA in PD-MCI patients, cognitively normal PD patients (PD-CN) and healthy controls (HC), to compare and disentangle key FC-traits that are altered in PD-MCI. Furthermore, we expect these specific connectivity patterns to be associated with performance on attention, executive function, memory, language, and visuospatial tasks.We hypothesize that cognitive function deficits in PD-MCI would be driven by altered patterns of inter-connectivity between the brain networks. Specifically, the FC in networks such as the SMN, FPN, DAN and VAN would reflect such cognitive decline and would, therefore, capture the FC differences between PD-MCI and PD-CN. Here, we aim to extract independent FC-traits by applying P = 0.017) and HC (P = 0.018), had fewer years of education than PD-CN (P = 0.003) and HC (P = 0.001) and higher scores in the MDS-UPDRS-III than PD-CN patients (P = 0.028). Cognitive assessments showed PD-MCI patients presented a multidomain deficit with higher deficits in attention and working memory, executive function, and memory domains than in language and visuospatial abilities  (23 PD-MCI and 19 PD-CN) and 21 HC  remained from the 71 initial subjects after data quality control for motion or artifacts in the resting-state fMRI images. Table Probabilistic-ICA was carried out extracting 65 independent FC-traits and their corresponding subject-specific connICA weights. Among these, only one FC-trait showed a statistically significant group effect Table . Post-hoThe FC-trait showed a large difference between PD-MCI and both PD-CN and HC Table . As depi29, and applying ConnICA28.The present study aimed to investigate FC changes in PD-MCI that occur in the major RSN. With the present approach we have been able to identify the characteristic differences of brain connectivity between PD-MCI and cognitively normal groups (HC and PD-CN). Using the connICA framework we were able to extract independent connectivity traits from a set of individual functional connectomes that represent interactions between multiple brain systems or networks at the FC level. Our findings revealed that PD-MCI patients exhibited a distinctive FC-trait characterized by functional coupling of the SMN with the DAN, VAN and FPN. We also linked the FC-trait to cognitive and behavioral tests used for proper diagnosis and cognitive subtype classification in PD-MCI in line with the MDS criteria level II. This FC-trait was associated with high-cognitive functions such as memory and working memory as well as motor function. This extraction was done in a completely data-driven approach by maximizing subjects’ identifiability31. Although the SMN is not considered to be an association network25, regions belonging to this network, such as the somatosensory cortex, are crucial for higher order processes 32. Indeed, the incidental memory test  and the delayed recall subtest of the RAVLT, both used to assess the memory cognitive domain, have a strong sensorimotor component. The ROCF asks subjects to replicate a previously observed figure, and the RAVLT is a verbal short-term memory test. Therefore, the correlation between our FC-trait and participants’ memory scores on these tests could reflect memory recall deficits related to SMN disruptions34.This distinctive FC-trait indicates that altered functional coupling of the SMN in coordination with the DAN, the VAN and the FPN may be a major indicator of PD-MCI. The differential FC pattern of this FC-trait was associated with attention and working memory, memory, and language tasks, suggesting cognitive deficits in multiple domains. Importantly, these were the domains that were most negatively affected in our PD-MCI population (Table S.1), reinforcing the relevance of this trait in the early phases of cognitive decline in PD. These results are consistent with previous studies showing that PD-MCI patients displayed disrupted inter-connectivity in the DAN or the SMN and connectivity reductions between the SMN and the cognitive control network37. FPN plays an important role in coordination with SMN in spatial working memory task performance38. Moreover, although the FPN is involved in both executive function and working memory, cognitive neuroscience studies suggest that PD mainly affects the frontoparietal areas involved in working memory whereas executive areas are preserved40. Likewise, the observed FC-trait was associated with the working memory tests corresponding to the attention and working memory domains but was not associated with tests examining executive function. The involvement of the DAN and VAN is also consistent with previous research findings. First, a negative connectivity between the SMN and DAN has been showed to adversely affect healthy subjects’ performance on tasks requiring attention and working memory41. Second, the DAN and VAN networks, in which cholinergic neurons play a crucial role, have been implicated in attention orienting tasks11. Both networks are typically compromised in PD-MCI and PD with dementia44.The attention and working memory assessments that also correlate with the observed FC-trait are representative of working memory46. The PD-MCI FC-trait found in the present study includes the DAN and posterior parietal areas linked to memory, language and visuospatial deficits related to the cholinergic system which, as indicated, is also affected in AD48. Finally, the FPN in PD-MCI was assumed to contribute to the dysexecutive cognitive profile49. However, some studies have also linked the FPN to memory deficits, more in line with its role in AD-MCI, where the FPN has been observed to be hypoactivated50. Notwithstanding, the main component of our FC-trait is the SMN, deterioration in which is exclusive to PD-MCI subjects since its origin comes from dopaminergic denervation resulting in executive function and working memory deficits51.Our FC-trait contains brain regions also found in MCI with AD patients. In AD-MCI the main affected brain areas include temporal, posterior parietal and hippocampus, which typically show reductions in FC or metabolic consumption52. Moreover, PD-MCI is associated with worse motor scores53, therefore this FC-trait could reflect an interaction between the motor impairments, represented by the SMN, and the cognitive deterioration, reflected in DAN, VAN and FPN. Hence, this FC-trait could be interpreted as an aberrant FC pattern between the SMN and the DAN, the VAN and the FPN in PD-MCI patients, reflecting motor and cognitive deficits related to cholinergic problems rather than dopaminergic depletion.Finally, MDS-UPDRS-III was also associated with this distinctive FC-trait. Given that PD patients were “ON medication” during the study, this relationship might be due to the motor progression of the disease, probably in aspects that show less improvement following dopaminergic treatment54. Nevertheless, motion in “OFF medication” PD patients undergoing fMRI has pervasive and confounding effects that it is preferable to avoid. Second, in the present work we used a static FC connectivity approach. Dynamic FC methods can be useful to obtain additional information such as dwell time or switching between networks55. Third, we analyzed data collected from two resting state runs with different temporal resolution , which can raise concerns regarding measurement-to-measurement repeatability. However, these runs were acquired in the same session on each subject, all other sequence parameters were matched as closely as possible. In addition, we applied two steps to minimize differences between each subject FC matrices: (1) The signal was filtered to keep the same spectral content. (2) we maximized the subject’s identifiability29 which eliminates the differences between the FC matrices of the two runs. In addition we performed a repeated measures ANOVA (rmANOVA) analysis, which accounts for double measurements as demonstrated Ge et al. 2017 where rmANOVA models differentiated intra- and inter-subject variation and computed group differences, and observed largely consistent spatial patterns of test–retest reliability between the human connectome project (HCP) and Genomics Superstruct Project (GSP) samples, despite their differences in scanning site and image protocols 58. Moreover, in this study the variation between sequences was minimized as images were obtained with the same MR scanner.Although this study has revealed new insights into FC patterns in PD-MCI through the application of an innovative analytic technique (connICA), some limitations should be noted. First, all patients were “ON medication”, which has been reported to modify RSFC59. The identification of these differential FC patterns contributes to improving our understanding of cognitive decline in PD and paves the way for further examinations FC differences underlying of the clinical and cognitive changes in subtypes of PD-MCI patients which could help identify risk of progression to dementia.In conclusion, using a data-driven methodology approach (connICA) that is optimal for examining FC interactions between RSN, we demonstrated that PD-MCI is associated with substantial RSFC changes in critical networks implicated in cognitive and motor deficits. A FC-trait was reliably extracted which reflected a distinctive functional connectivity between networks in the PD-MCI group comprising the SMN, DAN, VAN and FPN networks and was associated with cognitive performance on memory and working memory tasks, as well as with motor symptom severity. These results suggest that PD-MCI impairments can be induced by a progression of FC abnormalities present since the onset of the disease, which probably reflect dopaminergic deficits and other early events, as well as impairment in dorsal and ventral attention regions which are likely more related to cholinergic depletionSeventy-one right-handed participants including 20 PD-CN patients, 23 PD-MCI patients and 28 healthy controls (HC) were recruited at the Movement Disorders Unit at the Hospital Universitario Donostia .60. Exclusion criteria included history of head trauma, psychiatric or neurological disorders other than PD, other major medical comorbidities, alcohol or drug dependence or abuse, and being left-handed. PD with dementia was diagnosed according to the MDS Task Force criteria61, and patients fulfilling these criteria were excluded as well. All participants were screened for MRI compatibility according to standard procedures. Experimental procedures were explained to participants, and written informed consent was obtained prior to study participation, according to the Declaration of Helsinki. The study was approved by the Gipuzkoa Clinical Research Ethics Committee.PD was diagnosed according to the UK Brain Bank criteria26 when the following two criteria were fulfilled: (1) cognitive decline reported by either the patient or informant, or observed by the neurologist, that did not interfere significantly with the functional independence of the patient; (2) the patient scored more than 1.5 standard deviations below control values in at least two tests in the neuropsychological battery, either within a single cognitive domain or across different cognitive domains. Normative neuropsychological test values were taken from 32 healthy controls recruited among accompanying persons of PD patients. Z scores for the tests of each cognitive domain were calculated as follows: (test score—mean score of control sample)/(standard deviation of control sample). Z scores were used to diagnose PD-MCI. These were then averaged over both tests evaluating each domain to provide composite z scores that will be used latter for correlation analysis.Diagnosis of PD-MCI was made according to the MDS Task Force criteria (level II category)62 and MDS-UPDRS part III scales63 were used to evaluate motor features. As gait, freezing of gait and postural stability decline are often seen along with the cognitive decline, scores for these items from MDS-UPDRS III were evaluated separately 63, and then summed up in a new variable “MDS-UPDRS-III axial score”. The Hospital Anxiety and Depression Scale (HADS) was used to assess the presence of anxiety or depression symptoms and the 39-Item Parkinson’s Disease Questionnaire (PDQ-39) was used to evaluate health-related quality of life. Note that for HC subjects MDS-UPDRS-III, Hoehn and Yahr, and HADS scales were not acquired and therefore have a value of zero in all statistical analyses.The Hoehn and Yahr64. Executive function, comprising cognitive flexibility, visual scanning, motor function and verbal working memory, was measured by the trail making test B and the phonemic fluency test66. Memory, comprising the ability to retrieve items both immediately (short-term) and after a delay (long-term), was measured by the immediate recall subtest of the Rey–Osterrieth Complex Figure (ROCF) test33 for incidental memory, as well as the delayed recall subtest of the Rey Auditory Verbal Learning Test (RAVLT)34. Language, comprising semantic memory, lexical access and dysnomia, was measured using the semantic fluency and Boston naming tests67. Visuospatial function, comprising structural knowledge and space perception, was measured by the object decision and number location tests from the Visual Object and Space Perception Battery (VOSP)68.Neuropsychological evaluation was performed using the Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MOCA) for global cognition and a comprehensive neuropsychological battery including two validated tests for each of the five cognitive domains . Patients were under the first morning dose of antiparkinsonian medication during the MRI scanning session to minimize discomfort and movement2, 176 axial slices, voxel size = 1 × 1 × 1 mm3) and a T2-weighted Turbo Spin Echo . Two runs of T2*-weighted fMRI data were acquired during resting state, each with 10 min duration, with 1) a standard gradient-echo echo-planar imaging sequence (monoband)  and 2) a simultaneous multislice gradient-echo echo planar imaging sequence (multiband factor = 3) developed by the Center of Magnetic Resonance Research  . Single-band reference images were also collected before the multiband resting state acquisition for head motion realignment. During both acquisitions, participants were instructed to keep their eyes open and fixate on a white cross that they saw through a mirror located on the head coil, and not to think about anything specific. Field maps were also obtained to correct field distortions.The anatomical MRI images included a T1-weighted MPRAGE  = 7°, field of view (FoV) = 256 × 256 mmhttps://surfer.nmr.mgh.harvard.edu)69. Anatomical parcellations were aligned to the functional space using the single-band reference image. The T1-weighted images were also warped to the Montreal Neurological Institute (MNI) template MNI152_2009, and all the spatial transformations from the subject’s space to the MNI space were collapsed in a single spatial transformation. Resting-state fMRI (RS-fMRI) data was pre-processed using AFNI70. First, the volumes corresponding to the initial 10 s were removed to allow the signal to achieve steady state magnetization. Subsequently, the voxel time series were despiked to reduce large amplitude deviations and slice-time corrected. Inhomogeneities caused by magnetic susceptibility were then corrected using FUGUE (FSL) using the field map images. Next, the functional images were realigned to a base volume, which was the volume with the lowest head motion for the monoband datasets and the single-band reference image for the multiband datasets. Afterwards, a single simultaneous nuisance regression step was performed, the regressors were: 6th-order Legendre polynomials; Low-pass filtering with cutoff frequency of 0.25 Hz; Six realignment parameters plus their temporal derivatives; the first five principal components of voxels in deep white matter and the first five principal components of voxels in the lateral ventricles 71; the first five principal components of the brain's edge voxels72. The masks of white matter, lateral ventricles and edges of the brain were obtained based on the FreeSurfer tissue and brain segmentations, warped to the functional space73. In addition, scans potentially corrupted by artifacts were identified and censored when the Euclidean norm of the temporal derivative of realignment parameters (ENORM) was larger than 0.4 or the proportion of voxels adjusted in the despiking step exceeded 10%. Furthermore, time courses of the derivative of root mean square variance over voxels (DVARS) were also computed for each dataset74.Automated voxel based subcortical segmentation and cortical parcellation were extracted from the T1-weighted and T2-weighted images using the FreeSurfer image analysis software  whose data were included in all the analyses reported below. The ENORM and DVARS metrics after censoring did not differ statistically between the HC , PD-CN  and PD-MCI groups ; .Image quality was assessed using motion plots that included grayplots before and after nuisance regression considering the previously explained motion measures76 plus the 8 bilateral subcortical regions from the FreeSurfer brain parcellation77. This parcellation was chosen since we were interested in RSN such as SMN, DAN, VAN and FPN. The Schaefer functional has a one-to-one association between parcels and 17 large scale functional networks10. The Schaefer atlas is defined in the MNI volumetric space and was warped back to the subject’s functional space using the corresponding spatial MNI-to-functional spatial transformations, whereas the subcortical segmentations were computed in the native T1-weighted anatomical space and then also coregistered to the subject’s functional space.FC matrices Fig. A were ob28. ConnICA is two-step process , which quantifies the difference between the average within-subject FC similarity and the average between-subjects FC similarity in each session as a function of an increasing number of principal components. In this assessment, PCA was applied to the concatenated FC matrices for all K possible numbers of principal components; then the FC matrices were reconstructed. Finally, we selected the number of K principal components that maximized this differential identifiability; in our case, K = 65  was performed to reduce the dimensionality of the space spanned by the FC matrices. Since the two FC matrices computed per subject were obtained from fMRI data with two different sequences (monoband and multiband), we took this further step to ensure they were compatible and to maximize the identifiability of our participants´ FC matrices by finding the PCA reconstruction that best identified each individual’s FC matrix for both the multiband and monoband sequences 65 Fig. .Figure 328  An FC pattern representing different functional mechanisms (2) a vector of weights indicating how present the FC-trait is in each FC matrix of the input, which can be used to see group differences and correlations with cognitive and clinical test in statistical analyses.Next, we applied connICAOne-way ANOVAs were conducted to test for between-group  differences in demographic and clinical variables. Chi-square tests were used for the categorical variables, and Mann Whgitney tests were used for non-normally distributed variables. Significant differences in age and years of education were found for the PD-MCI group and, consequently, these were entered as covariates in all subsequent statistical analyses. To rule out any interactions between FC and gray-matter (GM) volume loss we ran Freesurfer’s mri_glmfit to check group differences in GM volume and cortical thickness. This analysis revealed no significant differences in the group comparisons .To examine group differences in the FC-traits, a rmANOVA was defined to explain the two weights for each FC-trait in each subject, with group  as the between-subjects factor, acquisition sequence  as a within-subject factor, and age, gender and TGM as covariates. Differences in the FC-traits between the groups were determined as statistically significant after controlling for multiple comparisons using the False Discovery Rate (FDR) for a q-value < 0.05. Simple effects post-hoc t-tests were then performed to examine the effects between pairs of groups.P < 0.05.In addition, regression one-way ANOVA analyses were performed to assess the association of the FC-traits, that reached significance in rmANOVA, with the neuropsychological and clinical evaluations. These one-way ANOVA models aimed to assess the weights of the FC-traits in terms of each of the following explanatory variables : z-scores for the 5 cognitive domains assessed, the MOCA, the MMSE, the MDS-UPDRS-III score and the MDS-UPDRS III axial score. They were compared against a baseline model with five variables of non-interest: age, type of MR sequence , average value of Euclidean norm of displacement parameters (avg. ENORM), total grey matter (TGM), and total intracranial volume (eTIV). F-statistics were computed for each explanatory variable separately in comparison with the five variables of non-interest, and statistical significance was set at Supplementary Table S1.