Despite a 53 % decline in under-five mortality (U5M) worldwide during the period of the Millennium Development Goals (MDGs), U5M remains a challenge. Under-five mortality decline in Ghana is slow and not parallel with the level of coverage of child health interventions. The interventions promoted to improve child survival include early initiation of breastfeeding, clean postnatal care, and skilled delivery. This study sought to assess the effectiveness of these interventions on U5M in Ghana.A quasi-experimental study was conducted using secondary data of the 2008 and 2014 Ghana Demographic and Health Surveys. Coarsened Exact Matching and logistic regression were done. The interventions assessed were iron intake, early initiation of breastfeeding, clean postnatal care, hygienic disposal of stool, antenatal care visits, skilled delivery, intermittent preventive treatment of malaria in pregnancy, and tetanus toxoid vaccine.There were 2,045 children under-five years and 40 (1.9 %) deaths in 2008. In 2014, the total number of children under-five years was 4,053, while deaths were 53(1.2 %). In 2014, children less than one month old formed 1.6 % of all children under-five years, but 47.8 % of those who died. Mothers who attended four or more antenatal care visits were 78.2 % in 2008 and 87.0 % in 2014. Coverage levels of improved sanitation and water connection in the home were among the lowest, with 11.6 % for improved sanitation and 7.3 % for water connection in the home in 2014. Fifty-eight (58), 1.4 %, of children received all the eight (8) interventions in 2014, and none of those who received all these interventions died. After controlling for potential confounders, clean postnatal care was associated with a 66% reduction in the average odds of death , while early initiation of breastfeeding was associated with a 62 % reduction in the average odds of death .Two  out of eight interventions were associated with a reduction in the average odds of death. Thus, a further decline in under-five mortality in Ghana will require an increase in the coverage levels of these two high-impact interventions.The online version contains supplementary material available at 10.1186/s12887-021-02934-3. Increasing usage of child health interventions has been advocated to achieve a rapid decline in mortality. However, the effectiveness of these interventions can be sub-optimal, and some interventions are associated with an increased risk of death of children.A quasi-experimental study design with coarsened exact matching was used to assess multiple interventions using secondary data.Only a few interventions were associated with a reduction in mortality.To achieve a further decline in mortality, coverage of early initiation of breastfeeding and clean postnatal care should be increased since their coverage levels are low.Attention should be paid to children at higher risk of dying, including those with preceding birth interval less than two years, neonates, multiple births, and children from polygamous homes.Under-five mortality U5M) remains a major public health problem despite a 53 % decline globally between 1990 and 2015 . Thus, iM remainsVariations in socio-demographic factors, including household, maternal, and child factors , 7, the While low coverage of interventions leads to low impact –14, the This was a quasi-experimental study design used to assess the effectiveness of eight  intervenThe study area was Ghana, a country in sub-Saharan Africa (SSA). There were ten  politicaRegarding the eating habits of Ghanaians, results from the literature are inconsistent. While Buxton  recordedThe study population was children under-five years old in the Ghana Demographic and Health Surveys (GDHS) data sets born between 2003 and 2014. The analyses were restricted to usual household members and last births. The last birth restriction was done because interventions including antenatal care visits, iron intake, and clean postnatal care were measured for only the last births. It would also reduce the effect of recall bias on the study results. Only usual household members were also used because some interventions were not measured for non-usual household members.Secondary data from the 2008 and 2014 GDHS were used. The data were collected by the Ghana Statistical Service (GSS). The GDHSs are complex household surveys, and the data were collected using a cross-sectional descriptive study design. Individuals were selected using a multistage cluster sampling technique . The dathttps://dhsprogram.com). The HR data sets were merged with the KR data sets so that household insecticide-treated nets/indoor residual spraying (ITN/IRS) information from the HR data set could be added to the KR data set. Household insecticide-treated nets/indoor residual spraying is an intervention and needed to be adjusted for in the analysis. The data sets of 2008 and 2014 were combined to obtain the final data set for the analysis.The Household Recode (HR) and Kids Recode (KR) files were used for this analysis. The KR file was the main data set, and it contained information about children under-five years old at the start of the data collection whose mothers were interviewed . The 20048 % (48 %) of observations had at least one missing value for the covariates to be adjusted for, and therefore, multiple imputation was done before the regression analyses were done. Imputation was done to preserve the sample size and representativeness of the data. Variables with missing data were birth interval (16 observations), number of co-wives (832 observations), mode of delivery of child (2 observations), child’s weight (19 observations), employment status of mother (13 observations), NHIS status (1 observation), maternal BMI , anaemia (204), height  and child given other milk within three days after birth (58). The information on the missing data is presented in Additional file The variables used in this analysis were socio-economic and demographic factors and interventions. Socio-economic factors were maternal and household variables, including maternal education, marital status, religion, ethnicity, household wealth, household size, number of children under-five years in a household, region, NHIS status, and place of residence. Demographic factors comprised child and maternal variables, including maternal age, child’s age and birth weight, birth interval, birth order, and multiple birth. Interventions were antenatal visit, IPT-p, iron intake, tetanus toxoid vaccine, skilled delivery, clean postnatal care, early initiation of breastfeeding, hygienic disposal of stool, water connection in the home, improved water source, improved sanitation, and ITN/IRS. The year of the survey was also included. The definition of variables is based on the 2014 GDHS report .Intermittent preventive treatment of malaria in pregnancy (IPT-p): Percent of pregnant women receiving 2 + doses of sulphadoxine-pyrimethamine/fansidar during pregnancy.Iron intake: Percent of pregnant women taking an iron supplement daily for at least 90 days.Tetanus toxoid vaccination: Percent of women who received two injections during the pregnancy of her last birth, or two or more injections (the last within 3 years of the last live birth), or three or more injections (the last within 5 years of the last birth), or four or more injections (the last within 10 years of the last live birth), or five or more injections at any time prior to the last birth.Skilled delivery: Percent of children born with a skilled attendant present, including a medical doctor, nurse/midwife, and community health officer/nurse.Clean postnatal care: Percent of neonates who received a preventive postnatal visit within 48 h of birth.Early initiation of breastfeeding: Percent of children who started breastfeeding within 1 h of birth.Hygienic disposal of stool: Percent of children’s stools that are disposed of safely. Stools are considered to be disposed of safely if: (1) the child always uses a toilet/latrine, (2) the feaces are thrown in the toilet/latrine, or (3) the feaces are buried in the yard.Antenatal care visit: Percentage of women with a birth in the last 5 years receiving antenatal care from a skilled provider for the most recent birth.Coarsened Exact Matching  was usedCEM puts observations into groups based on factors that influenced the decision to receive treatment. The groups were chosen by the first author to reflect clinically important categories and to be consistent with that in the GDHS reports. Observations in similar groups were matched, and treated and untreated individuals who did not have matches were deleted from the data set and the data analyzed as unmatched data. The covariates used in the matching process were variables that predicted intervention use from the literature review and were also associated with each intervention from the chi-square test done in this data analysis  by survival status (died or lived), and chi-squared test was used to assess the differences each for the years 2008 and 2014. Logistic regression was also fitted to assess factors associated with U5M after the multiple imputation using combined data of 2008 and 2014. The design features  of the GDHS were factored into all the analyses. Also, the difference in women’s population between the two time periods was accounted for in the analysis. De-normalized weights were used to account for the differences in the population of women in the reproductive age in the population at the time of each survey. The de-normalized weight was calculated as = ψ X ϕ15–49/ϕS15-49 . Where ψ reports , 34.Potential confounders adjusted for were those related to mortality and/or exposure from the conceptual framework/directed acyclic graphs (DAGs)  was the exposure, accounting for design features and controlling for potential confounders. Potential confounders adjusted for were those identified from the DAGs. Average treatment effects were reported as OR with 95 % CI and at a 2-tailed α level of 0.05 . AnalyseWhen logistic regression was fitted with the completed cases, the total number of observations was 5,224, and early initiation of breastfeeding, child’s age, multiple birth, birth interval, number of children under-five years old in the household, and polygamy (co-wives) were associated with U5M from the adjusted analysis.After multiple imputation, when maternal anaemia and Body Mass Index (BMI) were added, the sample size was reduced from 6,098 to 3,165 observations with very wide 95 % confidence intervals. The variable, water connection in the home, was omitted from the regression model. Body Mass Index and anaemia were not statistically significant, while household size, child’s age, multiple birth, maternal education, household indoor residual spraying and bed net ownership, and early initiation of breastfeeding were statistically significant. National Health Insurance Scheme status had borderline significance, p = 0.05. The confidence interval for multiple births ranged from 9.83 to 417.8.For the CEM analysis with completed cases, the sample size after matching for the regression analysis ranged from 4,914 to 6,043 observations. Only early initiation of breastfeeding was associated with U5M reduction from both the crude and adjusted analysis. Clean postnatal care and hygienic disposal of stool were significant from the crude analysis but not from the adjusted analysis.There were 2,045 children under-five years and 40 1.9 %) deaths in 2008. In 2014, the total number of children was 4,053, and deaths were 53 constituting 1.2 %  each intervention is targeted at and the quality of the implementation of the interventions. There is evidence of poor quality of skilled delivery contributing to lack of effect of skilled delivery on child mortality . DispariClostridium tetani, and therefore, low risk of neonatal tetanus, a resultant reduced risk of death from neonatal tetanus. Currently, Ghana is at the elimination state of neonatal tetanus, which means a lower risk of neonatal tetanus infection [Low incidence of disease that the intervention is targeted at could also explain the lack of statistical significance of the association between interventions and under-five mortality . With a nfection .With multiple interventions which have a direct and indirect effect on under-five mortality been implemented in the midst of socio-economic factors that also affect under-five mortality, Chowdhury  observedIn the midst of interventions, some socio-demographic factors remained associated with U5M. Children in households with three or more children under–five years were 81 % less likely to die than those with one or two children. Having more children under-five years could mean the mother will have more experience taking care of children, including identifying signs and symptoms of diseases. More children under-five years in a household have been associated with early care-seeking in Niger . ChildreHealth insurance membership offers financial access to healthcare and is associated with increased and timely healthcare-seeking –56. It cThe higher odds of death of younger children and multiple births have been documented . In thisAdditionally, children from mothers in polygamous marriages had twice higher odds of death. This finding is consistent with results reported in other studies , 59. RivLastly, children of birth intervals of less than two years also had twice higher odds of death compared to those of longer intervals. With shorter birth intervals, mothers might not have replenished nutrients used during the previous pregnancy resulting in under-nutrition . After dOn limitations, exact matching coarsened like other matching methods matches on measured variables  contained in the data set, unlike randomization that controls for measured and unmeasured potential confounders. However, balanced was achieved between the treated and untreated groups using the variables available in the data set. Additionally, potential confounders adjusted for in this multiple regression analysis comprised all measured potential confounders, and therefore, the effect of unobserved variables is ignorable.Also, 48 % of observations had at least one missing value. Multiple imputation was done to address the missing data. On the strengths of the study, nationally representative data were used, which had high response rates (over 90 %), and therefore, the study findings are generalizable. The pooling of the data also increased the power of the study and, thus, the validity of the results. The matching method that was used reduced model dependence and thus, increases the robustness of the analysis. Lastly, the restriction of the analysis to only the last births reduced the influence of recall bias on the study results.Of the eight (8) interventions assessed for effectiveness at reducing under-five mortality, two (2) showed effectiveness on mortality reduction. This, therefore, suggests that the scope and content of the current package of interventions targeted at reducing U5M will likely not achieve a rapid decline in mortality. At best, the mortality rate will be stagnant. To achieve a further decline in mortality, coverage of early initiation of breastfeeding and clean postnatal care should be increased. Further work should be done to understand the lack of association between some interventions, especially skilled delivery and U5M. Lastly, addressing issues affecting the health of children in polygamous homes and multiple births and improving National Health Insurance Scheme coverage could be beneficial.Additional file 1.Additional file 2.Additional file 3.Additional file 4.


Attention-Deficit/Hyperactivity Disorder (ADHD) and Autism Spectrum Disorder (ASD) are associated with motor impairments, with some children holding a comorbid diagnosis of Developmental Coordination Disorder (DCD). However, DCD is underdiagnosed in these populations and the volume abnormalities that contribute to explaining these motor impairments are poorly understood. In this study, motor abilities as measured by the Developmental Coordination Disorder Questionnaire (DCDQ) were compared between children with ADHD, children with ASD and typically developing (TD) children, aged 8–12 years old. Additionally, the association between the DCDQ scores  and regional volume abnormalities were explored in 6 regions of interest , within each group and across all participants. Children with ASD and children with ADHD showed impaired motor abilities in all the DCDQ-derived scores compared to TD children. Additionally, most children with ASD or ADHD had an indication or suspicion of DCD. Within the ASD group, coordination abilities were associated with the volume of the right medial frontal gyrus, and within the ADHD group, the total DCDQ score was associated with the volume of the right superior frontal gyrus. This study underlines the importance of routinely checking motor abilities in populations with ASD or ADHD in clinical practise and contributes to the understanding of structural abnormalities subtending motor impairments in these disorders. Attention-Deficit/Hyperactivity Disorder (ADHD) is defined by impairing levels of inattention, and/or hyperactivity-impulsivity . Autism The Developmental Coordination Disorder Questionnaire (DCDQ) is considered a valid screening tool for identifying children with possible DCD , 14 and At the structural level, voxel-based morphometry (VBM) studies have evidenced volumetric abnormalities in populations with ADHD and/or ASD in regions typically associated with motor performance, including the pre-central and post-central gyri and the inferior parietal cortex –19. A reOverall, there is limited research on the association between motor performance measures and brain volume in populations with ASD and/or ADHD . MahajanThe final sample of this study consisted of 22 children with ADHD, 16 children with ASD and 17 TD children aged 8–12 years old. The comparison of regional and total brain volumes between these groups of children is published elsewhere . ChildreParticipants were excluded if they had a history of prematurity, a neurological disorder, a genetic disease, a disabling somatic pathology, suffered from complications during labour with neonatal care unit hospitalisation, were left-handed, presented any contraindications to MRI or had a General Ability Index lower than 70 on the Wechsler Intelligence Scale for Children−4th Edition , 28. SevThe DCDQ-FC , 14 is a2, matrix size = 256 × 256 × 128 and voxel size = 0.94 × 0.94 × 1.2 mm3.Anatomical image acquisition was conducted using a Discovery MR750w 3.0T scanner  at UZ Brussel . Anatomhttp://www.neuro.uni-jena.de/cat12/CAT12-Manual.pdf), implemented in SPM12 (http://www.fil.ion.ucl.ac.uk/spm/) running on MATLAB . Image pre-processing and quality control is described elsewhere  = 0.32, p = 0.72].Imaging data were analysed using the Computational Anatomy Toolbox  and across all participants. Global scaling was used with total intracranial volume (TIV) and age was entered as a covariate. All analyses were conducted without IQ as a covariate .Following pre-processing and data quality control, individual subject smoothed grey matter volumes were entered into a second level analysis to estimate the correlation between the different DCDQ-FC scores and grey matter volume in the ROIs using a one-sample ovariate , 29. Masovariate . Within-p = 0.011) or the ADHD (p = 0.004) groups. Groups differed significantly on all the ADHD RS-IV scores . Total, Inattention and Hyperactivity scores were significantly higher in both the ADHD and the ASD groups compared to the TD group. These scores were not significantly different between the ADHD and the ASD groups. Additional demographic data analyses fractionating the ASD group in terms of the presence or absence of comorbid ADHD are reported in Sex was not significantly related to diagnosis and groups did not differ on age see . IQ was ps < 0.001). “Control During Movement,” “Fine Motor/Handwriting,” “General Coordination” and Total scores were significantly lower in both the ADHD and the ASD groups compared to the TD group  was still a significant when entering ADHD severity as a covariate in the ASD group contrast. However, the correlation between the DCDQ-FC Total score and GMV in the right post-central gyrus did not survived significance threshold within the ASD group.Importantly, the negative correlation between the DCDQ-FC General Coordination score and GMV in the right medial frontal gyrus within the ASD group at This study contributes to the investigation of motor impairments in ASD and ADHD. Children with ASD and children with ADHD exhibited impaired motor abilities in all the DCDQ-derived scores compared to TD children. Additionally, most children with ASD or ADHD had an indication or suspicion of DCD. Within the ASD group, coordination abilities were negatively associated with the volume of the right medial frontal gyrus, and within the ADHD group, the total DCDQ score was negatively associated with the volume of the right superior frontal gyrus. Across all participants, control during movement abilities were associated with GMV in the right superior frontal gyrus and coordination abilities were associated with GMV in the left inferior parietal cortex.Our results showed that an indication or suspicion of DCD was present in the majority of children with ASD and ADHD. Children with ASD exhibited lower scores in all the domains assessed by DCDQ-FC  when compared to TD children, hence evidencing an extensive motor impairment in ASD. Additionally, 81.25% of the participants with ASD had an indication of, or suspicion for DCD, similarly to Bhat . In a saAt the cerebral level, increased impairments in motor coordination were associated with increased GMV in the right medial frontal gyrus within the ASD group. The medial frontal cortex encompasses cortical areas within the median wall of the frontal lobe and has been associated with a wide range of functions including performance monitoring, action anticipation, response inhibition, egocentric spatial processing, and motor coordination –33. MediOn the other hand, increased motor impairments were associated with increased GMV in the right superior frontal gyrus within the ADHD group. The superior frontal gyrus is implicated in a variety of tasks including motor movement –37. In aOur findings should be interpreted in light of some limitations. Poor quality scans were excluded from our sample, decreasing the presence of artefacts but also reducing the size of our sample. Additionally, there was a high percentage of children in the ASD group who presented an ADHD comorbidity, consistent with recent comorbidity reports , but potFuture work should continue exploring the association between volumetric abnormalities and motor impairments in a larger sample of children with non-comorbid ASD, non-comorbid ADHD and children with both ASD and ADHD. Additionally, although the DCDQ is considered a reliable screening tool for detecting children who are at risk for DCD, recent evidence suggests that combining different sources of information when exploring motor abilities in children may be more informative. In particular, using tools such as the Movement Assessment Battery for Children M-ABC , 41 may In conclusion, this study contributes to the understanding of motor anomalies in ASD and ADHD, as assessed by the DCDQ. Our results evidence frequent motor impairments and a high risk for DCD in populations with ASD and ADHD, underlying the importance of routinely checking for their presence in patients with ASD or ADHD, as well as considering appropriate motor interventions including physical therapy in their treatment. Additionally, the results of this study showed that motor impairments in these populations are associated with volumetric abnormalities in different regions of the brain and contributes to understanding the pathophysiology of motor impairments in these disorders.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the ULB-Erasme University Hospital in Belgium . Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.AS participated in the conceptualization, data acquisition and analysis, and the writing of the article. TV participated in the conceptualization, data analysis, and writing of the article. PV, SB, PD, VD, GP, HR, LV, EW, and PP participated in the conceptualization, data acquisition, and the writing of the article. MS participated in the conceptualization and the writing of the article. ND participated in the conceptualization, data acquisition, supervision, and the writing of the article. IM participated in the conceptualization, data acquisition and analysis, supervision, project administration, funding acquisition, and writing of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study investigates the satisfaction and adequacy of citizens through the expected quality and perceived quality in the areas of planning and territorial viability, experience in the provision of municipal services and citizen experience in environmental issues, in order to provide tools for territorial decision making for the citizens’ well-being. In our research PLS software is used for the analysis of hypotheses. A questionnaire was delivered to a sample of 521 citizens, representing the spectrum of the population, and the statistical study of the responses yielded results on citizen satisfaction and loyalty. Our research includes the study of moderating effects on the causal ratio of perceived value and satisfaction in territorial planning and viability, the perceived quality in the provision of municipal services and the perceived quality in the citizen experience in the environmental management of the territory on the value relationship perceived by the citizen and general satisfaction. A second objective of the study is to see if there are significant differences in the hypotheses raised by gender by performing a multigroup analysis. This difference has been appreciated in two of the hypotheses. The study shows that the policies exercised by the territorial managers of the different areas have a significant influence on the value perceived by citizens, satisfaction and loyalty, which shape their general well-being. Areas for improvement in territorial policies and municipal services such as citizen security, air quality, public lighting and sports services have been identified. Knowing these shortcomings allows politicians to focus their efforts on improving the quality of life in cities. Territorial and environmental policies as well as public services in urban areas have become a decisive factor when it comes to attracting and arranging the population. Proper management of these policies is essential to achieve citizen satisfaction –3. It isThe correct planning of urban territorial policies, environment and municipal services promotes tourism, international sports activities and economic development , 11. TheThe citizen’s environmental experience is another important factor when deciding whether to establish residence in and recommend the city. Public institutions manage green spaces and the rules that regulate maximum noise levels (noise pollution), allocate funds for cleaning and green points for the selective collection of urban waste. These define what quality green space is , 18. TheFor a citizen to want to establish his residence in a metropolis, he considers the quality of life in it. The literature indicates that the larger the cities, the lower the level of satisfaction, due to insecurity and stress. Despite this, most people live in cities, due to the public services they offer in terms of quality and quantity. Rural areas are neglected in terms of basic services, their citizens having to move to the cities to satisfy medical, leisure, specialized supplies, etc .The literature indicates that in rich countries rural life is preferred, while in poor or developing countries life in urban areas is preferred  In generThe quality of life in urban areas is increasingly linked to political decisions about public services, the performance of municipal services has been one of the main concerns of public administrators and, therefore, has attracted considerable attention by the municipal administration. –26. In rWithin the public services we have to include the quality of public transport, urban security, public lighting, water quality, sewerage and sewerage, funeral services, fire fighting service, etc.; services that together make up the perceived quality of a reception comfortable and safe city where to establish residence , 32, 33.Our analysis is supported by an index used in satisfaction surveys around the world. The American Customer Satisfaction Index , 35.Our research brings a new perspective to the previous literature in relation to loyalty and citizen satisfaction in urban areas, since the expected quality is separated in territorial planning, municipal services and citizen’s environmental experience , 37. OncThere are various reasons why citizens decide to live in metropolises; despite sacrificing uses related to happiness and quality of life, such as air quality and environmental quality, to achieve other advantages such as professional satisfaction, culture, money and social mobility .The literature provides various studies that analyze these causal relationships, although it is true that few investigations are found that include modulating causal relationships between latent variables. The latent variables that were studied are citizen satisfaction and loyalty , 47. TheThe managers responsible for territorial policies in urban areas must have relevant information on the opinion of their citizens in order to plan actions that improve the expectations of potential new citizens attracted by them , 50, 51.The city of Guayaquil is an appropriate territory to carry out this research: it is a port city, which opens doors to the beaches of the Pacific and the Galapagos Islands; it is the largest city and the city with the highest population density in the Republic of Ecuador, with 2’698,077 inhabitants, which corresponds to 15.6% of the Ecuadorian population. It is considered as the economic capital of the country for the dynamism generated in commercial, industrial, services and agricultural activity, it also has the largest municipal infrastructure in the country, responsible for providing public services to the citizens through 27 administrative and operational areas and public companies that do the work of providing services under municipal jurisdiction.It is geographically divided into 16 urban and 5 rural areas. This research was carried out in the Tarqui urban parish, which houses 1’050,826 inhabitants, or 38.9% of the total population of Guayaquil and which is characterized by being a pole of urban development and greater economic growth, leading to a greater demand for quality public services.This research studies the satisfaction of urban citizens measured as well-being derived from the decisions made by territorial and municipal managers. It also analyzes the loyalty of citizens residing in urban areas, measured as the recommendation of the quality of public services managed by the administrations and their managers, as well as the recommendation of residents to other citizens, whether they are residents of urban areas or residents of other urban areas. In this way satisfied and loyal citizens contribute to the fight against the depopulation of vast urban areas, a problem that greatly worries public administrations. This study is also innovative because it uses a model of structured equations with moderating variables. That is, given the different values taken by the variables of perceived quality , how the direct relationship moderate between the perceived value of citizens in municipal services and citizen satisfaction measured as their well-being.The variables used to measure the fidelity of the citizens residing in Guayaquil, 1) expected quality territorial and road planning (EQTPR) 2) the expected quality provision of municipal services (EQMS), 3) expected quality experience of the environmental citizen (EQEEC), 4) perceived quality territorial planning and road (PQTPR), 5) perceived quality provision of municipal services (PQMS), 6) perceived quality experience of the environmental citizen (PQEEC), 7) the perceived value of the citizen (PV), 8) satisfaction (SATISFAC) and 9) loyalty (LOYALTY).The theoretical hypotheses  are raisHypothesis 1 (H1). The expectation of citizens in urban areas regarding the experience in territorial and road planning (EQTPR) influences directly and significantly the perceived quality of the city’s territorial and road planning experience (PQTPR).Hypothesis 2 (H2). The expectation of the experience of providing municipal services in urban areas (EQMS) influences directly and significantly the perceived quality of the experience of providing municipal services in the city (PQMS).Hypothesis 3 (H3). The expectations of the environmental experience of the citizen of urban areas (EQCE) influences directly and significantly the perceived quality of the environmental experience of the citizen in the city (PQCE).Hypothesis 4 (H4). The perceived quality of territorial and road planning in urban areas (PQTPR) influences directly and significantly the overall perceived value of the city (PV).Hypothesis 5 (H5). The quality of municipal service provision in urban areas (PQMS) influences directly and significantly the overall perceived value of the city (PV).Hypothesis 6 (H6). The perceived quality of the environmental experience of citizens in urban areas (PQCE) influences directly and significantly the general perceived value of the city (PV).Hypothesis 7 (H7). The perceived quality of the territorial planning experience in urban areas (PQTPR) directly and significantly modulates the direct relationship between the perceived value of the urban municipality (PV) and citizen satisfaction (SATISFAC).Hypothesis 8 (H8). The perceived quality of the experience in the provision of municipal services in urban areas (PQMS) directly and significantly modulates the direct relationship between the perceived value of the city (PV) and citizen satisfaction (SATISFAC).Hypothesis 9 (H9). The perceived quality of the citizen’s environmental experience in urban areas (PQCE) directly and significantly modulates the direct relationship between the perceived value of the citizen (PV) and the general satisfaction of the citizen in the city. (SATISFAC).Hypothesis 10 (H10). The global value perceived by the citizen in urban areas (PV) influences directly and significantly the general satisfaction of the citizen. (SATISFAC).Hypothesis 11 (H11). General citizen satisfaction in the urban municipal service (SATISFAC) influences directly and significantly the general loyalty of the citizen (LOYALTY).This research is aimed at establishing the causal relationships between latent variables based on a study of the expected quality with respect to the perceived quality, perceived value, level of satisfaction and loyalty of municipal public services in the urban sector Tarqui, characterized by being the largest in terms of territorial extension, as well as population density and economic growth of the city of Guayaquil.The data were obtained from an opinion, containing 80 questions, classified into 9 constructs, conducted at the household level. The universe of study is formed by 1′050.826 inhabitants that corresponds to the 38.9% of the total population of Guayaquil, and considering limitations of time and resources, we calculated a simple random sample representative of 528 households with a margin of error of 5% and level of confidence of 95%, achieving favorable responses of 521 families, surveying a representative from each household.The data were collected by a group of qualified interviewers from October 5 to 11, 2020, obtaining relevant sociodemographic information, as well as the levels of quality expected (expectation), perceived, satisfaction and loyalty of the respondents in each item contained in the questionnaire, implementing a Likert scale ranging from one to seven for each proposed statement .The sample covers the entire spectrum of age, level of education, professional category and income level . The valThe questionnaire consists of five sections:Section 1: Experience in territorial planning and road planning. Questions about Zoning and urban planning, Roads and pavements, Traffic organization and crossing, Public Transport Service, Parking services, Address information, Transport terminal services, Global satisfaction in the experience of territorial planning and road.Section 2: Experience in the provision of municipal services. Questions about Quality/Price Ratio, Drinking Water Quality, Wastewater and Sewerage Services, Garbage Collection and Environmental Cleaning Service, Parks and Gardens, Street and Road Lighting, Preservation of Historical and Cultural Structures, Cultural Activities, Social and Cultural Facilities, Cemetery Services, Social Activities, Firefighting Services, Municipal Police Services, Sports Services, Veterinary Services, Global Satisfaction in the experience of the municipal service provision.Section 3: Environmental experience of the citizen. Questions about Noise Minimization, Air Pollution, Green Areas, Recycling Points, Global Satisfaction in the User’s Environmental Experience.Section 4: Loyalty of the citizen. Questions about whether they would recommend the services provided by the Municipality of Guayaquil, if they would recommend to family or friends who live outside the city to move to live in Guayaquil, if they would recommend to family or friends who live outside the city to do tourism in Guayaquil, if they would recommend my friends or family to vote for the mayor of Guayaquil, if they would vote again for the mayor of Guayaquil.Section 5: Sociodemographic. Profile, Gender, age, level of education, professional category, level of family income.At all times, respondents were informed about the academic purpose of the study, in the same way that their responses would be anonymous. Before starting the questions, they were asked permission for their answers to be part of the study. To carry out the research, questions with a Likert scale were set. The survey haved 80 items, the sample data was collected at different times of the day. The survey was given to residents of Guayaquil. The sociodemographic profile was completed with closed questions. 521 valid questionnaires were obtained in the sample, which had a confidence level of 95% and a sampling error of 3.25%. The Warl PLS program has been used for the validation of the model. Once the methodology of the proposed structural equation model has been applied, results have been obtained that will be validated one by one.From the survey data, the sociodemographic profile has been calculated . 46.9% oThe proposed model was validated by studying the validity and reliability of the latent variables.The factor loadings and the limiting probability of the observed variables are shown in In order to study coliniality, the value of the variance of the inflation factor (VIF) was verified, whose value must be greater than five .The analysis of all these values validates the variables used, the factorial loads have values equal to or greater than 0.505 according to .The study of all these items does not show statistically significant loads at 99.99%, therefore we have considered the model valid and reliable.In order to measure whether the observed items strictly and appropriately measure the latent variable of which they are part, the composite reliability values as well as Cronbach’s Alpha are studied, verifying if they are greater than / equal to 0.7 .The convergent validity of a set of items is measured with the mean variance extracted (AVE) . FornellAfter having analyzed all the latent variables, considering their validity, as well as an adequate adjustment, it can be confirmed that the results obtained are reliable and justify their applicability.To finalize the hypothesis test, the factor loadings of each causal relationship were calculated .The hypotheses (H1), (H2), (H3), (H4), (H5), (H6), (H7), (H9), (H10) and (H11) were confirmed by the contract carried out. The factorial loads of the causal relationships, both direct and modulating, are shown in The satisfaction of citizens residing in urban areas is a determining factor for loyalty and is rooted in their territory. These urban citizens recommend and share their experience in this type of environment with family, friends and co-workers. In this way, the depopulation of urban areas can be fought with actions aimed at increasing the satisfaction of resident citizens in the various areas that the territory managers serve, in territorial planning and viability, in the experience as a user of municipal services and in the field of environmental policies carried out. The public administration has, among others, the objective of preserving urban areas, maintaining the sustainable value of these residential destinations that it manages and establishing and becoming attractive for new residents. In this way, the resources available to improve municipal services and policies to improve citizen satisfaction would be increased .The analysis of the fidelity of citizens in urban areas is important for the design and strategic planning of the territory. In this analysis, a model of structural equations was implemented in which modulating causal relationships were included on the relationship between satisfaction and perceived value. The Fidelity of the resident citizens in the urban residential destination of Guayaquil was analyzed. The results obtained in our analysis confirmed most of the causal relationships in the proposed model and can be used to improve the quality of life of citizens in urban areas. When citizens choose an urban population as a residential destination, once installed, they evaluate the gap between the expected quality of all areas of their population  and their perceived quality. To choose an area for residence, before setting up residence in a city, the citizen is informed about the quality of life in it, the services they offer and with all this information generates an idea of what life is like in the residential destination. If the perceived quality in your urban city is equal to or higher than the quality expected before choosing this destination to establish your residence, the perceived value in the set of municipal public services, and your experience as a user of territorial and environmental policies is high. This set of experiences, experiences and perceptions lead us to satisfaction measured as the well-being of the urban citizen, which leads us to recommend the urban area as a residential destination to other citizens , 77.Citizens of urban areas have very high expectations of public services, territorial planning and viability, as well as environmental experience, obtaining the maximum position in all measured dimensions. Once citizens have decided to establish their residence and have experienced, enjoyed and used the various public services, they perceive their quality. The main reasons why citizens of urban areas are satisfied and recommend family and friends to establish their residence in the urban area have been parking services, firefighting service, quality of drinking water, services wastewater and sewage, city cleaning and garbage collection, parks and gardens, preservation of historical and cultural monuments, cultural activities, social activities, sports services and air quality –81.Hypothesis 1. The quality expected in the territorial planning and the roads of the citizen residing in an urban area has a directly and significantly influence on their perceived quality of life. In Hypothesis 2. The expected quality of municipal services for citizens living in urban areas directly and significantly influences their perceived quality. Hypothesis 3. The expected quality of the user experience of the environmental citizen residing in urban areas has a directly and significantly influence on its perceived quality. Hypothesis 4. The perceived quality of territorial planning and the viability of the citizen residing in an urban Area directly and significantly influences the The perceived quality of territorial planning of the destination as a whole –89. Fig Hypothesis 5. Hypothesis 5. The perceived quality of municipal services by citizens living in urban areas has a directly and significantly influence on the perceived value of the destination as a whole –93. Fig Hypothesis 6. The perceived quality of the environmental experience by citizens living in urban areas has a directly and significantly influence on the perceived value –99. Fig Hypothesis 7 . The perHypothesis 8  This hypHypothesis 9  The percHypothesis 10 The value perceived by the resident citizen in an urban Area influences the satisfaction and well-being of the resident , 107. FiHypothesis 11 was also confirmed, which shows the direct influence of citizen satisfaction in the urban areas in which they establish their residence on their loyalty, recommending relatives, friends and acquaintances, whether they reside in a rural area or in a different urban area , 108–110This study outlines an achievable goal for public managers, who are in charge of territorial and viability planning, the municipal public services and the offer of public actions for an environmental experience of the citizen. If these elements improve, the experience of establishing their residence in an urban area can be maximized. This means that having satisfied urban citizens and well-being becomes one of the main actors in attracting and retaining people in urban areas. The results of this study are consistent with those found in previous researches that indicate that satisfaction positively influences the fidelity of citizens who establish their destination in an urban area and encourages them to recommend it. This study raises the most important factors to achieve the loyalty of the citizens of urban areas, generating positive synergies in the area, generating employment in various sectors, increasing demand by increasing the population that is installed in the urban destination.This study identifies several factors that citizens living in urban areas consider important when determining their residence in an urban destination. Firefighting services were the most valued. Urban spaces must be capable of combining municipal public policies that link the citizen with the metropolis. The brand created by this urban destination, together with the perceived quality of the different municipal service areas, are factors that can be used to attract people who want to settle in these places.The principal limitation of this study was related to the used sample with was obtained from the urban area of Guayaquil. It means this study is not generalizable to other populations. It would be desirable to study elsewhere where you can make comparisons of the results of the questionnaires. Another limitation is to carry out the study in a short period of time, and it would be convenient to do it periodically in order to analyze evolution. 16 Nov 2021 <div> PONE-D-21-28034 </div>  <div> Analysis of citizen satisfaction in urban areas </div>  <div> PLOS ONE <span style="color: rgb; font-family: verdana, geneva, arial, helvetica, sans-serif; font-size: 11.2px; background-color: rgb;"> Vergara-Romero </span> ,Dear Dr. Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. </div>  <div> Please consider all commentsplosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Dec 31 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at  </div> A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. 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If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. </font> The Reviewer #1: YesReviewer #2: Yes**********  <font color="black"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  <font color="black"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Dear authors,I read with interest your paper, although, after assuring the p-value and VIF were ok, I slightly "jumped" here and there the more technical parts trusting your academic reliability.https://www.sciencedirect.com/science/article/pii/S1877916621000059 would be of interest for your paper, especially for the introduction and conclusion when linking with the state of the art.I like the differences you mentioned between expected and perceived and I thought that some literature within this recent publication Your paper appears to me well structured even if some improvements in the theoretical parts related with quality of life in general, and the urban quality of life in particular would be beneficial.Reviewer #2: It is an interesting paper that focuses on the citizens’ satisfaction, the expected quality, and perceived quality. The method and results show credible. However, there are some questions that I think should be stated again.1. How is the state of arts of your research issue? There is no related works review.2. We have much research about citizens’ perception and satisfaction with urban management. What are your contributions, especially the theoretical contributions? Though you have shown in the section of the introduction, what’s the difference between yours and others?3. What’s the meaning of ‘experience in territorial and road planning’? Is it pointed to someone who has participated in the territorial and road planning of the urban?4. The part of ‘Multigroup Analysis’ seems no direct relationship with your research. While you just show the multigroup in gender, and how are other groups (education? Work? Income?) So, maybe you can give up this part.**********  <font color="black"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Please note that Supporting Information files do not need this step. </div> While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, AttachmentIt is an interesting paper that focuses on the citizens.docxSubmitted filename: Click here for additional data file. 9 Dec 2021Revision required [PONE-D-21-28034] - [EMID:847aab6a5cba99f6]Journal requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfhttps://journals.plos.org/plosone/s/submission-guidelines). In particular, the title should be "Specific, descriptive, concise, and comprehensible to readers outside the field" and in this case it is not informative and specific about your study's scope and methodology.2. Please consider changing the title so as to meet our title format requirement  psychology, utility and life satisfaction of urban living. It is really interesting and improves our manuscript.The following paragraphs have been introduced in the introduction and in the conclusions:Introduction: For a citizen to want to establish his residence in a metropolis, he considers the quality of life in it. The literature indicates that the larger the cities, the lower the level of satisfaction, due to insecurity and stress. Despite this, most people live in cities, due to the public services they offer in terms of quality and quantity. Rural areas are neglected in terms of basic services, their citizens having to move to the cities to satisfy medical, leisure, specialized supplies, etc [21].[21] L. S. D’Acci, “Preferring or Needing Cities? (Evolutionary) psychology, utility and life satisfaction of urban living,” City, Culture and Society, vol. 24, 2021, doi: 10.1016/j.ccs.2021.100375.The literature indicates that in rich countries rural life is preferred, while in poor or developing countries life in urban areas is preferred [22]. Citizens have the belief that all these wonderful things  attract them to the big cities without taking into account negative aspects  [23].[22] M. das Gupta, J. Zhenghua, L. Bohua, X. Zhenming, W. Chung, and B. Ha-Ok, “Why is son preference so persistent in East and South Asia? A cross-country study of China, India and the Republic of Korea,” The Journal of Development Studies, vol. 40, no. 2, pp. 153–187, 2003.[23] Kahneman, D., Kahneman, D., & Tversky, A. (2000). Experienced utility and objective happiness: A moment-based approach. 2000, 673-692.Conclusions: There are various reasons why citizens decide to live in metropolises; despite sacrificing uses related to happiness and quality of life, such as air quality and environmental quality, to achieve other advantages such as social mobility, money, culture and professional satisfaction [21].[21] L. S. D’Acci, “Preferring or Needing Cities? (Evolutionary) psychology, utility and life satisfaction of urban living,” City, Culture and Society, vol. 24, 2021, doi: 10.1016/j.ccs.2021.100375.Reviewer #2: It is an interesting paper that focuses on the citizens’ satisfaction, the expected quality, and perceived quality. The method and results show credible. However, there are some questions that I think should be stated again.1. How is the state of arts of your research issue? There is no related works review.The state of the art of this research focuses on the importance of the adequate provision of public services in urban areas analyzed from the causal relationship between expected quality and perceived quality, whose difference establishes a perceived value, in order to influence the level of citizen satisfaction, which generates loyalty or rootedness to the territory, as well as improvement in the quality of life. Thank you for this important remark. Next, we add reviews of related works.[7] S. P. M. Pereira and P. M. A. R. Correia, “Sustainability of portuguese courts: Citizen satisfaction and loyalty as key factors,” Sustainability (Switzerland), vol. 12, no. 23, 2020, doi: 10.3390/su122310163.[8] I. Almarashdeh, “The effect of recovery satisfaction on citizens loyalty perception: A case study of mobile government services,” International Journal of Electrical and Computer Engineering, vol. 10, no. 4, 2020, doi: 10.11591/ijece.v10i4.pp4279-4295.[9] A. Alkraiji and N. Ameen, “The impact of service quality, trust and satisfaction on young citizen loyalty towards government e-services,” Information Technology and People, 2021, doi: 10.1108/ITP-04-2020-0229.[16] X. mei Fu, J. hua Zhang, and F. T. S. Chan, “Determinants of loyalty to public transit: A model integrating Satisfaction-Loyalty Theory and Expectation-Confirmation Theory,” Transportation Research Part A: Policy and Practice, vol. 113, 2018, doi: 10.1016/j.tra.2018.05.012.[24] L. Ma, “Performance Management and Citizen Satisfaction With the Government: Evidence From Chinese Municipalities,” Public Administration, vol. 95, no. 1, pp. 39–59, 2017, doi: 10.1111/padm.12275.[32] S. Parnell and E. Pieterse, “The ‘right to the city’: Institutional imperatives of a developmental state,” International Journal of Urban and Regional Research, vol. 34, no. 1, pp. 146–162, 2010, doi: 10.1111/j.1468-2427.2010.00954.x.[50] G. van Ryzin, “The Measurement of Overall Citizen Satisfaction,” Public Performance & Management Review, vol. 27, no. 3, 2004, doi: 10.1080/15309576.2004.11051805.[51] M. H. Tammubua, “ANALISA PENGARUH SELF IMAGE CONGRUITY, RETAIL SERVICE QUALITY, DAN CUSTOMER PERCEIVED SERVICE QUALITY TERHADAP CUSTOMER LOYALTY YANG DIMEDIASI CUSTOMER SATISFACTION URBAN SURF/DISTRO DI JAYAPURA,” Jurnal Organisasi dan Manajemen, vol. 13, no. 2, 2017, doi: 10.33830/jom.v13i2.68.2017.2. We have much research about citizens’ perception and satisfaction with urban management. What are your contributions, especially the theoretical contributions? Though you have shown in the section of the introduction, what’s the difference between yours and others?Thank you for your comment. Although it is true that there are various studies of citizen satisfaction, most of them start from expectations and perceived quality in general, we have segmented into three dimensions this analysis of causal relationships between Expected Quality and Perceived Quality, which are: territorial planning and roads, Municipal services and environmental management in order to determine the level of satisfaction and loyalty independently based on constructs formed by items of the analyzed dimension, which show different coefficients between dimensions, which can be comparable and even generate future research. In this way, specific tools can be made available to public managers to improve the quality of life of citizens.Also, we propose an analysis based on moderating variables of the proposed causal relationships in order to know if there is a degree of influence by dimension of the constructs of perceived quality within the causal relationship that determines the degree of satisfaction.The results of this research by dimension generate theoretical contributions related to the importance of measuring the level of satisfaction and rootedness for each area that makes up public management, whose contributions will also be included in the conclusions as you rightly indicate.3. What’s the meaning of ‘experience in territorial and road planning’? Is it pointed to someone who has participated in the territorial and road planning of the urban?Thank you for your comment, theexperience in territorial and road planning, is the general result of the construct formed by a set of items that address issues related to the level of satisfaction in the provision of services inherent to territorial planning and roads. Some of the items that make up this construct are: Zoning and urban planning, Roads and pavements, Traffic organization, Public Transport Service, Parking services, Address information, Transport terminal services. The results of the set of items proposed for this construct in the citizen surveys generate a degree of general experience on the part of the citizens surveyed.4. The part of ‘Multigroup Analysis’ seems no direct relationship with your research. While you just show the multigroup in gender, and how are other groups (education? Work? Income?) So, maybe you can give up this part.Thank you very much for your support. In the multigroup analysis, we expected to find more significant differences between men and women.Following your recommendation, we have proceeded to delete the multi-group analysis paragraph.Attachmentresponse to Reviewer 2.docxSubmitted filename: Click here for additional data file. 17 Jan 2022Study of citizen satisfaction and loyalty in the urban area of guayaquil: perspective of the quality of public services applying structural equationsPONE-D-21-28034R1 <span style="color: rgb; font-family: verdana, geneva, arial, helvetica, sans-serif; font-size: 11.2px; background-color: rgb;"> Vergara-Romero </span> ,Dear Dr. We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Ahmed Mancy Mosa, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 26 Jan 2022PONE-D-21-28034R1 Study of citizen satisfaction and loyalty in the urban area of Guayaquil: perspective of the quality of public services applying structural equations Dear Dr. Vergara-Romero:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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CD35 polymorphisms and hepatocellular carcinoma (HCC). The current study was conducted to investigate the association between tag SNPs in CD35 and HCC susceptibility and postoperative recurrence, in an attempt to elucidate the gene-environment interactions in HCC. A total of 1233 Chinese Han people, including 647 healthy controls and 586 HCC cases, were sampled in this study. Six Tag SNPs  of CD35 were selected using the HaploView 4.2 program and genotyped by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Overall, the mutation genotypes CC/CG of CD35 rs7525160 significantly increased the risk of HCC. Stratification analysis indicated that CD35 rs7525160 CC/CG genotypes increased HCC risk in patients younger than 65 years and were closely related to the pathological type of poor prognosis of HCC. Cox proportional hazard ratio model analysis revealed that the rs7525160 CC/CG genotype remains a significant independent risk factor for postoperative recurrence of HCC. In conclusion, CD35 rs7525160 polymorphism may contribute to the susceptibility and prognosis of HCC in the Chinese Han population.CD35, an important molecule implicated in inflammation and immunity, is reportedly associated with several cancers. However, very few studies have investigated the relationship between  Hepatocellular carcinoma (HCC), which accounts for more than 80% of primary liver cancers worldwide, is estimated to be the fourth most common cause of cancer-related deaths, and poses a severe disease burden , 2. LiveThe complement system, which has been primarily viewed as the “first line of defense” against microbial intruders, participates in diverse processes, such as clearance of immune complexes, tissue regeneration, and lipid metabolism . HoweverComplement regulatory proteins comprise a large family that is essential for activation of the complement system . One of CD35 polymorphisms and HCC predisposition. Therefore, the current case-control study was conducted to investigate the association between tag SNPs in CD35 and susceptibility to and recurrence of HCC, in an attempt to explore the effect of gene-environment interaction on the risk for HCC.However, very few studies have investigated the relationship between This study strictly followed all relevant Chinese laws, regulations and rules, as well as the stipulations of the Declaration of Helsinki of the World Medical Association, and was approved by the Ethics Committee of Sichuan University (Ethics No. 2017-264).Newly diagnosed, untreated HCC patients admitted to the Department of Liver Surgery of the West China Hospital in Sichuan University , between May 2017 and March 2019, were enrolled in this study. Diagnosis of HCC was based on histological findings or a combination of elevated serum α-fetoprotein (AFP) and coincident imaging manifestations from two different imaging based diagnostic methods . Staging criteria for liver cancer were based on Barcelona-Clinic-Liver-Cancer (BCLC) stages  and the Written informed consent was obtained from each participant during recruitment. Baseline characteristics of each subject, such as age, sex, smoking history, drinking history, and family history of HCC, were obtained from the subjects, or their family members, and relevant information on HCC patients, such as tumor size, number of tumors, metastasis, Child–Pugh classification, and detection of microvascular invasion, was further improved by referring to medical records, laboratory tests, and imaging studies.CD35 was downloaded from the Human Genome Project and the International HapMap Project. HaploView 4.2 was used to select candidate tag SNPs of CD35 with an r2 threshold of 0.8, and minor allele frequency (MAF) greater than or equal to 0.05, based on Chinese population data from the HapMap database. Finally, six SNPs  were included in this study  strictly according to the manufacturer’s instructions. All extracted DNA specimens were diluted to 20 ng/μL in order to obtain a working concentration and stored at -20°C.Genomic DNA was extracted from whole blood samples using EDTA anticoagulant via the matrix-assisted laser desorption ionization-time of flight mass spectrometry method (MALDI-TOF), according to the manufacturer’s instructions. The detailed genotyping process is described in another study of ours   of ours . Primer All HCC patients who underwent hepatectomy were regularly followed up in the Outpatient Department of Liver Surgery of the West China Hospital in Sichuan University. During the first year after surgery, the patients were followed up every three months, and the patients with no recurrence for at least 2 years following surgery were followed up every six months instead. Abdominal ultrasonography and blood tests, including liver function tests and AFP levels, were performed at each follow-up. Any subject with a positive finding of AFP or abnormal ultrasonography was subjected to enhanced computed tomography (CT) or magnetic resonance imaging (MRI) of the upper abdomen immediately. Recurrence-free survival (RFS) was defined as the time interval from the date of hepatectomy to the date of recurrence or the deadline for the last follow-up. Overall survival (OS) was defined as the time interval from the date of hepatectomy to the date of death or the last follow-up.P<0.05. Each SNP frequency in controls was analyzed for deviation from the Hardy-Weinberg equilibrium (HWE) using the chi-squared goodness-of-fit test. Mann-Whitney U test (for continuous variables) and chi-square tests with Fisher’s exact test  were used to compare differences in the distributions of demographic characteristics between the HCC and control groups. The association between HCC risk and CD35 tag SNPs was estimated as odds ratios (OR) and 95% confidence intervals (CI) using a logistic regression model, adjusted by sex, HBV status, smoking, and drinking status where it was appropriate. The relationship between RFS/OS and CD35 tag SNPs was analyzed using univariate survival analysis and the Cox proportional hazard ratio model. Family history of liver cancer is defined as family members within three generations of patients with liver cancer. Ever/current smoking is defined as continuous/cumulative smoking for 6 months or more in a lifetime, and consumption of at least 100 cigarettes. Those who had consumed 12 standard drinking amounts  in the past 12 months were defined as ever/current drinkers . All tests were two-tailed, and statistical significance was set at drinkers .P<0.05). A total of 299 HCC patients underwent hepatectomy treatment and were followed up to December 2019, the maximum follow-up time being 30.7 months and a medium follow-up time of 18.0 months. The characteristics and clinical features of HCC patients treated with hepatectomy are summarized  and 647 controls (297 men and 350 women) remained. Baseline characteristics of the HCC cases and controls are summarized (CD35 in controls followed Hardy-Weinberg equilibrium predictions (P>0.05). Among the six SNPs of CD35, only the frequencies of GG, CG, and CC genotypes of rs7525160 in HCC and control groups were statistically significant ;  and environmental factors ; .We analyzed the polymorphism of CD35 rs7525160 CC/CG genotype may increase the risk of different clinicopathological types of HCC, especially among patients with AFP ≥ 400 ng/ml , tumor size > 5 cm , TNM stage III/IV , background cirrhosis , or portal vein tumor thrombosis   of 299 HCC patients who underwent curative hepatectomy. Patients who were treated with transcatheter arterial chemoembolization (TACE), radiofrequency ablation, or drugs only were excluded to eliminate the influence of different treatments on the prognosis of HCC. Kaplan-Meier analyses showed that the MRFS was 17.82 months with 95% CI of 16.104–19.544 months for CD35 rs7525160 CC/CG individuals, which was shorter than that for individuals carrying the GG genotype ; ; P=0.011], Child-Pugh Class B , Child-Pugh Class C , microvascular invasion , tumor BCLC stage B/C , AFP level ≥ 400 ng/mL , tumor size > 5 cm , multiple tumor number , TNM tumor stage III/IV , and portal vein tumor thrombosis  were factors that were possibly associated with HCC recurrence ;  of six tag SNPs of CD35 was associated with the risk of HCC in the Chinese Han population. Notably, compared with the GG genotype, the CD35 rs7525160 CC/CG genotype was associated with an increased risk for developing HCC.Several studies have confirmed that the n cancer –26, 33. CD35 rs7525160 CC/CG genotype was a risk factor for HCC in patients younger than 65 years.Diseases caused by genetic mutations usually occur at a relatively younger age; this is attributed to the vulnerability of youth to genetic effects rather than environmental exposure, as evidenced by several studies on young patients –36, indiCD35 rs7525160 in HCC patients was significantly different from that in controls, especially in patients with AFP ≥ 400 ng/ml, tumor diameter of > 5 cm, TNM stage III/IV, cirrhosis, and portal vein thrombosis, which suggested that CD rs7525160 CC/CG genotype may be related to the type of HCC with poor prognosis. To eliminate the effect of different treatments on the prognosis for HCC, we only studied the 299 untreated patients who had undergone hepatectomy to analyze the association between CD35 and RFS/OS. We found that the CD35 rs7525160 CC/CG genotypes were significantly associated with a shorter mean RFS for HCC patients, and rs7525160 was identified as an independent predictor of RFS in HCC patients by Cox analysis. However, no relationship between CD35 polymorphism and OS was found in this study, which may be due to OS and postoperative recurrence of HCC being affected by many factors, including the choice of treatment following recurrence and the economic status of patients, among others.Although the recurrence and fatality rates of HCC have decreased with the popularization of screening among high-risk groups and the improvement of treatment regimens , HCC remCD35 polymorphisms vary greatly between different ethnic groups. Therefore, our results cannot be applied to other populations, and data pertaining to multi-ethnic and multi-region populations of non-Chinese Han populations are felt to be required. Secondly, our study focused only on CD35 polymorphism, HCC susceptibility and prognosis at the genetic level, and therefore, further functional verification needs to be considered. Thirdly, a larger sample size is needed, especially when investigating the association between genetic polymorphism and HCC prognosis.This study was beset by several limitations that need to be addressed. First, our results are based only on the Chinese Han population. However, the allele frequency patterns of CD35 rs7525160 may be associated with increased HCC susceptibility in the Chinese Han population, especially in individuals younger than 65 years. In addition, our results indicated that the CD35 rs7525160 CC/CG genotype is closely related to a pathological type associated with poor prognoses for HCC and acts as an independent risk factor for a shorter RFS time following hepatectomy. Our results also suggest that CD35 rs7525160 may be used as a biomarker for screening as well as for prognostic prediction of HCC. Additional well-designed studies with larger sample sizes and long-term follow-up periods, that encompass different ethnic groups, may be required to better elucidate the causal relationship between CD35 polymorphism and HCC risk and prognosis.In summary, we found that The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Sichuan University (Ethics No. 2017-264). The patients/participants provided their written informed consent to participate in this study.LmL and QL performed the research and wrote the manuscript. LmL and ZS were responsible for the conceptualization, investigation, and validation of the study. QL and ZS analyzed the data. LxL and BC contributed to the sample collection. YP and YB reviewed and edited the manuscript. FL was in charge of supervision, conceptualization, and project administration. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China , and the Sichuan Science and Technology Program .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Uterine corpus endometrial carcinoma (UCEC) is a gynecological malignant tumor with low survival rate and poor prognosis. The traditional clinicopathological staging is insufficient to estimate the prognosis of UCEC. It is necessary to select a more effective prognostic signature of UCEC to predict the prognosis and immunotherapy effect of UCEC.CIBERSORT and weighted correlation network analysis (WGCNA) algorithms were combined to screen modules related to regulatory T (Treg) cells. Subsequently, univariate, least absolute shrinkage and selection operator (LASSO), and multivariate Cox regression analyses were used to identify the genes in key modules. The difference in overall survival (OS) between high- and low-risk patients was analyzed by Kaplan–Meier analysis. The Tregs-related risk signature (TRRS) was screened by uni- and multivariate Cox analyses. Afterward, we analyzed the expression difference of TRRS and verified its ability to predict the prognosis of UCEC and the effect of immunotherapy.Red module has the highest correlation with Tregs among all clustered modules. Pathways enrichment indicated that the related processes of UCEC were primarily associated to the immune system. Eight genes  were selected to construct TRRS. We found that this TRRS is a significantly independent prognostic factor of UCEC. Low-risk patients have higher overall survival than high-risk patients. The immune status of different groups was different, and tumor-related pathways were enriched in patients with higher risk score. Low-risk patients are more likely take higher tumor mutation burden (TMB). Meanwhile, they are more sensitive to chemotherapy than patients with high-risk score, which indicated a superior prognosis. Immune checkpoints such as PD-1, CTLA4, PD-L1, and PD-L2 all had a higher expression level in low-risk group. TRRS expression really has a relevance with the sensitivity of UCEC patients to chemotherapeutic drugs.We developed and validated a TRRS to estimate the prognosis and reflect the immune status of UCEC, which could accurately assess the prognosis of patients with UCEC and supply personalized treatments for them. Uterine corpus endometrial carcinoma (UCEC) is the third most common gynecological malignant cancer globally . In 2019With the continuous development of bioinformation technology, a great deal of methods has been employed to define biomarkers. WGCNA algorithm can identify highly correlated modules and genes for cancer based on the network construct of genes expression . This sthttps://portal.gdc.cancer.gov/)  data portal (er.gov/) . ClinicaCIBERSORT was employed to estimate the proportion of 22 immune cells for normal and tumor tissues . TherefoIntersecting Gene and Low-Tregs Groups. Second, we used Limma to define the differentially expressed genes with a p-value lower than 0.05 and log foldchange larger than 1 as the threshold  is a web-based program that has plenty information about protein and gene, in which investigators always use to do pathway and process enrichment analysis  primers are listed in Total RNA from 16 UCEC samples and 16 normal tissues was extracted on the basis of Trizol reagent (Invitrogen). Before reverse transcription to cDNA, 4× GDNA wiper mix (vazymer323-01) was employed to remove residual genomic DNA from total RNA. Complementary RNA was synthesized by using PrimeScript RT reagent kit. Real-time quantification was performed using the SYBR Premix Ex Taq Kit (TaKaRa DRR041). The relative expression level of the target gene was standardized by GAPDH and 2Five clinical characteristics such as age, stage, grade, histological type, and risk score were combined to establish a nomogram aims to calculate the OS of 1, 3, and 5 years of UCEC in the entire set. Calibration curves were employed to evaluate whether the established nomogram is reliable .A total of 552 UCEC samples and 23 normal samples were obtained from TCGA database, which was employed to verify the expression distinction between tumor and normal tissues. Furthermore, boxplots were drawn to analyze the relationship between gene expression and clinical factors. Finally, according to the level of expression, patients were split into two groups. Then, we verified the connection between TRRS expression and survival probability based on K–M analysis.Gene Set Cancer Analysis (GSCALite), a web server, aims to flexibly calculate the expression, mutation, and interaction of genes in different cancers . GSCALitThe areas under the curve (AUCs) of 1, 3, and 5 years on account of risk score and different clinical factors were calculated, respectively. Furthermore, the curve was employed to identify the total influence of risk score combined with clinical characteristics on survival probability. Overall survival rates of different clinical characteristics were calculated as well.https://tcia.at/home)]  molecules, effector cells, and immunosuppressive cells are the four main components to evaluate tumor immunogenicity. Through calculating the expression values of four kinds of immune genes, we derived a patient’s immunophenoscore (IPS). The IPS was achieved based on a scale range from 0 to 10. The higher the score, the stronger the immunogenicity. The IPSs , acquired from The Cancer Immunome Atlas [TCIA (t/home)] , were apThe connection between ICI expression and risk score of UCEC patients was identified based on Pearson correlation coefficient method. Boxplots display the results.p < 0.05 were considered significantly enriched. Immune scores, stromal scores, and estimate scores of each sample were received based on the “Estimation of Stromal and Immune cells in Malignant Tumors using Expression data” (ESTIMATE) algorithm  . The TMBhttps://www.cancerrxgene.org/)  (ne.org/) . The calhttp://cis.hku.hk/TISIDB/index.php) database were applied and inferred the correlation between ZSWIM1 expression and the TRRS of UCEC  was applied to perform statistical analysis in our study. All statistical tests were two-sided, and p = 4.756e−09, p = 6.05e−04, respectively) (p = 0.001)  as the threshold . Twelve genes were analyzed by LASSO, and eight of them were selected , p = 0.010), GOLGA7 , ST6GALNAC4 , CDC16 , ITPK1 , PCSK4 , p = 0.033)   Table 1.p < 0.05) by means of Wilcoxon signed-rank test (p < 0.001] and ZSWIM1  were combined with poorer OS of UCEC patients; however, CORO1B , CDC16 , PCSK4 , and ITPK1  expression level had a positively correlated with overall survival , histological type , and risk score  were related to UCEC prognosis. However, the outcome of multivariate Cox regression indicated that stage  is an independent prognostic factor of UCEC. Besides, the stage , grade , and risk score  were related to the prognosis of UCEC. However, in the train set, only the risk score  was independently correlated with the prognosis of UCEC patients , macrophage M1 , and macrophage M2 , T follicular helper cells , and gamma delta T cells  were positively associated with the risk score. While resting dendritic cells , monocytes , activating NK cells activated , CD8 T cells  were positively associated with the risk score. Specially, the risk scores were strongly negatively related to the expression of Tregs . This is consistent with the previous results  and PD1  were negatively correlated with TRRS in both train set and entire set (p < 0.05) . Consequently, we inferred that patients with low risk are more likely to trigger an immune response.Immune checkpoints (CTLA4 and PD1) are able to assess the response of patients to immunotherapy. The result showed that the expression of CTLA4  (p < 0.001)  and gemcitabine (p = 0.004) have higher sensitivity in the low-risk group, which indicated that chemotherapeutic drugs may have better curative effect in the high-risk group (p < 0.01) . Immunopotentiators, immunosuppressants, MHC molecules  can undertake the role of an independent predictor in several cancers. Consequently, TISIDB was applied to analyze the relationship between TILs and ZSWIM1expression. The correlation between ZSWIM1 expression and TILs in different descriptions of cancer is performed in UCEC is a common gynecological malignant tumor  with lowIn the immunotherapy of different kinds of cancer, blocking immune checkpoint has shown a wide range of effects . AntibodAt the beginning, CIBERSORT was employed to assess the proportion of T cells, which indicated that the more advanced the clinical stage and grade, the lower the expression of Tregs. This means that TRRS is a tumor suppressor gene. By using the WGCNA algorithm and correlation analysis, the red module was selected as the most relevant module. GO and KEGG enrichment analysis showed that the red module has the closest relationship with Tregs. Univariate, LASSO, and multivariate Cox regression were applied to calculate prognostic significances in risk score and some clinical factors.Genome-wide analysis of these eight genes indicated that these high frequency mutations and methylation had close relationships with gene expression, and they were involved in regulating the activity of injury response pathway. The high expression of these genes in tumor samples and early clinical stage indicates their potential use as biomarkers. The outcomes of qRT-PCR were similar to the trends detected in the TCGA dataset, which confirmed the prediction ability of the model to a certain degree. The AUC of the risk score showed the high sensitivity and specificity of TRRS. The AUCs of the risk score merged with clinical characteristics were higher, which suggests that using the TRRS together with clinical factors, we can give patients a better treatment guidance. In line with previous studies, the results of this study showed that age, stage, and grade were also connected with the prognosis of patients with UCEC. The nomogram shows that the TRRS can accurately assess the prognosis of UCEC . StudiesEight genes were selected for further analysis and had been used as prognostic markers in other diseases. CDC16 has a connection with multiple neurodevelopmental disorders , 51. It Genome-wide analysis of the genes showed that the mutation frequency of PCSK4 was the highest. The expression of the TRRS was positively correlated with CNV. Besides, the methylation of GOLGA7, PCSK4, ZSWIM1, ST6GALNAC4, and ITPK1 was downregulated. DNA methylation is an epigenetic mechanism to control the expression of oncogenic or tumor-suppressive genes. Scientists have studied the potential effectiveness of some methylated biomarkers in predicting cancer prognosis . HoweverPrevious studies have also found gene prognostic markers closely related to UCEC –70, but All in all, through a series of bioinformatics analysis, we constructed a TRRS as potential biomarkers and targets for immunotherapy of UCEC. Low-risk patients had better prognosis and higher response rate to ICI. In the future, TRRS is expected to help predict prognosis and assess the efficacy of immunotherapy for UCEC patients, which can provide individualized treatment.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/JB, JL, and RG conceived the study and participated in the study design, performance, and manuscript writing. JL, SY, and FS conducted the bioinformatics analysis. ZZ, MY, SN, and LC revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.