Perineal groove is a rare anoperineal congenital malformation disease that usually affects newborn females. It is unknown to many clinicians, which usually leads to misdiagnosis. The pathogenesis of perineal groove is not clear, and there are few cases reported in the current medical literature. Perineal grooves in two newborn babies were described in this report, and the literature on perineal groove was also reviewed and analyzed to improve the recognition of this disease. Congenital perineal groove is rare, and few cases have been reported. A previous report published by Kadowai et al.  describeThis infant's parents were both of Asian descent. The mother was gravida 1 and para 0 (G1P0). The ages of the mother and father were 19 and 24, respectively, and they were both healthy. Neither of the infant's parents had a family history of congenital abnormalities, and they were not consanguineous. The mother had no history of tobacco, alcohol or substance abuse during the pregnancy. The father had no remarkable medical history. There were no other abnormalities during pregnancy. The infant's gestational age was 37 weeks and 5 days. Apgar scores were not clear when the infant was born. Owing to a birth weight (BW) of 1930 g, the newborn baby, who was delivered by normal spontaneous vaginal delivery, was admitted to the neonatal intensive care unit (NICU) for further examinations. Physical examination of the perineum revealed a wet groove-like erythematous nonepithelized mucous membrane extending vertically downward from the posterior vaginal fourchette to the anterior anal verge when the infant's legs were flexed. The lesion was ~2 cm from the base of vaginal fourchette to the anterior rim of anus at the 12 o'clock position. The width of the lesion was ~0.1 cm, and the depth of the lesion was ~0.1 cm. The surface of the perineal groove had no fistula, secretions, bleeding, or infection. The urethral canal, vagina and anus were in the appropriate position, and the anal wink was intact. The spine and sacral area had no obvious abnormalities  with good prenatal care. Their first baby was induced owing to intrauterine fetal death, and the details were unknown. There was no family history of congenital abnormalities on either side of the family, and the parents were not consanguineous. The mother also had no history of tobacco, alcohol, or substance abuse. The mother's antenatal examination was uneventful except for gestational diabetes. During pregnancy, the mother's blood glucose was maintained between 6.1 mmol/l and 8.1 mmol/l without any drug treatment. The infant's gestational age was 37 weeks and 3 days. The newborn was delivered by vaginal delivery and her birth weight (BW) was 4910 g. Apgar scores were 6 and 8 at 1 and 5 min, respectively. Owing to dyspnea 2 h after the birth, the neonate also received further examination in the NICU. On examination, a perineal defect was noted. The groove extended vertically downward from the base of the vaginal fourchette to the anterior rim of the anus at the 12 o'clock position. The perineal groove was a moist red sulcus that was ~1 cm long, 0.1 cm wide and 0.1 cm deep. There were no signs of malformation, bleeding, fistula, secretions, or infection noted in the genital area . The diagnosis of congenital perineal groove depends on clinical examination. The pathogenesis is unknown. Conservative treatment is generally preferred for congenital perineal groove. Confusion regarding the diagnosis may lead to misdiagnosis and unnecessary surgical or medical intervention, and we report two cases of perineal groove to raise awareness of this unusual malformation. Understanding perineal groove as an abnormal perineum will help to avoid misdiagnosis and prevent excessive interventions or unnecessary surgical procedures. At the same time, as doctors, we can provide appropriate advice and follow-up recommendations to the parents of affected infants.This study was approved by the Ethical Review Committee of The First Hospital of Jilin University.HC drafted the manuscript. ZW diagnosed the malformation and revised the manuscript. HC and ZW contributed equally to this article and should be considered co-first authors. QZ and HZ diagnosed the malformation and provided advice regarding the manuscript. TX designed the topic and revised the manuscript. All authors read and approved the final manuscript. All authors agree to be accountable for the content of the work.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.


Objective: Preclinical studies have found both hyperactivity of hypothalamic- pituitary- adrenal (HPA) axis and synaptic degeneration are involved in the pathogenesis of Alzheimer’s disease (AD). However, the data on the relationship of activity of HPA axis and synaptic degeneration in humans are limited.Methods: We compared CSF cortisol levels in 310 subjects, including 92 cognitively normal older people, 149 patients with mild cognitive impairment (MCI), and 69 patients with mild AD. Several linear and logistic regression models were conducted to investigate associations between CSF cortisol and synaptosomal-associated protein 25  and other AD-related biomarkers.Results: We found that levels of cortisol in CSF were associated with SNAP-25 levels and tau pathologies but not amyloid-β protein. However, there were no significant differences in CSF cortisol levels among the three diagnostic groups.Conclusion: The HPA axis may play a crucial role in synaptic degeneration in AD pathogenesis. Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by a progressive decline of memory and cognitive function . AlthougSynaptic function has been regarded as a critical mechanism underlying cognitive impairment in the pathogenesis of AD . A previHere, the levels of cortisol in CSF among normal controls, patients with MCI and AD had been compared. Second, we examined the relationship of CSF cortisol levels and synapse degeneration. Finally, we investigated the associations between CSF cortisol levels and typical AD-related biomarkers, including hippocampal volumes, CSF Aβ42, t-tau, and p-tau levels.http://www.adni-info.org.Data used in the preparation of this study were extracted from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. The primary goal of ADNI has been to investigate whether neuropsychological testing, neuroimaging data, blood, and CSF biomarkers could be integrated to assess the progression of MCI and mild AD. This study was approved by local ethical committees. All subjects or authorized representatives provides written informed consent. Further information can be found at the website: Demographics were obtained from the ADNI database. In this analysis, there were 92 normal controls, 149 patients with MCI, and 69 patients with mild AD.1. Participants underwent comprehensive cognitive assessments, while we only selected Mini-Mental State Exam (MMSE) and Rey Auditory Verbal Learning Test  in the current analysis.APOE genotypes of the study participants were extracted from the ADNI databasehttp://www.adni-info.org. The CSF cortisol data were extracted from the ADNI database. Value are given in ng/ml.The levels of CSF cortisol were determined using Luminex xMAP immunoassay technology, which measures a range of inflammatory, lipid, metabolic, and other indices . Further® immunoassay system to determine the levels of SNAP-25 in CSF. All samples were run in triplicates. Values are given as pg/ml. The detailed protocol can be found at http://www.adni-info.org.Levels of SNAP-25 in CSF were determined at the departments of pathology and neurology at Washington University, St. Louis, United States. A sandwich ELISA that was developed using the ErennaLevels of Aβ42, t-tau, and p-tau in CSF were determined by the ADNI Core Biomarkers Team using xMAP Luminex platform and Innogenetics/Fujirebio AlzBio3 immunoassay kits. The detailed procedure for this determination is described elsewhere . Using aadni.loni.usc.edu. Hippocampal volume measurements were performed using FreeSurfer software2.The hippocampal volumes data were extracted from the ADNI database. The detailed neuroimaging methods used by ADNI have been described elsewhere . BrieflyF-test and x2 test were performed to investigate the mean differences of continuous variables and the distributions of categorical variables, respectively. Second, the Pearson correlation tests were used to investigate the correlations of CSF cortisol levels and other AD-related biomarkers in all participants. Finally, linear and logistic regression models were used to examine the association between cortisol and other biomarkers after adjusting several potential confounding variables. More specifically, logistic regression models were used to examine the association between amyloid status  and CSF cortisol levels (independent variable). Model 1 was unadjusted. Model 2 was adjusted for age, education, age, APOE genotype, MMSE scores and diagnosis. All statistical analyses were performed using R software. A two tailed P < 0.05 was considered to be statistically significant.First, the p < 0.001). In addition, there were significant differences in RAVLT immediate and delayed recall scores across the three diagnostic groups (p < 0.001). Consistent with the previous data, more than 50% of patients with MCI and AD carried at least one APOE 4 allele (P < 0.005). However, no significant differences in CSF cortisol levels were found between the three groups (P > 0.05).Table 4 allele . There wp > 0.05). In this analysis, we included 310 subjects, including 92 normal controls, 149 patients with MCI, and 69 patients with mild AD.As Figure r = 0.22, p = 0.009). Additionally, the correlations between CSF cortisol between tau protein were analyzed. Levels of cortisol in CSF were associated with CSF t-tau  and p-tau  levels. However, no correlations between CSF cortisol and Aβ42 levels were found , and AD-related biomarkers . Higher levels of cortisol in CSF were significantly associated with greater SNAP-25, t-tau, p-tau levels (5, and ADTo the best of our knowledge, the current study was the first to investigate the relationship of CSF cortisol levels and synapse degeneration among cognitively normal older people, patients with MCI and AD. In addition, we also found that levels of cortisol in CSF were associated with tau pathologies but not amyloid-β protein. However, no significant differences in CSF cortisol levels were found among the three diagnostic groups.Epidemiological studies suggested that chronic environmental stress plays an important role in the pathogenesis of AD . AdditioIn the present study, no significant differences in CSF cortisol levels were observed among three diagnostic groups. It has been reported that there were no differences in cortisol levels in plasma , salivarIn present study, we did not find significant correlations between CSF cortisol and Aβ-42 levels, which is consistent with a previous study . HoweverSeveral limitations should be noted. First, cross-sectional design limits our ability to explore the temporal relationship of cortisol and synapse degeneration and AD-related biomarker. Second, the findings of this study may not be generalizable to other populations. Finally, this study included only subjects with mild AD. Further study should also include moderate and severe dementia patients to clarify the relationship of severity of cognitive impairment and CSF cortisol levels.In conclusion, we found that CSF cortisol levels were associated with tau pathologies and synapse degeneration but not amyloid-beta, highlighting a potential role of cortisol in synapse degeneration in AD patients.JZ conceived and designed the studies. QW, WZ, and JZ performed the research, analyzed the data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Limited data have been available on the global practice of handwashing with soap (HWWS). To better appreciate global HWWS frequency, which plays a role in disease transmission, our objectives were to: (i) quantify the presence of designated handwashing facilities; (ii) assess the association between handwashing facility presence and observed HWWS; and (iii) derive country, regional and global HWWS estimates after potential faecal contact.First, using data from national surveys, we applied multilevel linear modelling to estimate national handwashing facility presence. Second, using multilevel Poisson modelling on datasets including both handwashing facility presence and observed HWWS after potential faecal contact, we estimated HWWS prevalence conditional on handwashing facility presence by region. For high-income countries, we used meta-analysis to pool handwashing prevalence of studies identified through a systematic review. Third, from the modelled handwashing facility presence and estimated HWWS prevalence conditional on the presence of a handwashing facility, we estimated handwashing practice at country, regional and global levels.P <0.001 for low- and middle-income countries, based on nine datasets. Third, we estimate that in 2015, 26.2%  of potential faecal contacts were followed by HWWS.First, approximately one in four persons did not have a designated handwashing facility in 2015, based on 115 data points for 77 countries. Second the prevalence ratio between HWWS when a designated facility was present compared with when it was absent was 1.99  Many people lack a designated handwashing facility, but even among those with access, HWWS is poorly practised. People with access to designated handwashing facilities are about twice as likely to wash their hands with soap after potential faecal contact as people who lack a facility. Estimates are based on limited data. One in four persons worldwide did not have access to a handwashing facility with soap and water on premises in 2015.In 2015, handwashing with soap occurred in about 26% after events of potential faecal contact, globally. In regions with high access to handwashing facilities, handwashing with soap was performed by about 51%, and in regions with more limited access, by about 22% after events of potential faecal contact.Though additional data and analyses are needed, quantifying the presence of handwashing facilities with soap and water on premises may be used to estimate actual handwashing behaviour.Important gaps exist for country-representative data on presence of designated handwashing facilities and on handwashing behaviour at household level, for all regions of the world and particularly for high-income countries.,Handwashing with soap (HWWS) is an important public health behaviour as it reduces exposure to faecal pathogens and other infectious agents, thereby reducing gastrointestinal,The proportion of people using a handwashing facility with soap and water on premises is a global indicator of the Sustainable Development Goals (SDG indicator 6.2.1b).,,Handwashing practice recorded during structured observations is considered the most reliable way to measure actual handwashing behaviours,The purpose of this analysis was to quantify the current state of global handwashing practice. To that end, we had the following three objectives: (i) to quantify presence of designated handwashing facilities at national, regional and global levels; (ii) to assess the association between presence of a designated handwashing facility and observed HWWS at crucial time points; and (iii) to derive country, regional and global estimates of HWWS practice after potential faecal contact.IJE online). We are following guidelines for accurate and transparent health estimates reporting (GATHER),We have summarized the key methodological approaches below; but as this work uses various methods and analysis steps, we have included a figure explaining the relationship between the three objectives . An addiWe extracted country data on household presence of a designated handwashing facility from the WHO/UNICEF Joint Monitoring Programme for Water Supply, Sanitation and Hygiene (JMP) global database. This compiles data collected through nationally representative household surveys and censuses, and represents proportions of households that have a handwashing facility with soap and water available on premises, i.e. within the dwelling, yard or plot (defined above as and named within this manuscript as a ‘designated handwashing facility’).n = 118, IJE online). IJE online) lists in column H whether the modelled estimate is based on own country values or whether it was extrapolated from the mean value.We modelled a continuous time series of estimates for 194 countries using a two-level (data points clustered within countries) linear multilevel model (MLM) with a random intercept by country. This approach allowed us to estimate values for countries with and without data points. The dependent variable (survey estimate of presence of a designated handwashing facility) was logit-transformed to restrict estimates between 0 and 1 (or between 0 and 100%). Covariates (fixed effects) included in the model were a continuous time variable , a factor variable indicating regional grouping  of the presence of a designated handwashing facility by urban and rural areas and for countries with data points were derived using the Monte Carlo method with random draws .As a sensitivity analysis, we modelled presence of a designated handwashing facility using only the more recent survey data since 2009, as before 2009 handwashing measurements in DHS and MICS had not yet been harmonized across countries.n of countries with a survey point for the year 2015 . The model was fitted n times, each time setting all survey points for icountry as missing (icountry formed the ‘test set’ in round n). Subsequently, icountry model prediction was compared with icountry actual survey value for the year 2015 and the root mean squared error across n rounds was calculated.The predictive performance of the model was assessed using cross-validation.We updated the results of our previous systematic reviewWe included any study that reported HWWS practice assessed with structured observations in adults and children. Though direct observation has shown to change participant behaviours,IJE online).Studies were selected for inclusion using a two-step review process. Titles and abstracts of all studies identified in the search were screened for relevance. The full text of each of the relevant articles was then reviewed, and studies were excluded if they did not provide observational data on the prevalence of observed HWWS. Data were extracted from each study using a standard protocol and included information on study setting (country), observation location (home or public setting), time frame of survey, population subgroup, sample size, a description of how handwashing prevalence was measured and specific prevalence estimates for any of the handwashing occasions, such as after toilet use or after cleaning up after a child. Characteristic of included studies and their references are listed in For analysing the association between presence of a designated handwashing facility and HWWS, studies were assembled from the systematic review that reported both observed presence of a designated place for handwashing and HWWS at critical times , assessed through structured observations. Additional study datasets were provided from subject matter experts. Studies needed to fulfil the following further criteria to be included in the analysis: (i) the study needed to be conducted at household-level; (ii) in case of post-intervention data, the control group needed to be identifiable; and (iii) the study needed to have been conducted within the past 20 years (1998–2017).,In case of intervention data, we included data from both intervention and control groups at study baseline if available  that could include a specific handwashing hardware, such as a tippy tap or washbasin, or be any set place that householders consider their designated handwashing place. Soap in this context includes any kind of soap or detergent but excludes mud, sand or ash. In most studies, householders were asked to show the place where household members usually washed hands and the presence of soap and water was observed. In two datasets, information on the designated handwashing facility was given for the primary and secondary (or tertiary) handwashing facility . In these datasets, the main exposure was present (coded 1) when soap and water were present at any of the observed handwashing facilities.The primary outcome was observed HWWS after potential faecal contact. Both hands needed to be washed with soap. Potential faecal contact in this analysis includes visiting the toilet, defaecation and cleaning a child’s bottom or changing its nappies, but does not include potential contact with animal faeces. Any handwashing occasion by any household member reported in the primary studies and related to the respective exposure was included in the analysis. Though the literature search had focused on observed HWWS after potential faecal contact, we also analysed the available data to investigate the association between presence of a designated handwashing facility and observed HWWS before food contact. Food contact in this analysis includes preparing, cooking or serving food, eating, and feeding or breastfeeding a child. We excluded handwashing observations when either the designated handwashing facility or HWWS could not be observed.,IJE online). As determinants for washing own hands might be different from washing children’s hands, we analysed data restricted to handwashing occasions among adults. More details on the model are included in IJE online).To estimate adjusted prevalences and prevalence ratios from binary outcome data, we used Poisson regression with robust standard errors.We used the ‘margins’ command in StataAs the selected studies for the analysis of the association between presence of a designated handwashing facility and observed HWWS were conducted exclusively in low- and middle-income countries, we conducted random effects meta-analysis on observed HWWS prevalence after potential faecal contact in high-income country studies identified in the systematic review, to approximate the proportion of HWWS after potential faecal contact in those countries.:IJE online). For estimating HWWS prevalence after potential faecal contact for high-income countries, the pooled HWWS prevalence from the meta-analysis (objective 2) was taken. To estimate the proportion of potential faecal events that were followed by HWWS at regional and global level for low-, middle- and high-income countries, we calculated population-weighted means of the country estimates. As we do not know the total number of potential faecal contacts by country and region, we assume an equal average number of faecal contacts by person across countries and regions, and use total population by country and region to calculate the regional and global estimates. The calculation of the 95% confidence intervals for these estimates at country, regional and global levels is described in IJE online).To estimate HWWS prevalence facility  and 4. TAs a sensitivity analysis, we re-calculated regional and global handwashing prevalence, applying the approach for low- and middle-income countries to high-income countries. We did so by using formula 1 for high-income countries, where datasets .IJE online). Only 29 countries had more than one data point available, making it difficult to determine trends , 14 countries in the WHO Region of the Americas (AMR), eight in the WHO Eastern Mediterranean Region (EMR), eight in the WHO European Region (EUR), five in the WHO South-East Asia Region (SEAR) and three in the WHO Western Pacific Region (WPR)] and one high-income country (Barbados).e trends .The root mean squared error (RMSE) from cross-validation was 11.1% for urban and 7.3% for rural areas, indicating that on average the model estimate for the year 2015 for a country without any survey point is respectively 11.1 and 7.3 percentage points away from the ‘true’ value (which is approximated here with the survey point for 2015 for the respective country).When modelling the presence of a designated handwashing facility based on uniformly collected data in MICS/ DHS from 2009 onwards , the estimated percentage of population with access to a designated handwashing facility changed only slightly .The systematic review identified a total of 42 studies of which 15 were conducted in high-income countries . Nine daIJE online).The eight studies (providing nine datasets), reporting both presence of a designated handwashing facility and structured observations of handwashing after potential faecal contact, were combined to estimate the association between presence of a designated handwashing facility and HWWS after potential faecal contact. Data from Zimbabwe urban and rural sites were different parts of one study from the same research group. The pooled dataset includes 3953 observed potential handwashing occasions after potential faecal contact. Of those, hands were washed with soap in 18.6% of potential handwashing occasions. In the presence of a designated handwashing facility, hands were washed with soap in 25% of potential handwashing occasions compared with 12% in the absence of a designated facility. The 28 observations with missing values in the variable indicating the presence of a handwashing place with soap and water  were excluded from the analysis (i.e. listwise deletion).P <0.001) for an association between presence of a designated handwashing facility and observed HWWS after potential faecal contact in structured observations. After taking account of the clustering of observations in households and countries (or studies), and after adjusting for study region, the prevalence of HWWS where a designated handwashing facility was present was 1.99  times higher than in the absence of a designated facility, Note that in the absence of a designated handwashing facility, householders could still fetch water and soap to wash their hands, use the neighbour’s facility etc..From the three-level Poisson model there was very strong evidence (P <0.001) for an association between having a designated handwashing facility and observed HWWS before food contact . After taking account of the clustering of observations in households and countries (or studies) and after adjusting for study region, the prevalence of HWWS in presence of a designated handwashing facility was present was 2.57  times higher than in the absence of a designated facility.There was also very strong evidence (P  <0.001) and of similar size compared with the analysis including data on children’s handwashing  and before food contact 2.42 ).After restricting to adult handwashing, there remained 2578 observed potential handwashing occasions after potential faecal contact and 7554 potential handwashing occasions before food contact. Of those, hands were washed with soap in 19.1% of potential handwashing occasions after potential faecal contact and in 4% of potential handwashing occasions before food contact. The association between presence of a designated handwashing facility and adult HWWS after potential faecal or before food contact remained very strong (IJE online) includes citations, number of observed HWWS events after potential faecal exposure, number of observed faecal contact events and characteristics for all 15 studies in high-income countries included in random effect meta-analysis. The pooled HWWS proportion after potential faecal contact was 51%  , which resulted in the choice of the pooled estimate over all 15 studies. We additionally examined the 15 studies for potential outliers.IJE online).IJE online) lists under column I how HWWS estimates were generated by country.Adjusted HWWS prevalences after potential faecal contact by presence of a designated handwashing facility and by region are given in HWWS prevalences after potential faecal contact, by country for low- and middle-income countries, are computed on the basis of the estimates of presence of a designated handwashing facility at country- evel (objective 1) and the adjusted regional predictions of HWWS after potential faecal contact by presence/absence of a designated facility .Using this approach, we estimate that, worldwide, 26.2% of potential faecal contacts were followed by HWWS in 2015 . CountryWhen calculating the regional (high-income countries) and global proportions of faecal contacts followed by HWWS using formula 1 as described above  also for high-income countries, estimates changed only slightly (48.7% instead of 50.6% for high-income countries and 25.9% instead of 26.2% for the world).This analysis shows that 27% of the world population—nearly two billion people—lack a designated handwashing facility (objective 1). People who have access to a designated handwashing facility, defined here as a specific place within the premises of a household which has both soap and water, are about twice as likely to wash hands with soap after potential faecal contact and 2.6 times as likely to wash hands with soap before food contact, compared with people without a designated facility (objective 2). Only 26% of potential faecal contacts  were followed by HWWS (objective 3).We provide country, regional and global HWWS estimates after potential faecal contact. These estimates are based on modelled estimates of survey data on the presence of designated handwashing facilities. The modelled estimates were converted to estimated HWWS practice using predicted handwashing prevalence in the presence or absence of a designated handwashing facility.Compared with our previous estimate of 19% HWWS after potential faecal contact,The estimates for presence of a designated handwashing facility are based on 115 data points. For 118 of 194 countries there was no data point, and for 48 countries there was only one data point. Trends over time were estimated based on the overall trend from available data points. For countries with no data, the mean for the respective region and income group was used. As such, the reliability of estimates for different regions and countries varies. Results from cross-validation showed, however, that the model predicted was sufficiently close to the actual survey data point even for countries with no data point. Our modelling approach offers several advantages, including a single model for all countries, a continuous time series and the use of information from other country data for countries with few data or no data.IJE online).Estimates for the association between presence of a designated handwashing facility and observed HWWS are derived by regions and are based on nine datasets from eight heterogeneous studies in low- and middle-income countries, covering various subgroups of populations in four out of six regions. For EMR and EUR regions, HWWS practice was assumed as the average adjusted predicted prevalence based on all included studies. As such, estimates of the association between presence of a designated handwashing facility and observed HWWS are based on limited evidence. However, the proportion of observed HWWS is consistently low across the nine datasets . The Afr2I statistic, a measure of inconsistency across study findings,Of the 15 studies conducted in high-income countries, there was only oneThis study provides important input to the SDG indicator 3.9.2 ,This analysis proposes an approach for estimating HWWS practice after potential faecal contact, including after toilet use, which is based on adjusted nationally representative household survey data and the probability of HWWS by presence/absence of a designated handwashing facility.,,We quantify the presence and importance of designated handwashing places as a crucial first step for achieving the desired handwashing behaviour. This analysis suggests that data on the presence of designated handwashing facilities at household level could be used to assess actual handwashing behaviour when handwashing facility presence is adjusted by the association with actual observed handwashing. This could be highly valuable for aggregated HWWS estimates (i.e. at country level) because data on handwashing facility presence are reliable and efficient and—whereas structured observations can estimate handwashing behaviour directly—structured observations are also too time consuming to be integrated in national data collection.HWWS is practised in around 26% of cases after potential faecal contact. To effectively promote HWWS, there is a need to increase handwashing facilities equipped with soap and water in or around the home. As HWWS remains limited even in the presence of a designated handwashing facility, efforts to promote handwashing behaviour need to be conducted simultaneously. This analysis uses nationally representative survey data and derived HWWS estimates, and is in line with individual HWWS observation studies. It suggests that the presence of a designated handwashing facility could be used to estimate actual handwashing behaviour. However, our estimates are based on limited data, i.e. 115 survey data points covering 77 countries for access to designated handwashing facilities and nine datasets from eight low- and middle-income countries, to analyse the association between presence of a designated handwashing facility and observed handwashing behaviour. There is a need for greater data availability on hand hygiene, in particular for: (i) data at household level reporting observed handwashing behaviour, including reporting of the presence of handwashing facilities for low-, middle- and especially high-income countries; and (ii) nationally representative data on presence of handwashing facilities for high-income countries and those low- and middle-income countries not yet covered by such data.This work was supported by the United Kingdom Department for International Development (DFID) [204700–101]. Those acknowledged have confirmed their agreement.dyy253_Supplementary_DataClick here for additional data file.


Here, we discovered a series of TM-IrO2 catalysts have a common activity trend for these oxidation reactions, and their activities are closely related with modified electronic states of IrO2, strongly affected by the types of the transition metal across the periodic table. For all oxidation reactions, Cr- and Mn-IrO2 achieved the highest oxidation catalytic activity, and sequentially decreased activities were obtained with Fe, Co, and Ni doped IrO2. For instance, the highest OER activity was achieved by Cr or Mn doping exhibiting the smallest overpotential η = 275~230 mV at 10 mA/cm2, while Ni-IrO2 showed rather larger overpotential (η = 347 mV) even compared with non-doped IrO2 (η = 314 mV). Scanning transmission X-ray microscopy and high-resolution photoemission spectra of TM-IrO2 indicated dopant metals modified the Ir-O interaction and thus increasing oxygen vacancy defects in IrO2. Strongly positive correlation was observed between the catalytic activities and vacancy states. The amount of defect related signals was observed the most for Cr- or Mn-IrO2, less so for Fe- or Co-IrO2, and unnoted for Ni-IrO2 compared with bare IrO2. Based on these catalytic activities and surface spectroscopic analysis results, vacancy defects induced by doping in TM-IrO2 NPs are proposed to contribute to enhance the oxidation activities.Catalytic activities of transition metal-doped IrO For instance, IrO2 has excellent electrocatalytic activities for oxygen evolution reaction (OER) in batteries or water-electrolysis and considered as a benchmarking material to compare the catalytic activities of others14. Defects or even amorphous structure on the IrO2 surface have been proposed as the active sites of electrochemical OER. In addition to the electrochemical oxidation reaction, IrO2 nanoparticles have been recently reported to display photocatalyst capability, in particular, to split water under UV irradiation through the excitation from the O-p band to the Ir-d band16.Iridium oxide 23. In IrO2, transition metal dopants can access various oxidation states which can disturb the bonding between Ir and O and consequentially modulate the electronic/chemical states of IrO2. Therefore, it can be hypothesized that these modified electronic states vary the intrinsic catalytic activity. Although the active sites of the IrO2 catalysts can be different depending on the catalytic reactions due to the different chemicals and reaction pathways, similar strategies such as doping or alloying have been successfully demonstrated to improve activity of IrO2. This can be valid if there is a related active factor of various reactions that controls catalytic activity on the IrO2 surfaces.Along with mechanistic studies of IrO2 for various reactions, we inserted various transition metal ions (TM+) into IrO2 NPs to enhance their own catalytic activities and then systematically compared the catalytic activities of these transition metal-doped IrO2 (TM-IrO2) NPs for different types of oxidation reactions. In particular, the cost-effective transition metals such as Cr, Mn, Fe, Co, and Ni were doped in IrO2 NPs by carrying out facile hydrothermal syntheses. Understanding the chemical/electronic structures of IrO2 NPs is important, but complexity in the electronic structures of the solid material can make it particularly difficult to determine the chemical state of the active surface species experimentally. Spectroscopic surface analysis techniques can provide the useful information to understand the surface states on IrO2 and modified IrO2 NP surface24. Therefore, the TM-IrO2 NPs were characterized by using scanning electron microscopy (SEM), Raman spectroscopy, and scanning transmission X-ray microscopy (STXM) and the influence of the dopant types on the electronic structures was studied. Then, the catalytic activities of the TM-IrO2 NPs were assessed for the OER in an aqueous solution by taking electrochemical measurements, for the photocatalytic oxidation of 2-aminothiophenol (2-ATP) in a gas phase media by performing high-resolution photoemission spectroscopy (HRPES), and for the thermal CO conversion to CO2 by measuring mass spectroscopy. We observed there is a common catalytic activity trend for all these different oxidation reactions. Espeically, highly enhanced catalytic activity was achieved when Cr or Mn was doped in IrO2 NPs in which modified electron states showed largest generation of vacancy defects proposing as a main contributor.To understand the possible relation of the active species and catalytic activity of IrO2 NPs and the five different TM-IrO2 NPs , 723 (B2g), and 752 cm−1 (A1g) are well associated with typical rutile IrO2 25. Compared with the non-doped IrO2 NPs, TM-IrO2 NPs was confirmed to have no significant changes in these Raman shifts, except additional minor peaks. These minor peaks are considered as dopant-related signals because they were observed near a Raman shift similar to that of its metal oxide bonds 30. In addition, SEM images were also obtained to determine the surface morphology of IrO2 and TM-IrO2 NPs which have mostly similar round partice shapes and small protrusion on the surface. Scanning transmission electron microscope (STEM) images and energy-dispersive X-ray spectroscopy (EDS) mapping images shows Ir and doped transition metal were uniformly distributed over the nanoparticles  was observed in the formation of an amorphous or defect structure on the surface31. Specifically, non-doped bare IrO2 NPs have four main peaks at 530.1, 532.9, 539.5, and 544 eV , which are matched with those of typical rutile IrO2 structures32. Strong and relative sharp absorption at 530 eV and 533 eV were reported to be induced by the Ir4+ and O2− in rutile IrO2 from a computed O K-edge spectrum, as well31.Figure 2 NPs have O K-edge features at similar position, peak shape and relative intensity near 530 eV was dramatically modified depending on the types of the dopants. In detail, both of the Cr-IrO2 and Mn-IrO2 NPs had particularly low intensity of peak a, and the broaden peak around 530 eV due to strong appearance of an additional peak (peak e) at a photon energy of 529 eV  Cr and Mn which changes the O states the most, (ii) Fe and Co which changes in a milder amount, (iii) Ni which changes least.Notably, although all of the TM-IrO eV Fig. . These drO2 Fig. . In othe2 NPs are compared in the O 1 s and Ir 4f regions , which might be associated with the defective oxygen structure in IrO2, and the other at ~533 eV 35. Similarly, a new set of minor peaks generated in the Ir 4f spectra at a higher binding energy state, which may have been attributed to Ir3+. The Ir3+ state has often been reported to be present in amorphous or anodized IrOx structures. Note that at both O 1 s and Ir 4f spectra, the intensities of the minor signals (i.e. Ov and Ir3+) were higher for Cr- or Mn-IrO2 NPs than those for the other dopant-containing IrO2 NPs, which provided a hint that these features are related to each other. These HRPES spectra indicate quite consistent dependence with those of the O K-edge XAS spectra.Next, HRPES spectra of TM-IrO2 NPs were evaluated in the alkaline 1 M KOH electrolyte condition  normalized by the catalyst loading was the same as that of the current density (mA/cm2). Cr-IrO2 and Mn-IrO2 showed the greatest increases in OER activity, Fe-IrO2 and Co-IrO2 showed modest increases, and Ni-IrO2 showed a rather decrease in activity, compared to the IrO2 NPs,  values of Cr-IrO2 and Mn-IrO2 were 267 mV and 279 mV, about 35 mV less than that of the IrO2 NPs. It is notable the enhanced overpotentials of these TM-IrO2 NPs are also comparable values with those of the high performing OER catalysts36 , the bound state (denoted as S2), and sulfonic acid (SO3H) (denoted as S3), respectively. Since sulfonic acid is formed as the result of the oxidation product of the thiol group38, and the unreacted one is observed at peak S1, we monitored the oxidation of 2-ATP by measuring the ratio of the intensity of S3 to that of S1. These oxidation also showed the same activity trends  Cr-IrO2 and Mn-IrO2 which had similarly high enhancement in activity, (ii) Fe-IrO2 and Co-IrO2 which had less enhancement, and (iii) Ni-IrO2 which had similar activity compared with non-doped IrO2. These results indicate there can be a common main contributor to catalytic activity, which is independent on the specific environmental conditions of each reation. In other words, changes in the electrical conductivity might appear important to determine electrocatalytic OER reaction activity but they are not expected to be critical for thermal CO oxidation. Or, doping in a photocatalyst material has been demonstrated to be effective to vary bandgaps or band positions affecting the photocatalytic acitivty, but it would not matter directly to electrochemical or thermal catalytic activity.By characterizing oxidation activites related to the electronic structures of bare IrO2 were modified especially in the O K-edge XAS signal related with O 2p and Ir 4d orbital interaction. A higher proportion of defect states appeared in Cr-IrO2 and Mn-IrO2 and decreased with the transition metal dopant in the order of Fe, Co, and Ni across the periodic table. Interestingly, the electronegativity values of Cr and Mn are close to each other as 1.66 and 1.55, respectively, and those of Fe and Co are similar as 1.83 and 1.88, respectivley, while Ni is slightly larger, 1.91. All of these values are smaller than the electronegativity value of Ir (=2.2). This electronegativity hypothesis and the lower level of hybridization between the doped metal d and O 2p orbitals may explain our results in a fairly straightforward and reasonable manner, but this explanation requires further studies to be proven.Based on the spectroscopic surface analysis studies, a reasonable explanation is that the dopant metals disturb the Ir-O interaction and create oxygen vacancy defective states which are crucial to determine the catalytic activity of various oxidation reaciton. It was observed that electronic states of TM-IrO2 nanoparticles were prepared and their catalytic activities were compared for three different types of oxidation reactions such as electrochemical oxygen evolution reaction, photocatalytic 2-ATP thiol oxidation reaction, and thermal CO conversion to CO2. Interestingly, Cr-IrO2 or Mn-IrO2 showed the most superior catalytic activity for all oxidation reactions followed by Fe-IrO2 and Co-IrO2, and Ni-IrO2 had similar or even decreased activity compared with non-doped IrO2 NPs. In other words, these five TM-IrO2 catalysts were classified into three group in terms of enhancement of the catalytic activity. For instance, the Cr- or Mn-IrO2 exhibited decreased OER overpotential at 10 mA/cm2 by 35~30 mV compared with non-doped IrO2, while the overpotential of Ni-IrO2 rather increased by ~30 mV. These activity trends were also in good agreement with our STXM and HRPES measurements that showing Cr-IrO2 and Mn-IrO2 had the most changes in O K-edge or Ir 4f spectra related to the oxygen vacancy sites of IrO2. Meanwhile, Fe- or Co-IrO2 had mild, and Ni-IrO2 had smallest evolution of defect-related features compared with IrO2. These combined experimental results suggest that, among five transition metals, Cr- or Mn- dopant in IrO2 are the most effective to generate defect structures by modifying the electronic/chemical states and thus contribute to enhanced activity for various oxidation reactions.In this study, transition metal doped IrO2IrCl6) and all dopant precursors (such as Cr(NO3)3∙9H2O, Mn(NO3)2∙xH2O, Fe(NO3)3∙9H2O, Co(NO3)3∙9H2O, and Ni(NO3)2∙6H2O) were purchased from Sigma-Aldrich, and sodium hydroxide (NaOH) was obtained from Samchun. All aqueous solutions were prepared using double distilled water (DDW).Potassium hexachloroiridate  was dissolved in water in a flask to produce 100 mL of the corresponding aqueous solution, to which 1 mL of an aqueous NaOH solution (25 wt%) was added. The flask was capped with a foil, the solution in the flask mixed, and immediately thereafter the foil cap was taken off and the flask was placed in a 90 °C oil bath for 20 min. The resultant solution was subsequently cooled to room temperature in an ice bath and stored in a capped vial for more than 24 hours. Upon being heated, the initially dark green solution became increasingly transparent, but then dark blue at 90 °C, as desired. An increasing quantity of particles were seen over time after the heating was completed. After being stored at room temperature, the upper layers of the heated solution separated from the upper level of the upper stream, and sediment eventually formed over this separation. But when heated to 90 °C, no such sediment was found.The IrO2 with different metal contents, the desired amount of dopant (TM(NO3)x∙nH2O solution) was determined in each case by calculation of mole fraction and then added into a solution of IrO2 NPs under continuous stirring. The mixed solution was transferred to a Teflon-lined autoclave, then placed in a convection oven preheated at 220 °C for seven hours. The product was washed and collected by precipitation several times with copious amounts of DDW.To prepare 5 wt% doped TM-IrO+ ion CW (514.5-nm-wavelength) laser. The morphologies of the samples were characterized by using a field-emission scanning electron microscope  operating at an acceleration voltage of 10 kV. Element distributions of Ir and dopant metal were characterized by using a scanning transmission microscope, energy-dispersive X-ray spectroscopy . A scanning transmission X-ray microscopy (STXM) was performed at the 10A beamline of the Pohang Accelerator Laboratory (PAL). A Fresnel zone plate with an outermost zone width of 25 nm was used to focus the X-rays onto the TM-IrO2 on the TEM grids. Image stacks were acquired using X-ray absorption spectroscopy (XAS) to extract the O K-edge spectra. High-resolution photoemission spectroscopy (HRPES) experiments were performed at the 8A1 beamline of the PAL with an electron analyzer . The binding energies of the core level spectra were determined with respect to the binding energy (EB = 84.0 eV) of the clean Au 4 f core level for the same photon energy.Raman spectra were obtained by using a spectrometer  with an Ar2 NP and TM-IrO2 sample (10 mg) was dispersed in a solution containing Nafion  and either absolute ethanol  or 2-propanol  by ultra-sonicating the mixture for 30 min. The prepared IrO2 NP or TM-IrO2 catalyst ink samples were drop-cast on a clean glassy carbon electrode (Alfa Aesar), and the area density of the loaded nanoparticle catalyst was controlled to be 2.8 ± 0.4 mg/cm2. The catalyst nanoparticles deposited on the glassy carbon electrodes were masked to have an exposed area of 0.5 cm2. The electrochemical OER activities were measured in 1 M KOH , pH 13.7, by carrying out cyclic voltammetry at a scan rate of 10 mV/sec using a potentiostat (Ivium Technologies). A coiled Pt wire and an Hg/HgO  electrode were used as a counter and reference electrode, respectively. All of the measured potentials were converted to being versus a reversible hydrogen electrode (RHE) using the equationThe electrocatalytic performances of the prepared nanoparticles were compared for the OER in 1 M KOH electrolyte. Each IrOs), electrochemical impedance spectra (EIS) were acquired in the frequency range 100 kHz – 0.1 Hz, and a typical Rs of ~5–10 Ω was obtained in 1 M KOH electrolyte.To compensate for the potential drop resulting from solution resistance  was purified by carrying out turbo pumping prior to dosing it onto the IrO2 NPs and the five TM-IrO2 samples. The dosing was carried out by using a direct dozer controlled by means of a variable leak valve. The resulting samples were then irradiated with UV light  through a quartz window of a vacuum chamber. The pressure of the chamber was maintained at 10−6 Torr during dosing, and the number of exposed molecules was defined by the dosing time in seconds, i.e., with one Langmuir (L) corresponding to one second of dosing under 10−6 Torr.2-Aminothio-phenol , and UV lamp was turned on through the view-port of UHV chamber to activate O2. The CO oxidation reaction was conducted for 1 hour and the amount of the generated CO2 was measured by a Hiden RC 301 mass spectroscopy (mass range ~300 amu). The reaction was repeated by five times at each condition and the average conversion rate was taken.The synthesized catalyst was dispersed in an aqueous solution (0.05 mg/10 mL) which was spin-coated to load on a copper substrate. Then, the catalyst loaded substrate was transferred into the ultra high vacuum (UHV) chamber for CO oxidation reaction. CO gas was flew into the chamber until the partial pressure became 10Supplementary files


Recent studies have shown that BMSCs have a putative ability to promote neurogenesis and produce behavioral and functional improvement. Our previous study demonstrated that co-treatment of G-CSF and BMSCs have beneficial effects on Parkinson's models. The main purpose of this research was to investigate the effects of these two factors on oxidative stress factors in the brain of Parkinson's rat. 6) and G-CSF (75 µg/kg) were used for treatment after creating the PD model. After four weeks, the brains of rats were removed and processed for immunohistochemical studies, such as TH-positive neurons as well as analysis of oxidative stress factors. Adult male Wistar rats (weighing 200–250 g) were used and randomly divided into five groups of seven each. To create the Parkinson's model, 6-OHDA was injected into the left SNpc. The BMSCs (2 × 10The results showed that the injected BMSCs could cross the BBB. The injected cells are also able to settle in different areas of the brain. Analyses of the brain oxidative stress factors showed that G-CSF and BMSCs reduced the expression of MDA and induced the activity of SOD, GSH-Px, and FRAP. Co-administration of G-CSF and BMSCs reduced the expression of pro-inflammatory cytokines and induced the activity of antioxidant enzymes; however, neurogenesis increased in the brain.  The main cause of this disease is the destruction of DA neurons in the pathway of nigrostriatal system. The symptoms of PD begin to appear when nearly 60% of the DA neurons of the SNpc is eliminated[2]. Different factors are involved in the development of PD. Oxidative stress is one of most the key factors affecting PD.Parkinson's disease is a widespread and popular neurodegenerative disorder worldwide[3]. BMSCs express unique surface markers such as CD29 and CD44, but they are not able to express hematopoietic cell markers, CD34 and CD45[4]. One of the advantages of BMSCs, in contrast to embryonic stem cells, is the low possibility of rejection after transplantation. Hence, the application of adult BMSCs rather than embryonic stem cells has been recommended[5]. BMSCs are capable of renovating themselves. They can differentiate into other cells, depending on their fetal origin[6]. G-CSF can pass the BBB[3], and in addition to the anti-apoptotic effect on the hematopoietic system, it has a neuroprotective impact on the 6-OHDA model of PD[7] and brain damage after hypoxia[8]. This neuroprotective effect is mediated by direct activity function of G-CSF on neurons. G-CSF has also strong anti-apoptotic activity in neuronal cells and induces the pathways of STAT3 and ERK 1/2/5 to promote the survival of the CNS[9]. However, in other cell types, G-CSF activates ERK5 pathway[11]. The greatest effect of the anti-apoptotic activity of G-CSF is in the PI3K/Akt pathway[12]. G-CSF induces neurogenesis in the CNS[13] and decreases systemic inflammatory reactions[14].G-CSF is a growth factor with the molecular weight of 19.6 kDa. Some cells such as monocytes, macrophages, endothelial cells, and fibroblasts secrete G-CSF in the body. By inhibiting apoptosis process, G-CSF is able to increase the survival and proliferation of neutrophil. Receptors of G-CSF have been indicated to be able to re-express extensively in the CNS[15]. Oxidative stress enzymes such as SOD, CAT, and GSH-Px act as causative factors in promotion of several neurodegenerative disorders, such as PD, Alzheimer, and amyotrophic lateral sclerosis[16]. Oxidative stress can initiate the mitochondria and endoplasmic reticulum dysfunction, leading to protein misfolding in neurons and then apoptosis. Mitochondria are important organells in the production of ROS in the cells[17]. Dysfunction of mitochondria results in decreased ATP production. The accumulation of oxidative stress causes changes in mitochondria, including deformation, calcium channel destruction, DNA damage, and eventually cell death[18]. Destruction of mitochondrial complex I is the main cause of ROS accumulation in DA neurons in PD, and following the destruction, the respiratory electron transport chain will be arrested, and the production of oxygen decreases, which in turn result in elevated levels of ROS products inside the cells[19]. Therefore, the irregular activity of mitochondria is the main source of intracellular ROS production that will lead to the destruction of DA neurons in PD[20]. The main purpose of this research was to investigate the effects of mesenchymal stem cells and G-CSF on oxidative stress in the brain of Parkinson's rat.Research has confirmed the key role of oxidative stress in PD pathogenesis. For instance, it has been shown that the increased level of oxidative stress markers in the SNpc induces nigral cell degenerationExperimental protocol6) by dorsal caudal vein one week after PD. The fourth group was treated with G-CSF (75 µg/kg) intraperitoneally for seven days, one week after PD. The fifth group received BMSCs (2 × 106) by dorsal caudal vein and G-CSF (75 µg/kg) intraperitoneally for seven days, one week after PD. All the rats were decapitated four weeks after the last behavioral test, and their brains were prepared for immunohistochemical investigations, including staining of TH-positive cells and analysis of stress oxidative markers.A total of 35 adult male Wistar rats, weighing 200-250 g at the beginning of the experiment, were provided by the Experimental Center of Semnan Medical University, Semnan, Iran. All the rats were housed at three or four rats per cage with free access to food and water. The temperature of the storage room was maintained at 20–23 °C and simulated daylight conditions (12-h dark and 12-h light). All the stages of testing were carried out in accordance with the Ethics Committee of Semnan Medical University Semnan . Rats were randomly divided into five groups (n = 7 in each group). The first group received only culture media (DMEM) as the control. The second group was given 4 µg of 6-OHDA in the left substantia nigra as the PD model. The third group received BMSCs  were used. Rats were then placed in a stereotaxic instrument . The skull was exposed by an incision in the scalp, and a single hole was drilled on the left side of the SNpc at the following positions according to the rat brain in stereotaxic coordinates: AP = -4.8 mm anterior to bregma, ML = -1.6 mm lateral to the midline, DV = 8.2 mm vertical from the dura. Next, 4 µl of 6-OHDA (2 µg/µl) dissolved in 2 mg/ml of ascorbic acid solution (Sigma-Aldrich) in saline was administrated to the SNpc with a 28-gauge Hamilton syringe. Next, 6-OHDA was injected to the left side at the rate of 1 µl/minBehavioral testingMotor imbalance was evaluated by the apomorphine-induced rotational test. All behavioral testings were performed by an observer blinded to the group. All the groups were tested for rotational behavior one week after the first surgery and also 1, 2, 3, 4 weeks after the treatment. The apomorphine hydrochloride of 2.5 mg/kg (Sigma-Aldrich) was used for the behavioral test. Complete rotations in the opposite direction of the lesion were counted for 30 minutes. The value was expressed as contralateral net turns/min.BMSCs culture in vitro2 culture flask containing Dulbecco's modified eagle medium  supplemented with 10% fetal bovine serum , 1% (v/v) penicillin/streptomycin (Gibco) in a 5% CO2 incubator at 37 °C. After two days, non-adherent cells were decanted, and adherent cells were used. The BMSCs were isolated on the basis of their ability to adhere to the flasks. Subsequently, incubation was continued, and the medium was changed at three-day intervals. BMSCs were allowed to grow until 70–80% confluency and then subjected to flow cytometry analysis.BMSCs were isolated under the sterile conditions from tibias and femurs of adult male Wistar rats . The bone marrow was flushed with Hank's balanced salt solution using a syringe with a 21-gauge needle. For removing debris, the cell suspension was filtered through a cell strainer (100 μm), and then all the cells were centrifuged and cultured into each 75-cmAnalysis of cell surface antigen markers4 cells/ml. Here, a combination of positive and negative markers were used. In this regard, CD34 and CD45 (as hematopoietic markers) and CD29 and CD44  were considered to characterize the isolated BMSCs. Cells were incubated directly with fluorescence-labeled monoclonal antibodies against CD29, CD34, CD44, and CD45 (Sigma Aldrich). Samples were analyzed using a FACSCalibur flow cytometry apparatus[22].The expression of surface markers on mesenchymal stem cells was performed after the third cell passage by flow cytometry technique. Briefly, cells from bone marrow source were harvested and centrifuged at 180 ×g at 4 °C for 3 min. The pellets were washed three times with a cold stain buffer, filtered through a cell strainer (100 µm) and re-suspended in cold stain buffer to the concentration of 2 × 10DiI labeling 6 cells  under 5% CO2 at 37 °C for 2 h. Then 2 × 106 cells were separated and injected into the vena caudal.CM-DiI is a carbocyanine membrane dye that exhibits increased fluorescence upon the insertion of its lipophilic hydrocarbon chains into the lipid membrane of cells. The high photostability and continual fluorescence of the dye serve as an effective dye for the recognition of neuronal structure. Fluorescence of CM-DiI-labeled BMSCs showed a strong red signal at 600 nm. For CM-DiI labeling of BMSCs, these cells were incubated with 5 µg CM-DiI /10Immunohistochemical and histological study2O2 at darkness for 8 min, washed several times with Tris (pH 7.4) and incubated in the citrate buffer (pH 7.6) at 98 °C for 10-15 min. The sections were then washed three times (each time for three min) with Tris (pH 7.4) and then blocked in 10% normal goat serum, 1% BSA, 0.3% Triton X-100 in PBS at 25 °C for 2 hours. Sections with the primary antibody  were incubated with 0.3% TBS/1% BSA at 4 °C overnight. The sections were then washed three times in TBS for 5 min. Biotin-conjugated secondary antibody incubation  was performed at room temperature (25 °C) for 2 h. After several washes in PBS, cells were incubated at 25 °C in 1% 3-3’-diaminobenzidine (Abcam) at darkness for 10 minutes. The sections were then mounted onto gelatin-coated slides and coverslipped after dehydration in ascending concentrations of ethanol-xylene solutions. The Dil-labeled BMSCs were prepared as described above and injected into the dorsal caudal vein of rats. The numbers of stained cell bodies were counted by an observer blinded to the history of treatment. NIH Image J software was used for counting the sections. After determining the area of the SNpc at low magnification (objective 4×) to avoid the repeated counting of neurons, immunopositive cells were counted only when their nuclei were optically visible. In order to investigate DA neuronal population within the SNpc, immunohistochemical study was conducted. Ater anesthesia, animals were intracardially perfused with saline and 4% fixative solution of paraformaldehyde in 0.1 M of phosphate buffer (pH 7.4). The brains were removed from the skull and fixed in 2.5% paraformaldehyde for one week. Coronal sections of 6 µm were then prepared. Tissue sections were blocked in 10% methanol and HMeasurement of oxidative stress markers[23]. The activities of MDA, SOD, and GSH-Px, and the content of FRAP in the midbrain were determined following the kit specifications (Abcam).At the end of the experiments, after deep anesthesia, rats were decapitated, brains were removed and dissected on an ice-cold glass plate. For assessing stress oxidative markers such as MDA, SOD, GSH-Px, and FRAP, after decapitation, the bilateral midbrain immediately was isolated from the brain stems. Samples were weighed exactly and prepared with 0.90% normal saline to give 10% tissue homogenate by super-audible cell disintegrator (Sonicater), then centrifuged at 3000 ×g/min at 4 °C for 20 min. The supernatant was collected and kept at -70 °C until use. The protein concentration of the substantia nigra was determined by Bradford methodStatistical analysisp < 0.05 was considered to be statistically significant.Results were presented as the units of activity per mg of protein (wet weight) or content. All data were expressed as mean ± SD. Data were analyzed by one-way ANOVA, followed by Tukey's post hoc test for DA neuron counts. A value of Flow cytometryFlow cytometry analysis of BMSCs after the third passage demonstrated that the cells were negative for surface expression of CD45 and CD34, but positive for surface expression of CD29 and CD 44, indicating the presence of stem cell markers on the surface of isolated cells .Behavioral results p < 0.01). The mean rotation in the PD group was 8.56 ± 2.1 circles/min. Rotation in the PD group was also higher than that of all the treatment groups, and the difference was significant, as compared to the other groups (p < 0.01). There was no significant difference in the number of rotations in BMSCs therapy and G-CSF (p < 0.01). In the group of G-CSF plus BMSCs, contralateral rotations significantly ameliorated in comparison to the other groups . In all the groups, the mean rotations at the end of tests were less than those at the beginning. In the G-CSF plus BMSC groups, the number of rotations at the end of treatment was lower than all other treatment groups and was close to the control group.The opposite rotation was induced by apomorphine hydrochloride in rats inside the cage . In the Immunohistochemistry+ cells in the 6-OHDA group in SNpc , and there were significant differences between the control and PD groups. The TH+ neurons in the G-CSF and BMSCs groups were 63.2 ± 7.2 and 75.3 ± 4.9, respectively, but 123.5 ± 7.3 in the G-CSF plus BMSCs group (p < 0.05). The highest number of TH+ neurons was observed in the G-CSF plus BMSCs group. There was a significant difference between the PD and other therapeutic groups (p < 0.001). The results confirmed significant difference between the G-CSF and BMSC groups (p < 0.05). TH staining showed significant preservation of DA neurons in all treatment groups. Therefore, it seems that G-CSF and BMSCs protect DA neurons in SNpc or may cause the migration and differentiation of stem cells from other areas of the affected site.TH staining revealed the significant reduction of THCM-DiI stainingCM-DiI staining was carried out to assess the location of transplanted cells. In the histological sections, it was found that the injected cells were distributed sporadically in different parts of the brain, and a large numbers of cells are located in the SNpc .Oxidative stress markersp ≤ 0.01). The levels of GSH-Px significantly decreased following treatment with 6-OHDA as compared with the control (3.12 ± 0.15 vs. 6.23 ± 0.12). GSH-Px amount in animals co-administrated with G-CSF plus BMSCs (5.16 ± 0.11) and G-CSF alone (4.69 ± 0.11) was closer to the control group (6.23 ± 0.12). There were no significant differences between BMSCs treatment and the PD group (4.14 ± 0.08 vs. 3.12 ± 0.15). Also, no significant difference was found between G-CSF plus BMSCs and the control groups (5.16 ± 0.11 vs. 6.23 ± 0.12). Based on the the data in p ≤ 0.05). The activity of SOD in PD group reduced by 44% when compared to the controls. However, in G-CSF and G-CSF plus BMSCs receiving groups , the activity increased compared to the control (23.21 ± 2.19). Also, there were no significant differences between the BMSCs-treated group and PD group (11.35 ± 2.11 vs. 10.2 ± 3.17). Results showed a significant decrease in the activity of FRAP upon 6-OHDA treatment compared to the control . FRAP level significantly increased in the G-CSF plus BMSCs (1.79 ± 0.3) and G-CSF administration alone 1.13 ± 0.1), but in the BMSCs-treated group, no significant change was found in comparison to 6-OHDA group.The results of oxidative stress tests showed changes in lipid peroxidation and antioxidant enzymes after the administration of BMSCs and G-CSF . MDA lev[23]. Studies have shown that in addition to neuroprotective effect, G-CSF has a potential role in the migration of progenitor cells from bone marrow to peripheral blood[24]. The neuroprotective effect of G-CSF has been attributed to the inhibitory activity of G-CSF on the main inflammatory mediators such as interleukin-1, tumor necrosis factor-alpha, and interferon gamma[9]. More importantly, G-CSF is able to cross the BBB and induces neurogenesis in the CNS, increases synaptic plasticity and influences neurons and glial cells in the injured area. G-CSF has also inhibitory actions upon pro-inflammatory cytokines while up-regulating neurogenesis[25]. Similar to G-CSF, stem cells are able to cross the BBB. G-CSF causes the migration of stem cells from the bone marrow to the bloodstream. Therefore, G-CSF is capable of migrating injected stem cells from the blood into the brain[5].The results of immunohohechemical staining showed that G-CSF is capable of migrating injected stem cells into the SNpc. It also changed the stress oxidative factors in the brain. The G-CSF is one of the most important components of the hematopoietic growth factor family6 BMSCs were injected via the dorsal caudal vein. This number of BMSCs, which was chosen to prevent clotting in the bloodstream, is usually used for in situ injection in the brain. The dorsal caudal vein was selected to prevent any damage to the brain.  Exogenous cells may be trapped in the shown that parenchyma of some internal organs such as the lungs, liver, and even in the spleen. Our previous study has the low number of injected cells (2 × 105 BMSCs) from the dorsal caudal vein with G-CSF (70 µg/kg) could not promote the migration of BMSCs into the brain[1]. In this project, after DiI labeling, we observed that, the labeled BMSCs were present in different parts of the brain and even in SNpc. We concluded that G-CSF facilitates the transit of the stem cells from the BBB and deposits into different parts of the brain. In addition, the injection of the BMSCs or G-CSF alone led to the behavioral improvement on animals, but no significant difference was observed between these two treatment groups. On the other hand, the number of TH+ neurons between the two groups did not show any significant difference. Therefore, we draw the conclusion that the injection of BMSCs or G-CSF (75 µg/kg) gives similar results. Treatment with the optimal dose of G-CSF (75 µg/kg) combined with BMSCs significantly increased TH+ neurons in comparison to all the treatment groups. Therefore, these two factors seem to have a synergistic effect. BMSCs will protect the DA neurons by modulating the activity of microglial cells as well as modulating their anti-inflammatory effects[26]. We suppose that BMSCs and G-CSFs can trigger certain factors or modulate expression of pro-inflammatory cytokines, which have a protective effect on DA neurons. The results showed statistically significant differences between the group of G-CSF plus BMSCs and BMSCs and G-CSF alone. It can be concluded that G-CSF is cooperated synergistically with BMSC. The co-administration of G-CSF and BMSCs facilitates the stability of the damaged area. G-CSF plays a role in healing the damaged nervous system by activating mechanisms such as anti-inflammatory pathways[27], anti-apoptotic effects, differentiation of adult stem cells, induction of angiogenesis and vasculogenesis, and renovation of the BBB[28] .In this study, 2 × 10[29]. We assume that G-CSF is the specific factor for the reduction of MDA and acts as an antioxidant. Therefore, it is likely that MDA levels are reduced by G-CSF through the boosting of release, migration and differentiation of BMSC, which ultimately results in the protection of damaged cells. Our investigations demonstrated that G-CSF significantly increased the level of SOD and GSH-Px compared to the PD group. The results also showed that the activity of the GSH-Px decreased in PD patients, but significantly increased in the G-CSF and G-CSF plus BMSC groups compared to the PD group. These results are in agreement with that of other studies showing that GSH-Px activity decreased in the substantia nigra of PD patients[30]. The results from our study revealed that the level of MDA increased in the brain of PD group, thereby increasing the lipid peroxidation in PD rats. Our results also showed a decrease in the level of MDA in G-CSF and G-CSF plus BMSCs groups. These results are consistent with previous studies that reported enhanced oxidative stress markers in the plasma, cerebrospinal ﬂuid, and the substantia nigra in PD2O2 in the SNpc. This part of the brain contains a high concentration of iron. All the conditions are gathered together for the transformation of H2O2 into OH by the Fenton’s reaction[31]. This free active radical can attack and destruct both lipids and proteins and then leads to the neuronal oxidative damage. In the brain, H2O2 is mainly catabolized by GSH-Px, a major enzyme in the development of neurodegenerative diseases[32]. Our study disclosed that G-CSF could result in the elevation of GSH-Px in SNpc. The key roles of G-CSF in CNS have been identified in new studies. G-CSF is able to bind to specific receptors (G-CSF receptor) in different cells such as monocytes, hematopoietic progenitor cells, neurons, platelets, endothelial cells, and small-cell lung cancer cells. With the activation of the G-CSF receptors, the signaling cascades including the Janus kinase/signal transducer and transcription activator, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Protein kinase B/Akt pathways will be stimulated. Activation of these cascades leads to the cellular proliferation, activation of anti-inflammatory and antiapoptotic processes and also mobilizes stem cells to target sites (sites of injury)[14]. According to these findings, G-CSF is a new, attractive and cost-effective factor for the treatment of neurodegenerative diseases. In Parkinson’s patients, reduction of erythrocyte GSH-Px activity could lead to the accumulation of HThe present data indicate that G-CSF plays more important role than the BMSCs in PD. Increased MDA and decreased SOD and GSH-Px cause mitochondrial and then neuronal destruction. These events could eventually participate in PD pahogenesis. The decreased activity of these enzymes is indirectly responsible for neuronal loss and probably has an essential role in the onset of PD. The balance of these enzymes will protect nerve cells. Based on our findings, G-CSF administration along with BMSCs injection elevates SOD and GSH-Px in PD; however, extensive research is needed to further understand the mechanisms involved in the development of this disease.


High-sensitivity cardiac troponin testing is used in the diagnosis of acute coronary syndromes but its role during convalescence is unknown. We investigated the long-term prognostic significance of serial convalescent high-sensitivity cardiac troponin concentrations following acute coronary syndrome.In a prospective multicentre observational cohort study of 2140 patients with acute coronary syndrome, cardiac troponin I concentrations were measured in 1776 patients at 4 and 12 months following the index event. Patients were stratified into three groups according to the troponin concentration at 4 months using the 99th centile  and median concentration of those within the reference range. The primary outcome was cardiovascular death.Troponin concentrations at 4 months were measurable in 99.0% (1759/1776) of patients , and were ≤5 ng/L (median) and >99th centile in 44.8% (795) and 9.3% (166), respectively. There were 202 (11.4%) cardiovascular deaths after a median of 4.8 years. After adjusting for the Global Registry of Acute Coronary Events score, troponin remained an independent predictor of cardiovascular death  with the highest risk observed in those with increasing concentrations at 12 months. Patients with 4-month troponin concentrations >99th centile were at increased risk of cardiovascular death compared with those ≤5 ng/L (29.5% (49/166) vs 4.3% (34/795); adjusted HR 4.9, 95% CI 3.8 to 23.7).Convalescent cardiac troponin concentrations predict long-term cardiovascular death following acute coronary syndrome. Recognising this risk by monitoring troponin may improve targeting of therapeutic interventions.ACTRN12605000431628;Results. In the assessment of patients with suspected acute coronary syndrome, international guidelines recommend serial measurement of cardiac troponin in accordance with the universal definition of myocardial infarction.Despite the widely accepted view that troponin concentrations typically return to baseline levels within 2 weeks, the exact time course of resolution of cardiac troponin following myocardial infarction remains unclear and several reports have described persistent elevations up to 7 weeks following initial presentation.There is now emerging evidence from posthoc analysis of clinical trial populations that increased troponin concentrations during the early and intermediate postmyocardial infarction period confer greater risk of recurrent cardiovascular events.From 2002 to 2009, 2140 patients were enrolled into the Coronary Disease Cohort Study (CDCS), a prospective, multicentre  observational cohort study in New Zealand designed to explore the determinants of long-term risk following acute coronary syndrome.10.1136/heartjnl-2019-315084.supp1Supplementary dataPatients underwent comprehensive clinical assessment including a 12-lead ECG, echocardiography and blood sampling at 1, 4 and 12 months following the index event, with additional annual review for at least 2 years online . EchocarSTAT assay , which has a limit of detection of 1.2 ng/L, coefficient of variation <10% at 3.0 ng/L and 99th centile upper reference limit of 16 ng/L for women and 34 ng/L for men.Following collection and centrifugation, EDTA-plasma blood samples were stored at −80°C until analysed. High-sensitivity cardiac troponin I concentrations were determined using the ARCHITECTThe primary endpoint was cardiovascular death following the 4-month visit. Secondary endpoints included all-cause death, non-cardiovascular death, fatal or non-fatal myocardial infarction, heart failure hospitalisation, the composite of cardiovascular death or non-fatal myocardial infarction and hospitalisation with non-fatal stroke.Patient characteristics were reported by groups determined from the 4-month troponin concentration rounded to the nearest integer value. Groups 1 and 2 comprised those below and above the median troponin concentration excluding those in group 3 with concentrations above the sex-specific 99th centiles. Cox regression results are reported as HRs per doubling of troponin concentration, both unadjusted and following adjustment for the Global Registry of Acute Coronary Events (GRACE) risk score at time of discharge following index hospitalisation.In determining the relationship between troponin concentrations and treatment, we defined optimal medical therapy at discharge as per current clinical guidelines.Clinical determinants of cardiac troponin concentrations were determined from linear mixed models. The slope  and intercept were included as random effects. Time varying covariates were centred within-person as per Curran and Bauer.The negative predictive values for the primary outcome were established across a range of troponin concentrations starting at 2 ng/L with a negative predictive value at 5 years of 97.5% evaluated as an acceptable threshold indicator of low risk.In a landmark analysis following the 12-month visit, the additional prognostic information provided by serial changes in troponin concentration was determined by including 12-month troponin concentrations in a Cox proportional hazards model adjusting for 4-month troponin concentrations. Patients were included in this analysis if they had troponin results available from all three study visits and had not experienced a recurrent myocardial infarction prior to the 12-month visit (n=1513).Data are presented as mean (±SD) or median (IQR) with 95% CI with two-tailed p values reported. Statistical analysis was performed using R V.3.4.3 . Additional details of the statistical analyses undertaken are described in the online After excluding patients with recurrent myocardial infarction prior to the 4-month visit, 1776 patients  had troponin concentrations measured at the second study visit (128 (IQR 116–140) days) and were included in the analysis . The aveMedian troponin concentrations declined at each successive visit over the first year with median values of 8.2 (IQR 4.2–17.8) ng/L, 6.1 (IQR 3.6–11.5) ng/L and 4.8 (IQR 2.9–8.6) ng/L at the 1-month, 4-month and 12-month visits, respectively  and 166 (9.3%) patients, respectively online . The medDuring follow-up, there were 375 deaths of which 202 were secondary to cardiovascular causes. A threshold of ≤5 ng/L applied at 4 months identified 45% of the study cohort  with a negative predictive value for cardiovascular death at 5 years of 97.1%  and experienced a slower decline in troponin (1.18-fold difference per SD in age; 95% CI 1.13 to 1.23, online In the evaluation of patients following hospitalisation with acute coronary syndrome, convalescent high-sensitivity cardiac troponin conveys powerful long-term prognostic information that adds substantially to currently recognised clinical risk predictors and scores. Indeed, even after adjustment for the GRACE score, we identified a fivefold increase in risk of cardiovascular death, associated with increasing convalescent troponin concentrations. This gradient of risk was continuous across the range of troponin concentrations and was greatest for cardiac events including cardiac death and heart failure hospitalisation.Our study has several important strengths. First, the availability of comprehensive outcome data with more than 200 cardiovascular deaths during up to 9 years of follow-up is a major asset and enabled us to define the long-term prognostic significance of convalescent measurement of troponin. Second, the deliberately broad inclusion criteria ensured recruitment of a diverse and representative cohort of real-world patients with acute coronary syndrome. The even distribution of patients across low-risk, intermediate-risk and high-risk groups as determined by the GRACE risk score offers strong evidence as to the generalisability of our findings. Third, detailed characterisation with repeated clinical, biochemical and echocardiographic assessments at three time points allowed us to explore patterns of change in convalescent troponin concentrations, and how these correlate with both patient characteristics and long-term outcomes.Our findings have clear potential to improve follow-up and management of patients discharged following acute coronary syndrome. We have shown that a single measurement of cardiac troponin during convalescence from an acute coronary syndrome can identify nearly half of patients as low-risk, with 0.5% annual incidence of cardiovascular death. A further 1 in 10 patients have persistent myocardial injury and are identified as very high risk, with annual event rates approaching 10%, for whom careful assessment and more intensive treatment may be warranted. Furthermore, we have demonstrated that for those at intermediate risk, repeated measurement of cardiac troponin concentrations at 12 months can be used to improve risk estimation and enable prognostic reclassification into either low-risk or high-risk groups.Convalescent troponin concentrations are not simply a surrogate for the extent of myocardial injury sustained during the index coronary event as evidenced by the weak correlation between peak troponin during the index hospitalisation and 4-month troponin. Neither are they simply a reflection of left ventricular function. Instead, they provide greater prognostic information than either of these traditional risk indicators and are additive to all other clinical, biochemical and echocardiographic risk factors that might typically be available to clinicians when making treatment decisions. Furthermore, within this real-world observational study, we have revealed a substantial treatment gap whereby less than half of patients with troponin concentrations >99th centile received all guideline-indicated medications. This gap likely reflects an underappreciation of risk by both clinicians and patients and signifies a clear clinical rationale for the prognostic information troponin offers.Although our findings are the first from a prospective observational cohort study, they are consistent with posthoc analyses of randomised controlled trials of both stable coronary diseaseTaken alongside these reports, our study has important clinical implications regarding the measurement of troponin following an acute coronary syndrome. The American Heart Association estimates that 720 000 individuals within the USA alone will experience a first fatal or non-fatal myocardial infarction during 2018.We recognise that our study has some limitations. The biomarker used for the index diagnosis of myocardial infarction varied between study centres and included the troponin T and troponin I assays available during the period of study recruitment. This precludes a direct comparison of the relationship between the peak high-sensitivity troponin concentration and those measured using the high-sensitivity assay during follow-up. However, peak creatine kinase concentrations were available in 91% of patients and have previously been demonstrated to correlate with size of infarct and clinical outcomes.We conclude that the measurement of high-sensitivity cardiac troponin in the convalescent phase following acute coronary syndrome identifies an important group of patients at high risk of recurrent cardiac events and death. Recognition of this increased risk may allow better targeting of secondary prevention therapies and could improve the clinical impact of future therapeutic trials in patients with acute coronary syndrome.In the diagnostic assessment of acute coronary syndromes, serial measurement of plasma cardiac troponin concentrations is recommended by European and North American guidelines. In this context, the detection of troponin concentrations above the 99th centile upper reference limit is associated with increased short-term risk and may help inform early management decisions regarding the need for hospital admission and inpatient coronary angiography. However, the prognostic value and optimal timing for measurement of convalescent troponin following hospital discharge remain unknown.We investigated the long-term prognostic significance of serial convalescent high-sensitivity troponin I concentrations following acute coronary syndrome in a large prospective multicentre observational cohort study with detailed clinical, biochemical and echocardiographic characterisation. We identified a strong association between 4-month troponin concentrations and cardiovascular death over 5 years that was independent of the extent of myocardial injury during the index event and all other established risk factors. Repeated measurement of troponin at 12 months provided additional prognostic information.Low plasma cardiac troponin I concentrations measured 4 months following an index hospitalisation with acute coronary syndrome identified nearly half of all patients as being at very low risk of long-term cardiovascular death. In contrast, 1 in 10 patients have persistent myocardial injury and are at very high risk. These individuals clearly warrant close follow-up, with concerted efforts made to optimise their medical care. For the remaining intermediate-risk patients, repeated measurement of troponin concentrations at 12 months enables prognostic reclassification into either low-risk or high-risk groups.


The aim of this study is to present our treatment protocol for impalpable testis. In a retrospective study we analyzed clinical data including diagnostic procedures, intraoperative findings, final diagnosis, treatment modality, and outcome of patients with impalpable testis who underwent surgery from January 2010 until December 2015. Ninety-one patients were admitted under the diagnosis of impalpable testis. In 39 patients ultrasound detected testis in the inguinal canal and orchidopexy was done. In 25 patients (48.08%) laparoscopy showed the entrance of the spermatic cord into the inguinal canal. Open exploration of the inguinal canal was done, testicular remnant removed, and appropriate testicular prosthesis implanted. Twenty patients (20/52) underwent orchidopexy of the abdominal testis (46.51%), 4 of which underwent Fowler-Stevens procedure in two stages, and in 16 patients deliberation of the testis and spermatic cord was sufficient to place the testis into the scrotum. Excision of the testicular nubbin is highly recommendable, as well as implantation of the testicular prosthesis at the time of orchiectomy. Cryptorchidism or undescended testis is one of the most common congenital malformations in male neonates and is related to a multifactorial process . IncidenDespite spontaneous descent within the first months of life, nearly 1.0% of all male infants still have undescended testes at 1 year of age . The mosFrom January 2010 to December 2015, 493 patients with undescended testis underwent surgery at the Institute for Children and Youth Healthcare of Vojvodina. Ninety-one patients (18.5%) had impalpable testes. A retrospective review was conducted. Data that we collected were diagnostic procedures used for a confirmation of a diagnosis of impalpable testis. Intraoperative findings, final diagnosis, treatment modality, and outcome were noted for every patient. The study was approved by Institutional Ethical Board.All patients with impalpable testis and negative ultrasound underwent abdominal laparoscopy. Open Hasson technique with a subumbilical incision was used to gain entry to the abdomen for laparoscopy, for insertion of the first port and creation of pneumoperitoneum. If the testis was identified intra-abdominally , two 3 oIn the case when no testis was identified or testicular remnant was present during inguinal exploration, the remnant was removed and an adequate testicular prosthesis was implanted during the same operation . The preStatistical analysis was performed using the SPSS program. Fisher exact test was used to compare the categorical data. P value less than 0.05 was considered statistically significant.Ninety-one patients were admitted under the diagnosis of impalpable testis. All patients underwent ultrasound examination that revealed an inguinal testis in thirty-nine patients. All of those patients underwent inguinal orchiopexy.In 52 patients, ultrasound did not detect testis in the inguinal canal. Those patients were treated for impalpable testis by laparoscopy. Patients were 1-17 years old (average 3.83). Forty-three patients (82.69%) had unilateral impalpable testis and eighteen of these patients had right-sided (41.86%) and 25 had left-sided impalpable testis (58.14%). Nine patients (17.31%) had bilateral impalpable testes with either positive or unclear stimulation test. Twenty-five patients (48.08%) underwent laparoscopic exploration that confirmed the entrance of the spermatic cord into the inguinal canal. Open exploration of the inguinal canal was done, testicular remnant removed, and appropriate testicular prosthesis implanted  underwent orchidopexy of the abdominal testis (46.51%), 4 of which underwent Fowler-Stevens procedure in two stages, and in 16 patients deliberation of the testis and spermatic cord was sufficient to place the testis into the scrotum. One of the patients with bilateral abdominal testes was operated in a single stage laparoscopy procedure, but required a subsequent open reorchidopexy on one side. In 7 patients (7/52) laparoscopy confirmed the entrance of the spermatic cord into the inguinal canal, where in three patients we found hypoplastic testis that we decided to leave in place, and in other 4 patients the remnant was removed, but prosthesis was not implanted due to a lack of either the appropriate size or parental consent.Our treatment protocol and results are presented in At the Institute for Child and Youth Healthcare of Vojvodina, a child with undescended testis is followed from birth to the age of 9 months, during which time clinical examination is performed at birth, at the age of 6 months, and at the age of 9 months. Hypertrophic contralateral testis could be correlated with absent or atrophic testis . NeverthAn important step in surgery is a thorough clinical examination once the boy is under general anesthesia, as a previously impalpable testis might be identified during this examination and subsequently the planned laparoscopic surgical approach can be changed to standard inguinal orchidopexy. However, even under anesthesia it can be difficult to palpate a testicular nubbin in the scrotum, especially in an obese child . Some stThe standard management of impalpable testes is surgical, which could be challenging and controversial. Orchidopexy is indicated if the testis is not located in the scrotum after 6 months as it improves fertility rates and allows close monitoring for the development of possible testicular masses . TherefoAnother controversial issue is the need for removal of testicular nubbin. Nubbin is a congenital condition in which no normal testicular tissue can be identified following exploration for a clinically impalpable testis . This inThe absence of a testis from the scrotal sac represents a psychologically traumatic experience in males of any age from childhood to the elderly , 23. TheThe timing of insertion of a testicular prosthesis in a child is not unequivocal. The psychological impact of an absent testicle in a child or adolescent is a good reason to consider implantation at the time of the initial surgery for a cryptorchid testis. The problem is that this may necessitate further surgery to insert a larger prosthesis when the child gets older. An alternative strategy is to delay the placement of the definitive prosthesis until the child reaches adolescence. If a child is content with the size of the initially implanted prosthesis, further surgery can be avoided. The underdeveloped scrotum that accompanies an undescended testicle may fail to accommodate the desired sized testicular prosthesis in adolescence.We believe that recent improvements in our protocol, including introducing the additional clinical examination in anesthesia and switch to initial inguinal approach, hold promise of improved outcomes. Even when inguinal exploration is being done, it is advisable to be ready to perform laparoscopic exploration of the abdomen in case of negative finding in the inguinal canal. Excision of the testicular nubbin is in our experience highly recommendable, as well as implantation of the testicular prosthesis at the time of orchiectomy.