Poor adherence is a major concern in the treatment of attention-deficit/hyperactivity disorder (ADHD). The objective of this study was to evaluate factors linked to early interruption of and low adherence to treatment with osmotic-release oral system methylphenidate hydrochloride (OROS-MPH) in pediatric patients with ADHD. A total of 1353 young people (age 6–17 years) with a diagnosis of ADHD who newly started OROS-MPH were extracted from the pharmacoepidemiological data of 3 million people in Japan. The cohort was retrospectively surveyed every month for 12 months. Ten possible risk factors were extracted from the data and analyzed by multivariable logistic regression. Sensitivity analysis was conducted to ensure the robustness of the analysis. The results revealed that treatment adherence was generally poor, with a tendency for discontinuation in the early stage. Multivariable logistic regression results showed that adherence is reduced by female sex, lower starting dose, and concomitant atomoxetine or hypnotics. These findings may help clinicians to predict the risk of poor adherence in the early stage of treatment and improve not only patients’ symptoms, but also their quality of life. The onset of ADHD occurs mostly in childhood, but 50% to 80% of diagnosed children continue to demonstrate ADHD symptoms into adolescence or adulthood5. The symptoms of ADHD and comorbid disorders can impair an individual’s ability to function in social circumstances, resulting in academic, familial, and social problems7.Attention-deficit/hyperactivity disorder (ADHD) is a common chronic neurodevelopmental disorder characterized by symptoms of inattention, hyperactivity, and impulsivity. Individuals with ADHD are at high risk of comorbid psychiatric disorders, such as oppositional defiant disorder, conduct disorders, depression, bipolar disorders, and anxiety disorders8, the management of children with ADHD should begin with psychosocial treatment. Additional administration of pharmacological therapy should be considered when psychosocial treatments are insufficient. However, as shown by fact-finding surveys on pharmacotherapy for ADHD, most physicians support the need for pharmacotherapy9.In the latest Japanese guidelines for the diagnosis and treatment of ADHD, released in 2016Four types of ADHD medication are currently available in Japan: osmotic-release oral system methylphenidate hydrochloride , atomoxetine , guanfacine hydrochloride (INTUNIV), and lisdexamfetamine dimesylate (VYVANSE). Of these medications, OROS-MPH is the most prescribed. Due to its addictive profile, the circulation management committee strictly controlled its prescription by limiting prescribers’ certification and the prescription duration until the ADHD proper distribution management system is established in December 2019. Accordingly, there is a high likelihood of proper prescription of OROS-MPH in Japan.8 recommended that termination of medication be considered when patients are stable for more than 1 year after they obtain a score of 61 or above on the Global Assessment of Functioning (GAF) scale. Therefore, patients need to adhere to the prescribed treatment regimens for at least 1 year.The latest Japanese guidelinesDespite the high reliability of proper prescription of OROS-MPH medication in Japan because of its strict control, there is an issue with patients’ unstable patterns of outpatient visits and oral intake in clinical practice. This leads to poor adherence or discontinuation of the treatment in the early stage, which could limit treatment effects and increase the risk of comorbid psychiatric disorders. There is a considerable need to understand the prescription patterns of ADHD medications and to identify risk factors associated with poor adherence in order to help patients to adhere to their recommended treatment regimen and to facilitate proper intervention. However, to our knowledge, no study has examined the actual prescription patterns of OROS-MPH and analyzed the possible factors associated with poor adherence in Japan. Therefore, in this study, we evaluated the current prescription trend of OROS-MPH in pediatric ADHD patients in Japan. We also analyzed the factors that could predict poor adherence in the early stage of the treatment, based on data from the claims database.All procedures were performed in accordance with the ethical guidelines for epidemiological research issued by the Japanese Ministry of Health, Labour and Welfare. This study was approved by the Ethics Committee of Akita University Graduate School of Medicine, Akita, Japan, which waived the requirement for obtaining written informed consent.10. All health insurance data included in the JMDC database are anonymized, and permission for secondary use of the data has been obtained from the subscriber.The data used in this research were provided by JMDC Inc. . JMDC Inc. has been collecting claims information from occupation-based health insurance agencies for corporate employees and their dependents since 2005The number of individuals registered in JMDC is about 3 million, which is approximately 2.5% of the entire Japanese population as of June 2016. Each record includes an encrypted personal identifier, age, sex, diagnoses, and prescriptions. The diagnoses are based on International Statistical Classification of Diseases and Related Health Problems, 10th revision (ICD-10) diagnostic codes. The prescription information includes WHO Anatomical Therapeutic Chemical (WHO-ATC) classification system codes, drug name, days of supply, dosage information, and mode of prescription. The date of service is specified up to the month and year.We selected outpatients who were 6 to 17 years when they were first prescribed OROS-MPH between December 2007 and May 2015. We selected only new users who had not been prescribed MPH within 1 year prior to their first prescription during our observation period. Then, we extracted individuals who could be observed for at least 1 year. We also excluded individuals who had a diagnosis of narcolepsy because they are prescribed methylphenidate hydrochloride and not OROS-MPH. A total of 1353 children were included in this study . The conventional 50% cutoff was used.16, male sex11, and ethnic background13, clinical characteristics, such as symptom severity16 and amount of symptom reduction18, quality of ADHD care, such as medication doses18, adverse events19, and concomitant medication11, and factors related to parents or families, such as belief in treatment12, poor family support20, and family history of ADHD14. In this study, we selected sex, age at first OROS-MPH prescription, clinician specialties, mean daily dosage prescribed in the first 3 months, and concomitant medication in the first 3 months.We selected the possible risk factors that might interfere with medication adherence. As mentioned in previous studies, many factors are related to medication continuation and adherence. These include specific child and adolescent characteristics, such as agePatient were divided into three age groups as follows: 6–12 years of age (elementary school students), 13–15 years of age (junior high school students), and 16–17 years of age (high school students or junior high school graduates) according to the Japanese school system.21. Clinician specialties were divided into three groups as follows: psychiatry/psychosomatic medicine, pediatrics, and others. The reasoning for this division of departments is that only members of the Japan Pediatric Society or the Japanese Society of Psychiatry are under the control of the circulation management committee.The mean daily dosage prescribed in the first 3 months was calculated by dividing the sum of the total prescription in the first 3 months by 90 days. The results were divided into three groups: a low-dose group (< 18 mg), middle-dose group (18–27 mg), and high-dose group (≥ 27 mg) according to the dosing schedule based on approvals granted by the Japanese regulatory authorityThe types of concomitant medications were divided into six groups: ATX, antipsychotics, hypnotics, antidepressants, antiepileptics, and anxiolytics  was calculated by correction for the population statistics issued by the statistics bureau of JapanP values < 0.05 were considered statistically significant.We used multivariable logistic regression analysis to assess the association between risk factors and poor adherence. We identified ten possible risk factors Table  and divi24. Sensitivity analysis was performed using the method provided by Fox et al.25.We also conducted sensitivity analysis to ensure the robustness of the analysis. We identified two factors from the literature: ADHD severity and adverse events. ADHD severity was thought to interfere with the mean daily dosage prescribed in the first 3 months, whereas adverse events are thought to interfere with concomitant hypnotic use. The OR for reduced adherence and the ratio of patients who might have possible confounding factors were determined from the literatureDuring the period from the launch of OROS-MPH in Japan (December 2007) to the last month of data acquisition from the Japan Medical Data Center , the estimated prescription rate gradually increased from 0.003 to 0.23% . The mean MPR became smaller as the age at first prescription increased, but there was no statistically significant effect of age on the mean MPR. In addition, clinician specialties were not found to be associated with adherence.The results of the multivariable logistic regression are shown in Table P = 0.000; OR, 2.499; 95% CI, 1.731–3.608; P = 0.000, respectively).Regarding the mean daily dosage prescribed in the first 3 months, adherence was likely to be poor if patients were prescribed doses lower than 18 mg per day and was significantly worse than if they were prescribed 18–27 mg per day and ≥ 27 mg per day  or hypnotics  was significantly associated with poor adherence.In terms of the types of concomitant medication in the first 3 months, concomitant use of ATX . Considering the original risk ratio of 1.52, it is necessary to be cautious about the risk of the occurrence of adverse events in relation to adherence and the concomitant use of hypnotics, and further consideration of this aspect is needed.29. However, the prevalence of ADHD drug use in pediatric patients was lower in Japan (0.4%)30 than in Norway and the United States (1.4–5.3%)32 but similar to that in Italy, France, and the UK (0.2–0.5%)35. In addition, the share of OROS-MPH in 2014 is lower in Japan (64%)30 than in Germany, the UK, and Norway (75–100%)36. Thus, both the prescription of ADHD drug and the share of OROS-MPH are low in Japan.First, this study revealed the actual status of OROS-MPH prescription. The trend for a gradual increase in prescription rates is similar to that of other countries8. Both OROS-MPH and ATX are equally listed as first-line drugs, in contrast to other guidelines that recommend psychostimulants as first choice39. Although there are differences in efficacy and tolerability among ADHD medications41, Japanese physicians may prefer ATX for the initial drug because it has no restriction.One of the reasons for this situation in Japan could be the strict control of OROS-MPH prescription by the circulation management committee to limit the qualification of prescribers and the prescription period. Another possible reason is the indication for the first-line drug of ADHD medication in the Japanese guidelines42. Although guidelines recommend patients take “drug holidays” when the use is extended over a long period39, some patients take these holidays on weekends or during school holidays from the early stage of the treatment.Second, we revealed the erratic prescription patterns of OROS-MPH and analyzed the possible factors that could predict the risk of low adherence in the early stage of the OROS-MPH prescription. This erratic pattern could be caused by patients forgetting to take the medication, intentional discontinuation, or “drug holidays”. The term “drug holiday” refers to the deliberate interruption of pharmacotherapy for a defined period and for a specific clinical purpose11 but less than the mean MPR of 0.64 found in the Netherlands43. Common adherence thresholds in previous studies were 0.844 or 0.745. However, as shown in our results, 90.8% of those with an MPR greater than 0.5 continued to take their medication until the end of the follow-up period. Therefore, we defined an MPR greater than 0.5 as an indication of adherence.The mean MPR in this study was 0.51 ± 0.32. This is similar to the mean MPR of 0.52 ± 0.30 for extended-release stimulants in Texas46. Although OROS-MPH can reduce both externalized and internalized symptoms19, improvements in externalized symptoms are more readily noticed by caregivers. Therefore, boys may more easily perceive the effects of the medication and thus be more likely to continue to take it, as shown in our results.Among possible risk factors for low adherence detected in our study, sex differences had a statistically significant influence on adherence. Compared with girls, boys with ADHD have a tendency to show more externalized symptoms47. Although the occurrence of adverse events might affect adherence, the adverse events that occur in the short term are mostly non-serious49 and lead to withdrawal in less than 10% of the patients who experience them49. In addition, lower doses are usually less likely to cause adverse events compared with higher doses.Regarding a mean daily dosage lower than 18 mg per day, an insufficient treatment effect due to an insufficient dose might be the reason for lower adherence50. Therefore, the patients who were prescribed ATX in the early stage could be in the process of treatment switching or augmentation. Patients undergoing switching or augmentation are likely to have lower adherence because of the risk of additive adverse effects caused by the combination use. Therefore, it is understandable that the patients with concomitant ATX use had lower adherence.In terms of concomitant medication, only ATX and hypnotics were linked to adherence. The common reasons for the prescription of concomitant ADHD medications are an inadequate response and intolerance to the previous treatment53. Therefore, patients with concomitant hypnotic use in the early stage are presumed to originally have had moderate or severe sleep problems. OROS-MPH may worsen sleep status as an adverse effect55, which may negatively affect adherence.Patients with concomitant psychotropic use had lower MPRs in our study. However, only hypnotics exhibited a statistically significant association with adherence. Sleep disturbances can appear as ADHD symptomsKenkō-Hoken). Therefore, there is a potential for bias in socioeconomic and family backgrounds, which could affect adherence20. In addition, there is a lack of clinical detail in the database, such as ADHD symptoms and severity, incidence of adverse events, and coexisting psychiatric diseases. Therefore, it was not possible to determine whether adherence was due to remission of ADHD, ineffectiveness of medication, or aggravated coexisting psychiatric diseases. Thus, we evaluated the effect of these factors by conducting sensitivity analysis. Another limitation is that this database represents the prescription records and not the actual oral intake records. Thus, there is a possibility that patients did not take the medication, even with the prescription. These limitations should be considered in future studies.There are limitations to the use of the JMDC dataset. It is one of the largest claims databases in Japan but includes only 1–2% of all Japanese inhabitants. Additionally, all participants were recipients (or the family members of recipients) of employee’s health insurance (Despite these limitations, the findings of our study reveal the current state of pharmacotherapy for ADHD using OROS-MPH in Japan and provide useful insights into its resolution. Due to the increased prevalence of ADHD and use of psychostimulants, the inappropriate use of and low adherence to psychostimulants have been major problems in Japan. Appropriate medications for young ADHD patients are important not only to improve ADHD symptoms, but also to prevent the resultant disabilities and difficulties in their daily lives.This is the first study to provide clinically important evidence of the relationship between adherence and the possible risk factors in OROS-MPH treatment for children and adolescents with ADHD in Japan using a large-scale claims database. These results can help clinicians to predict the risk of poor adherence in the early stage of treatment and improve not only patients’ symptoms, but also their quality of life.Supplementary Information.


Clinical studies found that medication for attention-deficit/hyperactivity disorder (ADHD) is effective in coexisting autism spectrum disorder (ASD), but current research is based on small clinical studies mainly performed on children or adolescents. We here use register data to examine if individuals with ADHD and coexisting ASD present differences in the prescribing patterns of ADHD medication when compared to individuals with pure ADHD.Data with information on filled prescriptions and diagnoses was retrieved from the Swedish Prescribed Drug Register and the National Patient Register. We identified 34,374 individuals with pure ADHD and 5012 individuals with ADHD and coexisting ASD, aged between 3 and 80 years. The first treatment episode with ADHD medications (≥ 2 filled prescriptions within 90 days) and daily doses of methylphenidate during a 3-year period was measured. Odds ratios (ORs) were calculated for the likelihood of being prescribed ADHD medication in individuals with and without ASD and Wilcoxon rank-sum test was used to compare group differences in dose per day.Individuals with ADHD and coexisting ASD were less likely to start continuous treatment with ADHD medication , were less likely to be prescribed methylphenidate, and were more commonly prescribed second line treatments such as dexamphetamine, amphetamine, or modafinil. No group difference was observed for atomoxetine. In adults with ADHD and coexisting ASD, methylphenidate was prescribed in lower daily doses over three years as compared to individuals with pure ADHD.The findings indicate that there are differences in the medical treatment of individuals with or without ASD. If these differences are due to different medication responses in ASD or due to other factors such as clinicians’ perceptions of medication effects in patients with ASD, needs to be further studied.The online version contains supplementary material available at 10.1186/s11689-020-09352-z. Attention-deficit/hyperactivity disorder (ADHD) is associated with extensive psychiatric comorbidity , includiIndividuals with coexisting ADHD and ASD are more severely impaired on a group-level, for example, regarding adaptive functioning and executive control . Due to Previous studies of ADHD medication in individuals with ASD are hampered by methodological problems, such as limited study samples with restricted length of the follow-up periods, typically only a few months. The length of follow-up is crucial, considering that ADHD medication is prescribed on a long-term basis . FurtherObservational pharmacoepidemiology studies, using data from health registers that cover all filled prescriptions, allows for analyses following prescribing patterns in larger groups, over extended time-periods, and in broader age-categories, and may be a valuable complement to results obtained from RCTs . To our The cohort was established by linking health care registers including the National Patient Register (NPR), with psychiatric diagnoses and dates with codes in accordance with International classification of diseases (ICD) from 1973  and 2001 ; the Total Population Register with information on year of birth on all residents in Sweden; and the Swedish Prescribed Drug Register (PDR), with information on all filled prescriptions from pharmacies in Sweden from July 1, 2005  with two or more diagnoses of ADHD with the first diagnosis of ADHD ICD-10, F90) appearing from July 1, 2005, and onwards, which matches the launch of the Swedish PDR, allowing us to capture new treatment periods. For flow-chart of the study inclusion, see Supplement Figure 0, F90 apFrom the cohort of individuals with ADHD, we identified those with two or more diagnoses of ASD appearing after age three, according to ICD-9 (299) and ICD-10 , but the first ASD diagnosis had to appear before January 1, 2011. ADHD and ASD are both neurodevelopmental disorders, and to be correctly diagnosed symptom onset is required from childhood. Therefore, the first ASD date that appears in the register is not an indicator of onset, but instead indicates when the diagnosis first was decided on in conjunction with the neuropsychiatric assessment. Subsequently, we considered individuals as ASD cases regardless if the first ASD date appeared before or after the first ADHD date .In Sweden, ADHD medications are usually prescribed in specialized psychiatric units, but may also be prescribed by medical doctors specialized in, e.g., pediatric neurology. Individuals must have a documented neuropsychiatric assessment that resulted in an ADHD diagnosis. Between the years of 2005 and 2013, the substances methylphenidate and atomoxetine were approved drugs for ADHD treatment in Sweden by the Swedish Medical Products Agency and recommended for first-line treatment . Prescription of dexamphetamine, and amphetamine substances  required a special application to the Swedish Medical Products Agency during the study period. Modafinil was not approved for ADHD treatment, but was prescribed off-label for ADHD. Short-acting and extended release formulations were dispensed for methylphenidate and amphetamines, while for dexamphetamine only short acting formulations were dispensed.From the PDR, we identified filled prescriptions for the following substances according to the ATC drug class: amphetamine (N06BA01), dexamphetamine (N06BA02), methylphenidate (N06BA04), modafinil (N06BA07), and atomoxetine (N06BA09). There were no dispensations recorded for lisdexamfetamine (N06BA12), guanfacine (C02AC02), dextromethamphetamine (N06BA03), and dexmethylphenidate (N06BA11) during the study period and these were not included in the analysis.We defined treatment periods for ADHD medication overall as “no treatment” (no filled prescription from any ADHD medication), “single dispense” (one filled prescription from any ADHD medication) and “continuous treatment” (a sequence of two filled prescriptions from one ADHD medication at separate dates where the gap did not exceed 90 days).Treatment periods were defined for each separate ADHD medication  as “no treatment” or “continuous treatment” . We fitted logistic regression models (SAS PROC LOGISTIC) and analyzed the odds for continuous treatment versus no treatment, which included those who had a single dispense. In a secondary analysis, we analyzed the odds for continuous treatment versus no treatment and continuous treatment versus one single dispense. Odds ratios (ORs) were calculated with the associated two-sided 95% confidence intervals (CIs), for the likelihood of being prescribed ADHD medication, comparing individuals with ADHD without coexisting ASD to individuals with ADHD with coexisting ASD. First, we fitted a model without any other covariates, except for sex and birth categories . Next, we included covariates adjusting for year of first ADHD diagnosis as a continuous linear covariate and the presence of psychiatric comorbidities (yes/no), which could be suspected to confound our OR estimates.Second, in those who started a continuous treatment, we examined whether individuals with ADHD and coexisting ASD are more likely to be prescribed second line treatment options . We fitted a logistic regression model for each type of ADHD medication in all individuals and separated into age-categories defined as in the first analysis.p values of < 0.05 corresponding to two-sided 95% confidence intervals not covering the value one were considered as statistically significant. Third, we examined whether individuals with ADHD and coexisting ASD were dispensed different average doses of ADHD medication. Due to non-normal distribution of the data, we applied the Wilcoxon rank-sum test (SAS PROC NPAR1WAY). Methylphenidate doses per day (mg/day) were calculated over six-month intervals, during a total time-period of three years. We also stratified into age-categories defined as in the first analysis. The Wilcoxon rank sum test is a non-parametric test that does not require the data to follow a particular data distribution, e.g., normal distribution, and is robust against single gross outliers . Two-sidSensitivity analyses were performed, by excluding individuals with their first ADHD diagnosis at age 40 or later, to ascertain that the results were not biased by possible differences regarding coexisting ADHD and ASD in young adults as compared to elderly. All statistical analyses were performed using SAS 9.4.We identified 39,386 individuals with a diagnosis of ADHD, of which 5012 (13%) had a coexisting diagnosis of ASD. The median age at first ADHD diagnosis was lower in individuals with ADHD and coexisting ASD , and the median age at first ASD diagnosis was 16 years (range 3-80, Table Individuals with ADHD and coexisting ASD were less likely to start continuous treatment with ADHD medication after adjusting for sex, age, year of first ADHD diagnosis and psychiatric comorbidities (OR, 0.80; 95% CI, 0.75-0.86; Table Among individuals who started a continuous treatment with ADHD medication, we analyzed the likelihood for starting a continuous treatment for different types of ADHD medications. Methylphenidate was the most commonly dispensed medicine, followed by atomoxetine Table . IndividWe then stratified the analysis into adults, adolescents, and children. Adults with ADHD and ASD were more likely to start treatment with dexamphetamine, modafinil, or amphetamine. Children diagnosed with ADHD and coexisting ASD were less likely to initiate treatment with methylphenidate but were more likely to initiate treatment with atomoxetine Table . No signWe studied doses of methylphenidate during a three-year observation period. By the end of the three years, 61% of the individuals with ADHD and 63% of the individuals with ADHD and ASD continued to receive dispensations from methylphenidate. Individuals with ADHD and coexisting ASD were prescribed statistically significantly lower doses of methylphenidate Table , Fig. 1.This population-based study with prospective follow-up on 39,386 individuals with ADHD with or without coexisting ASD found that individuals with coexisting ASD were at lower odds to start continuous treatment with ADHD medication and this was observed in adults, adolescents as well as in children. In those who had started treatment, children with ADHD and coexisting ASD were less likely to receive prescriptions from methylphenidate and more likely to receive prescriptions from atomoxetine, while adults with coexisting ASD were more likely to receive second line treatment choices, i.e., dexamphetamine, amphetamine, or modafinil. Furthermore, adults with coexisting ASD were prescribed, on average, lower doses of methylphenidate over a period of three years.Evidence for medical treatment of ADHD in ASD is limited and to our knowledge, there are no previous pharmacoepidemiological studies directly comparing dispenses and doses of ADHD medications in ADHD with or without coexisting ASD. However, most studies within the field conclude that ADHD medications, such as methylphenidate and atomoxetine, have effect on ADHD symptoms in ASD , 23–26. For the different medication types, we found that adults with ADHD and ASD were more likely to initiate treatments with dexamphetamine, modafinil, or amphetamine, and the results did not change after adjusting for a number of comorbidities including substance use disorder. In Sweden, during the period covered, ADHD patients were obliged to try out methylphenidate and/or atomoxetine with unacceptable side effects, or a marked insufficient effect, before being prescribed dexamphetamine or amphetamine. Thus, our findings lead us to speculate whether individuals with ASD may switch from methylphenidate to second line alternatives due to a worse response, or side effects from ADHD medication, as previously reported , 16, 17.Individuals with ADHD and coexisting ASD were on average prescribed lower methylphenidate doses and this difference was most pronounced in adults. The use of lower methylphenidate doses in ASD may be an indicator of differences in tolerance, treatment response, or that lower doses are sufficient when there is a treatment effect. One previous study found that individuals with ADHD and previous substance use disorder were prescribed 40% higher methylphenidate doses as compared to individuals with ADHD without substance use disorder . The finThe median age for the first ADHD and ASD diagnosis was relatively high, 18 years for individuals with ADHD and 16 years for individuals with ADHD and ASD. These results are however in agreement with previous studies using Swedish register data  and is mThe findings of the study must be interpreted in the light of several limitations. The data reflect filled prescriptions medications, but we do not have information on medications prescribed but were never dispensed, or whether the medications were consumed. Previous studies found that medical adherence in ADHD is low and may vary between 13 and 24% . HoweverThe study identified differences in the treatment with ADHD medication between adults, and to some extent adolescents  and children, with or without coexisting ADHD and ASD regarding treatment initiation, medication type, which could not be explained by other comorbid psychiatric disorders. Given that the prescribing patterns differed in individuals with coexisting ASD, the clinician needs to be aware not only of possible variances in response patterns or side effects from medications but also of differences regarding the individuals’ preferred choices of pharmacological treatment. The findings do not support a different treatment strategy regarding ADHD medication in individuals with coexisting ASD, but this patient population should be subject to more careful monitoring and dose-titration, and may be in greater need for clinical interventions and support other than pharmacological treatment. Further clinical studies are needed, particularly longitudinal studies, addressing potential differences in prescribing patterns, and pharmacological effects from ADHD medications that directly compares individuals with ADHD with and without coexisting ASD. Although the study is restricted to individuals in Sweden, the clinical diagnoses and the studied medications are now standard across the western world  and we bThis is the first study to investigate the prescribing patterns of ADHD medication in ADHD with and without coexisting ASD. The findings from this study indicate differences between individuals with and without ASD. If these are due to clinicians’ perceptions of medication effects in patients with ASD, or due to different medication responses in patients with ASD, need to be addressed in future studies. Meanwhile, patients with ASD should be subject to careful monitoring and dose-titration in the clinic.Additional file 1.


This work aims to investigate the effect of additional flue gas desulfurization gypsum (FGDG) on the properties of calcium sulfoaluminate cement (CSAC) blended with ground granulated blast furnace slag (GGBFS). The hydration rate, setting time, mechanical strength, pore structure and hydration products of the CSAC-GGBFS mixture containing FGDG were investigated systematically. The results show that the addition of FGDG promotes the hydration of the CSAC-GGBFS mixture and improves its mechanical strength; however, the FGDG content should not exceed 6%. Compamissions ,6.The main mineral phases of CSAC clinker are ye’elimite (e (C4AF) . Among t follows ,9,10:In the absence of gypsum,In the presence of gypsum, but in insufficient amounts,CH) is present,If the gypsum is sufficient and calcium hydroxide  is a critical parameter for the hydration of CSAC, which further determines the performance of CSAC. For Mg/y < 1.5, the CSAC shows the characteristics of rapid setting and hardening, while it is used for self-stressing when 1.5 < Mg/y < 2.5; however, it is expansive for Mg/y > 2.5 [The main hydration products of C3A⋅CS¯⋅H2, AFm annd so on . The hydstrength . From EqC3A⋅CS¯⋅H2, AFm an/y > 2.5 ,11. TherC3A⋅CS¯⋅H, AFm and of CSAC .In recent years, a number of supplementary cementitious materials (SCMs) have been blended with CSAC to reduce the total cost and modify the properties, such as fly ash ,15, lime2, Al2O3, Fe2O3, MgO, Na2O, K2O and other impurities [It is well known that the resource of natural gypsum is limited across the globe. Fortunately, flue gas desulfurization gypsum (FGDG), a by-product obtained from the desulfurization of coal-fired flue gas, supplies a potential resource of gypsum . The chepurities ,20,21. Tpurities ,22. UtilIn this work, FGDG was added in the CSAC-GGBFS blended mixture as additional gypsum, and the effect of FGDG content on the properties of the CSAC-GGBFS system was studied. This study contributes to the understanding of the hydration of this blended mixture and further improvement of its properties.2S, 13.35% C4AF and 58.68% Chinese standard 42.5R CSAC , which was used in this work, was prepared with 78% clinker, 16% anhydrite and 6% limestone. Moreover, the clinker was composed with 28% CFour mix proportions of blended cement paste were designed, as shown in The hydration heat of the mixture was measured using a TAM Air isothermal calorimeter , and the setting time was tested using the Vicat method, according to the Chinese standard GB/T1346-2011 . The min2S) is delayed. The figure also indicates that the height of the second peak decreases as the FGDG content increases from 0% to 9%. This illustrates that the early age hydration rate of CSAC-GGBFS can be reduced by the addition of FGDG; the greater the FGDG content, the stronger the reducing effect.The hydration heat flow and cumulative heat of CSAC-GGBFS cement pastes blended with different FGDG content are shown in As shown in The setting time of CSAC-GGBFS mixtures blended with different FGDG content is shown in The mechanical strength of CSAC-GGBFS mortars blended with different FGDG content is shown in The pore structure of CSAC-GGBFS mixtures blended with different FGDG content hydrated for 120 d is shown in From the above results, it can be seen that an appropriate quantity of FGDG is helpful in refining the pore size and reducing the total porosity of the CSAC-GGBFS mixture, which is the direct reason that the flexural strength and compressive strength increase as the FGDG content increases from 0% to 6%.ere used . This re2S, CH does not exist in the cement paste since it is consumed by the secondary hydration reaction (Equation (4)). In addition, the phases of AH3 and C–S–H gel are not detected by XRD, as they are amorphous; however, their occurrence can be determined by thermal analysis.According to 3; (Ⅲ) ~280 °C to ~660 °C: the dehydration of C–S–H gel and other hydration products; (Ⅳ) ~660 °C to ~740 °C: the decomposition of CaCO3. 3 can be consumed by the secondary hydration reaction (Equation (4)), and the amount of AFt increases [The TG-DSC curves of CSAC-GGBFS mixtures blended with different FGDG content hydrated for 120 d are shown in 3 is higher in the early hydration age, and it is then consumed by the hydration reaction (Equation (4)). In addition, the mass loss of stage (Ⅲ) increases continuously as the curing time increases from 1 d to 120 d; this indicates that the amount of C–S–H gel increases gradually. The total mass loss of cement paste increases from 20.53% to 27.64% as the curing time increases from 1 d to 28 d, and it reaches 29.38% at 120 d. According to the total mass loss, it can be seen that the hydration degree of CSAC-GGBFS-FGDG cement paste at 28 d is 94% of that at 120 d, which shows that the blended cement paste undergoes rapid hydration before 28 d.The TG-DSC curves and the mass loss at different stages for the CSAC-GGBFS mixture blended with 6% FGDG cured for 1 d, 28 d and 120 d are shown in 3 and unhydrated cement particles are loosely interwoven when the cement paste is hydrated for 1 d [The microstructure of the CSAC-GGBFS mixture blended with 6% FGDG hydrated for 1 d, 28 d, and 120 d is shown in  for 1 d . However for 1 d ,30,31. AThe effect of additional FGDG on the hydration of the CSAC-GGBFS system was investigated in detail. Some important conclusions are summarized as follows:(1) The duration of the induction period for the blended cement paste is prolonged by the addition of FGDG. The early age hydration rate decreases as the FGDG content increases from 0% to 9%; however, the later age hydration is promoted.(2) The addition of FGDG has little effect on the setting time of CSAC-GGBFS cement paste when its content increases from 0% to 9%, and the blended cement paste still exhibits the characteristic of rapid setting.(3) The flexural and compressive strengths of CSAC-GGBFS mortar slightly decrease as the FGDG content increases from 0% to 9% at 1 d and 3 d. However, after 28 d, the addition of FGDG has a positive effect on the mechanical strength of the CSAC-GGBFS mixture. The compressive strength of mortar at 120 d can be improved by nearly 20% when the dosage of FGDG is 6%.(4) The addition of FGDG can refine the pore size and reduce the total porosity of CSAC-GGBFS cement paste if its content is less than 6%. However, the pore size and total porosity increase when the FGDG content reaches 9%.(5) The addition of FGDG promotes the hydration of CSAC-GGBFS cement paste and increases the presence of hydration products, which further improves the mechanical strength of mortar at the later age. However, the FGDG content should not exceed 6%, or some micro-cracks appear in the cement paste. An appropriate increase in gypsum content benefits the hydration and mechanical strength of CSAC blended with SCM.


We aimed to investigate the effects of childhood adversity and its interaction with the polymorphisms in the monoamine oxidase A (MAOA), brain-derived neurotrophic factor (BDNF), and catechol-O-methyltransferase (COMT) genes on attention and hyperactivity disorder (ADHD) symptoms in a community sample of generally healthy youth. Participants (N = 432) completed questionnaires assessing ADHD symptoms  and adverse childhood experiences, such as adverse environments (AEs) and childhood maltreatment (CM). Salivary genomic DNA was used to test polymorphisms in MAOA, BDNF, and COMT genes. A gene score (GS) was created based on the number of risk allele in the studied genes. Multiple linear regressions were used to examine the genetic and environmental effects on ADHD symptoms. The univariate analysis indicated that CM was significantly associated with inattention , hyperactivity (0.25 [0.06–0.45]), and impulsiveness (1.16 [0.26–2.05]), while the GS was associated with hyperactivity (0.22 [0.11–0.33]) and impulsiveness (0.56 [0.06–1.05]). Only the GS remained significantly associated with hyperactivity (0.25 [0.12–0.37]) and impulsiveness (0.79 [0.20–1.38]) when the gene-environment interaction term was added in the model. No effects were found for AE and the gene-environment interaction term. In conclusion, CM was associated with ADHD symptoms in emerging adulthood. Genetic factors may also play a significant role in the association with these outcomes.  Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder that has heterogeneity in its etiology . Prior rEarlier studies have found that psychosocial and familial environmental adversity is likely to increase the risk of ADHD symptoms ,6 as welADHD has been genetically linked to neurological dysfunctions, including involvement in the dopaminergic, adrenergic, serotoninergic, and cholinergic pathways . For insADHD symptoms are not uncommon but are often overlooked among those entering adulthood . Higher A convenience sample of young people aged 16–22 years were voluntarily recruited from the campus or community via research announcements. Peeople were excluded if they had major physical  or psychiatric  diagnoses. Individuals with prior clinically confirmed diagnosis of ADHD were also excluded, as their symptoms may have onset in childhood and persist into adolescence or young adulthood. As such, our sample can be more representative of generally healthy young people. Participants and their guardians (if participants’ age was under age 20) were informed and gave consent as they completed the questionnaire. Those who were unable to provide informed consents were also excluded from the study. The entire procedure was approved by the institutional review board of our institution (B-BR-104086).ADHD symptoms were assessed using the six-item Adult ADHD Self-Report Scale (ASRS-6). The Chinese version of ASRS-6 has been validated in a young Taiwanese population . The scaA short Chinese version of impulsiveness scale developed from the Baratt Impulsive Scale to evaluate the participants’ impulsive characteristics has been validated in this local context . We adopWe adopted a modified version of the Adverse Childhood Experience International Questionnaire (ACE-IQ) to assess the ACE type and timing of the participants . The tooWe selected representative SNPs in the MAOA (rs1137070), BDNF (rs6265), and COMT (rs4680) genes. Salivary genomic DNA was extracted according to the standardized protocol (Geneius™ Micro gDNA Extraction Kit). Genotypes at the respective SNPs were determined for each sample using real time PCR that followed the standardized Taqman technology protocol (Applied Biosystems). Individuals with missing values in more than one SNP were eliminated from the analysis, as the combination effects of the three SNPs needed to be tested . For anaFirst, we tested individual associations between the selected SNPs and ADHD symptoms in three different inheritance models using linear regression analyses. In additive models, a beta coefficient (β) with 95% confidence interval (CI) was obtained for the minor allele vs the major allele. Further, based on an additive inheritance model, we created a gene score  by adding up the number of risk alleles, which was defined by the allele that was positively associated with the outcome variables. To test the genetic, environmental , and gene-environment interaction effects on the outcome variables, we built three different linear regression models. In Model 1, we tested the univariate effect of GS or ACEs on the outcome variables. Model 2 included GS and ACEs at the same time, while Model 3 further included the interaction term between the GS and ACEs. Missing data were imputed. As patterns of data did not differ before and after imputation, we only presented the results after imputing the data. All analyses were adjusted for age and gender, and a gender-stratified analysis was also conducted. Statistical analyses were performed using R Studio 1.2.1335. A total of 492 participants were recruited and, after eliminating those with missing values in more than one SNP, only 432 with a mean age of 20 (±1.41) years and 51% being males were considered valid for analysis . The meaWe further applied GS to test the genetic, environmental, and gene-environment interaction effects . In the The gender-stratified analysis showed the gender difference in the results . Among tThis study demonstrated that ACEs, particularly CM, may be associated with ADHD symptoms among a cohort of young people. Genetic factors, such as the MAOA, BDNF, and COMT genotypes, may also play a part in the associations among these outcomes. However, our analysis failed to demonstrate gene-environment interaction effects in this cohort.Our results extended the association between childhood experiences and ADHD symptoms emerging later in young adulthood. Further dividing the ACE subtypes, we found childhood maltreatment, rather than adverse environments, to be significantly associated with ADHD symptoms. This finding stood in line with a prior Danish national study, which found a significant association between co-occurring multiple types of CM and higher levels of self-reported ADHD symptoms . AnotherContrary to our a priori assumption, we did not find any gene-environment interaction effect in association with ADHD symptoms. Since the interaction term was included in the regression model, only the main genetic effect remained significant in the model. Based on these results, we tentatively argued that these studied genes independently influence ADHD symptoms. Our results were supported by some other studies. For example, Retz et al.  did not The present study has some limitations. First, the items on the questionnaire were self-reported and thus subject to perception bias. For instance, our ADHD symptoms were screened merely using validated questionnaires that usually have a very limited space for a full list of the ADHD symptoms. Thus outcome variables may be biased if some other core symptoms were overlooked in a brief questionnaire. Participants may also not be aware of ADHD symptoms that actually occurred earlier in childhood, although we have excluded those who had prior diagnosis of ADHD. As such, professional evaluations, such as social workers’ reports on ACEs and physicians’ interviews on ADHD symptoms, may ameliorate this sort of bias. Obtaining additional information may affect the association estimates and this requires further research to confirm our findings. Worthy of attention is that our sample was made up of generally healthy young people. These findings may not be generalizable to those who meet full diagnostic criteria of ADHD. The causal interpretation should be cautious in a population with high levels of ADHD symptoms and childhood adversity. Second, only three SNPs were chosen to test the genetic and gene-environment interaction effects with ACEs on ADHD symptoms. Unselected SNPs may also play a role either due to their direct contribution or via their interaction with ACEs. Analysis of gene-wide SNPs identified by a thorough sequencing of these genes may further elucidate the genetic and gene-environment interaction effects of target genes on the outcome of interest. We only adjusted for age and gender, while some other contextual factors  that may be related to ADHD symptoms, were not adjusted. These factors have long been thought of as salient environmental factors of ADHD symptoms and thus they may play a part in the gene-environment interaction effect. However, the effects of these additional contextual factors may not easily be disentangled from those of ACEs given our limited sample size. Further research of a larger participation may be needed to explore the complex interplay of environmental factors.CM was shown to have a significant impact on ADHD symptoms among a sample of young people, particularly in the case of females, while the combination genetic effects of MAOA (rs1137070 C > T), BDNF (rs6265 C > T), and COMT (rs4680 G > A), which reflected individual susceptibility, were significant in terms of the association with hyperactivity symptoms in both genders. Nonetheless, the gene-environment interaction was not observed in the present study. Our study highlights the roles of genetic and environmental factors in determining behavioral development in young people. The interplay between genes and environment, although non-significant in our analysis, can provide insight into predicting vulnerability to adult ADHD symptoms. Clinicians should be aware of the adverse impacts of ACEs on youth development because they may emerge as ADHD symptoms even over time. Genotypic information may help identify susceptible individuals who are at risk of ADHD symptoms; however, the clinical utility of genetic testing requires further research to validate such findings.


Human umbilical cord mesenchymal stem cell (hUC-MSC) therapy is considered as a promising approach in the treatment of intrauterine adhesions (IUAs). Considerable researches have already detected hUC-MSCs by diverse methods. This paper aims at exploring the quantitative distribution of CM-Dil-labeled hUC-MSCs in different regions of the uterus tissue of the dual injury-induced IUAs in rats and the underlying mechanism of restoration of fertility after implantation of hUC-MSCs in the IUA model.In this study, we investigated the quantification of the CM-Dil-labeled hUC-MSCs migrated to the dual injured uterus in Sprague Dawley rats. Additionally, we investigated the differentiation of CM-Dil-labeled hUC-MSCs. The differentiation potential of epithelial cells, vascular endothelial cells, and estrogen receptor (ER) cells were assessed by an immunofluorescence method using CK7, CD31, and ERα. The therapeutic impact of hUC-MSCs in the IUA model was assessed by hematoxylin and eosin, Masson, immunohistochemistry staining, and reproductive function test. Finally, the expression of TGF-β1/Smad3 pathway in uterine tissues was determined by qRT-PCR and Western blotting.p = 0.005), in the seroma  and in the epithelium . From the 2nd week of treatment, hUC-MSCs began to differentiate into epithelial cells, vascular endothelial cells, and ER cells. The therapeutic group treated with hUC-MSCs exhibited a significant decrease in fibrosis (TGF-β1/Smad3) as well as a significant increase in vascularization (CD31) compared with the untreated rats.The CM-Dil-labeled cells in the stroma region were significantly higher than those in the superficial myometrium (SM) (71.67 ± 7.98 vs. 60.92 ± 3.96, Our findings suggested that the distribution of the migrated hUC-MSCs in different regions of the uterine tissue was unequal. Most cells were in the stroma and less were in the epithelium of endometrium and gland. Injected hUC-MSCs had a capacity to differentiate into epithelial cells, vascular endothelial cells, and ER cells; increase blood supply; inhibit fibration; and then restore the fertility of the IUA model. Intrauterine adhesions (IUAs), also called Asherman’s syndrome, lead to the uterine cavity obliteration and have serious life consequences for women because they cause pelvic pain, menstrual aberrations, recurrent pregnancy loss, and mechanical infertility , 2. The HUC-MSCs can be collected easily and non-invasive; no ethical constraints for the umbilical cord is a discarded tissue and hold a high in vitro proliferative rate . MoreoveHUC-MSCs have exhibited considerable therapeutic potential for IUAs at present. However, the cell mechanism and cell distribution are far from well elucidated. This study aimed to determine the quantitative distribution of labeled hUC-MSCs in different regions of the injured uterine and the underlying mechanism of restoration of fertility after implantation of CM-Dil-labeled hUC-MSCs in a rat IUA model established by dual injury, which is significant for the further study of the treatment of IUAs.Following informed written informed consent from each donor mother, fresh hUCs were collected from term deliveries at an operating room. All clinical treatments involving patients were complied with the Declaration of Helsinki guidelines. This study was approved by the Local Institutional Review Board (registration number: 2019 - P041).3); transfer them into 10-cm tissue culture dishes (Corning); culture in MSC medium  consisting of a serum-free basic medium, serum-free nutritional supplement, and 100,000 U/ml of penicillin/streptomycin; and then maintain in a humidified 5% CO2 incubator at 37 °C. After approximately 12 days, the adherent cells were harvested by stem cell digestive juice  treatment for subculturing. Cells from passages (P) 2 and 5 were used for the following experiments.As previously described , after r6 cells were incubated with FITC-labeled anti-CD45, anti-CD90, PE-labeled anti-CD34, anti-CD73, APC-labeled anti-CD29, anti-HLA-DR, and PerC-labeled anti-CD105 in the dark at 4 °C for 30 min. After staining, the cells were fixed using paraformaldehyde, and the expression of the cell surface markers was detected using flow cytometry.hUC-MSC morphology was observed by an inverted microscope at P3, and representative pictures were captured at 10×. Moreover, the expression of hUC-MSC-related surface markers such as CD90 and CD105 and the lack of the HLA-DR and CD45 markers were evaluated by flow cytometry. In brief, after reaching 80% confluency, adherent cells were harvested and resuspended in PBS. Aliquots of 1 × 103 cells/well in the FBS medium. The assay was performed every 24 h during 7 days. The absorbance was measured at a wavelength of 570 nm by using a spectrophotometric plate reader .Add culture medium containing 2 mg/L CM-Dil  when hUC-MSCs reach 80% confluency after two rinses with sterile PBS. Then incubate for 10 min at 37 °C and 15 min at 4 °C. Next, the labeled cells were monitored for fluorescence using the Olympus BX51 microscope  followed by two washes with sterile PBS. Subsequently, the cells were detached and subcultured. Viability and proliferation of hUC-MSCs and CM-Dil-labeled hUC-MSCs at P3 were analyzed with the MTT method, according to the manufacturer’s instructions. They were seeded in 96-well plates at a concentration of 5 × 10n = 15); (2) rat, 2 weeks after surgery, receiving 500 μl PBS via intraperitoneal injection as the model group (n = 15); (3) rat, 2 weeks after surgery, receiving 2 × 106 CM-Dil-labeled hUC-MSCs in 500 μl PBS via intraperitoneal injection as the model with the hUC group (n = 25). All the study rats were euthanized on the 1st, 2nd, 3rd, and 4th weeks after hUC-MSCs injected.All procedures that involved animals were approved by the Local Institutional Animal Ethics Committee. Our rat model of IUAs was induced as previously described , 15. BriAfter retrieval of the uteri on the 1st, 2nd, 3rd, and 4th weeks after hUC-MSCs injected, they were embedded in OCT and sectioned (5-μm thick). Representative sections were stained with DAPI to examine the survival and migration of hUC-MSCs in vivo.Along the direction perpendicular to the long axis of the uterus, each uterus on the 1st week after hUC-MSCs injected was cut into roughly three equal pieces, then randomly equidistant selected three tissue masses in each rat. Each mass performed ten serial sections (5 μm) after frozen OCT-embedded tissue, then randomly equidistant selected 6 sections . CM-Dil-To assess the differentiation of the cells, the sections obtained from the samples on the 1st, 2nd, 3rd, and 4th weeks after hUC-MSCs injected were washed repeatedly with PBS solution to remove OCT, and then, to reduce non-specific background, sections were treated with 0.3% bovine serum albumin solution in PBS for 30 min. Samples were stained using cytokeratin 7 rabbit polyclonal , CD31 mouse monoclonal , and estrogen receptor alpha . The secondary antibody of Alexa Fluor® 488 Goat Anti-Rabbit antibody  and Alexa Fluor® 594 Goat Anti-Mouse antibody  were used and counterstained with DAPI. Visualization was performed using an optical microscope .Six weeks post-procedure, the uteri of the three groups were removed, washed with sterile PBS, fixed with 4% buffered formaldehyde, embedded in paraffin, serially sectioned at 5 μm thickness, and routinely stained with hematoxylin and eosin (H&E) and Masson stains. The sections obtained from the samples collected from the above were also used to detect the expression of CD31 (a vascular marker) on the uterine tissues by immunohistochemistry staining.The function of the regenerative endometrium was evaluated by the reproductive study for a period of 4 weeks. The rats of the three groups were mated with male SD rats. The day of vaginal plug presence was considered gestation day 0. Rats were euthanized at gestation days 15–18, and the presence of embryos were examined.Real-time PCR was performed using a PrimeScript RT reagent kit (Promega) on an Applied Biosystems 7300 Fast Real-Time Polymerase Chain Reaction System. The sequences of the specific primers used were as follows: transforming growth factor-β1(TGF-β1): 5′-ATTCCTGGCGTTACCTTGG-3′(forward), 5′-AGCCCTGTATTCCGTCTCCT-3′(reverse); Smad3: 5′-AGGAGAAGTGGTGCGAGAAG-3′(forward), 5′-GTGACCTGGGGATGGTAATG-3′(reverse). The mRNA expression of each sample on the 2nd and 4th week after hUC-MSC injection was determined after correction by GAPDH expression. The relative expression was calculated using the 2–ΔΔCT method.The tissues above were quickly removed and homogenized using RIPA buffer and phenylmethylsulfonyl fluoride  by sonication. Proteins from the supernatant were separated by electrophoresed in 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes . The PVDF membranes were incubated with primary antibodies, rabbit polyclonal anti-TGF-β1 , rabbit monoclonal anti-Smad3 , and rabbit polyclonal anti-β-actin  at 4 °C overnight after being blocked. The membranes were incubated with anti-rabbit IgG , a fluorescent labeled IRDye 800 secondary antibody, in the second day. Blots were digitally imaged with a LICOR Odyssey .p value of < 0.05 was considered to be a significant difference.Statistical analysis was performed using the Statistical Package for Social Science (SPSS) version 21.0 . Continuous variables were presented as mean ± standard deviation. One-way ANOVA was used for the homogeneous variance in three or more group comparisons followed by the LSD method between groups. Welch’s ANOVA was applied to the uneven variance in three or more group comparisons followed by the Games-Howell method between groups. A We isolated hUC-MSCs from WJ explants, the primary cultures of adherent cells with a mesenchymal-like morphology, and were subcultured on average at 12 days after plating. The cells maintained their morphology after subculturing . The CM-Dil-labeled cells in the stroma region were significantly higher than those in the SM , in the seroma  and in the epithelium . However, there were no differences between the stroma and the DM , or the DM and the SM .The uterus tissue consists of endometrium epithelium, stroma, SM, DM, and serosa, from the inside to the outside Fig. a. The frp = 0.003) calculated by Welch’s ANOVA. Also, the protein level of CXCR4 in the model with the hUC group was significantly higher than those in the normal group (p = 0.01) and the model group (p = 0.013) calculated by Games-Howell.To better confirm hUC-MSC migration to the injury uterus on the 1st week after hUC-MSC injection, CXCR4 expression around the damaged area was examined in terms of protein level. As shown in Fig. As Fig. The uterine tissue sections of the three groups on the 6th week post-procedure were used to evaluate the histological changes for confirming the efficacy of hUC-MSCs. H&E, Masson, and CD31 staining showed that after therapy, the endometrial thickness, the number of endometrial glands, and the CD31 positively stained vessels were increased, and the fibrous tissues of the stroma were decreased compared with the model group, and the manifestations were similar in both the normal group and the model with the hUC group Fig. a. The pr-β1 signaling showed the same trends as the expression of TGF-β1 (Fig. Gene expression of TGF-β1 was significantly downregulated in the model with the hUC group when compared with the expression in the model group on the 2nd and 4th weeks after therapy. Smad3 as a downstream effector of TGF-β1 Fig. a. TGF-β1-β1 Fig. b.Fig. 6At present, MSC-based cell therapy is a promising research for the treatment of various diseases. Among these MSCs, hUC-MSCs are preferred in that they can be collected easily, non-invasive, and no ethical constraints; they can secrete various cytokines and trophic factors; and they are immunoprivileged , 10. AboIn this study, CM-Dil-labeled hUC-MSCs were injected into the IUAs rats and red fluorescent cells were observed in the uterine sections. Jalalie et al.  also obsTherefore, we made a quantitative study on the distribution of CM-Dil-labeled hUC-MSCs on the 1st week after hUC-MSCs injected. The study has shown that CM-Dil-labeled hUC-MSCs were distributed unequally in different parts of the uterine tissue. More of the cells migrated to the stroma and myometrium regions, and almost exclusively no hUC-MSCs reached the epithelium of endometrium and gland. What is the reason for the unequal distribution of the hUC-MSCs? It may be due to the fact that the stroma and myometrium are rich in blood vessels than the epithelium, which is consistent with the study that MSCs were mainly distributed along the blood vessels . MoreoveOn the other hand, hUC-MSC is a kind of stem cells. It could colonize in the stromal region without any change. Also, it could differentiate into other kinds of cells under the specific niche. Then, we explored the characteristic of hUC-MSCs after implantation. To our surprise, we found that from the 2nd week of treatment, hUC-MSCs began to differentiate into epithelial cells and a small amount of ER cells. With the extension of treatment time, hUC-MSCs and CD31 were in the same position, and some of the co-expressed sites were in the location of vascular endothelial cells. To our knowledge, the formation of IUAs is relevant to some known reasons. The following are only a few examples:First, the decrease of ER, ERα, and ERbeta was colocalized in the nuclei of many stromal and glandular epithelial cells . ERα in Second is the angiogenesis disorder. The formation of IUAs, to a great extent, is related to intrauterine operation . Once thMeanwhile, our results have shown that the endometrial thickness, the number of endometrial glands, and the CD31 positively stained vessels were increased, and the fibrous tissues of the stroma were decreased in the model with the hUC group compared with the model group. And the fertility of the model with the hUC group was improved. Taken together, it seems at least possible that hUC-MSCs might exhibit restoration of meaningful fertility through differentiated into the above three types of cells and that during such migration, the mechanisms could be similar or different to those used for the paracrine effect.Further, TGF-β1 is the most important factor involved in the formation of fibrosis . Under nTo note, TGF-β is thought to have both pro- and anti-angiogenic properties, depending on the levels present. Low levels of TGF-β contribute to angiogenesis by upregulating angiogenic factors and proteases, while high doses of TGF-β stimulate basement membrane reformation, recruit smooth muscle cells, increase differentiation, and inhibit endothelial cell growth . It is cIn summary, this study shows that CM-Dil-labeled hUC-MSCs home in the dual injured rat uterus with most cells in the stroma and less in the epithelium of endometrium and gland. Injected hUC-MSCs have a capacity to differentiate into epithelial cells, vascular endothelial cells, and ER cells; increase blood supply and inhibit fiber formation; and then restore the fertility of the IUA model. A better understanding of how the hUC-MSC response to the uterine tissues may be beneficial for the further study of the treatment of IUAs.Additional file 1: Figure S1. There was no hUC-MSCs differentiation on the 1sth week after hUC-MSCs injected.


All subjects were genotyped for thirty SNPs within the HTR1A, HTR1B, HTR2A and TPH2 genes. Cortical and subcortical regions of interest (ROI) were defined and random forest (RF) machine learning was used for feature selection and classification in a five-fold cross-validation model with ten repeats. Variable selection highlighted the ROI posterior cingulate gyrus, cuneus, precuneus, pre-, para- and postcentral gyri as well as the SNPs HTR2A rs1328684 and rs6311 and HTR1B rs130058 as most discriminative between ADHD and HC status. The mean accuracy for the validation sets across repeats was 0.82 (±0.09) with balanced sensitivity and specificity of 0.75 and 0.86, respectively. With a prediction accuracy above 0.8, the findings underlying the proposed model advocate the relevance of the SERT as well as the HTR1B and HTR2A genes in ADHD and hint towards disease-specific effects. Regarding the high rates of comorbidities and difficult differential diagnosis especially for ADHD, a reliable computer-aided diagnostic tool for disorders anchored in the serotonergic system will support clinical decisions.Serotonin neurotransmission may impact the etiology and pathology of attention-deficit and hyperactivity disorder (ADHD), partly mediated through single nucleotide polymorphisms (SNPs). We propose a multivariate, genetic and positron emission tomography (PET) imaging classification model for ADHD and healthy controls (HC). Sixteen patients with ADHD and 22 HC were scanned by PET to measure serotonin transporter (SERT‘) binding potential with [ However, substantial fluctuation of prevalence was reported between and across nations, likely owed to disputed diagnostic criteria that are mostly based on behavioral symptoms rather than objective biomarkers. While pathognomonic for many psychiatric disorders, the lack of biomarkers for ADHD is particularly baneful due to the overlap of core symptoms with other frequent psychiatric disorders as mood, anxiety and personality disorders. Diagnosis of adult ADHD is further hindered by retrospective assessment of symptoms in the childhood. Disputes among opinion leaders on ADHD and debates over misuse of ADHD treatment like methylphenidate (MPH) have encouraged research exploring objective over subjective ADHD predictors, so far with modest success3.The most common neurodevelopmental disorder, attention-deficit and hyperactivity disorder (ADHD), affects up to 10% of children with symptoms often persisting throughout the whole lifespan and predisposes to comorbidities like major depressive disorder (MDD)5. Some candidate gene studies associated variants implicated in the monoaminergic neurotransmission with ADHD7, while the GWAS mostly highlighted genes that previously received less attention and are trickier to mesh with established etiologic theories8. However, genetic studies did not impact ADHD diagnosis or treatment yet9. Consequently, the translation to the clinic is lacking so far.Genetics were expected to resolve disparate findings and explain heterogeneity, especially in ADHD with heritability estimated to exceed 70%10. While misbalance of dopaminergic and noradrenergic neurotransmission is putatively the main biological substrate of ADHD, data on in vivo neuroreceptor binding are scarce due to the resource intensive nature of positron emission tomography (PET). While serotonergic neurotransmission is considered a pivotal substrate of affective disorders, the role of serotonin is not sufficiently understood in ADHD12. Data from animal models as well as pharmacological and genetic studies point toward involvement of serotonin in ADHD and atomoxetine, a well-established drug in ADHD treatment, has been demonstrated to block the serotonin transporter in addition to its noradrenergic properties13. Emotional dysregulation with mood swings and irritability, closely linked to serotonergic pathways, has lately been discussed as an additional core symptom of ADHD15. Additionally, comorbid mood disorders are frequent in ADHD. Nevertheless, only few PET studies have targeted the serotonin system in ADHD so far. Earlier studies on the serotine transporter (SERT) binding did not support differences while recently altered interregional connectivity of SERT binding in the hippocampus and precuneus of ADHD patients compared to control subjects was demonstrated16.Neuroimaging and data-driven diagnostics that naturally come along with it were considered a corrective to the issues of subjective symptoms and heterogeneity. Basic tools like electro-encephalography (EEG) as well as more advanced techniques as magnetic resonance imaging (MRI) have now been fully established in ADHD research10. However, no studies combined imaging and genetic predictors up to this point. A reliable diagnostic tool for ADHD may be especially relevant to precision medicine in psychiatry. Since serotonergic transmission has to some extent been demonstrated to pilot the disorders, the focus of this study was classification of ADHD and healthy individuals based on multimodal serotonergic data.With the influx of advanced statistics into neuropsychiatric research, a copious amount of machine learning studies targeted ADHD classification. Algorithms based on EEG and MRI features reported accuracies ranging from hardly above chance level to beyond 90%11C]DASB16.ADHD subjects derive from a previously reported study on SERT binding measured with DASB was used to quantify SERT binding as protocolled previouslyhttp://www.fil.ion.ucl.ac.uk/spm/)16. In summary, the means of all time frames without visually observable head motion was used for realignment of each time frame of the dynamic PET scans. All subjects also underwent MRI scans on a 3 Tesla Philips scanner . Summed PET images  from realigned data was co-registered to T1-weighted images. Next, spatial normalization of the T1-weighted images was performed. Transformation of the co-registered PET images into MNI standard space was achieved by application of the obtained transformation matrices to the dynamic PET data. Finally, computation of voxel-vise images of BPND values was carried out with PMOD image analysis software, version 3.509  and the multilinear reference tissue model with two parameters (MRTM2)19. The cerebellar grey matter without vermis and venous sinus was assigned as the reference region due to negligible availability of SERT in this region21.The protocol for data quantification was reported previously, including preprocessing carried out in SPM12  values were extracted for regions defined according to the automated anatomical atlas (AAL). Mean values were calculated from BPND for the left and right hemispheres. Thus, a total of 49 cortical and subcortical ROI was included in the analyses.Non-displaceable binding potential . The package “randomForest” was used for application of the eponymous algorithm (RF)23. In short, RF is an ensemble tree classification tool that randomly selects subsamples of observations and builds a decision tree for optimal splitting of these observations according to an outcome variable by a combination of predictors. For each split, the best performing predictor out of a random selection is applied. Generally, a higher number of predictors allowed for selection leads to optimal splits but also low diversity of the individual trees. Therefore, restricting the number of features can generate models that perform worse in the training set but are more flexible when exposed to new data. Here, 3000 trees were grown (ntree = 3000) for each model to enable multiple predictions for all patients. Classification was performed with a five-fold cross-validation (CV) design to allow optimal validation in absence of an independent test set24. If hyperparameters must be tuned, nested CV is the gold-standard technique to prevent data leakage from training to the validation phase. For RF only the number of features randomly selected at each split (mtry) can be optimized; however, there is a standard of using the square root of the number of predictors. To prevent overfitting, no optimization of mtry was performed for this analysis.Computations were performed with the statistical software “R”  were computed. Mixed model results were corrected for the number of tests and models with a corrected threshold of p ≤ 0.001. Based on these results, logistic regression models for each ROI and SNP with diagnosis as outcome variable and the respective ROI/SNP interaction term were computed. Logistic regression results were not corrected.In addition to the results produced by RF, a mixed model was computed with the “lme” package for “R”. Linear mixed regression models for BPUsing all available predictors in a five-fold CV model yielded an accuracy of 0.80 (±0.13). Restricting the predictors to either ROI or SNPs only, accuracies dropped to 0.58 (±0.15) and 0.62 (±0.15), respectively. Using only the top performing predictors boosted the accuracy to 0.76 (±0.13) and 0.71 (±0.16) for SNPs and ROI, respectively. Optimal results were achieved combining the selected SNPs and ROI, with an accuracy of 0.82 (±0.09) and positive and negative predictive values (PPV and NPV) of 0.80 and 0.83, indicating the probability of correct classification for and ADHD and HC, respectivelyFor a detailed overview of the classification outcome with all evaluation parameters Table .Table 2PHTR2A rs1328684 was selected by all models, followed by HTR1B rs130058 (82%). Other than that, only rs6311 (12%) and rs6313 (8%) were selected, which showed identical allelic expression. For a graphical depiction of importance measurements and probability rates of the top scoring predictors being selected in the training sets of each fold , precuneus 22%), middle temporal gyrus (22%), precentral gyrus (20%), paracentral lobule (18%), postcentral gyrus (18%) and temporal pole (18%). Among the SNPs, 2%, middlHTR1B SNP rs130058 , HTR2A SNP rs1328684 , HTR2A rs6313  and TPH2 rs1843809 . Significant results of the mixed models are presented in Table Mixed model revealed three-way associations for p = 0.022) as well as posterior cingulate gyrus  with HTR1B rs130058, none of which remained significant after correction. Significant results of the logistic regression models are presented in Table Logistic regression revealed interaction effects for the temporal pole 28. Altered DMN activation during sustained attention paradigms in ADHD could thereby be partly redeemed by methylphenidate31. Also both SNPs rs130058 and rs1328684 were implicated to mediate DMN abnormalities measured by MRI in a recent candidate gene study in PTSD32. HTR1B rs130058 was previously associated with ADHD as well as frequent comorbidities as substance dependence disorders34. Although HTR1B was highlighted as a top gene by candidate gene reviews in ADHD35, findings for rs130058 were overall inconsistent38. In this sample, ADHD patients showed an increased frequency of rs1328684 minor alleles (1.69 vs 1.22) and decreased frequency of rs130058 minor alleles (1.25 vs 1.75). Concerning other predictors, the prominent SNPs rs6311 and rs6313, which showed complete linkage in our sample, did hardly impact classification results. Among other positive reports, rs6311 was associated with ADHD in a rather large candidate gene study, but overall no consistent associations were reported so far 39.Predictor evaluation based on importance can adumbrate interaction effects that would not reach statistical significance in conventional association as thousands of predictor combinations are assessed for model building. Concerning the most prominent features in this analysis, SERT BP123I]FP-CIT, binding to dopamine and serotonin transporters, did not demonstrate serotonergic binding differences in 17 ADHD patients compared to HC40. These findings were supported by a PET study using the tracer [11C]MADAM that reported similar mean binding between eight ADHD patients and HC in several ROI, including prefrontal cortex, thalamus and putamen41. However, these results must be interpreted in the light of different radioligands and small sample sizes. The most extensive analysis of SERT binding in ADHD reported so far showed overall decreased BPND in 25 ADHD patients compared to age and sex matched controls applying the current gold-standard radioligand [11C]DASB. Strongest effects were observed in the striatum, insula and anterior cingulate cortex; however, these results did not withstand correction for multiple testing in the post hoc analyses. Interestingly, only interregional molecular correlations of SERT binding between the hippocampus and the precuneus withstood correction thresholds, indicating that an interplay of brain regions may better portray abnormalities of serotonergic transmission in ADHD16. These thoughts are in line with moderate to high accuracy despite the lack of single predictor association results in our sample.However, the literature on in vivo SERT binding is limited. A previous study using single-photon emission computed tomography and the radioligand DASB that could have been used for validation. Thus, we cannot rule out overoptimistic PPV and NPV in our model. Although 38 subjects can be considered a large sample for a PET neuroimaging analysis, the observation count is marginal for machine learning classification. The CV design with fold-specific feature selection, refrainment from further parameter tuning and averaging across repeats can be regarded as state of the art but cannot substitute low sample size and lack of external validationHTR1B as well as HTR2A activity, and especially put emphasis on the rs130058 and rs1328684 polymorphisms.To summarize, we propose a diagnostic prediction model for ADHD and HC based on multimodal serotonergic data. Thereby, we present the first PET based classification model for ADHD and expand on previous designs based solely on a single data type. We cannot yet advocate clinical applicability of this diagnostic model but present a step towards the goal of precision medicine in psychiatry. More importantly, our findings support different serotonergic profiles in ADHD and HC, reflected by distinct SERT and


Studies on gender differences in attention-deficit/hyperactivity disorder (ADHD) comorbidities in the Asian populations have been limited and previous studies have shown inconclusive findings. Singapore is a city-state country in Southeast Asia with a population of 5.7 million. This study examined gender differences in internalizing and externalizing problems in Singaporean children and adolescents with ADHD. The plausible social factors underlying the gender differences were discussed.A total of 773 participants  newly diagnosed with ADHD were recruited from the largest public child and adolescent psychiatric center in Singapore. Their internalizing and externalizing problems were assessed using the Child Behavioral Checklist and Teacher’s Report Form by parents and teachers respectively. Demographics and relevant social factors were collected using parent questionnaires.Females with ADHD were reported to have less delinquent and aggressive behavior but more depressive symptoms than their male counterparts, similar to findings in the Western literature. Gender remained a significant predictor of externalizing problem after controlling for other factors. Lower socioeconomic status and parental use of physical punishment were significant predictors of both internalizing and externalizing problems.Gender differences in ADHD comorbidities do exist in the Asian clinical population. The lack of externalizing symptoms in females with ADHD has made timely referral and diagnosis challenging. More research is needed in understanding the gender differences in ADHD and the biopsychosocial mechanism underlying the differences in order to improve the detection of ADHD in females. In Sing million . ADHD waIt is well known that ADHD affects more males than females and the gender ratio differs between the clinical samples and community samples. In a meta-analysis of 97 studies with community samples, the male to female ratio was reported to be about 3:1 . In the Before the change from ADHD ‘subtypes’ to ‘presentations’ in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders , past stOther than differences in core symptoms of ADHD, it has been reported that males and females with ADHD displayed different comorbidity profiles. In general, males with ADHD presented with externalizing problems such as conduct disorder and oppositional defiant disorder while females with ADHD presented with more internalizing problems such as anxiety and depression , 18–20. In addition to gender, age is another factor that moderates ADHD symptoms and comorbidities. In a prospective study of 128 boys with ADHD, decline in ADHD symptoms, especially hyperactivity and impulsivity, was noted as they grew up . HoweverTo date, most studies on gender differences on ADHD have been done in the western population. Not much is known about the gender differences in the developmental population in Asia. Due to cultural differences, the perception of hyperactivity, the stigma of ADHD and the threshold of seeking professional help may be different in the Asian population. The limited studies in Asia suggested no gender differences in terms of ADHD symptoms and comorbidities , 11. HowThis study aimed to examine the distribution of externalizing and internalizing symptoms between males and females with ADHD as well as between predefined age groups using parent-reports and teacher-reports through an Asian clinical sample, with adequate representation from each gender. The primary hypothesis was that females with ADHD would present with fewer externalizing and more internalizing symptoms compared to males with ADHD. We also hypothesized that the gender differences might be further moderated by age. The secondary aim attempted to investigate the effects of age, socioeconomic status, use of physical punishment and aggression between parents on internalizing and externalizing problems in ADHD. It was hypothesized that younger age, lower socioeconomic status, use of physical punishment and aggression between parents would be associated with more externalizing problems and older age would be associated with more internalizing problems.This was a retrospective study utilizing an existing database (reference number IMH/2005-0005) maintained by DDP at IMH. Children and adolescents with learning, emotional or behavioral problems were referred to DDP clinics by parents, schools or medical doctors . The regAll data was collected as part of routine assessments at the first clinic consultation. Written consents to use the collected data for research purposes were obtained from parents at the first clinic visit. Parents completed the Child Behavior Checklist (CBCL 6-18) while a school teacher who was familiar with the participant completed the Teacher’s Report Form (TRF).Both CBCL and TRF are commonly used behavioral measures describing a child’s behavioral, emotional, and social problems over the past 6 months . The verApart from CBCL, parents completed a standardized questionnaire which was adapted from the Ontario Child Health Study . The queParental education levels and whether they have experienced financial problems were extracted as proxy of socioeconomic status of the family. Their educational levels were categorized into two levels: secondary education or below and post-secondary education.To study the relationship between physical punishment and internalizing and externalizing problems, two relevant items were extracted. They were whether parents (1) spanked the young persons or (2) hit them with an object. To facilitate multivariate analysis, parents’ responses were coded as 0 = no physical punishment in disciplining, 1 = spanked or hit with an object, and 2 = spanked and hit with an object.Psychological aggression between spouses was assessed by asking parents whether these statements were true or not: “When you had disagreements with your spouse, you resolved it by (1) raising voices and yelling at each other; (2) refusing to talk; (3) insulting or swearing; (4) crying and (5) leaving the room to avoid continuing the argument”. These statements were extracted from the Conflict Tactics Scale which was designed to explore conflict and violence within a family . The resr = Z/√N, where N = number of observations [Distribution of demographic and clinical characteristics of the study population by gender was summarized using appropriate statistical measures. Internalizing and externalizing problem scores and syndrome scale scores of CBCL and TRF were compared between males and females. To study the effect of age on the above scores, the subjects were dichotomized into two age groups, 6 to 12 years old and 13 to 18 years old for comparison. The cut-off at 12 years old was chosen because it is the typical age when Singaporean students are being promoted from primary education to secondary education with significant changes in school environment. Normality checking was done by visual inspection of data distribution and Kolmongorov–Smirmov and Shapiro–Wilk tests. Mann–Whitney test was used for between genders and between age groups comparison in view of non-normal distribution of data and uneven group sizes with unequal variance. Effect sizes were calculated using the formula rvations . Multiplχ2 (1) = 4.95, p < 0.05. There were no statistically significant differences in fathers’ education level, ethnicity, type of housing, or parental occupation categories between the two groups.Among the 773 children and adolescents, there were 677 males and 96 females with a male to female ratio of 7:1. The gender ratio was consistent with those found in overseas and local clinical studies , 12, 32.The demographics and other characteristics of the participants were presented in Table U = 14,042, z = − 2.79, p = 0.005, r = − 0.12), less aggressive behavior  but they were more withdrawn/depressed . Males with ADHD had a significantly higher score for externalizing problems  but the gender difference in internalizing scores was not statistically significant, U = 15,659, z = − 1.52, p = 0.129, r = − 0.06. There was no statistically significant gender difference in attention problems.CBCL internalizing problems, externalizing problems, syndrome scales and total problems were compared between genders using Mann–Whitney test Table . As hypoU = 11,063, z = − 3.40, p = 0.001, r = − 0.15) and delinquent behavior . Males also had higher externalizing problem scores while females had higher internalizing scores but both differences did not reach statistical significance. In addition, males were noted to have more thought problems , social problems  and attention problems, U = 10,905, z = − 3.53, p < 0.001, r = − 0.15. Due to heterogeneity of the 12 items in thought problems, individual item was compared between genders using chi-squared tests with Bonferroni correction for multiple comparisons. No significant gender difference was noted in any item.Gender comparisons based on TRF showed similar results as CBCL Table . Males wU = 7190, z = − 2.63, p = 0.009, r = − 0.11) and externalizing problems, U = 7647, z = − 2.15, p = 0.032, r = − 0.09. The older group had significantly higher scores in withdrawn/depressed problems , anxious/depressed problems , attention problems  and delinquent behavior, U = 6804, z = − 3.05, p = 0.002, r = − 0.13.The effects of age on CBCL syndrome scales, internalizing and externalizing problems were summarized in Table F(9466) = 5.29, p < 0.001. Among the predictors, age , financial problems  and physical punishment  were significant predictors (Table F(9468) = 6.26, p < 0.001. Gender , father’s education level , psychological aggression between parents  and physical punishment  were significant predictors (Table In the multiple linear regression model predicting internalizing problem scores, the list of predictors were able to explain 9% of the variance, F966 = 5.29F966 = 5.29This study aimed to elucidate the gender differences in internalizing and externalizing problems in children and adolescents aged six to eighteen with ADHD using parent and teacher reports extracted from a clinical database. Consistent with studies in the West, females with ADHD presented with fewer externalizing problems than their male counterparts. In addition, females presented with more internalizing problems although it was not statistically significant.Gender remained a significant predictor of externalizing problem after controlling for other factors. In contrast to previous clinical studies in Asia where no gender difference was found in externalizing problems, the gender difference noted in our study could be due to two reasons. Firstly, our sample had more females which have increased the statistical power. Secondly, Singaporean parents, similar to most Asian parents, are highly concerned with academic success . SingapoFemales with ADHD were reported to have more internalizing problems but the difference did not reach statistical significance. This could be explained by several reasons. Firstly, only parent-reports and teacher-reports were used in assessing the internalizing problems. It has been shown that parent-reports were less sensitive and they tended to underestimate internalizing problems compared to self-reports , 36. SecIn the multiple linear regressions, the regression models could only explain a small percentage of the variance in internalizing and externalizing problems respectively, suggesting that some important predictors were not included in the regression models. After controlling for potential confounders, there was no evidence of a statistically significant association between gender and internalizing problems and only a weak and marginally significant association between gender and externalizing problems. Therefore, the gender differences observed in the univariate analysis could be contributed by gender differences in the relationship between these confounding factors and internalizing or externalizing problems. However, the gender effects of these confounding factors in the ADHD population are unclear. Their potential effects were discussed below.Adolescents were found to have more internalizing and externalizing problems than children. After controlling for other variables including gender, age remained a significant predictor of internalizing problems with older age having more internalizing problems. This could be due to the increase in academic demands and higher social expectation of impulse control with increasing age. Inattention, hyperactivity and impulsivity problems may become more impairing in secondary schools, leading to the development of more internalizing problems. Previous literature has shown that an earlier age of onset is associated with more externalizing problems and less internalizing problems . In thisIn this study, children and adolescents with ADHD of lower socioeconomic status had more internalizing and externalizing problems. Among studies on socioeconomic status and ADHD prevalence, it remained inconclusive whether low socioeconomic status could be a risk factor for ADHD . Our resConsistent with the literature, physical punishment such as caning and spanking was associated with more internalizing and externalizing problems . Based oAlthough males with ADHD had more thought problems than females with ADHD according to teacher-reports, no statistically significant gender difference was noted on individual items. Since these items described symptoms from a range of heterogeneous disorders including psychotic disorders and obsessive–compulsive disorder, more comprehensive assessment of these disorders is needed before we can establish their severity and prevalence for gender comparison. Furthermore, the low prevalence of thought problems in children and adolescents necessitates a larger sample for comorbidity study. The gender differences in thought problems observed by teachers in this study may reflect problems in inattention, impulsivity or hyperactivity e.g. “can’t keep his/her mind off certain thoughts”, “twitching”, “deliberately harm self”, “pick nose” and “strange behaviors”.Teachers reported more social problems in males than females with ADHD in the current study. It has been known that both genders with ADHD suffer from social problems including peer rejection and victimization –46. HoweUnlike most clinical studies, participants in this study were all newly diagnosed with ADHD and were treatment naïve. Hence, the results were not confounded by interventions. The study has also included the largest number of females with ADHD among studies on gender differences in ADHD in Asia to date. With more female subjects, we were able to capture the gender differences that were not reported previously among young persons with ADHD in Asia. The findings suggest that in the clinical population, males and females with ADHD could present with different comorbidity profiles. Awareness of the gender-specific comorbidity profiles and management addressing the specific needs is warranted.Although gender differences in ADHD symptoms and comorbidity have been reported more than two decades ago, the causes were not clearly understood. Structural and functional neuroimaging studies have shown numerous gender differences in neuroanatomy and neural network activities . For exaThe lack of externalizing symptoms in females with ADHD may have delayed referral to the professional. A study on barriers to treatment in ADHD has suggested that parents of daughters with high risks of ADHD were less likely to seek help than parents of males with similar problems . ParentaDuring assessment, clinician should be mindful of the gender differences in the symptoms presented. Gender specific threshold in symptom lists or diagnostic instrument could be a possible solution. However, there is a risk of overdiagnosis in females and it was suggested that the impairment criterion should remain the same for both genders . At the As most studies in female ADHD were based on clinical samples, characteristics of those that were not referred remained largely unknown. Future study using a community sample would help us understand the characteristics of this non-referred ADHD population. A community study would also promote understanding on the inherent gender difference on attention, hyperactivity and impulsivity in the general population. This information would be crucial in development of any gender-specific diagnostic tool or criteria. Apart from clinical criteria, future studies should explore gender differences in commonly used neuropsychological assessment in the ADHD population. This would allow more accurate interpretation of assessment.There were several limitations in this study. Firstly, the participants were not recruited by nationwide population sampling although they were recruited from the largest child and adolescent psychiatric center in Singapore. Secondly, database was incomplete due to missing data. Thirdly, the database was collected more than ten years ago but it was the largest and most comprehensive of the ADHD clinical population available at the time of the study. Since gender differences in ADHD in externalizing and internalizing problems have been consistently reported over the years, we expect minimal effect of the time gap on the generalizability of our results.This study shows that gender differences in comorbidity profiles do exist in Asian ADHD population with males having more externalizing problems than females. Although females were shown to have more internalizing problems than males in the literature, the difference was not significant in our sample. In addition, experiences of physical punishment and lower socioeconomic status were associated with more internalizing and externalizing problems. With other covariates being controlled, age was a better predictor of internalizing problems than gender while gender was a better predictor for externalizing problems than age.Our results showed that a multi-pronged approach is needed to improve detection of females with ADHD who usually present with fewer externalizing problems. At the parent and school level, we need to improve parental and teachers’ awareness of ADHD and promote help seeking behavior. At the clinician level, we need to re-examine whether gender-specific criteria are needed for diagnosis and whether certain neuropsychological assessments should have gender specific cut off scores.