Globalization of clinical trials has important consequences for trial planning and interpretation. This study investigated heterogeneity in patient characteristics and outcomes among world regions in the global idalopirdine Phase 3 clinical program.n = 2506). Patients received idalopirdine  or placebo, added to cholinesterase inhibitor treatment. Patients were categorized into the following regions: Eastern Europe/Turkey (n = 759), Western Europe/Israel (n = 709), USA/Canada (n = 444), South America/Mexico (n = 361), Asia (n = 134), and Australia/South Africa (n = 99). For each region, operational characteristics, baseline demographic and clinical characteristics, adverse events, and mean change from baseline to week 24 in clinical rating scale scores (placebo group only) were summarized using descriptive statistics.Data were pooled from three 24-week randomized controlled trials in patients aged ≥ 50 years with mild-to-moderate Alzheimer’s disease (AD)  and USA/Canada (17.5) than in other regions (16.9–17.1). Baseline AD Assessment Scale—Cognitive subscale (ADAS-Cog) scores ranged from 24.3 in USA/Canada to 27.2 in South America/Mexico. Baseline AD Cooperative Study—Activities of Daily Living, 23-item version (ADCS-ADL23) scores ranged from 58.5 in USA/Canada to 53.5 in Eastern Europe/Turkey. In the placebo group, adverse events were 1.6–1.7 times more common in Western Europe/Israel, USA/Canada, and Australia/South Africa than in Eastern Europe/Turkey. On the ADAS-Cog, Australia/South Africa and Western Europe/Israel showed the most worsening among patients receiving placebo , whereas South America/Mexico showed an improvement (−0.71 points). All regions worsened on the ADCS-ADL23, from −3.21 points in Western Europe/Israel to −0.59 points in Eastern Europe/Turkey.Completion rates were 0.86–0.90 in all regions. Heterogeneity among global regions was evident. Protocol deviations were twice as common in South America/Mexico as in USA/Canada (2.64 vs 1.35 per patient screened). Educational level ranged from 9.2 years in South America/Mexico to 13.4 years in USA/Canada. Regional heterogeneity—in terms of study conduct, patient characteristics, and outcomes—exists, and should be accounted for, when planning and conducting multinational AD clinical trials.NCT01955161. Registered on 27 September 2013.ClinicalTrials.gov, NCT02006641. Registered on 5 December 2013.ClinicalTrials.gov, NCT02006654. Registered on 5 December 2013.ClinicalTrials.gov,  N-methyl-d-aspartate (NMDA) receptor antagonist, memantine [Alzheimer’s disease (AD) is a highly prevalent, age-related, degenerative brain disease, associated with considerable burden for patients, their family members, caregivers, and society . As the emantine , 3. Whilemantine , 4, theremantine .Increasingly, clinical trials of potential new drugs in AD are conducted on an international basis, which allows for faster recruitment of patients, has lower costs, and provides the opportunity to pursue registration and sales in different regions , 7. HoweThe extent to which these regional factors can impact clinical study outcomes, and to which they should be considered in the design of multinational studies, is unknown. Using data from the multinational Phase 3 clinical program of idalopirdine, the aim of this analysis was to understand the effect of globalization on AD clinical trial design and outcomes by comparing study conduct, patient completion rates, patient characteristics, adverse events, and disease progression across regions.6 receptor antagonist—did not reduce cognitive loss compared with placebo over 24 weeks [Data were pooled from three 24-week, randomized, double-blind, placebo-controlled studies in mild-to-moderate AD: STARSHINE , STARBEAM , and STARBRIGHT . In these studies, treatment with idalopirdine—a selective serotonin 5-HTEach of the three studies had a similar design, comprising: a 2-week screening period during which patient eligibility was monitored by qualified medical staff employed by the sponsor; a 24-week, randomized, double-blind treatment period with regular safety and efficacy assessments; and a 4-week safety follow-up period (or enrollment into an open-label extension study). Patients were enrolled from 34 countries. The studies included male and female outpatients aged ≥ 50 years with a National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria diagnosis of probable AD , with a 23) and the AD Cooperative Study—Clinical Global Impression of Change (ADCS-CGIC). The ADCS-ADL23 is an informant-rated measure of functional impairment scored from 0 to 78, where a higher score indicates less impairment [23, ADCS-CGIC, and MMSE.The primary outcome measure of each study was the AD Assessment Scale—Cognitive subscale (ADAS-Cog), an 11-item, objective measure of cognitive impairment scored from 0 to 70, with a higher score indicating more impairment . Key secpairment , 14. Thepairment , 16. Othpairment . The MMSSafety was assessed via the reporting of adverse events, classified according to the Medical Dictionary for Regulatory Activities (MedDRA) version 19.0.For the present post-hoc analysis, patients were categorized into geographic regions according to shared culture, history, geography, and linguistic features, based on the work of Glickman et al.  and prevFor each region, operational characteristics of the clinical program were summarized using descriptive statistics, including the number of sites that screened at least one patient and that randomized at least one patient, the number of patients screened and number of patients randomized per month, and the randomization, completion, and protocol deviation rates.Baseline demographic and clinical characteristics were summarized descriptively by region for the all-patients-treated set (APTS), defined as all randomized patients who took at least one dose of double-blind medication. Statistical comparisons of baseline variables across all regions were calculated using Kruskal–Wallis tests for the continuous variables, and chi-squared tests for the categorical variables.The incidences of adverse events, serious adverse events, and deaths were summarized descriptively, per patient in the APTS, by treatment group and by region.Mean change from baseline to week 24 in the clinical rating scale scores was summarized descriptively using observed cases, by region, in the subpopulation of patients who were randomized to placebo and completed their respective study. For the ADCS-CGIC, which is itself a measure of change from baseline, the mean absolute value at week 24 was summarized using observed cases. Clinical outcomes were investigated in the placebo group rather than the total population to make the results more generalizable, and to prevent the bias that may occur due to a drug’s specific mode of action in relation to the genetic and environmental differences in patients across regions. Statistical comparisons of equality of variances were calculated using Levene tests, and pairwise comparisons between specific regions were calculated using Tukey tests. In case the assumption of equal variances was violated, sensitivity analyses based on Games–Howell post-hoc tests were performed. To evaluate whether regional differences in observed change from baseline to week 24 in the clinical rating scale scores were driven by regional differences in educational level, a linear regression analysis that adjusted for both region and years of education was used as a sensitivity analysis.Testing was done using a 0.05 significance level (two-sided) with no overall correction for multiple comparisons (regions were corrected for in the Tukey and Games–Howell tests). The statistical analyses were performed using SAS version 9.4 (SAS Institute Inc.).Operational characteristics of the clinical program across regions are presented in Table A total of 2506 patients received at least one dose of randomized treatment, split across the regions as follows: Eastern Europe/Turkey, 759 ; Western Europe/Israel, 709 (28.3%); USA/Canada, 444 (17.7%); South America/Mexico, 361 (14.4%); Asia, 134 (5.3%); and Australia/South Africa, 99 (4.0%).APOE) ε4 carriage, and MMSE score , but not for age or sex. Specifically, height was lowest in Asia (1.57 m) and South America/Mexico (1.59 m), and comparable in the other regions (1.63–1.64 m). Weight and BMI were lowest in Asia , and highest in Australia/South Africa  and USA/Canada . Educational level was lowest in South America/Mexico (9.2 years) and Asia (9.6 years), and highest in USA/Canada (13.4 years). The proportion of married patients was lowest in Eastern Europe/Turkey (58.2%) and highest in Asia (77.6%). Most commonly, caregivers were a spouse or partner in Western Europe/Israel, USA/Canada, Asia, and Australia/South Africa, and a child in Eastern Europe/Turkey and South America/Mexico. APOE ε4 carriage was most common in Australia/South Africa (80.6%), followed by Western Europe/Israel (63.1%), and was < 60% in the other regions, being lowest in Asia (51.5%).Baseline demographics by region are presented in Table At screening, the MMSE score was higher (indicating less cognitive impairment) in Eastern Europe/Turkey (18.0) and USA/Canada (17.5) than in the other regions (range 16.9–17.1). A similar pattern was seen at baseline. All regions showed a mean improvement in the MMSE score from screening to baseline. The degree of improvement ranged from 0.5 points in Eastern Europe/Turkey to 0.9 points in USA/Canada, South America/Mexico, and Australia/South Africa. For complete comparisons, see Table p < 0.0001). Median time since AD diagnosis varied from 1.3 years in Eastern Europe/Turkey to 2.2 years in Australia/South Africa. Median prestudy treatment duration varied from 1.0 year in Eastern Europe/Turkey to 1.3 years in Australia/South Africa and USA/Canada. Previous treatment with a ChEI other than donepezil (among patients currently treated with donepezil as required by protocol) ranged from 2.4% in Eastern Europe/Turkey to 7.8% in Western Europe/Israel. Previous treatment with memantine ranged from 1.0% in Australia/South Africa to 20.0% in USA/Canada.Clinical characteristics by region are presented in Table 23 , but not for the NPI. The baseline ADAS-Cog score was lowest (less cognitive impairment) in USA/Canada (24.3) and highest (more cognitive impairment) in South America/Mexico (27.2). The baseline ADCS-CGIC (severity) scores were lowest (less severely ill) in South America/Mexico (3.6) and Asia (3.7), and highest (more severely ill) in Eastern Europe/Turkey and Western Europe/Israel (both 3.9). The baseline ADCS-ADL23 score was highest  in USA/Canada (58.5), and lowest  in Eastern Europe/Turkey (53.5). Although not statistically different across all groups, the baseline NPI score was lower  in Asia (7.2) than in the other regions (10.1–11.7).Regional differences at baseline, also presented in Table The incidence of adverse events by region is presented in Table p < 0.0001), thus violating the assumptions of the Tukey test. A sensitivity analysis assuming unequal variances across regions (Games–Howell post-hoc test) confirmed the significant pairwise differences, except for USA/Canada and South America/Mexico (adjusted p = 0.075).Mean changes in the clinical rating scale scores from baseline to week 24 in the placebo group are presented in Table 23); the most worsening was seen in Western Europe/Israel (−3.21), and the least in Eastern Europe/Turkey (−0.59). The behavioral outcome (NPI) showed improvement in all regions (range −0.13 to −1.73) except for Western Europe/Israel (0.45). The MMSE score worsened in all regions (range −0.34 to −1.22) except for Eastern Europe/Turkey (0.10).All regions showed worsening on the functioning outcome (ADCS-ADLp = 0.0024), and the adjusted difference between Western Europe/Israel and South America/Mexico remained significant. Years of education was not a significant predictor for rate of decline for the other endpoints .When adjustments were made for education as well as region, years of education predicted a faster rate of decline on the ADAS-Cog  and the region with the lowest mean weight (Asia). BMI followed a similar regional pattern. Weight and BMI are important considerations in clinical trial design, since they can affect drug distribution and clearance  and may APOE ε4 carriers than has been observed in populations of biologically proven AD patients  [APOE ε4, which is higher in black populations than white populations [APOE ε4 status and risk of late-onset AD also varies among races, being strongest and most well established in European and Asian populations [Other than Australia/South Africa (80.6%), all regions had a lower proportion of 3 trial) . This coulations , 26. Theulations , 28.Several differences in diagnosis and treatment history were observed across regions, which may reflect differences in culture, societal awareness of AD, and standard of care. The time since AD diagnosis was almost a year longer in Australia/South Africa than in Eastern Europe/Turkey; this could suggest that patients in Eastern Europe/Turkey present to the clinic later in the disease , or that study inclusion criteria (such as the requirement for stable donepezil/ChEI treatment) selected for patients with a greater disease duration in some regions.Considering patient treatment history prior to enrollment, use of a ChEI other than donepezil (among those currently treated with donepezil) was low in all regions (< 10%), whereas prior memantine treatment varied from around 20% in USA/Canada and South America/Mexico to < 10% in all other regions. In the USA, memantine is often initiated as early as ChEIs, despite memantine not being approved for use in mild disease . In the Baseline clinical rating scale scores were heterogeneous among regions despite the fact that patients were monitored for eligibility during the 2-week screening period, and despite standardization of clinical rating scale usage. There were also differences in patient stability during the screening period, as shown by variation in the change in MMSE score from the screening visit to the baseline visit. Overall, at baseline, patients were least cognitively impaired in USA/Canada, and most impaired in South America/Mexico. Patients in USA/Canada also had the least impairment of functioning at baseline. The greatest impairment in functioning was observed in Eastern Europe/Turkey, which could reflect reporting behaviors of the different informants in this region, with a low proportion of married patients and with more patients being cared for by a child rather than by a spouse or partner. The suitability of activity of daily living (ADL) items to elderly patients differs across regions and can potentially affect functional metrics , 32. Fin23) was observed in Western Europe/Israel and Australia/South Africa, and the least decline was observed in Eastern Europe/Turkey and South America/Mexico. The variations may reflect differences in standard of care or accuracy of diagnosis among regions, as well as the proportion of APOE ε4 carriers. Such variations in placebo decline will influence the outcome of clinical trials, since low rates of decline among patients receiving placebo will make it more difficult to distinguish a treatment effect.Based on analysis of the change in clinical rating scale scores over the first 6 months in the placebo group, the greatest decline  at baseline as well as the least worsening in functioning, which could reflect: the low proportion who were married and differences between informants, as already discussed; cultural differences in the appreciation of the importance of different ADLs; and the positive effect on quality of life and standard of care that can be achieved from enrollment in a clinical trial. This latter point may also be reflected in the observation that Eastern Europe/Turkey was the only region to show improvement on the MMSE over 24 weeks in the placebo group. With regard to South America/Mexico, a similar pattern to that of Eastern Europe/Turkey was observed in terms of baseline ADCS-ADL23, change in ADCS-ADL23, and proportion of married patients. In addition, South America/Mexico was the only region to show improvement on the ADAS-Cog over 24 weeks (placebo group), despite having the most cognitive impairment at baseline; this may be linked to the lower level of education in this region and differing approaches to diagnosis.Overall, two regions stood out in their patterns of characteristics at screening/baseline and their subsequent outcomes/trajectories: Eastern Europe/Turkey and South America/Mexico. Eastern Europe/Turkey had the most impaired functioning  across regions. Awareness of such variations in adverse event reporting is important for industry sponsors of drug development programs, since reports from Eastern Europe and South America may not reflect the side-effect profile seen in other global regions.Other large multinational AD clinical programs  have also shown heterogeneity among geographic regions , 19. ComHeterogeneity may arise for many reasons, including differences in culture and standard of care for patients with AD. A heterogeneous patient population can be advantageous in a clinical program in order to show how an investigative agent affects a real-world AD population. However, it is possible that a heterogeneous patient population may indicate that patients without AD or with atypical AD are being included, making it more difficult to observe a treatment effect, and highlighting the importance in future AD dementia trials of recruiting patients with biomarker evidence of amyloid pathology and neurodegeneration, if proposed research diagnostic criteria are validated.This analysis of a large clinical trial program is limited by its post-hoc nature, since these studies were not designed to assess regional differences. In addition, homogeneity was not tested within the defined geographic regions, the population sizes were small in the Asia and Australia/South Africa groups, and the analysis did not control for multiple comparisons. These analyses are intended to provide observations of potential importance to those planning, conducting, and interpreting global clinical trials.These analyses—conducted on a large, global, clinical trial program—demonstrated that regional heterogeneity exists in multinational AD clinical trials, and that it should be accounted for, whether by limiting or excluding certain countries, increasing sample size to account for the variance, or finding additional means of increasing homogeneity . Sponsors must be familiar with regional differences in order to appropriately plan and power studies of global programs. Differences in training and vigilance may also be required among regions.


Pulmonary rehabilitation (PR), delivered as a supervised multidisciplinary program including exercise training, is one of the cornerstones in the chronic obstructive pulmonary disease (COPD) management. We performed a systematic review and meta-analysis to assess the effect on mortality of a supervised early PR program, initiated during or within 4 weeks after hospitalization with an acute exacerbation of COPD compared with usual post-exacerbation care or no PR program. Secondary outcomes were days in hospital, COPD related readmissions, health-related quality of life (HRQoL), exercise capacity , activities of daily living (ADL), fall risk and drop-out rate.We identified randomized trials through a systematic search using MEDLINE, EMBASE and Cocharne Library and other sources through October 2017. Risk of bias was assessed regarding randomization, allocation sequence concealment, blinding, incomplete outcome data, selective outcome reporting, and other biases using the Cochrane Risk of Bias tool.We included 13 randomized trials (801 participants). Our meta-analyses showed a clinically relevant reduction in mortality after early PR ) and at the longest follow-up ). Early PR reduced number of days in hospital by 4.27 days  and hospital readmissions ). Moreover, early PR improved HRQoL and walking distance, and did not affect drop-out rate. Several of the trials had unclear risk of bias in regard to the randomization and blinding, for some outcome there was also a lack of power.Moderate quality of evidence showed reductions in mortality, number of days in hospital and number of readmissions after early PR in patients hospitalized with a COPD exacerbation. Long-term effects on mortality were not statistically significant, but improvements in HRQoL and exercise capacity appeared to be maintained for at least 12 months. Therefore, we recommend early supervised PR to patients with COPD-related exacerbations. PR should be initiated during hospital admission or within 4 weeks after hospital discharge.The online version of this article (10.1186/s12890-018-0718-1) contains supplementary material, which is available to authorized users. Two studies were included and data from 86 participants were pooled in a meta-analysis evaluating HRQoL directly after end of early PR , 32 and P = 0.49). Unclear sequence generation, allocation concealment, blinding and selective outcome reporting led to downgrading of the confidence in our effect estimates  from five trials using 6MWT yielded a statistically significant mean difference in walking distance of 76.89 m, favoring early PR (95% CI: [21.34 to 132.45]) with high heterogeneity . However, we found a significant within-group effect of early PR after discharge  used the SWT to evaluate the walking distance after the end of treatment and showed a statistically significant mean difference in walking distance of 54.70 m, favoring early PR (95% CI: [30.83 to 78.57]). The subgroup analysis showed no difference in the effect between PR initiated during admission and after discharge (P = 0.40). However, we found a significant within-group effect of early PR during admission and after discharge  or Shuttle Walking Test (SWT)) after the end of treatment was investigated in eight studies , 36–39. P < 0.01)  Fig. . Due to N = 228; usual care: N = 212) [P = 0.37). Our confidence in the effect estimate was downgraded due to unclear sequence generation, allocation concealment, blinding of assessors and incomplete data outcome together with high risk of inconsistency (Table The effect of early PR on the drop-out rate at the end of treatment was investigated in eight studies providing data from 440 randomized participants (early PR: N = 212) , 37–39. N = 92; usual care: N = 89) [P = 0.30; I2 = 6.5%)  , 36, 38.5%) Fig. .None of the included studies reported results on the effect of early PR on ADL or the risk of falling.N = 322) with usual care (N = 312). Subsequent meta-analysis showed that supervised early PR after acute exacerbation of COPD reduced mortality and number of days in hospital together with a reduction in COPD related hospital admissions and an improvement of HRQoL and exercise capacity .The present review summarized the evidence from 13 RCTs including 634 participants with an exacerbation of COPD and compared the use of early PR  and 54.70 m in shuttle walking distance (SWD) immediately after early PR and an improvement of 90.27 m in 6MWD at the longest follow up , which aWe found no difference in drop-out rate between participants allocated to early PR compared with usual care. Thus, the effects were not driven solely by positive responders to PR, and secondly, the most severely affected patients will likely complete or drop-out to the same extent as usual care. As before mentioned, we did not include Greening et al. , since tIn this review ten studies implemented an extensive PR program which mostly showed large and consistent effects on mortality, days in hospital, COPD related hospital readmissions, HRQoL, and walking distance. The PR programs were not exactly similar within the reviewed studies, but the majority provided either many training sessions (more than 16 sessions) , 34, 38,, who reported a higher mortality in the unsupervised home-based rehabilitation group at 12 months compared with usual care group. The difference between groups however, was not related to the early rehabilitation intervention. Indeed, the per protocol analysis did not show a difference in mortality [Currently, the ideal timing of the onset of PR after AECOPD is highly debated. Based on low-quality of evidence, the ERS/ATS Task Force made a conditional recommendation against the initiation of PR during hospitalization since PR initiated during admission was found to increase mortality . This coortality , suggestortality . We did The results of the present review support the substantial and clinical important benefits of supervised early PR, indicating that this is an effective intervention with the purpose of reducing mortality following a hospitalization for AECOPD. Our meta-analysis shows that supervised PR during the recovery period after an AECOPD is superior to usual care in terms of improving prognosis, HRQoL and walking distance. Based on moderate to low quality of evidence, we conclude that supervised early PR reduces the risk of mortality, COPD-related hospital readmissions and the number of days in hospital, and lead to large and clinically relevant improvements in HRQoL and walking distance. Therefore, we recommend supervised PR to patients with COPD-related exacerbations. PR should be initiated during hospital admission or within 4 weeks after hospital discharge.Additional file 1:Search strategy. The full search strategy from the systematic multidatabase literature search performed in 2013 and 2017. (PDF 214 kb)Additional file 2:AGREE II. A critical group appraisal of: Pulmonary rehabilitation for patients with chronic pulmonary disease (COPD): an evidence-based analysis using the AGREE II Instrument. (PDF 54 kb)Additional file 3:AMSTAR (A Measurement Tool to Asses Systematic Reviews). An assessment of the methodological quality of the included systematic reviews. (PDF 10 kb)Additional file 4:Assessment of the included studies. Characteristics and risk of bias assessment of the included studies. (PDF 329 kb)Additional file 5:Table S1. Extensiveness of the PR programs in the included studies. (PDF 23 kb)


Introduction: Ovarian follicle growth is a key step in the success of assisted reproductive treatment, but limited data exists to directly relate follicle growth to recombinant FSH (rFSH) dose. In this study, we aim to evaluate FSH requirements for follicular growth during controlled ovarian stimulation.Method: Single center retrospective cohort study of 1,034 IVF cycles conducted between January 2012–January 2016 at Hammersmith Hospital IVF unit, London, UK. Median follicle size after 5 days of stimulation with rFSH and the proportion of antral follicles recruited were analyzed in women treated with rFSH alone to induce follicular growth during IVF treatment.Results: Starting rFSH dose adjusted for body weight (iU/kg) predicted serum FSH level after 5 days of rFSH , median follicle size after 5 days of rFSH, and the proportion of antral follicles recruited by the end of stimulation. Day 5 median follicle size predicted median follicle size on subsequent ultrasound scans , and hence time to oocyte maturation trigger . Insufficient rFSH starting dose that required >5% dose-increase was associated with increased variability in follicle size on the day of oocyte maturation trigger, and negatively impacted the number of mature oocytes retrieved.Conclusion: Weight-adjusted rFSH dose correlates with follicular growth during ovarian stimulation. Early recruitment of follicles using a sufficient dose of rFSH from the start of stimulation was associated with reduced variability in follicle size at time of oocyte maturation trigger and an increased number of mature oocytes retrieved. IVF treatment is increasingly used to help subfertile couples to conceive, with the number of cycles carried out annually increasing by ~5% per year . HoweverIn the physiological human menstrual cycle, a small ~30%) rise in serum FSH level, due to reduced estradiol negative feedback during the follicular phase 0% rise i, 5. As fSelecting the optimal starting dose of rFSH is challenged by the interindividual variation in response to the same dose. Clinicians use clinical experience as well as baseline variables such as age, antral follicle count (AFC), serum anti-Mullerian hormone (AMH), body mass index (BMI), and follicular phase serum FSH level in selecting an appropriate rFSH dose in patients presenting for their first treatment cycle , 13. DueThe majority of dosing algorithms relate rFSH dose to the number of oocytes retrieved, which assumes a homogenous response to the trigger of oocyte maturation . Howevern = 175) were completed during clinical trials investigating the use of kisspeptin as an oocyte maturation trigger and had more intensive monitoring of endocrine values such as serum FSH and LH during ovarian stimulation  in patients who had ovarian stimulation by Gonal F . Exclusion criteria were: use of additional/alternative medications for ovarian stimulation including urinary FSH, recombinant LH, or long-acting FSH preparations, or missing data on rFSH doses. Women were not excluded for having PCOS, endometriosis or for meeting Bologna/Poseidon criteria –25. A sumulation –28.Baseline demographic data such as height, weight, ethnicity, pre-treatment serum FSH (iU/L), LH (iU/L), AMH (pmol/L), estradiol (pmol/L) and total AFC on transvaginal ultrasound were collated. For the vast majority of patients, follicular phase AFC and FSH measurements occurred within 3 months of commencing stimulation. AFC was measured manually using 2D ultrasound. Follicle sizes were assessed by transvaginal ultra-sonographic measurement  conducted by up to nine experienced IVF physicians/ultrasonographers at Hammersmith IVF unit over the 4-year study period. The unit protocol is to conduct an ultrasound scan after 4–5 days of rFSH. Starting doses of rFSH (between 100 IU and 450 IU daily) were selected based on age, AFC and physician experience. Patients may have had either fixed (day 5 of rFSH) or flexible start (lead follicle of 14 mm) of GnRH antagonist administration due to a change in unit policy during the time of the study. Two thirds of patients started GnRH antagonist on day 5 of rFSH, 10% on day 7–8, 11% on day 9, and 13% on days 10–12. We collated data on rFSH doses throughout ovarian stimulation, follicle counts and sizes at each monitoring ultrasound scan. All follicle diameters at each scan were individually recorded and a median follicle size was calculated. Follicle sizes <6 mm in diameter were recorded as measuring 5 mm during analysis. Preliminary linear regression analysis suggested that an age of 35 years represented an inflection point for response to rFSH. Evaluation of follicular response following rFSH was assessed both in a subset of patients with predicted ‘good response’ (age 18–34 years and AFC >15) and those with predicted “poorer response” (age ≥35 years and AFC ≤15). Remaining patients were classified as intermediate responders (either age 18–34 years and AFC ≤15 or age ≥35 years and AFC >15).Patients who received kisspeptin to trigger oocyte maturation from randomized clinical trials approved by the Hammersmith and Queen Charlotte's Research Ethics Committee, London, UK –28. The Recombinant FSH (rFSH)  was used to induce follicular growth. Gonal F was started from day 2 or 3 in GnRH antagonist co-treated ICSI cycles. Premature ovulation was prevented by a GnRH antagonist- either cetrorelix  or ganirelix . The trigger was administered once 3 ovarian follicles were ≥18 mm in diameter and oocyte retrieval was conducted 36 h thereafter. The trigger of oocyte maturation was either hCG, choriogonadotrophin alpha , GnRH agonist—buserelin acetate , or kisspeptin-54  administered subcutaneously –28. DataSerum LH, FSH, estradiol, and progesterone were measured using automated chemiluminescent immune immunoassays . Interassay coefficients of variation were as follows: LH, 3.4%; FSH, 3.5%; estradiol, 3.4%; progesterone, 1.8%. Limits of detectability for each assay were as follows: LH 0.07 mIU/ml; FSH 0.05 mIU/ml; estradiol 70 pmol/l (19 pg/mL); progesterone 0.3 nmol/L (0.1 ng/mL). AMH was measured using an enzyme linked immunosorbent assay . The reference range was 2.2–48.5 pmol/L (0.3–6.8 ng/mL). The lower limit of detection was 0.6 pmol/l (0.08 ng/mL). The interassay coefficient of variation was 4.6%; the intraassay coefficient of variation was 4.0%.U test. For parametrically distributed data with multiple groups, one-way analysis of variance (ANOVA) was used, whereas for non-parametrically distributed data, the Kruskal-Wallis test was used. Simple linear regression modeling was performed with Pearson correlation coefficients for parametrically distributed data and Spearman correlation coefficients for non-parametrically distributed data. Categorical variables are presented as count . Multivariate generalized linear regression was used to assess the relationship between dose of Gonal F and follicular growth with the following variables assessed: age, AFC and pre-treatment serum FSH level. R2 coefficients of determination were derived using the formula R2 = 1 – /(null deviance). A p < 0.05 was regarded as signifying statistically significance.Statistical analysis was performed using GraphPad PRISM version 7.0, STATA version 14.0 and R version 3.5.1. D'Agostino & Pearson normality test was used to assess data distribution. Normally distributed data is presented as mean ±standard deviation (SD), and non-parametric data as median with interquartile range (IQR). Non-parametric paired data for two groups was compared by the Wilcoxon signed rank test. Non-parametric unpaired data with two groups was compared using Mann-Whitney n = 175).Overall, 1034 GnRH antagonist co-treated cycles were included, including 354 predicted “good responders” (age 18–34 years and AFC >15) and 183 predicted “poor responders”  had an average rFSH dose during treatment that was at least 5% higher compared to the starting dose and 82 (8%) had an average dose that was at least 5% lower than the starting dose, whereas the remaining 682 (66%) received an average dose within ±5% of the starting dose.r2 = 0.352, P < 0.001; p = 0.937; p < 0.0001).Serum FSH level at steady-state measured after 5 days of rFSH administration was associated with median follicle size on ultrasound . Serum FIn patients predicted to be “good responders” (aged 18–34 years and AFC >15), median follicle size following 5 days of treatment was associated with weight-adjusted rFSH dose . The pror2 = 0.58, p < 0.0001; r2 = 0.62, p < 0.0001; r2 = 0.22, p < 0.0001; Median follicle size after 5 days of rFSH was a useful measure of ovarian response, positively predicting median follicle size on day 7 . Me. Me22). r2 = 0.17, p < 0.0001) and the cumulative sum of all follicle sizes on the final scan before oocyte maturation trigger . The cumulative sum of follicle sizes is a better predictor of the number of mature oocytes retrieved than serum estradiol on the day of the final scan . However, as previously reported, the number of follicles sized between 12 and 19 mm is a better predictor still of the number of mature oocytes retrieved   .p < 0.0001)   and 2 daWe investigated the relationship between rFSH dose and follicle growth in data from 1,034 IVF cycles. Preliminary analysis revealed that recombinant FSH dose adjusted for body weight (IU per kg) was associated with steady-state serum FSH levels and median follicle size following 5 days of rFSH treatment. Median follicle size after 5 days of rFSH was a useful measure, positively predicting median follicle size on subsequent scans and thus time to administration of oocyte maturation trigger. Once antral follicles are recruited to grow, they become less FSH-dependent for their continued growth and accrue LH receptors . Indeed,Antral follicles represent a pool of follicles capable of being recruited to grow if sufficient FSH exposure is provided. The differential sensitivity of individual antral follicles within a cohort, enables the success of step-up protocols for inducing mono-follicular growth in ovulation induction cycles, by gradually achieving the minimum threshold for growth of the most sensitive follicles . In the During a multi-variate analysis, variables identified as being relevant were consistent with those identified to influence oocyte number in the CONSORT algorithm  , 33. AccIn accordance with the published literature, we observed that the dose-response relationship was lost in anticipated poor responders (AFC ≤ 15 & age ≥ 35 years) receiving doses of rFSH over 2.25iU/kg , 37. ThuSerum estradiol is used as a marker of adequate ovarian response by some clinicians, and rFSH dose increased if serum estradiol is deemed suboptimal, even if follicle growth appears adequate. However, this relationship appears to be indirect; serum estradiol at the end of stimulation was correlated with the cumulative sum of follicle sizes on the day of trigger, as well as the number of mature oocytes subsequently retrieved. Nevertheless, the number of follicles sized 12–19 mm at the end of stimulation was a better predictor of the number of mature oocytes retrieved than serum estradiol level  22)..22).We investigated the impact of dose-adjustment during stimulation. We hypothesized that an insufficient dose of rFSH could lead to recruitment of the most sensitive follicles, whilst the majority of follicles would remain dormant. A subsequent dose-increase would recruit another wave of follicles that would therefore result in increased variability of follicle size by the end of stimulation. We observed that a lower starting rFSH dose reduced the proportion of follicles sized 12–19 mm at the end of stimulation in keeping with data using a novel human rFSH  and withThe rFSH preparation studied  has a terminal half-life of 1 day and reaches steady state levels after 3–4 days . Thus, iIt has been established that there is insufficient evidence to suggest that rFSH dose significantly impacts live birth rates . This isStrengths of our study are the inclusion of a large number of cycles examined at a single center, thereby limiting heterogeneity due to protocol differences amongst different reproductive endocrinology treatment centers. Furthermore, monitoring was conducted using the same ultrasound machines  and no more than 9 experienced IVF practitioners measured follicle size during the period of the study. Only data from cycles that used rFSH  alone were included, thus limiting variability due to other FSH preparations. Limitations of the study include its retrospective design, and that due to heterogeneity in day of monitoring ultrasound scans, not all cycles could be used in every analysis. Serum FSH, LH, and AMH levels were only measured for a subset of cycles and thus any benefit to using serum FSH level over rFSH dose adjusted for weight could not be ascertained. Serum AMH and AFC both have similar diagnostic accuracy as ovarian response markers , althougTo conclude, we have identified a dose-response for starting rFSH dose to median follicle size following 5 days of treatment, and to the proportion of antral follicles recruited. Our data suggests that commencing stimulation with a sufficient rFSH dose from the start of the cycle reduces variability in follicle size to thus maximize the number of mature oocytes retrieved. Consistent with published data suggesting an upper threshold to useful FSH dosing, we find minimal further increase in response beyond a rFSH dose of 2.25iU/kg . These dThe datasets generated for this study are available on request to the corresponding author.Patients who received kisspeptin to trigger oocyte maturation from randomized clinical trials approved by the Hammersmith and Queen Charlotte's Research Ethics Committee, London, UK –28. The All authors provided contributions to study conception and design, acquisition of data or analysis and interpretation of data, drafting the article or revising it critically for important intellectual content, and final approval of the version to be published. Here are the most important contributions of each author: AA, RS, GT, AC, SL, RR, and WD designed the study. Data was collected by AP, AA, TH, GC, SC, PE, and MP. Analysis was carried out by AA, AP, and TK. WD takes final responsibility for this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This retrospective study was designed to compare prognostic relevance of magnetic resonance imaging (MRI) findings before and after neoadjuvant chemoradiotherapy (CRT). From 2002 to 2010, 399 patients who underwent surgery after CRT for rectal cancer (≥T3) and had adequate pre-CRT (mr) and post-CRT (ymr) MRI findings were examined. Factors examined included tumour (T), lymph node (N), mesorectal fascia (MRF), extramural venous invasion (EMVI), and tumour regression grade (TRG). Two Cox proportional hazard models were created using mr and ymr findings separately for overall survival (OS), disease-free survival (DFS), and local recurrence rate (LRR). Among mr findings, only mrEMVI was a significant prognostic factor for OS and DFS. Among ymr findings, ymrN, ymrMRF, and ymrEMVI were significant prognostic factors for OS and DFS, whereas ymrMRF and ymrEMVI were significant prognostic factors for LRR. C-indices tended to be higher for ymr findings than for mr findings . Survival outcomes of patients having all ymr risk factors were significantly poor . ymr findings showed better prognostic significance than mr findings. Among ymr findings, ymrN, ymrMRF, and ymrEMVI were independent prognostic factors for oncologic outcomes. Additionally, several studies have used MRI for assessing the response of rectal cancer to neoadjuvant therapy and have attempted personalised treatments such as local excision or induction chemotherapy3.Rectal magnetic resonance imaging (MRI) plays an important role in determining rectal cancer treatment. Practical guidelines recommend neoadjuvant therapy according to the initial clinical stage of rectal cancer, and MRI is one of the reliable tools for initial staging4. The postoperative T and N classifications are one of the traditional prognostic factors that have been used for a long period of time. The T and N staging of MRI have been used under the same context. Moreover, pathologic T and N classifications after neoadjuvant chemoradiotherapy (CRT) have been reported to represent the prognosis of patients5. However, the accuracy and prognostic significance of MRI restaging are controversial due to tissue changes such as fibrosis and oedema4.Various MRI findings of tumour (T), lymph node (N), mesorectal fascia (MRF), extramural venous invasion (EMVI), and tumour regression grade (TRG) are based on pathologic findings and known to be related to the prognosis of patients with rectal cancer6. Adam et al. have reported that postoperative CRM is an important prognostic factor in rectal cancer surgery7. MRI can accurately evaluate MRF involvement in rectal cancer8, and the MERCURY group reported that MRF involvement on MRI is associated with poor disease-free survival (DFS) and local recurrence rate (LRR)9.The MRF is a factor in determining the circumferential resection margin (CRM) during total mesorectal excision10. Smith et al. developed a scoring system to evaluate EMVI through high-resolution MRI11. Thereafter, several studies have demonstrated that grade 3 and 4 EMVI findings on MRI are associated with poor prognosis14.EMVI, defined as the presence of tumour cells in blood vessels outside the muscular layer, has been also known as a poor prognostic factor15. The MERCURY group applied a similar concept into MRI and showed that TRG on MRI after CRT could be a prognostic factor9.Pathologic TRG is an indicator of rectal cancer response to CRT, and patients with less regression have poor prognosis4. However, little is known whether the prognostic significance of MRI findings differ before and after CRT, and it is unclear which point of assessment is more appropriate for representing prognosis. Only few studies have evaluated the differences in prognostic significance of MRI findings before and after CRT16. Furthermore, it is also uncertain that these MRI findings are independent prognostic factors for each other because most of the previous studies did not include all  MRI findings14. Thus, this study aimed to determine the prognostic significance of pre-CRT MRI (mr) and post-CRT MRI (ymr) findings in patients with rectal cancer.Tissue changes after CRT could affect the accuracy and prognostic significance of MRI findingsThe median follow-up periods for OS, DFS, and LRR were 103 , 74 , and 81 (range 0–174) months, respectively. Distant metastasis (DM) occurred in 88 patients during the follow-up period. Local recurrence developed in 29 patients and was accompanied by DM in 24 patients. Nineteen patients experienced secondary malignancy in other organs, and secondary primary colon cancer occurred in two patients. At the end of the observation, a total of 102 patients died.Table In the analysis of OS, Harrell’s C-indices for EMVI were the highest among mr and ymr findings, being 0.656 and 0.680, respectively. For DFS, EMVI had the highest C-index (0.634) among mr findings, while N had the highest C-index (0.661) among ymr findings. For LRR, EMVI also showed the highest C-indices among mr and ymr findings, being 0.720 and 0.728, respectively . Among ymr findings, ymrN, ymrMRF, and ymrEMVI were significant variables, having HRs of 1.63 (p = 0.037), 1.76 (p = 0.023), and 1.73 (p = 0.029), respectively. Regarding mr and ymr findings for OS, Harrell’s C-indices for the final models were 0.635  and 0.682 , respectively  was the only significant variable among mr findings. Meanwhile, among ymr findings, ymrN , ymrMRF , and ymrEMVI  remained significant. Regarding mr and ymr findings for DFS, Harrell’s C-indices for the final models were 0.631  and 0.660 , respectively  and 2.42 (p = 0.021), respectively. Regarding mr and ymr findings for LRR, Harrell’s C-indices for the final models were 0.617  and 0.701 , respectively  patients, only three patients were ymrMRF(+) and ymrEMVI(+), and there was no significant difference between the DFS and OS groups Fig. .Figure 1In the ymrN(+) group, the 5-year OS was significantly worse in ymrMRF(+) and ymrEMVI(+) patients, that is, 52.4% , much lower than 83.3%  in patients without such findings (p < 0.006) Fig. . In the This study revealed that ymr findings tended to be more associated with the patient’s prognosis than mr findings. Among ymr findings, ymrN, ymrMRF, and ymrEMVI were independent prognostic factors for rectal cancer after CRT. When patients had ymrMRF and ymrEMVI, all survival outcomes were found to be significantly worse.et al. found that MRI stages II-III after CRT was a poor prognostic factor for 5-year DFS compared with MRI stage I17. In one study, among 92 patients with low rectal cancer with unsafe surgical resection margin on initial MRI, no patient was pathologically CRM(+) when they were assessed as “safe” on MRI after neoadjuvant therapy, while 23.9% of patients were pathologically CRM(+) when they remained to be assessed as “unsafe”16. Chand et al. showed that ymrEMVI is a significant prognostic factor on multivariable analysis13. In another study, the patient’s prognosis when ymrEMVI was positive after CRT was worse than when mrEMVI was positive alone14. All these studies have shown results consistent with our results, suggesting that post-CRT MRI findings have more prognostic impact than pre-CRT MRI findings and that ymrN, ymrMRF, and ymrEMVI were independent prognostic factors.One consistent finding of our study was ymr findings having better prognostic relevance than mr findings. Several studies have demonstrated the prognostic significance of post-CRT MRI findings, and a few of them have shown better clinical value of post-CRT MRI findings than pre-CRT MRI findings. Huh 18. Cho et al. reported that mrEMVI(+) predicts systemic recurrence in patients with rectal cancer even after good response to CRT followed by curative surgery19. In a pooled analysis of 269 patients who underwent CRT after neoadjuvant chemotherapy, mrEMVI(+) was the only significant prognostic factor among mr findings for distant progression-free survival20. mrEMVI(+) was also the only significant variable among mr findings for DFS on multivariable analysis in a study by Patel et al. in patients who underwent neoadjuvant chemotherapy21. All these studies showed similar results, suggesting mrEMVI as the most important MRI finding before CRT.Among mr findings, mrEMVI was the only significant prognostic factor in our analysis Table . The pro3. The RAPIDO trial enrolled patients with mrT3c/d or mrT4a/b, MRF(+), and EMVI(+) findings22. Our study revealed that ymrMRF(+) and ymrEMVI(+) were independent risk features. OS, DFS, and LRR were obviously worse in patients with both of these findings. In such patients, standard treatment might be insufficient and intensified treatment should be considered for better survival outcomes. Our previous study on consolidation chemotherapy for rectal cancer showed only marginal improvement in the down-staging rate that was explained partly due to the dilution effect of low risk patients23.[4] For future studies on induction or consolidation treatments, risk stratification based on our study will be helpful to evaluate treatment efficacy.Several trials have attempted to identify patients with high-risk features using MRI findings and apply intensified neoadjuvant therapy for better survival outcomes. The GRECCAR4 trial defined an unfavourable response as a tumour volume shrinkage of <75% on MRI or a ymrMRF(+) finding24. However, in our study, mrTRG was not significant on multivariable analysis using ymr findings, although it was significant for all survival outcomes on univariable analysis. Additionally, a recent study reported a low correlation between mrTRG and pathologic TRG25. Taken together, these findings suggest that mrTRG is a finding different from pathologic TRG and might not be an independent prognostic factor among other ymr findings. Thus, further study is needed to confirm the prognostic significance of mrTRG.mrTRG has been also reported as a significant prognostic factor in previous studies27. Moreover, in this study, one experienced radiologist reviewed every MRI finding with consistency. Second, emerging MRI prognostic factors such as diffusion-weighted images and diffusion coefficient values were not considered in the analysis. This was partially due to the absence of corresponding MRI findings in part of the study period. Furthermore, we paid attention to MRI findings based on pathology and included all of them in the analysis. Third, the MRI resolutions were different because 1.5-T and 3.0-T MRI systems were used during the study period. However, one study has shown that both 1.5-T and 3.0-T MRI systems have similar accuracy28.Our study has some limitations. First, we did not address interobserver disagreement in the analysis. However, mrMRF, ymrMRF, and ymrEMVI were reported to have fair to good interobserver agreement in previous studiesIn conclusion, this study found that MRI findings after CRT tended to correlate better with the patient’s prognosis than MRI findings before CRT. mrEMVI(+) was the only significant prognostic factor among mr findings, whereas ymrN(+), ymrMRF(+), and ymrEMVI(+) were independent risk factors among ymr findings. In addition, ymrMRF(+) and ymrEMVI(+) showed an additive effect on the patient’s prognosis. Therefore, MRI findings after CRT could be helpful in distinguishing high-risk patients.Institutional review boards of the National Cancer Centre, Korea reviewed this study and waived the requirement for informed consent on the basis of its retrospective design and minimal risk to the participants (NCC2018-0266). However, method was conducted in accordance with the committee’s approved guidelines to protect patients’ health information.We reviewed the medical records of 533 patients who underwent CRT for biopsy-confirmed rectal cancer (≤15 cm) from May 2002 to December 2010. The inclusion criteria were as follows: (1) primary rectal cancer without DM or other concomitant malignancy, (2) T3 or T4 classification on initial MRI; (3) adequate mr and ymr data, and (4) curative radical surgery after CRT. Among the 533 patients, 134 were excluded from analysis due to incorrect inclusion (n = 47) and inadequate MRI data (n = 87). The remaining 399 patients were included in the study. Details of the patient selection are summaried in Fig. All patients underwent workups before and after CRT. Rectal MRI with or without rectal ultrasonography was used for local staging. Presence of DM was checked with chest and abdominopelvic computed tomography (CT), and additional positron emission tomography (PET) was performed if necessary. Digital rectal examination (DRE), laboratory tests including carcinoembryonic antigen (CEA), and colonoscopy with biopsy were also checked before CRT. Tumour response was assessed at 6–8 weeks after CRT completion using a protocol similar to pre-CRT workups.et al.11 mrTRG was assessed based on the grading system presented by Patel et al.9.All patients underwent rectal MRI before and after CRT. A phased array body coil (USA Instruments) using the standard imaging protocol was used. One hour before the MRI examination, one bisacodyl suppository  was administered for bowel preparation. Thirty minutes prior to the MRI, 20 mg of scopolamine butylbromide  was intravenously injected to reduce colonic motility, unless otherwise contraindicated. MR sequences were obtained via the standard T2-weighted, fast spin-echo imaging at three planes  using the following parameters: echo time (TE)/repetition time (TR) of 80–110/2500–8600 ms, slice thickness of 3–5 mm, a 1-mm intersection gap, echo train length of 16–32, matrix of 224 × 224 to 800 × 538, no fat saturation, and a field of view of 150 × 150 to 360 × 360. Axial, T1-weighted, three-dimensional spoiled-gradient-echo sequence was also performed. 1.5-T MRI  and 3.0-T MRI  systems were used for assessment. One radiologist with 11 years of experience reviewed all rectal MRI scans. The radiologist was blinded from clinical information of patients. All readings of reviewed rectal MRI scans included information on T, N, MRF, EMVI, and mrTRG. N(+) was defined when there was a lymph node with any findings of larger than 5 mm in short diameter, irregular margin or heterogeneous signal intensity. MRF(+) was defined as a tumour, lymph node, EMVI, or tumour deposit located <1 mm from the MRF. Scores of 3 and 4 were considered EMVI(+) using the scoring system presented by Smith A dose of 50.4 Gy was administered for 5.5 weeks as long-course radiotherapy. Neoadjuvant chemotherapy was administered concurrently with radiotherapy. The common chemotherapy regimens were 5-fluorouracil (n = 205) and capecitabine based (n = 109); other regimens included oral tegafur/uracil (n = 53), irinotecan (n = 29), and oxaliplatin based (n = 1) regimens. Two patients underwent radiotherapy alone due to avoidance of chemotherapy.TME surgery was performed for all patients at 42 days  after CRT completion and 2  days after post-CRT MRI assessment. Abdominoperineal resection was performed in 56 (14.0%) patients, and the remaining 343 patients underwent a sphincter-preserving operation. In 14 patients, adjacent pelvic organs, mainly vagina with or without uterus, were resected due to suspicious tumour invasion. All patients were considered for adjuvant chemotherapy regardless of the postoperative histologic findings. Adjuvant chemotherapy commenced at 3–6 weeks after surgery in 381 (95.5%) patients. Fluoropyrimidine (n = 309), tegafur/uracil (n = 37), oxaliplatin (n = 33), and irinotecan based (n = 2) regimens were used for adjuvant chemotherapy.All patients were instructed to visit the hospital every 3 months for the first 2 years, every 6 months for the next 3 years, and every year thereafter. DRE, laboratory tests including CEA, and chest X-ray were performed at every visit. Abdominopelvic CT with or without chest CT was taken every 6 months, and colonoscopy was performed at 1, 3, and 5 years after surgery. The follow-up duration was shortened if there was any sign of recurrence, and rectal MRI or PET was added if necessary. Recurrence was confirmed by biopsy or radiologic findings on serial imaging. Tumour recurrence within the pelvis was considered as local recurrence, whereas that outside the pelvis was defined as DM.The Kaplan-Meier method and log-rank test were used to evaluate survival outcomes. The survival time was calculated from the date of surgery to the first event or date of last follow-up for censored data. The events for each survival outcome were defined as follows: death from any cause for overall survival (OS); any recurrence, second primary colorectal or other malignancy, or death from any cause for DFS; and recurrence within the pelvis for LRR.Univariable analyses of MRI findings were performed using Cox regression for OS, DFS, and LRR. Age, sex, tumour location, and CEA were included as clinical variables. Subsequent stepwise multivariable Cox regression was performed to derive two final models for OS, DFS, and LRR using the mr and ymr findings, separately. Backward elimination method was applied, and among clinical and MRI variables, only significant variables (p < 0.05) were included for making final predictive models in the univariable analysis. Harrell’s C-index was used as a measure of predictive accuracy for the final model determining the prognostic value of the mr and ymr findings. Statistical analyses were performed using the R Project for Statistical Computing software version 3.2.3 .The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary table1 and 2


In the most available db mouse line, wild-type (WT) mice have a mutation in the dedicator of cytokinesis 7 gene, named misty, which was recently revealed to be involved in neuronal development. Therefore, we established a line of db mice without the misty mutation using natural mating. Adult (8 weeks of age) homozygous db/db mice displayed significantly higher core body weight (BW) and FI and significantly lower core BT than WT mice. However, postnatal (2 weeks of age) db/db mice displayed similar BW and milk intake and significantly lower core BT than WT mice. Correspondingly, adult and postnatal db/db mice exhibited altered mRNA levels of hypothalamic orexigenic and anorexigenic peptide in adults but not in pups. Additionally, db/db mice displayed significantly lower mRNA levels of brown adipose tissue uncoupling protein 1 at both ages. In conclusion, the db mouse line without the misty mutation clearly showed the different involvements of the Lepr long form in FI and BT in pups and adults.Different involvement of leptin signaling in food intake (FI) and body temperature (BT) in pups and adults has been suggested. However, the leptin receptor (Lepr) long-form-deficient ( Leptin and the leptin receptor (Lepr) are important for physiological and pathological states related to body weight (BW) regulation in humans and rodents –5. To dain vivo, two mutant mouse strains with ligand (ob/ob mice) or receptor deficiency (db/db mice) have been widely used as obesity and type 2 diabetes animal models, respectively [ob/ob mice, the effects of leptin administration were examined to elucidate the role of leptin in appetite and thermogenesis [db/db mice, the genetically induced rescue of Lepr was assessed to elucidate the role of leptin signaling in the hypothalamus and sympathetic nerves [ob/ob and db/db mice displayed similar but slightly heavier BWs to their respective controls including heterozygotes and wild-type (WT) mice [To reveal the role of leptin and Lepr ectively . In ob/oogenesis , 11. Addc nerves –14. Leptc nerves . HoweverWT) mice –18. LeptWT) mice . PhysiolWT) mice . In a leWT) mice , 20.db/db mice might be useful to examine the age-dependent influence of Lepr deficiency [db/db mice under the BKS.Cg background are generally identified by coat color because of proteins coded by the dedicator of cytokinesis 7 (Dock7) gene, which is near the Lepr gene [db gene express the wild-type Dock7 gene, leading to obesity and black coat color. Thus, mice with a wild-type Lepr gene display a homozygous mutation in the Dock 7 gene, named misty (m/m), leading to a lean body type and grey coat color [m/m mice have lower BWs than WT mice. Therefore, to examine the lack of Lepr signaling on BT and FI regulation in an age-dependent manner, especially in pups, a line of db mice without the influence of the misty gene should be established.ficiency . Howeverepr gene , 23. Speat color . Recentlat color , 26. As db mouse line without the influence of misty, we examined FI and BT in pups and adults. In addition, the age-dependent expression of orexigenic and anorexigenic genes in the hypothalamus and uncoupling protein 1 (Ucp1) in the BAT was examined. Furthermore, thyroid function, which is associated with FI and BT regulation, was surveyed. In the present study, the roles of the long form of Lepr, which is specifically deficient in db/db mice, in the age-dependent regulation of FI and BT were elucidated.Using a db gene, a mutation in the Lepr gene, without a mutation in the Dock7 gene (misty). First, we purchased heterozygote male mice  that had a black coat color and lean body type . As displayed in M db/M+, possibly because of natural recombination during breeding at the companies. A male M db/M+ mouse was mated with female M db/m+ mice, producing M db/M db, M db/m+, M db/M+, and M+/m+. The mating for the experiments was permitted by the company (Charles River). From the genotyping results of the pups, we obtained a pair of Lepr gene heterozygotes, who had the wild-type Dock7 gene (M db/M+). Male and female M db/M+ mice were mated to obtain WT (M+/M+: +/+), heterozygous (M db/M+: db/+), and homozygous (M db/M db: db/db) mice. We chose a colony with 5–8 litters and genotyped the mice  at 1 week of age. Mice BWs were measured weekly from 1 to 8 weeks of age. Mice were individually recognized by black pen markings on the surface of their hand and foot. At 3 weeks of age, the mice were removed from their dams, separated by sex, and placed into a cage with littermates of the same sex. At 4 weeks of age, mice were separated and then kept individually in one cage. Male +/+ mice and db/db littermates at 2 and 8 weeks of age were further examined as pups and adults, respectively. db/db mice at 8 weeks of age were designated as adults, although this is younger than generally defined, because older mice display a loss of hyperinsulinemic conditions [All mice were maintained at a constant room temperature of 22 ± 2°C, with a 12 h light cycle (07:00–19:00 h) and free access to water and standard chow . Briefly, we generated a mouse line carrying the nditions , 22. Micnditions . The plaAll animal experiments and procedures used in the present study were approved by the Ethics Committee for Animal Experimentation at Kagoshima University , which is standardized to the Japanese national guidelines for animal experiments. Additionally, the principles of laboratory animal care were followed.db, a mutation in the Lepr gene, was obtained using the PCR method with mismatched primers [Lepr gene was amplified with forward and reverse primers (Supplementary Hpy166II restriction enzyme (New England Biolabs Japan Inc.). The PCR products digested were analyzed by agarose gel  for identification of the three genotypes . The Dock7 gene was amplified with one forward and two reverse primers  was performed.Previously,  primers , 29. In For adults, the weight of the diet was measured at 9:00 h. FI was then calculated as the difference in diet weight over 4 consecutive days. To measure milk intake of mice at 2 weeks of age, dams were separated from pups for 3 h. The pups were weighed 3 h after separation and again after 1 hour of suckling. Body weight gain during 1 h of suckling was utilized to estimate milk intake .Core BT was measured with a high traceable thermometer D642 . The thermocouple electrode was introduced 15 mm into the rectum of the animals.Plasma glucose and triglyceride (TG) levels were measured with a commercial kit . Plasma insulin, leptin, and corticosterone concentrations were measured with the respective ELISA kits . Plasma hormones related to thyroid function, such as triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH), were measured by ELISA .μg total RNA with oligo(dT)20  following the manufacturer's instruction. Gene expression was determined by quantitative RT-PCR with gene-specific primers using the SYBR Green Master Mix  on a TAKARA detection system TP800 . Relative mRNA levels were determined by the 2-∆∆CT method, with Gapdh as a reference gene. Primer sequences are provided in Supplementary RNA from the hypothalamus and BAT was extracted using a Trizol reagent  following the manufacturer's instruction. Isolated RNA was treated with DNase  to remove genomic contamination. First-strand cDNA was synthesized using 5 t-tests were used as appropriate. P < 0.05 was considered significant.Values are presented as the mean ± standard error (SE). Significant differences between groups in M db/m+) (db with wild-type Dock7 (M db/M+). By further mating, we then generated mice carrying db without the Dock7 mutation. For the experiments, we produced mice by cross mating in the line.We classified mice with a black coat color and lean body type from the two companies as heterozygotes  . Because+Lepr/+Lepr) and homozygous mutants (db/db: dbLepr/dbLepr) in male (a) and female (b) mice fed with a regular chow diet after weaning at 3 weeks of age. There were no significant differences in BW between male and female WT and db/db mice for the initial 6 and 5 weeks after birth by two-way (repeated measurement) ANOVA. Both male and female db/db mice displayed significantly higher BWs than WT mice in 7 and 6 weeks of age after birth, respectively.db/db mice displayed a similar BW change, indicating that mice ingested a similar amount of milk during the 1 h suckling period at 2 weeks of age. Seven WT and 7 db/db male mice were obtained from the same four mothers. The mean litter size was 7.7 ± 1.1 and 7.9 ± 1.2, indicating no significant difference. Furthermore, 5 WT and 5 db/db female mice were obtained from the same three mothers and one different mother. The mean litter size was 7.6 ± 1.1 and 7.0 ± 1.0, indicating no significant difference. Therefore, the milk availability between genotypes was not influenced by the litter size. However, both sexes of db/db mice displayed higher FI than WT mice at 8 weeks of age.As displayed in db/db and WT mice was similar at 2 weeks of age and significantly different at 8 weeks of age. The heart weight was significantly different between mice at 2 and 8 weeks of age. In BAT, db/db mice displayed significantly higher weights than WT mice, and mice at 8 weeks of age displayed higher weights than mice at 2 weeks of age.Organ weights, including the brain, heart, liver, WAT, and BAT, were measured in male mice . There wdb/db and WT mice were similar at 2 weeks of age and significantly different at 8 weeks of age. In leptin, db/db mice at both ages displayed a significantly higher value than WT mice at both ages, respectively. Additionally, plasma TG levels were comparable between the two genotypes at 2 and 8 weeks of age, although mice at 8 weeks of age displayed a significantly higher value than mice at 2 weeks of age. For hormones related to thyroid function, db/db mice showed significantly lower T4 but similar T3 and TSH levels in plasma compared with WT mice.Only males were examined. There was a significance in the interaction between age and genotype in glucose, insulin, leptin, and corticosterone. With a multiple comparison, the values in glucose, insulin, and corticosterone of db/db mice at 2 and 8 weeks of age  without misty . Regarding the main clinical manifestations such as BW, milk and food intake, and BT, although there may be a slight difference in degree, mice of both sexes displayed a similar tendency of the manifestations. Therefore, further examinations  were performed in males only.The present study revealed the distinct roles of Lepr in FI and BT regulation in pups and adults using an established mouse line carrying db genotyping was reported [db gene was used to establish a pure db mouse line. This system using a novel restriction enzyme that recognizes the mutated sequence might be easier and more reliable than previous systems . Therefore, recombination of the genes is likely in more than 1 out of 50 births [M db/M+, and then a mouse line carrying db without the Dock7 mutation was established (db and homozygous wild-type Lepr 1 week after birth. BW changes and FI were examined (db/db mice displayed a higher BW after the weaning period but not in pups. Correspondingly, adult, but not pup, db/db mice displayed higher FI. The BW was not significantly different between WT and db/db mice at 2 weeks of age, and the brain, liver, and WAT weights were similar in db/db mice and WT mice. These data suggest that there is a similar role for Lepr signaling between BW regulation and adiposity regulation.A method using mismatched primers for reported , 29. A n0 births . As expeablished . In addiexamined . db/db mdb/db mice had lower BT in pups and adults. The results were consistent with those in Zucker rats with a leptin receptor mutation. Zucker rats displayed a lower core BT at 1 week after birth and increased FI later in life [ob mice with leptin deficiency displayed thermogenic defects and similar milk intake, compared with control mice, at around 2 weeks of age, the preobese phase [In contrast,  in life . ob micese phase , 18.Ucp1 in BAT (db/db mice. This finding was consistent with reports based on leptin administration experiments including an ob/ob mouse study [Lepr gene expression. Alternatively, the leptin signaling cascade from Lepr may be blunted. The mechanisms underlying this regulation such as the methylation state of gene promoters involved in leptin signaling should be examined [The findings of the present study regarding FI and BT regulation were corroborated by the results on gene expressions of the hypothalamus and BAT. These findings were consistent with the expression of orexigenic and anorexigenic peptide genes in the hypothalamus and 1 in BAT . In pupsse study , 33. Becse study , 33. Aftexamined –36.db/db mice with long form Lepr deficiency, suggesting that Lepr, particularly the long form, is consistently involved in BT regulation. In adult mice, POMC is important for BT regulation by mediating leptin action [Pomc expression levels were similar in db/db mice, suggesting that the decrease in BT is not explained by the reduced signaling of melanocortin-4 receptor involved in POMC (db/db mice indicates that Lepr is involved in BT regulation in association with the lower expression of UCP1 at both age, but the precise underlying mechanism has not been elucidated (db/db mice, where there was a significant difference between the genotypes under less significant interaction of age and genotype (db/db mice had a significantly lower expression of PPARγ (db/db mice may underlie the different mechanism between pups and adults [Ucp1 and Pparγ genes [db/db mice displayed lower T4 but similar TSH levels in both ages, suggesting that central hypothyroidism is involved in the reduced BT.In contrast, BT was lower in pup and adult n action , 3, 37.  in POMC . Reduceducidated . This gegenotype . Postnatof PPARγ . This fid adults . For exarγ genes , 40. In rγ genes . In the Ucp1 expression in postnatal db/db mice is unclear, it may be related to the lower BT in these mice. Based on our results, the leptin surge may be more involved in BT regulation than in FI regulation.The leptin surge or increased leptin in the postnatal rodents was reported, but its physiological importance remains unclear , 19. Thedb/db and +/+ mice with the BKS background without misty. In the other studies using the db/db mouse line with the BKS background, which is relatively prone to diabetes [m/m or m/+ mice were used as controls [misty mutation may influence BW and findings related to sympathetic nerve activity. To compare the results from the present study, especially, in adult mice with that of other studies with mice harboring the misty mutation, we should consider the results from the control mice, which may be influenced by misty.To the best of our knowledge, the present study is the first to compare diabetes , m/m or controls , 43. Thefa rats and ob mice, especially in the preobese phase corresponding to 2 weeks of age. Using a db mouse line without the misty mutation, the different appearances of FI and BT at different ages were presented at the molecular levels, suggesting the age-dependent involvements of the leptin receptor long form in FI and BT.In conclusion, the age-dependent phenotypes were corroborated by the results of gene expressions of the hypothalamus and BAT, which have not been fully elucidated in


We then conducted a series of bioinformatic investigations on the identified loci. Combining the evidence from both the MTAG analysis and the in silico replication, we identified three novel loci at the genome-wide significance level (α = 5.0 × 10−8): 3q23 , 7p15.3 , and 9q33.3 . Each of the three lead SNPs was associated with at least one of eight brain-related traits including intelligence and educational attainment. Credible risk variants, defined as those SNPs located within 500 kb of the lead SNP and with p values within two orders of magnitude of the lead SNP, were enriched in DNase I hypersensitive site region in brain. Nine candidate genes were prioritized at the three novel loci using multiple sources of information. Gene set enrichment analysis identified one associated pathway GO:0048009, which participates in the development of nervous system. Our findings provide useful insights into the genetic basis of HC and the relationship between brain growth and mental health.As an important trait at birth, infant head circumference (HC) is associated with a variety of intelligence- and mental-related conditions. Despite being dominated by genetics, the mechanism underlying the variation of HC is poorly understood. Aiming to uncover the genetic basis of HC, we performed a genome-wide joint association analysis by integrating the genome-wide association summary statistics of HC with that of its two related traits, birth length and birth weight, using a recently developed integrative method, multitrait analysis of genome-wide association (MTAG), and performed  The size of infant head circumference (HC) is thought to affect brain development in later life . It is aDespite being influenced by maternal nutrition during gestation, infant HC is dominantly determined by inheritance . A previAs a measure at birth, HC is related to birth length (BL) and birth weight (BW). They have robust links with each other in epidemiology and are all associated with common factors such as fetal sex, maternal factors, and gestational age . Many stin silico replication in large-scale GWAS summary results of adult intracranial volume. We will then conduct genomic feature enrichment analysis and candidate gene prioritization. We will also perform gene set enrichment analysis to highlight relevant pathways.Recently, a new statistical method multitrait analysis of genome-wide association (MTAG) was proposed, which integrates the information contained in correlated traits to boost genetic association signals for each individual trait . In the In this study, we performed a joint GWAS analysis by integrating the information carried in the GWAS summary results of HC, BL, and BW traits, with the main purpose of identifying responsible genomic loci for HC.http://egg-consortium.org) and retrieved the part of results that included participants with European ancestry only for each trait.All GWAS meta-analyses for the three individual traits were carried out by the Early Growth Genetics (EGG) consortium, and basic characteristics of individual studies are detailed in respective publications Table 1 http://genome.ucsc.edu/cgi-bin/hgLiftOver), and deprecated SNPs were dropped (213 for BL and 256 for HC); 3) for BL summary results with no allele frequency information, allele frequency was estimated based on the HapMap Phase 2 + 3 European individual genotypes, and SNPs with missing allele frequency were excluded  criteria were applied to retain eligible single nucleotide polymorphisms (SNPs): 1) Duplicated SNPs were dropped within each trait ; 2) SNP genomic coordinate for BL and HC was transformed from GRCH 36 to GRCh 37 with LiftOver excluded ; and 6) https://github.com/bulik/ldsc). We applied the linkage disequilibrium score (LDSC)-based regression analysis to the summary results of each trait to evaluate its genetic architecture. LDSC takes GWAS summary statistics as input and partitions overall inflated association statistics into one part attributable to polygenic architecture and another part due to cryptic relatedness and population stratification . LDSC cahttp://ldsc.broadinstitute.org/ldhub/) to the GWAS summary results of HC and BL traits, and of HC and BW traits, to study the genetic correlations between HC and the other two traits β2, where f is allele frequency and β is regression coefficient.All the three traits are standardized  in their original studies. Therefore, we estimated individual variant effect size as the variance it explains with the formula 2in silico replication in intracranial volume. This is because HC is both phenotypically and genetically highly correlated with intracranial volume (Each lead SNP from the newly identified loci was subject to independent l volume . Their gl volume . Some prl volume .http://enigma.ini.usc.edu/research/download-enigma-gwas-results/) with permission.The summary results of intracranial volume come from a large-scale GWAS meta-analysis conducted by the CHARGE and the ENIGMA consortia in 26,577 subjects of European ancestry and were downloaded through the ENIGMA’s website , where adult height is believed to closely link to the BL . We kept in mind that about 67,000 participants overlapped between this analysis and the BW results used in the present study. Again, the replication significance was evaluated in terms of both effect direction and Bonferroni corrected one-sided p value.Although the focus of the present study was HC, it did produce re-estimated MTAG results for BL and BW. We performed t height  , cognitive impairment (CI)  and Parkinson disease (PD) . We explored the association of the SNPs identified in the present study for HC with multiple mental health and intelligence-related traits. A total of eight traits were collected, which could be classified into two categories. The first category contains two normal traits intelligence  and educWe assessed the enrichment of the HC SNPs into different genomic features with the GWAS Analysis of Regulatory or Functional Information Enrichment with LD correction (GARFIELD) . GARFIELhttps://www.ebi.ac.uk/birney-srv/GARFIELD/). Genomic features include DNase I hypersensitive sites and genic features from three tissues  to capture the cell-type-specific feature enrichment. The assessment was conducted at four p value thresholds . To account for correlation between genomic features, the effective number of genomic features was first estimated by GARFIELD, which was 35.32. Statistical significance was then declared at a Bonferroni-corrected threshold, i.e., 1.42 × 10−3 (0.05/35.32).Genomic feature information was accessed from the software website  as those located within 500 kb of the lead SNP, and with lead SNP . This islead SNP .We incorporated five sources of information to seek evidence of a gene’s causality: i) being the gene nearest the lead CRV, ii) containing a missense coding CRV, (iii) being the target gene for any cis-eQTL CRV, iv) being prioritized by DEPICT, or v) being prioritized by SMR analysis.https://gtexportal.org/home/) . Significance threshold was set at 0.05.Cis-eQTL information was obtained from two datasets: GTEx (v7) (g/home/)  and the g/home/) . Cis-eQTg/home/)  to annotDEPICT is an integrative tool that employs predicted gene functions to systematically prioritize the most likely causal genes at associated loci . SignifiSMR is another prioritization method that integrates summary-level data from GWAS with cis-eQTL data to identify genes whose expression levels are associated with trait because of pleiotropy . Three chttp://www.geneontology.org/). A gene-based association test was examined to determine individual gene association signals. A gene set association signal was then calculated by integrating all gene signals within the set. We used competitive model to test whether genes in a gene set are more strongly associated with the phenotype than other genes and multitesting problem was corrected by permutation test.We performed gene set enrichment analysis with MAGMA (v1.6) . MAGMA sAfter a series of inclusion criteria, we finally included 1,977,400 SNPs for analysis. Using HC summary association statistics at these SNPs, the LDSC estimated a SNP attributable heritability 0.23 (s.e. = 0.05) for the trait. Meanwhile, the SNP attributable heritability for BL and BW was 0.169 and 0.098, respectively. There are two loci 12q15 and 12q24 being significant at the genome-wide significance level in the original HC summary results. After excluding the two lead SNPs (rs1042725 and rs7980687) and their neighboring SNPs within 500 kb to either direction, the heritability is 0.22 (s.e. = 0.04), showing that most of the SNP-attributable heritability is hidden in the current GWAS setting.When applied a LDSC-based genetic correlation analysis, the genetic correlation coefficient between HC and BL is estimated to be as high as 0.55 (s.e. = 0.12) and that between HC and BW is 0.37 (s.e. = 0.08), in line with previous research  , with the exact same two lead SNPs (rs1042725 and rs7980687) and stronger signals. To search for additional loci, we evaluated MTAG results of all SNPs excluding those within 500 kb of a locus that was reported previously. These identified 76 SNPs in 9 distinct loci that are significant at the GWS level. Of the nine lead SNPs, four are not significant in the original HC study at the nominal level (α = 0.05) but extremely significant for BW and BL, indicating that the significant signals are likely to be driven solely by BL and/or BW instead by HC. As a QC criterion, we discarded these four loci.The two originally significant loci remain significant at the genome-wide significance level . Both previously identified SNPs are significant (one-sided p = 3.22 × 10−9 and 0.03) in the replication results, demonstrating their robustness. Of the five newly identified loci, all are consistent in effect direction between HC and intracranial volume. Among them, one-sided p value at three SNPs further achieves the Bonferroni-corrected significance level (α = 0.01), while rs3795128 has a weaker but nonsignificant signal (p = 0.02). Taken together, SNPs from three loci were convincingly associated with HC in both discovery and replication stages. The three lead SNPs explain 0.6% of HC phenotypic variance. Results are listed in We then sought for in silico replications in adult height and BW, respectively. As shown in For BL and BW, new loci beyond the originally significant ones are identified, too. For BL, a total of 539 SNPs from 27 distinct loci are significant at the GWS level. Of them, only one was reported by the original study, while the remaining 26 loci are novel  to several mental health and intelligence-related traits and diseases. We collected GWAS summary results for eight traits. −3), any of the five SNPs is associated with at least one trait. The two known SNPs rs1042725 and rs7980687 are both associated with educational attainment (rs1042725 p = 7.63 × 10−6 and rs7980687 p = 3.01 × 10−25). In addition, rs1042725 is associated with subjective well-being (p = 6.63 × 10−4), and rs7980687 is associated with intelligence (p = 1.44 × 10−6). The three newly identified SNPs are associated with educational attainment (rs9846396 p = 1.44 × 10−7), intelligence (rs12534093 p = 3.25 × 10−4) or loneliness (rs7048271 p = 1.82 × 10−4). There is also a hint for the association of rs12534093 with loneliness, although the signal (p = 9.74 × 10−3) does not reach the significance threshold.At the Bonferroni corrected significance level  associated with educational attainment have been identified to be significant at the GWS level , with diT = 5.0 × 10−8), significant enrichment is observed in up to 13 features. Notably, SNPs are enriched in the DNase I hypersensitive site peak and a broader DNase I hypersensitive site category hotspot in only one  of the three tissues being investigated, but not in fetal hippocampus and cerebellum. At footprint feature, which is defined as a paucity of cleavage because protein-bound DNA is typically protected from DNase I cleavage, SNPs are observed to be enriched in brain  and brain hippocampus . SNPs are also enriched in exon  and intergenic regions  with lower ORs. At T = 1.0 × 10−7, significant enrichment is found in the same 13 features with similar ORs. At T = 1.0 × 10−6 and 1.0 × 10−5, however, enrichment is observed in a fewer number of features .We investigated the enrichment of the identified SNPs into a variety of genomic features in three brain-related tissues including brain/fetal brain, fetal hippocampus, and cerebellum. The results are shown in ZBTB38 and STRBP) are prioritized by more than one source. Both genes are closest to respectively lead SNPs and are the cis-eQTL target gene of the latter. What is more, previous research demonstrates that STRBP at 9q33.3 is differentially expressed between patients with Down’s syndrome and controls  receptor binding to one of its physiological ligands (http://amigo.geneontology.org/amigo/). The role of this gene set includes regulating the development and growth of central nervous system . As a QC criterion, we discarded these four loci because they might represent false positive signals. As stated by the authors of MTAG, this false positive finding could happen when SNP is nonsignificant for one trait but significant for others in finite samples. Indeed, none of these SNPs could be replicated by intracranial volume (p = 0.02) in this dataset. We prioritized four genes  for this SNP using same gene prioritization strategy. Among them, PLCG1 is the most promising candidate gene at 20q12 detected by multiple sources because it is the closest to lead CRV (rs3795128) and contains a missense CRV (rs753381) that is in high linkage equilibrium  with lead CRV. In a previous case–control study, missense mutations in PLCG1 were reported to participate in the pathogenesis of cutaneous T-cell lymphomas by enhancing downstream signaling towards nuclear factor of activated T-cell activation. We therefore hypothesized that rs3795128 was linked to T-cell lymphomas through its strong LD with the missense mutation in PLCG1  in intracranial volume at Bonferroni-corrected threshold, although it was nominally significant , IGF-1 is a phylogenetically ancient neurotrophic hormone and plays crucial roles in central nervous system development and maturation . In astrin silico replication in a genetically highly correlated trait intracranial volume. We believe that the replication results imply important clues towards true associations. Second, the unbalanced sample sizes used in the MTAG analysis may elevate false positive rate, as described by the MTAG authors , the Natural Science Foundation of Jiangsu Province of China (BK20150323 to Y-FP), a project funded by the Priority Academic Program Development of Jiangsu higher education institutions (PAPD), and the Undergraduate Innovation Program of Jiangsu Province (KY2019881B to X-LY).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Social participation benefits health. We assessed the relationship between self-reported visual impairment (VI) and glaucoma versus seniors’ social participation.Data from individuals aged ≥65 years responding to the Canadian Community Health Survey Healthy Aging 2008/2009  was analyzed. Participation in eight social activities by seniors with and without self-reported VI or glaucoma was compared.Seniors with VI had significantly reduced participation (p<0.05) in sports/physical activities (18.0% vs. 33.6%), family/friendship activities outside the household (39.7% vs. 53.0%), service club/fraternal organization activities (11.4% vs. 18.4%), volunteer/charity work (13.4% vs. 24.9%), educational/cultural activities (16.2% vs. 24.5%), and other social recreational activities (21.6% vs. 31.0%) compared to those without VI. Differences in participation in church/religious activities (40.6% vs. 44.5%) and community/professional association activities (15.3% vs. 18.0%) were non-significant between seniors with and without VI. Seniors with glaucoma versus those without had significantly reduced participation (p<0.05) in family/friendship activities (46.6% vs. 52.9%), sports/physical activities (26.0% vs. 33.6%) and volunteer/charity work (20.4% vs. 24.9%). No participation in any social activity was significantly higher among seniors with VI versus those without , but was similar among seniors with and without glaucoma . After adjusting for the effects of age, sex, education, household income, ethnicity, job status and chronic diseases  2.0–5.8), seniors with VI but no glaucoma were more likely not to engage in any social activities compared to seniors without VI and no glaucoma. Seniors with glaucoma but without VI had a similar level of non-participation .Significantly reduced social participation was found across six community activities among seniors with self-reported VI and in three activities among those with self-reported glaucoma. Policies and programs that help seniors with VI or glaucoma engage in social activities are needed. Globally, loss of central vision, or visual impairment (VI), impacts 70 million people over the age of 70. The top Global Strategy and Action Plan on Ageing and Health introducing the concept of “healthy ageing” as the focal point for governments to develop policies to meet the needs of the aging population.[In 2016, the WHO endorsed the pulation.,10 Sociapulation.,10 This pulation.–15, motopulation., depresspulation.,19, and pulation.In the literature, many studies on vision disorders have focused on the link between the element “impairment” (e.g. glaucoma) and the element “activity limitation” (e.g. limitation in driving activity). For instance, reports have shown that VI  is associated with significantly decreased activities of reading and mobility–25 and tGiven our rapidly growing aging population, the number of seniors affected by vision disorders will increase in the coming years.,40 UnderPopulation-based survey data from the Canadian Community Health Survey—Healthy Aging (CCHS-HA) 2008–2009 was analyzed. The CCHS-HA was a health survey run by Statistics Canada, a government of Canada agency commissioned with collecting population-level data. IndividuData on self-reported VI was obtained by asking:without glasses or contact lenses?Are you usually able to see well enough to read ordinary newsprint with glasses or contact lenses?Are you usually able to see well enough to read ordinary newsprint Are you able to see at all?without glasses or contact lenses?Are you able to see well enough to recognize a friend on the other side of the street with glasses or contact lenses?Are you able to see well enough to recognize a friend on the other side of the street Answers to the above questions were grouped into 5 mutually exclusive groups by Statistics Canada:No visual problems;Problems corrected by lenses ;Problems seeing distance with or without correction;Problems seeing close with or without correction; andProblems seeing close and distance, or no sight at all.In this study, we defined self-reported VI as impairment uncorrected by lenses for distance vision (group 3), near vison (group 4) or both, or no sight at all (group 5).Respondents were classified to have glaucoma if they self-reported having glaucoma that had lasted or was expected to last six months or more and that had been diagnosed by a health professional.Social participation was assessed by asking how often in the past 12 months respondents had participated in eight areas of activity. The eighDuring analyses, answers of ‘at least once a day’, ‘at least once a week’, or ‘at least once a month’ were grouped together as ‘participation’ to avoid sparse data. Answers of ‘at least once a year’ or ‘never’ were considered as ‘non-participation’. However, for participation in ‘family or friendship activities outside the household’, ‘participation’ was defined differently, where answers of ‘at least once a day’ or ‘at least once a week’ were deemed as ‘participation’ due to the common occurrence of this activity.Data on age, sex, ethnic background (Caucasians vs. non-Caucasians), highest level of education (without vs. with post-secondary degree), total household income (under middle level vs. middle level or higher), current job status (part-time or full-time workers vs. non-workers) and chronic conditions (having at least one of 25 chronic conditions surveyed vs. none) other than VI and glaucoma was self-reported. RespondeWe analyzed the raw de-identified data housed at the Research Data Centre (RDC) at the University of Toronto where participant’s confidentiality was strictly protected. Survey weights provided by Statistics Canada were used in all analyses. These weights accounted for the complex survey design and sample selections, adjustments for nonresponse, seasonal effects, and poststratification. WeightedIn Canada, an estimated 163,100 (3.8%) seniors had self-reported VI, 312,000 (7.2%) had self-reported glaucoma and 30,900 (0.7%) had both self-reported VI and glaucoma in 2008/2009. The characteristics of respondents are shown in Seniors with self-reported VI experienced significantly reduced participation in six of eight activities surveyed: family/friendship activities, sports/physical activities, service club/fraternal organization activities, volunteer/charity work, educational/cultural activities, and participation in other social recreational activities compared to those without VI p<0.05; . When paWhen the patterns of social participation were examined among people aged 45+, except for participation in church/religious activities, differences in all other activities between individuals with and without self-reported VI were statistically significant . No partSeniors with glaucoma experienced significantly reduced participation in family/friendship activities, sports/physical activities and volunteer/charity work compared to those without glaucoma p<0.05; . When paThe patterns among individuals aged 45+  differedWhen potential confounding effects of age, sex, highest level of education, total household income, ethnic background, current job status and chronic conditions other than self-reported VI and glaucoma were controlled for, seniors with self-reported VI and no glaucoma were more likely not to be involved in any social activities compared to seniors with no VI and no glaucoma (adjusted odds ratio (aOR) = 3.4, 95% CI 2.0–5.8; Among individuals aged 45+, low levels of education and income and self-reported VI and no glaucoma were similarly associated with an increased risk of no participation in any of the eight social activities . HoweverThis study examines the association between self-reported VI and glaucoma versus senior’s participation in a wide range of social and community activities. We report that seniors with self-reported VI had significantly reduced participation in sports/physical activities, family/friendship activities, service club/fraternal organization activities, volunteer/charity work and educational/cultural activities. Among seniors with self-reported glaucoma, we observed significantly reduced engagement with family/friends activities, sports/physical activities and volunteer/charity work. Seniors with self-reported VI and no glaucoma were more likely not to be involved in any social activities compared to those without self-reported VI and no glaucoma.Our findings on reduced social participation in the elderly with self-reported VI are in line with previous reports.–38 In a In the US, Crews et al. studied seniors aged 70 years or older and reported that seniors with VI had significantly reduced OR for visiting friends in the past 2 weeks (0.7), phoning friends (0.8), attending church (0.7), going to movies (0.6), eating out (0.7), and exercising (0.7) compared to seniors with no vision and no hearing loss. These fiIn the UK, Liljas studied VI among only senior men with VI, assessed with the question “using glasses or corrective lenses if needed, can you see well enough to recognize a friend at a distance of 12 feet/4 yards (across a road)?”. This VI We suggest potential reasons for seniors with VI having reduced participation may include reduced ability to ambulate (particularly in unfamiliar environments), fear of falling, transportation issues and psychological distress .–51 The rOur study shows that compared to seniors without glaucoma, those with glaucoma experience significantly reduced participation in sports/physical activities, family/friendship activities and volunteer/charity work. This finding may be explained partly by reports that individuals with glaucoma walk more slowly, are more likely to bump into objects , have a There are limitations of this study. First, VI and glaucoma was self-reported, not clinically measured. Bias related to self-reporting cannot be ruled out. However, it is the individual’s self-reported vision (not their best corrected vision measured in clinics) that maintains an individual's day-to-day function. We feel self-reported VI may best reflect real-life situations. This concept is also in agreement with the revised definition for VI by WHO where the words ‘best corrected’ be replaced by “presenting”.,58 For gA study strength is the nationwide and randomly selected sample. Common biases from clinic-based studies such as unrepresentative and/or smaller samples have been overcome. Furthermore, participation in social activities was investigated using a standardized set of eight questions.In conclusion, the number of seniors with VI or glaucoma will likely increase substantially over the next twenty years. We repor