Eye-drop administration errors occur in the majority of patients and increase the risk for treatment failure or systemic adverse events. While lacking knowledge is the principal error cause, most patients overestimate their skills and are unaware of often substantial knowledge gaps. Therefore, the impact of including motivational patient education on long-term eye-drop administration skills of patients was investigated.This is a cluster-randomized controlled trial in German community pharmacies. Patient education in both groups comprised observation of the patient during eye-drop administration to identify individual errors, pharmaceutical counseling, and teach-back evaluation of the training. In the intervention group, motivational communication techniques were included to increase error awareness and readiness for patient education. In addition, intervention patients were trained on repeated errors until administration was performed correctly. In contrast, patients in the control group only received feedback on erroneous administration steps without another assessment and reinforced training.In total, 152 adult patients were eligible to the study and 91 patients (intervention group N = 46) agreed to participate in a 1-month, 6-month, and 12-month follow-up. Patient education significantly increased the proportion of patients correctly administering eye-drops from 6%  at baseline to 35%  at the 1-month follow-up, and 64%  at the 6-month follow-up irrespective of group allocation. In some patients previously resolved errors recurred during follow-up visits. This emphasizes the need for periodical reevaluation of patient administration skills and the provision of prevention strategies besides education.Patient education that included demonstration of administration skills and verbal and written counseling on observed errors improved eye-drop administration skills irrespective of the communication technique applied. Whereof, high drop-out rates limited the power to detect a difference between groups. In particular, periodic demonstration of administration skills seemed important for sustainable improvement of administration skills. However, further error prevention strategies such as additional education materials or support by a caregiver may be necessary in some patients. The eye-drop administration process comprises multiple action steps with various opportunities for error that can cause treatment failure or adverse drug events.–7 Howeveth, 2014 and registered in the German Clinical Trials Register (DRKS00008941). The trial protocol can be accessed as supporting information. The study was registered after recruiting of the community pharmacies and inclusion of pharmacy staff but before the first patient was included to the study. The authors confirm that all ongoing and related trials for this intervention are registered.The study was part of a patient education program to improve administration of several error-prone dosage forms. It was approved by the Ethics Committee of the Medical Faculty of Heidelberg University (S-492/2014) November 26www.randomization.com. AL allocated community pharmacies to intervention or control group according to sequence of agreement with the head of the pharmacy. Pharmacy staff were not blinded to group allocation.In April 2015, German community pharmacies were invited by the Chamber of Pharmacists Baden-Wuerttemberg to participate in a multicenter, parallel-group, cluster-randomized, controlled trial to improve counseling on correct medication administration. Cluster-randomization into intervention or control group was performed on pharmacy level to prevent contamination between intervention and control group. Computer-generated randomization was performed using Pharmacy staff in both groups were informed about the study procedure and received training on correct medication administration in an approximately 60-min seminar. In the intervention group, the seminar was expanded to approximately 120 min to additionally teach suitable communication techniques including the spirit of motivational interviewing. There wePatients who correctly administered their eye-drops were not included in the study but were asked whether the completed checklist could be kept anonymously to determine the total number of patients assessed for administration errors. Patients with at least one missing or incorrect administration step were trained according to group allocation. Patients who used eye-drops for the first time were immediately counseled. In both groups, pharmacists used information leaflets to structure the training session. Counseling in the intervention group additionally included aspects of the previously performed communication training that aimed at increasing the patients’ awareness of the importance of correct eye-drop administration and, thus, motivation for a permanent change of their eye-drop administration routine. Subsequently, teaching success was evaluated by observing patients administering placebo eye-drops. If an error re-occurred, patients in the intervention group were trained on individual errors with repeated observations until administration was performed correctly, whereas patients in the control group only received feedback on erroneous administration steps without another assessment. Afterwards, pharmacy staff informed patients about the study and invited them for follow-up visits after one month, six months, and twelve months to re-evaluate administration skills. At follow-up visits, pharmacy staff assessed administration skills of patients and provided counseling when necessary, followed by a teach-back evaluation. For evaluation of practicability, pharmacy staff in both groups started recording the time as soon as an eligible candidate for the study was identified until patient education was completed. The time difference allowed estimation of the net duration of the patient education including the baseline assessment and evaluation of teaching success.Community pharmacies with premises for confidential consultation were included after pharmacy staff gave their written informed consent. Adult customers (age ≥ 18 years) who obtained eye-drops were cognitively and physically capable to participate and self-administered eye-drops were considered eligible for the study. In case a caregiver administered the eye-drops for the patient, the caregiver would be eligible for study participation and trained on correct administration technique. Study information was given to the patients after the first training session in order to simulate usual counseling procedures during drug dispensing without study-specific interruptions, such as signing informed consent. If participants gave their written informed consent, a pseudonymous identifier was allocated to the previously completed checklist to allow matching of follow-up visits with the baseline assessment. This procedure was approved by the Ethics Committee of the Medical Faculty of Heidelberg University (S-492/2014). All patients included to the follow-up gave their written informed consent. Pharmacy staff recruited patients from May 2015 until August 2016. The follow-up continued until September 2017.Placebo eye-drops were provided by the pharmacy of Heidelberg University Hospital in a polyethylene dropper bottle containing 3 ml sterile 0.9% saline without preservatives. In both groups, pharmacy staff used information leaflets, which contained recommendations for correct eye-drop use and were previously validated for comprehensibility .The primary outcome measure was the comparison of the number of patients who correctly performed eye-drop administrations between intervention and control group before counseling after six months.Secondary outcomes were (i) the number of patients who correctly administered their eye-drops before and after patient education at the first encounter as a measure for the immediate impact of patient education on administration skills, (ii) the type of administration errors at baseline, and (iii) the number of patients who correctly administered their eye-drops after one month and six months compared to baseline as a measure for the sustainability of patient education. In addition, (iv) the patients’ perspective on their administration skills was obtained by self-evaluation of drug administration before patient education and compared to their demonstrated skills.Sample size calculation was based on a randomized trial that compared a teach-to-goal intervention for the correct use of a metered-dose inhaler with a brief intervention comparable to routine training. AssumingDuring conduct of the study it became evident that participating pharmacies had difficulties with recruiting patients. Therefore, after approximately one year, the study was stopped and included patients were analyzed.Comparisons between groups were calculated using a chi-squared test. Before–after comparisons were calculated using a McNemar test. Statistical analysis was performed using IBM SPSS Statistics Version 24. The 12-month follow-up was not analyzed because of low numbers of patients available for the 12-month follow-up at the time of the end of the study. The level of significance was set at p ≤ 0.05. Because of the exploratory character of the study, p-values were interpreted accordingly.A total of 28 German community pharmacies were randomly allocated to intervention (N = 11) or control group (N = 17) . Four coIn the intervention group, 56 regular eye-drop users were assessed for administration errors and 9 patients were immediately taught about correct administration because they used eye-drops for the first time. In the control group, 82 patients used eye-drops regularly and were eligible for baseline assessment, while 13 first-time eye-drop users were counseled. Seven patients in the intervention group and one patient in the control group administered their eye-drops correctly at baseline and, thus, received no counseling and were not included to follow-up visits. Hence, 152 patients were eligible for the study and 91 patients (60%) agreed to participate in follow-up visits: The proportion of patients agreeing to participate was 79% (46 out of 58 eligible patients) in the intervention group and 48% (45 out of 94) in the control group (p ≤ 0.001). Most of the included patients (80%) reported not having received previous education on correct eye-drop administration . In the The majority of patients used eye-drops regularly  and rateOverall, 15 intervention and 29 control patients attended both follow-up visits . Errors Correct medication administration is crucial to effective drug treatment. The majority of our patients could not administer their eye-drops correctly although most of them used their medication regularly. Knowledge-based errors such as omission of eyelid closure combined with nasolacrimal occlusion seemed unknown or forgotten by most patients. However, both steps crucially influence treatment outcome by increasing ocular drug availability , 21, 22 This study has several limitations. The results are limited by the small number of patients, which impaired the evaluation of the impact of a motivational communication technique on patient administration skills. High drop-out rates were possibly promoted by the long follow-up of six months, which was chosen to evaluate whether a time-consuming intervention would pay off in the long run because the training effect would last longer. However, studies should determine the appropriate interval to re-evaluate administration skills and need for education. In addition, cluster-randomization possibly introduced a bias for example when patients in one pharmacy have previously received patient education while patients other in another pharmacy have not . For exaIn conclusion, in both groups patient education improved eye-drop administration but high drop-out rates limited the power to detect differences between groups. The joint exploration of errors by pharmacy staff and the patient, handing over of comprehensible information material, and teach-back evaluation were the key components for effective counseling. In particular, periodic observations of individual administration skills seemed crucial to sustained teaching success. However, some patients probably require further error prevention strategies such as additional education materials or external support with eye-drop administration.S1 FigThe specific procedure comprised an assessment of administration skills, tailored counseling, and teach-back. At the first encounter, patients, who administered their drugs for the first time, were immediately counseled without baseline assessment of administration skills. Patients, who demonstrated correct administration at follow-up visits, received no further counseling at that visit.(TIF)Click here for additional data file.S2 FigPatients who regularly used eye-drops could rate their administration skills as “very good” (black), “good” (squared), “sometimes good, sometimes poor” (grey), “less good” (hatched), “not good” (striped), or “I don’t know” (white).NP: non-participating patients, i.e., patients who correctly administered eye-drops and, thus, received no counseling or patients who received patient education but refused to participate in the follow-up visits.(TIF)Click here for additional data file.S3 FigGraphs are ordered according to number of errors and recurrence of already resolved errors at the 1-month or 6-month follow-up. Numbers indicated erroneous administration steps: 1 = hand washing, 2 = instilling a single drop, 3 = instillation into the conjunctival sac, 4 = eyelid closure for approximately one minute, 5 = nasolacrimal occlusion, 6 = dropper tip was not touched.(TIF)Click here for additional data file.S4 FigGraphs are ordered according to number of errors and recurrence of already resolved errors at the 1-month or 6-month follow-up. Numbers indicated erroneous administration steps: 1 = hand washing, 2 = instilling a single drop, 3 = instillation into the conjunctival sac, 4 = eyelid closure for approximately one minute, 5 = nasolacrimal occlusion, 6 = dropper tip was not touched.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


This study was aimed to compare serial long-term postoperative changes in quality-of-life (QoL) between photoselective-vaporization (PVP) using 120W-High-Performance-System and holmium-laser-enucleation (HoLEP) in benign-prostatic-hyperplasia (BPH) patients and to identify factors influencing the QoL improvement at the short-term, mid-term and long-term follow-up visits after surgery. We analyzed 1,193 patients with a baseline QoL-index ≥2 who underwent PVP (n = 439) or HoLEP (n = 754). Surgical outcomes were serially compared between the two groups at up to 60-months using the International-Prostatic-Symptom-Score (I-PSS), uroflowmetry, and serum PSA. We used logistic regression analysis to identify predictors of QoL improvement (a reduction in the QoL-index ≥50% compared with baseline) at the short-term (12-months), mid-term (36-months), and long term (60-months) follow-up after surgery. In both groups, the QoL-index was decreased throughout the entire follow-up period compared with that at baseline. There were no significant differences in postoperative changes from the baseline QoL-index between the two groups during the 48-month follow-up, except at 60-months. The degree of improvement in QoL at 60-months after HoLEP was greater than that after PVP. A lower baseline storage-symptom-subscore and a higher bladder-outlet-obstruction-index (BOOI) were independent factors influencing QoL improvement at the short-term. No independent factor influences QoL improvement at the mid- or long-term. As expectations for QoL are increasing with increased life expectancy, many elderly men with LUTS become less tolerant5 and complain about LUTS, leading to poor QoL, although LUTS due to BPH is not life threatening. Thus, one of the aims of treatment for BPH patients is improving QoL through improving LUTS6, and, to improve LUTS and QoL in men with severe LUTS, surgical treatment would be useful5.Lower urinary tract symptoms (LUTS) caused by benign prostatic hyperplasia (BPH) is known to be a highly prevalent disease with increasing age8. Recently, as alternatives for TURP, laser surgeries such as photoselective vaporization of the prostate (PVP) and holmium laser enucleation of the prostate (HoLEP) have been increasingly performed9, because of fewer perioperative complications, which are related to perioperative QoL10. Specifically, in several studies, PVP and HoLEP were reported to have, at least, noninferior efficacy, less bleeding, and less catheter duration than TURP11. Thus, PVP was recommended in patients with a high cardiovascular risk and high bleeding risk12; additionally, HoLEP has an advantage regarding hemostasis13. As less complications would be expected to lead to better QoL as mentioned above10, PVP and HoLEP could be the good option to LUTS patient who pursuit QoL.For decades, standard treatment for BPH was transurethral prostatectomy (TURP)15. Additionally, few studies have investigated which factors could influence postoperative improvements in QoL after the two laser surgeries. Therefore, the aim of this study was to compare serial long-term postoperative changes in QoL between PVP using a 120-W GreenLight high-performance system (HPS) and HoLEP in patients with BPH and to determine the factors influencing the improvement of QoL at the short-term, mid-term, and long-term follow-up visits after surgery using a serial long-term follow-up database.To the best of our knowledge, few studies have directly compared postoperative treatment outcomes between PVP and HoLEP, focusing on improvement in QoL during a serial long-term follow-up period, although a few short-term follow-up studies showed no significant differences in the postoperative improvement of QoL during a one-year follow-up after surgeryIn comparing the baseline characteristics between the PVP and HoLEP groups, the PVP group had a higher level of serum PSA, a smaller prostate volume, a higher voiding symptom score (VSS), a higher storage symptom score (SSS), a higher total International Prostate Symptom Score (I-PSS), a longer operation time, and greater energy applied during the surgeries than the HoLEP group Table . HoweverRegarding the baseline urodynamic data, the HoLEP group showed a smaller postvoid residual urine volume (PVR), a smaller maximum cystometric capacity (MCC), a higher bladder outlet obstruction index (BOOI), a higher bladder contractility index (BCI), and a higher percentage of patients with involuntary detrusor contraction (IDC) than the PVP group Table .Qmax), PVR, and bladder voiding efficiency (BVE), was maintained during the entire follow-up period after PVP or HoLEP, except for Qmax at 60 months after PVP. However, the values of VSS and SSS starting from 36 months after the PVP were increased compared with those at 12 months after surgery, although their decrease was sustained up to 60 months after surgery compared with that at baseline. Meanwhile, the values of VSS starting from 24 months after the HoLEP were increased compared with those at 12 months after surgery, whereas those of SSS at 24, 36, 48 and 60 months after surgery were not different from 12 months postoperatively. The Qmax values in both the PVP and HoLEP groups were deteriorated starting from 24 months compared with those at 12 months after surgery. However, the increase in the Qmax value at all follow visits after the HoLEP was maintained up to 60 months compared with that at baseline, whereas the Qmax value at 60 months after the PVP was decreased to the baseline level. The incidence of transient urinary incontinence after HoLEP was higher than that after PVP  analyses, univariate analysis showed that the serum PSA level, preoperative prostate volume, baseline SSS, BCI, BOOI on baseline urodynamic study (UDS), operation time, and energy applied during surgery were associated with QoL improvement at one year after surgery. Multivariate analysis revealed that lower baseline SSS and higher BOOI were independent factors influencing QoL improvement at the short-term follow-up visit after surgery Table . In term4, one of the goals for the treatment of BPH was focused on improving QoL16. Laser surgery for BPH has been expected to be alternatives to the gold standard of BPH surgery (TURP or open prostatectomy) because, compared with TURP, it showed comparable efficacy and lower postoperative morbidity as mentioned above17. Specifically, Xue et al. reported that PVP provided equivalent efficacy and fewer bleeding complications for 3 years after surgery, compared with TURP18. HoLEP, an enucleation surgery for BPH, was reported to have similar efficacy and lower perioperative complications with a significant level of evidence, than TURP8. However, there has been a scarcity of studies mainly focusing on QoL improvement after laser prostatectomy. Because the treatment outcomes of BPH surgery greatly affect patients’ QoL, this study may extend the current knowledge regarding them. The results of our study are summarized as follows:Postoperative improvement of QoL was maintained up to the long-term follow-up period after PVP or HoLEP.No significant differences were found between the two groups in postoperative changes from baseline of the QoL index during the 48-month follow-up period after surgery. However, the degree of improvement in QoL at 60 months after HoLEP was greater than that after PVP.Lower baseline SSS and higher BOOI were independent factors influencing QoL improvement at the short-term follow-up visit after surgery. However, because the follow-up duration was longer, no independent factor influenced QoL improvement at the mid- and long-term follow-up visits after surgery.Because LUTS strongly influences QoL negativelyet al. reported that the postoperative improvement of QoL was sustained up to 36 months after TURP or PVP-120 W-HPS18. Additionally, Gilling et al. compared the surgical outcomes at 1, 3, 6, 12, 24 and 92 months between TURP and HoLEP19. According to their results, the postoperative improvement of QoL was maintained up to 92 months after TURP and HoLEP, without a difference between the two surgeries19. Meanwhile, although laser surgery is expected to have a positive effect on the postoperative improvement of QoL in men with BPH, few studies have focused on the comparative analysis of serial long-term treatment outcomes for QoL between PVP and HoLEP. Previously, a minority of studies showed no difference in the postoperative improvement of QoL at one year after surgery between PVP using 120 W HPS and HoLEP15. Interestingly, our study with long-term follow-up data showed that the degree of improvement in the QoL index at 60-month follow-up visits after HoLEP might be superior to that after PVP, perhaps in part because of a difference in the long-term durability of surgical outcomes. This result could be supported by our finding that the degree of improvement in voiding symptoms, peak flow rate or reduction in the serum PSA level at the long-term follow-up visits after HoLEP was higher than that after PVP. In accordance with these findings, recent literature by Hermann et al. noted that transurethral endoscopic enucleation of the prostate, such as HoLEP, offer some advantages in terms of morbidity13. However, further long-term follow-up studies comparing PVP using the 180 W XPS laser and HoLEP are necessary to draw a solid conclusion because PVP using the 180 W XPS laser has recently been performed in men with BPH.Our study has shown that the postoperative improvement of QoL was maintained up to the long-term follow-up period, irrespective of the type of laser surgery. In agreement with our results, Xue et al. showed that transient storage symptoms were developed more frequently after GreenLight enucleation of the prostate than after standard PVP or anatomic PVP20. They suggested that this might be due to microinjury of capsules and per-capsule innervation caused by more coagulation in the capsular bleeding spot in GreenLight enucleation of the prostate20.Interestingly, according to our data, the degree of improvement in voiding symptoms during the early postoperative period (1 to 3 months) after PVP or HoLEP was much greater than that in storage symptoms. Thus, storage symptoms appear to improve gradually with time after surgery, whereas voiding symptoms do dramatically from the immediate postoperative period after surgery. This may be attributed to an irritative effect of laser vaporization despite enucleation during laser prostatectomy. Thus, laser energy applied during laser prostatectomy might be beneficial with respect to hemostasis, but some patients could pay the price of postoperative storage or irritative symptoms in the early postoperative period. The irritative effect may have impacts on the surgical outcome for storage symptoms in the early postoperative period. In accordance with this, Cindolo 22. Accordingly, when the learning curve for the efficiency of HoLEP was assessed by enucleation efficiency (a ratio of retrieved tissue weight/enucleation time) in our study, the enucleation efficiency appeared to be stationary after approximately 50 cases  in our study had sufficient experience in endoscopic surgery for BPH. However, it is difficult to directly compare the studies because of differences in the baseline characteristics and study populations, as well as in definitions of learning curve.A factor to be considered when evaluating the surgical outcomes of PVP or HoLEP is the surgeon’s expertise. Particularly regarding the learning curve of HoLEP, some literature has shown that HoLEP has a steep learning curve with a stationary state at 20–50 caseset al. showed that a BOOI greater than 40 was a predictor of treatment success, including QoL at six months after HoLEP24. Additionally, the patients with less severe storage symptoms before surgery appear to have a higher possibility of postoperative improvement in QoL, perhaps because, as our data showed, the lower baseline SSS was significantly correlated with a lower SSS and a lower QoL index at one year after PVP or HoLEP . Interestingly, based on univariate analysis, several factors, including a higher BOOI and preoperative prostate volume, were associated with postoperative QoL improvement at the mid-term follow-up visit, no independent factor of QoL improvement was found on multivariate analysis. Furthermore, no influential factor of QoL improvement was found at the long-term follow-up visit, even on univariate analysis. Thus, postoperative QoL improvement at the short-term follow-up period after PVP or HoLEP appears to be maintained up to five years, without any specific factor influencing its improvement at the mid-term or long-term follow-up visit.To our knowledge, this is the first study that reported the factors influencing the postoperative improvement of QoL serially at the short-term, mid-term, and long-term follow-up visits after PVP or HoLEP. Our LR analyses showed that a lower baseline SSS and a higher BOOI were independent factors influencing postoperative QoL improvement at the short-term follow-up visit after PVP or HoLEP. Given that BPH surgeries such as PVP and HoLEP have been designed to relieve BOO, it may be reasonable to assume that patients with a higher BOOI have a higher probability of improvement in QoL after surgery. In accordance with our result, Ryoo There are a few limitations in our study. First, because our study was retrospective, there were differences in a few baseline parameters between the two groups. Second, we used PVP using 120 W-HPS; however, in 2010, PVP using 180 W-XPS was introduced. In the future, long-term follow-up studies comparing PVP using the 180 W XPS laser and HoLEP are necessary to validate our result, although the long-term follow-up data of PVP using 180 W-GreenLight XPS might still be limited. Nevertheless, our data have some clinical implications to understand the outcomes of QoL-related LUTS after laser prostatectomy, such as PVP or HoLEP. Our results may help effectively counsel patients about expectations for two representative laser prostatectomies or surgical outcomes for QoL related to LUTS. Additionally, our results may be used to counsel patients on which ones can benefit the most from PVP or HoLEP in terms of QoL-related LUTS, particularly at the short-term follow-up period after surgery.In conclusion, our data confirm that both PVP and HoLEP have durable efficacy in QoL improvement throughout the five-year follow-up period. HoLEP might provide more improvement in QoL at the long-term follow-up point than PVP. A lower baseline SSS and a higher BOOI appear to independently influence QoL improvement at the short-term follow-up visit after PVP or HoLEP. When the follow-up duration is longer, no factor appears to influence QoL improvement independently at the mid- and long-term follow-up visits. Subsequent prospectively controlled comparative studies with a larger study population and a long-term follow-up on PVP using 180 W GreenLight XPS and HoLEP would be needed to validate our findings.This study was approved by the Institutional Review Board at both Seoul National University and Seoul Metropolitan Government Seoul National University Boramae Medical Center. All methods in this study were carried out in accordance with relevant guidelines and regulations. The need for written informed consent was waived because of the retrospective nature of this study. We retrospectively reviewed the data of 1,569 patients who had undergone PVP-120 W-HPS (n = 564) or HoLEP  because their LUTS/BPH was unresponsive to medications from January 2008 to March 2014 at our institution. We excluded 320 patients with previously diagnosed urethral stricture, cancer of the bladder or prostate or urethra, history of other urologic surgeries, or incomplete data. Among the remaining 1,249 patients, 1,193 patients  with a baseline QoL of the I-PSS of at least 2 before surgery were included in this study.25. Briefly, PVP was performed using the planned vaporization-resection technique, with a 120 W GreenLight HPS laser at a setting of 80 W for vaporization-resection and 100 W for vaporization. HoLEP was performed by one of two surgeons (JSP or SJO) in the usual manner as previously mentioned26. Briefly, enucleation was performed mainly using three-lobe techniques with a 26 Fr resectoscope, a 550-μm laser fiber, or an 80-W holmium:YAG laser at a setting of 2 J 50 Hz or 2 J 40 Hz. Morcellation of the enucleated prostatic adenoma was performed with a morcellator. The patients received follow-up visits serially at 1, 3, 6, 12, 24, 36, 48 and 60 months after surgery. At each follow-up visit, the patients were evaluated for the I-PSS, serum PSA, and uroflowmetry.Before the surgeries, all patients received preoperative evaluations for LUTS secondary to BPH, including medical history, physical examinations, the I-PSS, urinalysis, serum PSA, transrectal ultrasound for prostate, and a multichannel UDS. A surgical method, either PVP or HoLEP, was chosen based on the surgeon’s preference. PVP was performed by a single surgeon (HS) as described in a previous studyWe defined ‘QoL improvement’ as a reduction of 50% or more in the QoL index at each follow-up visit compared with that at the baseline to investigate patients with a definite effect after surgery to confirm strong factors leading to favorable outcomes. Bladder voiding efficiency (BVE) was equated as (voided volume) × 100/(voided volume + PVR).To compare the preoperative characteristics and surgical outcomes between PVP and HoLEP, we used independent t-test and chi-squared or Fisher’s exact test. For comparisons between baseline variables and postoperative outcome parameters, we used paired t-test. Additionally, the repeated-measures ANOVA test was performed to adjust for the impact of time on the QoL outcomes. To identify the factors influencing the ‘QoL improvement’ at the short-term (one year after surgery), mid-term (three years after surgery), and long-term (five years after surgery), we applied LR analyses. Variables with p < 0.05 in the univariate LR were included in multiple LR. p < 0.05 was considered as statistically significant in all analyses. Statistical analyses were performed using IBM SPSS version 21.0.Supplementary information


Caenorhabditis elegans to produce transcription profiles for seven specific embryonic cell populations from gastrulation to the onset of terminal differentiation. The expression data for these seven cell populations, covering major cell lineages and tissues in the worm, reveal the complex and dynamic changes in gene expression, both spatially and temporally. Also, within genes, start sites and exon usage can be highly differential, producing transcripts that are specific to developmental periods or cell lineages. We have also found evidence of novel exons and introns, as well as differential usage of SL1 and SL2 splice leaders. By combining this data set with the modERN ChIP-seq resource, we are able to support and predict gene regulatory relationships. The detailed information on differences and similarities between gene expression in cell lineages and tissues should be of great value to the community and provides a framework for the investigation of expression in individual cells.We have used RNA-seq in  Caenorhabditis elegans embryo develops from one cell to 558 cells, the transcriptional profile of each cell undergoes unique changes to drive differentiation into distinct cell types and tissues. Although the invariant lineage of the cell divisions has been mapped down to each division and apoptotic event  to isolate fluorescent cells at multiple time points from synchronized embryos containing early and specific tissue/lineage markers . We obseC. elegans, we exploited strains expressing fluorescent reporters in muscle , intestine (end-1p::mCherry), neurons (cnd-1p::mCherry), pharynx (pha-4::GFP), and hypodermis (nhr-25::GFP) (end-1p::mCherry). The lineages in which these genes are expressed, as determined by 4D microscopy, include most of the cells of the animal with the exception of the D and P4 lineages  and ABala (ceh-32p::mCherry). ABa produces a variety of neurons, pharynx, and hypodermis, whereas ABala produces a subset of neurons and glial cells  . To assay gene expression in each of these tissues over time, we sampled synchronized populations of embryos at 90-min intervals for five time points . Our initial time point was ∼120 min after embryo isolation, shortly after the fluorescent markers were first visible and about halfway through gastrulation. The last time point (∼480 min after isolation) was approximately when the embryo reached the threefold stage, when most cells are beginning terminal differentiation, but before the cuticle forms; when this outer layer is present, it complicates cell recovery. For each sample, we also collected a set of unlabeled cells. To obtain well-synchronized populations, we harvested young adult animals from a synchronized population just as the first eggs were appearing . At the initial time point, the bulk of these embryos had between eight and 26 cells, with 15-cell embryos being the most abundant ; this synchrony persisted through successive time points . To obtain time- and tissue-specific exon usage, we sampled the full length of both nonpolyadenylated and polyadenylated transcripts (see Methods). Lastly, we devised a novel PCR duplicate detection method to identify the fraction of PCR duplicates and remove them from our sequencing output without distorting the expression values of highly expressed genes. Our method estimates the PCR duplicate rate in low-coverage genome regions where duplicates would be rare by chance and removes these duplicate reads as well as proportional numbers of detected duplicate read pairs in high-coverage regions .To obtain transcription profiles of the major tissues and organs of 25::GFP) . Becausecnd-1 samples, which selected about 250 cells from the embryo). For each sample, expression levels were calculated .In total, we produced time series data for five different tissue/organ-specific cell populations and two lineage-specific populations, each of which is henceforth described as a tissue for the sake of simplicity. Each time series had two biological replicates, except for the ABa and muscle series, which had three and four replicates, respectively. After removal of rRNA and PCR duplicate reads, our 85 samples had a total of more than 1 billion paired-end reads, giving an average of 12 million read pairs mapped to the transcriptome for each sample and a minimum of 4 million read pairs. With this depth of coverage and approximately 20,000 mRNA molecules per post-mitotic cell , each mRlculated  in transSupplemental Fig. S7). This high fraction of histone transcripts makes clear the demands that the rapid division rates place on the C. elegans embryo. Nevertheless, because the representation of histone transcripts and rRNA content vary considerably between tissues and time points and because they are not included in other studies that use poly(A)-selected transcripts, we excluded reads mapping to histones (and rRNA) in both the calculation of the TPM and the remainder of the analysis.As we noted with whole-animal rRNA-subtracted samples , reads dSupplemental Fig. S8). Our data confirmed 93,164 WormBase (WS245)-annotated introns and also confirmed 14,017 introns not annotated in WormBase WS245 that we detected previously  and robust expression for 11,408 genes (TPM ≥ 15 in at least one sample) . Associated with this increase of DE genes, we note a shift in the distribution of expressed genes over time, with an increase of about 1000 in the number of modestly expressed genes (TPM > 6 and < 100) and a corresponding drop in nonexpressed or poorly expressed genes (TPM < 6) . In comparing the different tissues at similar time points, the intestinal samples consistently have the most DE genes, followed by muscle and neurons. As expected, the two lineage samples show fewer DE genes compared against tissues with which they share cells .To evaluate the dynamics of gene expression between tissues and over time, we compared expression among all pairwise combinations of the samples using DESeq2 . In part2FC) value ≥1.0 for the highest-expressed tissue over the second highest-expressed tissue and an adjusted P-value of ≤0.1, while excluding samples that shared common cells . In total, 2700 genes were scored as DE in a single tissue . The intestinal and muscle series have the highest number of DE genes with 1182 and 694 genes, respectively. These DE genes fall into the types of Gene Ontology categories expected for the cell types present in the samples .To find genes specifically enriched in a single series at particular times, we used DESeq2 to identify genes with a log base twofold change . We also looked for genes whose expression varied minimally over time within each series and found 1572 genes that are stably expressed in one or more tissues over time .An additional 54 genes showed differential expression for one series at one time point and a different series at a later time point in development, thus changing the tissue specificity over time. Often these appeared to represent earlier onset of expression in one tissue of a zygotically expressed gene, and the pattern is seen across replicates . To visualize the clusters, t-SNE  samples. Adjacent clusters have enriched intestinal expression but with different temporal patterns. Other groups of clusters have genes enriched in muscle, pharynx, hypodermis, and neuronal cells as indicated .Among genes that are more broadly expressed, we see temporal patterns. Genes in cluster 31 have decreasing expression across all tissues and represent maternally expressed genes. The labeled adjacent clusters also have genes that are decreasing in expression over time with fairly uniform expression among all tissues. Conversely, >70% of the genes in clusters 4, 14, 30, 33, 43, and 57 are expressed stably in every tissue type. Adjacent clusters 12, 13, 46, and 53 meet these criteria for 40% of their genes. A high proportion of the broadly and stably expressed genes are in operons: 913 (58%) genes in operons are among the 1572 stably expressed genes compared with 3128 genes in operons in the 11,408 genes analyzed . In contrast, 32 genes have gene expression restricted to muscle, including the known muscle genes unc-89 (obscurin), dys-1 (dystrophin), and the muscle-specific integrin pat-2. The specialized proteins that these genes encode presumably reflect the very different demands that contraction places on the adhesome structures in muscle. Another 18 genes are enriched in muscle but have low to moderate expression in other tissues, including tln-1 , unc-112 (kindlin), and unc-97 (PINCH), indicating either shared usage or tissue-specific isoforms. Another set of genes is expressed only at lower levels in muscle with primary expression in one or more other tissues. These include genes primarily expressed in the intestine—plc-3 (phospholipase C), pkc-2 (protein kinase 2), and act-5 (actin)—and neurons tiam-1 (TIAM) and jnk-1 . Our findings also shed new light on prior genetic studies. For example, the gene pxl-1 (paxillin), when mutated, results in death in the larval stage L1 owing to failure of the pharynx to pump . The RNA levels of a downstream gene could differ from its upstream gene because of post-transcriptional mechanisms but also because of the use of independent promoters for the downstream gene. To detect the latter, we assayed the fraction of splice leader SL1 trans-splicing, which occurs predominantly at the initiation of the transcript, whereas SL2 is used predominantly in downstream operon genes , in accord with the notion that the gene has an independent promoter as well as being part of an operon. We also noted that the fraction of SL2 used in downstream genes decreased as expression increased, confirming our prior results . Differentially used exons are more likely to be conserved in Caenorhabditis briggsae orthologs . Also, we find that DE genes are ∼10% more likely to contain differentially used splice junctions.Because we obtained sequence reads from the entire length of transcripts, we were able to use JunctionSeq  to assaySupplemental Table S7). In contrast, splice sites producing exons of different lengths showed the least specificity, with 70% showing no evidence of differential usage. Possibly, exons differing only by a few bases are functionally similar, with little advantage conferred by tissue-specific expression or they may be functionally unique, but their largely shared sequence may make detecting differences more difficult .Of the different types of alternative splicing, mutually exclusive exons show the most specificity, with ∼43% showing differential usage among tissues (supported by both exon and splice junction reads) and an additional 23% showing evidence from either exon or splice junction reads alone  . Further, there are 89 initial exons and 24 initial splice junctions that are significantly more frequently used  in one tissue than in all others, and there are 49 initial exons and 13 initial splice junctions that were significantly less frequently used in one tissue than in all other tissues .Of the 2601 WormBase-annotated genes (WS245) with alternative start sites, 916 genes (1614 first exons and splice junctions) show some pairwise differential usage of the first exons . Of the 2601 genes with alternative start sites, 1423 of these showed evidence of splice leader usage, including some SL sites that were tissue-specific. We also found broadly expressed genes that were only trans-spliced in one tissue and genes in which the acceptor site, and sometimes the splice leader type, varied between tissues . These results reveal novel complexity in the regulation of splice leader usage.In 1 or SL2 , more re-spliced . We idenlev-11) (unc-52) (fln-2), one of two genes encoding a filamin protein in the worm genome. The hypodermal isoform has an alternative start site and lacks the first 10 exons included in the pharyngeal isoform, as well as exons 42 and 43  present in the hypodermal isoform. Intestinal fln-2 codes for a protein most similar to the hypodermal isoform but contains only 13 IG/Fil domains. The ABa lineage produces cells in both pharyngeal and hypodermal tissues and appears to have both forms.Alternative splicing events sometimes involve multiple exons, producing substantially different proteins in one tissue versus another. We see examples of this in integrin adhesome genes, including previously published examples such as tropomyosin (lev-11)  and perl(unc-52) . We also2 and 43 . In additln-1, also displays tissue-specific isoform usage that results in substantially different proteins. It has an alternative 3′ exon that is used almost exclusively in muscle . As a result, the shorter, muscle-specific isoform lacks the three VL actin binding domains and one of the two I/L WEQ binding domains present in the longer isoform. The shorter form also lacks the single Talin-½ domain that is predicted to facilitate disengagement of TLN-1 from the integrin. We speculate that in muscle integrin adhesion complexes (dense bodies), the absence of this dissociation domain contributes to the stability of the dense body in contrast to the less stable complexes found in migrating cells. The adhesome components thus differ substantially not only in the genes used but also in the specific isoforms produced by differential processing.A second integrin adhesome gene, C. elegans by Supplemental File S5). Although some support was found for six of the sites, strong support was found for editing in only one gene, Y105E8A.3, with no resulting change in the amino acid. No support was found for the C-to-U editing event suggested by RNA editing is the post-transcriptional modification of RNA by nucleotide modification/substitution, insertion, or deletion, increasing the number of different transcripts that can be produced  by changSupplemental File S5). A total of 17 of the 48 edits resulted in an amino acid change , and one, in C07E3.9, resulted in a premature stop truncating the final 27 amino acids.By using an analysis pipeline similar to that of The combined tissue and temporal specificity of the data provide an opportunity to examine the regulation of gene expression. We used the data to ask (1) if genes potentially sharing upstream regulatory regions were more likely to show similar expression patterns, (2) which transcription factors are expressed in which tissues and in what order, and (3) which transcription factor binding sites were more likely to be associated with tissue-specific genes.We first asked if divergently transcribed genes , which might be influenced by common regulatory elements, were more likely to have similar patterns of expression than other gene orientations (head-to-tail and tail-to-tail) or random pairs. We considered downstream genes in operons separately, because for the most part their expression is expected to be similar to the initial gene. We then compared the expression of gene pairs in 35-dimensional space for each class of genes.t-test . Of these, 689 have robust gene expression in at least one of our samples (TPM ≥ 15). In total, 178 transcription factors show differential expression based on DESeq2 analysis and 259 transcription factors based on fuzzy k-means clustering . In muscle, known muscle transcription factors are present such as hlh-1  domains and ELT-2 binding sites in its promoter region, clusters with genes sharing late-onset intestinal expression and is a transcription factor with almost exclusively intestinal gene expression beginning ∼400 min into development. This places F55B11.4 downstream from both end-1 and elt-2 in the intestinal transcriptional regulatory pathway, with presently unknown regulatory targets. For genes in which the expression is tissue-specific, we can also use the whole-embryo gene expression data  domains and an HLH-1 binding site in its promoter region, is enriched in muscle; in the whole-embryo data, it has a temporal profile almost identical to that of unc-120, which follows hlh-1 and precedes ceh-18.The temporal resolution of our data also provides information about the order in which transcription factors act within a tissue. For example, the different intestinal clusters reflect different temporal patterns of expression. The GATA factor end-1  is imporustering , 3. The  pathway , clusterion data , which hC. elegans transcription factors have binding site information as a result of ChIP-seq experiments performed as part of the model organism Encyclopedia of Regulatory Networks (modERN) project (http://epic.gs.washington.edu/modERN/). We analyzed the binding sites for these transcription factors to determine which transcription factors showed a bias toward binding in the regulatory region of DE genes. For each transcription factor, we compared the percentage of DE genes in a given tissue that had a ChIP-seq peak to the percentage of non-DE genes with a ChIP-seq peak, and calculated the significance of the difference (Methods). Out of 183 transcription factors, 127 showed either a significantly enhanced presence in front of DE genes or a significantly reduced presence in front of DE genes in any given time point of a tissue , including genes expressed exclusively or mainly in muscle, some primarily in other tissues, and some broadly expressed across tissues and times. We also saw that alternative splicing produces very different forms of the genes fln-2 and tln-1 in different tissues, allowing for further specialization of adhesome function.The integrin adhesome genes provide an example of some ways our data set can inform the study of larger complexes. The essentiality of some of the genes in the complex for muscle function has inhibited the genetic analysis of these genes in other tissues. These other roles include cell migration mediated by focal adhesion-like structures, distal tip cell migration, axon extension, pharyngeal muscle cell adhesions, neuronal point contacts found in growth cones, and anchor cell invasion. By examining their expression across the various tissues, we are able to see distinct groups of genes with substantially different expression patterns  was added to the embryos at a ratio of 1 mL Chitinase to 0.5 mL embryo suspension, and the embryos were transferred to 30-mm petri dishes to incubate before embryo dissociation. For details, see Supplemental Methods.To dissociate the embryos into single cells, 100 µL of 15 mg/mL Pronase (P6911 Sigma-Aldrich) was added to ∼1.5 mL of embryo/egg buffer suspension. The cell suspension was drawn from the dish using a 3 cc syringe and used to wash the dish to isolate all embryos. Embryos were repeatedly pulled through a 21-gauge needle 20×, in a 1.5-mL Eppendorf tube, and then incubated at room temperature for 5 min. Lastly, the cell suspension was drawn repeatedly through a 21-gauge needle until a suspension of single cells was confirmed via microscope. Cells were sorted using a FACS ARIA III to isolate a minimum of 150,000 cells per sample and chilled during sorting. For details, see Total RNA was isolated using TRIzol LS/chloroform extraction of total RNA, followed by EtOH precipitation and column-based cleanup using Direct-zol tubes (Zymo Research). Ribosomal RNA was reduced in each sample using Ribo-Zero (Illumina) and purified using SPRI beads (Agencourt RNAClean XP). Immediately following the RNA cleanup, first-strand cDNA synthesis was performed using SuperScript IV (Invitrogen), followed by second-strand synthesis using the second-strand synthesis module (NEB). Shearing of cDNA was performed using a Covaris LE220 and end repaired using the NEB end repair module. Y-Adapter ligation to A-tailed cDNA was followed by QPCR-based barcoding of samples.Supplemental Table S20). Transcript-level expression was calculated using BitSeq . TPM values were averaged for the replicates of each tissue's time point to obtain a TPM for each tissue. To test saturation of sequencing, we used the program subSeq  along with the DESeq2 adjusted P-value . We also used a chi-square test to assess whether our calculation of a gene being stably expressed was independent of the gene's presence in an operon.By using DESeq2 differential expression analysis, we identified genes expressed predominantly in a single tissue within each time point using TPM values as input. For each gene, we report the ratio of the largest TPM expression value to the second largest TPM expression value  under accession number PRJNA477006. Expression profiles across tissues and time points for individual genes are available at http://genome.sfu.ca/gexplore .All sequencing data from this study have been submitted to the NCBI BioProject database (


Adjuvant paclitaxel and trastuzumab has been shown to be an effective regimen with low risk of cancer recurrence and treatment-related toxicities in early-stage node-negative, HER2-positive breast cancer. We investigated the cost-effectiveness of this regimen.A Markov-based microsimulation model with six health states is used to simulate four adjuvant therapy options for women with early-stage node-negative, HER2-positive breast cancer at different age groups. The four treatment arms are 1) adjuvant paclitaxel and trastuzumab (TH), 2) doxorubicin, cyclophosphamide, paclitaxel and trastuzumab (ACTH), 3) docetaxel, carboplatin and trastuzumab (TCH), and 4) no adjuvant trastuzumab (NT). Data from randomized trials were used to estimate treatment efficacy. Societal perspective was used in this cost-effectiveness analysis. Costs were measured in 2016 US dollars (US$) and quality-adjusted life-years (QALYs) was used for health outcomes. Sensitivity analyses were performed to evaluate the impact of uncertainty in parameter estimation.We found that 40-year-old women undergoing TH treatment would have an average of 16.17 QALYs for the cost of $178,650 when lifetime horizon is used. Compared to NT, TH has incremental cost-effectiveness ratios ranged from $10,584 (ages 40–49) to $84,981 (age 80+) per additional QALYs. The sensitivity analysis showed that TH is cheaper and leads to higher QALYs compared to both ACTH and TCH for all age groups and time horizons.TH is cost-effective for all age groups in the base case scenario and in the sensitivity analysis. In order to reduce the parameter uncertainty, clinical trials with longer follow-up times are needed. Breast cancer remains the most common cancer among the US women with an estimated 252,710 diagnoses and over 40,000 deaths in 2017 . TrastuzBecause the majority of patients enrolled in the early trastuzumab studies had higher-risk disease with larger, node-positive tumors, and with concerns of toxicities from adjuvant chemotherapy, there has been increasing interest in de-escalation of therapy for low-risk disease. Tolaney and colleagues recently reported disease-related outcomes and low toxicities with a novel paclitaxel and trastuzumab (TH) adjuvant therapy regimen in early-stage node-negative tumors with risk of disease recurrence of less than 2% at 3 years . DespiteWith the clinical benefit of adjuvant trastuzumab alongside the escalating costs of cancer treatments, we sought to assess the cost-effectiveness of the TH regimen as compared to standard of care, ACTH and TCH regimens for small, node-negative HER2-positive breast cancer in the United States using a Markov modeling approach. We also included an arm of no adjuvant trastuzumab (NT), based on retrospective reports of outcomes reported in this context ,9.A Markov state-transition microsimulation model was developed in MATLAB 2016 software to evaluate the cost-effectiveness of four adjuvant treatment strategies for early-stage node-negative, HER2-positive breast cancer  Figs. ThWe presented the results in terms of incremental cost-effectiveness ratios (ICERs) using two different effectiveness measures: cost per quality-adjusted life-years (QALYs) gained and cost per life-years (LYs) gained. Following the recommendations of the Panel on Cost-Effectiveness in Health and Medicine, we performed our analysis using the societal perspective and discounted both future costs and future QALYs at 3% in the base case analysis . We prefOur model considers five age groups  with early-stage node-negative HER2-positive breast cancer. Age groups 40–49 and 50–59 represent the median age group of the participants in the addressed randomized trials , we extrThis cost-effectiveness analysis evaluates and compares the clinical and cost outcomes of four alternative adjuvant therapies. The first arm represents the no adjuvant trastuzumab (NT) which is based on the control arms of the NSABPB-31 and NCCTG9831 clinical trials . The secACTH and TCH regimens were considered as comparative programs . These tThe Markov model is essentially divided into two sub-models: adjuvant chemotherapy sub-model and follow-up sub-model . Markov Once a patient completes adjuvant therapy or quits adjuvant therapy prematurely, she moves directly to the follow-up sub-model and starts in the “alive with relapse-free” state (state 4). This sub-model consists of three states. Two of these states are identical to those in the adjuvant chemotherapy sub-model (states 4 and 6). The third state is “alive with relapse” (state 5). Upon a breast cancer recurrence or a new primary breast cancer event, the patient undergoes the same treatment she had in the adjuvant chemotherapy sub-model one more time and remains at the “alive with relapse” state until death. This assumption is made only for local recurrence or a new primary breast cancer which would be treated with curative intent with adjuvant therapy. Due to limited data, we assumed similar relapse probability for all age groups in each of the adjuvant therapy arms. Patients are followed until they die .We adjusted all rates to estimate the monthly health-state transition probabilities . We usedIn this study, we considered toxicity events resulted from using trastuzumab in the adjuvant therapy. Low and high toxicity rates were derived from the respective randomized trials ,7. We asWe used latest published studies to estimate the quality-of-life for the adjuvant therapies, low and high toxicities, and breast cancer relapse . For lowUsing the medical care component of the consumer price index, we converted all costs into 2016 US dollars . For eacWe utilized several methods for validation. First, we ensured the face-validity of the model by consulting our clinician co-author to share his clinical expertise on the model’s structure, assumptions, and the input parameters. Additionally, all health-related parameters were derived directly from the relevant clinical trials. Finally, a cross-validation was performed by comparing our model’s results for the no adjuvant trastuzumab (NT) treatment strategy with those of the study by Kurian et al. .We utilized different methods to perform sensitivity analyses in order to test the robustness of the base case results. We conducted one-way and probabilistic sensitivity analyses to account for uncertainty in model parameters. We varied the model parameters based on the distributions as reported in Tables Furthermore, we evaluated the cost-effectiveness for different age groups in 40 to 80 years old. We determined the age-based ICER assuming all parameters are kept constant except for the mortality rates, patient utility without the treatment, and the monthly cost of being at the “alive with relapse-free” state. We also evaluated the cost-effectiveness of considered treatments when different time horizons are used.The results of the base case analysis over a lifetime horizon for age group 40–49 years  show thaTime horizon has a significant impact on the ICER . The lonThe time horizon represents the maximum time that the TH is effective in reducing the probability of relapse, the regimen leads to no reduction in relapse beyond the time horizon. Abbreviations: ICER, incremental cost-effectiveness ratio; NT, no adjuvant trastuzumab; QALY, quality-adjusted life year; TH, adjuvant paclitaxel and trastuzumab regimen.In the base case analysis and with a WTP threshold of $50,000 per QALY, TH regimen is cost-effective for time horizons of 12 years or longer and for patients aged between 40 and 79. However, gains due to treatment become smaller and the TH regimen becomes less cost-effective as the patient is older. Nonetheless, TH regimen continues to be cheaper and leads to higher QALYs compared to both ACTH and TCH regimens for all age groups see .We presented the results of one-way sensitivity analysis for the 40–49 age group with lifetime horizon using a tornado diagram . The resOur sensitivity analysis on the impact of increasing only the cost of treatment based on the TH regimen shows a very steady increase in the ICER of TH regimen compared to the NT arm; whereas a relatively sharp increase in the ICER compared to both ACTH and TCH regimens see . At 20% The probabilistic sensitivity analysis showed that the likelihood of the cost-effectiveness of TH adjuvant therapy as compared with NT was 100% with a $50,000/QALY threshold see  Figs.In this paper, we used a Markov microsimulation model to compare four strategies for treating women with early-stage node-negative, HER2-positive breast cancers. Our base case analysis shows an ICER of $10,584 per QALY for TH compared to NT when we assume that the impact of TH on relapse rates is observed throughout the lifetime of a patient . Our probabilistic sensitivity analysis confirms that the TH regimen is cost-effective in the base case for the 40–49 year–old age group, indicating the robustness of our model and findings. TH regimen is cheaper and leads to higher QALYs compared to both ACTH and TCH regimens for all age groups in the base case scenario as well in the sensitivity analysis due to the relatively high probability of toxicity events and high costs associated with ACTH and TCH.Several studies reported the cost-effectiveness of widely accepted breast cancer therapies and found favorable ICERs ,15,20. FOne-way sensitivity analysis shows that although discount rate, the probability of relapse, and cost of TH are the most influential parameters affecting the ICERs, these parameters still do not have any major impact on the ICERs. For instance, even if the cost of TH regimen is increased by 60%, TH remains cost-effective.Our study’s major limitation is related to the limited availability of follow-up data on the efficacy of treatment regimens. Namely, our study uses the trial data from the study by Tolaney et al. , in whicThe discovery of trastuzumab has revolutionized the treatment of HER2-positive breast cancer with significant improvements in disease-free and overall survival. However, this does come at a significant cost to the society. With excellent outcomes of low-risk HER2-positive early breast cancer, de-escalation of therapy has generated interest both from an individual patient perspective as well as the society as a whole. This has led to efforts to reduce adjuvant therapies and the evolution of regimens like TH which we hereby show to be cost-effective with an ICER of $10,584 per QALY compared to NT much below the $50,000 per QALY, a commonly used benchmark for cost-effectiveness –27. AlthIn conclusion, based on the current data, the TH regimen has a low relapse risk and improved overall survival in early-stage node-negative, HER2-positive breast cancer, alongside being cost-effective compared to regimens like ACTH and TCH. While awaiting long-term survival data, this regimen is increasingly being considered in clinical use.Adjuvant paclitaxel and trastuzumab (TH) is cost-effective for women with early-stage node-negative, HER2-positive breast cancer aged between 40 and 79 years old in the base case scenario as long as the time horizon is longer than the 12 years. Similarly, TH remains cost-effective for women aged between 40 and 49 in the sensitivity analysis. Moreover, TH is cheaper and leads to higher QALYs compared to both ACTH and TCH regimens for all age groups, therefore clinicians should consider utilizing TH for treating women with early-stage node-negative, HER2-positive breast cancer due to its effectiveness and low toxicity.S1 Checklist(DOCX)Click here for additional data file.S1 TableThe table shows the distribution of cost in dollars, QALYs, and ICERs.(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigThe square represents the decision node, yellow circles represent chance nodes, the purple circles represent Markov nodes, and the green triangles represent the outcome of each random event.(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(A) Age group 40–49. (B) Age group 50–59. (C) Age group 60–69. (D) Age group 70–79. (E) Age group 80 and older.(TIF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S6 FigScatter plot of incremental costs (in thousand dollars) and QALYs based on 10,000 samples.(TIFF)Click here for additional data file.S7 Fig(TIFF)Click here for additional data file.


Infections caused by extended-spectrum β-lactamase–producing Enterobacterales (ESBL-E) among hospitalized neonates in sub-Saharan Africa pose significant clinical challenges. Data on prevalence and acquisition of ESBL-E carriage among hospitalized neonates in the region are few, and risk factors for transmission are not clearly defined.In a cohort study of consecutive neonatal admissions to Kilifi County Hospital from July 2013 through August 2014, we estimated ESBL-E carriage prevalence on admission using rectal swab cultures and identified risk factors using logistic regression. Using twice-weekly follow-up swabs, we estimated the incidence and identified risk factors for ESBL-E acquisition in hospital using Poisson regression.The prevalence of ESBL-E carriage at admission was 10% (59/569). Cesarean delivery, older neonatal age, and smaller household size were significant risk factors. Of the 510 infants admitted without ESBL-E carriage, 238 (55%) acquired carriage during their hospital stay. The incidence of acquisition was 21.4%  per day. The rate was positively associated with the number of known neonatal ESBL-E carriers and with the total number of neonates on the same ward.Carriage of ESBL-E was common among neonates on admission, and in-hospital acquisition was rapid. The dissemination and selection of ESBL-E appears to be driven by hospital exposures, operative delivery, and neonatal ward patient density. Further attention to infection control, patient crowding, and carriage surveillance is warranted. We report a prospective hospital-based longitudinal study that estimates the extended-spectrum β-lactamase–producing Enterobacterales (ESBL-E) carriage prevalence among neonates on admission, the incidence of acquisition of ESBL-E carriage in hospital, and the risk factors for neonatal prevalent and incident ESBL-E carriage. Outbreaneonates  and are neonates .It is known that neonatal ESBL-E carriage can be a precursor to invasive infections , 8, but Klebsiella pneumoniae over a decade at KCH [At Kilifi County Hospital (KCH), we have observed sporadic outbreaks of ESBL-E bacteremia among neonatal admissions over several years. These infections often have a poor outcome . We have at KCH .Here we report a prospective, hospital-based, longitudinal study at KCH to estimate ESBL-E carriage prevalence among neonates on admission, the incidence of acquisition of ESBL-E carriage in hospital, and the risk factors for neonatal prevalent and incident ESBL-E carriage.Neonatal admissions were eligible for recruitment into the study if they were admitted to the high-dependency unit between 1 July 2013 and 29 August 2014 or to the neonatal rooms in the general pediatric ward between 16 August 2013 and 29 August 2014. The high-dependency unit consists of an open ward with 6 beds for older children and 2 small rooms for neonatal admissions. The neonatal rooms are of equal size and have a combined bed capacity of 8. In the general pediatric ward, there are 2 neonatal rooms with a combined bed capacity of 24, including 5 incubators, 4 small beds, and 15 cots. KCH practices comprehensive obstetric care, as defined by the World Health Organization, with cesarean delivery services available.Epidemiological and clinical data were collected on admission and entered in real time into an electronic medical record system. Rectal swabs were collected on admission (day 0), at days 2, 4, and 6, and twice weekly thereafter until an ESBL-E was isolated or until hospital discharge or death, whichever came first. Rectal swabs were collected using premoistened viscose-tipped swabs and placed in Amies transport media . The number of neonates in each room, bed location, and antimicrobial use of all participants was recorded daily. Blood culture is performed routinely at admission on all children hospitalized at KCH . ClinicaRectal swabs were inoculated onto 5% horse blood agar and MacConkey agar supplemented with 8% gentamicin. Cefotaxime (30 μg) and ceftazidime (30 μg) antibiotic disks  were added on the second and fourth streaking zones on the blood agar plate to detect bacteria resistant to third-generation cephalosporins. Blood agar plates were incubated in a carbon dioxide incubator while MacConkey agar plates were incubated in an aerobic incubator for 24 hours at 35°C ± 2°C. Oxidase-negative, gram-negative rods were subcultured and identified using standard techniques . Antimicrobial susceptibility testing was performed using the disk diffusion method according to the Clinical and Laboratory Standards Institute 2014 guidelines [The binomial confidence interval (CI) around a prevalence estimate is widest (for a fixed sample size) when the estimate is 50%. We calculated that a sample size of 555 neonates would be required to estimate a 50% prevalence of ESBL-E carriage with a precision of ±5%.Carriage on admission  was defined as a positive culture on the first rectal swab, provided it was obtained within 48 hours of admission. Logistic regression was used to determine risk factors for carriage at admission. The factors considered were infant age, sex, maternal age, infant weight at admission, current method of feeding, place and mode of delivery, prematurity , number living in the same household, type of toilet, and main source of water. Multivariable logistic regression models were fitted after confounders had been identified . Infant Kaplan-Meier curves were used to describe the time to acquisition of carriage in hospital among neonates who did not have ESBL-E carriage at admission. In this analysis, follow-up time, which was measured in days after admission, was censored at the earliest of (1) time of the first ESBL-E culture-positive swab, or (2) time of last swab collection if the neonate died or was discharged and had remained negative throughout the course of admission. For neonates who acquired ESBL-E, the date of acquisition was assumed to be the midpoint between the date of the last negative swab and the date of the first positive swab.We calculated the rate of ESBL-E acquisition per 100 days at risk. Poisson regression was used to identify predictors of the acquisition rate and to test for interactions. The potential predictors were both time-invariant  and time-varying . A multivariable Poisson regression model was fitted to investigate the effect of crowding on ESBL-E acquisition. The model included as covariates the ward, the number of neonates on the ward, and the number of known ESBL-E carriers on the ward.We defined multidrug resistance as resistance to at least 1 agent in 3 or more antimicrobial categories . StatistThe Kenya Medical Research Institute National Ethical Review Committee approved the study (SSC 2301). Informed consent was obtained from all parents/guardians before enrollment.During the study period, 1014 neonates were admitted to KCH, and the parents/guardians of 597 neonates gave consent for them to participate in the study . The medKlebsiella pneumoniae, 25 Escherichia coli, 8 Enterobacter cloacae, and 1 Klebsiella oxytoca. Multiple colonization, that is, colonization with ≥2 ESBL-E isolates from 1 participant, was found in 6 of 59 neonates (10%).Of 597 who gave consent to participate, 5 parents/guardians withdrew consent and 23 neonates had no swab collected. The prevalence of ESBL carriage at admission was 10% (59/569). From the 59 neonates with ESBL-E carriage at admission, there were 65 isolates consisting of 31 Among the 510 noncarriers on admission, 55% (283/510) acquired ESBL-E during their hospital stay. The incidence of ESBL-E acquisition was 21.4  per 100 child-days of observation, and the median time to acquisition among these patients was 3  inpatient days . Nine neMost ESBL-E isolates were multidrug resistant, with resistance to chloramphenicol, trimethoprim-sulfamethoxazole, quinolones, and gentamicin being common; none were resistant to imipenem and only 5% were resistant to amikacin .P < .001). After adjusting for prematurity and place/mode of delivery, increasing infant age was positively associated (P < .001) with ESBL-E carriage at admission, with odds ratios (ORs) of 1.72  and 3.88  among neonates aged 1–2 days and 3–28 days, respectively, relative to the odds of ESBL carriage among neonates admitted on the day of birth. Being born at term was not associated with ESBL-E carriage after adjusting for the effect mediated through age on admission. We did not estimate an adjusted OR for place/mode of delivery and number of people in the same household, as the associations were not confounded  in this study, but it is known that such isolates are present in Kenya [Being a hospital-based study, focusing on sick newborns, our estimates of ESBL-E prevalence at admission cannot be generalized to community prevalence. It is theoretically possible that low-level ESBL-E carriage was more prevalent at admission than we were able to determine—below the detection rate by culture methods, but then amplified by selection pressure from the use of antibiotics in hospital until detectable. We also were only able to recruit 68% of eligible neonates, limiting the generalizability of our findings . Of notein Kenya , 29. UseIn conclusion, our findings reveal a high incidence of ESBL-E colonization, which is endemic in this setting, among hospitalized neonates. Further work to investigate the association between ESBL-E acquisition and both cesarean delivery and crowding, perhaps including restrictions on room capacity and more deliberate cohorting of older neonates and those born in hospital through cesarean delivery, is needed. Given the link between ESBL-E carriage and outbreaks of potentially fatal ESBL-E infection, our data emphasize the importance of routine surveillance and hospital infection control.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciy976_suppl_Supplementary_Table_S1Click here for additional data file.


Moderate-to-vigorous physical activity (MVPA) in old age is an important indicator of good health and functional capacity enabling independent living. In our prospective twin cohort study with 616 individuals we investigated whether long-term physical activity assessed three times, in 1975, 1982 and 1990 (mean age 48 years in 1990), and other self-reported health habits predict objectively measured MVPA measured with a hip-worn triaxial accelerometer (at least 10 hours per day for at least 4 days) 25 years later (mean age of 73 years). Low leisure-time physical activity at younger age, higher relative weight, smoking, low socioeconomic status, and health problems predicted low MVPA in old age in individual-based analyses . However, quantitative trait modeling indicated that shared genetic factors explained 82% of the correlation between baseline and follow-up physical activity. Pairwise analyses within monozygotic twin pairs showed that only baseline smoking was a statistically significant predictor of later-life MVPA. The results imply that younger-age physical activity is associated with later-life MVPA, but shared genetic factors underlies this association. Of the other predictors mid-life smoking predicted less physical activity at older age independent of genetic factors. High participation in moderate-to-vigorous physical activity (MVPA) at older ages is an indicator of good physical fitness and health, and consequently predicts reduced risk of disability and death in the older population4.Reduced physical activity in old age predisposes strongly to disability while exercise-based rehabilitation improves measured and self-rated function among individuals with various chronic diseases5. Low physical activity7 and other lifestyle factors, such as smoking and use of alcohol10 predict or are associated with later disability and impaired mobility. However, no data exist describing whether long-term leisure-time physical activity during adulthood predicts objectively measured physical activity/mobility in old age. Non-communicable diseases and performance and activity limitations develop slowly, so it is important to investigate the long-term predictors of later-life physical activity levels.Some observations suggest that midlife low physical activity, obesity, and poor health status predict sedentary lifestyle in old age11. Thus, both genetic factors, including the possibility of genetic pleiotropy, and childhood environment-related factors may predispose to different clusters of risk factors and associated diseases13. By studying outcomes in twin pairs discordant for exposure to different health habits and health outcomes, the possible confounding role of genetic and shared early childhood experiences can be considered. Twin pairs almost always share the same childhood family environment. Dizygotic (DZ) pairs share, on average, half of their segregating genes (like non-twin siblings), while monozygotic (MZ) pairs are genetically identical at the sequence level. Co-twin control analyses among discordant MZ twin pairs allow for stronger estimates of causal influences compared to associations seen in unrelated individuals.Twin, family, and molecular genetic studies provide evidence for a role of genetic factors in obesity, many non-communicable diseases, fitness, and participation in physical activity, but the identity of specific genes for physical activity remains largely unknownIn this study, we show how prospectively collected self-reported long-term leisure-time physical activity and other health habits from ages 31 to 48 years predict objectively measured physical activity and sedentary behavior a quarter of a century later at a mean age of 73. This prospective twin cohort study in Finland comprised 616 individuals , who responded to baseline questionnaires in 1975, 1981, and 1990, and participated in accelerometer monitoring at follow-up . Primary exposure was long-term leisure-time physical activity; the mean MET index expressed as the mean sum-score of leisure-time physical activity MET-hours per day in 1975, 1981 and 1990 (LT-mMET index). Covariates were body mass index (BMI), work-related physical activity, smoking, heavy alcohol use and health status in 1990, and socioeconomic status. Follow-up physical activity was measured with a hip-worn triaxial accelerometer (at least 10 hours per day for at least 4 days) to obtain daily mean MVPA, daily steps and other characteristics of physical activity or sedentary behaviour. Our data demonstrate how the younger-age physical activity is associated with later-life physical activity, but shared genetic factors underlie the association of mid-life physical activity with later-life MVPA over a 25 year period.Mean age of the participants was 48.3 years (range 45.9–51.4) at the time of response to the 1990 questionnaire and 72.9 years (range 71.1–75.0) for objective physical activity monitoring. Among those who responded to all the baseline LT-mMET questions in 1975, 1982 and 1990 , the LT-mMET index including leisure-time and commuting activity was similar in those who participated in the follow-up accelerometry study (n = 616) and those who did not participate for various reasons (n = 1 030) . However, among all cohort members having data on baseline predictors, there was a statistically non-significant tendency towards lower LT-mMET index among both men and women who died before the follow-up examination compared to those participating in the follow-up measurement of physical activity. Current smoking and socioeconomic status (not a white collar worker) at baseline statistically significantly associated with death during follow up among both men and women. Respectively, high body-mass index, heavy use of alcohol, non-sedentary work and health status  at baseline statistically significantly associated with death during follow-up among men. Baseline participant characteristics by LT-mMET index tertiles are shown in Table 2 2.0% after age- and sex adjustment, P = 0.002), more MVPA , more daily steps  and also higher intensity of 10 minute continuous physical activity (Peak-10 min MET) during the monitoring week   or for health status  differed in their follow-up MVPA volumes  and other covariates explained one fifth (20.3%) of the variation in objectively measured moderate-to-vigorous activity in older age in this prospective twin cohort study. According to pairwise analyses, much of the association was driven by shared genes underlying mid-life physical activity and later objectively measured activity. Smoking contributed independent of genes.16. In cross-sectional and shorter-term follow-up studies, low physical activity is associated with lower fitness, more frailty, higher disability, and poor health20. No long-term randomized trials have addressed whether changes in health behavior in middle age lead to late-life differences in physical activity. Also, observational follow-ups on this topic are rare, and we are not aware of other studies relating long-term leisure-time physical activity differences in younger adulthood to objectively measured physical activity/inactivity in later years21.In line with our findings, high physical activity is associated in cross-sectional or longitudinal designs with high previous physical activity, low BMI, low work-related physical loading, and good health statusIn our individual-based analyses, we found significant predictors for later-life physical activity, but could not replicate all of the results in pairwise analyses among the predictor-discordant MZ twin pairs. The outcome is a reminder that genetic or other familial factors may explain why associations are often seen between younger-age physical activity and later-age health-related factors and, consequently, mobility.23. Our quantitative trait modelling was in agreement with the results of the pairwise analyses. Smoking affects both pulmonary and cardiovascular health and increases systemic inflammation, all of which may decrease the ability to exercise. We cannot exclude the possibility that smoking is also a marker for other lifestyle factors that predict less physical activity.Smoking at baseline also predicted less MVPA at follow-up in pairwise analysis among MZ twin pairs, which is evidence for an association not explained by genetic factors. These results are in line with our earlier finding that MZ twin pairs discordant for smoking show a clear difference in overall mortality while pairs discordant for physical activity participation do not24.The strengths of our study include that we had physical activity data from three different baseline time-points, a nationally representative large twin cohort, very long-term prospective data, and novel valid analysis of the follow-up physical activity and sedentary behavior profileOur study has also limitations. Our baseline predictor assessments relied on self-reported questionnaire data. We lack comprehensive data on dietary factors or clinical examinations at baseline. Although our study was large enough for the individual-based analyses, the number of MZ twin pairs discordant for some of the predictors was quite low providing only moderate statistical power for some analyses. At follow-up, most twins were community dwelling, so there were very few individuals with severe mobility limitations. Individuals who have survived to the mean age of 73 years represent a selected group, which may weaken the statistical power to detect the influence of some predictors of the capacity for being physically active.25. Also, paying attention to body weight control and physical activity since early childhood may be a good strategy to increase later life activity levels. In addition, promoting other health habits such as not smoking and avoidance of heavy drinking may influence later life physical activity capacity. Finally, it is good to keep in mind that randomized controlled intervention studies have shown that among individuals whose physical functioning is reduced due to chronic diseases appropriately tailored exercise therapy improves their physical function and mobility2.The public health community has been trying for years to get people more active at the population level, but with limited to moderate success. Our study increases understanding on this challenge. As participation in physical activity and its tracking has a familial component  it is understandable that it is challenging to activate middle-aged and older adults having been previously physically inactive. As the genetic components contributing to the correlations between mid-life LTPA and later life MVPA or Peak-10min MET were high unlike that for daily steps, recommending less vigorous  activities for older individuals may be a successful strategy. Be it noted that objective accelerometry-based monitoring records absolute physical activity intensities and previously inactive low-fit older individuals may not be able to participate in MVPA in terms of absolute intensity although they may be able to participate in physical activity which is moderate-to-vigorous relative to their fitness levelOur follow-up study among twins showed that middle-age low leisure-time physical activity, obesity, smoking, low socioeconomic status, and health problems predicted low objectively measured MVPA at older age in individual-based analyses. According to pairwise analyses, smoking seemed to causally predict less physical activity in later years while other associations were more likely attributable to shared genetic factors and childhood environment.26. Written informed consent was obtained from all participants, and the study was approved by the Ethics Committee of the Hospital District of Southwest Finland on 20 May 2014. All study methods were performed in accordance with the Helsinki Declaration and good clinical and scientific practice.This MOBILETWIN study is an ancillary to the older Finnish Twin Cohort Study26. A baseline questionnaire was sent to all twin candidates in 1975. Among those whose home addresses could be identified (93.5%) in 1975, the response rate for twins was 87.6%. A subsequent questionnaire was mailed in 1981 to all of the verified twins. The corresponding response rate among those responding in 1975 and alive in 1981 was 90.7%. A third questionnaire was sent out in 1990 to all twin individuals aged 33–60 (birth cohorts 1930–1957) years who had responded to at least one of the earlier questionnaires 27.The study is based on a nationwide sample of all same-sex twin pairs born before 1958 with both co-twins alive in 1975For the current physical activity study (MOBILETWIN), twins from the 1940–1944 birth cohorts were selected .The postal questionnaires in 1975 and 1981 were very similar, but the questionnaire in 1990 was slightly different in some parts; however, they all included questions on physical activity, occupation, work-related physical activity, smoking, use of alcohol, and physician-diagnosed diseases  and physical activity during commuting. The index was calculated by assigning a MET score to each activity and by calculating the product of that activity: intensity × duration × frequency28. The MET index was expressed as the sum-score of leisure-time physical activity MET-hours per day. To estimate the mean volume of physical activity during the three baseline survey years, the average of the MET index values obtained in 1975, 1981, and 1990 was computed. This new leisure-time mean MET value (LT-mMET index) was then divided into three activity tertiles labelled low (LT-mMET index 0–1.54 MET h/day), medium (1.54–2.92 MET h/day), and high (2.92–26.13 MET h/day) using the same tertiles as in an earlier study31. Twin pairs were classified as discordant for physical activity if one co-twin was in the low-activity tertile and the other co-twin was in the high-activity tertile.As other predictors and covariates, body mass index (BMI), self-reported work-related physical activity, smoking status, use of alcohol and health status in 1990, and socioeconomic status were used. After preliminary analyses, to maximize statistical power for pairwise twin and multivariate analyses, covariates were dichotomized by merging classes not differing for baseline and follow-up physical activity levels.22 ignoring the type of employment. A response to the first option “mainly sedentary work, which requires very little physical activity” was classified as sedentary work, while all other responses  were classified as non-sedentary work. Majority of the participants who were sedentary workers worked outside home (94.9%) or at home (2.1%) while in the non-sedentary category there were more individuals who worked at home (8.6%), were on disability pension (5.5.%) or were unemployed (2.8%). Three socioeconomic status categories  were defined by years of education and amount of physical activity at work27. The blue collar and intermediate groups were combined in the analyses because their baseline and follow-up physical activity was similar. Great majority of the white collar workers worked outside home (97.0%) while in the others category there were more individuals who worked at home (6.5%), were on disability pension (3.8%) or were unemployed (2.2%). Smoking status, originally coded into four categories33, was dichotomized (current daily vs. others) for the main analyses. Alcohol use was expressed as a dichotomous variable of heavy drinking occasions  at least monthly35. Somatic health status  was defined as having/not having a disease diagnosed by a physician, serious injury/illness, or permanent work disability, according to self-report items in 199027.BMI was calculated based on self-reported height and weight. In 1990 work-related physical activity of their current or latest work was classified by the respondents using a categorical variable evaluated with a four-point ordinal scale38.Physical activity was measured with a hip-worn, light triaxial accelerometer . The device and instructions for use were mailed to the participants, who were asked to use the accelerometer during waking hours for 7 consecutive days. Participants mailed the device back to UKK Institute for data analysis, and they were later provided with their own results. The analysis of raw acceleration data was based on novel algorithms that employ the mean amplitude deviation (MAD) of the resultant acceleration during a 6 s epoch and the angle for posture estimation (APE) of the body, metrics that provide a consistent assessment of the intensity of physical activity and separate accurately sedentary and stationary behaviors from any physical activity2 during walking or running on an indoor track36. This strong association allowed for conversion of MAD values to incident energy consumption (MET). The MET values for each minute were calculated as the one-minute exponential moving average of epoch-wise MAD values. According to standard use24, cut-off points for different activities were set as 1.5–3 MET for light activities, 3–6 MET for moderate activities, and over 6 MET for vigorous activities, and corresponding mean daily total times were determined. Mean daily sedentary time was defined as MET under 1.5 during lying down or sitting. Mean daily standing time was analyzed separately. Average daily step count and the most intensive continuous 10-minute period of physical activity (Peak-10min MET) during the monitoring week were also documented.MAD was also validated through directly measured incident VOP = 0.30) and daily steps  were seen between these 616 individuals and the 175 individuals who did not have complete physical activity data for the calculation of baseline LT-mMET but participated in the accelerometer study.Altogether, 791 twin individuals wore the accelerometer for at least 10 hours per day for 4 days. On average, they wore the device 6.73 days  and 14:01:44 h:min:sec/day (95% CI 13:56:31–14:04:37). A total of 616 had complete data for calculating MET indices from all of the 1975, 1981, and 1990 questionnaires. No significant differences in MVPA  as a measure of variance accounted for. The analyses were done with twins treated as individuals; however, because the observations obtained from twin pairs may be correlated, robust estimators of variance (the cluster option in Stata) were used39. All basic analyses yielding R2 values were adjusted for age and sex. To obtain R2 only for the studied variable, the variable was entered the model after the basic model and then the difference in R2 (∆R2) was calculated. Multivariate models were adjusted for BMI, smoking, alcohol, work-related physical activity, health status, and socioeconomic status. Square root-transformation for MVPA, logarithm-transformation for Peak-10min MET, and cubic root transformation for LT-mMET were used for regression analyses because these variables were not normally distributed.Descriptive statistics were calculated with bootstrapping (1000 samples unless otherwise noted) and are given as medians and interquartile ranges (IQRs) or 95% confidence intervals (CIs). We used linear regression analyses to define R squared  were done using Wilcoxon matched-pairs signed-rank test for whether pairs discordant for specific baseline characteristics or health habits differed in the objectively measured physical activity variables at follow-up.Quantitative trait modeling was done using the MET variables from 1975, 1981, and 1990 to analyze whether they were direct risk factors or whether the association with the follow-up physical activity variables was mediated by genetic or other environmental factors. The quantitative trait modeling is described in Supplementary Methods and Results, and only the main results are given above.All the MOBILETWIN Study data is not allowed to be placed in open archives due to data protection issues of possibly identifiable twins. Researchers are encouraged to contact the last author (U.M.K.) and we will make every effort to accommodate additional analyses.Supplementary Info