NMB facilitates intubating conditions in general anesthesia. However, it is associated with significant residual postoperative paralysis and morbidity.To investigate the rate of underdiagnosed residual NMB based on two TOFR criteria (< 0.91 and < 1.00).We performed a retrospective study adhering to STROBE guidelines. We included patients undergoing ENT surgery using single-dose neuromuscular block for balanced general anesthesia from June to December 2018. We collected demographic and anthropometric data, ASA score, NMBA dose, TOFR recordings at 5, 30 and 60 min and end of the surgery, anesthesia and surgery time, and administration of reversal agent. Statistical analysis included descriptive and dispersion measures statistics, curve and cross tables for residual NMB on different TOFR criteria with sub-analysis for AR, RR, and OR in patients over 65 years old.We included 57 patients, mean age 41; 43 females and 14 males. Mean anesthetic and surgical time were 139.4 and 116.1 min, respectively. All the patients received rocuronium under a mean ponderal single-dose of 0.48 mg/kg. Residual NMB rates were 29.9 and 49.1% for a TOFR < 0.91 and < 1.00, respectively. Older adults had an OR of 6.08 for residual NMB.The rate of residual NMB was 29.9 to 49.1%, depending on the criteria used . Patients above 65 years old had an increased risk of residual NMB (6.08 OR) and clinical symptoms related to residual NMB (11.75 OR). We recommend future research aiming to provide a specific surveillance protocol for patients above 65 years old, including shorter-action NMB, early reversal, and prolonged surveillance using the TOFR criteria of < 1.00 to identify patients at risk of residual NMB readily.The online version contains supplementary material available at 10.1186/s12871-023-02027-x. • The rate of residual NMB in our practice was in the range of 29.9 to 49.1% depending on the criteria used .• Patients above 65 years old are at an increased risk of residual NMB (6.08 OR) due to increased NMBA duration of action, as demonstrated by the curves of NMB during surgery.• Older patients also had an increased risk (OR 11.75) of clinical symptoms related to the residual neuromuscular block.• We recommend providing specific surveillance protocols for patients above 65 years-old, including the use of shorter-action NMB, early reversal, and prolonged surveillance in the postoperative period.• We recommend using the TOFR criteria of < 1.00 to readily identify more patients at risk of residual neuromuscular block, especially in older people.• We recommend the use of reversal agents in patients at high risk of residual neuromuscular block, such as patients above 65 years-old, especially in those cases where patients have difficultness for reaching a TOFR criterion of 1.00 because of an increased risk of residual neuromuscular block and its complications.The online version contains supplementary material available at 10.1186/s12871-023-02027-x. Neuromuscular block agents facilitate intubating conditions in general anesthesia patients by immobilizing vocal cords , 17. HowWe performed a retrospective observational cross-sectional study adhering to STROBE guidelines. The protocol was approved by the General Hospital of Mexico Ethics and Research Committee by the number: DI/20/101/03/77; informed consent was waived because of being a retrospective study. We reviewed the clinical files of patients undergoing Ear-Nose-Throat (ENT) surgery from June 2018 to December 2018 in the General Hospital of Mexico. Inclusion criteria were adult patients (over 18 years) with an American Society of Anesthesiologists score (ASA) risk between I and III, having TOFR monitoring throughout balanced general anesthesia  using neuromuscular block with rocuronium  at a single dose. Exclusion criteria considered any muscle relaxant other than rocuronium, patients requiring additional muscle relaxant doses during surgery, muscle-related diseases or allergies, magnesium sulfate or antiepileptics medication, or illnesses affecting TOFR evaluation and incomplete medical records. We collected demographic and anthropometric data, ASA score, NMBA and dose, TOFR registers at 5 min, 30 min, 60 min, and end of the surgery, anesthesia time in minutes, surgery time, and administration of reversal agent. We performed descriptive statistics (frequencies and percentages) for demographic data; we used mean dispersion measures  for continuous variables. We used graphics to demonstrate the curve of NMB during surgery and residual NMB as the outcome in percentages. We used cross tables for residual NMB using two criteria, including TOFR < 0.91 or TOFR < 1.00. We made a sub-analysis for residual NMB using both criteria on patients under and above 65 years old. Table All TOFR Recordings performed were done using an acceleromyographic portable Drager TOFScan® locating two electrodes on the wrist and the distal forearm of the non-dominant hand to stimulate the first finger abductor and register its movement using the second or third finger as reference Fig. , TOFR meWe included 57 patients and excluded no patients; 43 females (75.4%) and 14 males 24.6%). The mean age was 41.74 years old (16.62 standard deviation (SD), 95% confidence interval (CI) at 37.33–46.15), and the mean body mass index (BMI) was 26.56 kg/m2 . Patients more frequently had an ASA score of II , followed by I , and lastly, III . The mean anesthetic time was 139.42 min , while the mean surgical time was 116.14 min . All 57 patients received a rocuronium at a mean ponderal single-dose of 0.50  mg/kg. Mean TOFR values were as follows: 5 min 0.029 , 30 min 0.19 , 60 min 0.67 , and at the end of surgery (outcome) 0.97 . Figure 4.6%. TheCurrent standards for quality care in anesthesia were first reported approximately in 2010 to increase patient safety during general anesthesia . Many meThe use of NMBA has significantly impacted current clinical practice for patients, anesthesiologists, and surgeons in many ways, including improved first-pass intubation for experts in elective conditions and non-experts in emergency conditions, aid for difficultness in facemask ventilation, enhanced surgical conditions for open and laparoscopic intermuscular approaches such as those of abdominal and pelvic procedures  in such Despite almost 80 years of experience in using NMB, several studies continue to report high incidence rates of residual neuromuscular block with the accompanying side effects and risks that it provides. Residual neuromuscular block increases the risk of pulmonary aspiration events and the hypoxic ventilatory response . RespiraMechanomyography (MMG) is the gold standard for monitoring neuromuscular function. Nevertheless, acceleromyography (AMG) is increasingly being used in a clinical setting as it is relatively inexpensive, easy to set up, and able to detect neuromuscular blocks with accuracy . HoweverAdditionally, to the possible incomplete understanding of the NMB effects and pharmacokinetics, a significant rate of underused monitoring is reported worldwide either by lack of devices, protocols, or culture to use monitoring , 16. As Underdiagnosing RNMB is not surprising considering the lack of implementation and expertise on NMB monitoring and the variations in device parameters and cut-off criteria, which make unneeded reversal even plausible, as was the case in one of our cohort patients. Adding to the already high rate of RNMB poses a significant risk of complications, especially in high-risk populations such as older people and patients with serious comorbidities. Reversal agents significantly reduce this collateral damage; nevertheless, they are not without risk. Reversal agents can have serious detrimental effects in the postoperative period. Traditionally, we achieve the reversal of neuromuscular block by administering acetylcholinesterase inhibitors . These dAdditionally, the type of reversal agent is also essential; there exists evidence that sugammadex reduces the risk of pulmonary complications by up to 30% . Based oSuch problems make suboptimal treatment  account for preventable complications. Increasing the importance of these phenomena is the observed prolonged times of action of NMB agents in elderly populations . Our stuLimitations, given the retrospective nature of the findings and the small sample size, we could not verify the process of TOF Baseline calibrations; nevertheless, it is a routine in our service practice.The rate of residual NMB in our practice was in the range of 29.9 to 49.1% depending on the criteria used , more probably the higher limit, in line with literature ranges. We do recommend extubating patients based on a TOFR criterion of 1.00, especially on the elderly, to avoid clinically relevant residual NMB or its complications. Patients above 65 years old are at an increased risk of residual NMB (6.08 OR) due to increased length of action, as demonstrated by the curves of NMB during surgery. Older patients also had an increased risk (OR 11.75) of clinical symptoms related to the residual neuromuscular block. Therefore, we recommend providing specific surveillance protocols for patients above 65 years-old, including the use of shorter-action NMB, early reversal, and prolonged surveillance in the postoperative period. In addition, we recommend the use of reversal agents in patients at high risk of residual neuromuscular block, such as patients above 65 years-old, especially in those cases where patients have difficultness for reaching a TOFR criterion of 1.00 because of an increased risk of residual neuromuscular block and its complications. We will look to develop future research prospectively to corroborate these results.Additional file 1.


Diagnosing central precocious puberty (CPP) requires an integrated approach based on clinical, biochemical and instrumental data. The diagnostic gold standard is represented by GnRH (gonadotropin-releasing hormone) stimulation test. Some undoubted limitations of this procedure led the international scientific community to look for cheaper and less invasive alternative diagnostic methods, such as luteinizing hormone urinary levels (uLH) measurement. This study aims to define the reliability of urinary LH levels as a biomarker of pubertal development, both concerning the initial diagnostic management and the monitoring of patients with central precocious puberty undergoing therapy with GnRH analogues. Furthermore, the study plans to detect the potential association between LH peak serum (pLH) and urinary LH in patients undergoing diagnostic tests with GnRH and to identify a possible cut-off of uLH that may be suggestive of ensued successful hormonal stimulation.The study includes 130 female patients with suspected precocious puberty or in follow-up during suppressive therapy. After the collection of the informed consent, the patients underwent clinical evaluation, auxological assessment, and hormone assays .U < 0.05), and uLH collected in prepubertal group control.Two uLH cut-off values have been identified: the first of 0.25 UI/L [C.I. 95% 0.23–0.27], able to distinguish between pubertal and pre-pubertal patients, the second of 0.45 UI/L  suggestive of occurred hormonal stimulation in patients with diagnosis of CPP at GnRH test. All 30 patients with CPP in follow-up during suppressive therapy presented uLH values ≤ 0.45 IU/L (puLH assays on the first morning urine specimen could be considered a low-cost and minimally invasive tool for precocious puberty diagnosing and monitoring, making possible to be easily performed even by a general pediatrician. Thus, this could help referring only selected patients to pediatric endocrinologists. After an appropriate validation, this approach could reasonably reduce hospital attendance and costs of performing more invasive procedures, with a more significant emotional impact on the pediatric patient. The diagnostic approach to the patient with suspected precocious puberty is based on the integrated evaluation of clinical, biochemical, and instrumental data . No singIn central precocious puberty (CPP), the activation of the hypothalamic-pituitary-gonadal neuro-endocrine axis has to be detected by a stimulation test with GnRH (gonadotropin-releasing hormone), the diagnostic gold standard . This anDue to these limitations, the international scientific community has been looking for alternative diagnostic methods for several years, and luteinizing hormone urinary levels (uLH) measurement is one of the most appealing –7. ULH iLH and FSH are secreted in a pulsatile pattern under GnRH stimulation. During puberty, the frequency and amplitude of this secretion increase progressively. In the early stages of pubertal development, the amplification of the secretory pattern of these hormones, particularly the LH’s one, can be observed mainly during the night and then becomes evident during the daytime. As a result, in both sexes, the differential between night-time and daytime secretion peaks of luteinizing hormone (pLH) is clearer in Tanner stages 2 and 3, then becoming progressively less evident with the progression and the end of pubertal development , 9.This study aims to assess the feasibility of using urinary LH, assayed on the first-morning urine, as a biomarker of pubertal development, both in the initial diagnostic approach and the monitoring of patients with central precocious puberty undergoing therapy with GnRH analogues (GnRHa).This study aims to identify a possible diagnostic role of LH urinary assay in female patients with a clinical suspicion of precocious puberty, as well as evaluate its possible use as a biomarker of therapeutic goal in patients treated with GnRHa.Two groups of patients were studied: prepubertal subjects  and girls with clinical suspicion of precocious puberty; the second included patients already being treated with GnRHa for a diagnosis of true precocious puberty.Currently, international guidelines consider the GnRH stimulation test the diagnostic gold standard for central precocious puberty, with a serum LH peak cut-off (pLH) of 5 IU/L , 10.Basal serum LH levels (sLH) are less validated, even though values above 0.3 IU/L can be considered predictive of hypothalamic-pituitary-gonadal axis activation. On the contrary, under 0.2 IU/L sLH values predict no clinical progression toward puberty .The primary aim of the study, in the first group of patients, is to find a possible urinary LH cut-off capable of discriminating between pubertal and prepubertal patients. Those subjects enrolled with anamnestic, clinical, biochemical, and instrumental features of hypothalamic-pituitary-ovarian axis activation were considered pubertal.Moreover, the study aims to detect the potential association between LH peak serum and urinary LH in patients undergoing diagnostic tests with GnRH and identify a possible cut-off of uLH that may, on its own, be suggestive of ensued successful hormonal stimulation.In the second group of patients, this study also aims to demonstrate the potential role of uLH as a therapeutic monitoring tool for true precocious puberty. The aim is to establish if the mean uLH values in this group are comparable to the uLH values in the group of controls, confirming a good hormone suppression ensured by the therapy.The retrospective monocentric study analyzed clinical, laboratory, and instrumental data of 130 female patients referred to the Outpatient Clinic for Auxology, Diabetology, Endocrinology and Pediatric Gynaecology of the I.R.C.C.S. Policlinico San Matteo in Pavia. The subjects were selected between September 2020 and September 2021, considering those with suspected precocious puberty or in follow-up during suppressive therapy with GnRHa.After the collection of informed consent by their parents, the patients underwent the following: (i) clinical evaluation, including assessment of the pubertal stage according to Tanner staging; (ii) auxological assessment: height and weight measurement, calculation of body mass index, growth rate, and genetic target; all data were subsequently compared with reference curves for sex, age, and ethnicity; (iii) hormone assays, including basal levels of LH (sLH), FSH (sFSH), and oestradiol (E2), possibly followed, in patients with suspected precocious puberty, by GnRH stimulating test; (iv) urinary LH assay on the first morning urine sample (uLH), collected after waking up (first voided specimen). In particular, the collection of first morning urine samples took place between 08:00 a.m. and 12:00 a.m. to better reflect the nocturnal peak of LH secretion , 12. AllThe first study group included 100 female subjects aged between 6 and 10 years. Of these, at clinical evaluation 60 girls (age 6.0–10.00) were considered pubertal, with subsequent diagnosis of central precocious puberty, pubertal advancement, or physiological puberty as defined by the Italian Society of Pediatric Endocrinology and Diabetology (SIEDP) guidelines . All patThe second study group included 30 patients (age 6.00–11.92) diagnosed with central precocious puberty confirmed by GnRH test (pLH > 5 IU/L). All enrolled patients received suppressive therapy with GnRHa (triptorelin) for at least 6 months. The instructions given to these patients for urine sampling were the same as in the first group: collection occurred in the morning, immediately after waking up (between 8:00 and 12:00 a.m.), and before further biochemical and/or instrumental investigations were carried out. No patients were excluded from the study because of inadequate urine specimen collection.All patients not in the control group underwent venous blood sampling for basal pituitary gonadotropin (LH and FSH) assays. Following the diagnostic procedure recognized by the SIEDP  in suspeUrinary LH was assayed on the first-morning urine sample (first voided specimen) to assess the night-time secretory peak of luteinizing hormone and subsequently corrected for the subject’s urinary creatinine. Urines were stored without preservative materials at 4 °C until the assays were carried out. All tests were performed by the end of the collection day. In particular, the samples were not frozen in order to avoid the physical degradation of LH subunits . The uriDescriptive statistical analysis was performed for all variables. Categorical variances were given as absolute values and percentages, while continuous variables were defined as means ± standard deviation (SD). The processing of the collected data was carried out using STATA statistical software (version 16.1).cut-off value of urinary LH, the area under the curve (AUC) ROC was calculated with a corresponding confidence interval [C.I. 95%]. With 100 patients and a ratio of pre-pubertal to pubertal subjects of 0.67, it was possible to elicit as significant a difference between an area under the ROC curve of 0.95 (excellent) and an area of 0.85 (very good) with a type I error of 5% and a power of 90%.For the definition of the Student’s t-test for the comparison of symmetrically distributed parametric means, while the Mann-Whitney U-test was used for asymmetric variables.A statistically significant association between the parameters under investigation was established through the chi² exact test) was used to test hypotheses involving two dichotomous nominal variables on small samples.In contrast, Fisher’s exact test  with an average age of 7.35 years ± 1.04, while 60 patients were pubertal (60%) with an average age of 8.08 years ± 0.98.According to the Tanner clinical staging, patients were also distributed into four distinct subclasses: 36% of subjects with Tanner stage 1, 42% with Tanner stage 2, 21% with Tanner stage 3, and only 1% with Tanner stage 4. Patients with complete pubertal development, Tanner stage 5, were not assessed. Specifically, all 60 pubertal patients presented thelarche (Tanner stage B2 or higher); among the 40 prepubertal patients, only 17 (42.5%) showed mammary granulation. On non-parametric analysis, according to Fisher, this distribution was statistically significant (p < 0.05).The basal serum LH concentration (sLH) in the 95 patients who had venous sampling averaged 0.48 IU/L ± 0.33; considering the two subgroups, the mean sLH was 0.14 IU/L ± 0.05 in prepubertal patients and 0.68 IU/L ± 0.46 in pubertal patients (p < 0.05).ULH levels, considering all the enrolled patients, averaged 3.14 IU/L ± 3.00, with a respective distribution of: 0.16 IU/L ± 0.05 in prepubertal patients and 5.12 IU/L ± 4.05 in pubertal patients (p < 0.05)  who underwent the GnRH test for the diagnosis of central precocious puberty, the mean peak LH (pLH) reached in the prepubertal group after pharmacological stimulation was 3.2 IU/L ± 1.02 (≤ 5 IU/L), while for the pubertal group it was 14.05 IU/L ± 12.85 (> 5 IU/L) (p < 0.05).When analyzing the raw data on the GnRH test results, it was found that 76.5% of the patients with pLH equal to or less than 5 IU/L were considered to be prepubertal, whereas almost all of the patients considered to be pubertal had a pLH greater than 5 IU/L  (p < 0.05). , the uLH value = 0.25 IU/L [C.I. 95% 0.23–0.27] was identified as the best cut-off for discriminating the actual onset of puberty , ROC curve analysis of urinary LH values (collected before the test was performed) identified uLH = 0.45 IU/L [C.I. 95% 0.20–0.70] as the In Table The second study group included a total of 30 female patients with a mean age of 9.00 ± 1.45 years. All subjects underwent stimulation testing with GnRH and were diagnosed with true precocious puberty (pLH > 5 IU/L). Assumed the rapid progression of the clinical developmental in these girls, suppressive therapy with GnRHa (triptorelin) was started for all of them.At the beginning of the treatment, according to Tanner’s staging, 20 patients (66.6%) had stage 2; 9 patients (30%) had stage 3; 1 patient (3.4%) was in stage 4. None of the patients was assigned to a Tanner stage 1  or 5 (fully developed).cut-off. All 30 patients on GnRHa therapy presented uLH values ≤ 0.45 IU/L  had uLH values ≤ 0.25 IU/L and only 8 patients (26.7%) had values above this In Table In accordance with previously published clinical studies , 15–17, Integrated clinical, biochemical, and instrumental data provided by the 100 patients examined in the first study group allowed us to identify a uLH value capable of discriminating between pubertal and prepubertal patients accurately: in particular, the cut-off of 0.25 IU/L, not only has a high positive and negative predictive value but owns a good narrow confidence interval [C.I. 95%: 0.23–0.27] too. This result is further supported by what is daily observed in our clinical experience diagnosing central precocious puberty: in this study case series, urinary concentrations of luteinizing hormone above 0.25 IU/L are associated with clinical, biochemical, and radiological findings compatible with effective activation of the hypothalamic-pituitary-ovarian axis.The study was able to document a statistically significant discriminating power of urinary LH regarding the pLH increment for patients who underwent stimulating tests with GnRH. The cut-off we identified, hypothetically able to independently predict the dynamic test response, is 0.45 IU/L (ROC test with an AUC of 0.99).Moreover, the reliability of the obtained best cut-off is supported by using a standardized test for sLH measurement: uLH assay is carried out by translating the serum LH test into the urinary matrix. As underlined by Bourguignon et al. , a possiTo confirm the importance of an adequate urine collection procedure, it should also be remembered that the circadian rhythm of sLH, and consequently of uLH, is particularly marked in prepubertal girls up to Tanner stage 3 .Although home urinary samples could underlie some limitations concerning the correct collection procedure, as reported by Xu et al. , it reprThis encouraging statistical result contributes to the proposed validation of uLH as a routine diagnostic procedure to distinguish between prepubertal and pubertal subjects, and it is associated with a relatively wide confidence interval [C.I. 95%: 0.20–0.70], which minimally affects the predictive sensitivity of the test under investigation. This analytical issue could be relieved by expanding the sample size case series or drawing a similar study on a homogeneous but independent population, possibly involving other qualified centres.In a clinical context, this statistical aspect translates into greater difficulty interpreting the few uLH values between 0.25 and 0.45 IU/L, needing integration with clinical and instrumental data. While values of uLH less than or equal to 0.25 IU/L indicate a prepubertal stadium, and values greater than 0.45 IU/L predict activation of the hypothalamic-pituitary-gonadal axis (pLH greater than 5 IU/L), values in the above range, taken individually, cannot be related immediately to a specific group of patients and must be appropriately contextualized.In the second group of patients, consisting of female subjects suffering from rapidly progressive precocious puberty, treated with GnRH analogues (triptorelin), the study shows that uLH can be used to monitor therapeutic efficacy. In this case, the average uLH values found in patients undergoing treatment were identical to those reported for the control group involving prepubertal patients.Moreover, this result appears to have a high statistical significance, especially compared to the average uLH values recorded in pubertal patients not treated with GnRH analogues. According to previous data published by Zhan, Zhung and Tripathy , 20, 21,One of the undoubtedly most promising data relates to the percentage achieved (100%) of prepubertal and pubertal patients on triptorelin therapy who present urinary LH values below the cut-point of 0.45 IU/L, as a result of actual functional inactivity of the endocrinological hypothalamic-pituitary-ovarian axis.In a broader perspective, urinary LH values could thus be considered valid biomarkers of clinical response to suppressive therapy with GnRH analogues.Potential study limitations could be some procedural problems inherent in the urine collection and storage method and the lack of a confirmatory study on a homogeneous subject sample.The choice of topic for this study was undoubtedly influenced by the particular interest aroused among those involved in pediatric endocrinology about the increased incidence of precocious puberty during the SARS-COV2 pandemic: the forced sedentary lifestyle imposed by lockdown periods has forced many children to abstain from physical activity, habitually consume harmful comfort food and spend many hours in front of bright screens, factors that seem to determine early pubertal onset –26.The limitations provided by the lockdown, including the restrictions on access to specialist public health clinics for non-urgent problems, allow the possibility of seeking alternative diagnostic strategies, such as urinary dosing of luteinizing hormone to diagnose and monitor central precocious puberty. According to the most up-to-date recommendations, the GnRH stimulation test remains the gold standard for identifying the hypothalamic-pituitary-gonadal axis activation; this test is, however, characterized not only by a certain degree of invasiveness and high costs but also by the apparent need to keep the patient in the hospital for several hours, an event that should be reduced to what is strictly necessary, especially during a pandemic period like this.The primary objective of this study is to validate a low-cost, minimally invasive method for precocious puberty diagnosing and monitoring that can be rapidly and easily performed, even outside hospitals, such as uLH assays on the first voided specimen of the morning urine (taking care to illustrate to the parent the correct way to collect the sample).Referring to urinary LH levels only, even in a territorial setting, a general pediatrician could select patients with suspected precocious puberty to send to an endocrinological third-level centre. After an appropriate validation by the most authoritative scientific societies of pediatric endocrinology, this approach could reasonably reduce hospital attendance and the costs of performing more invasive procedures with a more significant emotional impact on the pediatric patient.Regarding the need to validate the obtained results, an essential path to attribute a globally recognized diagnostic role to urinary LH, it is important to point out that despite the high statistical accuracy of the best cut-off obtained on this population, an expansion of the sample size is still necessary to reduce the range of interpretative uncertainty concerning values between 0.25 IU/L and 0.45 IU/L as much as possible.Although the high sensitivity and specificity of the cut-point of 0.25 IU/L to identify pubertal patients in a mixed population , as well as the high power of this assay in predicting serum pLH greater than 5 IU/L in the GnRH test for urinary LH values greater than 0.45 IU/L, it remains challenging to define pubertal patients who are in an intermediate condition between these two biochemical realities.Finally, about the possibility of using the urine-based luteinizing hormone test for the therapeutic monitoring of drug-treated precocious puberty patients, this study further highlights the usefulness of observing the hormone suppression brought by using GnRH analogues in a non-invasive way.In particular, in assessing therapeutic effectiveness, the role of urinary LH even seems to exceed its power as a therapeutic and diagnostic predictor. In pubertal patients examined at least six months after the start of triptorelin therapy, urinary hormone levels are not only significantly similar to those of prepubertal patients. However, they are also stably below the cut-off of 0.45 IU/L, indirectly suggesting complete suppression of the hypothalamic-pituitary-gonadal axis. The need to investigate this research topic through further studies, once its usefulness in clinical practice has been recognized, becomes even more urgent given the scarce availability of relevant studies in scientific literature.


Pagrus major). Twelve experimental diets were formulated. Control diet (CON) was designed to contain 60% FM. Other experimental diets were formulated by replacing 50% of FM from the CON with soy protein concentrate (SPC), corn gluten (CG), meat meal (MM), and/or chicken byproduct meal (CBM). Four diets were designed including one of SPC, CG, MM, or CBM as FM replacer and designated as SPC, CG, MM, and CBM. Six other diets were formulated by adding two ingredients as SPC and CG, SPC and MM, SPC and CBM, CG and MM, CG and CBM, or MM and CBM, and designated as SCG, SMM, SCM, CMM, CCM, and MCM, respectively. The 12th diet (MIX) was formulated by including SPC, CGM, MM, and CBM. Triplicate fish groups (50.2 ± 0.1 g) were hand-fed for 12 weeks. Weight gain (WG) of fish was significantly improved by MM and MCM diets compared to CG, SCG, CMM, and CCM diets. WG of CON, SPC, CM, SMM, SCM, and MIX groups were comparable with MM and MCM groups. The lowest WG was observed in CG and CMM groups. Feed efficiency (FE) was significantly higher in MM group compared to SPC, CG, SGC, and CMC groups. FE of MCM group was significantly higher than CG and SCG groups. Fillet linolenic acid (C18:2n–6) level in CG group was significantly higher than CON, MM, CM, SCM, CCM, and MCM groups. Serum lysozyme activity was significantly higher in MCM and MIX groups. Therefore, a high level of dietary CG reduces the growth performance and feed utilization of red seabream. A mixture of MM and CBM seems to be more efficient in replacing FM from red seabream diet.The experiment was conducted to evaluate alternative protein ingredients in a low-fish meal (FM) diet for red seabream ( Alternative protein ingredients have been evaluated in fish feed during the last few decades because of limited supply, high demand, and increasing price of fish meal (FM). Different types of alternative protein sources such as plant-based byproducts, terrestrial animal byproducts, insect meals and single-cell proteins are currently used in the aquafeed sector –4.Pagrus major) diets through a number of research works. SPC could be used to replace some extent of FM from red seabream diets [Soy protein concentrate (SPC) has been evaluated as an alternative protein source in red seabream (am diets . Thereafam diets –8. Howevam diets .Acanthopagrus schlegelii) diet and 60% of FM protein from gilthead seabream (Sparus aurata) diet [Corn gluten (CG) is also incorporated in red seabream diets when formulating low-FM diets , 10. Howta) diet , 14.Epinephelus coioides) diet, 80% of FM was successfully replaced with a mixture of MM and blood meal [Lates calcarifer). MM successfully replaced approximately 70% of FM from olive flounder  diet [Meat meal (MM) is produced using byproducts discarded from meat processing plants and slaughterhouses. It contains a high level of protein as it is produced without bones . Informaood meal . Williamood meal  evaluateus) diet . Nutrienus) diet .Centropristis striata) diet [Oncorhynchus mykiss) fed non-FM diets [Chicken byproduct meal (CBM) is also produced from processing waste such as necks, feet, and intestines. An early study reported that CBM could replace dietary FM up to 100% in yearlings and up to 70% in juvenile stage of red seabream without adverse effects . Howeverta) diet , enhanciFM diets , and maiFM diets .Red seabream is a carnivorous fish species cultured in the Eastern Asia region. Average red seabream production in South Korea was estimated as 5,400 tons per year . The lim−1) as shown in 3Twelve experimental diets were formulated to be isonitrogenous (45% crude protein) and isocaloric  and transferred to the fish and shellfish holding facilities at the Gangneung–Wonju National University, Marine Biology Center. Fish were acclimatized to experimental facilities and conditions for 2 weeks while feeding a commercial diet . After 2 weeks acclimation period, red seabream, averaging 50.2 ± 0.1 g, was stocked at a density of 15 fish per tank in 36 fiberglass tanks having a 300 L capacity. Tanks were in a flow-through system supplied with continuous seawater flow and aerated with sandstones. Each tank was randomly assigned to one of the three replicates of 12 dietary treatments. Fish were fed one of the experimental diets to apparent satiation  for 12 weeks. The uneaten feed was collected 30 min after feeding, dried in an oven at 105°C for 6 hr and reweighed to determine precise feed intake. The photoperiod was controlled to closely resemble the natural day length of the season. Water temperature (18.9 ± 0.3°C) was monitored every day and the other quality values including dissolved oxygen , pH (7.56 ± 0.3), and salinity (33.1 ± 0.4 ppt) were monitored throughout the feeding trial.g for 10 min and stored at −70°C. Blood samples were allowed to clot at room temperature for 30 min prior to separating serum samples. After blood sampling, fish were stored frozen at −20°C for proximate analyses. The remaining fish in each tank were killed, the total length of fish was measured to the nearest 0.1 mm and their viscera and livers were dissected and weighed to determine hepatosomatic (HSI) and viscerosomatic indices (VSI). The fillet of fish was also sampled for the analysis of proximate composition, amino acids, and fatty acid levels.At the end of the feeding trial, all remaining fish in each experimental tank were starved for 18 hr, counted, and bulk weighed for the calculation of survival rates, growth performance, and feed efficiency including weight gain (WG), daily feed intake (DFI), feed efficiency (FE), and protein efficiency ratio (PER). Six fish were randomly sampled from each tank and anesthetized with 2-phenoxyethanol (200 ppm). Blood samples were collected from three fish per tank using heparinized syringes to separate plasma for biochemical analyses and from the other three fish using nonheparinized syringes to separate serum samples for immune parameter analyses. Both plasma and serum were separated by centrifugation at 5,000 Micrococcus lysodeikticus  as a substrate [3), amylase (AMYL), and total cholesterol (TCHO) levels with dri-chem slides  purchased from FUJiFILM Co., Japan.Moisture and ash levels of the experimental ingredients, diets and fillet samples of fish were analyzed according to standard methods . Crude pubstrate . Serum sP < 0.05. Data were presented as mean ± standard error (SE). Percentage data were arcsine transformed before statistical analysis.Data were subjected to one-way analysis of variance. Then, Duncan's  multipleGrowth performance, feed utilization, and biometric parameters of red seabream are shown in Proximate composition of red seabream whole-body and fillet were presented in n−6) level of fish fed CG diet was significantly higher than CON, MM, CM, SCM, CCM, and MCM groups. CM group exhibited significantly lower linolenic acid compared to fish fed SPC, CG, SCG, and CMM diets. However, the deposition of other fatty acids was not significantly influenced by alternative ingredients.Fillet fatty acid profiles of red seabream are presented in Fillet essential amino acid levels fish fed experimental diets are presented in Biochemical parameter in red seabream fed experimental diets are presented in Growth performance of red seabream was significantly improved by MM and MCM feed although DFI of fish groups was not significantly different after 12 weeks of feeding trial. Therefore, it is obvious that efficiency of feed was improved by MM alone or together with CBM. Experimental diets were formulated to be isonitrogenous and isolipidic resulting in only slight variation in total amino acid and fatty acid compositions although total omega-3, omega-6, and HUFA levels were lower in all low-FM diets . TherefoMicropterus salmonids) fed animal or plant protein sources. They suggested that reduced feed intake due to low palatability of plant protein sources was the main reason for the retarded growth performance. In the present study, DFI was not significantly changed with diets indicating that feed palatability was not affected by plant protein sources during the trial period. Murashita et al. [MM used in the present study was prepared using meat byproducts without bones. The protein level was higher in MM (84%) as a result of its protein-rich raw material. MM also exhibited high-pepsin digestibility which was comparable to sardine FM and SPC . Rahman a et al.  found tha et al. . TherefoPsetta maxima), when they were fed a diet containing 20% CG [Morone chrysops × M. saxatilis) and juvenile Ussuri catfish (Pseudobagrus ussuriensis) when they were fed high level of CG [Plant protein sources were also reportedly effective in FM replacement in red seabream diet , 30. Espg 20% CG . Howeverel of CG , 43. Dieel of CG . Especiael of CG , 8, 29. Fillet proximate, fatty acid, and amino acid compositions were not significantly affected by single or combinations of protein sources in diets except for linoleic acid levels. Red seabream fed feeds containing plant protein sources exhibited high-linoleic acid levels compared to those fed diets containing only animal protein sources. Linoleic acid level in SPC and CG is considerably higher than the other protein sources . TherefoPlasma biochemical parameters of red seabream were not significantly affected by the experimental diets . AccordiSerum lysozyme activity was significantly increased in red seabream fed MCM and MIX diets although SOD activity was not significantly affected by diets. Lysozyme activity of red seabream is increased when their diet contains favorable nutrition levels. High-lysozyme activity is usually observed in fish groups having high-growth performance and feed utilization , 47, 50.In summary, the results of the present study clearly indicated that a high level of CG in low-FM diets reduces the growth performance and feed utilization of red seabream. SPC can be efficiently used as a protein ingredient when mixed with other alternative protein sources to replace FM. MM and CBM are suitable FM replacers in red seabream diets. Especially, a mixture of both MM and CBM seems to be more efficient than a single ingredient in diets. Therefore, we concluded that MCM and MIX diets are more efficient in replacing FM from red seabream diet compared to other diets tested in the present study. Future studies should be conducted to evaluate the effects of different levels of ingredient mixtures used in both MCM and MIX diets for red seabream.


We aimed to determine the effects and tolerability of repetitive transcranial magnetic stimulation (rTMS) on apathy in patients with neurodegenerative conditions, mild cognitive impairment (MCI), stroke, and traumatic brain injury (TBI) via systematic review.We conducted a systematic search in major electronic health databases, including PubMed, Scopus, and PsycINFO, covering the period from inception to June 2023. Comparative clinical trials and cohort studies, and studies with before-after designs were considered for inclusion. We used the Cochrane Risk of Bias and the National Institutes of Health (NIH) tools to assess methodological quality.Out of 258 records identified, 14 studies met our eligibility criteria  and 3 studies utilized before-and-after designs) with a total of 418 patients  included in the review. The overall methodological quality of the included studies was assessed to be fair to good. The stimulation parameters used varied considerably across the studies. The summary findings of our review indicate the following observations on the effects of rTMS on apathy: (1) the results of all included studies in Alzheimer’s disease investigating the effects of rTMS on apathy have consistently shown a positive impact on apathy; (2) the majority of studies conducted in Parkinson’s disease have not found statistically significant results; (3) a single study (RCT) on patients with primary progressive aphasia demonstrated significant beneficial effects of rTMS on apathy; (4) the trials conducted on individuals with MCI yielded varying conclusions; (5) one study (RCT) in chronic stroke suggested that rTMS might have the potential to improve apathy; (6) one study conducted on individuals with mild TBI did not find a significant favorable association on apathy; and (7) the use of different rTMS protocols on the populations described is generally safe.The feasibility of utilizing rTMS as a treatment for apathy has been suggested in this review. Overall, limited evidence suggests that rTMS intervention may have the potential to modify apathy among patients with AD, PPA, MCI and chronic stroke, but less so in PD and mild TBI. These findings require confirmation by larger, well-designed clinical trials. Apathy is manifested by a deficiency in goal-directed activities or motivation and blunted emotional responsiveness and spontaneity, which could manifest on a spectrum depending on the underlying disorder, severity, and apathy dimension involved  , 2. The n = 3,626) reported that cognitively impaired geriatric population with apathy are at 3.1-fold increased risk of one-year mortality compared to their counterparts without apathy  as a method that can induce excitatory or inhibitory changes in the underlying cerebral cortex in a nonintrusive manner and potentially induce long-term neuroplastic changes has received a great deal of attention , 15. In  effects .rTMS has known applications in the field of psychiatric disorders, particularly in the treatment of treatment-resistant depression , 18. RecOur rationale for including various neurocognitive disorders is to explore the feasibility of utilizing the rTMS intervention in a number of neurocognitive disorders. Since the mechanisms of action of different NIBS modalities for these disorders may be distinct, we concentrated our investigation on the effects of rTMS in order to give a more in-depth analysis of the studies that utilized this intervention while also providing a clearer scope of the review.2.2.1.We considered including randomized controlled trials (RCT) as well as prospective or retrospective cohort studies that incorporated a control group. Furthermore, we considered research that utilized a single-group, pre-post design. Investigations utilizing alternative/less rigorous research methodologies were eliminated from consideration. Human studies that used rTMS as an intervention (at least 5 consecutive sessions administered) for the improvement of apathy symptom and compared its effects to those of sham therapy, medication-only, no-treatment control, or to baseline measures were taken into consideration for inclusion. We covered populations with neurodegenerative disorders, cognitive impairment, stroke, or traumatic brain injuries in this review, regardless of age, sex, social construct of race/ethnicity, comorbidities or medications taken. We excluded studies that relied on secondary data or lacked complete accessibility to full-text reports. We also removed replicated reports, abstract or poster-only publications, outcomes lacking a corresponding description of the background and methods, animal studies, and reviews.2.2.The present review centered on the impact of rTMS on apathy within the aforementioned populations. We considered apathy as a condition presenting as a diminished or absent motivation that cannot be attributed to a decline in consciousness, cognitive impairment, or emotional distress, caused by observable alterations in affect, behavior, and cognition . To quan2.3.The databases of MEDLINE by PubMed, Scopus and PsycINFO were searched to include studies from inception until April 2023 for potentially relevant records. Given our objective to conduct a comprehensive review to identify relevant studies that may be associated with the concept of apathy, we incorporated search terms that pertain to various dysexecutive syndromes, including anhedonia, abulia, akinetic mutism, among others. The applied search terms were as follows: ((neurodegenerative OR neurodegeneration OR “neurologic degenerative”) OR  OR ) AND ((“non-invasive brain stimulation” OR “noninvasive brain stimulation” OR “NIBS”) OR ) AND (apathy OR anhedonia OR abulia OR “akinetic mutism” OR hypoactivation OR “psychomotor retardation” OR “executive dysfunction” OR “executive disorder”).The process of selecting studies was carried out utilizing the Covidence platform. Two reviewers (AIE and TH) conducted an independent assessment of the titles and abstracts of all records retrieved, utilizing the screening criteria. The full texts of all pertinent trials that met the screening criteria were obtained and evaluated by 2 reviewers (AIE and TH) based on the eligibility criteria. In the event of inconsistencies in meeting the screening and eligibility criteria, a third reviewer (MB) was consulted for further discussion. All studies that fulfilled the eligibility criteria were included in this review.2.4.The risk of bias in the included studies was assessed by 2 reviewers (AIE and TH), with a third reviewer (MB) serving as an arbitrator in the event of any discrepancies. The Risk of Bias (RoB) Tool 1 developed by Cochrane was employed for evaluating randomized controlled trials (RCTs)  while thTwo reviewers (AIE and JKT) independently conducted the data extraction process. The obtained data encompassed various aspects such as the study design, patients’ characteristics, trial settings, treatment and comparator regimen features, and pertinent outcomes determined by various apathy scales and detecting any adverse events. We gathered data on the incidence of variables of interest among participants, particularly the number of patients who were positive on a dichotomous variable and the total number of individuals in each group. We also collected relevant available data on important continuous variables, including mean/median pretreatment/posttreatment values and mean differences, and measures of dispersion such as standard deviations (SD)/standard error (SE)/95% confidence intervals (CI), and the number of patients in each treatment group. The presentations and analyzes of all data were done qualitatively.3.3.1.clinicaltrials.gov website which was unpublished; the narrative results were found from the funding agency website  and methodological designs in the included studies.n = 143) (n = 187), 3 studies involving patients with MCI (n = 56) (n = 13) (n = 15) (n = 20) (n = 143) , 46, 47 n = 143) , 39, 44 (n = 56) , 37, 40;(n = 15) , and pri(n = 20) . The maj(n = 20) , 37, and(n = 20) ) and Nor(n = 20) , 45, Uni(n = 20) , 39–41),(n = 20) , 38 and 3.2.Nearly all of the included studies employed high-frequency (HF) rTMS as an intervention , 46, 47,3.3.There was a considerable degree of variation in the outcome measures that were used to evaluate apathy in the included studies see . Seven s3.4.A considerable proportion of the studies exhibited an unclear risk with regards to selection bias, primarily stemming from inadequate disclosure of the randomization process and allocation concealment , 39–44. 3.5.3.5.1.n = 20; trial registration: NCT02190084), the effects of HF rTMS (n = 9) at left DLPFC administered for 20 sessions over 4 weeks was compared to sham stimulation (n = 11) in terms of the AES-clinician version (AES-C) among patients with AD diagnosed using the Diagnostic and Statistical Mental Disorders-Text Revision (DSM-IV-TR) criteria. Pertinent baseline clinical variables including age, sex, education profile, concomitant medications and comorbidities, as well as pretreatment baseline measures including AES-C were similar in both groups. Following a period of 4 weeks of post-intervention monitoring, a significant difference was observed between rTMS  and sham groups [−0.8 (−4.8 to 3.2)] with regard to the AES-C score change  using analysis of covariance (ANCOVA). After 4 weeks, within-group analyzes indicated a statistically significant improvement in AES-C following rTMS treatment, with an average reduction of 11.0 points from the initial measurement (p < 0.001) while within-group sham treatment did not lead to a significant change in AES-C (p = 0.662). However, after 8 weeks and 12 weeks, the effects were not statistically durable in the within-group rTMS analyzes using repeated measures mixed model [−3.5 (−9.6 to 2.6)] and .In a randomized, participant-, rTMS personnel-, and outcome assessor-blinded study by Padala et al.   and at 12 weeks (p = 0.002); however, no significantly different results were found between rTMS + tDCS regimen and either rTMS-only or tDCS-only (both p > 0.017). Significant improvements in NPI-AS compared to baseline were observed within the rTMS+tDCS group after 4 (p < 0.001) and after 12 weeks (p = 0.001).Hu et al.  conducten = 30) without a control group to test the impact of NeuroAD procedure (HF rTMS + cognitive training) on AI scores in patients with dementia due to probable AD followed up to 4 years (β coefficient (95% CI), −8.7 (−11.1 to −6.3)], 3 months [−7.2 (−9.7 to −4.7)], 12 months [−6.8 (−9.5 to −4.1)], at a mean of 28 months [−5.5 (−8.5 to −2.5)] .Moreno et al. performed a retrospective, pre−/posttest designed study  followed over 6 months in a prospective study with a single-group, pre−/posttest design to test its effect on AI scores (p = 0.0125).Nguyen et al. administered the NeuroAD regimen in AD patients (I scores . The tre3.5.2.n = 61) followed over 6 months (n = 20) (2); M1 + sham (n = 14) (3); left DLPFC + sham (n = 12) (4); double sham (n = 15); the treatment course consists of 5 sessions per week over 2 weeks. Baseline characteristics such as age, sex, disease duration, Unified Parkinson’s Disease Rating Scale (UPDRS) scores, among others, appear to be similar across the groups. Using repeated-measures ANOVA, the results showed no significant difference among the treatment groups in AES scores at 1 week, 1 month, 3 months and 6 months after treatment compared to baseline . The authors also concluded that there was no evidence of a synergistic effect of stimulating both the M1 and DLPFC .Brys et al. conducted a randomized, participant-, study personnel-, and outcome assessor-blinded, controlled trial with an aim to determine the effects of HF rTMS on AES compared to sham on patients with PD (6 months . The rTMn = 16) vs. sham (n = 8) on AES and LARS scores among PD patients (n = 24) with a follow-up period of 10 days via implementing a randomized, participant- and outcome assessor-blinded, controlled trial (In NCT00955032 (“ReStore Study”), the investigators tested the impact of HF rTMS  performed a randomized, participant- and outcome assessor-blinded, controlled trial comparing LF rTMS (n = 34), HF rTMS (n = 34) and sham (n = 34) arms on their effects on improving the NMSQ-AS scores in the PD population followed over 20 weeks (p = 0.55) and at week 20  did not show statistically significant differences.Shirota et al.  using the Wilcoxon test. The findings of the study also suggest that there was a significant difference in the posttreatment SAS scores between the apathetic group and the non-apathetic group (p < 0.0001); however, this difference can be attributed to the substantial difference in their baseline SAS scores as well (p < 0.0001).Wei et al. conducted a randomized, participant-blinded, controlled trial with an aim to test HF rTMS’ impact on SAS scores in PD patients with a follow-up period of 2 weeks . The stu3.5.3.n = 27) undertook a randomized, participant- and outcome-assessor-blinded, cross-over trial study with an objective to determine the effect of active-site HF rTMS (n = 20) vs. control-site (n = 7) stimulation on NPI-AS scores in patients with PPA followed over 16 weeks  . The control-site stimulation was administered over the vertex, which was described elsewhere (p = 0.03) analyzed using the Mann–Whitney U test.Pytel et al. (3580954) , 55. Baslsewhere . The sti3.5.4.n = 21) and Cirillo et al. (n = 20) conducted controlled trials on the impact of HF rTMS on AES compared to sham among the MCI population (reported to be diagnosed using the NIA-AA workgroup guidelines on MCI diagnosis due to AD), with randomization, and blinding of participants and outcome assessors , n = 12] and healthy controls  at 4 weeks of intervention (adjusted p = 0.213). A similar conclusion was also observed in Cirillo 2023, where the rTMS  and sham  groups did not vary substantially in terms of AES scores at 4 weeks (adjusted p = 0.983). Furthermore, Cirillo et al. (p = 0.586). All analyzes were conducted using ANCOVA utilizing pretreatment score as a covariate.Both Esposito et al.  performed a randomized, participant-, study personnel/rTMS technician-, and outcome assessor-blinded, cross-over trial examining the effect of left DLPFC HF-rTMS compared to sham on AES-C among the MCI cohort diagnosed using the Petersen’s criteria   , 57. The3.5.5.p = 0.02) in chronic stroke patients analyzed using the Mann–Whitney U test. The within-group rTMS arm demonstrated a significant improvement from pre- to posttreatment AS scores , whereas the sham stimulation group nonsignificant pre−/posttreatment changes in the AS score  evaluated using the Wilcoxon signed-rank test.Sasaki et al. undertook a randomized, participant- and outcome assessor-blinded controlled study to examine the effects of HF rTMS on AS scores compared to sham among chronic stroke patients who were followed over 5 days. The treatment regimen consisted of 5 sessions administered consecutively over a span of 5 days. The targeted area for stimulation was the upper-middle region of the forehead, extending from the external auditory meatus to a point 30° above the orbitomeatal line  to medial prefrontal cortex (mPFC)). Age, sex, stroke subtype, and laterality of the brain lesion were all comparable between the rTMS and sham groups. The number of years between stroke onset and initiation of treatment was also comparable in both the rTMS group  and the sham group (5.3 ± 5.7 years). The findings of the study indicate that there was a statistically significant difference in the extent of change observed in the AS score between the group that received rTMS (47.5% ± 31.9) and the group that received sham stimulation  conducted a pilot study with pre-post design (no control group) with an aim to examine the effect of LF rTMS on right DLPFC to improve AES scores in patients with mild TBI followed over 3 weeks (p = 0.334) after 3 weeks.Meek et al. , MCI, chronic stroke, and traumatic brain injury. In summary, our review reveals the following insights (1): all included AD trials indicate beneficial signal on apathy for those exposed to rTMS (2); the prevailing body of studies on PD has yielded non-significant results (3); a single study (RCT) involving patients with PPA demonstrated significant positive effects (4); the MCI trials yielded varying conclusions (5); one study (RCT) on chronic stroke found rTMS may improve apathy (6); one study on mild TBI found no significantly favorable association; and (7) the implementation of various rTMS protocols on the populations described is generally safe.The findings from the 2 included randomized, sham-controlled trials suggested that rTMS may be beneficial in terms of improving apathy among AD , 41. TheOn the other hand, inconsistencies were observed in the conclusions of the included studies regarding the MCI population. Results from the two published articles that describe an overlapping MCI cohort , 37, an In the PD population, 4 randomized, sham-controlled trials were identified, which investigated the effects of rTMS on apathy , 39, 44.In the RCT conducted by Pytel et al. on individuals diagnosed with PPA, the results indicated that theimplementation of active-site rTMS intervention yielded positive outcomes in terms of ameliorating apathy . In addiBiological marker-based diagnosis for AD and MCI as reported by Jack et al. and Dubois et al. plays a crucial role in giving context to the observed effects of rTMS in the included AD and MCI studies . The incIn terms of the MCI diagnosis, it is also important to take into account how the studies diagnosed patients with MCI, as this condition represents a heterogeneous group that includes amnestic MCI  and non-amnestic MCI  . In PadaOverall, the inclusion criteria used in the diagnosis of AD dementia and MCI in the included studies did not mention the use of relevant AD biomarkers, which may diminish the internal validity of the results. It is crucial to acknowledge that distinct neurodegenerative disorders may have variable responses to rTMS due to heterogeneity in their pathological underpinnings.The majority of the patients enrolled in the included AD studies had late-onset AD (LOAD) i.e., ≥ 65 years). We theorize that the age of presentations in AD and MCI may also affect response to various rTMS parameters. Individuals with sporadic early-onset AD (EOAD) exhibit a more accelerated clinical deterioration and experience more pronounced deficits in attention, language, visuospatial abilities, and executive functions  years. W. Furthern = 9) open-label, before-and-after study on FTD in which the majority of participants had bvFTD and were treated with 10 sessions of HF rTMS over bilateral DLPFC showed improvement in the Montreal Cognitive assessment, letter and digit cancelation tests, Stroop reading time and error number, and Frontal Behavioral Inventory caregiver’s impression of daily functioning , which encompasses several clinical presentations, including the behavioral variant FTD (bvFTD). bvFTD manifests with a remarkably high incidence of apathy, ranging from 62 to 89% , 74. In ctioning . HoweverOne notable and prevalent challenge in clinical practice is distinguishing apathy and depression . Apathy The rating tools used to measure apathy in the included studies varied significantly which limits comparison of results across the studies. Various apathy scales are utilized in clinical and research environments, each exhibiting unique psychometric characteristics. In a previous systematic review of measurement properties of apathy tools that involved 57 studies, AES and LARS were recommended for measuring apathy in older adults and people living with dementia due to sufficient content validity, reliability, construct validity, structural validity and internal consistency . In PD pThe presence of apathy has been associated with diverse dysfunctions in brain circuitry that vary depending on the specific condition. Apathy is typically distinguished by a decrease in activity primarily within prefrontal network . Aside fThe precise mechanism by which rTMS affects apathy remains unclear. We speculate that the underlying mechanism by which rTMS influences apathy may involve alterations in the overall connectivity of the brain, which in turn may lead to a beneficial alleviation of apathy symptoms. Previous studies showed that the utilization of rTMS targeting the DLPFC had been observed to modulate the release of endogenous dopamine in various brain regions, including the mesostriatal and mesolimbic areas, as well as the anterior cingulate cortex and orbitofrontal cortex . AdditioThe careful consideration of the site of stimulation for rTMS holds significant importance. A significant number of the included studies opted to utilize HF rTMS specifically targeting the left DLPFC. This choice is supported by a substantial body of literature that has established a robust level of evidence for the efficacy of this approach, notably in the treatment of depression . It is tThere are several methodological concerns present in the included studies that warrant recognition, as they pose a potential threat to the internal validity of their findings. Nearly all of the clinical trials included indicated that randomization was implemented; however, they overlooked to specify the precise method of randomization, including the allocation concealment technique, employed in the trials. Fortunately, the majority of studies included exhibited a similarity of the baseline characteristics among the treatment arms which limits potential confounding variable effects ; however, as mentioned earlier, the included studies did not take into consideration the possible confounding effects of change in depression levels on the link between rTMS and apathy. In addition, several trials implemented blinding techniques for participants, yet they often overlook the masking of study personnel/rTMS technicians, thereby elevating the risk of performance bias. We recognize the difficulty in implementing the blinding of rTMS technicians as these would require the utilization of a blinded sham-coil that should also be visually and auditory similar to the blinded active-coil. Nevertheless, it deserves mentioning that due to the absence of anticipation in the control group, unblinding of participants/personnel can result in differential behaviors in the treatment arms  . In triaOverall, the comparability across studies in the included studies and the aggregation of relevant effect estimates are hindered by the variability in population characteristics, differences in the rTMS protocols and control groups, diverse outcome scales used to assess apathy and measures of treatment effect, and methodological disparities. The presence of these substantial variabilities hinders our capacity to provide precise recommendations regarding the most effective and optimized rTMS parameters to address apathy for each population discussed in this review. Therefore, for future studies, it is imperative to establish a suitable correlation between the underlying pathophysiology of apathy, the site of stimulation, and the patterns of stimulation.Our review proposes the potential feasibility of rTMS as a therapeutic intervention for apathy despite the recognized limitations. Current limited evidence indicates that the use of rTMS holds promise in modulating the apathy signal in individuals with AD, PPA, MCI, and chronic stroke, but possibly less so in PD and mild TBI. Considering the acknowledged limitations of the included studies, it is essential for future research endeavors to prioritize the conduct of clinical trials with more robust designs aimed at confirming or refuting the preliminary findings presented in this review.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.AE: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. TH: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. JT: Data curation, Formal analysis, Investigation, Project administration, Software, Validation, Writing – original draft, Writing – review & editing. MB: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing. AB: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.


This study was to compare the clinical presentations and survivals between the non-small cell lung cancer (NSCLC) patients with occult lymph node metastasis (OLNM) and those with evident lymph node metastasis (ELNM). We also intended to analyze the predictive factors for OLNM.Kaplan–Meier method with log-rank test was used to compare survivals between groups. Propensity score matching (PSM) was used to reduce bias. The least absolute shrinkage and selection operator (LASSO)-penalized Cox multivariable analysis was used to identify the prognostic factors. Random forest was used to determine the predictive factors for OLNM.A total of 2,067 eligible cases  were included. The rate of OLNM was 21.4%. Patients with OLNM were tend to be female, non-smoker, adenocarcinoma and had smaller-sized tumors when compared with the patients with ELNM. Survival curves showed that the survivals of the patients with OLNM were similar to those of the patients with ELNM both before and after PSM. Multivariable Cox analysis suggested that positive lymph nodes (PLN) was the only prognostic factor for the patients with OLNM. Random forest showed that clinical tumor size was an important predictive factor for OLNM.OLNM was not rare. OLNM was not a favorable sign for resected NSCLC patients with lymph node metastasis. PLN determined the survivals of the patients with OLNM. Clinical tumor size was a strong predictive factor for OLNM.The online version contains supplementary material available at 10.1186/s12885-023-11189-3. Non-small cell lung cancer (NSCLC) is a leading contributor to cancer-related mortality worldwide –3. Over To date, it is evident that the survivals of the patients with OLNM are inferior to those of the patients without lymph node metastasis , 13, 15.Against this background, this study focused on the resected NSCLC patients with OLNM from a large Chinese cohort. The main object was to systematically characterize and evaluate the survival outcomes of this category of patients. The second object was to explore the predictive factor for OLNM. We anticipated that our study might provide a more comprehensive understanding of this population.Between 1999 and 2018, a series of 7,931 consecutive resected patients were evaluated from our center. The well-managed dataset used in this study was reported before [The eligible patients were categorized into five groups: N0 group, occult N1 group, evident N1 group, occult N2 group and evident N2 group according to the results of clinical and pathological results of nodal status.This study was approved by The Ethics Committee of Peking University People’s Hospital (the approved number: 2020 PHB 421–02). The Ethics Committee of Peking University People’s Hospital waived off the informed consent due to the retrospective nature.In routine, chest and abdomen computed tomography (CT) and brain magnetic resonance imaging (MRI) were performed to determine the clinical tumor-node-metastasis (TNM) stage. Positron emission tomography (PET) imaging was not mandatory in our center because it is expensive and has not been covered by medical insurance in mainland China. In general, lymph node with short axis diameter >  = 1 cm in the CT scan or with maximal standardized uptake value >  = 2.5 in the PET was consider malignant. Once the patients were suspicious of clinical N2 category, invasive nodal evaluation modalities such as endobronchial ultrasonography transbronchial needle aspiration or mediastinoscopy were recommended but not mandatory. After surgery, the formalin-fixed paraffin-embedded tissue sections of harvested lymph nodes with hematoxylin and eosin staining were reviewed by one junior and one senior pathologist of the Department of Pathology in the Peking University People’s Hospital.The surgical approach and surgical extent were discussed and decided at a multidisciplinary team meeting. All included patients underwent lobectomy or pneumonectomy and systemic lymph nodes dissection. Systemic lymph nodes dissection was defined as mediastinal lymph node dissection of at least three stations, and station 7  must be dissected. Regarding N1 station lymph nodes, the station 10, 11 and 12 were dissected intraoperatively, and the station 13 and 14 were dissected by pathologists from the excised specimen, but this procedure was not mandatory. In addition, at least 6 lymph nodes were harvested. Adjuvant therapies were performed according to the National Comprehensive Cancer Network (NCCN) guidelines , usuallyFollow-up information was mainly obtained through telephone calls and hospital visits. In general, postoperative follow-up was performed every three months for the first two years, every six months for the next three to five years and annually thereafter . Physicath edition of the TNM staging manual was used in this study [The clinicopathologic information and treatment data were extracted from the electronic medical record system, which included age, sex, smoking status, family tumor history, body mass index (BMI), comorbidity, staging methods, tumor location, clinical tumor size, clinical tumor (T) category, clinical nodal (N) category, forced expiratory volume in 1 second (FEV1%), diffusion capacity for carbon monoxide (DLCO%), the American Society of Anesthesiologists (ASA) physical status grade, surgical approach, surgical extent, histology, visceral pleural invasion (VPI), lymphovascular invasion (LVI), examined lymph nodes (ELN), positive lymph nodes (PLN), pathologic tumor size, pathologic T category, pathologic N category, pathologic tumor-node-metastasis (TNM) stage, postoperative complications, adjuvant therapy and hospital stay. The 8is study . Complethttp://www.r-project.org) and the IBM SPSS Statistics . The Shapiro–Wilk test was used to analyze the normal distribution of the continuous variables, and non-normally distributed continuous variables were presented as median (range). The Mann–Whitney U test was used to compare the non-normally distributed variables. Categorical variables were presented as percentages and were compared using Pearson Chi-square test or Fisher’s exact test. OS and DFS were analyzed by the Kaplan–Meier method with log–rank test. A one to one propensity score matching (PSM) was used to reduce the bias caused by the baseline confounders in the occult N1 & evident N1 pair and the occult N2 & evident N2 pair using the R package “MatchIt” [P < 0.05 was considered statistically significant.All statistical analyses were conducted via the R version 4.1.1 , non-smokers  and smaller-sized tumors (P = 0.022) in the occult N1 group when compared with those in the evident N1 group. In addition, more patients in the evident N1 group underwent PET  and open surgery . Regarding the patients with N2 category, there were more young patients , females  and non-smokers  in the occult N2 group when compared with those in the evident N2 group. More patients were suffered from preoperative comorbidities in the evident N2 group . The clinical tumor sizes of the patients with occult N2 metastasis were smaller than those of the patients with evident N2 metastasis . Adenocarcinoma (ADC) occurred in a sizable fraction of patients with occult N2 metastasis . Patients in the evident N2 group had more tumors with advanced T categories (P = 0.031). After PSM, the variables in the occult N1 & evident N1 pair and the occult N2 & evident N2 pair were all balanced well , smoking (P = 0.001), DLCO% (P < 0.001), surgical approach (P = 0.012), histology (P = 0.034), pathologic T category (P < 0.001), pathologic N category (P < 0.001) and PLN (P < 0.001) were independent prognostic factors for OS (Table SP < 0.001), smoking (P = 0.001), DLCO% (P < 0.001), pathologic T category (P < 0.001), pathologic N category (P < 0.001) and PLN (P < 0.001) were proven as the independent prognostic factors for DFS  and PLN (P < 0.001) were independent prognostic factors for OS, and PLN (P < 0.001) was an independent prognostic factor for DFS  was the strongest predictor, followed by surgical extent (P < 0.01), age (P < 0.05) and evaluation of nodus status (P < 0.05)  Fig. .Fig. 4RaOur comprehensive analysis of the patient with OLNM demonstrated that the rate of OLNM was not rare (21.4%). Patients with OLNM were tend to be female, non-smoker, ADC and had smaller-sized tumors when compared with the patients with ELNM. Survival curves showed that irrespective of whether it was before or after PSM analyses, the survivals of the patients with OLNM were similar to those of the patients with ELNM. LASSO-penalized multivariable Cox analysis suggested that pathologic N category (N0 vs. occult N1 vs. evident N1 vs. occult N2 vs. evident N2) was a prognostic factor for both OS and DFS in the entire cohort. Only PLN was proven as the prognostic factor for the patients with OLNM. At last, random forest showed that clinical tumor size, surgical extent, age and evaluation of nodal status were the predictive factors for OLNM. Our study provided comprehensive knowledge of NSCLC patients with OLNM and possess a certain clinical reference value.In forerunning clinical series, Gwozdz et al.  and BeyaPrevious studies supported the notion that the survivals of the patients with OLNM were superior to those of the patients with ELNM because the former group usually had lower tumor burden , 16, 26.It is curious to observe that PLN was the only prognostic factor for the patients with OLNM. To date, although tumor burden has not been adopted in the current TNM staging system , ample eThe results of random forest suggested that clinical tumor size was the strongest predictive factor for OLNM, which was confirmed by the previous studies , 9. In tOur study had some limitations. First, only a small portion of patients underwent PET or invasive staging tools (23.1%). Therefore, high false-negative rate of clinical N category might occur in our cohort. In developing countries, preoperative staging by PET or invasive tools is still not common. We hoped patients’ data with definite N category could be applied to validate our conclusion. We optimistically foresaw that with the development of economy and medical technologies, the number of patients with OLNM would de decreased. Second, our study included patients from a time period from 1999 to 2018, which is long time span. There have been huge strides in the development of preoperative evaluation tools and treatment modalities. Therefore, patients in the late period might receive more standard and powerful evaluations and therapies, which could contribute to bias. Third, the variables included in the random forest model were not enough. Further efforts on more detailed information collection such as imaging features and tumor markers are encouraged to improve the performance of the predictive model. Third, although the number of cases included in this study was larger than those of most previous studies, only a small number of patients could be gathered for some subgroups. Therefore, multicenter studies with high quality databases could validate our results. At last, this is a retrospective single-center study, although PSM were performed, the inherent bias was inevitable.However, notable strengths of this study included that the rigor and efficient statistical methodologies  used in the study made our results more reliable. The sample size was large, and therefore, our results had certain clinical reference value. In addition, the included patients were treated by a standardized surgical protocol at single institution which could reduce bias. At last, the dataset that we used is well-managed, which includes well-annotated clinicopathologic data and complete follow-up information.OLNM was not rare. OLNM was not a favorable sign for NSCLC patients with lymph node metastasis. PLN determined the survivals of the patients with OLNM. Clinical tumor size was a strong predictive factor for OLNM.Additional file 1: Table S1. The covariates distribution in the occult N1 & evident N1 pair and the occult N2 & evident N2 pair after PSM. Table S2. LASSO-penalized multivariable Cox analysis of the entire cohort.Additional file 2: Figure S1. Prognostic factors selection for OS (A and B) and DFS (C and D) of the entire cohort using the LASSO regression model. LASSO coefficient profiles of 21 included factors against the log (Lambda) sequence for OS (A) and DFS (C). Tuning parameter (Lambda) selection in the LASSO model used 10-fold cross-validation via minimum criteria . LASSO, least absolute shrinkage and selection operator; OS, overall survival; DFS, disease-free survival.