By applying orthopedic criteria to analyze the amplitude of the curvature at the apex of the kyphosis, the curve pattern, the sagittal balance and sex bias, we demonstrate that pkd2l1 knock-outs develop a phenotype reminiscent of Scheuermann’s disease. Altogether our work consolidates the benefit of combining genetics and analysis of spine deformities in zebrafish to model idiopathic spine disorders in humans.Scheuermann’s disease, also referred to as Scheuermann’s kyphosis, is the second most frequent spine deformity occurring in humans after adolescent idiopathic scoliosis (AIS), both with an unclear etiology. Recent genetic studies in zebrafish unraveled new mechanisms linked to AIS, highlighting the role of the Reissner fiber, an acellular polymer bathing in the cerebrospinal fluid (CSF) in close proximity with ciliated cells and mechanosensory neurons lining the central canal of the spinal cord (CSF-cNs). However, while the Reissner fiber and ciliary beating have been linked to AIS-like phenotypes in zebrafish, the relevance of the sensory functions of CSF-cNs for human spine disorders remains unknown. Here, we show that the thoracic hyper-kyphosis of the spine previously reported in adult  In humans, spine disorders are characterized by defects of the vertebral alignment that can be classified according to their cause. While congenital deformities of the spine are observed at birth and involve malformations of the vertebral bodies contributing to a regionalized curvature of the spine3, syndromic deformities are acquired during life and are associated to diverse pathological states6. In contrast, idiopathic deformities of the spine are defined by atypical curves without underlying malformations that arise spontaneously from an unknown origin7.The spine is a segmented structure consisting of vertebrae intercalated with intervertebral discs that shares the same architecture and developmental origin in amniotes and bony fish9. It is a one-dimensional deformity occurring in the sagittal plane, associated with no vertebral malformations and consisting of a pronounced hyper-kyphosis of the thoracic spine that arises during the growth period in otherwise healthy patients. Its etiology remains unclear but growth irregularities and multiple genetic factors are reported to be involved in this disease10. AIS is the most frequent form of idiopathic spine disorders affecting almost 4% of the population7 and is defined by a three-dimensional misalignment of the vertebrae occurring during the period of growth.The two most frequent idiopathic deformities of the spine occurring in humans are adolescent idiopathic scoliosis (AIS) and Scheuermann’s disease. Scheuermann’s disease affects 0.4–10% of the population13. The CSF contains the Reissner fiber, an acellular thread conserved in many vertebrates that runs from the roof of the third ventricle down to the caudal end of the central canal and that is formed by the cilia-dependent aggregation of the SCO-spondin glycoprotein15. The reduction of cilia motility in c21orf59 mutants leads in juvenile zebrafish to three-dimensional spine curves recapitulating the features of AIS16. Remarkably, zebrafish mutants devoid of the Reissner fiber develop similar spine curves characterized by an increased misalignment of the vertebrae both in the sagittal and coronal planes19. This body of work revealed that defects in the assembly of the Reissner fiber is a key mechanism driving spine deformity across different genetic models of AIS.While the pathogenesis of AIS is still a subject of debate in humans, zebrafish allowed for substantial advances in the identification of novel underlying mechanisms. They involve the cerebrospinal fluid (CSF), a protein-rich solution filling the cavities of the nervous system whose constant circulation is generated by the coordinated beating of motile cilia projecting from the walls of the brain and spinal cord cavities20. To detect spine curvature, CSF-cNs require the transient receptor potential channel Trpp2 (formerly Trpp3) or Pkd2l121 enriched in their apical extension22 and the Reissner fiber23. pkd2l1 zebrafish mutants in which the mechanosensory function of CSF-cNs is abolished in the larva21 develop at the adult stage an increased curvature of the spine that occurs in the sagittal plane and increases in severity between 12 and 19 months of age22. Thus, Pkd2l1 is required both for the mechanosensory function of CSF-cNs and in the maintenance of spine alignment throughout life.In the spinal cord, the Reissner fiber is confined in the central canal where it forms a sensory system with CSF-contacting neurons (CSF-cNs), a population of interoceptive neurons projecting their apical extension into the CSFpkd2l1 zebrafish mutants is not associated with vertebral malformations but is idiopathic and restrained to the sagittal plane. By applying orthopedic criteria to analyze spine misalignments in pkd2l1 mutants, including the amplitude and location of hyper-kyphosis in the thoracic spine, curve pattern, sagittal balance, and sex bias, we further confirm that the loss of the mechanosensory function of CSF-cNs in pkd2l1 mutants is associated with a deformity reminiscent of Scheuermann’s disease.This body of work highlights the influence of the cellular niche formed around the Reissner fiber, together with ciliated cells driving CSF flow and CSF-cNs, on spine alignment. However, while the Reissner fiber and ciliary beating have been directly linked to AIS-like phenotypes in zebrafish, the relevance of the mechanosensory function of CSF-cNs for spine deformities in humans remains unknown. Here, we show that the pronounced curvature of the spine reported in 1. pkd2l1icm02/icm02 mutants develop an accentuated curvature of the thoracic spine22. To clarify whether hyper-kyphosis in pkd2l1 mutants is idiopathic, we combined micro-computed tomography and Alizarin-S staining on skeletal preparations of 18 months old adult zebrafish  exhibited no deviation in the coronal plane similarly to their wild-type siblings, confirming that they do not develop three-dimensional misalignments of the spine.The morphoanatomy of the zebrafish spine, as in humans, is characterized by a natural longitudinal curvature at the thoracic level, referred to as kyphosis26. Thus, we investigated whether the phenotype of adult pkd2l1icm02/icm02 mutants could arise from vertebral malformations. In human, complex syndromic hyper-kyphosis, such as in Klippel-Feil syndrome, is associated with a reduced cervical length27. Given that the length of the cervical segment can vary with the body size of the animals, we used the cervical ratio defined as the length of the cranio-cervical segment normalized by the total axial length . Moreover, the number of thoracic vertebrae ranged between 8.1 ± 0.1 and 8.6 ± 0.2 in wild-type and pkd2l1icm02/icm02 mutants, respectively . This is in accordance with the axial formula previously reported for rib-bearing vertebrae observed in the adult zebrafish spine28 and suggests that the phenotype of pkd2l1icm02/icm02 mutants does not correlate with spine patterning defects.Several zebrafish mutant models of congenital syndromes recapitulate pronounced curvatures of the thoracic spinepkd2l1icm02/icm02 mutants using Alizarin-S staining. Similar intensities of Alizarin-S were observed in wild-type and mutant siblings  even at the site of curvature . This was accompanied by a slight but non-significant cranialization of the kyphotic curve in pkd2l1icm02/icm02 mutants . Interestingly, heterozygous pkd2l1icm02/+ animals showed an intermediate phenotype characterized by a 1.45- and 1.95- fold increase of the main and greater kyphosis angles, respectively, compared to wild-type siblings . Overall, pkd2l1 zebrafish mutants exhibited a typical 1.5- to 2.3- fold increase of the thoracic Cobb angle, similar to the ratios reported in patients with Scheuermann’s disease37.In humans, Scheuermann’s disease is identified by the accentuation of the natural kyphosis of the thoracic spine and is characterized by a T1-T12 Cobb angle ranging between 50° and 70° and higher for severe casespkd2l1 zebrafish mutants phenocopy the sagittal imbalance reported in patients with Scheuermann’s disease37. The orthopedic evaluation of curve patterns in patients relies on sagittal parameters taking into account measures in reference to the pelvis such as the pelvic incidence, the sacral slope and the pelvic tilt39. The absence of limbs and pelvis in zebrafish precludes the assessment of sagittal alignment according to the orthopedic standards in humans. Thus, we evaluated the degree of deviation of the kyphotic curve relative to the entire spine by quantifying the curve pattern index, defined as the ratio between the kyphosis height and the cranio-spinal length40 . We also evaluated the distribution of the kyphotic curve along the spine by measuring the Harrington index, commonly used to follow curve progression in patients42. We observed a significant 2.2- fold and 1.8- fold increase of the median Harrington index in pkd2l1icm02/icm02 and pkd2l1icm02/+ animals compared to wild-type siblings, respectively . Sagittal imbalance in patients with Scheuermann’s disease includes the appearance of lumbar lordosis36 to compensate for thoracic hyper-kyphosis and maintain a stable center of gravity as well as postural control43. Thus, we quantified the frequency at which lordosis occurs in the caudal spine . However, we did not observe significant differences in the length of the caudal compensatory curves amongst lordotic pkd2l1icm02/icm02 and pkd2l1icm02/+ animals compared to wild-type siblings . Taken together, our results show that the phenotype of Pkd2l1-defective mutants evokes the sagittal imbalance observed in patients with Scheuermann’s disease.We next examined whether 48. Thus, we investigated whether the phenotype of pkd2l1 mutants is gender gender-biased. We compared the distribution of the main kyphosis angle . In contrast, pkd2l1icm02/icm02 males exhibit a 1.5- fold higher median kyphosis angle and a 1.3- fold higher median curve pattern index compared to females of the same genotype . Similarly, pkd2l1icm02/+ males exhibit a 2.5- fold higher median kyphosis angle and a 1.4- fold higher median curve pattern index compared to females of the same genotype . Our results show that the severity of hyper-kyphosis is sexually biased towards males in pkd2l1icm02/icm02 mutants. Altogether, our results confirm that Pkd2l1-defective mutants develop a hyper-kyphosis of the thoracic spine that is exaggerated for males and therefore reminiscent of Scheuermann’s disease.Spine hyper-kyphosis in Scheuermann’s disease patients is more severe in men than in womengle Fig. a and thegle Fig. b betweenpkd2l1 mutants is restricted to the sagittal plane and is not associated with vertebral malformations. By employing an orthopedic-based approach on micro-computed tomography data, we show that Pkd2l1-defective animals, previously shown to lack CSF-cN mechanosensory response at larval stage21, exhibit a round hyper-kyphotic curve at the thoracic level that is associated with a sagittal imbalance involving a caudal lordosis of the spine. We also report that hyper-kyphosis in mutants is more severe in males. Based on these observations, we propose that the closest human pathology is Scheuermann’s disease and that the pkd2l1 knock-out is a relevant model for this idiopathic spine deformity.Here, we show that the hyper-kyphosis of the thoracic spine observed in adult pkd2l1 animals develop a hyper-kyphotic phenotype characterized by increased kyphosis angles, curve pattern and Harrington indexes compared to wild-type siblings, but of intermediate severity compared to homozygous mutants of the same age. This indicates a dose-dependent effect of the Pkd2l1 channel on the alignment of the spine, suggesting that a controlled amount of Pkd2l1 protein addressed at the apical extension of CSF-cNs may be required to sustain their mechanosensory function. This working model is supported by evidence that the spontaneous activity of CSF-cNs is reduced in heterozygous pkd2l1icm02/+ embryos and nearly abolished in homozygous mutant siblings22.Our results also show that adult heterozygous 50 and is accompanied by degenerative changes in vertebral structures50. Here, the orthopedic characterization of spine hyper-kyphosis in pkd2l1 mutants relies on comparisons with wild-type siblings of the same age, limiting the bias related to the spontaneous occurrence of spine misalignments in mutants. Moreover, we report that no vertebral dislocations nor obvious changes in bone mineralization, assessed qualitatively via Alizarin-S staining, occur in pkd2l1 knock-outs compared to their wild-type siblings, making unlikely that mutant animals develop spine misalignments of degenerative origin. Hyper-kyphosis in pkd2l1 mutants has originally been reported over three independent generations22 and observed as early as 12 months of age. However, Scheuermann’s disease is diagnosed around the pubertal period and in young adults, making the onset of hyper-kyphosis in zebrafish pkd2l1 mutants distinct from that characterizing the human pathology. This discrepancy might be linked to the fact that unlike humans, zebrafish grow throughout their life span49 and suggests that compensatory mechanisms might occur to sustain a normal alignment of the spine in pkd2l1 mutants prior to adult stage. Future investigations will be needed to decipher whether targeting CSF-cNs specifically during the juvenile period of growth is sufficient to drive similar spine alignment defects.Zebrafish are considered adult when they reach sexual maturity around 3 months of age and are subject to spontaneous spine deformities with elderliness. The severity of the curvatures increases linearly in animals over 20 months of age33. Indeed, urp1;urp2 double mutants were recently reported to develop AIS-like phenotypes is zebrafish52. Moreover, a downregulation of Urp1/2 peptides expression level was reported in AIS mutant models with defective Reissner fiber acting upstream of CSF-cNs19. In the embryo, pkd2l1 mutants retain a normally assembled Reissner fiber and express urp1 and urp2 at similar levels compared to wild-type siblings32 suggesting that the mechanosensory function of CSF-cNs does not control Urp1/2 expression level. Our work further supports that mechanosensation and neuropeptide expression, both taking place in CSF-cNs, are two mechanisms that influence differentially the alignment of the spine, which may explain why pkd2l1 mutants develop spine deformities distinct from AIS-like phenotypes.Our study shows that the lack of mechanosensation in CSF-cNs at larval stage correlates with spine hyper-kyphosis reminiscent of Scheuermann’s disease in adults. In zebrafish, CSF-cNs are also involved in spine deformities reminiscent of AIS, which rely on the maintenance of a controlled expression level of Urp1 and Urp2, two neuropeptides expressed from the embryonic life to adulthood in these sensory neurons46. Families with an autosomal dominant inheritance pattern and a high penetrance are also documented57. Mutations in COL2A1 and COL9A3 encoding for collagens enriched in intervertebral disks58 were recently reported in patients with Scheuermann’s kyphosis and associated with a defective growth of the cartilage endplates at the vertebrae margins60. Interestingly, recent findings report that murine intervertebral disks are enriched with UTS2R61, a receptor for Urp1/2 peptides expressed by CSF-cNs, which is asymmetrically expressed in the paravertebral muscles of AIS patients62. This raises the question of whether CSF-cNs could influence the development of intervertebral discs or muscles surrounding the vertebrae during growth.The main hypothesis underlying the etiology of Scheuermann’s disease is genetic. Indeed, a higher occurrence of this deformity has been reported in monozygotic twins, indicating a genetic contribution to the etiology of the disease23. Two hypotheses have been proposed to explain the selective activation of the sensory neurons located on the concave side of the curvature: either a direct contact between the Reissner fiber and CSF-cNs or local enhancements of CSF flow near the apical extension of the cells23. Interestingly, MRI studies in Scheuermann’s disease patients reported frequent epidural lipomatosis and syringomyelia54 that are well known to impair CSF pressure and flow. Moreover, a dedicated study shows that 41% of Scheuermann’s disease patients recapitulate the typical features of spinal epidural lipomatosis55, known to produce increased epidural pressure and consecutively increase CSF pressure56. This suggests a possible correlation between CSF circulation and Scheuermann’s disease.Our work links zebrafish mutants in which mechanosensation is defective in CSF-cNs with Scheuermann’s disease. But what could be the underlying mechanisms? In the zebrafish larva, the Reissner fiber is required for the asymmetric mechanosensory activation of CSF-cNs during tail bendingBy characterizing the relevance of zebrafish mutants defective in the mechanosensory function of CSF-cNs for Scheuermann’s disease, our work highlights the influence of the niche formed around the Reissner fiber, known to be essential for spine morphogenesis. Dysfunctions in the Reissner fiber or in CSF-cN’s mechanosensory functions lead to spine misalignments reminiscent of AIS and hyper-kyphosis, respectively, suggesting a common origin linked to this axial sensory system for eliciting distinct idiopathic spine disorders.pkd2l1icm02/+ parents resulting in a progeny (one generation) of wild-type (25%), heterozygous (50%) and mutant siblings (25%). Animals were maintained in a 14 / 10 h light cycle at 28 °C until 6 days post-fertilization and then raised in 20L aquarium at 25 °C until 18 months-old. Animal handling and protocols were carried out in accordance with the European Communities Council Directives (2010/63/EY) and French law (87/848) and approved by the Paris Brain Institute (Institut du Cerveau). An approval agreement for experimentation on adult animals was obtained from the ethic committee of the French Ministry for research (APAFIS agreement 2018071217081175).Animals were generated from incrosses of Full spine X-ray radiographs were obtained as part of patients’ care course  in accordance with the Declaration of Helsinki of the World Medical Association revised in 2013 for medical research involving human subjects. An approval agreement for the collection and publication of X-ray radiographs from human patients was obtained from the ethic and data protection committee of the Assistance Publique des Hôpitaux de Paris (APHP agreement 20220916143818).pkd2l1icm02 mutation was genotyped by PCR at 61 °C using forward (TGTGTGCTAGGACTGTGGGG) and reverse (AGGGCAAGAGAATGGCAAGACG) primers to generate a 400 bp product. Wild-type sequences were cleaved by Sac1 to produce a 350 bp and a 50 bp band, while the mutant sequences were resistant to digestion21.Genotyping was performed from genomic DNA isolated from fin clips (for adults) or whole larvae Fig.  by overn40. Acquisitions were performed on a Quantum FX Caliper  with a 59 µm spatial resolution using a 30 mm field of view. Imaging required up to 3 min per animal. Rigaku software  was used for real time 3D reconstructions and RadiAnt DICOM Viewer  for image processing. The quantitative analysis of spine alignment was performed on 3D reconstructions oriented in the sagittal plane except for Fig. Animals were anesthetized using 0.02% Tricain  and transferred into imaging chambers filled with Tricain-containing fish water in order to avoid positioning artifacts due to a contact with a dense surfaceThe cranio-cervical length was calculated from the tip of the jaw to the caudal-most vertebra of the spine, excluding the tail fin set Fig. a, b. The2O2 for 24 h, washed 3 times during 10 min in tap water and incubated in 30% borax for 24 h. Soft tissues were then digested in a solution containing 2% borax and 0.5% Trypsin  over one day and one night. Skeletal tissues were stained using a solution containing 1 mg/mL Alizarin S  and 1% KOH. Samples were washed 3 times during 10 min in tap water and incubated in a 2% borax solution over two nights. Samples were then stored in glycerol until imaging. Stained skeletal preparations  and fixed in 4% paraformaldehyde . After evisceration, samples were depigmented in a solution containing 1% KOH and 3% H15. 3 and 6 days post-fertilization larvae were euthanized in 0.2% Tricain , fixed 2 h in 4% PFA, 3% sucrose at 4 °C, and washed 3 times during 10 min in 1X PBS. Skin from the rostral part of the trunk was removed and samples were blocked overnight in 0.7% Triton X-100, 1% DMSO, 10% NGS and 2 mg/mL BSA. Larvae were then incubated overnight at 4 °C in a solution containing 0.5% Triton X-100, 1% DMSO, 1% NGS, 1 mg/mL BSA and L1P1b anti-serum63 diluted at 1:200. Alexa Fluor Plus 488 goat anti-Rabbit IgG (H + L) secondary antibodies  were diluted at 1:500 in a solution containing 0.7% Triton X-100, 1% DMSO, 10% NGS and 2 mg/mL BSA and incubated 2.5 h at room temperature. Larvae were mounted laterally in IBIDI Antifade Mounting medium and imaged on a confocal spinning disk microscope  equipped with a 20X water immersion objective. Samples were subsequently unmounted and subjected to genomic DNA extraction and genotyping. Images were processed using Fiji64. Maximal Z-projections of 10 microns in depth are represented in Fig. Immunohistochemistry was performed as described ini.e. IQR) where whiskers denote the maximal and minimal values of the distribution. Values in Fig. p < 0.05; **p < 0.01, ***p < 0.001; ns, p > 0.05.Values in Figs.


Producers require tools that are easy to implement in order to adopt adequate mitigation techniques to minimize the adverse effects of heat waves on animal wellbeing and animal performance. This study assesses four different thermal comfort indices in order to predict the tympanic temperature of feedlot steers. The temperature humidity index adjusted by solar radiation and wind speed (THIadj), the heat load index (HLI), and THIPST using pen surface temperature instead of air temperature. Respiration rates (RR), tympanic temperatures (TT), and panting scores (PS) were also collected in each year and from each location. During 2007, mean values of soil temperature, PST, outgoing shortwave radiation, and TT were greater than in 2008 (p < 0.011). However, HLI, relative humidity, and incoming and outgoing long-wave radiation were greater during 2008 (p < 0.012). The TT was positively correlated with THIPST and THIadj , whereas RR had a moderate correlation with THI, THIadj, and HLI . Thermal comfort indices showed a positive correlation with TT, especially the THIPST. These relationships vary with location. However, all of the thermal indices showed weak relationships with the observed RR. This would confirm the different roles that TT and RR have as indicators of heat stress. The THIPST was the best index for predicting TT across years.Climatic data from different years and experiments conducted in Nebraska were used to estimate four comfort thermal indices and to predict the risk of heat stress and its relationship with pen surface temperature (PST). These included the temperature–humidity index (THI), the adjusted THI (THI The microclimate surrounding cattle exerts a powerful effect on animals’ health, productivity level, and survival . Thus, aThe uncomfortable microclimate for cattle is exacerbated in the summer season by the increasing and recurrent heat waves, which impact the well-being, performance, and survival of feedlot cattle around the world ,11. The Heat stress has been defined as a multifactorial problem limiting animal efficiency and negatively impacting animal health . Mellor In 1970, the THI was adopted by the livestock industry in the USA as a basis for the Livestock Weather Safety Index , which dExperiments carried out in 2007 in Concord were conducted at the University of Nebraska Haskell Agricultural Laboratory, previously reported by Mader et al. , locatedadj), and heat load index (HLI), which were calculated using the following equations:All of the data correspond to three experiments conducted during the summer season in two experimental feedlots of the University of Nebraska (2007 and 2008) located in Concord and Scottsbluff, NE, USA, and reported by Arias . Meteoro(a) Temperature–humidity index :THI = 0.−1), and SWin is the incoming shortwave radiation (W·m−2);(b) Adjusted temperature–humidity index ,27:THIad(c) Heat load index :HLI(BG>2HLIBG> = 8.82 +The black globe temperature was estimated for Scottsbluff by using the following equation :BG = (1.(d) Estimated respiration rate :RRe = (5The black globe temperature was used to estimate HLI in Scottsbluff .PST) was also estimated. The THIPST was calculated using pen surface temperature instead of air temperature. Additionally, respiration rates (RR), TT, and panting scores (PS) were also collected in each year and location. Finally, net solar radiation (NR), incoming shortwave solar radiation (SWin), outgoing shortwave solar radiation (SWout), incoming long-wave radiation (LWin), and outgoing long-wave radiation (LWout) were collected by two precisions spectral pyranometers , whereas incoming and outgoing long-wave radiation were collected using two precision infrared radiometers . Simultaneously, net solar radiation was collected using an REBS Net Radiometer model Q − 7.1 . All radiation measurements were collected hourly in an adjacent empty pen in the feed yard.In addition, a THI index based on pen surface temperature (THI® logger in order to collect TT for a period of six days (11 to 17 July). In experiment 2, a total of 35 steers received the same kind of loggers (14 to 19 July). Steers receiving the devices were chosen randomly each year. The iButtons loggers were programmed to collect TT once every minute and then compiled it into hourly readings. The devices were inserted manually into the ear canal of each animal as described by Arias, et al. [Experiments 1 and 2 : in the first experiment, 34 crossbred steers (Angus × Hereford) were fitted with an iButton, et al. . Additio® loggers between 22 and 25 July 2008. Data from TT, RR, and PS were collected following the same procedures previously mentioned.A score of PS was assigned using the scoring system presented in ® and SAS®, whereas Excel for Mac® was used for plotting mean values. Additionally, comparisons between years and locations were conducted for the thermal indices and the main environmental variables by ANOVA. The relationship between the observed and calculated respiration rates was also investigated, as well as the relationship between PS and observed and estimated respiration rates. These relationships were conducted by using a subset of data that contained the average value of the environmental and physiological variables collected between 12:00 and 15:00 h, because this was the time at which PS and RR were collected. The final analyses included correlation (PROC CORR and PROC MEANS in SAS) and regression analysis (JMP). The experimental and observational unit in each analysis was a steer and the significance level was established at 5%.Analyses of the relationships between thermal comfort indices and physiological stress indices were investigated. These analyses were conducted using JMPadj, THIPST, and SWin. During 2007, the mean values of soil temperature, pen surface temperature, SWout, and TT were greater compared to those measured in 2008 (p < 0.011). In comparison, the mean values for HLI, RH, LWin, and LWout were greater during 2008 (p < 0.012). In 2008 there was a greater variation in the climatic variables. For instance, RH had a range of 72.0 vs. 59.3% for the years 2008 and 2007, respectively. Soil temperature and PST had a range of 12.3 vs. 9.5 °C and 57 vs. 36.5 °C, respectively, whereas TT and the Ta had a range of 3.2 vs. 2.1 °C and 19.5 vs. 24.3 °C for years 2008 and 2007, respectively. There were differences in the soil temperature, RH, THI, THIadj, SWout, LWin, LWout, and HLI (p < 0.015) between locations, with greater values at Concord than at Scottsbluff. In addition, Ta tended to be greater in Concord (p = 0.07), whereas pen surface temperature tended to be higher in Scottsbluff (p = 0.07), which was a sandier soil.PST and THIadj .There was more variation in RH in Scottsbluff, which may be due to the precipitations recorded during the week of data collection. Correlation coefficients for all of the thermal comfort indices, TT, and RRe are presented in adj, THI, and THIPST , whereas correlation with TT and HLI was lower .Similarly, RRe was correlated positively with all of the thermal indices, but mainly with THIPST, and THIadj , whereas a weak relationship was observed with HLI (0.22). However, HLI showed a better correlation coefficient with RRe. A simple linear regression analysis showed that THI explained 44 and 87% of the variability of TT and RRe, respectively. Likewise, when THIadj was used as a predictor the results improve lightly . However, the best predictor for TT was obtained with THIPST (53%), which in turn explained 72% of the variation in RRe. Finally, HLI showed lower values with 0.38 and 0.10 for RRe and TT, respectively. In general, TT was explained better by all thermal indices in Concord than in Scottsbluff, except for HLI. In addition, RRe explained more variability of TT in Scottsbluff than in Concord. Interestingly, in both locations, the best variable explaining TT was the THIPST. A similar response was observed when data from both locations were pooled. On the other hand, the THI and THIadj were the best thermal comfort indices that explained the RRe in Concord, whereas the THIPST and HLI were the best ones in Scottsbluff.adj showed similar patterns during a 24 h period, with HLI reaching the maximum value early in the morning (09:00 h). The HLI remained very constant until 17:00 h, after that, it started to decrease quickly. In addition, THI and THIadj showed similar patterns but with slightly higher values for THIadj during the period of the day with higher solar radiation, that is, between 10:00 and 17:00 h. This highlights the importance of SWin in THIadj estimation. It is important to mention that there was a lag of maximum TT regarding the maximum values of all comfort thermal indices. This lag was greater in Concord than in Scottsbluff (4 vs. 1 h).p < 0.0001). p < 0.0001) when data from both locations and years were analyzed (p < 0.0001).The accuracy of the equation used to calculate RRe was assessed by comparing these data with collected respiration rates (RR) during the summer of 2008. A low correlation was observed for RRe and RR  and PS are presented in Thermal comfort and heat stress indices have been the subject of in-depth studies in man and livestock ,30. Howeadj. This could be explained in part because Ta was similar between years, even though there were higher RH values during 2008, which apparently did not impact the THI indices. In addition, no differences were observed in WS and SWin between years, which are the factors used by Mader et al. [PST in 2007 was due to the higher values recorded for soil surface temperature for that year. On the other hand, HLI was greater during 2008 because of higher RH and black-globe temperatures . Thus, none of the thermal comfort indices assessed were able to match the difference observed for TT between years. Only THIPST showed a better result that could explain the differences in TT observed. Based on the Livestock Weather Safety Index [PST (77.7) corresponded to a condition of “alert”, whereas all of the other thermal indices were considered “normal”.No differences were observed between years for THI and THIr et al.  to adjusty Index , the greadj, THIPST, and HLI were statistically greater in Concord than in Scottsbluff. Nevertheless, the indices presented normal values (<74), and HLI was considered safe (<86). The greater values of THI and THIadj could be associated with the higher values of RH recorded in Concord. However, an analysis of the number of hours of thermal indices above “danger” for the THI (>79) and “extreme caution” for HLI (>92) showed that, in 2007, there were more hours under these categories. Simple correlation analyses for the number of hours of thermal indices over critical values were positively correlated with TT in 2007 . These relationships were less clear during 2008 due to fewer hours over those critical values. Only the THIPST showed a consistent relationship trend for Concord and Scottsbluff during 2008 . For the experimental periods, THIPST presented a greater number of hours over the “danger” category in 2007 (Concord) and 2008 (Scottsbluff), but in 2008 (Concord), THIadj was the index with a greater number of hours in that category. It is important to recall that the five domains’ concept implies not only providing the basics to animal survival but also reaching a condition of well-being. Thus, tools which are easy to adopt and use, like those here proposed, are of high relevance to producers for their animals to reach a welfare enhancement status.When data were compared across locations, no differences were observed in TT, even though THI, THIThe high correlations observed between RRe and the thermal comfort indices could be due to the equations used to estimate RR and THI sharing at least two of the same environmental variables (RH and Ta). The use of Eigenberg’s equation (RRe) to obtain respiration rates in this analysis could be confounding the real relationship of the comfort indices with the respiration rates of cattle, because most of them are based on the same environmental variables.adj, and HLI . Likewise, the correlation coefficients between PS and the thermal indices were 0.29, 0.19, and 0.25 for THI, THIadj, and HLI, respectively (p < 0.0001). The correlation coefficients between TT and PS and between TT and RR were lower . However, the correlation coefficient between RR and PS was high . Non-significant correlations were observed for THIPST with PS or RR, but this was significant with tympanic temperature Therefore, only the relationships of TT with the indices and the estimated RR should be considered. The same observation is valid for the linear regression analysis herein conducted for the RRe. Assessing the relationships of RRe and RR with TT, a new set of correlation coefficients for RR and the variables of interest were calculated. These correlations were conducted using the observed RR collected during the afternoon. The results showed a moderate correlation with THI, THIadj with TT, but similar values of correlation for HLI. In their experiments, da Silva et al. [adj index. The same authors reported that the best correlation coefficients among thermal indices and RR were obtained with HLI and the equivalent temperature index [2 for TT and THI (0.26) to those reported by da Silva et al. [2 for THI, THIadj, and THIPST . Differences among the studies discussed can have many potential explanations, e.g., micrometeorological conditions in each location due to relief and microrelief, wind exposition, and soil texture (sandy soil in Scottsbluff). Changes in soil density imply changes in the proportion of porous space and associated changes in the water retention of a particular soil. Both are important factors related to the heat flow capacities of a particular soil and its potential transfer to animals. Nevertheless, no reports regarding the relationship between pen surface temperature and animal production or animal physiology have been published up to now. Nevertheless, the exchange of heat at the ground surface exposed to sunshine establishes the conditions of an important part of the animal’s environment [In a study conducted by da Silva et al.  assessina et al.  did not re index  were 0.5re index  reporteda et al. . Howeverironment . In addiBody temperature and RR lags have been previously reported, ranging from 1 to 4 h ,34. EvenPST index, although these relationships seem to vary with the location. Thermal indices showed weak relationships with the observed RR, even though they were significant and positive, confirming the different roles that TT and RR have as indicators of heat stress. The THIPST was the unique index that matched well with the increase in the TT observed across the years. In addition, Eigenberg’s equation was a useful tool. However, it underestimated the true RR. Further research that assesses the effects of thermal indices based on pen surface temperature should be conducted in the future.The thermal comfort indices showed a high and positive correlation with TT, especially the THI


The morphometric features of the biceps groove were measured to investigate their correlation with the injury of the pulley and the long head of the biceps tendon (LHBT).A total of 126 patients undergoing arthroscopic rotator cuff repair surgery had their morphological features of bicipital groove evaluated on a 3D reconstruction model of the humeral head. The groove width, groove depth, opening angle, medial wall angle, and inclination angle of the bicipital groove were measured for each patient. During the surgery, the type of injury to the biceps pulley and the degree of long head of biceps tendon injury were assessed. The correlations of these injury assessments with bicipital groove measurements were analyzed.The average groove width was(12.3 ± 2.1) mm. The average groove depth was(4.9 ± 1.4) mm. The average groove inclination angle was 26.3° ± 8.1°. The average opening angle was 89.8° ± 18.4°. The average medial groove wall angle was 40.6° ± 7.9°.Sixty six patients had injury of the biceps pulley structure, and their Martetschläger classifications were as follows: type I injury in 12 patients, type II injury in 18 patients, and type III injury in 36 patients. The Lafosse grades of Lesions of LHBT were as follows: 72 cases were grade 0 injury, 30 cases were grade I injury, and 24 cases were grade II injury. We found no significant correlation between the opening width, depth, inclination angle, opening angle, and medial wall angle of the morphological features of bicipital groove and injuries of the pulley and the LHBT. The correlation between pulley structure injury and lesions of LHBT was statistically significant.Lesions of LHBT show strong correlation with pulley injuries.This study does not find a correlation between the injury of the pulley or the LHBT and bicipital groove morphology. Lesions of the LHBT are a common source of pain in the shoulder anterior –3, and oBefore LHBT enters the biceps groove, there exists the long head tendon pulley structure, which is a soft tissue stable structure. The relationship between pulley structure injury and LHBT lesions has attracted increasing attention in recent years . Trauma,In this study, patients undergoing arthroscopic shoulder surgery were subjected to a preoperative CT scan, and a 3D model of the proximal humerus was constructed. The width, depth, angle of inclination, angle of opening, and medial wall angle of the biceps groove were measured. Their correlation with pulley structure and LHBT lesions was also investigated.This retrospective study was approved by the institutional review board at our institution.The study participant gave written, informed consent to participate in the experiment.Between January 2021 and June 2021, patients who were diagnosed with rotator cuff tear who had undergone MRI and shoulder arthroscopic surgery were selected. During this time frame, there were 142 patients with rotator cuff tears, and 16 patients were excluded, because they had inflammatory diseases, contraindications to surgery, and irreparable rotator cuff tears. A total of 126 patients  were included. There were 40 cases in the left shoulder and 86 cases in the right shoulder. The patients were 31–78 years old, with an average of 61.05 ± 11.91 years old, and the disease duration was 0.1–3 years, at an average of 1.6 ± 2.26 years.The shoulder joint of the patient was CT scanned, with the affected shoulder placed on the side of the body, and a 3D model of the biceps groove was constructed based on CT scan data. A 64-slice CT scanner  was used, covering the upper end of the humerus. The layer thickness was 0.625 mm, the layer spacing was 0.95 mm, and each pixel of the obtained image was 512 × 512. After scanning, the CT images were preprocessed in CT workstation, and CT data obtained were stored in the DICOM format.DICOM scan data were imported into Mimics 21.0 software. Threshold analysis was performed using the threshold tool, which was set at 245. The humeral boundary was isolated, the joining images were excised using the zone growth tool, and the excess structures were isolated. The layers were edited one by one, with the remnants replenished and the noise removed. The model was further optimized in Geomagic Warp reverse engineering software. The software was used to fill holes, remove noise, and repair boundaries to smooth the model, resulting in a 3D model of the humeral head containing the biceps groove.The depth of the biceps groove was defined as the distance from the highest point of the tuberosity to the bottom of the biceps groove , 12. It Groove width (WG)On S1 plane, the width of the groove refers to the straight line distance between the vertices of the large and small tuberositys.(2)Groove depth (DG)The depth of the biceps groove is the length of a straight line perpendicular to the vertex of the tuberositys.(3)Opening Angle of biceps groove (OA)The lowest point of the biceps groove was selected, and a tangent line was made along the lateral wall of the tuberositys. The angle between the two points was the opening angle of the biceps groove.(4)Medial Wall Angle of biceps groove opening (MWA)MWA refers to the angle between the tangent line passing through the bottom of the intertubercular groove and the medial wall of the intertubercular groove, as depicted in Fig. Lines are drawn tangential to top and bottom of groove and tangential to medial and lateral border. 1 = groove width; 2 = groove depth;3 = opening Angle of biceps groove;4 = medial Wall Angle of biceps groove opening.In this study, the angle between the two lines of the large and small tuberositys and the transverse line of the humerus shaft was defined as the opening inclination angle of the intertubercular groove. Since the connection of the vertices of the large and small tuberositys is not in the same plane in 3D, the connection L1 of vertices of the large and small tuberositys were directly projected, and the projected line L2 and the vertical segmentation plane S1 of humerus bone were established, in UG (Unigraphics NX Siemens USA) software. The angle between L2 and S1 was measured to obtain the opening inclination angle of the intertubercular groove, as demonstrated in Fig. Arthroscopy is considered the gold standard for evaluating pulley and Lesions of LHBT. In this study, arthroscopy was performed by the same senior shoulder surgeon to observe the degree of Lesions of LHBT in the glenohumeral joint and classify the pulley structure injury.Classification of pulley structure injury.A-Type I:Lesion of the medial pulley B-Type II: Lesion of the lateral pulleyC-Type III:Lesion of the medial and lateral pulley slingsThe method proposed by Martetschläger  was used2.Classification of Lesions of LHBTA-Grade 0: no injury to long head tendon of bicepsB-Grade I: minor injury C- Grade II: major injury (extensive absence or erosion of more than 50% of the tendon)The method proposed by Lafosse  was usedP values < 0.05 were considered statistically significant.The relationship between themorphological features of the biceps groove and injuries to the biceps pulley and the long head tendon of the biceps were analyzedusing the Spearmann correlation test. If data were ordinal and categorical, correlation analysis was performed using gamma test.Statistical analyses were performed using SPSS version 23.0 . The width of the biceps groove opening ranged from 8.9 mm to 17.2 mm, averaging 12.5 mm ± 2.1 mm. The biceps groove depth ranged from 3.2 mm to 9.2 mm, averaging 4.9 mm ± 1.4 mm. The inclination angle of the biceps groove ranged from 12.7° to 40.4°, averaging 26.3° ± 8.1°. The biceps groove opening angle ranged from 40.9° to 112.7°, averaging 89.8° ± 18.4°. The inner wall angle ranged from 30.9° to 64.3°, averaging 40.6° ± 7.9°.Sixty patients showed no pulley structure injury, while the remaining 66 patients showed pulley structure injury.According to Martetschläger classification system , twelve There were 54 patients with LHBT structure injury. According to the Lafosse classification system, 30 patients had a grade I injury and 24 patients had a grade II injury.P < 0.01), with a gamma coefficient of 0.639. These quantities exhibited a positive correlation trend.According to the gamma test, the correlation between pulley structure injury and biceps long head muscle injury was statistically significant (P > 0.05). Table All patients were divided into LHBT-injured and non-LHBT-injured groups. After examination, we found no statistical differences in the width, depth, inclination angle, opening angle, and medial wall angle of the biceps groove between the two groups (P > 0.05). Table Spearman correlation test was performed between lesions of LHBT degree and biceps groove morphology, and we found that the correlation between Lesions of LHBT degree and the width, depth, inclination angle, opening angle, and medial wall angle was not statistically significant (P > 0.05). Table All 126 patients were divided into the injured pulley structure group and the non-injured group. After examination, there was no statistically significant difference in the width, depth, inclination angle, opening angle, and medial wall angle of the biceps groove between the two groups (P > 0.05). Table Spearman correlation test was performed between the pulley structure injury type and themorphological features of the biceps groove, and the correlation between the pulley structure injury type and the width, depth, inclination angle, opening angle, and inner wall angle was not statistically significant , the coracohumeral ligament (CHL), the supraspinatus muscle, and the subscapularis tendon, which is a soft tissue stabilizing device before the biceps long head tendon enters the bone pulley groove. Pulley structure injury is an important reason for the anterior pain of the shoulder joint –18. ReceThis study has many shortcomings. All patients included in this study had rotator cuff tears, shoulder impingement, or other serious cases requiring surgery, and these diseases are high-risk factors of LHBT and pulley injuries  and coulIn conclusion, there are variations in the morphological features of bicipital groove, but the correlation between the pulley structure and Lesions of LHBT was not found in this study.


Surgical-assisted accelerated orthodontics (SAAO) has become very popular recently. Therefore, this study aimed to investigate the extent to which researchers adhere to Item 19 (harms) of the Consolidated Standards of Reporting Trials (CONSORT) in the published studies in the field of SAAO. In addition, the study evaluated the possible association between harm reporting and the human development index (HDI) of the recruited research sample country, CiteScore-based quartile (CSBQ) of the publishing journal, invasiveness of the surgical intervention (ISI), and the type of orthodontic tooth movement (TOTM). Moreover, it aimed to summarize the different possible harms and complications that maybe encountered in the course of SAAO.Electronic searching of six databases was conducted for SAAO-related English RCTs published between January 2000 and April 2022. For the RCTs that did not report harms, information was sought by contacting the corresponding authors. Descriptive statistics of the evaluated variables were performed. The association between 'harm reporting' and the HDI of the research team, the BDRQ of the publication journal, the ISI, and the TOTM were investigated. Binary logistic regression was used, and the odds ratios (ORs) with 95% confidence interval (CIs) of the evaluated variables were obtained. Moreover, the risk of bias of the included RCTs was assessed using the RoB2 tool.p < 0.018) for invasive methods compared with minimally invasive ones). There was a significant positive correlation between harm reporting and both the CSBQ of the publishing journal and the HDI of the recruited research sample country . On the contrary, a non-significant association was found between harm reporting and the type of OTM (p = 0.695). The incidence of harms associated with SAAO was approximately 17.5%.Among the 91 included RCTs, 54 RCTs (59.3%) did not adhere to reporting harm associated with the SAAO. The non-adherence was significantly associated with the ISI (OR 0.16; CI 0.03–0.73; Assessment was restricted to English RCTs related to SAAO.The adherence to reporting harms in the field of SAAO was deficient. Efforts should be made by authors, peer reviewers, and editors to improve compliance with the CONSORT guidelines regarding harms reporting. Additionally, there is a wide spectrum of harms that could be associated with SAAO that the practitioner should pay attention to and alert the patient to the possibility of their occurrence.The online version contains supplementary material available at 10.1186/s40510-023-00457-4. Several methods for accelerating orthodontic tooth movement (OTM) have been proposed to reduce treatment time and achieve patient satisfaction . AccelerCurrently, surgical interventions can be considered one of the most applied and tested acceleration methods that have shown promising results in shortening the duration of orthodontic treatment , 3. SurgTo formulate high-quality protocols and to enhance the quality of reported RCTs, the SPIRIT  , as wellThis MES was constructed under 'Guidelines for reporting meta-epidemiological methodology research' .Harms are defined as the totality of possible adverse consequences of an intervention or therapy. They are considered as the direct opposite of benefits, against which they must be compared . In thisHuman Development Index (HDI) was adopted in the current study to classify the country of the recruited research sample of the included RCTs. HDI is a socio-demographic variable, which was introduced by the WHO in the 1990s . It helpIn the current study, to evaluate the impact of the publication journal of the included research paper, the CiteScore was used . The quaOrthodontic tooth movement that is assisted by one of the SAAO methods includes: retraction of , decrowding of anterior teeth, impacted canine traction, and intrusion of molar or incisors.Surgical acceleration methods were divided into: (1) Invasive methods require full mucoperiosteal flaps, suturing with the potential associated surgical side effect . It inclResearch articles were screened for eligibility using the following criteria:All randomized controlled trials that were published between January 2000 to April 2022, as well as included healthy human participants of both gender who underwent fixed orthodontic treatment assisted with one of the surgical acceleration interventions  compared with a non-accelerated group, or an accelerated group with another protocol or surgical technique, with no restriction for age, type of malocclusion and racial group. On the other hand, the non-RCTs, retrospective trials, animal trials, case reports or case series, and non-English language trials were excluded. The included RCTs in which the majority of their outcomes were directly related to the assessment of harms associated with the provided intervention , were deemed ineligible for inclusion in our study.Electronic searching of the Cochrane Library, PubMed®, Scopus®, Web of Science™, and Google™ Scholar was conducted. Moreover, in addition to the search in Google™ Scholar to determine any relevant papers in the grey literature, a search in the OpenGrey was also performed. The systematic search was done by two authors (DTA and MYH). The search was restricted to articles published between January 2000 and April 2022. For additional sources and to ensure that no relevant research paper was left out, the reference list of the selected papers was also checked. More details about the search strategy used are given in Additional file The potential eligible trials were screened separately by two authors (DTA and MYH). In case of any conflict, the third author (KD) was consulted to reach a resolution. The selection was carried out according to the following methodology: firstly, records from each database were imported into Endnote™ X9, and then merged into a single data unit to remove duplicate records and to facilitate retrieval of related articles. Then after deleting duplicate records, the titles and abstracts of the remaining articles in the Endnote™ list were only checked. Thereafter, the full text was evaluated if reading the title and abstracts did not help in deciding the eligibility of the article, as well as if the paper appeared to fit the inclusion criteria. Data were extracted by the same two investigators, then they were organized into a pre-designed table. From each study, the following data were obtained: the first author's name, year of publication, country where the study was carried out, Human Development Index (HDI) of the country of the recruited research sample , journal of publication, and the CiteScore (CS) of the journal, the Quartile to which the journal belongs (Q) according to Scopus® indexing ; a journal was labeled 'Not indexed' when it is was not found in both Scopus® and Web of Science™ databases. In addition, the following pieces of information were gathered: the type of OTM , invasiveness of the SAAO , adherence to harm reporting , and the method of reporting harms .For more information about the harms associated with SAAO, emails were sent to the corresponding authors of the included RCTs that did not report harm. After introducing ourselves and explaining the main purpose of this study, the authors were asked to help answer the following question: "Did any of your patients suffer harm from the surgical technique applied in accelerating orthodontic tooth movement?".The risk of bias in the included RCTs was assessed by two reviewers (DTA and MYH) using the RoB-2 tool . In cases). Then, the strength of the relationship according to rs was defined as: 0.00–0.19 “very weak correlation”, 0.20–0.39 “weak correlation”, 0.40–0.59 “moderate correlation”, 0.60–0.79 “strong correlation”, and 0.80–1.0 “very strong correlation”. Chi-square and Fisher’s exact tests were applied to examine the association between harm reporting and both invasiveness of the SAAO, and the type of OTM. Then, the strength of the relationship was detected using Cramer's V (φc) as follows: from 0.00 to less than 0.10 “negligible association”, from 0.10 to less than 0.20 “weak association”, from 0.20 to less than 0.40 “moderate association”, from 0.40 to less than 0.60 “relatively strong association”, from 0.60 to less than 0.80 “strong association”, and from 0.80 to 1.00 “very strong association”. The threshold of statistical significance was determined at an alpha value of 0.05. In addition, to investigate the prediction model of reporting harms and estimate the odds ratios (ORs), binary logistic regression analysis was used. All statistical tests were carried out using the Statistical Package for the Social Sciences, version 23.0 .Descriptive statistics including the percentage and frequencies for each studied variable were calculated. Mann–Whitney test (U) was used to compare the harm reporting and the year of publication. The association between the categorical variables was detected. To investigate the association between harm reporting and CiteScore-based quartile of the publishing journal and HDI of the recruited research sample country, the rank biserial correlation was applied by using Spearman's Rho test  as presented in Fig. SAAO research teams of the included RCTs were distributed in seventeen countries worldwide. Moreover, nine of these countries are located in Asia. Two countries from Asia occupied the top ranks in the number of SAAO published papers, where 23 (25.3%) RCTs were conducted in India, and 15 (16.5%) SAAO trials were carried out in Syria. On the other hand, 18 (19.8%) trials were accomplished in Egypt, and 7 (7.7%) RCTs came from Turkey.The number of SAAO papers conducted in the 'medium' human development countries was 56 trials (61.5%). In contrast, 23 RCTs (25.3%) and 12 RCTs (13.2%) were performed in both 'very high' and 'high' human development countries according to HDI data, respectively.The American Journal of Orthodontics and Dentofacial Orthopedics, the Angle Orthodontist journal, and the European Journal of Orthodontics were the top three journals to publish SAAO-related papers, with 9, 8, and 5 published RCTs, respectively. Moreover, there were about 33 journals that published one article related to the topic of our current review.However, 34.1% of the publishing journals belonged to the first quartile (Q1) in the hierarchy of journal's scientific impact according to Scopus®, whereas 22.0% of the included papers were published in non-indexed and unclassified journals in the main two bibliographic databases.Orthodontic retraction of incisors, canines, or the upper six teeth together was the most applied orthodontic procedure in the included RCTs . Regarding the invasiveness of the included interventions, the majority of the included trials involved minimally invasive surgical interventions (60 RCTs: 65.9%). Concerning the adherence to reporting of harms, 37 trials (40.7%) included information about the occurrence or absence of harms during the trial course. Six of them (6/37 RCTs: 16.21%) declared that harms had actually occurred with details about these events, whereas the rest  reported the absence of any harms. On the other hand, 54 trials (59.3% of the whole included RCTs) did not report this element. Among the studies that reported harms, 18 trials (48.64%) allocated a specific paragraph under a definite subheading in the Results section, whereas in the rest of the papers , the information about possible harms or injuries were given inexplicitly in the context of the Results or Discussion sections of the manuscript. More details about the characteristics of included RCTs can be found in Table Of the 91 included RCTs, only 2 studies were judged as 'low risk of bias'. On the other hand, 55 RCTs were assessed as having 'some concern of bias', while the other 34 RCTs were at 'high risk of bias' . More information about the descriptive statistics of the proportions and the association between HR and other variables can be found in Table An association between HR and the invasiveness of SAAO was tested. However, the result presented a moderate, positive significant association between the two variables (χ2(8) = 23.731, p = 0.003), which explained 31% of the variation of harms reporting (Nagelkerke R2) and correctly predicted 71.4% of cases. The Hosmer and Lemeshow goodness-of-fit test showed that this model had a good fit (p > 0.05). However, the odds of non-adherence with the harm reporting in invasive surgical methods was 0.16 times the odds of non-adherence in minimally invasive surgical methods  papers. These included gingival recession , gingivaIn recent years, the trend toward the application of OTM acceleration methods, especially surgical ones, has increased. Undoubtedly, any intervention carries potential risks. Therefore, before making any treatment decision, it is necessary to weigh the potential benefits and risks in order to provide the best possible treatment for the patient . This leThe number of English RCTs carried out in the field of surgically accelerating OTM clearly increased, particularly in the last 5 years. The increasing desire of orthodontists to reduce the duration of orthodontic treatment to fulfill the requirements of adult patients , maybe oOf the included RCTs, the percentage of non-adherence to reporting harms has reached 59.3%. This result is not surprising, as a low level of HR adherence is well recognized across other fields of dentistry , 58. In According to our findings, there was no significant difference between the RCTs that reported harm and those that did not, with regard to the year of article publication. This result is in agreement with the findings of Khan et al. study , which fRegarding the HDI, a positive correlation between the HR and the HDI was found. This means RCTs conducted in developed countries are more transparent in reporting SAAO-related harms. However, this contrasts with the results of Contopoulos-Ioannidis et al., who reported in their MES that poor reporting of harms is a global problem regardless of where the RCTs were conducted .Concerning the CSBQ of the publishing journal, the correlation between HR and the CSBQ was positive. However, many high-quartile journals request authors to present the CONSORT checklist with manuscript submissions , which cMinimally invasive procedures (MIPs) dominated the acceleration techniques applied in the included studies. However, MIPs are flapless, therefore the pain and discomfort associated with these techniques are minimal, which positively affects the patient's acceptance . In addiOn the other hand, the odds ratio of not reporting harms in RCTs that applied invasive surgical acceleration methods was 0.16 times higher than in RCTs that applied minimally invasive methods. However, surgical acceleration methods, regardless of the technique applied, are fairly invasive, and therefore can be associated with complications . FurtherRegarding the type of OTM, no association between HR and the type of OTM applied in the included RCTs, was found. Of the included RCTs, orthodontic retraction  was the most frequently applied orthodontic procedure in the included studies. The reason for this may be that retraction cases require a total treatment period of about 2 years . TherefoBias defines as "systematic error, or deviation from the truth, in results" . In our Regarding the harms associated with SAAO, whether mentioned in studies or reported by authors via email, most were found to fall into the two categories of SAAO-related PROMs harms and SAAO-related gingival harms. However, concerning harms associated with invasive SAAO, DAD can be accompanied by loss of tooth vitality . While PThe fact that SAAO-related harms occurred and did not mention in the published articles may indicate one of two things: either there is ignorance regarding the importance of item 19 of CONSORT, or this information has been deliberately hidden out of fear of refusal to accept the article’s publication.In future trials, researchers must have an obligation to report harms that may be associated with their implementation of the surgical acceleration method. In addition, this notification must be sufficient by stating the nature of the damage, when it occurred, and how it was managed. Additionally, journal editors and reviewers should be more stringent about “harms reporting” as this has an impact on whether or not to adopt the proposed intervention.Only English RCTs related to SAAO were included in this study. This study focused on the investigation of adherence to reporting harm associated with SAAO and did not address the other non-surgical acceleration methods. Moreover, other factors such as the number of medical centers involved in the RCT and the nature of funding for the RCT that may have a potential impact on the transparency of reporting harms, were not studied in this meta-epidemiological study.The proportion of adherence to reporting harms in the field of SAAO was substandard (40.7%). Authors, peer reviewers, and editors must be stricter regarding compliance with the CONSORT guidelines regarding harms reporting. This will allow to weigh the benefits and harms of surgical acceleration techniques and thus optimally adopt the best procedure with the least complications or adverse effects. Patients should be fully aware of the complications that may accompany undergoing SAAO. Likewise, researchers must be fully aware of the harms associated with SAAO. On the other hand, it should be known that some factors may play a role in the adherence to reporting harm in the field of SAAO such as the scientific strength of the publishing journal assessed by the CiteScore-based quartile, the HDI of the recruited research sample country, and the invasiveness of the surgical intervention.Additional file 1: Table S1. Electronic Search Strategy.Additional file 2: Table S2. Excluded studies after reading the full text and the reasons beyond exclusion.Additional file 3: Fig. S1. Risk of bias summary of the included RCTs. Low risk of bias (the + sign); some concern of bias (the - sign); high risk of bias (the X sign).Additional file 4: Fig. S2. Summary of the percentages of the risk of bias assessment for each domain.Additional file 5: Table S3. Details of the risk of bias assessment of the randomized controlled trials using the ROB-2 tool.Additional file 6: Table S4. Responses from authors regarding the SAAO-related harms.


When the donor GGT peak value was dichotomized using a cut-off of 160 IU/L, the odds ratio was 1.90 . When the graft-loss rates were investigated, significantly higher rates were reported in LT cases with donor GGT ≥160 IU/L. In detail, 90-day graft-loss rates were 23.2% vs. 13.9% in patients with high vs. low donor GGT, respectively (log-rank p = 0.004). Donor GGT was also added to scores conventionally used to predict outcomes . In all cases, when the score was combined with the donor GGT, an improvement in the model accuracy was observed. Conclusions: Donor GGT could represent a valuable marker for evaluating graft quality at transplantation. Donor GGT should be implemented in scores aimed at predicting post-transplant clinical outcomes. The exact mechanisms correlating GGT and poor LT outcomes should be better clarified and need prospective studies focused on this topic.Background: Growing interest has been recently reported in the potential detrimental role of donor gamma-glutamyl transferase (GGT) peak at the time of organ procurement regarding the risk of poor outcomes after liver transplantation (LT). However, the literature on this topic is scarce and controversial data exist on the mechanisms justifying such a correlation. This study aims to demonstrate the adverse effect of donor GGT in a large European LT cohort regarding 90-day post-transplant graft loss. Methods: This is a retrospective international study investigating 1335 adult patients receiving a first LT from January 2004 to September 2018 in four collaborative European centers. Results: Two different multivariable logistic regression models were constructed to evaluate the risk factors for 90-day post-transplant graft loss, introducing donor GGT as a continuous or dichotomous variable. In both models, donor GGT showed an independent role as a predictor of graft loss. In detail, the log-transformed continuous donor GGT value showed an odds ratio of 1.46 (95% CI = 1.03–2.07;  Liver transplantation (LT) represents the best curative therapy for a large number of acute and chronic end-stage liver diseases  and hepaHowever, it has been documented that adopting expanded-criteria donors should be correlated with an increased risk of poor post-LT outcomes . TherefoRecently, growing interest has been focused on the potential detrimental role of donor gamma-glutamyl transferase (GGT) peak at the time of organ procurement regarding the risks of graft discard  and poorThis is a retrospective international study based on prospective databases of patients receiving a first liver transplantation. The primary study endpoint was to investigate whether high donor GGT values were correlated with reduced post-LT graft survival.The study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines . All autn = 384); Tor Vergata University Rome, Italy (n = 364); Catholic University Rome, Italy (n = 339); and Ghent University Hospital, Belgium (n = 309). A total of 1335 patients were enrolled. All the centers involved did not present a specific common protocol in the acceptance of donors with high GGT values.Participants included four centers: Sapienza University Rome, Italy (All enrolled cases were adults (≥18 years), consecutively receiving a first LT from January 2004 to September 2018. Exclusion criteria were pediatric transplant patients (age < 18 years) and retransplantation. All the cases investigated included only LTs performed using grafts from deceased donors. No living donors or domino transplants were considered in the analysis.Data collected in the study included recipient age; HCC, acute liver failure; HCV; HBV; alcohol-related cirrhosis; NASH, MELD; cold ischemia time; regional vs. extra-regional graft share; distance LT center procurement; use of airplane by the procurement team; donor age; donor sex; split/partial liver; anoxia as the cause of donor death; cerebrovascular accident as the cause of donor death; length of donor ICU stay; donor BMI; donor history of diabetes; donor history of arterial hypertension; previous donor surgery; donor history of tobacco abuse; donor history of alcoholic abuse; donor anticore positivity; donor hemodynamic instability before procurement; cardiac arrest in the donor before procurement; vasoactive score (VAS); donor sodium peak (mEq/L); donor AST peak (IU/L); donor ALT peak (IU/L); donor total bilirubin peak (mg/dL); donor INR peak; donor platelets peak; and last available donor GGT at the time of procurement (IU/L). All the donor blood tests collected during the ICU stay were evaluated, and the peak values were collected. In the case of GGT, the last available value was considered. The donor GGT peak value was not collected due to a high number of missing data.Ninety-day graft loss was defined as any episode of loss of liver function due to patient death, re-listing, or retransplantation reported within three months of liver transplantation. The last follow-up date was 31 March 2021.Continuous variables were reported as medians and the first–third quartile (Q1–Q3). Dummy variables were reported as numbers and percentages. For each variable, missing data involved less than 10%, being handled using the maximum-likelihood estimation method. The Mann–Whitney U test was used for comparisons between groups in the case of continuous variables, and Fisher’s exact text was adopted in the case of categorical variables.The best cut-off of donor GGT for the risk of 90-day graft loss was established using ROC curves and the Youden index.Multivariable logistic regression models were constructed using the backward Wald method to identify the risk factors for 90-day graft loss, high donor GGT, and macrovesicular steatosis at the time of procurement. Beta-coefficients, standard errors, odds ratio (OR), and 95% confidence intervals (95% CIs) were reported.The additive predictive ability of donor GGT in terms of risk of 90-day graft loss was calculated using the Akaike information criterion (AIC). The AIC represents an estimator of prediction error and thereby the relative quality of statistical models for a given data set. In other terms, the lowest AIC value identifies the best model, namely the model that best fits reality. The AIC is calculated using the following formula: p-value < 0.05 was considered statistically significant in all analyses. Statistical reports and plots were performed using the SPSS statistical package version 27.0 .Kaplan–Meier survival curves were used to estimate graft and patient survival rates. Log-rank analysis was conducted to compare sub-groups. A n = 1210, 90.6%) group or high donor GGT  group. Donor-related characteristics in the investigated population are reported in A total of 1335 transplanted patients were enrolled. Adopting the Youden index, the best cut-off of donor GGT value at the time of procurement for the risk of 90-day graft loss was established using the ROC curves, corresponding to 160 IU/L. The cohort was split into two groups according to this value: low donor GGT , younger age , and more donors with anoxia as the cause of death . Donor history of alcoholic abuse only merged statistical relevance, with more cases reported in the high donor GGT group . Donors with high GGT at procurement had more episodes of cardiac arrest before procurement  and a lower median VAS score . As for blood analysis, median transaminase peaks were higher in the donors with higher GGT (p < 0.0001). Median total bilirubin peak merged significance .As for the recipient- and transplant-related features, no significant differences were reported when comparing the two groups. In detail, no differences were reported in terms of recipient age, the main indication for transplantation, MELD laboratory score at the time of transplantation, and cold ischemia time. Conversely, some differences were reported when the donor-related characteristics were compared. In detail, the patients in the high donor GGT group had donors with a longer ICU stay . The most relevant independent risk factors for graft loss were MELD  and donor total bilirubin peak , followed by the use of a split/partial liver, donor history of alcoholic abuse, donor age, and VAS score.The role of different transplant- and donor-related features was investigated to explore the predictors for 90-day graft loss. Two different multivariable logistic regression models were constructed, introducing donor GGT as a continuous versus dichotomous variable . In bothp = 0.006) was confirmed. Also in this model, MELD was confirmed to be the most relevant risk factor for graft loss .When the donor GGT value measured at the time of procurement was dichotomized according to the previously identified cut-off of 160 IU/L, its independent predictive role with an OR = 1.90 . In all cases, when the conventional score was combined with donor GGT at the time of procurement, an improvement in the model accuracy was observed compared to the conventional model in which the score was evaluated by itself. In detail, adding donor GGT to the MELD score achieved the best model to fit with reality, with an AIC = 2179.50. All the other scores presented greater AIC values, therefore showing an inferior accuracy  .p < 0.0001) and a previous history of donor alcohol abuse . Donor age only merged statistical significance as a risk factor for high donor GGT .According to the data reported in p = 0.004)  .n = 461), intending to investigate the risk factors for macrovesicular steatosis > 30%. According to the data reported in p = 0.006), duration of donor ICU length of stay , and donor GGT . Donor history of type 2 diabetes and bilirubin peak only showed a trend towards statistical significance.A sub-analysis was performed in all donors with available biopsy at the time of procurement  showed that donor GGT was significantly associated with an increased risk of liver discard, with GGT >200 IU/L showing a hazard ratio (HR) of 2.74 . In the sub-group of transplanted cases , GGT < 20 IU/L was a protective factor for the risk of graft loss  [A study based on US data (= 0.045) .Of great interest is two recently published studies from North America and Europe explicitly exploring the risk of graft discard that did not incorporate this marker, pointing out the potential risk of underestimating its role in international databases ,23.N = 5723) intended to develop the Eurotransplant DRI, in which the classical parameters composing the American DRI [p = 0.005). After having integrated these variables, the predictive ability for the risk of graft loss significantly increased when the Eurotransplant vs. the American DRI were compared (c-indices: Eurotransplant-DRI 0.624 vs. DRI 0.614) [A significant European analysis (ican DRI  were intI 0.614) .N = 678) investigated the impact of donor GGT in terms of post-LT early allograft dysfunction (EAD); by adding donor GGT  to donor BMI, macrovesicular steatosis, and cold ischemia time, an equation was created to enable the prediction of EAD with a c-index of 0.68 [A study from Germany ( of 0.68 .N = 1152) explored the impact of donor GGT on the risk of 90-day graft failure, identifying a cut-off of 170 IU/L. In detail, the authors observed an increased risk of graft loss (39% at one year) in patients receiving livers from donors with a history of alcohol abuse and GGT ≥ 170 IU/L [An experience from France (p = 0.006). As reported, adding the donor GGT to previous scores improved the concordance for diagnosing worse outcomes after transplantation.These experiences align with our results, in which a donor GGT ≥160 IU/L correlated with an increased risk of 90-day graft loss .These data are in line with our results, in which a correlation between macrosteatosis and increased GGT was reported: when investigated in biopsied grafts as an independent risk factor for a macrosteatosis > 30%, donor GGT appeared to be a relevant risk factor . It is not completely clear why such a correlation exists. Donors with a prolonged ICU stay are often excluded from organ donation because of a supposed deleterious effect of a lengthy ICU stay. We can only assume that different mechanisms might be involved, like an increased risk of infection, hemodynamic instability, and vasoconstriction derived from inotropic drugs, all concurring in a deleterious ischemic effect. Further studies are needed to clarify these aspects better.A study from Switzerland based on 89 pediatric LTs receiving grafts from deceased donors showed a correlation between a longer donor ICU stay and increased GGT . We alsoThe potential clinical implications of the findings reported in the present study should be of relevance in terms of pre-transplant evaluation and risk stratification. In fact, donor GGT is typically not used as a selection parameter for donor graft quality, and many scores used in the selection process avoid incorporating it. On the opposite, our study showed that the integration of donor GGT into commonly used scores plays a role in improving the risk stratification of the recipients. Therefore, a proposal for further exploring donor GGT in this setting should be of great relevance.Moreover, the use of donor GGT as a relevant parameter to consider during donor evaluation should modify the ordinary management approach, requiring a graft biopsy per protocol in the presence of multiple risk factors . In our series, approximately 10% of our donors showed high GGT, with 54/125 (43.2%) of grafts selected after a biopsy. In all cases of a history of alcohol abuse, the biopsy was used for selecting the graft parenchyma quality. Further analyses on larger populations are required to investigate in detail the role of biopsy in the selection process of donors with high GGT.The study presents some limitations. First, the study is retrospective and multicentric, presenting some selection-bias risk. Second, no protocol biopsies were performed in all the grafts at the time of procurement, limiting our analysis of the correlation between GGT and macrosteatosis. However, the biopsies were performed in the real-life clinical routine only when some doubt about graft quality was raised. No data were available on the initial donor GGT value, limiting the possibility of investigating a potential effect of GGT rising during the donor ICU stay. No analyses on metabolomics or proteomics were available on the donor and liver immediately after implantation. Unfortunately, no research in the literature exists on this specific topic. Further prospective studies are needed to better clarify the mechanisms connected between a GGT rise and liver damage. Lastly, a possible bias for generalizability exists. In fact, the increased rate of donors with a high GGT value may vary in other countries in accordance with the varying rates of alcohol use, NAFLD, and high BMI. Therefore, further studies coming from other countries are needed to confirm the impact of the donor GGT in other geographical settings.In conclusion, donor GGT could represent a valuable marker for evaluating graft quality during transplantation. GGT should be implemented in the scores for predicting post-transplant clinical courses. GGT was correlated with more severe macrosteatosis and typically rose in donors with extended ICU stay. The exact mechanisms correlating GGT and poor LT outcomes should be better clarified, and there is a need for prospective studies focusing on this topic. Based on the current results, further exploration of the underlying mechanisms and clinical implications would contribute to a better understanding of the role of donor GGT in liver transplantation, potentially leading to improved outcomes for transplant recipients.


P < 0.001). White blood cell changes were positively correlated with lymphocyte changes . In the subgroup with increased white blood cells (n = 80), there were 62 cases with increased lymphocytes, 1 case with unchanged lymphocytes, and 17 cases with decreased lymphocytes after PEG-rhG-CSF treatment. There was significant difference in the count of lymphocytes and white blood cells (P < 0.001). In the subgroup with 6 mg of PEG-rhG-CSF (n = 66) and the subgroup with 3 mg of PEG-rhG-CSF (n = 22), the changes of white blood cell and lymphocyte counts before and after treatment were statistically significant (P < 0.001). The two were positively correlated in the 6 mg PEG-rhG-CSF subgroup, with correlation coefficient r = 0.34 (P = 0.002). PEG-rhG-CSF can increase the count of lymphocytes and white blood cells in patients with malignant tumors, and the increase of lymphocytes is positively correlated with the increase of white blood cells.We investigated the effect of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) on lymphocytes and white blood cells of patients with malignant tumors. After PEG-rhG-CSF treatment, the count of lymphocytes increased in 66 cases, remained unchanged in 2 cases, and decreased in 20 cases. The difference in lymphocyte count before and after treatment was statistically significant ( The exclusion criteria were: 1) patients with repeated myelosuppression in previous chemotherapy; 2) patients with uncontrollable infection; 3) patients with previous hematopoietic stem-cell transplantation or organ transplantation; 4) patients with previous radiotherapy treatment; 5) patients with bone marrow invasion; 6) patients with kidney metastasis, adrenal metastasis, or abnormal renal function, such as abnormal creatinine clearance rate and blood urea nitrogen; 7) patients with other serious complications; and 8) patients with hematologic system disease.This is a retrospective observational study. Initially, 183 patients who received PEG-rhG-CSF in the Department of Oncology of the Eighth People’s Hospital of Jinan from January 2021 to June 2022 were screened. Finally, 88 patients with nonhematologic malignant tumors were enrolled in this study. The inclusion criteria were as follows: 1) patients with histopathologically or cytologically confirmed nonhematologic malignant tumors; 2) patients with age equal to or greater than 18 years; 3) patients with Eastern Cooperative Oncology Group Performance Status (ECOG-PS) score less than or equal to 2; 4) before chemotherapy, white blood cells ≥4.0 × 102.1.1α = 0.05, 1 – β = 0.90, ρ0 was determined as 0.2 and OR was determined as 3.079. The calculated sample size was 82. Finally, 88 cases were enrolled in this study.The sample size was calculated using the following formula:Informed consent: Informed consent has been obtained from all individuals included in this study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies, and in accordance with the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of the Eighth People s Hospital of Jinan (No. 2020053). This study has been registered at Chinese Clinical Trial Register (ChiCTR2200063211).2.2For primary or secondary prevention of chemotherapy-related neutropenia, patients were subcutaneously injected with PEG-rhG-CSF  on day 3 after chemotherapy.2.3According to the pharmacokinetic parameters of PEG-rhG-CSF , we chos2.4Clinical data were collected including 1) demographics (sex and age), 2) pathological diagnosis, 3) ECOG PS score, and 4) blood routine results.2.5t-test. Pearson regression analysis was used for correlation analysis. P < 0.05 was considered as statistically significant.SPSS 19.0 statistical software was used. The measurement data were expressed as mean ± standard deviation, and compared with paired sample 33.1The baseline characteristics of patients are shown in 3.2P < 0.001)  .3.3x) as the explained variable and white blood cell change (y) as the explanatory variable: y = 0.0202*x + 0.2043 (P = 0.001). The regression coefficient was highly significant (1% significance level), which indicates that lymphocyte change showed a significant linear relationship with white blood cell change. On average, for every 1 × 109/L increase in white blood cells, lymphocytes would increase by 0.02 × 109/L.Pearson regression analysis was conducted with lymphocyte change (+ 0.2043 . The res3.4P < 0.001), as shown in Among the 80 cases with increased white blood cells after treatment with PEG-rhG-CSF, 62 cases had increased lymphocytes, 1 case had no significant change in lymphocytes, and 17 cases had decreased lymphocytes. The difference in lymphocyte count was statistically significant before and after PEG-rhG-CSF in the subgroup with increased white blood cells after treatment (3.5x) as the explained variable and white blood cell change (y) as the explanatory variable: y = 0.0344*x + 1.1674 , which suggests a significant linear relationship between lymphocyte change and white blood cell change. For every 1 × 109/L rise in white blood cells, lymphocytes will increase by 0.03 × 109/L.In the subgroup with increased white blood cells after PEG-rhG-CSF treatment, Pearson regression analysis was performed with lymphocyte change (+ 1.1674 . The res3.6P < 0.001). Similarly, in the subgroup with 6 mg of PEG-rhG-CSF, white blood cell count and lymphocyte count were positively correlated, with a correlation coefficient of r = 0.34 and P = 0.002.A total of 66 patients were treated with 6 mg of PEG-rhG-CSF. As shown in 3.7P < 0.001) and lymphocytes (P = 0.012) before and after treatment, as shown in P > 0.05).A total of 22 patients were treated with 3 mg of PEG-rhG-CSF. There were also statistically significant changes in white blood cell count  is often used to treat neutropenia in clinic. PEG-rhG-CSF has been confirmed to be as effective as hG-CSF in preventing and treating chemotherapy-related myelosuppression and FN . Howeverr = 0.36, P = 0.001). Pearson regression analysis showed a highly significant regression coefficient (1% significance level). On average, for every 1 × 109/L increase in white blood cells, lymphocytes will increase by 0.02 × 109/L. This indicates that PEG-rhG-CSF can increase lymphocyte counts in patients with malignant tumors. Single-cell analysis by Franzke et al. showed that CD4 + T cells and CD8 + T cells also expressed G-CSF receptors after stimulation with short-acting G-CSF, and that G-CSF could regulate T cells directly through these receptors [PEG-rhG-CSF has a definite effect on granulocytes. However, there are limited studies on the effects of PEG-rhG-CSF on immune cells , immune microenvironment, and immune status in patients with malignant tumors. This study investigated the effect of PEG-rhG-CSF on lymphocytes and white blood cells in patients with malignant tumors. Our results found that lymphocyte counts were significantly increased in 66 cases after PEG-rhG-CSF treatment. The white blood cell increase was positively correlated with lymphocyte increase , the dose of PEG-rhG-CSF for subcutaneous injection can be adjusted to 3 mg per cycle [r = 0.34, P = 0.002. The 22 patients in the PEG-rhG-CSF 3 mg subgroup showed statistically significant changes in lymphocyte count and white blood cell count before and after treatment. However, there was no correlation between the changes in white blood cell count and lymphocyte count before and after treatment in the 3 mg subgroup. This may be caused by the small number of samples in the 3 mg subgroup. It should be further clarified in future studies by increasing the sample size.Clinically, the commonly used dose of PEG-rhG-CSF for subcutaneous injection is 6 mg per cycle. If the body weight of patients is less than 45 kg or if the patients have a “white blood cell-like reaction”  antibodies and chimeric receptor lymphocytes, for example, have been widely used as first-line class IA and recommended drugs for advanced lung cancer, esophageal cancer, and other solid tumors ,15,16. DThere are many theories on the mechanism of poor efficacy and drug resistance of immunotherapy. Some scholars consider that the internal factors of tumor cells (such as activation of driver genes and inactivation of oncogenes) and external factors may lead to drug resistance . In addiLong-term stimulation by tumor antigens can cause T cells to gradually lose their original ability to recognize antigens, activate and proliferate, or secrete interleukin-2, leading to T cell depletion . The immIn order to improve the efficacy of immunotherapy, some new combination immunotherapy models have been proposed, including immunotherapy combined with radiotherapy and double immunotherapy ,25,26. OThe following were the limitations of this study: 1) although the sample size was large, the selective bias and recall bias of data selection were high; 2) the disease types of the study population were heterogeneous; 3) this study did not perform further detailed analysis of lymphocyte subgroups and did not conduct a T-cell antigen receptor group library study; and 4) follow-up study was not performed. Further prospective studies with specific tumor type and detailed analysis of lymphocyte subgroups  are needed.5In summary, PEG-rhG-CSF at both 3 and 6 mg per cycle can increase the count of lymphocytes and white blood cells in patients with malignant tumors. Immunotherapy combined with PEG-rhG-CSF may serve as a new combined immunotherapy mode to prevent granulocytopenia and FN, increase the number of lymphocytes in patients with malignant tumors, reverse lymphocyte depletion, and then improve the efficiency of immunotherapy.