The t-tau and p-tau181 levels of CSF-derived EV were positively correlated with the t-tau and p-tau181 levels of total CSF in former NFL players, respectively, but not in the CTRL group. 429 unique proteins were identified from CSF-derived EVs and quantified by TMT-10 plex method. The identified protein molecules were significantly enriched for the extracellular exosome molecules, Alzheimer’s disease pathway and Age/Telomere Length ontology as determined by DAVID Gene Ontology analysis. Ingenuity pathway analysis of the differentially expressed EV proteins revealed enrichment of canonical liver/retinoid X receptor activation pathway. Upstream effect analysis predicted MAPT (tau) as an upstream regulator in former NFL players. These data will be useful for understanding the EV-mediated disease spread and development of novel EV biomarkers for CTE and related disorders.Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of repetitive mild traumatic brain injury, such as American football players. Initial neuropathologic changes in CTE include perivascular deposition of phosphorylated microtubule-associated protein tau (p-tau) neurofibrillary tangles and other aggregates in neurons, astrocytes and cell processes in an irregular pattern often at the depths of the cortical sulci. In later stages, the p-tau depositions become widespread and is associated with neurodegeneration. Extracellular vesicles (EVs) are known to carry neuropathogenic molecules, most notably p-tau. We therefore examined the protein composition of EVs isolated from the cerebrospinal fluid (CSF) of former National Football League (NFL) players with cognitive and neuropsychiatric dysfunction, and an age-matched control group (CTRL) with no history of contact sports or traumatic brain injury. EVs were isolated from the CSF samples using an affinity purification kit. Total tau (t-tau) and tau phosphorylated on threonine181 (p-tau Chronic Traumatic Encephalopathy (CTE) is a neurodegenerative tauopathy associated with repetitive mild traumatic brain injury, including concussion and subconcussion. CTE was first described in boxers in the 1920s and 1930s as “punch drunk,”  or “demeCurrently, CTE cannot be diagnosed during life. A recent study provided preliminary support for the positron emission tomography (PET) imaging p-tau ligand flortaucipir to detect CTE in living former National Football League (NFL) players . HoweverExtracellular vesicles (EVs), including exosomes (50–150 nm), ectosomes/microvesicles (150–1000 nm), and apoptotic bodies (1000–5000 nm) are released from neurons, glia, and various other cells into the extracellular space . They coThe CSF samples were obtained from the National Institutes of Health–funded study, “Diagnosing and Evaluating Traumatic Encephalopathy using Clinical Tests” (DETECT) at Boston University School of Medicine (BUSM). Participants included in the current study were 15 former NFL players with cognitive and neuropsychiatric symptoms and a control group (CTRL) of 16 asymptomatic same-age men without a history of contact/collision sports or traumatic brain history. The DETECT study procedures, including lumbar puncture and neuropsychological assessment, have been described elsewhere , 2018. AThe EV fraction was isolated from CSF samples using the MagCapture Exosome Isolation Kit PS . Briefly, the CSF samples were centrifuged at 1,200 g for 20 min at 4°C, then the supernatant was centrifuged at 10,000 g for 30 min at 4°C. The supernatant was filtered by 0.22 μm Spin-X centrifuge tube , then the EV fraction was isolated from the flow-through using the MagCapture Exosome Isolation Kit PS, according to the manufacturer’s instructions.® Mammalian Protein Extraction Reagent (#78503 PIERCE) was added to the isolated EV fraction using MagCapture Exosome Isolation Kit PS with HaltTM Protease and Phosphatase Inhibitor Cocktail  and was mixed by vortexing for 15 min. The lysed EVs were filtered by 0.45 μm Spin-X centrifuge tube . The EV t-tau and p-tau181 were measured using modified the Human Total Tau Simoa kit  on the Simoa HD-1 analyzer (Quanterix). Briefly, this kit is an updated version of the Simoa assay. It uses a monoclonal capture antibody that reacts with an epitope in the mid-region of all tau isoforms in combination with a detection antibody that reacts with an epitope at the N-terminus of t-tau for t-tau immunoassay or in PHF-tau  for p-tau181 immunoassay. We used t-tau or human tau (p-tau181) as standards in the Human Tau ELISATM kit or Human Tau [pT181] phosphoELISATM ELISA kit  for each assay, respectively  with Fujirebio  immunoassay kit-based reagents  at least 1:10 to obtain particles within the target reading range for the Nanosight 300 machine , which was 10-100 particles per frame. Using a stage pump system four 30-s videos were taken for each sample at 21°C. Analysis of particle counts was carried out in the Nanosight NTA 3.2 software  with a detection threshold of 5.Ice-cold 100% (w/v) trichloroacetic acid (TCA) (#T6399 Sigma-Aldrich) was added to the isolated EV fraction to a final concentration of 20% of TCA, then the mixed sample was incubated for 30 min on ice and was centrifuged at 15,000 g for 20 min at 4°C. The pellet was then washed twice with ice-cold acetone (#179124 Sigma-Aldrich). After drying, the pellet was resuspended in Laemmli sample buffer (#1610747 Bio-Rad) with 5 mM dithiothreitol (# 43815 Sigma-Aldrich), reduced for 20 min at 65°C, and alkylated with 15 mM iodoacetamide (# I1149 Sigma-Aldrich) for 20 min at room temperature in the dark. Subsequently, the samples were run in a 4–20% gradient gel (#4561096 Bio-Rad) until the dye front was 10 mm from the top of the gel. The gels were washed twice with distilled water, fixed with 100% Methanol, and stained with GelCode Blue Stain Reagent (#24590 Thermo Fisher Scientific) for 16 hrs. Each lane was then individually removed from the gel. Gel pieces were then transferred to 1.5 mL tubes and destained twice using 50% acetonitrile  in 25 mM HEPES (pH 8.8) at 22°C, for 15 min with shaking, and dehydrated with 100% acetonitrile for additional 10 min with shaking, for a total of three times. The destained gel piece was dried up using SpeedVac Concentrators (Thermo Fisher Scientific). The gel pieces were digested with proteomic grade trypsin  in 25 mM HEPES overnight at 37°C. The digested peptide was extracted with 70% acetonitrile/1% formic acid, and were removed the gel by Ultrafree-MC Centrifugal Filter (#UFC30L Millipore United States). The digested peptides were reconstituted in 25 μL of 200 mM EPPS (pH 8.0) and vortexed for 5 min.Tandem mass tag (TMT) labeling was performed according to the manufacturer’s instructions (#90409 Thermo Fisher Scientific). In brief, 4 μL of TMT reagent (20 ng/μL) was added to the digested peptides. Following incubation at room temperature for 1 h, the reaction was quenched with 2 μL of 5% hydroxylamine for 15 min. The TMT-labeled peptide samples were pooled at a 1:1 ratio across 10 samples. The combined sample was added into 100 μL of 20% formic acid, 2 mL of 1% formic acid, desalted via StageTip, dried by vacuum centrifugation, and resuspended in 5% acetonitrile and 5% formic acid for nano-liquid chromatography and tandem mass-spectrometry (NanoLC-MS/MS).5; maximum injection time 100 ms). Precursors for MS2/MS3 analysis were selected using a Top10 method. MS2 analysis consisted of collision-induced dissociation  35; maximum injection time 150 ms). Following acquisition of each MS2 spectrum, we collected an MS3 spectrum using our recently described method in which multiple MS2 fragment ions were captured in the MS3 precursor population using isolation waveforms with multiple frequency notches. MS3 precursors were fragmented by high energy collision-induced dissociation (HCD) and analyzed using the Orbitrap .NanoLC–MS/MS analysis was conducted by an LTQ-Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) equipped with a Proxeon EASY-nanoLC 1200 liquid chromatography pump . Peptides were separated on a 100 μm inner diameter microcapillary column packed with ∼40 cm of Accucore 150 resin . We loaded 4 μL onto the column and separation was achieved using a 180 min gradient of 8 to 23% acetonitrile in 0.125% formic acid at a flow rate of ∼550 nL/min. The analysis used an MS3 based TMT method, which has been shown to reduce ion interference. The scan sequence began with an MS1 spectrum  target 5 × 10Homo sapiens UniProt database . This database was concatenated with one composed of all protein sequences in the reversed order. Searches were performed using a 50 ppm precursor ion tolerance for total protein level profiling  to the mzXML format, as well as to correct monoisotopic m/z measurements . Databasrofiling . The prorofiling . For TMTXD015358 . Protein181, proteomics data, and demographics data using IBM SPSS software ver.25. The Gene Ontology of identified proteins were elucidated by DAVID Bioinformatics Resources 6.8. The Protein networks and pathway analysis were generated using Ingenuity Pathway Analysis (IPA)1. The Venn diagram and Heatmap analysis were generated using Venny_2.12 and MeV_4_83.Statistical analysis was conducted using IBM SPSS software ver.25 and GraphPad Prism 6. Between group comparisons were analyzed by non-parametric Mann-Whitney U or one-way ANOVA followed by Bonferroni correction for multiple comparisons. Bivariate correlation analysis examined differences between former NFL players and controls in EV t-tau, EV p-tau181 immunoassays, the 10 former NFL players and 8 CTRLs isolated EVs were lysed with M-Per lysis buffer and then measured the levels of t-tau and p-tau181 in EVs or total CSF were measured by ultrasensitive or conventional immunoassay   . For theMethods) . The isop = 0.9628). The mode size distribution peaked at 86 nm in former NFL players CSF and at 88 nm in CTRLs (p = 0.9628) (181) in isolated EVs and total CSF from the 10 former NFL players and 8 CTRLs by ultrasensitive immunoassay or multiplex Luminex assay. Both t-tau and p-tau could be detected in both former NFL players and CTRLs CSF-derived EVs and total CSF samples. However, there was no significant difference between former NFL players and CTRLs     (data not shown).To measure the concentration and size of EVs in CSF, we examined former NFL players and control CSF-derived EVs using nanoparticle tracking analysis NTA, . The EV  0.9628) . To chartal CSF) . The lev 0.0001) . Unexpec 0.0524) , but notp value of <0.05. Next, we performed semi-supervised hierarchical clustering of differentially expressed proteins across the individual proteomics dataset using Pearson Correlation metric and Average Linkage clustering as parameters for the Raw (proteins). Proteins cluster if they are differentially expressed between former NFL players and CTRLs (p = 0.0259).To generate EV protein profiling of former NFL players’ CSF, we analyzed EV proteins isolated from the former NFL players and CTRL cohorts by NanoLC-MS/MS using TMT-based labeling . We idennd CTRLs . The TGMnd CTRLs , in the To assess enriched canonical pathways, upstream, and functional networks in the differentially expressed 61 EV proteins in the former NFL players (fold-change: >1.2 or <0.83), we analyzed the individual sample datasets using Ingenuity Pathway Analysis (IPA). Among top 10 differentially regulated canonical pathway, liver/retinoid X receptor (LXR/RXR) activation pathway, which was associated with ALB, C4A, FGA, S100A8, TF, TTR and SERPINA1, was upregulated in the former NFL players . Interes181 and NanoLC–MS/MS analysis in CSF-derived EVs from former NFL players for the first time. We found that t-tau and p-tau181 levels of CSF-derived EV were positively correlated with the t-tau and p-tau181 levels of total CSF in former NFL players, but not in the CTRL group. We also identified 429 unique proteins from CSF-derived EVs, which were significantly enriched for the extracellular exosome molecules, Alzheimer’s disease pathway and Age/Telomere Length ontology as determined by DAVID Gene Ontology analysis. Ingenuity pathway analysis of the differentially expressed EV proteins revealed enrichment of canonical liver/retinoid X receptor activation pathway. Finally, upstream effect analysis predicted MAPT (tau) as an upstream regulator in former NFL players.In this study, we performed digital ELISA analysis of t-tau and p-tau181 levels. One explanation for this finding is that EVs in CSF might contain an N-terminal-deleted fragment of tau, which might be undetected by the tau ELISA kit. We used a commercial Human Tau ELISATM kit and modified Human Tau [pT181] phosphoELISATM kit for this study. The total tau ELISA kit uses a capture antibody that reacts with mid-region of tau and a detection antibody that recognizes the N-terminal region. However, the modified phosphoELISA kit uses a capture antibody that recognizes the mid-region of tau and a detection antibody that reacts with p-tau181. When t-tau and p-tau181 in the CSF from the same former NFL players and CTRLs were measured by Fujirebio kit, which uses mid-region capture antibody for t-tau or p-tau181 specific antibody in mid-region for p-tau and mid-region-specific detector antibody for both t-tau and p-tau detection, the CSF level of t-tau was higher than p-tau. As for tau levels in the CSF-derived EVs, the t-tau levels in the CSF-derived EV samples was lower than expected and accounted for less than 20% of p-tauThere is accumulating interest in proteomic analysis of CSF and CSF-derived EVs to identify biomarkers for neurodegenerative disorders. Here, we report the first proteomic analysis of CSF-derived EV samples isolated from former NFL players. Our proteomic analysis reports a slightly higher enrichment rate of EV-specific molecules in our CSF-derived EV preparation than previous reports, despite the lower number of identified molecules . The Mag181 in CSF-derived EVs and a combination of several cell type-specific molecules are potential monitoring biomarkers in former NFL players at risk for CTE. Further investigations are necessary to confirm these biomarkers, including additional validation sets, and to clarify the implications of the correlation of this marker with disease, and to elucidate the mechanisms of EV secretion from the brain cell types in former NFL players.In summary, we have quantified tau levels and proteomic profiles in CSF-derived EVs from former NFL players in this study, identified unique enrichment of Alzheimer’s disease pathway and Age/Telomere Length ontology, canonical liver/retinoid X receptor activation pathway, and predicted MAPT (tau) as an upstream regulator in former NFL players. Fluid and neuroimaging biomarkers have been mainly focused on tau: Tau PET imaging has been developed as an important tool for CTE diagnosis  and totahttp://www.proteomexchange.org).The datasets generated for this study can be found in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015358 (The studies involving human participants were reviewed and approved by the Boston University School of Medicine. The patients/participants provided their written informed consent to participate in this study.SM, RS, and TI designed the research. SM, MJ, HT, and AD performed the research. SM, MJ, SG, TT, and TI analyzed the data. RS provided the CSF samples. SM and TI wrote the manuscript. SM, MJ, AD, SI, RS, and TI edited the manuscript.TI has sponsored research agreement from Aethlon Medical, Inc., for this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Macaca fascicularis) at baseline and two-week follow-up.Recent advancements have been made toward understanding the diagnostic and therapeutic potential of cerebrospinal fluid (CSF) and related hydrodynamics. Increased understanding of CSF dynamics may lead to improved detection of central nervous system (CNS) diseases and optimized delivery of CSF based CNS therapeutics, with many proposed therapeutics hoping to successfully treat or cure debilitating neurological conditions. Before significant strides can be made toward the research and development of interventions designed for human use, additional research must be carried out with representative subjects such as non-human primates (NHP). This study presents a geometric and hydrodynamic characterization of CSF in eight cynomolgus monkeys (2 respectively. Subarachnoid space cross-sectional area and hydraulic diameter ranged from 7–75 mm2 and 2–3.7 mm, respectively. Stroke volume had the greatest value of 0.14 ml at an axial location corresponding to C3-C4.Results showed that CSF flow along the entire spine was laminar with a Reynolds number ranging up to 80 and average Womersley number ranging from 4.1–7.7. Maximum CSF flow rate occurred ~25 mm caudal to the foramen magnum. Peak CSF flow rate ranged from 0.3–0.6 ml/s at the C3-C4 level. Geometric analysis indicated that average intrathecal CSF volume below the foramen magnum was 7.4 ml. The average surface area of the spinal cord and dura was 44.7 and 66.7 cm Cerebrospinal fluid (CSF) is a clear, colorless fluid with water-like mechanical properties that bathes the entire brain and spinal cord. CSF plays a role in the protection of neural structures, metabolic homeostasis of the central nervous system (CNS), autoregulation of cerebral blood flow, and immunological support for neural tissue. CSF moves freely in an oscillatory manner with approximately zero net flow, and in synchrony with cardiac-related intracranial pulsations and respiration –5. RecenThe importance of CSF dynamics has been investigated in several CNS conditions including syringomyelia  AlzheimeStudies have examined the possible role of CSF as a conduit for distribution of radiolabeled tracers  and therMacaca fascicularis). Cynomolgus monkeys are a useful model for such studies since they are relatively compact compared to other NHP species, and share physiologic and cognitive similarities to humans. Despite being a frequently studied species, CSF hydrodynamic properties have not been studied or reported. At present, we do not know how NHP CSF hydrodynamics compare to humans or if they are consistent across animals and/or over time. The aim of the present study was to a) develop an MRI-based method to quantify intrathecal CSF dynamics in cynomolgus NHPs, b) use this method to quantify intrathecal CSF dynamics and geometry in a series of NHPs (N = 8), and c) measure the reliability of MRI-derived measures over a 2-week time interval.While intrathecal delivery of drugs or biologics to the CNS offers a promising treatment option, the dearth of knowledge has slowed therapeutic development and potentially confounded the analysis of therapeutic effectiveness. A common animal model used to test intrathecal therapeutics is the NHP, with one of the most common species being the cynomolgus monkey . This study did not unnecessarily duplicate previous experiments and alternatives to the use of live animals were considered. Procedures used in this study were designed with the consideration of the well-being of the animals.MRI measurements were obtained for eight (NHP 01–08) healthy five-year-old adult cynomolgus monkeys  from Charles River Research Models, Houston TX with a weight of 4.4 ± 1.2 kg (mean ± standard deviation). NHP 01 was male. All other NHPs were female (02–08). These animals were purpose-bred and experimentally naïve. Each NHP was scanned with an identical protocol at baseline and at follow-up after a 2-week time interval.All MRI measurements were acquired at Northern Biomedical Research  on a Philips 3T scanner . Prior to MRI scanning each NHP was prepared using standard procedures and precautions. NHPs were positioned in the scanner in the supine position without assistance from artificial respiration. During each scan, heart rate and respiration was monitored continuously with ~ 1 liter/minute of oxygen and 1–3% isoflurane anesthetic administered via endotracheal tube for sedation.Table 1). The anatomical region scanned was ~30 cm in length, which included the intrathecal SAS below the lower brain stem extending caudally to the filum terminale. This comprised a total of ~720 images with 0.5 mm slice spacing, 1.0 mm slice thickness, and 0.375 mm isotropic in-plane resolution.Total scan time to quantify CSF space geometry and flow (including NHP MRI preparation) for each NHP was ~1 hour after the protocols were in place. A stack of high-resolution axial T2-weighted MR images of the complete spinal subarachnoid space (SAS) geometry was acquired for each NHP using a VISTA (31 minutes) protocol (Table 1). Slice location for each scan was oriented approximately perpendicular to the CSF flow direction with slice planes intersecting vertebral discs. More details on the PC-MRI protocol are given in Martin et al. [Thru-plane  CSF flow was measured by phase-contrast MRI (PC-MRI) images collected at six axial locations along the spine for each NHP. Axial locations were located at the foramen magnum (FM), C2-C3, C5-C6, T4-T5, T10-T11, and L3-L4 and required ~3 minutes scan time per location. Flow images were acquired with a retrospective ECG triggered sequence with 24 heart phases, 0.45 mm isotropic in plane resolution, and 5 mm slice thickness . The manual segmentation tool was used most frequently with view of the three orthogonal planes. Detailed information on the segmentation procedure is provided by Martin et al. [The high-resolution T2-weighted anatomic MRI images were semi-automatically segmented using the free open-source ITK-snap software  , which pn et al. . Once thFig 2) using GTFLOW software  by the following procedure previously described in [Fig 2A). Individual pixel velocities within each ROI were exported to a .CSV  file for further analysis using MATLAB software . CSF flow waveform within the ROI, Q(t), was computed with Q(t) = ∑ApixelVpixel(t), where Apixel is the area of one MRI pixel, Vpixel(t) is the velocity for the corresponding pixel at any time, and Q(t) is the summation of the flow for each pixel within the ROI. The CSF flow waveform was offset to ensure zero net flow over the flow cycle since CSF flow in the spine has approximately zero net flow (oscillatory).CSF flow was quantified at six axial locations along the spine , along the spine. The axial phase-contrast slice locations were not identical for each NHP due to differences of the exact vertebral levels across cases. Thus, the z-location of each slice was manually measured based on the distance of that slice caudal to the FM (Table 2). The six distinct flow rates were smoothed in a spatial-temporal fashion using MATLAB and a 2D “fit” function with the fit-type designated as “smoothing-spline”. Since heart rate variability was present between the PC-MRI scans, the CSF flow waveform timing was normalized to the average heart rate for all NHPs. An average spatial-temporal CSF waveform was determined. CSF pulse wave velocity, PWV, was computed based on the slope of the arrival time of peak CSF flow along the spine [The following method was applied to generate a smooth spatial-temporal CSF flow distribution, he spine .Several geometric and hydrodynamic parameters were calculated based on the 3D segmentation and flow analysis using our previously published methods . Using tThe following parameters were computed based on overall spine geometry: Total SAS surface area, SAsas, was calculated as the sum of surface area of spinal cord, SAc, and dura, SAd. Spinal cord nerve roots were not included in the surface area calculation of the cord since these small features were not possible to accurately visualize by MR imaging. Total volume of the SAS, Vsas, was computed by subtracting the volume of the spinal cord, Vc from the volume of the dura, Vd. An overall average, maximum, and minimum value was then computed across all NHPs. Total SAS length, Lsas, from the FM to the SAS termination was quantified.The following parameters were determined for each 1 mm interval along the spine (z-location): Axial distribution of the SAS cross-sectional area, Asas(z), was based on cross-sectional area of the spinal cord at that location, Ac(z), and dura, Ad(z). Similarly, hydraulic diameter, Dh(z) = 4Asas(z)/Psas(z), was determined based on the wetted perimeter, Psas(z), with the perimeter computed as the sum of the spinal cord, Pc(z), and dura, Pd(z), perimeters at each z-location. Axial distribution of CSF stroke volume was computed as SV(z) = ∫|Q|dt, where |Q| is the absolute value [Qsys(z) and Qdia(z), and the CSF flow rate amplitude was given by Qa(z) = Qdia(z)−Qsys(z). Spatial mean thru-plane velocity at peak systole was computed as v is the kinematic viscosity of CSF at body temperature, 0.693 mPa∙s [ω is the angular velocity (ω = 2π/T) of the volume flow waveform with T equal to the heart rate.te value . Peak sy93 mPa∙s . WomerslLsas measured for all NHPs. After normalization, the mean axial distribution for each parameter was computed across all NHPs. The mean axial distribution was then used to obtain an average, maximum, and minimum parameter value along the spine based on all NHPs.To allow parameter comparison across NHPs, each parameter’s axial distribution for each NHP was normalized to the average Reliability was assessed by obtaining MRI measurements for each NHP at baseline and 2-week follow-up while ensuring identical methods during both collection intervals. To quantify measurement reliability, we performed a regression of baseline versus follow-up parameters computed at each axial location along the spine. All computations and plots were generated using MATLAB software .Table 3). Overall, the MRI protocol allowed quantification of all proposed geometric and hydrodynamic parameters. These parameters had a relatively similar axial distribution across all NHPs analyzed and were similar at follow-up for each NHP. CSF flow was laminar in all NHPs with the greatest degree of CSF motion observed in the cervical spine. Average results showed that maximum Re and α was 80 and 7.7, respectively. Asas and Dh ranged from 7–75 mm2 and 2–3.7 mm, respectively. Maximum SV ranged from 0.14 ml in the cervical spine to roughly 0 ml in the lower lumbar spine for all NHPs.Results were obtained for a total of eight NHPs at baseline and a 2-week follow-up time point . The minimum value for Asas and Dh was 7 mm2 and 2 mm, respectively (Table 3). These values occurred at ~70 mm caudal to the FM, a location approximately corresponding to T2-T3 (Table 2). Maximum difference in Asas and Dh between NHPs at any axial location (omitting the FM) was ~30 mm2 and 4 mm, respectively.Average Q(t) of each NHP quantified along the spine had a similar waveform shape, magnitude and axial distribution . Q(t) shape showed a well-defined systolic peak at 100 to 300 ms (negative flow) followed by a diastolic peak that varied based on the heart rate (similar to cardiac blood flow). Qsys ranged from 0.35–0.87 (ml/s) at the C3-C4 level for all NHPs. Q(t) at the FM was markedly smaller than at C3-C4. Caudal to C3-C4, Q(t) had a decreasing trend in magnitude moving down the spine. The CSF flow was found to be nearly zero in all PC-MRI scans before eddy current offset correction. Maximum average CSF flow offset was 13% relative to the arithmetic mean of the absolute CSF flow.Q(t) distribution across all NHPs showed a relatively smooth decrease in amplitude along the spine and had relatively small, if any, wave reflections from the SAS termination . Spatial temporal Q(t) distribution showed that maximum CSF flow rate occurred ~25 mm caudal to the FM . Q(t) shape and magnitude were similar from ~125 mm to the SAS termination.Average spatial-temporal SV ranged from ~0 to 0.14 ml along the spine and had the greatest value at the axial location corresponding to C3-C4 . Difference in SV between NHPs was a maximum of ~ 0.1 ml at the upper cervical spine (C3-C4).Q(t) along the spine . This phase shift is thought to be representative of intrathecal space stiffness or compliance and can be quantified in terms of PWV. Based on the time of Qsys at the FM versus the lumbar spine, PWV was estimated to be vary from 0.73 to 3.45 m/s among NHPs with an average value of 1.13 m/s (Table 3).A noticeable phase shift was observed in the Re had a decreasing trend moving caudally along the spine . Re varied from 80 in the cervical spine to 0 at the most caudal region, with the maximum value located at C3-C4 level. Local difference in Re among the NHPs was a maximum of ~75 and located within the cervical spine. α ranged from 4 to 7.7, with a maximum value located near the FM .Fig 6E). Asas; axial locations with largest Asas  demonstrated reduced velocities compared to areas with smaller Asas and their respective increased velocities.The peak value of the Figs 7 and 8. The results correspond to all eight NHPs and are plotted for the entire spine model between baseline and follow-up (from FM to the SAS termination).There was relatively good agreement between the baseline and follow-up MRI scans across all parameters confirming the reproducibility of the method. Differences between geometrics and hydrodynamic parameters obtained from the baseline to the follow-up MRI scan were quantified using regression analysis as shown in Pd , Pc , and Psas . Correlation was stronger for the Ad  and Ac , but was slightly weaker in Asas .Strong correlation was observed from the linear regression analysis for Fig 8) showed that the value of discrepancy between baseline and follow-up results could be higher for hydrodynamic parameters. There was a relatively weak correlation for α and Dh . Relative to geometric results, there was additional discrepancy for flow parameters related to R2 = 0.72, slope = 0.93), Qa , and SV , though not to the degree as the α and Dh.The second set of regression plots (Macaca fascicularis). Results show that CSF geometry and dynamics can be reliably detected using non-invasive MRI measurements and that results are consistent for cynomolgus monkeys of a similar size and age.This study presents a method and results for detailed characterization of intrathecal CSF geometry and hydrodynamics in cynomolgus monkeys . Re was computed to represent the ratio of steady inertial forces to viscous forces and help indicate whether laminar flow (Re<2300) was present at each phase contrast slice location . A laminar CSF flow indicates that the flow is smooth with relatively little lateral mixing. This is different from a turbulent flow, where chaotic changes in pressure and velocity occur and can lead to a large increase in lateral mixing. Thus, CSF flow is expected to remain laminar throughout the CSF flow cycle as the Re remained sub-critical  for all NHPs analyzed. However, it is possible that disease states that result in strongly elevated CSF flow velocities (jets) could result in turbulence [Our results show that CSF moves in a smooth oscillatory manner along the entire spinal axis of cynomolgus NHPs. Chaotic velocity or pressure fluctuations are not expected and transverse CSF velocities (non-streamwise) are likely small compared to axial velocities. CSF dynamics were found to be most active in the cervical spine near the C3-C4 vertebral level with a maximum rbulence .α varied in the same fashion as Dh with a minimum and maximum value of 4.1 and 7.7 . α, was computed to quantify the ratio of unsteady inertial forces to viscous forces that impact the CSF velocity profile shape [α<1, the CSF velocity profiles will be parabolic in shape. α>10 will result in relatively flat or plug-like velocity profiles [α [Fig 2C) as the MRI resolution was not fine enough to accurately capture the relatively thin boundary layer expected in a blunt flow profile.Inertial effects are expected to dominate the SAS CSF flow field for normal physiological flow rates, frequencies and CSF fluid properties. le shape . For α<1profiles . This mefiles [α . Our prefiles [α . It is dPWV~ 1.13 m/s . This wave appeared to be damped along the spinal axis and had relatively little reflection due to the SAS termination. This PWV is similar to the study previously reported by our group [PWV studies have been conducted for humans. Williams obtained simultaneous invasive recordings of ventricular and lumbar CSF pressure in humans during various maneuvers such as coughing and valsalva [PWV can be estimated to range from 8–4 m/s, after coughing. Kalata et al. used high-speed PC-MRI to quantify the CSF velocity wave speed in a small portion of the cervical spine (~20 cm) and found it to be 4.6 ± 1.7 m/s [PWV to be ~3 m/s [PWV varied from 2.5 to 13.5 m/s depending on dura elasticity [in vitro models and found CSF wave reflections to be present [Fig 5B).With each heartbeat, a cardiac-induced CSF pulse wave was found to travel in the cranial-caudal direction (downwards) at a rate of ur group  for one valsalva . From th 1.7 m/s . Anothere ~3 m/s . Martin asticity . They al present . Similar present –44. ResuPWV has been found to have important implications in several vascular diseases [PWV could also have implications on perivascular transport in context of syringomyelia [PWV in the spine and its relevance CNS physiology in health and disease.Arterial diseases ,46. Spingomyelia –49. FurtAc, Pc, and Dh in a cynomolgus monkey has not been reported in the literature. This may be due, in part, to the relatively long time period  required to obtain the high-resolution MRI images (375 μm isotopic) used to segment the CSF space in this study. Geometric parameters such as Ad, Ac, Asas, Pd, Pc, and Psas were shown to vary significantly along the spine. Hydrodynamic parameters such as Dh, Re, α, Qa and SV also varied significantly along the spinal canal due to the changes in geometry. CSF flow measurements in the cervical spine by MRI were used to estimate flow values of hydrodynamic parameters. The variation in Asas is significant ~7 to 75 mm2 , which indicates fluid acceleration may be significant in the spinal cavity near the skull and base of the spine. Dh ranged from ~1.5–4.5 mm in all NHPs analyzed. The axial distribution of SAS geometry in the cynomolgus monkey had a similar trend as that quantified in humans for Ac, Pc, and Dh [To the best of our knowledge, axial variation in spinal SAS geometry in terms of , and Dh , albeit , and Dh .Vsas for all NHPs in this study was ~7.41 ml. To our knowledge, Vsas has not been measured in NHPs. However, total NHP CSF volume is typically considered to range from 12–15 mL in CSF dosing studies [Average  studies . Our res studies . Recent  studies ,53. DetaAd, Ac, Asas, Pd, Pc, and Psas in Fig 7) and to a lesser degree for hydrodynamics based parameters . The reason for lower degree of reliability for hydrodynamic parameters is likely because these parameters incorporate input from both flow and geometry, both of which will have associated error and/or natural physiologic variation in NHPs. It should also be noted that we do not expect all parameters to remain identical at the 2-week follow-up time point as CSF flow can be altered due to posture, sedation, and other factors that were not specifically controlled to be identical across MRI scans in the present study. Nevertheless, the degree of reliability is presented to give a benchmark for how much these parameters can change under normal conditions. A previous study by Martin et al. showed a high degree of inter- and intra-operator reliability for MR-based geometric and hydrodynamic parameters derived from the SAS for a single patient with Chiari malformation and a healthy control subject [To help understand parameter reliability, we collected MRI images for 8 NHPs at two time points separated by a two-week time interval. Results showed a relatively strong degree of parameter reliability for all geometric-based parameters  with reliability assessed between baseline and a two-week follow-up time point. Results showed laminar CSF flow along the entire spine with maximum CSF flow rate at the C3-C4 vertebral level and peak systolic CSF flow rate and stroke volume at C3-C4. The methods presented demonstrate a reliable method for CSF quantification in NHPs, which may extend in future studies to Homo sapiens.This study presents a detailed geometric and hydrodynamic characterization of intrathecal CSF for eight cynomolgus monkey .(XLSX)Click here for additional data file.


There is a need for clinically useful biomarkers of disease activity in clinically isolated syndrome (CIS) and relapsing remitting MS (RRMS). The aim of this study was to assess the correlation between neurofilament light chain (NFL) in cerebrospinal fluid (CSF) and serum and the relationship between NFL and other biomarkers, subsequent disease activity, and brain volume loss in CIS and RRMS.A panel of neurodegenerative and neuroinflammatory markers were analyzed in repeated CSF samples from 41 patients with CIS or RRMS in a prospective longitudinal cohort study and from 22 healthy controls. NFL in serum was analyzed using a single-molecule array (Simoa) method. “No evidence of disease activity-3” (NEDA-3) status and brain volume  were recorded during 4 years of follow-up.n = 63, r 0.74, p < 0.001), but CSF-NFL showed an overall stronger association profile with NEDA-3 status, new T2 lesions, and brain volume loss. CSF-NFL was associated with both new T2 lesions and brain volume loss during follow-up, whereas CSF-CHI3L1 was associated mainly with brain volume loss and CXCL1, CXCL10, CXCL13, CCL22, and MMP-9 were associated mainly with new T2 lesions.NFL levels in CSF and serum correlated significantly  contains supplementary material, which is available to authorized users. Multiple sclerosis (MS) is a chronic disease characterized by inflammation and degeneration of the central nervous system (CNS). Relapses, new T2 lesions, and disability progression are conventional signs of disease activity in MS. To aid personalized treatment, prognostic biomarkers and biomarkers of disease activity are needed. Assessing brain volume loss and biomarkers in cerebrospinal fluid (CSF) or serum (S) could potentially give a more comprehensive evaluation of disease activity –4.Brain atrophy is present early in the disease course , 6 and aThis study aimed to assess the correlation between S-NFL and CSF-NFL, to evaluate NFL levels in relation to other biomarkers and disease activity parameters and to identify parameters associated with number of new T2 lesions and brain volume loss during 4 years of follow-up in a longitudinal cohort of patients with newly diagnosed CIS and RRMS.Forty-one patients with CIS or RRMS were consecutively enrolled in a prospective longitudinal cohort study of CIS and newly diagnosed MS at the Department of Neurology, University Hospital of Linköping, Sweden. All patients fulfilled the revised McDonald criteria from 2010  for CIS n = 20 at 1 year, n = 12 at 2 years, and n = 7 at 4 years), while patients with relapses, brain MRI activity, or sustained disability worsening were classified as showing evidence of disease activity (EDA-3) . All 41 patients were evaluated at 1 year and 39 patients were evaluated at 2 and 4 years of follow-up. At 2 years, one pregnant patient did not undergo MRI and one patient had left the study. At 4 years, one additional patient had left the study, while the previously pregnant patient completed 4-year follow-up.All clinical assessments regarding relapses and EDSS were performed by the same neurologist (IH), and all MRI examinations were thoroughly reviewed by the same neuroradiologist. Patients that showed no relapses, no brain MRI activity (no new or enlarging T2 lesions or Gadolinium-enhancing lesions), and no sustained disability worsening (EDSS progression) during follow-up were classified as showing “no evidence of disease activity”-3 (NEDA-3) (g for 10 min.) and the supernatant was aliquoted and immediately frozen and stored at − 70 °C.All CSF sampling was carried out by the same neurologist (IH), and CSF was always collected 8–12 a.m. Serum samples were collected directly after CSF collection. One aliquot of the CSF sample was used for cell counting, CSF/serum albumin ratio, IgG index, IgG synthesis index, and isoelectric focusing, all according to clinical routines performed at the Department of Clinical Chemistry, Linköping University Hospital. Within 1 hour, the remaining CSF was centrifuged  according to the manufacturer’s instructions, except that an additional lower standard point was added to the standard curve. The measurements were performed using Luminex®200™ . For data acquisition, the software program xPONENT 3.1™  was used, and for data analysis, MasterPlex® Reader Fit was used. The detection limits were 16 pg/mL for CXCL1, CXCL10, and CCL22; 3.2 pg/mL for CXCL8; and 3.9 pg/mL for CXCL13. Values below the detection limit were assigned half the value of the detection limit.CSF NFL concentration was measured using the NF-light assay according to instructions from the manufacturer . CSF NFH concentration was measured using the Phosphorylated NEFH (Human) ELISA Kit according to instructions from the manufacturer . CSF MMP-9 concentration was measured using the Human MMP-9 Base Kit according to instructions from the manufacturer . CSF GFAP concentration was measured using an in-house ELISA as previously described . CSF CHIS-NFL concentration was measured using the NF-Light kit from UmanDiagnostics , transferred onto the Simoa platform using a homebrew kit , as previously described in detail . The lowAll subjects were examined on a 1.5-T Philips Achieve MRI scanner  using an eight-channel phased array head coil. Quantitative MRI images were acquired using QMAP sequence . BPF wasU test was used to compare two groups and non-parametric bivariate correlation analysis (Spearman) was used for correlation analyses. The relationship between NFL in CSF and serum was examined using bivariate linear regression and Spearman correlation analysis, as well as Pearson correlation analysis when sample size was > 50. Friedman’s test with Dunn correction for multiple comparisons was used to compare repeated measurements of immunological markers in patients over time. Repeated measures ANOVA with Bonferroni correction for multiple comparisons was used to compare repeated BPF measurements in patients over time. Multiple linear regression analysis was used to evaluate brain volume loss over time and number of new T2 lesions over time. Logistic regression was used when investigating NFL and other markers in relation to NEDA. Receiver operating characteristic (ROC) curves were derived from logistic regression to investigate the discriminatory power of NFL and other markers between patients with and without disease activity during follow-up. Because of multiple testing, a stringent p value of ≤ 0.01 was considered to be significant in Mann-Whitney U tests, t tests, repeated measures ANOVA, and linear regression analyses. In Spearman correlation analyses, a very stringent significance level of Spearman’s rho ≥ 0.5 and p ≤ 0.01 was used, which entailed a maximum type II error of 0.21 when n = 41 and 0.27 when n = 37. For comparisons within the CIS group, where patient numbers were small and comparisons few, a p value of ≤ 0.05 was considered significant in Mann-Whitney U tests. Area under curve (AUC) values were compared using the DeLong method, in MEDCALC®, with the significance level set at 0.05. All p values were based on two-tailed statistical tests.Statistical analyses were performed using SPSS for Windows, version 23. Analyzing data sets with non-Gaussian distribution, the Mann-Whitney n = 22, Spearman’s rho 0.59, p = 0.004), in patients at baseline , in samples from patients at baseline and HC combined . R2 from bivariate linear regression with S-NFL as dependent variable and CSF-NFL as independent variable (n = 63) was 0.54, p < 0.001, equation y = 0.007x + 10.1. A correlation plot is presented in Fig. S-NFL and CSF-NFL correlated in HC  were compared with healthy controls. Both S-NFL and CSF-NFL were significantly higher in patients than in controls, see Fig. R2 0.01, p = 0.5 for CSF-NFL and R2 0.003, p = 0.7 for S-NFL in simple linear regression). In healthy controls, on the other hand, age was a significant factor in relation to both CSF-NFL and S-NFL levels . S-NFL levels, just like recently reported for CSF-NFL levels [p = 0.9 for S-NFL and p = 0.4 for CSF-NFL) or between patients with and without a relapse starting within 2 months prior to sample collection at baseline (p = 0.7 for S-NFL and p = 0.1 for CSF-NFL). However, when including not only baseline samples but also follow-up samples, thereby increasing total n from 41 to 161, RRMS patients had higher CSF-NFL (p = 0.01), but not S-NFL (p = 0.6), than CIS patients, and NFL levels were higher in relapse patients than in non-relapse patients (p = 0.004 for S-NFL and p ≤ 0.001 for CSF-NFL).In patients, neither CSF nor serum NFL could be explained by age , but did not differ significantly at 1 and 4 years (p > 0.05). With a cutoff value of ≥ 450 ng/L for baseline CSF-NFL, the sensitivity of identifying patients with disease activity was 95% at 1 year, 93% at 2 years, and 81% at 4 years, with specificity 50, 67, and 57%, respectively. With a cutoff value of 14.2 ng/L for baseline S-NFL, sensitivity of identifying patients with disease activity was 76% at 1 year, 74% at 2 years, and 72% at 4 years, with specificity 50, 50, and 57%, respectively. Thus, low levels of CSF-NFL entail a high probability of NEDA during follow-up.Area under curve (AUC) for baseline levels of NFL in relation to disease activity during 1, 2, and 4 years of follow-up were 0.75, 0.65, and 0.69 for S-NFL, and 0.81, 0.85, and 0.73 for CSF-NFL; ROC curves are presented in Fig. p ≥ 0.05). CXCL10 was the only marker with all three  AUCs above 0.8.For prediction of NEDA-3/EDA-3 during 1 and 2 years of follow-up, baseline CXCL10 and baseline CSF-NFL had the highest ROC-based AUCs (0.81–0.85) and for prediction of NEDA-3/EDA-3 during 4 years of follow-up, baseline MMP-9, CXCL10, CCL22, and CXCL13 had the highest AUCs (0.80–0.83), see Additional file p ≥ 0.05). These data show that NFL levels in CSF during follow-up, especially mean levels, are highly associated with ongoing disease activity expressed as NEDA-3/EDA-3.When evaluating associations with disease activity, both in the cross-sectional and the longitudinal perspective, AUCs from follow-up levels as well as mean levels of follow-up levels were assessed, in addition to AUCs from baseline levels  and BPF at 2 and 4 years were significantly lower than at 1 year (p < 0.001 and 0.004). However, BPF at 4 years was not lower than at 2 years (p = 0.9). Time from baseline MRI to follow-up MRI (mean ± SD) was 12 ± 1 months, 24 ± 1 months, and 44 ± 5 months for 1-, 2-, and 4-year follow-up, respectively.One patient was excluded at all four time points due to very low BPF associated with congenital hydrocephalus. Three patients were excluded due to missing data. BPF data from the 37 patients that completed all four BPF measurements are presented in Fig. p = 0.001), but not two  and four  years of follow-up.Mean BPF decrease was 0.49/year from baseline to 1 year, 0.50/year from baseline to 2 years, and 0.36/year from baseline to 4 years. Mean BPF decrease from baseline to 4-year follow-up was significantly higher in patients with EDA-3 than in patients with NEDA-3 during 1 year  and a peak adjusted R2 of 0.41 (p = 0.001) was noted for a model including age, CSF-mononuclear cells, NFH, MMP-9, CHI3L1, and T2 lesions at baseline. As for association with BPF decrease, adjusted R2 peaked with a model including baseline T2 lesions, baseline OPN, and 1-year CHI3L1 , and the single parameter that performed best was 1-year CHI3L1 . For prediction of new T2 lesions, no model reached the significance level p ≤ 0.01, whereas for association with new T2 lesions, adjusted R2 peaked with a model including mean CCL22, 1-year CSF-NFL, and baseline CXCL13 , and the single parameter that performed best was 1-year CSF-NFL . Details of linear regression statistics are given in Additional file To identify the most important parameters for prediction (using baseline parameters only) of, and for association (follow-up data included as well) with, BPF decrease and new T2 lesions, linear regression modeling of BPF decrease and new T2 lesions during 4 years of follow-up was performed. Details about these procedures are given in Supplemental data. For prediction of BPF decrease, baseline CHI3L1 was the single parameter that performed best (n = 19), CXCL10, NFL, and MMP-9 in CSF were significantly higher (p ≤ 0.05 in Mann-Whitney U tests) in patients that converted to RRMS during 4 years of follow-up (n = 12) than in non-converters (n = 7), whereas S-NFL (p = 0.10) and the other markers (p = 0.07–0.90) did not differ. For the 12 CIS patients that converted to RRMS during the study, multiple regression modeling of time to conversion resulted in a highly significant model  when including baseline CSF levels of CXCL1, CXCL10, CXCL13, and NFL, and number of T2 lesions at baseline).Within the CIS group at baseline (r 0.74) that is in line with what has been reported by others [In the present study, we report a correlation between NFL levels in CSF and serum  or with new T2 lesions . In linear regression modeling of new T2 lesions, the combination of 1-year CSF-NFL, CCL22 (mean level) and CXCL13 (baseline level) performed better than 1-year CSF-NFL alone, suggesting an added value of combining several markers, which was recently shown for chemokines and OPN . Of noteExpanding NEDA-3 to NEDA-4 and NEDA-5 by including brain volume loss and biomarkers could improve assessment of disease activity in MS. Number of T2 lesions and T2 lesion volume in conventional MRI correlate poorly to clinical disease manifestations and disease progression in MS , althoug, thereby exploring association over time instead of prediction. When examining, for example, the association between mean level of NFL during follow-up and NEDA-3 status during follow-up, the impact of treatment is negligible or small unless treatment affect NFL and NEDA-3 status very unevenly. Study strengths are the control group, consisting of sex- and age-matched healthy individuals, that the patient group consisted of well-characterized CIS and MS patients examined and thoroughly followed up prospectively by the same neurologist in a standardized way and that all patients were untreated at baseline. Also, a broad panel of potential biomarkers was included. The present study provides 4-year follow-up data on CSF-NFL in a patient cohort that we have previously presented 2-year follow-up data from, and in addition, we report S-NFL data as well as brain volume analyses for the entire study period (4 years). Taken together, this is the first study that reports longitudinal data on these markers, including NFL levels in CSF and serum, in relation to both NEDA-3 and brain volume loss in a clinical setting study.Limitations of this study include the cohort size, varying treatment and lack of MRI data from healthy controls. Varying treatment regimens in the cohort could lower the prognostic value of baseline biomarker levels. However, despite the fact that some patients received disease modifying drugs during follow-up, baseline levels of some proteins clearly showed prognostic value. We also focused on NEDA status, new T2 lesions, and BPF decrease in relation to follow-up levels and mean levels of NFL, etc.We show that although serum and CSF levels of NFL correlate significantly, CSF-NFL predicts and reflects disease activity better than S-NFL during follow-up in our cohort of patients with CIS and RRMS. CSF-NFL is associated with both new T2 lesions and brain volume loss during follow-up. Our findings further add to the accumulating evidence that CSF-NFL is a useful biomarker in CIS and RRMS and should be considered in the expanding NEDA concept. Our findings also support S-NFL as a potential biomarker in CIS and RRMS. Other biomarkers that need further attention include CXCL10 and CHI3L1 for prediction of NEDA-3 and brain volume loss, respectively.Additional file 1:Table S1. Neurodegenerative and neuroinflammatory markers in CSF  over time in patients with CIS and RRMS. (DOCX 23 kb)Additional file 2:Table S2. Neuroinflammatory and neurodegenerative markers with respect to separation of patients with and without disease activity during follow-up. (DOCX 17 kb)Additional file 3:Linear regression modeling details. (DOCX 12 kb)


Patients who continued low-dose oral prednisolone maintenance therapy after methylprednisolone pulse therapy showed sustained improvement in OMDS and CSF CXCL10 and neopterin levels lasting for 2 years. In contrast, OMDS and the CSF marker levels in patients who discontinued treatment returned to pre-treatment levels. This rebound phenomenon was also observed in patients who discontinued oral prednisolone therapy independently of pulse therapy. Our findings suggest that CSF CXCL10 may serve as a therapy-response and therapy-predictive marker for HAM/TSP. In addition, since decrease in CSF CXCL10 level was associated with good functional prognosis, CSF CXCL10 is a potential surrogate marker for treatment of HAM/TSP.Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a debilitating, progressive disease without effective treatment; therefore, development of disease modifying therapy that improves long-term functional outcomes is an unmet need for patients. However, it is virtually impossible to consider this as a primary endpoint in clinical trials owing to the prolonged disease course. Therefore, development of surrogate markers that help predict the effectiveness of new interventions is essential. Currently, several candidate surrogate markers have been identified for HAM/TSP. Cerebrospinal fluid (CSF) C-X-C motif chemokine 10 (CXCL10) is involved in the pathogenesis of HAM/TSP and was shown to correlate with disease progression. However, it remains unclear whether changes in CSF CXCL10 levels are observed in response to treatment and whether these correlate with prognosis. Here we investigated several markers, including CSF CXCL10, in this respect. Data pertaining to patient characteristics and results of motor function evaluation and CSF examination of 13 HAM/TSP patients who received steroid treatment were retrospectively analyzed. Osame motor disability scores (OMDS), 10 m walking time, and CSF levels of CXCL10, neopterin, total protein, cell counts, and anti-HTLV-1 antibody titer were compared before and after steroid therapy. Levels of all CSF markers, with the exception of cell count, were significantly decreased after treatment. Nine of the 13 patients (69.2%) showed improvement in OMDS and were considered responders. Pre-treatment CSF levels of CXCL10 and anti-HTLV-1 antibody titer in responders were higher than those in non-responders ( Human T-cell leukemia virus type 1 (HTLV-1) is a human pathogenic retrovirus . A propoAn ideal surrogate marker should qualify the following criteria :1.good correlation with disease progression;2.biological plausibility that shows association with the true endpoint;3.correlation with the true endpoint independent of treatment; and4.the effect of treatment on the surrogate marker correlates with its effect on the true endpoint.Several candidate surrogate markers in the context of HAM/TSP have already been identified; these include Cerebrospinal fluid (CSF) markers , anti-HTLV-1 antibody titer, cell count, and total protein) and HTLV-1 proviral load . Among tCorticosteroids are commonly used to treat HAM/TSP in endemic areas . These aIn this study, we retrospectively investigated the clinical course and the time course of CSF marker levels (including CXCL10) in 22 HAM/TSP patients . Next, we sought to identify patients who are more likely to benefit from steroid therapy; for this purpose, we compared the pre-treatment marker levels between patients who showed improvement in Osame motor disability scores (OMDS) post-treatment and those who did not show improvement post-treatment. Furthermore, in order to examine the linkage between the clinical course and the chronological changes in CSF marker levels, we investigated three groups: patients who received prednisolone maintenance therapy after methylprednisolone pulse therapy, patients who did not receive prednisolone maintenance therapy, and independently of pulse therapy, patients who discontinued oral prednisolone therapy.The study was approved by the Institutional Review Board of St. Marianna University School of Medicine (#1646) and Fukuoka University Hospital (#14-2-08). Prior to the collection of blood or CSF samples, all participants provided written informed consent for analysis of their samples for research purposes as part of their clinical care.This was a retrospective observational study. All subjects were diagnosed with HAM/TSP based on the World Health Organization criteria . The clin = 6) received oral prednisolone therapy . HTLV-1 proviral load was measured using real-time PCR, following DNA extraction from PBMCs, as previously described  and HTLV-1-proviral load in peripheral blood mononuclear cells (PBMCs). In both hospitals, lumbar puncture for CSF examination is performed before and about 2 weeks after the start of treatment (mean ± standard deviation (SD): 2.5 ± 0.9 weeks from the first day of pulse therapy). CSF samples and PBMCs were prepared as described previously . Brieflyescribed .p-values are two tailed, and the threshold of significance was set at 0.05.The Wilcoxon signed rank test was used to compare the pre- and post-treatment biomarker levels in the same patient. The Mann–Whitney U test was used to compare baseline values of biomarkers between two patient groups (responders and non-responders). Fisher’s exact test was used to examine the relationship between additional oral prednisolone therapy and the change in OMDS during the observation period. Statistical analyses and graph composition were performed using R version 3.2.2  or GraphPad Prism 7 . All The median age (range) of 13 HAM/TSP patients  who received methylprednisolone pulse therapy in the treatment group was 67 (33–72) years; the median (range) baseline OMDS was 6 (3–9) . We defiThe percentage of patients whose OMDS improved by ≥1 after 1 month from the start of steroid therapy was 57.1% (four of seven patients) at the Fukuoka University Hospital and 83.3% (five of six patients) at the St. Marianna University Hospital; the overall percentage was 69.2% . Among tn = 11 or 12). As shown in p = 0.0005, p = 0.0005, p = 0.0059, and p = 0.0078, respectively). CSF cell counts also tended to decrease; however, the difference was not significant (p = 0.0645). When comparing the pre- and post-treatment levels for each hospital, significant reduction was observed only for two out of the five CSF markers (CXCL10 and neopterin) (data not shown). In contrast, none of the five markers showed a significant reduction in HAM/TSP patients (n = 5) who were not treated with steroids or interferon-α . CSF neopterin concentration was not significantly different (p = 0.187); however, after exclusion of one non-responder (outlier), CSF neopterin was significantly higher in responders (p = 0.009). CSF cell count showed a higher tendency in responders (p = 0.070). There was no significant difference between the two groups with respect to total protein level (p = 0.796); nevertheless, the total protein level was high in some of the responders. Although the number of patients who we investigated was limited, the HTLV-1 proviral load in PBMCs did not differ significantly between responders and non-responders (p = 0.667).HTLV-1-associated myelopathy/tropical spastic paraparesis patients who showed improvement in OMDS were defined as responders (9 out of 13 patients). In order to identify predictors of therapeutic response, we compared the pre-treatment marker levels between responders and non-responders . In addin = 8) and the group that did not receive oral prednisolone (n = 5). The OMDS in seven of eight HAM/TSP patients who received maintenance therapy improved compared with that at baseline; the improvement was maintained afterward in all but one patient (no. 6) who showed no change throughout 2 years (p = 0.035). We were able to obtain longitudinal data pertaining to CXCL10 and neopterin levels in CSF from seven of eight patients who received maintenance therapy and three of five patients who did not receive maintenance therapy. As shown in Next, the clinical course and the time course of CSF marker levels for approximately 2 years after initiation of treatment were compared between the group that received maintenance therapy with oral prednisolone after methylprednisolone pulse therapy  in the spinal cord . TherefoThis study has mainly two limitations. The observation period of 2 years was relatively short considering the prolonged clinical course of HAM/TSP. Second, there were many missing data due to retrospective data collection. Future prospective study involving data collection over a longer period of time may provide more definitive evidence. However, a prospective study may pose a challenge as denial of maintenance therapy to patients with high disease activity is ethically difficult after the importance of maintenance therapy becomes apparent.In conclusion, the present study suggests that CSF CXCL10 may serve both as a marker and predictor of therapeutic response in patients with HAM/TSP. In addition, since it is likely that the therapeutic effect on CSF CXCL10 is associated with the effect on long-term functional prognosis of HAM/TSP patients, CSF CXCL10 has a potential to serve as a surrogate marker for treatment of HAM/TSP.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Institutional Review Board of St. Marianna University School of Medicine (#1646) and Fukuoka University Hospital (#14-2-08). The patients/participants provided their written informed consent to participate in this study.KT, YY, and YT conceived and designed the study. KT, TS, JT, SF, JY, and YY collected the clinical data. KT and TS analyzed the results. KT, TS, NY, NA, JY, AC-R, MN, YH, and YY drafted and corrected the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objectives: Elevated neurofilament light chain (NFL) levels within the cerebrospinal fluid (CSF) are a biomarker representing axonal neurodegeneration in rapid progressive neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). It is unclear to what extent the levels of NFL increase in the CSF (CSF-NFL) in a chronic neuroinflammatory process with axonal neurodegeneration, as found in primary progressive multiple sclerosis (PPMS).Methods: We used a multicenter approach to statistically compare CSF-NFL levels between PPMS patients (n = 50), ALS patients (n = 50), and healthy controls (n = 50). Clinical findings, including disease duration, expanded disability status scale (EDSS), electrophysiological recordings such as visual evoked potentials or spinal and cerebral MRI, and previously administered treatment were selected as experimental parameters retrospectively.Results: Median [range] CSF-NFL concentrations in PPMS patients were significantly higher than in the controls , and significantly lower compared to ALS patients . There was no correlation between CSF-NFL and disease duration (p = 0.5), EDSS (p = 0.2) or treatment (p = 0.3).Conclusion: We conclude that CSF-NFL may mirror the proposed slow axonal degeneration in PPMS, but does not reflect the disease severity. The pattern underlying the concentrations of neurofilament light chain (NFL) in the cerebrospinal fluid (CSF), referred to as CSF-NFL, in diseases with slow progressive axonal degeneration, including primary progressive multiple sclerosis (PPMS), are not clear. Aggressive axonal injury after acute inflammatory events in patients with multiple sclerosis (MS), and rapidly progressive neurodegenerative diseases with a predominant affection of the central motor system such as amyotrophic lateral sclerosis (ALS), result in a clear pathological increase of the CSF-NFL levels –3. In coHere we conducted a cross-sectional multicenter study measuring the CSF levels of the stable protein NFL  in patien = 50 patients with clinically definite PPMS according to the McDonald criteria 2010  recruited in Magdeburg, comprising cases with non-specific complaints who underwent lumbar puncture (LP) during a routine diagnostic examination conducted to rule out any neurological condition. None of the controls suffered from a neurological disorder (neuroinflammatory or neuromuscular), in particular not from MS, peripheral polyneuropathies, muscle or motor neuron disease, nor did they display any specific abnormalities during the neurological examination  and Kiel D525/16) and a general commitment of the Charité Berlin Clinic for external analyzes of retrospective data in line with an external German ethics vote. We included CSF samples from (i) ria 2010  recruitecriteria  recruitemination , 16. In  and a ge and KielClinical scoring [Expanded disability status scale (EDSS)]  was perfEDSS, T2-weighted MRI conducted close to the LP, and CSF levels were selected as parameters to verify the PPMS diagnosis, which was based on the clinical progression over the course of 1 year. These parameters were observed together with the existence of cerebral or spinal cord T2-intense lesions or oligoclonal bands (OCB) , and resVEP evaluations were documented to describe axonal integrity loss in PPMS according to previously defined diagnostic criteria of PPMS . LatenciDisease duration was defined as the time in months between symptom onset and the LP.alb) and oligoclonal bands (for PPMS patients only) were measured. Every sample was stored at −80°C and shipped on dry ice for CSF-NFL measurement. CSF-NFL levels were determined in Magdeburg using commercially available ELISA kits . Intraassay coefficient of variance is 7.4% and interassay coefficient of variance is 6%  applying pairwise Dunn-Bonferroni post-hoc testing). Spearman's rank correlations were performed between CSF-NFL and age, between CSF-NFL and further CSF measures , as well as between CSF-NFL and clinical scores (EDSS). P-values < 0.05 were deemed to be statistically significant.Statistical analysis was conducted using SPSS 21 (IBM). Groups were compared with respect to categorical (using a χ2-test) and continuous variables (using a p = 0.2) related differences between PPMS, ALS and HC, whereas the values for the CSF cell count, protein and Qalb varied between the diagnostic groups Table p < 0.001]. For PPMS, disability severity was high (median EDSS 6) at the time of the LP. The PPMS patients recruited from the three different University centers did not differ with respect to age, sex, CSF protein, oligoclonal band positivity, median EDSS or disease duration. There were, however, inter-center differences regarding the prevalence of spinal cord lesions (p = 0.017), and the values for CSF cell count and Qalb. Several PPMS patients, n = 11 (28%), received immunomodulatory and immunosuppressive therapy (“treatment attempt”), comprising Rituximab (n = 2), and intravenous (n = 3) or intrathecal (n = 6) methylprednisolone. The Berlin cohort contained significantly more treated patients than the Magdeburg and Kiel cohorts (p = 0.001) Table The demographics, clinical and CSF data of the cohorts are shown in Table p = 0.03), Qalb , and CSF protein , while there was no correlation with sex  or CSF cell count .When considering the whole sample, we found small- to medium-effect size correlations between CSF-NFL and age , and post-hoc analysis revealed lower median [range] levels for PPMS compared to ALS , ALS compared to HC  and PPMS compared to HC (p = 0.015) Figure p = 0.3) Figure p = 0.05), and also not between study centers (p = 0.5); CSF-NFL concentrations were not related to disease duration  or EDSS  Figure There was a significant group effect of CSF-NFL levels when comparing PPMS to ALS p < 0.00, and posWe investigated CSF-NFL levels in clinically definite PPMS patients suffering from a mainly long-standing disease without inflammatory disease activity. To our knowledge this is the largest cohort of patients investigated to date addressing the question whether CSF-NFL levels can mirror disease associated axonal neurodegeneration and its value as a clinical biomarker in PPMS patients. Our analysis reveals that PPMS diagnosis relates to significantly different levels of CSF-NFL than the levels found for the other two groups, and affirms a moderate PPMS CSF-NFL level situated in-between values for HC and ALS patients.Since several studies have shown contradicting results of lower  or higheThe subjects included in our PPMS cohort are likely to suffer from corticospinal tract (CST) demyelination, typically found in long-standing PPMS . This isWe conducted a direct CSF-NFL comparison between PPMS and ALS, with both diseases sharing clinical signs resulting from the degeneration of the long cerebral and spinal cord tracts in the central motor system . These cMoreover, when considering our previous work on CSF-NFL, PPMS patients presented significantly lower levels not only compared to ALS patients but also compared to patients with a slow neurodegenerative disease like frontotemporal dementia (FTD) , emphasiIn line with recent PPMS studies we did not find any relationship between the clinical impact, as measured using the EDSS, and CSF-NFL , 7, 8. TThe PPMS patients recruited at the three different Universities differed with respect to (i) CSF cell count and album quotient, which were greater in the Kiel cohort, and (ii) treatment frequency, which was highest in the Berlin cohort. These findings lead us to assume that the Kiel patients display more  inflammatory activity (without corresponding relapses and MRI contrast-enhancing lesions), and we could also deduct that the Berlin University regularly applies off-label drugs in accordance with positive case series reported by the Berlin group , 35. HowLimitations of the present study include the absence of MRI measurements obtained exactly at the same point in time as the LP was performed. We therefore cannot completely exclude the presence of Gd-enhancing lesions. Moreover, longitudinal studies are needed to evaluate if NFL is a suitable biomarker to predict and adjudicate disability progression in PPMS. In addition, future studies should address the questions (i) if CSF-NFL could also aid to differentiate potential PPMS disease mimics, e.g., hereditary spastic paraplegia, and (ii) if composite measurement of DTI CST involvement, viewed together with CSF-NFL levels, may provide a better correlate with the overall functional state of PPMS patients. A further point of interest would be to measure the contribution of the inflammatory vs. neurodegenerative components influencing the CSF-NFL levels, through contrasting PPMS patients with acute inflammatory (MRI-) disease activity with those patients in progressive disease stages and the possible relations to therapeutic approaches.In our current study we were able to demonstrate that CSF-NFL levels can clearly discriminate between slowly progressive neuroinflammatory (PPMS) and more rapid neurodegenerative (ALS) processes, but do not correlate with assessments of PPMS severity on a clinical disease scale. We conclude, that CSF-NFL mirrors the inflammation driven neurodegenerative aspect in MS and thus, CSF-NFL might not be an ideal biomarker in PPMS.We confirm that we have read the Journal's position on issues involved in ethical Publication and affirm that this report is consistent with those guidelines.MP has access to all the data and takes responsibility for the data, accuracy of the data analysis, and interpretation of the data and drafting the manuscript for intellectual content. SS, DB, JK, FL, KR, SM, SV Design and conceptualization of the study; revising the manuscript for intellectual content; RC Design and conceptualization of the study; revising the manuscript for intellectual content and language improvement. PK Design and conceptualization of the study; revising the manuscript for intellectual content, Study supervision.MP received speaker honoraria from Roche, Genzyme and Novartis and travel/accommodation/meeting expenses from Novartis, Biogen Idec, Genzyme and MERCK Serono. KR has received research support from the German Ministry of Education and Research  Merck Serono and Novartis as well as speaking fees and travel grants from Guthy Jackson Charitable Foundation, Bayer Healthcare, Biogen Idec, Merck Serono, Sanofi-aventis/Genzyme, Teva Pharmaceuticals, Roche and Novartis. SM receives honoraria for lecturing, and travel expenses for attending meetings from Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, Celgene, Diamed, Genzyme, MedDay Pharmaceuticals, Merck Serono, Novartis, Novo Nordisk, ONO Pharma, Roche, Sanofi-Aventis, Chugai Pharma, QuintilesIMS and Teva. His research is funded by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgesellschaft (DFG), Else Kröner Fresenius Foundation, German Academic Exchange Service, Hertie Foundation, Interdisciplinary Center for Clinical Studies (IZKF) Muenster, German Foundation Neurology and Almirall, Amicus Therapeutics Germany, Biogen, Diamed, Fresenius Medical Care, Genzyme, Merck Serono, Novartis, ONO Pharma, Roche, and Teva. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cryptorchidism or undescended testes is the most common disorder of the male endocrine glands in children. With the advancements in laparoscopic techniques and instruments, laparoscopic orchidopexy has become the standard procedure in the management of nonpalpable undescended testes.  To evaluate and determine the therapeutic role, sensitivity, and specificity of laparoscopy in localizing nonpalpable testes and the mean operative time, the conversion rate (and reasons thereof), postoperative wound infection, postoperative stay, and time taken for return to daily activities following laparoscopic orchidopexy or orchidectomy.  This was a prospective study carried out in the Postgraduate Department of Surgery, Government Medical College, Srinagar, J&K, India, from May 2008 to August 2011. All patients who presented to the outpatient department with complaints of absent testes were examined, and the ones with nonpalpable testes were included in the study.  The mean operative time for bilateral and unilateral nonpalpable testis was 102.76 and 53.67 minutes, respectively. Minor postoperative wound infections were noted in 4 of our patients. Mean duration of hospital stay was 14.23 hrs for unilateral cases and 16.27 hrs for bilateral cases. Patients who underwent laparoscopic orchidopexy resumed their normal activities within 4 ± 1 days.  Laparoscopy clearly demonstrates the anatomy and provides visual information upon which a definitive decision can be made for further management of the undescended nonpalpable testis. Cryptorchidism or undescended testes is the most common disorder of the male endocrine glands in children. Physical examination of the testis can be difficult, and further evaluation should be considered if a normal testis cannot be definitely identified. Reasons for treatment of cryptorchidism are increased infertility, testicular malignancy, testicular torsion, trauma, and psychological stigma of an empty scrotum .About 1% incidence of cryptorchidism is reported by 1 year of age, and about 20% of children with cryptorchidism may have one or both nonpalpable testes. The condition of the nonpalpable testis may fall into one of the following categories: agenesis, vanishing testis, intra-abdominal testes, or inguinal testes . In expeApproximately two-thirds of neonates born with an undescended testis will undergo spontaneous testicular descent, typically by 4 to 6 months postnatally, mediated by postnatal testosterone surge .Majority of the intra-abdominal testes are located less than 2 cm from the internal ring. The intra-abdominal testis may be associated with a closed or an open deep inguinal ring. In the closed ring variant, the processus vaginalis does not develop, the gubernaculum is absent, and the internal ring is closed. In the open ring variant, a patent processus vaginalis exists in the internal inguinal ring and the gubernaculum is present. Vanishing testis is one in which testicular vessels and vas deferens are found on surgical exploration but a testis is absent. Supporting evidence for testicular infarction includes the common finding of hemosiderin, and calcium deposits in testicular remnants found on exploration . One in To determine the sensitivity and specificity of laparoscopy in localizing nonpalpable testesTo evaluate the therapeutic role of laparoscopy in nonpalpable testesTo determine the mean operative time, the conversion rate (and reasons thereof), postoperative wound infection, postoperative stay, and time taken for return to activities of daily lifeThis study entailed the following aims and objectives:This was a prospective study carried out in the Postgraduate Department of Surgery, Government Medical College, Srinagar, from May 2008 to August 2011. All patients who presented to the outpatient department with complaints of absent testes were examined, and boys with nonpalpable testes were included in the study. These boys were examined as outpatients, at the time of admission and after general anesthesia to exclude palpable testes. Patients with nonpalpable testes were subjected to diagnostic and operative laparoscopy. The primary 5 mm camera port was introduced by open technique , pneumoperitoneum was created (10 mmHg) with the patient in a 30° head-down Trendelenburg position, the area of the internal inguinal ring was inspected bilaterally, and subsequent trocars  were placed under laparoscopic (30° telescope) visualization into the right and left lumbar region in line with the umbilical port. Subsequent surgery was planned according to the laparoscopic findings following the algorithm for the management of the nonpalpable testis .A diagnostic laparoscopy was done for rest of the abdominal quadrants in all cases. The inguinal ring was first examined to evaluate its patency, and then the bilateral iliac areas and pelvis were inspected. If intra-abdominal blind ending cord structures were found, no further exploration was performed, and a diagnosis of intra-abdominal vanishing testes was made.If an intra-abdominal testis was found, it was classified as high  or low  depending on its position to the internal ring.With gentle traction on the testis, the most distal gubernacular attachment was divided with electric hook cautery, and gubernaculum was used as a handle for mobilizing the testis. Once the testis had been adequately mobilized, confirmed by performing stretch maneuver where the testes are stretched to the contralateral deep inguinal ring, the dissection was further carried out medially all around the ipsilateral deep inguinal ring to complete the herniotomy. Peritoneal coverage between the vas deferens and urinary bladder was dissected. Usually, a neotunnel was created between the medial umbilical ligament and the inferior epigastric artery (Prentiss maneuver) to gain extra length for reaching the scrotum. The blunt tip of the laparoscopic dissector was passed over the pubic bone into the ipsilateral hemiscrotum, thus creating a pathway. A dartos pouch was prepared in the hemiscrotum. A suction cannula was passed along the passage which was created transabdominally by laparoscopic dissector, and grasping the tip of the suction cannula, a long curved clamp was passed into the abdominal cavity and stretched in all directions to create adequate passage for bringing the testes down. The gubernacular tissue was grasped and pulled, thus delivering the testes out into the scrotal dartos pouch, and the length was checked by deflating the abdomen. Releasing the pneumoperitoneum gave additional length. Any tension found on the pedicle was released by further mobilization of the peritoneal flaps. Testes were fixed in the scrotum using 5-0 chromic catgut suture. At the end of the procedure, the abdomen was examined for any bleeding, the pneumoperitoneum was released, and trocars were removed. Trocar sites were closed. Children were discharged home next day. All the patients were followed at 6 weeks and 6 months.We used diagnostic and operative laparoscopy in the management of 52 patients with 61 undescended testes. 26 patients had 28 palpable testes (45%), and 26 patients had 33 nonpalpable testes (55%). Boys with the palpable testis at any point were excluded from the study. The study sample size of our series was 26 patients with 33 nonpalpable testes. Ratio of palpable testis to nonpalpable testis was 1 : 1.17, respectively.The youngest and the eldest patient in our study group were 1 and 18 years of age, respectively. Majority of patients, i.e., 12, were in the age group of 4–7 years. Of these 33 nonpalpable testes, 7 (21%) were on the right side, 12 (36%) on the left side, and 14 (42%) bilateral. On ultrasonography, nonpalpable testes were localized at various sites as intra-abdominally , near deep inguinal ring, in inguinal canal, and in superficial inguinal pouch and could not be localized in 11 (33.33%), 9 (27.2%), 3 (9%), 3 (9%), and 7 (21.2%) cases, respectively.n=33).On diagnostic laparoscopy, the deep inguinal ring was found to be open or closed in 30 (90.9%) and 3 (9.1%) cases, respectively. The number of testes found normal on diagnostic laparoscopy was 18 (54.5%) followed by 11 hypoplastic (33.3%) and 3 atrophic (9.09%) testes. Blind ending vas deferens and vessels (vanishing testis) were found in 1 (3.03%) patient only.Majority of high intra-abdominal testis were hypoplastic (58.8%), highlighting the deleterious effect of increased abdominal temperature on development of the testis.The operative procedures performed were laparoscopic orchidopexy, laparoscopic orchidectomy, open orchidopexy (conversion), open orchidectomy (conversion), and diagnostic laparoscopy in 24 (72.7%), 5 (15.1%), 2 (6.06%), and 1 (3.03%) cases, respectively. All of the patients were managed by single-stage procedure.Laparoscopy was successful (diagnostic or therapeutic) in 30 (90.90%) cases, and the procedures included laparoscopic orchidopexy, laparoscopic orchidectomy, and diagnostic laparoscopy. Mean operative time for 14 bilateral undescended testes was 102.76 (with SD of 2.37 and SEM of 5.08) minutes while as for 19 unilateral cases, it was 53.67 (with SD of 5.38 and SEM of 4.02) minutes.Postoperative complications included surgical emphysema , type 1 The median age group of our study subjects was 5.2 years with patients from 1 to 18 years of age included in our study. Satar and Hvistendahl have reported similar median age group in their respective studies as 5.4 and 5.7 years [42% of our patients had bilateral undescended testis, and the incidence of bilateral undescended testis was relatively higher as compared to those reported by Ismail (13.5%), Zubair (33%), and Hassan (21%) –11. ThisThe USG (abdomen) could localize 78.78% of nonpalpable testes in our study. Ismail reported that USG was helpful in localization of the nonpalpable testis in 100% of his patients, as he used colour Doppler USG in his study .We found the diagnostic yield of laparoscopy was 100% and the therapeutic yield was 96.9%, as we could localize and manage 32 nonpalpable testes while one case was diagnosed as vanishing testes on laparoscopy. El-Gohary has reported 100% accuracy in laparoscopic assessment of the testis .The closed deep inguinal ring with vas deferens and vessels traversing it were found in 28% of our study subjects. The significance of this fact was that these patients had testis in the superficial inguinal pouch (ectopic testis). Godbole (33%) and Masao (35%) have also reported similar findings of the closed deep inguinal ring from their studies , 14.Our study revealed normal testes in 54.5% of cases followed by hypoplastic testes in 33.3% of cases. Morphology of testis was correlated with localization of testis which revealed that features of hypoplasia were high in intra-abdominal testis. Similar findings were noted in the study of Humphery and Boekmann . One patWe performed orchidopexy in 26 out of 33 nonpalpable testes (78%) out of which laparoscopic orchidopexy was done in 24 (72.7%) testis, and conversion to open orchidopexy was necessitated in 2 (6.06%). The reasons for conversion were dense adhesions of testis with surrounding structures. Orchidectomy was done in 6 (18%), of which 3 testes were atrophic and 3 testes were hypoplastic (could not be mobilized adequately as determined by the stretch test). Masao and Khan et al. have reported laparoscopic orchidopexy in 33% and 35% cases , 16. TheThe mean operative time in our study was 53.67 minutes (SD of 2.37 minutes) for unilateral testis and 102.76 minutes (SD of 5.38 minutes) for bilateral nonpalpable testis. Mark and Davidson reported 15 minutes as average time for laparoscopy in unilateral nonpalpable testis . As our No major surgical complications were observed in our patients. Postoperative surgical emphysema was seen in 2 of our cases, which resolved spontaneously. One of our patients developed superficial surgical site infection, and one patient developed scrotal hematoma. The patients were on regular follow-up. No evidence of testicular atrophy was seen in those patients with follow-up till the study time. Various other authors have also reported similar findings as regards to complications following laparoscopic management of nonpalpable undescended testes , 18, 19.The average postoperative hospital stay was 14.23 hours (SD of 2.37 hours) for unilateral undescended nonpalpable testes and 16.27 hours (SD of 5.38 hours) for bilateral undescended nonpalpable testes. Most of our patients were discharged on the next day because no immediate postoperative surgical complications were seen. Time taken to return to daily activities was more in patients who underwent orchidopexy (4 ± 1 days) as compared to patients who underwent orchidectomy (2 ± 1 days). Koyama reported a hospital stay of less than one day, and Desai discharged his patients on the next day .In one (3%) of our cases, no testis was found as it was a vanishing testis. The morphology of testes as determined by laparoscopy was 96.9%. Laparoscopic procedures were performed in 90.9% of our cases. Open orchidectomy/orchidopexy was done in 9.1% of cases. Patients with vanishing testis (3%) had the advantage of avoiding unnecessary groin exploration. Zubair and Godbole have reported that unnecessary exploration or negative exploration can be avoided in 20% and 42% cases, respectively , 13.Our study showed that patients with intra-abdominal testis and vanishing testes benefited from laparoscopy due to the fact that this technique provided them with a definitive diagnosis, direct surgical approach according to the location of testes, and avoidance of unnecessary abdominal exploration in case of vanishing testis. Laparoscopy clearly demonstrates the anatomy and provides visual information upon which a definitive decision can be made.


Treatment-resistant thin endometrium (TTE) during in-vitro fertilization is a relatively uncommon and challenging problem.The primary aim of the study was to assess the TTE rate during frozen embryo transfer (FET) cycles and the secondary aim was to evaluate the effect of intrauterine instillation of granulocyte colony stimulating factor (G-CSF) in these cases. In this cross-sectional study, all of the women who underwent FET cycles with hormonal endometrial preparation in Royan Institute from June 2015 to March 2018 were evaluated and all of the cases with TTE diagnosis  with TTE diagnosis were detected. Finally, 20 eligible patients were included. The changes of endometrial thickness after G-CSF therapy were significant (p It was found that the rate of TTE during the FET cycle is very low and intrauterine perfusion of G-CSF has a potential effect to increase the endometrial thickness in these patients; however, the rate of cancellation was still high and poor pregnancy outcomes were observed. Endometrial growth is an essential step in endometrial receptivity and embryo implantation in assisted reproductive technique cycles (1). A thin endometrium of “G-CSF is a glycoprotein and known as colony stimulating factor 3 produced in vascular endometrium, macrophages, and other immunocytes that operate not as a growth factor but also as a cytokine (5). G-CSF promotes endometrial stromal cell decidualization via cyclic adenosine monophosphate mediator by apocrine and paracrine action and induces proliferation and differentiation of the human endometrium” (5). In medicine recombinant, G-CSF was used in immunology for various indications for a long time (6). It is a pro-angiogenic mediator and increases type 2 T helper cell cytokine secretion, recruitment of dendritic and T regulatory cells . According to previous clinical studies, G-CSF can be considered as a factor preventing repeated miscarriages and implantation failures (8-10), and it has no impact on the chromosomal constitution of human embryos (11). From the first case report study by Gleicher and co-workers in 2011 (4), until now, five studies in IVF (12-16) and four in FET cycles  have evaluated the effect of intrauterine infusion of G-CSF on thin endometrium; since the reported results have been controversial.The present study was conducted at the Royan Institute to assess the treatment-resistant thin endometrium rate during FET cycles with hormonal endometrial preparation (as primary aim) and to evaluate the effect of intrauterine instillation of G-CSF in these cases (as secondary aim).endometrial preparation at the Royan Institute from June 2015 to May 2018, and all eligible cases with treatment-resistant thin endometrium diagnosis were included. The women aged 20-40 years with previously canceled at least one cycle because of thin unresponsive endometrium  cycles with hormonal  et al. in their study (12). In summary, the content of the G-CSF ampule (300 mcg/1ml)  daily and low-dose aspirin from the second or third day of the menstrual cycle. The Endometrial thickness measurement  was carried out by ultrasonography, 9 or 10 days later. When the endometrial thickness was The research protocol was approved by the Institutional Review Board and the Ethics Committee of the Royan Institute (Ethics code/94/178). Each participating women signed informed consent.t-test to compare means endometrial thickness in patients before and after treatment according to the results of the Kolmogorov-Smirnov normality test for endometrial thickness. The level of statistical significance was set at p Data was statistically analyzed by using Statistical Package for the Social Sciences  software version 20. Categorical data were expressed as number and percentage and numerical data as mean and standard deviation. The statistical analyses for parametric data were performed with the use of a paired During the study period, 8,363 FET cycles were evaluated and a total of 30 infertile patients (0.35%) with treatment-resistant thin endometrium diagnosis were detected, finally, 20 eligible patients were satisfied to receive the G-CSF therapy. Two patients did not agree to participate and eight patients did not meet the inclusion criteria of the study. The basic and demographic characteristics of patients are presented in Table I, Table II shows the changes in endometrial thickness after treatment with G-CSF. The mean of endometrial thickness at the time of infusion was 5.35 In the present study, the incidence rate of thin endometrium during FET cycle in our institute was estimated to be 0.35%, which is almost similar to the rate of thin endometrium in IVF cycles (< 1%) reported by Al-Ghamdi and colleagues (19). It has been found that intrauterine injection of G-CSF in patients with unresponsive thin endometrium diagnosis can improve the endometrial thickness, but it failed to find out its effect on pregnancy. However, multiple factors affect pregnancy and endometrial receptivity and endometrial thickness are only a quantitative criterion; poor pregnancy outcomes in the present study can be due to that the averages of women's age and BMI were higher than those in other studies.et al. in a small number of patients with unresponsive thin endometrium did not find significant effect after intrauterine infusion of G-CSF (20). Recently, Barad and colleagues in a randomized clinical trial assessed 73 normal patients during IVF and concluded that in normal IVF patients, G-CSF has no impact on the endometrial thickness, implantation rates, and clinical pregnancy rates (13).After the first case report study by Gleicher and co-workers (4), four case-series studies have evaluated the effect of intrauterine infusion of 300 μg of G-CSF in patients with thin endometrium diagnosis at the day of ovum pickup and reported a significant positive impact on improving endometrial thickness . However, Check et al. in a prospective study found no significant differences in the endometrial thickness, implantation, and clinical pregnancy rates between patients treated with 100 μg of G-CSF and the control subjects (3). Besides, Eftekhar and co-workers in a non-randomized clinical study assessed 68 patients with thin endometrium and reported no significant improvement in endometrial thickness; it is worth noting that the clinical pregnancy rate was higher in the G-CSF group than that in the control group (18). Later, Xu and colleagues in a prospective study divided 30 patients with thin endometrium diagnosis in two treatment subgroup  (17). After treatment in both the groups, the endometrial thickness increased significantly. The G-CSF with endometrial scratch subgroup associated with nominally higher rates of clinical pregnancy and live birth in comparison with the G-CSF only subgroup; though the differences were non-significant (17). Finally, it concluded that endometrial scratch did not damage the G-CSF action for thin endometrium and lean toward a better pregnancy and live birth rates. They recommended embryo transfer cancellation and G-CSF therapy in subsequent FET cycles for patients with a thin endometrium (17). In contrast, Kunicki and co-workers (6) evaluated 62 women with thin unresponsive endometrium (29 patients treated with a G-CSF infusion and 33 cases who opted out of the study considered as controls). It was found that G-CSF infusion leads to an improvement in endometrium thickness but has no beneficial effect on clinical pregnancy and live birth rates. Therefore, although the case series and non-randomized studies are promising, the only published randomized clinical trial failed to find any effect on clinical outcome. Reliable evidence for the beneficial effect of G-CSF in improving endometrial receptivity is still limited (5) and more data is warranted for a definitive conclusion in this regard.A few studies evaluated the efficacy of intrauterine instillation of G-CSF for improving endometrial thickness in FET cycles, and yet the findings remain controversial. At first, Li The first limitation of the study is a relatively small cohort size; although, in most studies conducted in this area , the patients' number in the experimental group (G-CSF treatment) was less than 40, due to the low prevalence of treatment-resistant thin endometrium (0.1-2%). Therefore, the evaluation of this number of patients can be valuable and represent a "small piece" to increase the research about this type of treatment. The second limitation is the lack of a control group. The strength of this study was to evaluate the incidence rate of thin endometrium during FET cycles since there is very limited data in this regard.It was found that the rate of treatment-resistant thin endometrium during the FET cycle with hormonal endometrial preparation is very low and G-CSF has a potential effect to increase the endometrial thickness in these patients; however, the rate of cancellation was still high and poor pregnancy outcomes were observed.There are no conflicts of interest to declare.