The different molecular profiles of cerebrospinal fluid (CSF) between ventricular and lumbar compartments remain elusive, especially in the context of leptomeningeal metastasis (LM), which affects CSF flow. We evaluated CSF metabolomic and proteomic profiles based on the compartments and the diagnosis of spinal LM, proved by MRI from 20 paired ventricular and lumbar CSF samples of LM patients, including 12 spinal LM (+) samples. In metabolome analysis, 9512 low-mass ions (LMIs) were identified—7 LMIs were abundant in all lumbar versus paired ventricular CSF samples, and 3 LMIs were significantly abundant in all ventricular CSF. In comparisons between spinal LM (+) CSF and LM (−) CSF, 105 LMIs were discriminative for spinal LM (+) CSF. In proteome analysis, a total of 1536 proteins were measured. A total of 18 proteins, including complement C3, were more highly expressed in all lumbar CSF, compared with paired ventricular CSF, while 82 proteins, including coagulation factor V, were higher in the ventricular CSF. Of 37 discriminative proteins, including uteroglobin and complement component C8 gamma chain, 4 were higher in all spinal LM (+) CSF versus spinal LM (−) CSF. We further evaluated metabolic pathways associated with these discriminative proteins using the Gene Ontology database. We found that 16/17 spinal LM (+) pathways, including complement activation, were associated with lumbar discriminative proteins, whereas only 2 pathways were associated with ventricular-discriminative proteins. In conclusion, we determined that metabolite and protein profiles differed between paired lumbar and ventricular CSF samples. The protein profiles of spinal LM (+) CSF showed more similarity with the lumbar CSF than the ventricular CSF. Thus, we suggest that CSF LMIs and proteins could reflect LM disease activity and that LM-associated differences in CSF are more likely to be present in the lumbar compartment. Leptomeningeal metastasis (LM) is a devastating complication of cancer that occurs in 1–15% of solid tumor patients ,3 with aInterestingly, positive cytological results in LM patients depend on the CSF sampling compartment. For example, lumbar CSF samples were more likely to yield higher positive cytology rates compared with ventricular CSF samples in patients with LM, especially in those who had spinal symptoms ,9. BecauConstituents of the CSF, such as metabolites, extracellular vesicles, extracellular DNA, and microRNA (miRNA), have been studied as possible biomarkers for CNS disease ,16,17,18In this study, we analyzed the metabolomic and proteomic profiles of ventricular and lumbar CSF in 20 paired samples from LM patients and compared the profiles of 12 LM (+) spinal CSF samples versus 7 LM (−) spinal CSF samples. We expected that characterization of biomolecules in lumbar CSF from patients who had spinal LM compared with lumbar CSF from patients who had not spinal LM would reveal a biomolecule that was characteristic of local LM activity. We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) database metabolic pathway analysis and Gene Ontology database molecular pathway analysis with LMI and proteomic profiles and estimated causal associations between metabolic and proteomic profiles using pathways mapping. We determined whether discriminative profiles of lumbar CSF compared with ventricular CSF (based on topographic differences according to CSF compartments) were similar to profiles of spinal LM (+) samples versus spinal LM (−) samples, with the assumption that the lumbar CSF carries more LM-related biomolecules than the upstream ventricular CSF.Our strategy to study the comparative profiling of different CSF sampling site is illustrated in Gadolinium-enhanced brain MRI verified LM for 15 patients (75%) and was suggestive of LM for the other 5 patients. Accompanying parenchymal brain metastasis was found in 14 patients. Spine MRIs were evaluated in all but 1 patient, and 12 patients (63%) presented with diffuse/nodular enhancement of the spinal cord or cauda equina.3  in ventricular CSF and was significantly lower than the lumbar CSF . The CSF protein level was also significantly lower in ventricular CSF  compared with that of lumbar CSF . The median glucose concentration of ventricular CSF was 72 mg/dL , significantly higher than the median lumbar CSF concentration of 50 mg/dL  (p = 0.047).Comparisons of CSF profiles between paired ventricular and lumbar CSF samples are shown in In comparisons of CSF profiles between spinal LM (+) and LM (−) lumbar CSF, protein levels were significantly higher in spinal LM (+) samples than LM (−) samples  versus 32 mg/dL , m/z) and retention time (min) and the peak area of each LMI was normalized using a per-sample scaling factor (see details in the p < 0.05). Conversely, a total of 5265 LMIs (55%) were higher abundant in the ventricular CSF than the lumbar CSF, and 223 LMIs (4.2%) were statistically significant (p < 0.05). However, no LMIs showed a threshold level that differentiated lumbar and ventricular CSF completely.In a same CSF sample, we performed metabolomic and proteomic analyses using appropriate experimental methods B. In metWe then analyzed the differences in peak area between paired lumbar and ventricular CSF samples . A totalp < 0.05, p < 0.05, We searched for LMIs that distinguish spinal LM (+) lumbar CSF from spinal LM (−) lumbar CSF, which would reflect the local disease activity of LM at the lumbar spinal cord . In grouWhen we defined a discriminative LMI as having a summed sensitivity and specificity >160% at the threshold value for each LMIs showing the highest sum of sensitivity and specificity, a total of 105 LMIs were discriminative for the spinal LM (+) CSF. Among these, 6 LMIs were higher in the spinal LM (+) samples at a summed sensitivity and specificity >180% B, and anp-value) involving the lumbar discriminative LMIs were as follows: 1-carbon metabolism, mediated by folate; sphingolipid metabolism; alanine, aspartate, and glutamate metabolism; histidine metabolism; arginine and proline metabolism; arginine biosynthesis; aminoacyl-tRNA biosynthesis; ubiquinone and other terpenoid-quinone biosynthesis; vitamin B6 metabolism; and tyrosine metabolism  differences for ventricular versus lumbar and spinal LM (+) samples versus spinal LM (−) . In thisp = 0.34).We determined which metabolic pathways were shared among CSF compartments . Thirty-A total of 1536 proteins were measured, and their abundance ratios were calculated in 14 out of the 20 paired ventricular and lumbar CSF samples available for proteomics , were more highly expressed in all lumbar CSF samples compared with ventricular CSF B, 41 proThe proteins that were discriminative for spinal LM (+) lumbar CSF versus spinal LM (−) lumbar CSF were evaluated by groupwise comparisons based on the abundance ratio . In thisNext, we selected discriminative proteins using the same criteria as used for discriminative LMIs (a summed sensitivity and specificity >160%). Thirty-seven proteins were discriminatively higher in spinal LM (+) CSF compared with LM (−) CSF samples. Among these, 4 proteins, including uteroglobin (P11684) and complement component C8 gamma chain (P07360), were higher in the spinal LM (+) samples at a summed sensitivity and specificity ≥180% B, and anWe evaluated metabolic pathways that were associated with these discriminative proteins using the Gene Ontology database to determine which pathways were associated with the CSF compartment or with spinal LM. We selected discriminative proteins that showed differential expression in ≥12 out of 14 paired samples for lumbar and ventricular compartments using the summed sensitivity and specificity ≥160% for spinal LM (+) CSF .p-value) associated with the lumbar discriminative proteins were the following: the complement activation classical pathway, complement activation, regulation of complement activation, negative regulation of endopeptidase activity, platelet degranulation, receptor-mediated endocytosis, innate immune response, complement activation alternative pathway, acute-phase response, and fibrinolysis . We also determined whether this association applied for the spinal LM (−) pathways for each CSF compartment. Among 18 spinal LM (−) pathways, 7 pathways, including central nervous system development, were shared with the ventricular-discriminative protein associated pathways, and 6 pathways, including extracellular matrix disassembly, were common to the lumbar discriminative protein associated pathways. Among these pathways, five pathways  were common to spinal LM (−) and both CSF compartment pathways. Thus, the spinal LM (−)-associated pathways did not show any bias based on the CSF compartment.Comparing spinal LM (+) pathways with each CSF compartment, we found that 16/17 (94%) of spinal LM (+) pathways, including complement activation and hyaluronan metabolic process, were also associated with the lumbar discriminative proteins C, whereaAnalysis of these discriminative proteins in the KEGG metabolic database and annotated metabolic pathways  indicatep < 0.0001).We determined whether discriminative proteins of spinal LM (+) CSF, which reflects local LM disease activity, were preferentially expressed in either lumbar or ventricular CSF. We determined the distribution of significantly expressed spinal LM (+) and LM (−) proteins for each CSF compartment . A totalWe determined the distribution of significantly expressed proteins that were shared by the CSF compartment and spinal LM (+) proteins  and verified the similarity of profiles of spinal LM (+) samples of CSF from the lumbar and the ventricular compartments.Although CSF circulates throughout the CNS, CSF profiles differ by site, especially in the case of CNS diseases ,11,12. UIn LM, where CSF flow is frequently disturbed, there are large differences in both the cellular and extracellular components of CSF between the ventricular and lumbar compartments. Two studies reported false-negative CSF cytology results when the sampling site was not associated with LM-related symptoms and recommended sampling the CSF at the compartment nearest to the location of LM. For example, lumbar CSF showed higher positive rate if the patients presented with symptoms of conus involvement versus ventricular CSF when patients had cranial neuropathy in LM ,29. GajjRecently, cancer cell-derived components, such as microRNAs, exosomes, and extracellular DNA, have been analyzed to monitor cancer metastasis ,31,32. WCSF is produced by filtering blood through a specialized capillary, the so-called ‘blood–CSF barrier’, which naturally blocks the passage of cells and reduces plasma content at a scale of 1/103 to 1/106 depending on the active transport system and molecular characteristics, such as size and lipid solubility . As CSF To minimize the bias of protein expression levels based on normalization methods, it is essential to define brain-specific proteins that are consistently present at enriched levels. However, we found no housekeeping proteins, such as cellular β-actin or GAPDH, in the extracellular compartment of the CSF. Begcevic et al.  performeIn a comprehensive analysis of the proteome of the CSF extracellular compartment by Chiasseri et al., they found that extracellular vesicle (EV) proteins were more abundant than soluble (supernatant) fraction proteins . AlthougMajor limitations of our LMI study are that non-targeted LMIs were not verified by targeted MS using standards for candidate molecules and the extracted profiles were without external validation of large number of CSF samples. Previously, we used an advanced MS technique to identify thousands of LMIs in CSF and designated LMI profiles to differentiate LM from parenchymal brain metastasis and primary brain tumors . We are Integrate discriminative proteomics and metabolomics without a validation method can be seen as a fragile approach. Thus, although our metabolite–protein interaction network is somewhat speculative, it provides a starting point for understanding metabolite–protein interactions. In this study, we identified three interaction nodes, including the coagulation cascade, glycerophospholipid metabolism, and sphingolipids metabolism. In our metabolic pathway analysis of spinal LM (+) CSF in comparison with spinal LM (−) CSF, platelet degranulation and blood coagulation were among the top 10 enriched pathways, and coagulation factors IX and XII were discriminative proteins in spinal LM (+) CSF. Many glycerophospholipids have been suggested as markers of cancer  since thWe collected 20 ventricular/lumbar CSF-paired samples from 20 LM patients from our archives of CSF for biomarker studies . For sampling, (1) lumbar CSF was sampled via lumbar puncture at the time of diagnosis or from lumbar drainage just before starting ventriculolumbar perfusion chemotherapy , and (2)g, 20 min) within 1 h of collection, and the supernatant was aliquoted. A 50 µL sample of each supernatant was used for MS analysis. The remaining samples were centrifuged again at 10,000× g for 30 min and kept frozen at –80 °C for further study . Nitrogen gas was used to dry the supernatant for MS analysis. The extracted metabolites were dried and then reconstituted in 0.1% formic acid and subjected to LC–MS/MS analysis. We used a Shimadzu Nexera X2 system  coupled to a Sciex Triple TOF 5600+ system ; the front end was equipped with a DuoSpray ion source. For the ultraLC separation, the sample was loaded into an Atlantis T3 sentry guard cartridge . Separation was performed in an Atlantis T3 column . The MS system was set to perform one full scan (50 to 1200 m/z range) followed by LC–MS/MS of the 10 most abundant parent ions .The metabolites in the CSF were extracted using a modified Bligh and Dyer method . In briem/z, mass–charge ratio) and RT (min) column common to all samples.Peaks Table Using the MarkerView Software: A list of LC–MS peaks (.peaks file) was created from a measurement file (.wiff file) for every sample using the MarkerView software . The parameters for this process were as follows: minimum retention time (RT), 0.00 min; subtraction offset, 10 scans; subtraction multiplication factor, 1.3; noise threshold, 10; minimum spectral peak width, 10 ppm, and minimum RT peak width, 5 scans. Next, a table of peaks was created by importing the .peaks files into the MarkerView software, for all samples simultaneously, using the following parameters: RT tolerance, 0.01 min; mass tolerance, 10.0 ppm; intensity threshold, 10; maximum number of peaks, 20,000; and area reporting using the option of ‘area integrated from raw data, not from original peak finding.’ The peaks table  consisted of one peak area column per sample, and a mass value , specific compounds were found for the given m/z, listed in rank order based on the MS and MS/MS data.Using metabolite databases from the Human Metabolome Database  were precipitated using cold acetone. Precipitated samples were reduced with 10 mM DTT, alkylated by iodoacetamide (IAA), and digested with mass spec grade trypsin for 12 h at 37 °C. Digested peptides were desalted using C18 spin columns  according to the manufacturer’s instructions.TMT labeling: Peptide samples were reconstituted in 100 mM triethylammonium bicarbonate (TEAB), labeled using TMT11plex reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, each prepared TMT reagent was transferred to the peptide sample, the mixture was incubated for 1 h, quenched by the addition of 8 μL of 5% hydroxylamine, and incubated for 15 min at room temperature. Each set of 11 TMT-labeled peptide samples was pooled and dried using a vacuum concentrator.Mid-pH reverse-phase liquid chromatography fractionation: The pooled 11 plex TMT-labeled sample was separated using Agilent 1260 Infinity HPLC system . An Xbridge C18 analytical column  and a guard column  were used for peptide separation. Solvents A and B were 10 mM TEAB in water (pH 7.5) and 10 mM TEAB in 90% acetonitrile , respectively. Peptide fractionation was performed using a 115 min gradient at a flow rate of 500 μL/min, as follows: 0% solvent B (10 mM TEAB in 90% acetonitrile) for 10 min, from 0 to 5% solvent B over 10 min, from 5% to 35% solvent B over 60 min, from 35% to 70% solvent B over 15 min, 70% solvent B for 10 min, from 70% to 0% solvent B over 10 min. A total of 96 fractions were collected every minute from 15 to 110 min and were pooled into 24 non-continuous peptide fractions . For each pooled fraction, 5% was dried for global proteome analysis, and 95% of 24 peptide fractions were further combined into 12 fractions, dried using a vacuum concentrator, and stored at −80 °C.3+-NTA beads were washed, resuspended in 1:1:1 ACN/MeOH/0.01% acetic, and aliquoted into 12 microcentrifuge tubes, and washed with 500 μL binding buffer. Resuspended peptide samples were transferred to tubes of Fe3+-NTA beads, incubated for 30 min, washed, eluted using 1:1 ACN/2.5% ammonia in 2 mM phosphate buffer (pH 10), and acidified with 10% TFA to pH 3.5–4.0 before vacuum drying.Phosphopeptide enrichment: Each TMT-labeled 12-peptide fraction was resuspended in 500 μL binding buffer (80% ACN/0.1% TFA). The Ni-NTA magnetic agarose beads  were washed with deionized water, reacted with 100 mM EDTA (pH 8.0), and reacted with freshly prepared 10 mM aqueous FeCl3 solution. The Fem/z with a mass resolution of 120,000 (m/z 200). The AGC target value was 3 × 106. The 10 most intense peaks with charge state ≥2 were fragmented in the higher energy collisional dissociation (HCD) collision cell with a normalized collision energy of 32 and tandem mass spectra were acquired in the Orbitrap mass analyzer with a mass resolution of 45,000 at m/z 200.LC–MS/MS analysis (Proteomics): A total of 12 TMT-labeled 12 phosphopeptide fractions and 24 global peptide fractions were analyzed by a Q Exactive HF-X hybrid quadrupole-orbitrap mass spectrometer  coupled with an Ultimate 3000 RSLCnano system (Thermo Scientific). The peptides were loaded onto a trap column (100 μm × 2 cm) packed with Acclaim PepMap100 C18 resin and peptides were eluted with a gradient from 5% to 36% solvent B (0.1% formic acid in ACN) for 180 min at a flow rate 300 nL/min. The eluted peptides, separated by the analytical column , were sprayed into the nano-ESI source with an electrospray voltage of 2.3 kV. The Q Exactive HF-X Orbitrap mass analyzer was operated using a top 10 data-dependent method. Full MS scans were acquired over the range of 350–2000 Protein abundance ratio: From 14 paired (lumbar and ventricular) CSF samples with a pooled mixture comprising all 14 paired samples, quantitative proteomes were measured using the Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). The abundance of each protein was expressed as a ratio (abundance ratio = abundance in each sample/average of abundance in the pooled mixture). The range of abundance ratios was 0.01–100. An abundance ratio of more than 100 was recorded as 100. An abundance ratio of 0.01 was used for no abundance or an abundance ratio of less than 0.01. The abundance ratio was then converted to common logarithms; thus, the range of logarithmic abundance ratios was −2–~2.Database search: Database searching of all raw data files was performed in Proteome Discoverer 2.3 software (Thermo Fisher Scientific). SEQUEST-HT and MS Amanda 2.0 were used for database searching against the Swiss-Prot Human database. Database searching against the corresponding reversed database was also performed to evaluate the false discovery rate (FDR) of peptide identification. The database searching parameters included precursor ion mass tolerance 10 ppm, fragment ion mass tolerance 0.08 Da, fixed modification for carbamidomethyl cysteine (+57.021 Da/C) and TMT tags , and variable modifications for oxidation (+15.995 Da/M) and phosphorylation . We obtained an FDR of less than 1% on the peptide level and filtered with the high peptide confidence.m/z, mass–charge ratio), retention time (min), and peak area. The non-zero peak areas in the table were converted to common logarithms. Metabolites showing a topographical difference between the paired (lumbar and ventricular) samples were identified using their logarithmic peak area. The steps for assessing an individual metabolite were as follows: (1) for each metabolite, the number of samples whose peak area in lumbar CSF was higher (or lower) than in ventricular CSF was counted; (2) individual metabolites showing consistency of 80% or greater (16 or greater of the 20 paired samples) in the topographical difference were set aside for subsequent analysis. Additionally, to obtain information on proteins, quantitative measurements using the Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) were performed on 14 paired samples of lumbar and ventricular CSF. Proteins showing a topographical difference between the paired (lumbar and ventricular) samples were identified using the logarithmic abundance ratio. The same steps as above were applied to discover individual protein candidates.To obtain information on LMIs, non-targeted LC–MS measurements using the TripleTOF 5600+ system  were performed on 20 paired samples of lumbar and ventricular CSF. Then, a table of metabolite peaks was made. The peaks table contained data on the mass value  from LM (−) groups) were identified using the logarithmic peak area in lumbar CSF. The steps for assessing an individual metabolite were as follows: (1) for each metabolite, a discrimination threshold was determined, with an increment of 0.01, such that the sum of the sensitivity and specificity was highest— when more than one adjacent threshold showed the same discrimination performance, the thresholds were averaged; (2) individual metabolites showing good discrimination (a summed sensitivity and specificity of 160% or higher) were set aside for subsequent analysis. Additionally, proteins having good discriminative ability were identified using the logarithmic abundance ratio in lumbar CSF. The same steps as above were applied to discover individual protein candidates by replacing metabolite peak area with protein abundance ratio.https://www.metaboanalyst.ca/, accessed on 13 September 2021). Sets of discriminative metabolites were used as input data for the enrichment analysis module. We obtained KEGG pathways for the analyzed data and GlobalTest’s p-values. Protein enrichment analysis was performed in DAVID Bioinformatics Resources 6.8 . Sets of discriminative proteins were used as input data for functional annotations analysis. We obtained KEGG pathway annotations among several annotation results and provided Fisher’s exact p-values.Metabolite enrichment analysis was performed in Metaboanalyst 5.0  or SPSS 18.0 . Prism 6  was used to analyze data and generate figures.CSF profiles, including cell count, chemistry, metabolites, and proteins, were compared in two ways. For paired samples analysis, the paired ventricular and lumbar samples were compared using Wilcoxon signed rank-sum test. Second, the CSF profiles were compared between groups of different compartments (ventricular versus lumbar) or those with or without spinal LM findings on spinal MRI using Mann–Whitney U test. A We determined that profiles of metabolites and proteins differed between paired lumbar and ventricular CSF. We also identified LMIs and proteins that were significantly increased in spinal LM (+) CSF compared with spinal LM (−) CSF. The profiles of spinal LM (+) CSF showed similarity with lumbar CSF compared to ventricular CSF. Based on these results, we suggest that CSF LMI and proteins could reflect both different CSF compartments and localized LM disease activity, and differences in CSF associated with LM were more likely be present in the lumbar compartment. Further studies with larger numbers of samples and targeted MS are needed to validate these findings.


Background: Survival outcome after developing brain metastasis is poor and there is an unmet need to identify factors that can promote brain metastasis. Granulocyte-colony stimulating factor (G-CSF) is given to support neutrophil recovery after myelosuppressive chemotherapy to some patients. However, there is emerging evidence that neutrophils can promote metastasis, including through the formation of neutrophil extracellular traps (NETs), scaffolds of chromatin with enzymes expelled from neutrophils to the extracellular space. In animal models, G-CSFs can induce NETs to promote liver and lung metastasis. The primary objective of this study was to test the association between G-CSF use and the later incidence of brain metastasis.Methods: Patients with de novo Stage IV breast cancer, without known brain metastasis at the time of initial diagnosis, were identified from electronic medical records covering the period from 1/1/2013 to 12/31/2020 at Northwell Health. Univariate and multivariate logistic regression models were used to test the association between variables of interest, including G-CSF use, and brain metastasis.Results: A total of 78 patients were included in the final analysis. Among those 78 patients, 24 patients (30.8%) had received G-CSF along with chemotherapy at least once. In logistic regression models, G-CSF use was not a significant factor to predict brain metastasis . Interestingly, in multivariate logistic models, pulmonary embolism (PE)/deep venous thrombosis (DVT) was a significant predictive factor of brain metastasis  (38.5% vs 21.5%).Conclusions: The use of G-CSF was not associated with increased risk of brain metastasis in patients with de novo Stage IV breast cancer. Interestingly, PE/DVT, which can be associated with elevated NETs, was associated with brain metastasis. Further studies are warranted to determine whether DVT/PE with or without elevated NETs levels in the blood, is predictive of developing brain metastasis in patients with de novo Stage IV breast cancer. In the United States, breast cancer is the most common newly diagnosed non-cutaneous cancer and the second most common cause of cancer-related death in women Granulocyte colony stimulating factor (G-CSF) has been recommended by the National Comprehensive Cancer Network (NCCN) in vitroG-CSF clearly has clinical benefits in the short-term, but the long-term consequences of G-CSF treatment are less clear. A clinical study showed that high expression levels of endogenous G-CSF in triple-negative breast cancer are associated with poor overall survival c-JUN upregulation and increased G-CSF secretion. This lead to increased neutrophil recruitment and increased formation of breast cancer brain metastasis, while blocking G-CSF resulted in fewer brain metastasis in multiple mouse models NETs were originally identified as a mechanisms of microorganisms killing In non-metastatic setting, the majority of the patients are expected to receive G-CSF as a part of standard care chemotherapy regimen. Thus, it is difficult to compare long-term outcome for patients who receive chemotherapy(s) with or without G-CSF in a retrospective study. In contrast, G-CSF is not routinely used in metastatic setting. Approximately 15-30% of patients with metastatic breast cancer develop brain metastasis In addition to their ability to promote metastasis, formation of NETs has also been linked to increased risk of thrombus formation including deep venous thrombosis (DVT) and pulmonary embolism (PE) de novo stage IV disease without brain metastasis at the time of diagnosis.Given the mounting evidence that G-CSF promote breast cancer metastasis in mouse models by acting on neutrophils to induce NETs as well as through other mechanisms, we hypothesized that patients who received G-CSF along with chemotherapy(s) have higher incidence of brain metastasis than those who did not receive G-CSF. The primary objective of this study was to test for a potential association between G-CSF use and the incidence of brain metastasis. The secondary objectives were to test the association between G-CSF use and brain metastasis free survival and overall survival (OS) and to test for an association between PE/DVT and brain metastasis and G-CSF use in patients with de novo stage IV breast cancer who 1) did not have confirmed brain metastasis at the time of diagnosis, and 2) had been treated with at least one cycle of any type of chemotherapy(s) with or without G-CSF at the Northwell Health Cancer Institute. G-CSF used was determined by each treatment physician based on clinical guidelines This retrospective chart review study was approved by the Institutional Review Board (protocol number: 20-0879) at the Feinstein Institutes for Medical Research at Northwell Health and a waiver of informed consent was granted based on the study's retrospective nature. We reviewed the electronic medical record (EMR) covering the period from 1/1/2013 to 12/31/2020 to identify patients with From the EMR, we extracted age at diagnosis, ethnicity, menopausal status at diagnosis, histological type , hormone receptor (HR) status, the human epidermal growth factor 2 (HER2) receptor status, smoking status, existence of pulmonary embolism (PE) and/or deep venous thrombosis (DVT) which was defined any PE/DVT found after the initial diagnosis, hormonal therapy, HER2 targeted therapy, immunotherapy, and G-CSF use. HR positivity was defined as positivity for estrogen receptor (ER) and/or progesterone receptor (PR) by immunohistochemistry staining. ER and PR positivity was defined based on the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline. HER2 positivity was defined as a HER2/CEP17 fluorescence in situ hybridization (FISH) ratio of ≥2.0 and/or an immunohistochemical (IHC) staining score of 3+ Standard descriptive statistics and frequency tabulation were used to summarize data. The chi-square test and Fisher's exact test were used to evaluate the association between two categorical variables. The Kruskal-Wallis test was used to compare the distributions of continuous variables among different groups. Univariate and multivariate logistic regression models were used to investigate the association between each variable and incidence of brain metastasis. Univariate and multivariate cox proportional hazard models were used to investigate the association between each variable and brain metastasis free survival (BMFS) and OS. BMFS was defined as the time from initial diagnosis to that of brain metastasis or last follow-up. OS was defined as the time from the date initial diagnosis to that of death or last follow-up. Patients who have not experienced the event (brain metastasis or death) was censored at the time of last follow-up. Variables with p-values<0.05 in univariate analysis and G-CSF use were included in multivariate analysis. A Kaplan-Meier estimate with a log-rank test was used for BMFS. All tests were two-sided and p-values <0.05 were considered statistically significant. All analyses were carried out using STATA ver. 14 software.A total of 78 patients were included in the final analysis. Among these 78 patients, 24 patients (30.8%) received G-CSF. There were no significant differences in variables of interest between patients treated with G-CSF and those who were not treated with G-CSF, except for HER2 targeted therapy: G-CSF use was associated with HER2 targeting therapy. Fourteen of 24 patients (58.3%) who were treated with G-CSF received HER2 targeting therapy and 18 of 54 patients (33.3%) who were not treated with G-CSF received it (P=0.04). Baseline patient characteristics are show in Table A total of 23 patients (29.5%) developed brain metastasis during the follow-up period. Thirteen of 54 patients treated without G-CSF use (24.1%) and 10 of 24 patients treated with G-CSF use (41.7%), a difference was not statistically significant (P=0.28). Among 13 patients who were treated without G-CSF and developed brain metastasis, 6 patients had PE/DVT (46.1%) and among 10 patients who were treated with G-CSF and developed brain metastasis, 2 patients had PE/DVT (20%). In univariate logistic regression models, G-CSF use was not a significant predictor of brain metastasis  (Table There was no significant difference in BMFS between patients treated with or without G-CSF (Log-rank P=0.35) . G-CSF wWith regard to OS, G-CSF use was not a significant variable  in univariate cox proportional hazard models . HER2 pode novo stage IV breast cancer. Interestingly, PE/DVT having occurred during follow-up period was associated with high incidence of brain metastasis.In this retrospective study, we found no association between G-CSF use and the development of brain metastasis in patients with newly diagnosed P < 0.001) In the metastatic setting, G-CSF is currently used to support neutrophil recovery after myelosuppressive cytotoxic chemotherapies, prophylactic to maintain dose density or to treat febrile neutropenia Recently, it has been shown that one mean by which neutrophils can promote cancer progression is through the formation of NETs. This is of particular interest because G-CSF can induce neutrophils to form NETs Our current study, including 78 patients, showed that patients who received G-CSF demonstrated numerically high incidence of brain metastasis compared to those who did not (24.1% versus 41.7%), but the difference was not statistically significant P=0.28). This result remained similar in logistic regression models . A recent paper analyzing a total of 468 patients showed that the levels of NETs in brain metastases were not significantly increased over the levels in the primary breast tumors and furthermore, the serum levels of NETs were not significantly elevated in patients with brain metastasis compared to patients without any type of metastasis . This rede novo stage IV breast cancer because earlier stage breast cancer almost always utilize G-CSF in neoadjuvant or adjuvant therapy but this may not represent the pattern of metastasis in other settings of patients with breast cancer. Third, sample size is small which is partially because our patients at Northwell system are taking annual screening regularly and tend to be diagnosed with earlier stage. Due to the good compliance with annual screening, the number of patients with de novo stage IV was relatively small and brain metastasis, our event of interest, is relatively rare. In our study, the total sample size is 78, with 13 of 54 having not been treated with G-CSF (24.1%) and 10 of 24 treated with G-CSF (41.7%) developed brain metastasis. With our sample size, the power to detect the 17.6% difference (41.7% vs 24.1%) using α=0.05 is 0.65. With α=0.05 and β=0.8, calculated sample sizes are 150, 79, and 47, respectively to detect 15% difference (40% vs 25%), 20% difference (40% vs 20%), or 25% (40% vs 15%). Based on these calculations, we cannot exclude that G-CSF use causes an increase in brain metastasis incidence, up to about a 25% difference between treated and untreated.There are several limitations in this study. First, this is a retrospective study. Second, it was necessary to focus on de novo Stage IV breast cancer, although a larger study is necessary to exclude an association. Interestingly, PE/DVT, which can be associated with elevated NETs, was associated with brain metastasis and further studies are warranted to determine more directly whether the presence of NETs in blood and DVT/PE are predictive of developing brain metastasis in patients with de novo Stage IV breast cancer.In conclusion, with the noted limitations, it is encouraging that the use of G-CSF was not associated with brain metastasis in patients with Supplementary figure and tables.Click here for additional data file.


Lepus granatensis) is a popular small game species in the Iberian Peninsula, and it has never been reported to be affected by sarcoptic mange. An adult female Iberian hare with overt skin lesions on forelimbs and ventral thorax, suggestive of sarcoptic mange, was culled in Quart de les Valls municipality in the Valencian Community, Spain, in 2019. Skin scrapings were digested in 10% KOH solutions to confirm the presence of mites. Ten Sarcoptes microsatellite markers were used to characterize the genetic structure of mites obtained from the hare, and from sympatric and allopatric wild rabbits (Oryctolagus cuniculus) and red foxes (Vulpes vulpes). A total of 56 alleles were counted across the 10 microsatellite loci. Six private alleles were found at four loci . The multivariate analysis characterized three main clusters, corresponding to mites collected on foxes originating from Catalonia, foxes from Valencia and the hare plus wild rabbits. To our knowledge, this is the first reported case of sarcoptic mange in the Iberian hare. The origin was molecularly traced back to contacts with endemically infected wild rabbits. We encourage further investigations on cross-transmissibility of S. scabiei between wild rabbits and the diverse representatives of Lepus spp.The Iberian hare (  •Crusted scabies in an Iberian hare was detected for the first time in Valencia, Spain in 2019.•S. scabiei for the Iberian hare, rather than sympatric predators.Wild rabbits might be a natural source of infection of •Sarcoptes scabiei infection.Microsatellite markers are efficient epidemiological tools for tracing the source of  The species is widely distributed in agricultural areas and open fields at altitudes ranging between sea level and 1,200 m above the sea level (Lepus europaeus) and the vulnerable Broom hare (Lepus castroviejoi)  (The Iberian hare (ea level . In nortoviejoi) . The Ibeoviejoi) . Lepus gtimidus) . Howevertimidus) . The immtimidus)  could reSarcoptes mites collected on the new host.The aim of this study was to report the first case of sarcoptic mange in the Iberian hare, and to describe the molecular profile of the 2During a surveillance campaign for the detection of myxomatosis in hares in the Valencia Community, Spain, an adult female Iberian hare was found dead with overt skin lesions compatible with sarcoptic mange in the Quart de les Valls municipality , Valencian Community, Spain.The necropsy was carried out at the Veterinary Faculty of the CEU Cardenal Herrera University. Skin samples from forelimbs and ventral thorax were taken and digested in a 10% KOH solution at 37°C overnight for the specimen isolation. Samples were then centrifuged at 3,500 rpm for 5 min, and the supernatant was removed and floated with a saturated sucrose solution for 5 min . To collS. scabiei mites   . CapillaThe genetic profile of mites isolated from the hare of this study was compared to those of mites previously collected from wild rabbits and red foxes in Spain.ade 4.0  and expected heterozygosity (He), allelic richness (R) and the Hardy-Weinberg Equilibrium (HWE) analysis, were carried out with software R 4.0 using the package et 2.1.3 .K = 1–20) were run. The estimation of clusters was performed using the DK method of Evanno. Individual mites were then associated to the correspondent inferred cluster . Observed heterozygosity and allelic richness ranged between 0.07 (Sarms 37) and 0.25 (Sarms 35) and between 0.61 (Sarms 33) and 0.88 (Sarms 41), respectively  to 10 (Sarms 33). Six private alleles were found across the 10 microsatellite loci, distributed among four loci . A significant deviation from HWE was detected throughout all loci, except for Sarms 34, 35, 37 (ectively .Table 2DK = 2), the Bayesian assignment test revealed the presence of two geographically separated Sarcoptes-derived clusters  markers have been widely used in igations  and in figations . In the According to the Bayesian assignment test , the cluSarcoptes mite populations  should be also carefully assessed under this perspective.We encourage further field and experimental investigations on possible No funding was received for this study.Not applicable.JC, LR, and BM designed the study. AMM coordinated the collection of the carcass and performed the post-mortem examination. SL and RV collected mite samples from Catalonia. BM performed the molecular analysis. JC drafted the first manuscript. JC, LR, VLL, SL, RV, BM and AMM finalized the manuscript. All authors read and approved the final manuscript.Raw data used and analyzed during the present study are available from the first and the corresponding author upon reasonable request.The authors declare that they have no competing interests.


Equus asinus). The CSF samples were collected from fifty healthy donkeys at the lumbosacral (LS) and atlanto-occipital (AO) sites.Diseases of the central nervous system are a well-recognized cause of morbidity and mortality in equine. Collection and analysis of cerebrospinal fluid (CSF) give information about the type and stage of degenerative and inflammatory diseases in central nervous system (CNS). The present research aimed to assess the clinical complications of CSF collections and to establish range values of cytological and biochemical parameters of CSF in adult healthy donkeys (P < 0.05). The CSF sodium concentration (123 to 160 mmol/L) was approximately like that of serum, but potassium (1.5–3 mmol/L) was lower than that of serum (P < 0.01). Urea concentrations (1.1–2.9 mmol/L) were markedly lower than serum (P < 0.001). Concentrations of CSF total proteins, and albumin ranged from 0.1 to 0.6 g/dL, and from 0.002 to 0.013 g/dL, respectively. The albumin quotient ranged from 0.06 to 0.56.Hypothermia, tachycardia, ataxia and recumbency may develop post-puncture. Erythrocytes were noticed in 35 of 50 CSF samples. Total nucleated cell counts ranged from 0 to 6 cells/μL, and lymphocytes predominated the cells (61%). The concentration of glucose (1.2 to 5.3 mmol/L) was lower than that of serum (Equus asinus) that can provide a basis in comparison when evaluating CSF from donkeys with neurologic diseases.Transient hypothermia, tachycardia, ataxia and recumbency may develop as clinical complications of CSF puncture procedures. The collection site has no impact on the constituents in CSF. Furthermore, this study presented the range values for normal cytological and biochemical constituents of CSF in donkeys ( Diseases of the central nervous system are a well-recognized cause of morbidity and mortality in equine . Many diIn equine, CSF can be collected via the atlanto-occipital (AO) or lumbosacral (LS) spaces . Many faEquus asinus); [2] determine the cytological and biochemical constituents of CSF analysis.At present, according to the authors’ knowledge, there are no literatures describing the clinical complications of CSF puncture procedures in donkeys. Therefore, the objectives of the current research were as follows: [1] describe the clinical observations and possible complications that may develop after CSF collection in clinically healthy adult donkeys (Equus asinus), of both sexes . The average age was 8 ± 1.5 years, and weight was 110 ± 2.8 kg (mean ± standard error). All animals were clinically healthy and showed no signs of CNS or other neurological diseases during physical examination. The body score of animals ranged from 2 to 3 [The present research was ethically approved by the Animal Care and Welfare Committee of Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt. All national and institutional guidelines for the care and use of animals were followed during study procedures. All animals were housed and cared according to the Egyptian animal welfare act . Moreover, informed consent was granted by the owners of the donkeys. The current study was carried out on 50 adult clinically healthy donkeys  0.03 mg/kg acepromazine , and 15 min thereafter, each donkey was injected IV with 1 mg/kg xylazine 2% . The anesthesia was maintained by intramuscular injection of 2 mg/kg ketamine . From each donkey, two CSF samples were collected, the first was obtained from lumbosacral site, and the second sample was collected from atlanto-occipital place . Both co2 × r × 1.118, where RCF = relative centrifugal force, RPM = number of rotations per minute, r = centrifuge radius, thereafter the sera were harvested and analyzed for biochemical indices.A one-time blood sample was collected from each donkey by venipuncture from the jugular vein immediately following CSF collection. After that, the blood samples were centrifuged for 15 min according to centrifuge equation: RCF = (RPM/1000)For cytological analysis, the samples of CSF were examined immediately after collection. Total nucleated cell count was determined using a hemocytometer as reported elsewhere . For dift-test was used. To compare between AO and LS methods, linear regressions were carried out, and R square and regression coefficients were estimated, as well as Bland-Altman analysis was conducted. For all statistical procedures, the degree of significance was set at P < 0.05.Data are presented as means ± standard error (SE) and the analysis was carried out using SPSS software . The normal distribution of all data was tested using Kolmogorov-Smirnov test. To compare the effect of puncture site (AO vs. LS) on each parameter, paired sample P < 0.05). However, the rectal temperature returned to the normal physiological values 2 h post-collection of CSF samples. In contrast, heart rates showed significant increases post-puncture (P < 0.05), while the respiratory rates showed insignificant changes (P > 0.05). Five donkeys suffered from hind limb ataxia and recumbency and were unable to stand by themselves after AO puncture. However, they walked sound without difficulties 2 h thereafter. The feed and water intakes were normal after collection, however, three donkeys showed local pain, discomfort, shaking of the tail . The results of biochemical findings of serum and CSF in clinically healthy donkeys were presented in Table P < 0.05), while sodium showed no significant changes (P > 0.05). In comparison with their serum values, the CSF concentrations of potassium was lowered (P < 0.01), while chloride was higher (P < 0.01). The CSF concentrations of calcium, phosphorous, magnesium and blood urea were lower than their corresponding values serum (P < 0.01). The puncture site had no significant influences on the studied biochemical parameters. Table P > 0.05). The correlation coefficients (r) for cytological and biochemical variables in AO CSF and their corresponding parameters in LS CSF ranged from moderate to strong positive relationships . Table The collection and analysis of CSF can provide valuable information about the central nervous system. Evaluation of CSF is an important tool, and together with the history, clinical examination, neurologic examination, and other ancillary test procedures may help in the diagnosis and prognosis of neurologic disease in equine .In the present work, CSF samples, in both techniques, were collected from anesthetized animals with analgesic drug as reported in methodology. It had been recommended induction of general anesthesia for AO puncture  and sedaIn the current study, physical examination revealed lowered body temperature after collection of CSF, indicating a clinical complication and a physiological response in stressed donkeys. In a previous study , the autAnalysis of CSF is a general index of CNS health and often provides useful information about the type of neurologic lesion that is present . In the In both puncture techniques, values of RBCs ranged from 0 to 8 cells/μL, indicating minor blood contamination. Furthermore, blood contamination in this study was not felt to be significant based on clarity, color and small number of RBCs, as well as the all turbid and red-tinged CSF samples were excluded from the analytical procedures to avoid misinterpretation of obtained data. In contrast, no RBCs were observed in CSF samples of another breed of donkey (miniature) . Such diIn the current study, results of cytology, including total nucleated cells and differential cell count of both punctures, are consistent with previous findings in horses , 29. CSFBiochemically, the CSF glucose concentrations for the donkeys in this study were slightly higher than those previously reported in miniature donkeys . These dIn the present study, the concentration of CSF total proteins was nearly in agreement with a previous report in horses , and in In general, some cytological and biochemical constituents of CSF in this study are varied from those reported in miniature donkeys  and horsCollection of CSF from both sites at the same time and comparing the findings may be helpful in cases in which neuroanatomic localization of the lesion is difficult . In the Equus asinus) that can provide a basis in comparison when evaluating CSF from donkeys with neurologic diseases.Minor physical changes in the form of transient hypothermia and tachycardia, as well as ataxia and recumbency may develop as clinical complications of puncture procedures for CSF collection with rapid subsequent recovery. The puncture site had no effect on the cytological and biochemical constituents of CSF samples. The current study presented the normal values for cytological and biochemical constituents of CSF in donkeys (


Due to the growing number of young patients at risk of germ cell loss, there is a need to preserve spermatogonial stem cells for patients who are not able to bank spermatozoa. Worldwide, more and more clinics are implementing testicular tissue (TT) banking programs, making it a novel, yet indispensable, discipline in the field of fertility preservation. Previously, TT cryopreservation was predominantly offered to young cancer patients before starting gonadotoxic chemo- or radiotherapy. Nowadays, most centers also bank TT from patients with non-malignant conditions who need gonadotoxic conditioning therapy prior to hematopoietic stem cell (HSCT) or bone marrow transplantation (BMT). Additionally, some centers include patients who suffer from genetic or developmental disorders associated with prepubertal germ cell loss or patients who already had a previous round of chemo- or radiotherapy. It is important to note that the surgical removal of TT is an invasive procedure. Moreover, TT cryopreservation is still considered experimental as restoration methods are not yet clinically available. For this reason, TT banking should preferably only be offered to patients who are at significant risk of becoming infertile. In our view, TT cryopreservation is recommended for young cancer patients in need of high-risk chemo- and/or radiotherapy, regardless of previous low-risk treatment. Likewise, TT banking is advised for patients with non-malignant disorders such as sickle cell disease, beta-thalassemia, and bone marrow failure, who need high-risk conditioning therapy before HSCT/BMT. TT retrieval during orchidopexy is also proposed for patients with bilateral cryptorchidism. Since patients with a medium- to low-risk treatment generally maintain their fertility, TT banking is not advised for this group. Also for Klinefelter patients, TT banking is not recommended as it does not give better outcomes than a testicular sperm extraction later in life. Over the last decades, the overall rate of childhood and adolescent cancer incidence has increased. The death rates, on the other hand, have strongly decreased due to major advances in oncological treatments. Nowadays, long-term survival is expected in about 80% of young cancer patients . In Eurovia testicular sperm extraction (TESE)  . If thesn (TESE) . For preSince the first experimental fertility preservation program for (pre)pubertal boys in 2002, more centers around the world have started to collect and cryopreserve immature TT from boys at risk of losing their SSCs . An intein vitro spermatogenesis  auto-transplantation of the thawed TT, 2) auto-transplantation of isolated SSCs from the TT, and 3) ogenesis , 12. Recogenesis . When trogenesis . Althougogenesis  and two ogenesis , 16 were patient . However rodents  and furtogenesis .Despite the progress that has been made over the past few years, TT banking remains an experimental method as these fertility restoration techniques are currently not yet implemented in clinical practice , 20. MorAll taken together, TT banking remains a relatively new technique of which many aspects still require further investigation. To safeguard the well-being of the young patients, and to avoid unnecessary surgery, strict selection criteria are required for this procedure. In this review, the risk of infertility for the most frequent conditions and treatments will be described based on the most recent findings. Moreover, as the indications for TT banking vary between centers, this review aims to revise the current inclusion criteria and gives advice on which patients are (still) eligible for TT banking.The most common indication for TT banking continues to be childhood malignant disease. Within this category, the most prominent disorders are hematological cancers, central nervous system tumors, Ewing family tumors, rhabdomyosarcoma, osteosarcoma, neuroblastoma, and other non-central nervous system solid tumors . MalignaAlthough the malignancy itself plays a role, the fertility decline in cancer patients predominantly depends on their treatment with surgery, chemotherapy, local or total body irradiation, and/or a combination of those. The cancer treatment can either cause direct damage to the germ cells and/or the somatic cell populations of the testis , or it cTumor resection surgery involving the reproductive organs can affect the patient’s fertility potential. Performing a unilateral orchiectomy in patients with testicular cancer results in decreased germ cell numbers, and may put them at risk for prematurely reduced Leydig cell function –61. In tChemotherapy is one of the most commonly used treatments to cure cancer. Its principle is to target the rapidly dividing cancer cells. Nevertheless, chemotherapy does not only target the cancerous cells, it also targets other healthy dividing cell populations . SertoliChemotherapeutic agents can be categorized as low-, medium-, or high-risk depending on their expected degree of gonadal damage. It is important to note that high cumulative doses and the combination of different cytotoxic agents can significantly increase the risk for long-term fertility problems . BesidesAnother frequently used cancer treatment is radiotherapy. Analogous to chemotherapeutic agents, radiation targets the cancerous cells but has also off-targets like the gonads. The testes are one of the most radiosensitive tissues of the male body . Damage Testicular irradiation and TBI with doses of 10-12 Gy have been reported to harm Sertoli cells. The damage to the Sertoli cell is linked with smaller testicular volumes at adulthood and high FSH and low INHB levels , 82, 83.Impaired fertility has also been reported after cranial radiotherapy using doses of 35–45 Gy. Cranial radiotherapy does not directly damage the gonads, but it is known to cause neuro-endocrine imbalances when the HPG axis lies within the irradiation field . This miAs shown in Targeted cancer treatments are gaining popularity in the field of oncological research and clinical application. In contrast to conventional chemo- and radiotherapy, targeted therapies will specifically act on the cancerous cells instead of all rapidly dividing cells . Many taNowadays, HSCT is not only a curative treatment option for patients with malignant hematological diseases, but also for patients with SCD, thalassemia, bone marrow failure, or other non-malignant conditions . HSCT reSCD or sickle cell anemia is the most common non-malignant condition for which allogeneic HSCT is recommended . Each yeFor many years, it is known that the SCD itself can cause delayed puberty as well as disturbed semen parameters and sperm abnormalities at adulthood –106. TheHydroxyurea (HU) therapy is another commonly used treatment to prevent vaso-occlusive pain episodes, acute chest syndrome, hospital admissions, and the need for blood transfusions in SCD patients. HU is an FDA-approved non-alkylating antineoplastic agent that can increase the concentration of fetal hemoglobin and thus prevent the sickling of the red blood cells with the mutated adult hemoglobin , 112. OnFor patients with a severe SCD phenotype and end-organ damage, allogeneic HSCT is recommended , 118. ThBeta-thalassemia is the second most common hereditary hematological disorder, with about 60,000 new symptomatic cases each year . Beta-thB-TM patients need lifelong blood transfusions to avoid skeleton deformities, hepatomegaly, and splenomegaly . As discDespite the declined morbidity and mortality with periodic blood transfusion and iron chelation, HSCT remains the only curative option for patients with B-TM . TransplChildhood bone marrow failure (BMF) is another well-represented group of benign conditions for which immature TT is banked . The terSimilar to the previously discussed blood disorders, first-line treatment often consists of blood transfusions to counteract the anemia, as well as chelation therapy to limit iron overload. Platelet transfusion can be administered to prevent or treat bleeding due to thrombocytopenia . In the Fanconi anemia often causes genital abnormalities as cryptorchidism, hypospadias, seminiferous tubule hypoplasia, and small testes. The disease itself is also shown to affect the HPG-axis, with hypogonadism, delayed or accelerated puberty, and fertility issues as previously discussed , 140. ThSome other benign pathologies for which TT banking is recommended because of the fertility problems linked to high-risk treatment are inborn errors of metabolism for which other available therapies are less effective, and severe immune diseases that are not responsive to immunotherapy , 141. WeThe third and last category of childhood conditions that may affect fertility and for which immature TT has been banked over the last years consists of genital, testicular, and sexual disorders . As thisUndescended testis or cryptorchidism is a pediatric condition in which one  or both testes  fail to descend into the scrotum. Cryptorchidism is one of the most common congenital anomalies that affects about 1-4% of full-term and 30% of preterm newborns worldwide . This coSome researchers suggested that hormone therapy with human chorionic gonadotrophin, GnRH, or LH-releasing hormone could have an additional protective effect on the fertility of boys with cryptorchidism. However, the results of these studies are not convincing and therefore, hormonal therapy is not advised . To decrNevertheless, the risk of azoospermia remains 18–46% in patients with bilateral cryptorchidism despite early surgical treatment , 158. ToWith an incidence of as much as 1-2/1,000 newborn males, Klinefelter syndrome (KS) is the most frequent sex chromosome abnormality in the human . About 8With this in mind, TESE during early adolescence has been proposed in the hope of having better success rates compared to TESE in adulthood –172. Thein vitro spermatogenesis (IVS), unfeasible. Additionally, as the biopsies only rarely contained spermatogonia and the TT biopsy procedure may further reduce the number of spermatogonia, TT banking in KS patients remains a highly controversial subject . Patients undergoing medium- to low-risk therapy and their parents should be informed about the possible impact of the therapy on the reproductive function, but TT banking is not advised in these patients. It is proposed to retrieve a TT biopsy during orchidopexy in patients with bilateral cryptorchidism. For Klinefelter patients, however, TT banking is not recommended because it generally does not give better outcomes than a TESE later in adult life. Also for patients with other testicular disorders, a TT biopsy is not recommended as the benefits usually do not outweigh the risks.ED performed the literature search and manuscript writing, while AB and EG contributed to the critical revision of this review. All authors were involved in the conception and design of this review and approved the submitted version.This review paper was written with financial support from the Research Programme of the Research Foundation-Flanders (FWO), Kom Op Tegen Kanker (KOTK), and Wetenschappelijk Fonds Willy Gepts (WFWG).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Metformin has favorable effects on cardiovascular outcomes in both newly onset and advanced type 2 diabetes, as previously reported findings from the UK Prospective Diabetes Study and the HOME trial have demonstrated. Patients with type 2 diabetes present with chronically elevated circulating cardiac troponin levels, an established predictor of cardiovascular endpoints and prognostic marker of subclinical myocardial injury. It is unknown whether metformin affects cardiac troponin levels. The study aimed to evaluate cardiac troponin I and T trajectories in patients with diabetes treated either with metformin or placebo.This study is a post-hoc analysis of a randomized controlled trial  that included 390 patients with advanced type 2 diabetes randomized to 850 mg metformin or placebo up to three times daily concomitant to continued insulin treatment. Cardiac troponin I and T concentrations were measured at baseline and after 4, 17, 30, 43 and 52 months. We evaluated cardiac troponin trajectories by linear mixed-effects modeling, correcting for age, sex, smoking status and history of cardiovascular disease.This study enrolled 390 subjects, of which 196 received metformin and 194 received placebo. In the treatment and placebo groups, mean age was 64 and 59 years; with 50% and 58% of subjects of the female sex, respectively. Despite the previously reported reduction of macrovascular disease risk in this cohort by metformin, linear mixed-effects regression modelling did not reveal evidence for an effect on cardiac troponin I and cardiac troponin T levels . A statistically significant time-treatment interaction was found for troponin T  but not troponin I concentrations .In this post-hoc analysis of a 4.3-year randomized controlled trial, metformin did not exert a clinically relevant effect on cardiac troponin I and cardiac troponin T levels when compared to placebo. Cardioprotective effects of the drug observed in clinical studies are not reflected by a reduction in these biomarkers of subclinical myocardial injury.Trial registration ClinicalTrials.gov identifier NCT00375388.The online version contains supplementary material available at 10.1186/s12933-022-01482-z. While its clinical use as a first-line pharmacological means to temper hyperglycemia and increase insulin sensitivity now well exceed 60 years , metformFindings of the cornerstone long-term UK Prospective Diabetes Study (UKPDS) and the Diabetes Prevention Program Outcome Study are often referenced to substantiate the drug’s cardioprotective effects in populations with newly diagnosed diabetes and prediabetes. The UKPDS, a non-placebo-controlled, open label trial, was the first study showing a reduction of cardiovascular disease (CVD) and cardiovascular (CV) mortality of 40% in obese patients with newly diagnosed type 2 diabetes using metformin compared to controls , 4.The randomized, placebo-controlled trial hyperinsulinemia: the outcome of its metabolic effects (HOME) was designed to investigate whether treatment with metformin, in addition to insulin for glycemic control, could decrease cardiovascular disease in established diabetes patients. As a result, we have previously reported the reduction of macrovascular disease risk observed in the 4.3 year follow-up period . The stuSpecifically, high-sensitivity cardiac troponin I (hs-cTnI) and cardiac troponin T (hs-cTnT) emerged in the last decade as circulating biomarkers with prognostic value. Chronically elevated troponin levels were found to be indicative of subclinical myocardial injury and associated with increased risk of CV outcomes and all-risk mortality in the general population , 7 as weWith an aim to elucidate the mechanism by which metformin exerts its cardioprotective effects, we hypothesized that metformin treatment may reduce circulating hs-cTnT and hs-cTnI levels. The scope of the current study was to investigate both biomarkers’ trajectories in patients with type 2 diabetes treated with insulin combined either with metformin or placebo.The HOME trial was a randomized trial that included 390 patients with advanced type 2 diabetes mellitus and took place from 1998 to 2002 . The trial was conducted in the outpatient clinics of three non-academic hospitals in the Netherlands: Bethesda General Hospital, Hoogeveen; Isala Diaconessen Hospital, Meppel; and Aleida Kramer Hospital, Coevorden. Refer to Fig. Participant eligibility criteria and further details are reported elsewhere . BrieflyStudy participants provided written informed consent prior to inclusion. The medical ethical committees of the participating hospitals approved the trial protocol. The study was conducted in accordance with Good Clinical Practice  and with the Declaration of Helsinki .Blood samples were drawn at baseline and after 4, 17, 30, 43 and 52 months, and aliquots of serum were stored at − 80 °C until analysis. Serum high-sensitivity cardiac troponin T  and high-sensitivity cardiac troponin I  concentrations were measured. The assays were calibrated and quality controlled as intended by the manufacturers. Limit of blank (LoB) and limit of detection (LoD) were 2 ng/L and 3 ng/L, for cardiac troponin T (Roche package insert 08469814500 version 3.0); 1 ng/L and 2 ng/L for cardiac troponin I (Abbott package insert 08P13), respectively. The hs-cTnI assay achieves a 10% coefficient of variation at 4.7 ng/L and a 20% coefficient of variation at 1.3 ng/L. The hs-cTnT assay achieves a 10% coefficient of variation at 13 ng/L and a 20% coefficient of variation at 6.8 ng/L.Continuous variables were expressed as mean and standard deviation (SD), or median and interquartile range (IQR) for non-parametric variables. Categorical data were reported as n (%). The data and analyses presented concern all randomized patients following the intention-to-treat principle. Cardiac troponin value distributions were skewed and were log-transformed prior to analysis. Upon log-transformation, three influential data points were visually identified , were considered clinical outliers and were therefore not included in the analysis. Baseline and over time comparisons of within-group median cardiac troponin values were done using the paired samples Wilcoxon test. All statistical analysis was performed using R version 3.6.1.2 test. In addition, troponin I values were dichotomized using sex-specific risk stratification cut-offs defined by the manufacturer, with values exceeding 4 and 6 ng/L categorized as moderate to high risk for female and male subjects, respectively (package insert version B3P2F0).We applied a conditional modeling strategy by constructing linear mixed-effects regression models for continuous, within-subject clustered, cardiac troponin I and T data points. The model included fixed effects for treatment assignment (placebo as reference), time as a continuous variable and the interaction between these indicators. Models including random intercept effects only and a combination of random intercept/random slope effects were fitted and compared using the maximum likelihood technique. Identified optimized models were fitted using the restricted maximum likelihood approach, including age, sex, smoking status and cardiovascular history as fixed covariates. Covariance structures for residual errors of longitudinally clustered measures were selected based on the Akaike information criterion (AIC). A sex-stratified analysis was performed by fitting optimized models to each of the sexes individually. Influential data points were identified by determining the Cook’s distance. Optimized models were also fitted to the dataset excluding data points with Cook’s distance > 0.01. Model estimates were reported as antilog values. To facilitate visual interpretation of the effect of metformin on the prevalence of elevated troponin levels, troponin values for which within subject repeated measurements were available at baseline, 4 months and 52 months, were dichotomized at the study’s population sex-specific 75th percentiles. Differences between prevalence of elevated troponin values across treatment groups at each time point were evaluated using the χm = 100), using all variables from the optimized analytical model for MI-MAR and excluding treatment allocation for MI-MNAR. No auxiliary variables were used. Algorithm convergence was assessed by examination of the potential scale reduction factor and using integrated diagnostic plots  was found, although not statistically significant . The time-treatment interaction was a statistically significant determinant of troponin T  but not troponin I concentrations , meaning that the favorable effect of metformin on troponin T concentrations increased slightly over time with respect to placebo treatment.Figure ɸ reported in Additional file Tables Upon removal of influential data points (Cook’s distance), favorable effects on the course of troponin levels of both metformin treatment and time-treatment interaction were confirmed. The constant treatment effect on troponin I concentrations determined in this subset of observations was not statistically significant , with no change over time . The time-treatment effect on troponin T was statistically significant , but not the constant treatment effect , analogous to the findings obtained without exclusion of influential data points.Refer to Additional file p value for the time-treatment interaction in women neared the commonly used threshold of p < 0.1 for statistical significance of interaction terms. The direct treatment effect on troponin I concentrations was not statistically significant in either sex. For troponin T concentrations, the time-treatment interaction was a significant determinant only in women.A sex-stratified analysis reported in Additional file We dichotomized all troponin I and troponin T values using the sex-specific 75th percentile in the study population at baseline as a threshold to define elevated levels of the biomarker . Additional file Dependent variable missingness was monotone and was 19.5% and 19.3% for cardiac troponin I and cardiac troponin T, respectively. All predictor variable values were available apart from cardiovascular history for one subject (0.25% of data points). Given the missing at random assumption cannot generally be verified and for the current study type discontinuation can be anticipated to be heterogeneous due to dependence on pathology and treatment side-effects, robustness of the primary analysis’ estimates was tested through complete case analysis and multiple imputation (MI) under both missing at random and missing not at random assumptions.Results from full mixed-effects modelling using only complete cases and multiple imputation under both missing at random and missing not at random missingness mechanisms are reported in Additional file The current report covers results of the first post-hoc analysis of a long-term randomized placebo-controlled trial on the effects of metformin on cardiac troponin I and T trajectories. Thorough statistical analysis of hs-cTnI and hs-cTnT levels in the HOME cohort, which previously reported reduced macrovascular disease risk as a result of metformin treatment, did not reveal evidence of a clinically relevant effect of metformin treatment on these cardiac biomarkers, when compared with placebo. Our results corroborate and extend previously published work by Srivastava et al. which investigated short-term effects of metformin on circulation concentrations of cardiac troponin T, reporting no significant effect in a population of relatively recent-onset diabetes . The autBaseline troponin levels in the current study cohort were high compared to those in the general population and other type 2 diabetes populations , 14, 20.A sex-specific sub-analysis applying the optimized conditional model revealed that the identified trend for the time-treatment interaction was not statistically significant in males. A previous study by Lyons et al. reported altered myocardial glucose metabolism in men but not women treated with metformin, with male subjects exhibiting decreased glucose metabolism and increased myocardial fatty acid (FA) metabolism. In women, metformin was found to reduce myocardial FA metabolism and reduction of left ventricular mass was more pronounced . Female As highlighted previously, few studies were specifically designed to investigate metformin’s effect on cardiovascular endpoints, of which only the HOME study framework allowed for longitudinal biochemical analysis of cardiac circulation markers to quantify its cardioprotective effects and potentially elucidate underlying mechanisms. We have previously investigated the effects of metformin on N-terminal pro B-type natriuretic peptide (NT-proBNP) plasma levels and found no effect when compared to placebo . While mThe current study was a post-hoc analysis, and its strengths include the randomized, placebo-controlled, double-blind design, the lengthy follow-up period during which serum samples were collected frequently and a direct comparison of cardiac troponin I and T. Also, given the study was conducted in a non-academic setting, patient recruitment is reflective of actual peripheral diabetes management.Our study also has some limitations. First, imbalance between treatment groups after randomization. Although the mixed model analysis approach taken is robust against baseline differences, we cannot completely rule out residual confounding and the imbalance hampered straightforward data visualization and interpretation. Second, the power analysis of the HOME trial was based on the studies’ original primary micro- and macrovascular endpoints ; neverthIn conclusion, our findings show that in patients with long-lasting type 2 diabetes that are intensively treated with insulin, the concomitant use of metformin does not affect circulating cardiac troponin I and T as compared to placebo when tested in a trial cohort that previously reported reduction in cardiovascular disease associated with the drug. Metformin exerts its cardioprotective effects in a way that does not alter cardiac troponin levels, a finding relevant to elucidate its exact mechanism in the future.Additional file 1: Figure S1. Prevalence of Troponin T and I levels > 75th percentile.Additional file 2: Figure S2. Prevalence of Troponin I levels exceeding the manufacturer’s low risk threshold.Additional file 3: Table S1. Troponin I and Troponin T linear mixed-effects model metrics. Table S2. Sex-stratified mixed models of Troponin I and Troponin T. Table S3. Prevalence of elevated cardiac troponins in placebo vs. metformin. Table S4. Random slope/random intercept models as sensitivity analysis for Troponin I. Table S5. Random slope/random intercept models as sensitivity analysis for Troponin T.


Several difficulty grading systems have been developed as a useful tool for selecting patients and training surgeons in laparoscopic procedures. However, there is little information on predicting the difficulty of laparoscopic donor nephrectomy (LDN). The aim of this study was to develop a grading system to predict the difficulty of LDN.Data of 1741 living donors, who underwent pure or hand-assisted LDN between 1994 and 2018 were analyzed. Multivariable analyses were performed to identify factors associated with prolonged operative time, defined as a difficulty index with 0 to 8. The difficulty of LDN was classified into three levels based on the difficulty index.P < 0.001), BMI > 28 , pure LDN , multiple renal arteries  and multiple renal veins  were independent risk factors influencing prolonged operative time. The difficulty index based on these factors was calculated and categorized into three levels: low (0–2), intermediate (3–5), and high (6–8) difficulty. Operative time was significantly longer in the high difficulty group (225 min) than in the low  and intermediate difficulty group . The conversion rate was higher in the high difficulty group (4.4%) than in the low  and the intermediate difficulty group . No significant difference in major complications was found between the groups.Multivariable analyses identified that male (odds ratio [OR] 1.69, 95% CI 1.37–2.09, We developed a novel grading system with simple preoperative donor factors to predict the difficulty of LDN. This grading system may help surgeons in patient selection to advance their experiences and/or teach fellows from simple to difficult LDN. The advantages of minimally invasive surgery over open donor nephrectomy (DN) are well established; reductions in length of stay, pain, and convalescence; faster return to normal activity . Pure anRecently, several practical difficulty scoring systems have been developed in the field of laparoscopic procedures –7, and cThe aim of the present study was to develop a grading system to predict the difficulty of LDN in a high-volume center of living kidney donation and kidney transplantation in Western Europe. Moreover, we aimed to propose the benchmark operation of LDN.A retrospective review was performed using a prospective kidney transplant database including 1741 consecutive living donors who underwent LDN at the Erasmus MC, University Medical Centre Rotterdam, The Netherlands, between January 1998 and December 2018. The present study was approved by the Ethics Committee of the Erasmus MC (MEC-2019-0373), and was conducted in accordance with the tenets of the Declaration of Helsinki. This type of retrospective analysis does not require informed consent from the individual patients.Using the kidney transplant database, the following donor data were collected: age, gender, body mass index (BMI), relationship between donors and recipients, the technique of LDN (pure or hand-assisted), the side of LDN (right or left kidney), operative time, blood loss, conversion rate, number of renal artery (single or multiple), number of renal vein (single or multiple), the incidence of postoperative major complications (Clavien-Dindo grade ≥ III ), and po2O pressure and a 30° video endoscope. The colon was mobilized, and perirenal fat was divided using an ultrasonic device . The ureter, the renal artery, and the renal vein were identified and dissected, afterwards a Pfannenstiel incision was made. The ureter, the renal artery, and renal vein were divided and the donor kidney was extracted using the endobag through the Pfannenstiel incision. In case of conversion from LDN to hand-assisted DN, a Gelport  was inserted via the Pfannenstiel incision.Details of surgical techniques of LDN were previously reported . Donors 2O pressure. Dissection around kidney and identifying/ dissecting of the renal vessels and ureter were similar to pure LDN. The kidney was extracted manually via the Gelport.Hand-assisted LDN were performed through the retroperitoneoscopic approach. First a Pfannenstiel incision was made to create a retroperitoneal space and a Gelport was inserted. Three trocars were replaced with a triangular shape, and carbon dioxide was insufflated retroperitoneally to 12-cm HAmong donor factors, donor BMI was one of factors for the decision of procedures . Hand-asTo construct a difficulty grading system, univariate and multivariable analyses were performed to identify donor factors that were significantly associated with prolonged operative time. Factors associated with prolonged operative time were investigated with the Cox proportional hazards model showing odds ratio (OR) and 95% confidence interval (CI). The multivariable models to estimate surgical difficulty were used to generate a simple difficulty grading system , in whicn = 57). The incidence of postoperative major complications and mortality were 1.2% (n = 22) and 0%, respectively. Mean LOS was 3.4 days.The characteristics of the 1741 donors undergoing LDN between 1994 and 2018 are depicted in Table P < 0.001), BMI > 28 , pure LDN , multiple renal arteries  and multiple renal veins  were independent risk factors related to prolonged operative time . Multivariable analyses identified that male  and the intermediate difficulty group . The incidence of conversion rate was higher in the high difficulty group (4.4%) than in the low  and the intermediate difficulty group . No significant difference in major complications was found between the groups.Outcomes based on the difficulty classification were shown in Table Based on the difficulty index and the difficulty classification, we propose the benchmark operation of LDN, as demonstrated in Fig. The present study demonstrates a novel difficulty grading system for LDN by analyzing 1741 living donors in a high-volume center. Our difficulty grading system was developed by assessing easily available preoperative donor factors such as gender, BMI, the technique of LDN, and the number of renal artery and vein. The difficulty classification according to the difficulty index can predict the estimated operative time and the risk of conversion. In contrast, the incidence of postoperative major complications was similar regardless of the difficulty classification. In addition, we proposed the benchmark operations based on the difficulty classification evaluated by the difficulty index.Several difficulty indexes, consisting of patient characteristics, anatomy, and tumor characteristics, have been used to classify the difficulty of several laparoscopic procedures –8. OperaThe benchmark operations of LDN were constructed based on previous reports and our experiences. The number of procedures requiring to perform LDN proficiently and independently remains controversial, therefore we defined inexperienced surgeons as performing < 15 cases of LDN, and experienced surgeons as performing > 30 cases according to the previous reports , 18. DefA critical question is how to design training in the most efficient manner possible, while developing excellent skills and long-term retention. The graduated responsibility in surgical training is built on a balance between mastery of skills and the ability to recognize potential pitfalls. A recent review provides an evidence-based overview to optimize the learning curve . An optiThe present study has several limitations to be disclosed. We performed a retrospective study in a prospective database in a single center. There might be a potential donor selection bias. Regarding the selection for the technique of LDN, it was decided by considering not only surgeon experience but also donor factors. However, the selection was not randomly assigned, therefore an invariability might be created in the analysis. Although conversion has been used as a predictor of difficulty score among several operative surrogates in other fields , 21, thiWe proposed a novel grading system with simple preoperative donor factors to predict the difficulty of LDN. Our difficulty index well reflects the estimated operative time and can easily categorize LDN as low, intermediate, or high difficulty. Moreover, this grading system can be helpful to guide surgeons in patient selection as well as advancing their experiences and/or teach fellows from simple to difficult LDN in a clinical practice. However, this system should be prospectively validated.


Hepatocyte growth factor (HGF) plays a role in neuronal survival and development, and has been implicated in neurodegenerative diseases. We sought to examine the associations of the CSF HGF with Alzheimer’s disease (AD) pathology and cognitive function.A total of 238 participants  and 148 mild cognitive impairment (MCI)) who had measurements of CSF HGF were included from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. Multiple linear regression models were utilized to explore the cross-sectional associations of CSF HGF with AD biomarkers  in non-demented participants. Moreover, linear mixed-effects regression models were utilized to explore the longitudinal associations of HGF subgroups with cognitive function. Mediation analyses were utilized to explore the mediation effects of AD markers.MCI individuals had significantly increased CSF HGF compared with the CN individuals. Results of multiple linear regressions showed significant correlations of CSF HGF with CSF Aβ42, pTau, and tTau in non-demented participants. Higher level of baseline CSF HGF was associated with faster cognitive decline. Influences of the baseline CSF HGF on cognition were partially mediated by Aβ42, pTau, and tTau pathologies.High concentrations of HGF in CSF may be related to faster cognitive decline. The cognitive consequences of higher CSF HGF partly stem from AD pathology, which suggests that the CSF HGF may be an attractive biomarker candidate to track AD progression.The online version contains supplementary material available at 10.1186/s12883-021-02356-9. Alzheimer’s disease (AD) is pathologically characterized by aggregated amyloid plaques and neurofibrillary tangles (NFTs) . Amyloidhttp://adni.loni.usc.edu. The ADNI (launched in 2003) was led by Dr. Michael W. Weiner. All participants were recruited from more than 50 sites across the Canada and United State. The ADNI aim to develop clinical, biochemical, and imaging biomarkers for tracking AD. ADNI was approved by all regional ethical committees, and contained written informed-consent documents of all participants.Alzheimer’s Disease Neuroimaging Initiative (ADNI) data are deposited in a open access repository and can be accessed at n = 90), early MCI or late MCI (n = 148) participants. Cerebral amyloid-β accumulation generally precedes the dementia stage by many years. We excluded individuals with dementia (due to AD), since the aim of this study was to examine detection of the earliest accumulation of amyloid-β. All participants underwent assessments of CSF HGF and CSF AD biomarkers at baseline. All participants were adults aged 55 to 90 years. The specific ADNI diagnostic criteria for distinguishing CN, MCI, and AD participants were summarized in Additional Table From ADNI, we included only cognitively normal (CN) , and pTau (pg/ml)). The within-batch precision values were < 10% .In ADNI, CSF procedural protocols have been described previously. INNOBIA AlzBio3 immunoassay  was used to measure CSF AD biomarkers (including CSF Aβhttp://adni.loni.usc.edu). Analytes were removed if the mean percentage difference was greater than 35% or the test-retest sample was less than 7 or the mean absolute percentage difference was greater than 60% or if the Bland-Altman slope and intercept significantly differed from zero. Participants who had extreme outliers (< 3-fold or > 3-fold standard deviations (SD) from the mean value) were removed. Finally, 5 participants were removed from the data set.CSF HGF was measured using a multiplex panel. The multiplex panel is based upon Luminex immunoassay technology and has been developed by Rules Based Medicine (MyriadRBM) to measure a range of inflammatory, metabolic, lipid and other disease relevant indices. Quality control (QC) data that is specific for the CSF samples included in this study are the test/retest results for the 16 randomly selected CSF samples , ADNI memory (ADNI-MEM), and ADNI executive function (ADNI-EF)) were used to evaluating cognitive functions in this study. The ADNI-MEM was developed from the Logical Memory Test, Rey Auditory Verbal Learning Test, MMSE, and Alzheimer’s Disease Assessment Scale cognitive subscale (ADAS-Cog) . ADNI-EFAPOE ε4 allele, carriers of two APOE ε4 alleles, and APOE ε4 non-carriers.The APOE genotyping was performed by Hhal restriction enzyme digestion, polymerase chain reaction (PCR) amplification, and standard gel resolution and visualization processes , 21. Quahttp://adni.loni.usc.edu/methods/mri-tool/mri-analysis). White matter hyperintensities volume (WMHV) were measured using the Bayesian approach for the segmentation of high-resolution 3D MPRAGE T1-weighted and T2-FLAIR sequences [All subjects were examined using 3.0-Tesla MRI scanner. The procedure is described in previous studies , 23. Allequences .APOE4 status, and diagnosis). We used Tukey HSD post hoc test to perform pairwise multiple comparisons. We used linear mixed-effects regression models to explore the longitudinal relationship of HGF subgroups with cognitive function . GraphPad Prism 8.00 and R version 3.6.2 software were used for statistical analyses and figure preparation.We used ANOVA, non-parametric Kruskal-Wallis H test, and Chi-squared test to compare the baseline clinical and demographic characteristics. In addition, pearson correlation tests (for continuous variables) were used to explore the associations between the baseline CSF AD biomarkers  and CSF HGF. All the non-demented participants were grouped by tertiles of CSF HGF at baseline . We used multiple linear regressions to explore the associations of HGF subgroups (independent variable) with CSF AD biomarkers and cognition (dependent variables) . We estimated the mean rates of change  for the whole samples [APOE4 status). We used bootstrapping  methods to estimate the 95% CI [P < 0.05 was considered significant.Longitudinal rates of change in cognitive function  were computed by using linear mixed models  compared to CN participants   who had measurements of CSF AD biomarkers  and HGF were included. Demographical and clinical characteristics are described in Table P = 0.00077), CSF pTau , and CSF tTau  in non-demented participants , the HGF tertiles were significantly associated with baseline CSF Aβ42 , pTau , and tTau  , ADNI_MEM , and ADNI_EF  compared to group A (the lowest tertile) , individuals in group C (the highest tertile) showed faster decline in MMSE  in our analyses. Second, we did not discuss the association between the longitudinal changes of CSF HGF and AD pathology due to the limited data. Third, this study was conducted with modest samples sizes, and the generalizability of our conclusions might be restricted by sources of studied populations of ADNI which recruited participants from volunteers. Further large-scale community-based longitudinal studies are warranted to validate these associations.The present study showed that the CSF HGF levels were significantly increased in MCI individuals. High concentrations of HGF in CSF may be related to AD pathology and faster cognitive decline in non-demented participants. AD pathology may act as a key mediator for influences of HGF on cognitive function. The CSF HGF may be an attractive biomarker candidate to track AD progression.Additional file 1: Table 1. Specific components of ADNI classification criteria to distinguish CN, Early MCI, Late MCI, and AD. The specific ADNI diagnostic criteria for distinguishing CN, MCI, and AD participants were summarized in Additional Table 1. Table 2. Tukey post hoc test for multiple comparisons between CSF HGF groups and CSF AD biomarkers and cognitive function. Using post hoc tests (Tukey HSD), it was found that CSF Aβ42 was reduced among Group C participants compared to Group A participants. CSF pTau and CSF tTau were increased among Group C participants, as compared to Group A and Group B participants. Table 3. Multiple comparisons between CSF HGF and longitudinal changes in cognitive function. After controlling for a range of potential confounders , individuals in group C (the highest tertile) showed faster decline in MMSE , ADNI_MEM , and ADNI_EF  compared to group A (the lowest tertile). Figure 1. Associations of baseline CSF HGF with CSF AD biomarkers in non-demented participants (stratified by diagnosis). The baseline CSF HGF is significant associated with the CSF Aβ42 in MCI , but not in CN . Figure 2. Associations of baseline CSF HGF with CSF AD biomarkers in non-demented participants (stratified by diagnosis). The baseline CSF HGF is significant associated with the CSF pTau . Figure 3. Associations of baseline CSF HGF with CSF AD biomarkers in non-demented participants (stratified by diagnosis). The baseline CSF HGF is significant associated with the CSF tTau .