Pseudomonadaceae (44.61%), Moraxellaceae (19.54%), Mycobacteriaceae (11.80%); the genera were Pseudomonas (45.42%), Acinetobacter (19.91%), Mycobacterium (12.01%). There were no detectable differences in the CSF microbial composition of the samples from the two groups. Sequencing of bacterial DNA present in the CSF was possible only after increasing PCR sensitivity. The results of 16S rRNA sequencing showed the presence of a microbial community in the CSF in cattle with neurological disorders. Further studies, in which CSF samples from healthy animals and samples from the environment are included as controls, are needed.Bacterial infection of the central nervous system (CNS) in cattle requires prompt and adequate antimicrobial treatment. The current gold standard for antemortem etiological diagnosis is cerebrospinal fluid (CSF) culture, which often yields false negative results. CSF has long been considered a sterile district in healthy patients, but this notion has been recently challenged. For this pilot study, we used 16S rRNA gene sequencing to investigate the microbial composition of CSF of cattle presenting with CNS disorders and to compare it between subjects with CNS infections and with CNS disorders of other nature. The study sample was 10 animals: 4 presenting with CNS infectious-inflammatory diseases and 6 with other CNS disorders, based on definitive diagnosis. Since the initial round of a standard 16S rRNA PCR did not yield sufficient genetic material for sequencing in any of the samples, the protocol was modified to increase its sensitivity. Bacterial genetic material was identified in 6 animals and 2 groups were formed: an infectious inflammatory (n = 3) and a noninfectious inflammatory group (n = 3). The most frequently expressed bacterial families were  Escherichia coli, Trueperella pyogenes, Histophilus somni, Pasteurella multocida and Listeria monocytogenes; mixed aerobic-anaerobic bacterial infections are reported as well  infection in cattle incurs economic losses due to livestock mortality, neurological impairment, and reduction of animal performance and productivity. Neurological diseases may also be zoonotic  collection can be easily and safely performed in field conditions; it is the most direct antemortem method of diagnosing CNS disease, in which advanced diagnostic imaging in large animals is much less feasible Nagy . InfectiThere is increasing evidence for 16S rRNA gene sequencing in human medicine as a rapid and accurate means to detect bacteria in the CSF, thus overcoming the limitations of culture-based bacterial detection  exploring whether there were any detectable differences in the microbial distribution between the two groups.The study sample was obtained from animals referred to the Neurology Service of the Veterinary Teaching Hospital, University of Turin, because of neurological signs suggestive of a CNS disorder. All underwent general physical examination, neurologic examination by a board-certified neurologist (ADA), CSF sampling in the field and analysis within 1 h after collection, blood biochemical analysis and necropsy when possible. CSF analysis also included CSF bacterial culture.The final diagnosis was reached based on signalment, results of the neurological examination, blood and CSF analysis, response to treatment, and postmortem histopathology when performed. Information on therapies administered before referral was recorded. Two groups were formed according to the final diagnosis expressed according to the VITAMIN D classification Jaggy . The oneThis prospective pilot study was conducted in accordance with current animal welfare regulations . Samples were collected during the routine analysis required to perform the diagnostic procedure. Written informed consent was obtained from the owners to authorize veterinary assessment and treatment of their animals.CSF was collected in the field from the lumbosacral space, as described by Mayhew . If neceDisposable sterile spinal needles  were used depending on the size of the animal. After correct positioning of the needle, the sample was obtained by gentle aspiration with a sterile syringe. The CSF sample was then divided into two aliquots and placed in empty sterile tubes. The tube cap was previously sterilized with alcohol and a new sterile hypodermic needle was used to transfer the CSF into each tube. The first aliquot, intended for routine analysis and macroscopic evaluation, total nucleated cell count (TNCC), erythrocyte count, and morphological evaluation, was analyzed within 1 h of collection and the second aliquot was stored at -80 °C until 16S rRNA sequencing analysis.DNA extraction from CSF was performed by using three different extraction kit, including: QI Amp® DNA Mini Kit , Bacterial Genomic DNA Isolation Kit  and DNAzol™ . The DNAzol™ extraction protocol was the one that led to a suitable final DNA concentration for the Next Generation Sequencing (NGS) library production.DNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).In order to generate a suitable library, enrichment PCR reactions were performed towards the 16S rRNA V3–V4 hypervariable region, using the forward primer (5’ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGCWGCAG) and the reverse primer (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). PCR Illumina standard conditions  were applied according to the manufacturer’s instructions for an initial analysis (first phase) and then modified (second phase) to achieve a more sensitive amplification as follows: 12.5 µL of 2x KAPA HiFi HotStart ReadyMix, 10 µL of each primer and 8 µL of 12.5 ng/µL of microbial DNA underwent initial denaturation at 95 °C for 3 min, then 25 cycles at 95 °C for 30 s, at 55 °C for 30 s, at 72 °C for 30 s, at 75 °C for 5 min and then maintained at 4 °C. Controls were added in each extraction and amplification step. Samples in which no genetic material was amplified were excluded from further analysis. The enriched 16S rRNA PCR library was then sent for normalization and quantification (Biomolecular Research Genomics) before proceeding with sequencing on an Illumina iSeq100 platform (Illumina) with a paired-end (PE) 2 × 300 bp protocol.Reads were checked for sequence quality using FastQC (version 0.11.5) Andrews , trimmedStandard descriptive statistics are reported as median and interquartile range (IQR) for continuous variables and as percentage and frequency for categorical variables. Statistical analysis was performed using Excel  .Ten animals were enrolled in this pilot study: 4 with a CNS infection and 6 with CNS disorders of other nature. The median DNA concentration in the CSF of the animals was 90 ng/µL (IQR 4.85–725).Standard PCR yielded no genetic material from any of the samples. The modified 16S rRNA PCR amplification produced no bacterial genetic material in samples from 4 out of 10 patients: 1 with CNS infection-inflammation and 3 with a noninfectious inflammatory CNS disorder. These 4 were excluded from further analysis; the remaining 6 were: 4 males (66.67%) and 2 females (33.33%) of Piemontese , Holstein Friesian , Blonde d’Aquitaine , and mixed breeds , ranging from 3 days to 3 years of age ; the neuroanatomical localisation was: forebrain , diffuse intracranial , lumbosacral spinal cord segments , and multifocal .The final diagnosis based on the VITAMIN D classification Jaggy  was: infIn the infectious-inflammatory group, neonatal meningitis/meningoencephalitis(-myelitis) of suspected bacterial origin was diagnosed in 2 calves < 1 month that presented with 1–3 day history of neurological signs, which occurred following enteritis and omphalophlebitis in 1 calf (Case 1) and enteritis and arthritis in the other (Case 2). CSF analysis revealed mononuclear pleocytosis in both cases. One older calf (Case 3) (7 months old) presented with clinical signs consistent with meningitis/meningoencephalitis and 1–3 day history of seizures. CSF analysis showed marked mixed pleocytosis and increased total protein concentration. The patient was euthanized a few hours after the referral due to deterioration of neurological signs and onset of status epilepticus. Necropsy highlighted yellow-greenish thickening of the meninges at the level of the cerebellum, multifocal abscesses and mononuclear vasculitis at the level of the pons and cerebellum, edema, and inflammatory infiltrate in the brain parenchyma. All brain areas showed signs of predominantly mononuclear inflammation. The polymicrobial growth in the culture of brain samples was a mixed flora of slow-growing bacteria that in a standard culture system caused the failure to identify the single microorganisms. The noninfectious inflammatory group included 3 patients. A 3-month-old calf (Case 5) presented with central blindness and abnormal compulsive behaviour. CSF analysis was normal. Hypovitaminosis A was suspected; vitamin A therapy resolved all the clinical signs but not the blindness. A 3-year-old (Case 6) patient presented because of recurrent epileptic seizures. CSF analysis was normal and blood analysis revealed hypocalcemia. The patient died shortly after referral; necropsy was unremarkable. Hypocalcemia was the assumed cause of the neurological symptoms.A 3-day-old calf (Case 7) presented with abnormal facial conformation and inability to stand on forelimbs and hindlimbs since birth. The diagnosis was complex congenital malformation of the head consistent with hydranencephaly or holoprosencephaly associated with other congenital facial abnormalities disclosed on post mortem autopsy ; those obtained after elimination of host material, elimination of poor quality sequences, and pairing between forward and reverse sequences (indicated as merged) were about 70% of the starting counts . The sequences compared to the database were about 45% of the total as only the real sequences, not the chimeric ones, were considered . The samples from Case 3 produced the lowest number of reads , while the highest reads count was obtained from sample 5 .Each ASV resulting from DADA2 denoising step was then compared to the SILVA database to obtain a classification into a microbial taxonomy. A total of 17 phyla, 27 classes, 71 orders, 115 families and 150 genera were identified and the most present are reported in Table Corynebacterium kroppenstedtii (49.70%), Lactobacillus algidus (14.05%), Pseudomonas pertucinogena (8.50%), Cutibacterium granulosum (6.61%), Bifidobacterium bifidum (6.37%), Treponem porcinum (5.19%), Corynebacterium maris (3.07%), Sphingobacterium jejuense (2.71%), Pseudomonas formosensis (2.60%). Negativicoccus succinicivorans and Peptoniphilus duerdenii accounted for < 1% of the identified species.Only 11 out of 728 ASVs were identified at the species level, accounting for 1.51% of the ASVs and 0.69% of total abundance. The most abundant were: Both alpha and beta diversity analyses did not detect any differences between the microbial communities of the two groups. Alpha and beta diversity analyses values are reported in Additional file 1. The rarefaction curves reached a plateau, indicating good representation of the microbial community. The rarefaction curve plots are reported in Additional File 2.Acinetobacter johnsonii (43.46%), Chryseobacterium profundimaris (10.73%), Pseudomonas helmanticensis (4.94%), Psychromonas aquatilis (4.54%), Afipia massiliensis (4.50%), Carnobacterium gallinarum (2.92%), Staphylococcus hominis (2.39%), Anaerococcus nagyae (1.64%), Acinetobacter indicus (1.46%), Brevundimonas naejangsanensis (1.37%), Rothia endophytica (1.21%), and Corynebacterium pilbarens (1.06%). Six of the 11 ASVs assigned to a species by QIIME were likewise identified by BLAST and there was correspondence between them.By blasting the ASVs versus the NCBI database, we assigned 169 ASVs to a species, 105 of which were ASVs previously recognized by QIIME with a CL > 97% and 64 ASVs with a CL < 97%. The former accounted for 14.28% of ASVs and 11.61% of total abundance, the most abundant identified species were: With the present study we evaluated for the first time the feasibility of 16S rRNA gene sequencing of DNA isolated from CSF of cattle with CNS disorders. Also, we evaluated the CSF microbiota composition in patients with CNS infection-inflammation and CNS disorders of other nature.Despite the attempt to increase sensitivity by means of modified PCR, not all samples led to bacterial genetic material detection. Possible explanations are the very low CSF biomass, since the amount of CSF that can be collected is usually very small (a few mL) and the concentration of the genetic material in CSF is low . While it has been reported that the most variable regions are sufficient to identify genera, they are unlikely to adequately discriminate between species  and non infectious inflammatory (NON INF) groups. Observed features, Shannon’s diversity index, Simpson index and Faith’s Phylogenetic Diversity values are reported as mean ± standard error and their corresponding P value.Table S2 Beta diversities calculated for the infectious inflammatory (INF) and non infectious inflammatory (NON INF) groups. Comparisons based on unweighted unifrac distance, weighted normalized unifrac distance and Bray-Curtis dissimilarity are reported through their pseudo-F ratio statistics and P Value.Additional file 2: Panel 1 Rarefaction curve showing the Shannon Alpha Diversity estimation as a function of sequencing depth for all the six analyzed subjects rarefaction curves plot.Panel 2 Rarefaction curve showing the Shannon Alpha Diversity estimation as a function of sequencing depth for the subjects grouped into the two classes.Additional file 3: Table S3 Table containing ASVs identifiers, taxonomic assignment, taxonomic assignment confidence level, ASVs abundance for the 6 subjects.11259_2022_9949_MOESM1_ESM.docx (PDF 217 kb)11259_2022_9949_MOESM2_ESM.docx (PDF 333 kb)11259_2022_9949_MOESM3_ESM.xlsx (PDF 61 kb)


Pediatric behavioral health emergencies (BHE) are increasing in prevalence, yet there are no evidence-based guidelines or protocols for prehospital management. The primary objective of this scoping review is to identify prehospital-specific pediatric BHE research and publicly available emergency medical services (EMS) protocols for pediatric BHE. Secondary objectives include identifying the next priorities for research and EMS protocol considerations for children with neurodevelopmental conditions.This is a scoping review comprised of a research literature search for publications from 2012-2022 and an internet search for publicly available EMS protocols from the United States. Included publications contain data on the epidemiology of pediatric BHE or describe prehospital management of pediatric BHE. EMS protocols were included if they had advisements specific to pediatric BHE.A total of 50 research publications and EMS protocols from 43 states were screened. Seven publications and four protocols were included in this study. Research studies indicated an increase in pediatric BHE over the last decade, but few papers discuss current prehospital management (n=4). Two EMS protocols were specific to pediatric BHE or pediatric agitation, and the other two EMS protocols focused on adult populations with integrated pediatric recommendations. All four EMS protocols encouraged nonpharmaceutical interventions prior to the use of pharmacologic restraints.Although there is a substantial rise in pediatric BHE, there is sparse research data and clinical EMS protocols to support best practices for prehospital pediatric BHE management. This scoping review identifies important future research aims to inform best practices for the prehospital management of pediatric BHE. Over the past decade, there has been a steady rise in the incidence of pediatric behavioral health emergencies (BHE) in the United States (US). From 2007 to 2016, the number of pediatric patients seen for emergency department (ED) visits for deliberate self-harm increased by 329%, and visits for all pediatric mental health disorders rose by 60% [The rise in pediatric BHE is a concern for all health professionals, including prehospital emergency medical services (EMS). Every year in the US, EMS clinicians treat one to two million pediatric patients. They also serve as the first point of contact for many children with BHE . Among EIn contrast with adult BHE, pediatric BHE patients may have unique neurodevelopmental conditions and needs which may not be addressed in adult EMS protocols or in routine EMS clinician training. The increase in prehospital pediatric BHE also presents an operational and clinical stress point for EMS agencies and clinicians. Given the overall rise in pediatric BHE, evidence-based guidelines (EBGs) and/or evidence-based protocols (EBP) for the prehospital management of pediatric BHE may help optimize patient outcomes and EMS clinician safety. However, to date, an EBG for pediatric BHE has not been developed and is needed in the US. Therefore, the objective of this study was to perform a scoping review of published research related to the prehospital management of pediatric BHE and an internet search of publicly available statewide EMS pediatric BHE protocols or guidelines.Materials and methodsScoping Review Search & Selection CriteriaWe searched the published research literature using strategies created by a medical librarian (CA) and other authors  for papers relevant to pediatric BHE in the prehospital setting. The search was performed in PubMed and Google™ for English language manuscripts published between January 1, 2012 and May 1, 2022. That date range was chosen because of the supporting literature demonstrating a rising incidence of pediatric BHE within the last decade . The seaTo search for publicly available statewide EMS protocols/guidelines, , a Google™ search was performed in November 2020 by one author (LM) to identify U.S. (United States) state EMS protocols related to the management of pediatric BHE and/or agitated pediatric patients. The decision was made to search for US state protocols as they may be more likely publicly available on the internet in contrast to local agency protocols. Key search terms included but were not limited to pediatric agitation, restraints, and the Stabilization, Acknowledgment, Facilitate Understanding, Encouragement, Recovery/Referral (SAFER) method . Forty-tMajor findings of the research studies and specific management actions identified in the review of the EMS protocols/guidelines were abstracted and summarized (EZ). For research studies, information related to publication year, setting, patient characteristics, and interventions were abstracted. For EMS protocols, the protocol/guideline source, and procedure and medication recommendations were abstracted. All authors then reviewed the preliminary findings.ResultsThis scoping review identified a total of 50 research studies and 43 EMS protocols for screening, of which a total of seven research studies and four state EMS protocols were included for in-depth analysis Figure . Of the The remaining four research publications included in this review discussed prehospital interventions for pediatric BHE patients Table . Those aSummaries of two states with publicly available pediatric-specific state EMS BHE protocols  to moderate (prospective study). Of the seven studies, two discussed the safety and efficacy of droperidol, whereas published EMS protocols contain ketamine, midazolam, diazepam, and haloperidol. Thus, there may be an opportunity for consolidation of sedation medication options within EMS protocols and an opportunity for further research looking at the prehospital use of droperidol in comparison to other sedation medications currently used for pediatric BHE patients. Of note, the use of droperidol greatly decreased in 2001 after its black box warning for QTc prolongation but was reintroduced in the US in 2019 after clarification that its black box warning applied to doses of droperidol that are greater than 2.5 milligrams (mg) ,13. In 2With regard to assessment and appropriate monitoring, the study by Page et al. discussed the use of a sedation assessment tool (SAT) before and after giving pharmaceutical treatment for agitation . StudiesOne case study of this review demonstrates the importance and challenges of pediatric neurodevelopmental conditions in the management of pediatric BHE. Autism spectrum disorder (ASD) is not unique to the pediatric population; however, children with ASD often have behavioral health emergencies. Patients with ASD and other neurodevelopmental conditions can have increased pain tolerance, inability to express pain, inability to communicate expressively, and impaired receptive communication. These factors can hamper the prehospital assessment, management, and transport of a pediatric patient with ASD or other neurodevelopmental conditions, especially when a caregiver is not available to assert the patient’s baseline behavior and attest to any home medications that may also cause agitation . Given tIn this study, we only identified four states with state protocols that addressed pediatric BHE. Interestingly, all of the included EMS protocols contained common themes including the importance of addressing medical emergencies that can cause agitation or altered mental status, and situational “de-escalation” before the use of medications. The corresponding research literature in this scoping review did not contain evidence for various types of non-pharmacologic de-escalation techniques. Research on the identification and examination of non-pharmaceutical de-escalation techniques in the prehospital setting is needed. Non-pharmacologic techniques are especially impactful for EMS providers who are unable to administer medications in the prehospital setting.This study has limitations that merit consideration. The quality of the evidence of this scoping review is variable and our search was limited to only 10 years. Older studies may have provided evidence for management strategies in pediatric BHE missed in our date range of literature review. As in systematic reviews, this study may be subject to publication bias as well. In addition, the review of publicly available state EMS protocols for pediatric BHE is not a complete review of all US EMS protocols, as not all states have statewide EMS protocols. Therefore, it is most likely that there are local/regional EMS protocols that contain pediatric-specific BHE recommendations.Although there is a rise in pediatric BHE, there is sparse research data to support prehospital EMS practices and protocols. There is also a notable absence of literature regarding more pediatric-specific neurodevelopmental conditions that can also exhibit behavioral health issues and non-pharmacologic techniques for EMS to incorporate into the assessment and management of prehospital pediatric BHE. What little evidence does exist suggests that drugs such as droperidol could be considered for incorporation into EMS protocols for chemical sedation when non-pharmacologic methods fail. Given the rising incidence of pediatric BHE, research and clinical quality assurance must be performed and results expeditiously disseminated to support EMS agencies and EMS clinicians to optimize patient outcomes and provider safety.


We report the electroencephalography (EEG) showing an intermittent generalized polyspike pattern in EEG due to an aseptic meningoencephalitis in a 71-year-old soporous patient. Initially, she presented with word-finding disturbances and later with generalized tonic–clonic seizures. The cerebrospinal fluid (CSF) showed pleocytosis of 99 leukocytes/μL (primarily neutrophils) and an increased protein level of 1240 mg/L (CSF/serum glucose ratio and lactate unremarkable). Pathogens and autoimmune antibodies in CSF were not found. Brain imaging was unremarkable. After antibiotic, antiviral and anticonvulsive therapy, the pattern in the EEG was no longer detectable. The patient was discharged to go home due to absence of any residues.


Infiltration of malignant cells into the central nervous system in hematological malignancies correlates with poor clinical outcomes. Investigations into the penetration of venetoclax into the central nervous system have been limited. We report venetoclax pharmacokinetics in plasma and cerebrospinal fluid samples from a Phase 1 study in pediatric patients with relapsed or refractory malignancies that demonstrate venetoclax ability to cross into the central nervous system. Venetoclax was detected in cerebrospinal fluid (CSF) samples, with concentrations ranging from < 0.1 to 26 ng/mL  and a plasma:CSF ratio ranging from 44 to 1559 . Plasma:CSF ratios were comparable among patients with AML and ALL and no clear trend was observed in the ratios over the course of treatment. Moreover, improvement in central nervous system (CNS) involvement status was observed in patients who had measurable concentrations of venetoclax in the CSF. CNS resolution was observed for up to six months while on treatment. These findings highlight the potential role of venetoclax and provide the opportunity to further investigate its utility in improving clinical outcomes for patients with CNS complications. Central nervous system (CNS) involvement, where malignant cells infiltrate the CNS, meninges, or cerebrospinal fluid (CSF), is a complication that occurs across hematological malignancies, and correlates with high-risk features and poor clinical outcomes Venetoclax is a first-in-class, potent, oral B-cell lymphoma (BCL-2) inhibitor that is approved for treatment of chronic lymphocytic leukemia (CLL) and adult AML To penetrate into the CNS, venetoclax would need to overcome physicochemical and physiological barriers. Venetoclax is a “large, small molecule drug” (molecular weight of 868.44), which is hypothesized to limit passage through the blood-brain barrier's (BBB) tight junctions. Moreover, venetoclax is a substrate of several efflux transporters such as P- glycoprotein (P-gp) and breast cancer resistance protein (BCRP) that are expressed within the BBB and can actively pump venetoclax outside the CNS The study reported here (NCT03236857) was conducted in accordance with Good Clinical Practice Guidelines and the ethical principles that have their origin in the Declaration of Helsinki. The protocol was approved by the institutional review board at each study site and each patient provided written informed consent before any study-related procedures were performed. Participants were < 25 years of age (with preference to pediatric patients < 18 years) with relapsed or refractory malignancies, had adequate hepatic and renal function. Additional details on inclusion and exclusion criteria are reported elsewhere Pediatric patients received daily doses of 400 mg or 800 mg  of venetoclax. Doses were adjusted upon concurrent administration of moderate or strong CYP3A4 inhibitors to achieve comparable venetoclax exposures. Chemotherapy was administered based on the type of malignancy as specified in the study protocol . Intrathecal chemotherapy was allowed during the screening period and when clinically indicated throughout the study. Intrathecal therapy included cytarabine and hydrocortisone, with or without methotrexate. Intensive plasma PK sampling from pediatric patients was performed in Cycle 1 of treatment, followed by sparse plasma PK sampling on scheduled visits throughout the study. Venetoclax was measured in CSF as a surrogate for BBB penetration. CSF samples for PK assessments were collected at screening and while on treatment during scheduled visits if a lumbar puncture was performed as standard of care. CSF and blood PK samples were collected at the clinic. On days when a clinic visit was scheduled, patients were instructed not to administer venetoclax until they arrived at the clinic to facilitate trough PK sampling. The venetoclax concentrations in the CSF and plasma samples were determined using liquid-liquid extraction followed by liquid chromatography (LC) with tandem mass spectrometric detection (MS/MS) The analysis included samples from 46 patients with relapsed or refractory AML (n = 24), ALL (n = 18) or other hematological malignancies (n = 4). The median age of the patients was 8.5 years (range < 1 year - 25 years). Twenty (43%) of the patients were male.Plasma and CSF samples from pediatric patients receiving venetoclax at either 400 or 800 mg  in combination with chemotherapy were evaluated. A total of 92 CSF samples were collected. Venetoclax was detected in most of the CSF samples. The mean venetoclax concentration in CSF was 3.6 ng/mL with concentrations ranging from < 0.1 to 26 ng/mL  out of the 46 patients included in the analysis demonstrated CNS involvement either at screening (n = 5) or after initiation of therapy (n = 4). Of those, seven patients had subsequent CSF assessments after initial detection of CNS involvement. All seven patients demonstrated resolution of CNS involvement at their subsequent assessments while on treatment. The venetoclax CSF concentrations in these patients (n = 7) ranged from 0.30 to 7.7 ng/mL (mean 2.7 ng/mL). The remainder two patients who demonstrated CNS involvement did not have further CSF assessments after initial detection and hence it could not be determined if the CNS status improved or not. Noteworthy, for patients with more than one calculated plasma:CSF ratio, no clear trend was observed in the ratios over the course of treatment  of treatment of venetoclax in multiple primary AML specimens treated ex-vivo was well below 10 nM (8.7 ng/ml) In conclusion, these findings demonstrate the ability of venetoclax to penetrate into the CNS. This distribution into the CNS, coupled with its potent apoptotic effects suggest clinical utility in management of CNS complications in hematological malignancies.


Nonetheless, Cys-DA/DOPAC ratio  and hsp27 levels  were significantly higher. To the best of our knowledge, the c.2614G>T SYNJ1 mutation has not been previously reported. Our patient does not produce fully functional spatacsin and synaptojanin-1 proteins. In this line, our results showed decreased DA and DOPAC levels in the patient’s CSF, indicating loss of DAergic neurons. Many factors have been described as being responsible for the increased cys-DA/DOPAC ratio, such as MAO inhibition and decreased antioxidant activity in DAergic neurons which would increase catecholquinones and consequently cysteinyl-catechols. In conclusion, haploinsufficiency of spatacsin and synaptojanin-1 proteins might be the underlying cause of neurodegeneration produced by protein trafficking defects, DA vesicle trafficking/recycling processes, autophagy dysfunction, and cell death leading to hsp27 upregulation as a cellular mechanism of protection and/or to balance impaired protein trafficking. We report a 49-year-old patient suffering from spastic paraplegia with a novel heterozygous mutation and analyzed the levels of heat shock proteins (hsp)-27, dopamine (DA), and its metabolites in their cerebrospinal fluid (CSF). The hsp27 protein concentration in the patient’s CSF was assayed by an ELISA kit, while DA levels and its metabolites in the CSF, 3,4-dihydroxyphenylacetic acid (DOPAC), Cys-DA, and Cys-DOPA were measured by HPLC. Whole exome sequencing demonstrated SPG-11 c.1951C>T and novel SYNJ1 c.2614G>T mutations, both heterozygous recessive. The patient’s DA and DOPAC levels in their CSF were significantly decreased (53.0 ± 6.92 and 473.3 ± 72.19,  Hereditary spastic paraplegia is a rare syndrome comprising a group of neurodegenerative diseases that are genetically heterogeneous. Spastic paraplegia is caused by distal degeneration of lateral corticospinal tract axons causing a typical phenotype consisting of progressive and bilateral spasticity and weakness of the lower limbs ,2. The pSmall heat-shock proteins (sHsps) are a family of chaperone proteins . ChaperoBoth Parkinson’s disease and spastic paraplegia are protein-misfolding diseases, characterized by a reduction in DA due to loss of dopaminergic neurons and abnormal oxidation of DA . 5-cysteThe study project was approved by the Santa Lucia Hospital Clinical Ethics Committee (ref. CEI.22-18_SPG11s). Written informed consent was obtained from the patient’s legal ward to carry out the study with his biological samples and for the publication of any potentially identifiable images with scientific divulgation interest or data included in this article.The patient completed large clinical examinations and tests from 2015 to 2020, such as determinations for toxic, metabolic, infectious, autoimmune, and inflammatory processes and had undergone genetic testing for common hereditary neurodegenerative disorders. The Spastic Paraplegia Rating Scale (SPRS) was registered upon admission (March 2020) and 1 year before . AdditioAs previously described elsewhere by the authors , DNA wasSamples of cerebrospinal fluid were solved in lysis buffer. Protein concentrations were measured by using a bicinchoninic acid kit . The hsp27 protein concentration was assayed by a hsp27 enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions as previously published . The detg, filtered through a 0.20 μm membrane filter, and transferred to a high-performance liquid chromatography (HPLC) device. The detection limits for catechols were about 10 pmol/L or 10 fmol per assayed mL of CSF. The amount of DA in the CSF samples was measured as previously described [v/v) acetonitrile/water to achieve a final concentration of 1000 μg/mL for the preparation of the internal standards. The CSF concentrations of biogenic amines and their metabolites were expressed as “pg/mg”.HPLC equipment, which included a dual pump and Nanospace SI-2/3001 pump , was used to assess the amounts of DA and its metabolites in the CSF samples. Dopamine and its metabolites standards were obtained from Sigma  and from the Chemical Synthesis and Drug Supply Program of the National Institute of Mental Health. Previously, the CSF samples were centrifuged for 10 min at 4 °C with 12,000× escribed . Brieflyp < 0.05.Data are expressed as the mean ± SEM. Statistical analysis was performed by using IBM SPSS 20.0  software with one-way ANOVA followed by the Bonferroni post hoc test and graphs designed by using GraphPad Prism 8.4.3 for Windows . The results were considered significant when A 49-year-old Mediterranean European Caucasian male with a history of epilepsy treated with valproate since he was 17, mild intellectual disability without any impact on his daily life, and bladder dysregulation was admitted to the neurology department in March 2020 for gait impairment and progressive cognitive decline in the last 5 years. His parents were 74 and 72 years old, both European Caucasian and healthy with no history of consanguinity or Parkinsonism. The patient has one sister and one brother , both of whom are healthy. When registered, his newborn metabolic and hearing screening tests were normal. Speech–language and early psychomotor development, such as walking and sphincter control, were normally developed at the age of 1 and 2.5 years, respectively. He finished his primary studies and then lived with his parents and worked in a family business.The patient was studied by the neurology department 1 year before he was admitted for progressive cognitive impairment and bradykinesia–rigidity syndrome. This was attributed to a long-term side effect of valproate. Nonetheless, no improvement was observed once valproate was suspended nor when the L-DOPA test was carried out. Suspecting atypical Parkinsonism, a Wilson disease study and genetic tests of Charcot–Marie–Tooth (CMT), atypical Parkinsonisms , and atypical spinal–muscular atrophies  were carried out. All of these genetic tests were normal.Upon admission, neurological examination showed poor language ability, hypometric saccades in all directions, mild weakness in the right arm and in the left extremities, mainly in the foot, generalized hyperreflexia, non-stop ankle clonus, extensor plantar reflex, and generalized spasticity with impossibility to stand-up and walk. The Spastic Paraplegia Rating Scale (SPRS) scored 36 indicating severe disease. Brain MRI A–D showe1,5 = 9.802, p < 0.001) and right  striates and putamen nuclei compared to a series of five cases in which the tests were considered normal by experienced radiologists . Accordingly, the patient’s DA and DOPAC levels in their CSF  compared to the healthy controls . Furthermore, while no differences between the CSF samples were found in the Cys-DA levels  compared to the control samples (0.143 ± 0.007). Finally, the levels of hsp27  compared to the controls (589.2 ± 36.4).The SPECT DA Transporter (DAT) scan of the brain patient E,F,H shoheir CSF G,I were A levels J, the raA levels K was sigof hsp27 L in the We found heterozygous recessive mutations in the SPG-11 c.1951C>T) and SYNJ1 (c.2614G>T) genes. The present c.1951C>T SPG-11 mutation was previously described as a recessive compound heterozygous mutation in an isolated patient from Romania  and a Po51C>T andBiochemically, spatacsin is a 280-kDa protein implicated in the axonal trafficking of vesicles and lysosomal homeostasis during autophagy  resultinox, the hsp27 activity was significant in preventing amyloid fibrillar and amorphous aggregation of proteins [Finally, the CSF levels of hsp27 were enhanced in the patient. Small heat-shock proteins are well known because their cellular function is to stabilize and prevent protein misfolding and aggregation . A recenproteins . Thus, wIn conclusion, we report for the first time a known heterozygous recessive SPG11 c.1951C>T mutation and a not-reported heterozygous recessive SYNJ1 c.2614G>T mutation in a European Caucasian patient with clinical spastic paraplegia of late onset. We suggest that this complex genetic interaction causing haploinsufficiency of both proteins might be underlying the impairment in protein/vesicle intracellular trafficking and consequently the upregulation of hsp27. Finally, the diagnosis of spastic paraplegia should be considered in patients with unexplained neurodegeneration especially when accompanied by a history of atypical Parkinsonism.


All five patients who received a prior anti-BCMA CAR T responded to ide-cel, and survival outcomes were best for this subgroup. In conclusion, treatment with ide-cel yielded meaningful clinical responses in real-world patients exposed to a prior BCMA-TT, though response rates and durability were suboptimal compared to those not treated with a prior BCMA-TT.Most patients with multiple myeloma experience disease relapse after treatment with a B-cell maturation antigen-targeted therapy (BCMA-TT), and data describing outcomes for patients treated with sequential BCMA-TT are limited. We analyzed clinical outcomes for patients infused with standard-of-care idecabtagene vicleucel, an anti-BCMA chimeric antigen receptor (CAR) T-cell therapy, at 11 US medical centers. A total of 50 patients with prior BCMA-TT exposure  and 153 patients with no prior BCMA-TT were infused with ide-cel, with a median follow-up duration of 4.5 and 6.0 months, respectively. Safety outcomes between cohorts were comparable. The prior BCMA-TT cohort had a lower overall response rate (74% versus 88%;  Prior to the development of therapies targeting the B-cell maturation antigen (BCMA), patients with multiple myeloma refractory to immunomodulatory agents (IMiD), proteasome inhibitors (PI), and anti-CD38 monoclonal antibodies had limited therapeutic options and an estimated median overall survival (OS) of 5.6–13 months . The antGiven the emergence of BCMA-targeted therapies with three distinct mechanisms of action, treatment of patients with disease progression after receiving a BCMA-targeted therapy (BCMA-TT) has become an unmet need. The registrational trials for the approved BCMA-TT excluded patients who had received a prior BCMA-TT, resulting in limited data characterizing outcomes for such patients , 5–7. ReIn the pivotal phase 2 KarMMa trial, patients infused with ide-cel achieved an ORR of 73%, ≥ complete response (CR) rate of 33%, median progression-free survival (PFS) of 8.6 months, and median OS of 24.8 months , 12. ThiThis was a retrospective multicenter observational study of patients with RRMM for whom treatment with commercial ide-cel was planned at one of 11 US medical centers. Each center obtained independent institutional review board approval and informed consent per institutional requirements. While data was collected for all patients who underwent leukapheresis with the intent to manufacture ide-cel from April 1, 2021 to May 1, 2022, patients included in this study were those who underwent ide-cel infusion and had at least a day 30 response assessment completed by the time of data cut-off. Patients who died after ide-cel infusion but prior to response assessment were included in the safety and survival analyses, but not in the response analysis.2 and fludarabine renally-dose adjusted per institutional guidelines. Cytokine release syndrome (CRS) and immune-effector cell-associated neurotoxicity syndrome (ICANS) were graded as per the American Society for Transplantation and Cellular Therapy (ASTCT) criteria [Following leukapheresis, there were no restrictions on the use of bridging or radiation therapy. Lymphodepleting chemotherapy was administered on days -5 through -3 with cyclophosphamide 300 mg/mcriteria . Hematolcriteria .P-values of <0.05 were considered significant. Statistical analyses were conducted using STATA 17 .Baseline characteristics and outcomes related to efficacy and safety were summarized with descriptive statistics. Differences between groups were evaluated using chi-squared or Fisher’s exact tests for categorical variables, or Kruskal-Wallis rank-sum tests for continuous variables. Definitions for duration of response (DOR), PFS, and OS are provided in Appendix 1, as are the methods for the multivariable logistic regression analysis examining the association of prior BCMA-TT with best ORR , and best CR rate (≥CR versus < CR). The Kaplan–Meier method was used to estimate DOR, PFS, and OS, and survival outcomes amongst subgroups were compared using the log-rank test. Multivariable Cox proportional hazard regression models were used to analyze the association of prior BCMA-TT with PFS and OS while adjusting for the selected characteristics outlined in Appendix 1. Proportional hazards assumptions via Schoenfeld residuals were examined under these parameters with no deviations detected for the global tests. High-risk cytogenetics violated the proportional hazards assumption for the OS model and was included as a strata term in that model. All statistical tests were two-sided and As of May 1, 2022, 239 patients underwent leukapheresis with the intention to manufacture ide-cel. Of the 56 patients who had received a prior BCMA-TT, five patients did not proceed with ide-cel infusion (one manufacturing failure and four due to interim progression/death), and one patient was pending infusion at data cutoff. Thus, 91% of patients who had received a prior BCMA-TT  were able to complete ide-cel infusion  compared to those who had not (n = 153). The median age of the prior BCMA-TT cohort was 66 years, 66% were male, 19% had an Eastern Cooperative Oncology Group performance status (ECOG PS) ≥ 2, 27% had R-ISS stage III disease prior to infusion, 50% had extramedullary disease, and 36% had high-risk cytogenetics as defined by t, t, or del(17p). Patients in both cohorts were heavily pretreated, and the prior BCMA-TT cohort patients were more likely to have penta-refractory disease  and had received a greater median number of prior therapies  compared to the cohort who had not received a prior BCMA-TT.Patient baseline characteristics are presented in Table Regarding the specific type of BCMA-TT received before ide-cel, 38 (76%) patients received an ADC, of which the vast majority was commercially available belantamab mafodotin. Seven 14%) had received a prior bispecific antibody and five (10%) had gotten a prior CAR T, with all prior bispecifics and CAR therapies received on a clinical trial. Amongst the prior CAR T subgroup, two patients had received ide-cel as their prior CAR construct on early phase trials, two had received an allogeneic CAR, and one received an autologous CAR with a non-viral transduction method. Further details about the clinical course for the five prior CAR T patients can be viewed in Supplemental Table 4% had reAdverse events are summarized for both cohorts in Table p = 0.064), which translated to a higher rate of thrombopoietin mimetic use  for the prior BCMA-TT group. The rate of documented infection was similar between cohorts at 34% for the prior BCMA-TT cohort and 39% for those with no prior BCMA-TT.Within the first 30 days after ide-cel infusion, grade ≥ 3 anemia, neutropenia, and thrombocytopenia occurred in 38%, 76%, and 64% of the patients in the prior BCMA-TT cohort respectively. There was a trend towards higher rates of grade 4 thrombocytopenia in the prior BCMA-TT cohort  was driven by a significant increase in death related to myeloma disease progression for this cohort . There was no significant increase in the incidence of death due to non-relapse mortality in the prior BCMA-TT cohort .At data cutoff, 14 patients who had received commercial ide-cel died in the prior BCMA-TT cohort  and best response of ≥CR  compared to the cohort who had not received a prior BCMA-TT , a longer amount of time from their last BCMA-TT exposure to apheresis , and a longer time to ide-cel infusion  compared to non-responders (Table p = 0.076) and best response of CR or better  compared to the patients receiving ide-cel >6 months after their last BCMA-TT exposure, this did not reach statistical significance. The nine patients receiving ide-cel within three months of their last BCMA-TT also had a numerically lower ORR  compared to those receiving ide-cel >3 months after the last exposure.Best ORR to commercial ide-cel was evaluable for 49 patients in the prior BCMA-TT cohort and 144 patients for the no prior BCMA-TT group. Patients in the prior BCMA-TT cohort had a lower ORR . The median DOR by specific type of prior BCMA-TT were 7.4 months, 2.8 months, and not reached for prior ADC, bispecific, and CAR T respectively .The median DOR was shorter in the prior BCMA-TT cohort compared to the no prior BCMA-TT cohort . Having attained a response of ≥PR to the initial BCMA-TT was not associated with an increased likelihood of attaining a response of ≥PR or ≥CR to commercial ide-cel. In a multivariable analysis including all patients who received ide-cel (Table p = 0.003).A univariate analysis of the patients who had received a prior BCMA-TT . The median PFS by specific type of prior BCMA-TT were not reached, 3.2 months, and 2.8 months for the prior CAR T, prior ADC, and prior bispecific groups respectively  and inferior OS . In addition, ECOG PS ≥ 2 was predictive of both inferior PFS and OS, while high-risk cytogenetics and age < 65 were associated with worse PFS  represents the largest cohort described for a specific anti-BCMA therapy after prior exposure to another BCMA-TT. The prior BCMA-TT cohort consisted of heavily pre-treated patients  with aggressive disease characteristics . Ide-cel manufacturing and infusion were feasible in this real-world cohort of patients who had received prior anti-BCMA therapy given the comparable rates of manufacture failure (1.8% versus 2.7%) and ide-cel infusion in the intention-to-treat population (91% versus 92%) when compared to the cohort who had not received a prior BCMA-TT.This consortium previously reported on a cohort of 159 patients with RRMM who received ide-cel in the real-world setting, which demonstrated comparable safety and efficacy outcomes compared to those observed on the KarMMa trial despite 75% of patients not meeting the eligibility criteria . The patDespite a low ORR of 21% to the prior BCMA-TT, treatment with commercial ide-cel yielded an impressive ORR of 74%. Although the ORR was significantly lower than that observed in the no prior BCMA-TT cohort (74% versus 88%), the ORR observed with ide-cel compares favorably with those reported for other BCMA-targeted therapies in this setting. Cohort C of the prospective CARTITUDE-2 study consisted of 20 patients infused with cilta-cel after prior exposure to a non-cellular BCMA-TT . Cilta-cel treatment resulted in an ORR of 60%, median DOR 11.5 months, and median PFS of 9.1 months . While OResponse rates to ide-cel in our prior BCMA-TT cohort also compare favorably to the current commercially available non-BCMA therapies for patients with triple-class refractory myeloma who have also received a prior anti-BCMA therapy. Preliminary data from the STOMP study included 11 patients who had progressed after a prior BCMA-TT and were treated with heterogeneous selinexor-containing regimens. The ORR was 64% for this trial population, but a real-world study of patients with disease progression after BCMA CAR T noted lower rates of response to salvage selinexor-based therapy , 17. GivAlthough the ORR to ide-cel for patients in our prior BCMA-TT cohort was relatively high, this group of patients had a significantly lower ORR (74% versus 88%), rate of ≥CR (29% versus 48%), median DOR (7.4 versus 9.6 months), and median PFS (3.2 versus 9.0 months) compared to our real-world cohort without prior BCMA-TT exposure. Patients in the prior BCMA-TT cohort had received a greater median number of prior lines of therapy and were more likely to have penta-refractory disease, which may partially explain the observed worse efficacy outcomes. However, multivariable analysis demonstrated that treatment with a prior anti-BCMA therapy was an independent predictor of worse PFS and OS, as well as a lower likelihood of having a best response of ≥CR. Future prospective studies investigating anti-BCMA CAR T in patients with prior BCMA-TT exposure should give consideration to combination strategies, consolidation approaches, or maintenance therapies in order to extend the duration of clinical benefit. As standard-of-care anti-BCMA CAR T therapies remain a limited resource due to manufacturing slot allocation, it is important to consider potential prognostic and predictive factors such as the specific type and timing of prior BCMA-TT exposure in patient selection for commercial CAR T.n = 5), our study indicates that sequential anti-BCMA CAR T can be an efficacious strategy and prior anti-BCMA CAR T treatment should not be an exclusion criterion for future prospective studies of investigational anti-BCMA CAR T-cell therapies. Several ongoing studies have demonstrated high response rates to investigational GPRC5D-targeted CAR T-cell therapies after prior anti-BCMA CAR T, further validating the potential utility of sequential cellular therapies in RRMM [The subgroup of patients who had received an anti-BCMA CAR T prior to ide-cel had the best outcomes in terms of ORR (100%), median DOR, PFS, and OS  compared to patients who had received a prior ADC or bispecific. While these results should be interpreted with caution given the small sample size , with DOR ranging from 1.9 to 6.8 months and a median PFS of 1.0 months  Both patn = 7). Also, the response rate to the prior bispecific was 0% and thus not representative of the expected ~60% ORR seen on clinical trials for several BCMA-targeted bispecific antibodies [While the ORR to ide-cel for the patients who had received a prior anti-BCMA bispecific was high at 86%, these patients numerically had the lowest median DOR, PFS, and OS compared to those receiving a prior ADC or CAR T. The median DOR was significantly lower for patients receiving a prior bispecific compared to an alternative type of BCMA-TT (2.8 versus 8.9 months). This should be interpreted with caution due to small sample size n = . Also, ttibodies , 25. Thetibodies . In the tibodies . In a detibodies .While the aggregate data regarding sequencing of T-cell redirecting therapies seems to support the use of CAR T prior to bispecific antibodies, this strategy may not always be feasible due to access limitations related to CAR T manufacturing and the need for administration at highly specialized centers. For patients being considered for anti-BCMA CAR T after a prior BCMA-TT, the timing between sequential BCMA-TT may be a potential predictor of response. Patients in our study who responded to ide-cel had a significantly shorter duration of exposure to their prior BCMA-TT, and a significantly longer amount of time from their last BCMA-TT exposure to both apheresis and ide-cel infusion when compared to non-responders. Similar findings related to prior BCMA-TT timing were observed with cilta-cel in the CARTITUDE-2 cohort C analysis when stratifying by responders versus non-responders . AlthougBiallelic BCMA loss has been described as a resistance mechanism, albeit relatively uncommon , 27. A rStrengths of this study include analysis of the largest multi-institutional cohort of patients receiving a specific anti-BCMA CAR T after exposure to another BCMA-TT, who would not have been eligible to receive ide-cel on the KarMMa trial. Limitations of our study include the retrospective design, small sample sizes for each individual type of prior BCMA-TT, response assessment per investigator discretion without mandating confirmatory testing or imaging, and limited duration of follow-up at time of data cutoff. Another limitation that may impact future generalizability is that the majority of our prior BCMA-TT cohort received belantamab mafodotin, which has since been withdrawn from commercial use.In summary, treatment with commercial ide-cel after prior BCMA-TT resulted in a relatively high ORR, but significantly lower ORR, median DOR, and median PFS compared to patients not receiving a prior BCMA-TT. The timing of ide-cel infusion in relation to the last exposure to a prior BCMA-TT may be predictive of the likelihood of response, and the small subgroup of patients who received sequential anti-BCMA CAR T had the best response and PFS outcomes.Supplemental Material


The use of donor oocytes in assisted reproduction has seen a significant rise worldwidein the last two decades. Postponement of motherhood and premature ovarian insufficiencyare the main reasons for the increase in the number of in vitro fertilization cycleswith donor oocytes. The present study aims to characterize donor oocyte cycles toanalyze factors that may have an effect on live births and clinical pregnancyoutcomes.Data were obtained from a single Assisted Reproduction Center in southern Brazil.Recipient demographics (n=148 patients) and cycle characteristics  were analyzed. Statistical analysis wasperformed using chi-squared and t-test as appropriate.On average, recipients that reached gestation were significantly younger than the onesthat did not. We also observed a significant positive effect of constant dose estrogentherapy on pregnancies.Patient age and response to estradiol therapy are important factors in the attainmentof the best possible outcomes in cycles with donor oocytes. Cycles with donor oocytes were first performed in the 1980, as a treatment for prematureovarian insufficiency (POI). In vitro fertilization (IVF) with donor oocytes has gained moreacceptance for the treatment of age-related diminished ovarian reserve (DOR).The use of donor oocytes has been widely accepted around the world. According to the HumanFertilization and Embryology Authority (HFEA), UK’s independent fertility treatmentregulator, 3,924 women underwent in vitro fertilization cycles with donor oocytes in 2016, a105% increase in relation to 2006 . DatafrThe main indication for the use of donor oocytes is POI, characterized by menopause beforethe age of 40 years . GeneticToday, donor oocyte cycles represent the most successful assisted reproduction technology(ART) treatment worldwide. Pregnancy rates resulting from donor oocyte cycles continue torise, currently with a live birth rate of about 50%, making it the most successful ARTprocedure .Beyond the undisputable beneficial effects for women unable to use their own oocytes toachieve gestation, the use of donor oocytes in ART treatments brings unique opportunities tostudy and understand factors affecting classical IVF cycles. In donor oocyte programs theoocyte source and the endometrium represent independent entities contributing to embryoimplantation and gestation. In this sense, several studies have been carried out tounderstand the role that endometrial, embryonic and/or hormonal factors play on IVF outcomes. It has The present retrospective study describes the characteristics of donor oocyte cycles andanalyzes the data relative to these cycles to determine the factors that might have affectedclinical pregnancy and live birth outcomes in our assisted reproduction center.The study was conducted in compliance with Resolution No. 466/12 of the Ministry ofHealth  and submThe study presented minimal risks related to the identification of participants based onthe data extracted from their medical charts. The authors of the study pledged to maintainthe confidentiality of patient identification information and ensuring patient anonymity.For data collection, a report was generated with information referring to the variablesunder study, and each patient was identified by a sequential number defined according tothe date of completion of their donor oocyte cycles. A Data Use Disclaimer and a DataLending Agreement was signed by the technical director of the institution where the studytook place.The outcomes of 213 embryo transfers in donor oocyte cycles corresponding to 148 patientsand their treatment characteristics were analyzed. Embryos were generated byintracytoplasmic sperm injection (ICSI) using fresh or cryopreserved donor sperm. Age ofthe oocyte donors ranged from 25 to 30 years. Semen parameters were not taken into accountin our analysis.For purposes of analysis, egg recipients were split into three groups according to age and particular attention was given to the putative effectof preimplantation genetic testing for aneuploidies (PGT-A) on recipient pregnancy andimplantation rates. Additional analyses were performed by grouping cycles according tooutcomes of clinical pregnancy (positive or negative) and live birth (positive ornegative).The parameters considered in statistical analysis were cycles with positive clinicalpregnancy with the presence of a gestational sac, regardless of whether it ended in livebirth or miscarriage, and cycles with negative clinical pregnancy (negative humanchorionic gonadotropin – hCG – test) and biochemical pregnancy . Cycles ended with the delivery of a healthy baby were categorized aspositive deliveries; other outcomes were considered as negative deliveries.Recipients with ovarian function and menses received depot gonadotropin-releasing hormone(GnRH) antagonist for pituitary desensitization. All women underwent endo-metrialstimulation with oral or vaginal estradiol (E2). Initial estrogen administration wasperformed at a constant dose  for all patients. CD treatment was maintaineduntil embryo transfer (ET) in women presenting a satisfactory response (growth) of theendometrial lining. Patients that did not respond to initial E2 stimulus receivedpersonalized, increasing dose (ID) E2 therapy. The ID of E2 was administered as follows:oral E2 started at 6mg/day from day 1 until the day of the first ultrasound scan. Whenendometrial growth was not satisfactory, 8mg/day of E2 was recommended. This E2 dose wasmaintained until ET. One patient used vaginal ID estrogen with a dose similar to the oralID protocol.th week of gestation in case of apositive hCG test performed 14 days after embryo transfer. Minor variations in endometrialpreparation protocol can occur based on medical preferences or patient needs.E2 supplementation was offered for 17 to 21 days, depending on the day the embryos wereavailable for transfer and whether endometrial growth was satisfactory. Progesteronesupplementation was performed with 1 (400mg) or 2 (800mg) intravaginal capsules(Utrogestan) or intravaginal progesterone gel (Crinone), both administered every 12h fromthe day of embryo transfer until the 12Gamete manipulation, oocyte insemination, embryo culture, PGT-A biopsy and embryocryopreservation were performed according to standard protocols used in the IVF laboratoryof our assisted reproduction center.The blastocysts were scored according to the Gardner and Schoolcraft grading system(th and 75th percen-tiles).To associate categorical variables, the Chi-squared test or the Chi-squared test withYates correction in 2x2 tables was used. Quantitative variables with a normal distributionwere compared between groups using Student’s t-test for independent samples and the oneswith an asymmetric distribution were compared based on the Mann-Whitney test. Factors thatshowed significant associations in bivariate analysis for at least one of the outcomeswere included in Poisson regressions with robust variance. A significance level of 5% wasconsidered.Data were entered into Microsoft Excel and later exported to IBM SPSS version 20.0 forstatistical analysis. Categorical variables were described by frequencies and percentages.The normality of the quantitative variables was evaluated using the Kolmogorov-Smirnovtest. Quantitative variables with a normal distribution were described by mean values andstandard deviation, while variables with an asymmetric distribution were described usingmedian values and interquartile ranges  were aged 40 years or older during IVF treatment and the most frequentdiagnosis was diminished ovarian reserve (89.9%). For patients that presented more than oneinfertility factor, the main factor was considered for analysis. The number of mature donor oocytes used in each cycle varied from 6 to 10 and the rate ofblastocyst formation per donor oocyte cycle was 66.3%. In the 213 cycles analyzed, the overall clinical pregnancy rate was 47.6% (n=101) and thelive birth rate was 41.3% (n=88). vs. 45.3 years; p=0.036). There was also a signif-cantlyhigher rate of live birth among women who underwent more than one transfer cycle and asignificant increase in the number of clinical pregnancies in patients given constant doseoral E2. None of the other parameters showed a significant impact on clinical pregnancy orlive birth rates.However, when pregnant and non-pregnant patients were compared for age, statisticalanalysis revealed that pregnant patients were younger than non-pregnant ones  of the donor egg recipients were aged 40 years or older. Thisfinding agrees with previous reports  for implanting donor oocytes inthe patients included in this study. The European Society for Assisted Reproduction  and a reIn the present study, the source of oocytes, fresh or frozen, did not seem to exert asignificant impact on pregnancy or live birth rates in donor oocyte cycles. This may havehappened due to the smaller number of frozen donor oocyte cycles, when compared with thenumber of fresh donor oocyte cycles. However, our results are in agreement with a recentreport by Although questionable, the use of PGT-A in donor oocyte cycles is a common practiceworldwide. PGT-A was applied in 19,2% of the cycles in the present study showing no positiveeffect on pregnancy and live birth rates, in agreement with previous reports  of the donor oocyte cyclesanalyzed in this study. Although the Resolution No. 2.168/2017 of the Federal Board ofMedicine  allows tThe limitations of our study are related to the fact that we did not assess oocyte donorcharacteristics or semen quality in the donor oocyte cycles analyzed. There arecontroversies in literature on whether donor factors, such as BMI, age and lifestyle exertan effect on donor oocyte procedures and outcomes. A similar situation occurs when semenparameters are considered in this mode of infertility treatment. The two parameters shall beinvestigated in a future study, where specific donor factors, E2 administration protocolsand semen quality represent the central point of our research.The present study describes the putative cycle parameters that contributed to successfuldonor oocyte cycle outcomes. The source of oocytes, fresh or cryopreserved, did not affectpregnancy or live birth rates. Donor egg recipient age seems to have an impact on pregnancyrate, an effect possibly related to the endometrial aging process. E2 CD administration mayexert a positive effect on pregnancy rates. However, further studies are needed to explorethis hypothesis. In view of the high implantation and pregnancy rates, the transfer of asingle embryo is advised in this mode of infertility treatment. In addition to expanding theability to treat infertility, donor oocytes represent a valuable tool to study isolatedovarian and endometrial factors and their impact on reproductive outcomes.


Interestingly, individuals with cognitive impairment had higher HBA levels after TG infusion than those with normal cognition. These results suggest that interventions that increase plasma ketones may lead to higher brain ketones in groups at risk for AD and should be confirmed in larger intervention studies.Brain glucose hypometabolism is an early sign of Alzheimer’s disease (AD), and interventions which offset this deficit, such as ketogenic diets, show promise as AD therapeutics. Conversely, high-fat feeding may exacerbate AD risk. We analyzed the metabolomic profile of cerebrospinal fluid (CSF) in a pilot study of older adults who underwent saline and triglyceride (TG) infusions. Older adults , age 65.3 ± 8.1, and 9 with cognitive impairment (CI), age 70.9 ± 8.6) underwent a 5 h TG or saline infusion on different days using a random crossover design; CSF was collected at the end of infusion. Aqueous metabolites were measured using a targeted mass spectroscopy (MS) platform focusing on 215 metabolites from over 35 different metabolic pathways. Data were analyzed using MetaboAnalyst 4.0 and SAS. Of the 215 targeted metabolites, 99 were detectable in CSF. Only one metabolite significantly differed by treatment: the ketone body 3-hydroxybutyrate (HBA). Post hoc analyses showed that HBA levels were associated with age and markers of metabolic syndrome and demonstrated different correlation patterns for the two treatments. When analyzed by cognitive diagnosis group, TG-induced increases in HBA were over 3 times higher for those with cognitive impairment (change score CN +9.8 uM ± 8.3, CI +32.4 ± 7.4,  Alzheimer’s disease (AD) is the most common cause of dementia. One AD risk factor identified in epidemiologic studies is a ‘Western diet’, which is characterized by high intake of saturated high-fat foods (HFF) ,2. AnimaAnother consequence of AD is brain glucose hypometabolism . DespiteAnalysis of the CSF metabolome can offer biochemical insights into brain function . This boThe Institutional Review Boards of the University of Washington and the Veterans Affairs Puget Sound Health Care System approved the study. Subjects were recruited from the Memory Disorders Clinic of the VA Puget Sound Health Care System, and through advertisements in local newspapers. All potential subjects underwent a physical examination, clinical laboratory testing, and APOE genotyping using PCR amplification with primer sequences and methods as per Hixson et al. . InclusiThis was a randomized cross-over design involving an intravenous saline versus a triglyceride infusion in older adults, followed by a lumbar puncture. All study subjects were blinded to the intervention and arrived at the research unit after a 12 h fast. Bilateral 20-gauge IVs were placed, and blood was drawn at 0 h and 5 h. The participants received a 5 h infusion of normal saline (0.9% 15 cc/h) or 20% triglyceride emulsion at 45 cc/h  on different days in random order. The infusion consisted of emulsified fat particles of approximately 0.4 micron in diameter, similar to naturally occurring chylomicrons, and contained 10% safflower oil and 10% soybean oil as well as egg yolk phosphatides [vol/vol) methanol in water (including isotope-labeled standards) to a 50 μL aliquot of the sample. After vortexing, samples were kept on ice for 20 min and then spun at 4 °C for 15 min at high speed. Supernatant (150 μL per sample) was transferred to a new tube and dried down by using a speed vacuum at 30 °C. Dried samples were stored at −80 °C.Metabolites were extracted by first thawing the CSF on ice and adding 300 μL of 80%  acetonitrile, 2.0% methanol, and 0.2% acetic acid) and B (10 mM ammonium acetate in 92.9% (vol/vol) acetonitrile, 4.9% H2O, 2.0% methanol, and 0.2% acetic acid). After the initial 2 min isocratic elution of 90% (vol/vol) B, the percentage of solvent B decreased to 50% (vol/vol) at t = 5 min. The composition of solvent B was maintained at 50% (vol/vol) for 4 min (t = 9 min), and then the percentage of B gradually went back to 90% (vol/vol) to prepare for the next injection. The metabolite identities were confirmed by spiking a pooled serum sample used for method development with mixtures of standard compounds. The extracted multiple reaction monitoring peaks were integrated by using MultiQuant 3.0 software . Of the 215 metabolites targeted, up to 99 metabolites were detected. We also targeted and detected 34 isotope-labeled metabolites that were used to both monitor sample preparation and run conditions as well as to determine absolute concentrations for the corresponding endogenous metabolites. For quality control (QC), a sample of CSF pooled from the study samples and a pooled human serum were analyzed every 10 samples and at the beginning and end of the runs. For the pooled serum sample, 116 metabolites and 34 labeled internal standards were detected with a median CV value of 3.9%. For the pooled aliquots of the CSF samples, we detected up to 90 metabolites and 32 labeled internal standards with a median CV value of 4.5%.Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed according to methods developed at the University of Washington’s Northwest Metabolomics Research Center as per Skill et al. ,19. The Data were analyzed using MetaboAnalyst 4.0 online software  for dataOf the 215 targeted metabolites, 99 were detectable in the CSF, along with 28 of the utilized 32 isotope-labeled internal standards for absolute quantification. Targeted and detected metabolites marked with an * are shown in the Next, we looked specifically at glycolysis and TCA cycle intermediates. We classified 35 metabolites as being involved in glycolysis or the TCA cycle. Of these metabolites, 19 were present in CSF and most of these were TCA cycle intermediates or sugars such as glucose. We detected only two glycolysis cycle intermediates: glucose 1-phosphate (G1P) was not present in any participant’s CSF after the saline infusion but was detected in two samples after TG infusion. Glyceraldehyde-3-phosphate (D-GA3P) was detected in all samples. In contrast, several TCA cycle intermediates were detected in all CSF samples including citrate, aconitate, succinate, fumarate, and oxaloacetate .An average of 88 different metabolites were detected in each CSF sample, and this did not differ between saline and TG infusion . A total of 12 metabolites were present in <50% of samples and thus were excluded from MetaboAnalyst , which are both shown in Using Benjamini–Hochberg robust correction for multiple comparisons, only one significant metabolite was identified: an increase in the ketone body 3-hydroxybutyrate (HBA) after the TG infusion .p = 0.0198). Examination of the means showed that fasting HBA was similar by diagnosis, but TG-infusion induced increases in HBA were nearly 3 times higher for those with any cognitive impairment ; however, numbers of MCI and AD subjects were very small . For the TG samples, HBA did not significantly correlate with acetoacetate but correlated with pyruvate .Next, we used the Pattern Hunter feature of MetaboAnalyst to examine which metabolites correlated with CSF HBA levels . This feIn this group of older adults, of the 215 possible metabolites measured in this targeted platform, 99 were detectable in CSF in at least some samples, and 81 were detectable in all samples. By and large, metabolite levels did not vary between the two interventions within individuals, indicating that CSF metabolites remain relatively stable over a short intervention. However, the HCA of the data, as shown in the heat map of p value cutoff value of 0.05, and only one met a more stringent cutoff of p < 0.01 [We detected 99 metabolites in CSF overall, with an average of 88 per sample. These findings were expected because, due to the nature of CSF production, this fluid contains many fewer detectable proteins and metabolites than plasma. For example, the same methodology run on plasma would typically measure about 120–140 metabolites . In othep < 0.01 . A studyp < 0.01 .In terms of the types of metabolites noted, many CSF metabolites belonged to nucleic or amino-acid-related metabolites or precursors. In addition, a total of 116 metabolites were never detected in any of the CSF samples, representing 54% of detectable metabolites in our targeted profile. Unlike the lipidomics analysis from this same intervention, which revealed numerous lipids and classes of lipids which changed in response to the infusion , here weMoreover, in our study, glucose, pyruvate, and lactate were widely present, but lactose, mannose and galactose sugars were not detectable, consistent with these sugars requiring further processing before brain metabolism . Many phOnly one metabolite met our statistical threshold as differing between the two conditions—the ketone body HBA. HBA is one of several ketone bodies synthesized in the liver from acetyl-CoA and can be used as an energy source by the brain when blood glucose is low ,21. ThisDiets and supplements which induce ketosis are being investigated as treatments for AD . HealthyWe recently showed that after a 6-week modified ketogenic diet study in older adults, lower plasma ketones were noted for participants with MCI compared with those with normal cognition, and we speculated this could be due to either greater ketone uptake into target tissues or decreased ability to generate ketones . This spUnlike the lipidomic signature , we did The findings of this study are limited by a small sample size, which particularly limits our ability to look for subgroup effects by sex, diagnosis, or APOE genotype. Early in the study we had to change to a different manufacturer for the lipid infusion; however, the two infusions had the same components per the manufacturer. Findings would be strengthened by plasma metabolite analysis (not currently available in our data set), as well as metabolic analysis of the infusion. For future studies such as this one, we recommend analysis of the infusion product itself to identify all potential compounds.In this small pilot study, we showed that unlike the lipidomic signature, the CSF metabolic signature remained remarkably stable after a triglyceride infusion in older adults at risk for AD. Most TCA metabolites were present in CSF, whereas glycolysis intermediates were mostly absent. The ketone body HBA was the only significant metabolite that changed in response to TG infusion, and those with cognitive impairment showed a more significant increase in CSF HBA levels. Metabolite studies such as the one presented here are feasible to explore metabolic hypotheses about ketone bodies in human brain, and more generally may be useful for understanding brain fuel metabolism.