To improve outcome prediction following subarachnoid haemorrhage (SAH), we sought a biomarker integrating early brain injury and multiple secondary pathological processes in a prospective study of 42 non-traumatic SAH patients and 19 control individuals. Neurofilament light (NF-L) was elevated in CSF and serum following SAH. CSF and serum NF-L on Days 1–3 post-SAH strongly predicted modified Rankin score at 6 months, independent of World Federation of Neurosurgical Societies (WFNS) score. NF-L from Day 4 onwards also had a profound impact on outcome. To link NF-L to a SAH-specific pathological process, we investigated NF-L’s relationship with extracellular haemoglobin. Most CSF haemoglobin was not complexed with haptoglobin, yet was able to be bound by exogenous haptoglobin i.e. haemoglobin was scavengeable. CSF scavengeable haemoglobin was strongly predictive of subsequent CSF NF-L. Next, we investigated NF-L efflux from the brain after SAH. Serum and CSF NF-L correlated positively. The serum/CSF NF-L ratio was lower in SAH versus control subjects, in keeping with glymphatic efflux dysfunction after SAH. CSF/serum albumin ratio was increased following SAH versus controls. The serum/CSF NF-L ratio correlated negatively with the CSF/serum albumin ratio, indicating that transfer of the two proteins across the blood–brain interface is dissociated. In summary, NF-L is a strong predictive marker for SAH clinical outcome, adding value to the WFNS score, and is a promising surrogate end point in clinical trials. et al. show that neurofilament light levels predict long-term neurological outcomes following subarachnoid haemorrhage, if measured in CSF or serum within the first 72 hours. The measure improves accuracy of outcome prediction after the World Federation of Neurosurgical Societies score has been taken into account.Garland  Subarachnoid haemorrhage (SAH) leads to an increase in intracranial pressure, transient cerebral ischaemia, and a subsequent prolonged multifactorial insult including neurotoxicity from haemoglobin released from the blood clot, delayed cerebral ischaemia, and inflammation . To predIn trying to explain a larger proportion of outcome variance, we took advantage of neurofilament light (NF-L), an intracellular neuronal protein released during damage to neurons. NF-L can be detected in both CSF and serum and is a well-established biomarker for neuronal injury in neurodegenerative and neuroinflammatory conditions . CSF was centrifuged at 1500 relative centrifugal force (RCF) for 10 min at 21°C and frozen within 1 h of sampling. Blood samples were routinely taken on the same days as the CSF samples. SAH patients were followed up at 6 months to determine clinical outcome as assessed by the modified Rankin Scale (mRS)  and institutional (ERGO 41084 and ERGO 10492) approvals. Paired CSF and serum were collected daily on the first 3 days and on alternate days thereafter over a 14-day period post-ictus in 44 patients following original Fisher grade 3–4 (CSF NF-L was measured by ELISA (UmanDiagnostics). Briefly, samples were thawed at 21°C, and centrifuged at 1750 RCF for 5 min at 21°C. Samples were diluted 1:2 with sample diluent and added in duplicate to microplate wells coated with a monoclonal capture antibody specific for NF-L. Samples were then incubated with a biotinylated anti-NF-L monoclonal detection antibody. The detection complex was completed with the addition of horseradish peroxidase-labelled streptavidin and tetramethylbenzidine (TMB) substrate. Sample concentrations were calculated from a standard curve, fitted using a four-parameter logistic curve. The intra-assay coefficient of variation (CVs) was 1.8% and the inter-assay CV was 10.9%, calculated according to ISO 5725-2 standards.d-galactopyranoside substrate produces a fluorescent signal proportional to the concentration of NF-L present. Duplicate measurements were taken of each sample. Sample concentrations were calculated from a standard curve, fitted using a four-parameter logistic curve. Intra-assay CVs were <11%, as determined by two quality controls (low and high concentration) according to ISO 5725-2 standards.Serum NF-L was measured by single molecule array (Simoa) on an HD-1 analyser (Quanterix), according to the manufacturer’s instructions (Lot: 501832). Briefly, samples were thawed at 21°C, vortexed, and centrifuged at 10 000 RCF for 5 min at 21°C. Onboard the instrument, samples were diluted 1:4 with sample diluent and bound to paramagnetic beads coated with a capture antibody specific for human NF-L. NF-L bound beads were then incubated with a biotinylated anti-NF-L detection antibody in turn conjugated to streptavidin-β-galactosidase complex that acts as a fluorescent tag. Subsequent hydrolysis reaction with a resorufin β-Ultra-performance liquid chromatography (UPLC) was used to separate CSF components in a Tris-saline mobile phase, coupled to absorbance measurement at 415 nm to identify haem-containing species , the probability of a type I error, was 0.05. Two-tailed hypotheses were considered throughout.Statistical analysis and graph preparation were performed using SPSS (v24) and GraphPad Prism (v7.01), with data expressed as median ± interquartile (IQR), mean ± standard error, or 95% confidence intervals (CI). The NF-L reference range was defined as the mean ± 1.96 standard deviations (SD) of the control population log-normal distribution. Area-under-the-curve (AUC) analysis was undertaken using the trapezoid method, and normalized per day. Normality and heteroscedasticity were routinely determined across all datasets. Where necessary, logarithmic transformation was used to normalize data. Model comparison for multivariate linear and ordinal regressions was performed using the F-test. In regression modelling, WFNS was dichotomized into low grade (i.e. 1–3) and high grade (i.e. 4–5). In ordinal regression, the proportional odds assumption was fulfilled. The Nagelkerke’s pseudo RThe data that support the findings of this study are available from the corresponding author, upon reasonable request, subject to institutional agreements and ethical approvals.P = 0.926 and P = 0.511 for CSF and serum NF-L, respectively)  .P = 0.031) for poor outcome. The odds ratio for WFNS was 10.4 . CSF NF-L and WFNS independently explained the 6-month mRS, such that CSF NF-L improved model fit compared to WFNS alone .In view of the NF-L peak at Day 2, we first investigated whether early NF-L measurement between Days 1 and 3 could explain additional variance in outcome alongside WFNS. Multivariable ordinal logistical regression was conducted, with mRS at 6 months post-ictus as the dependent variable, and NF-L as predictor, controlling for WFNS. Age, gender, premorbid history of hypertension, Fisher scale, size and location of ruptured aneurysm lumen, and treatment modality were not significant covariates in univariate analysis. High CSF and serum NF-L were defined as higher than the upper limit of the respective control population-based reference ranges. High CSF NF-L significantly predicted a worse mRS , with anP = 0.04). The odds ratio for WFNS was 3.2 . Serum NF-L and WFNS independently explained the 6-month mRS, such that serum NF-L improved model fit compared to WFNS alone .Similarly, high serum NF-L significantly predicted a poor neurological outcome at 6 months post-ictus , with anP = 0.035 and P = 0.037 for CSF and serum NF-L, respectively), improving model fit .Both CSF and serum NF-L independently predicted outcome when added to the Jaja core model predictors  or serum . However, when both early and late CSF NF-L AUC were analysed together, only late CSF NF-L retained significance .Multivariable ordinal regression controlling for WFNS showed that NF-L level in the late epoch contributed to outcome additional to WFNS, whether it was assessed in CSF   .To substantiate the relationship of NF-L to pathological processes that are (i) specific to SAH; (ii) delayed; and (iii) potentially reversible, we selected extracellular haemoglobin as an example for several reasons. First, haemoglobin is specific to SAH. Second, it is released gradually and its CSF concentration increases with time during the first 2 weeks post-SAH, as clot lysis proceeds ; neither age nor sex contributed significantly.To investigate the relationship between CSF scavengeable haemoglobin and NF-L levels we used multiple linear regression, controlling for WFNS, age and sex . With thP = 0.024), suggesting glymphatic efflux dysfunction after SAH.A circulating biomarker, such as serum NF-L, would be of immense value following SAH, since CSF would only be accessible in a minority of patients. Serum NF-L concentration would be expected to follow CSF NF-L due to brain–blood efflux, and indeed this was the case with the maximum CSF NF-L predicting the subsequent maximum serum NF-L over the sampling period . The serNF-L (62 kDa) has a similar molecular weight to albumin (60 kDa), so we investigated whether NF-L efflux and albumin influx across the blood–brain interface, albeit in different directions, were synchronized, since this may provide insight into the routes of exchange of these two molecules. Albumin influx from the blood into the CSF occurs via the choroid plexus and the blood–brain barrier . Since at-test between the control population and the maximum Qalb in the SAH patients shows a highly significant difference (There is evidence that blood–brain barrier permeability increases after SAH . Figure fference .NF-L may exit the brain into the circulation via a number of routes, including CSF drainage via the cribriform plate, nerve roots and dural lymphatics, the intramural peri-arterial pathway and reflux across the gliovascular membrane complex at the blood–brain barrier . This fiSAH results in a long-term deficit in neurological function, yet outcome prediction is challenging. Here we take the novel approach of combining WFNS as a clinical marker of early brain injury, and NF-L as an independent biomarker integrating multiple post-injury processes, to best model long-term neurological outcome in SAH patients. An early CSF or serum NF-L estimation, within the first 1–3 days after SAH, adds value to the WFNS, in the prediction of 6-month outcome following SAH. A serum biomarker is significantly advantageous over a CSF biomarker, since CSF may not be readily available in all patients. External ventricular drain placement is required for clinical management in only a third of patients (This study extends and clarifies research examining neurofilament subunits, namely heavy (The early prognostic effect of NF-L suggests that its release is likely to be linked to early brain injury. The latter triggers a number of prolonged processes such as inflammation and oxidative stress while other processes have a delayed onset such as delayed cerebral ischaemia, haemoglobin toxicity, ferrotoxicity and other as yet unidentified mechanisms. Hence it is not surprising that CSF NF-L AUC from Day 4 onwards had a bigger impact on the 6-month outcome, compared to CSF NF-L AUC on Days 1–3. While it is not clinically practical to measure NF-L levels repeatedly, AUC analysis here provided biological insight. In particular, the dramatic contribution of CSF NF-L AUC in the late epoch suggested that significant neuronal damage occurs in a delayed manner, and this impacts significantly on outcome. It is very possible that early brain injury directly following SAH may interact with secondary processes to determine outcome.One of the prolonged secondary pathological processes likely to be integrated in the NF-L rise after SAH, is the gradual release of haemoglobin from the clot. Extracellular haemoglobin is directly neurotoxic (Not surprisingly, the clinical significance of raised serum NF-L followed that of CSF NF-L. A number of factors are likely to affect the relationship between CSF and serum NF-L, as NF-L generated intrathecally is transferred to the circulation, including blood–brain transfer rate and circulating half-life. Nevertheless, serum NF-L correlated with CSF NF-L, in keeping with the prognostic value of NF-L in both compartments.The blood–brain barrier was compromised after SAH, as confirmed by the high Qalb in SAH patients compared to control subjects; this has been previously reported  causes bThe serum/CSF NF-L ratio can be considered to be a marker of the rate of solute drainage from the brain. This ratio was decreased in SAH patients versus controls, in keeping with glymphatic efflux dysfunction, which has been recently demonstrated in an animal model of SAH (The highlight of this work is the prognostic usefulness of CSF and serum NF-L in predicting clinical outcome after SAH. Further work is required to progress these findings in larger more generalizable cohorts, including SAH patients with lower Fisher grades. We introduce the novel concept of including a biomarker integrating an array of early and secondary processes, in predictive modelling of SAH outcome. NF-L is used here as an example; other radiological or biochemical markers may be better, or their addition may improve prediction. The use of such variables has potential to transform modelling of long-term clinical outcome following SAH.awaa451_Supplementary_DataClick here for additional data file.


The decision to use an anonymous gamete donation in fertility treatment could have significant long-term psychological and social effects for all stakeholders involved. In light of the growing recognition of donor-conceived children’s right to know their genetic parentage, this entails profound ethical implications. This review aims to carve out the full spectrum of recipients’ motives and experiences related to donor anonymity which could serve as an analytical framework for future ethical and sociological research on issues of donor anonymity. This review was conducted following a seven-step approach for systematic reviews of empirical bioethics literature. The characteristics and quality of the studies included in this review were reported. Data analysis was conducted using qualitative content analysis and was informed by sociological functionalist theorizations of ignorance. The 53 studies selected showed a diverse spectrum of characteristics concerning date and country of study, methodology, family type of participants, sample size, and the timing of data collection in relation to the stage of treatment. A total of 22 categories of motives and experiences of recipients concerning donor anonymity were identified inductively and grouped into five main categories. Donor anonymity was identified as a eufunctional form of ignorance, by which the recipients experienced or intended to control, regulate, or protect inter-stakeholder relations. Interpreting recipients’ motives and experiences concerning donor anonymity as a form of ignorance directed toward particular stakeholders helps reframe the discourse on donor anonymity. It is a fruitful approach that can be refined further and applied in future research. This review identified possible directions for future investigations on motives for donor anonymity: the need for more thorough inquiries into the change in recipients’ preferences over time, such as in the form of longitudinal studies and research on the perspective of non-biological parents. Choosing to conceive a child through a sperm or oocyte donation is a significant decision for aspiring parents to make in their quest for parenthood. However, such a decision would also lead them to make important choices around areas such as the donor type, that is, whether to use a known, anonymous, or identity-release donor. A known donor, as the name suggests, is known to the recipient at the time of treatment, such as in the case of oocyte donation by a sister of the recipient. An anonymous donor is one whose identity is not known to the recipient—such as in the case of a sperm donation via a sperm bank in a country with mandatory donor anonymity, where only non-identifiable information is passed on to the recipients. An identity-release donor is one whose identifying information will be released to the (adult) child upon request. The implications of the choice of donor type such as possible ethical dilemmas and potential long-term psychological and social effects for the recipients themselves, the donor-conceived offspring, the donor, and the relationship among these stakeholders are subject of an ongoing debate .Along with a growing recognition of the rights of donor-conceived children to know their genetic parentage, donor anonymity has been coming under increasing scrutiny in a growing number of jurisdictions since the 1980s . As a rede facto in a position to choose between an anonymous and non-anonymous donation. This may be because the recipients reside in a jurisdiction that imposes no legislative constraints on their choice concerning donor anonymity (such as in the US). For example, it has been suggested that many recipients of oocyte donation in the US “have strong preferences regarding the use of anonymous versus directed donors and will express and exercise these preferences when given the opportunity” anonymity cover a broad spectrum of countries, timeframes, and samples in various settings and accordingly present strongly varying findings . The firA thorough understanding of motives and preferences, and the experiences and retrospective reflections related to the recipients’ choice between a known, anonymous, and identity-release donor plays a crucial role in the following four contexts.First, since the 1980s many countries have observed a growing trend toward a policy of increased openness in ART involving gamete donation, both in the form of encouraging disclosure of the mode of conception to the donor-conceived offspring as well as promoting legislative measures to ban donor anonymity . This leSecond, in many such cases, public perception and the political deliberation process tend to be determined by a focus on the perspective of the donor-conceived offspring and on donor-related issues, such as the question of how to prevent a decline in gamete donation, which was assumed would occur following the abolition of donor anonymity. The perspective of recipients, however, seems far less represented in these discourses  and precThird, pre-treatment psychological counseling is an important part of many ART programs based on gamete donation. Effective counseling with the aim of sensitizing aspiring parents to the implications of their choices concerning donor anonymity is closely related to an understanding of the concerns of aspiring parents in relation to donor identifiability and anonymity as well as intra-familial mechanisms and dynamics potentially triggered by the recipients’ choice of donor type. A comprehensive understanding of the full range of patterns, motives, and mechanisms underlying the recipients’ preferences concerning donor anonymity can be considered instructive in offering the recipients successful psychological guidance throughout the challenging decision-making process.Fourth, the phenomenon of CBRS puts aspiring parents in a position to have extended options concerning their choice from among the options available to them. Although there are various factors underlying a decision to seek treatment abroad, such as the type of treatment sought, the type of recipient, the country of origin and destination and their disparities concerning the availability of donors, waiting times, treatment costs, or success rates , some fiResearch has shed light on a number of important aspects of recipients’ attitudes and preferences concerning donor anonymity in country-specific contexts by focusing on various recipients such as heterosexual or lesbian couples or single mothers by choice. A systematic overview of the possible motives and experiences of recipients from a more universal perspective remains, to the best of the author’s knowledge, a desideratum. Therefore, this review aims to carve out the full spectrum of recipients’ motives and experiences related to donor anonymity by a systematic examination of the existing qualitative research. Intended to be a preliminary step to further investigation, this review does so by proposing a theory-driven approach that could serve as an analytical framework in future ethical and sociological research on issues of donor anonymity.This systematic review was conducted following the seven-step approach proposed by The review question was specified using the methodology, issues, participants (MIP) model outlined by Based on the results of preliminary scoping searches and on the understanding that research relevant to the review question would be situated at the intersections of multiple disciplinary fields like psychology, sociology, and medical ethics, I decided to pursue a broad approach to identify studies from a wide range of backgrounds. Therefore, the following seven databases covering a broad spectrum of disciplines were included in the literature search: Web of Science Core Collection, SCOPUS, MedLine, PubMed, CINAHL, PsycINFO, and ProQuest Central.After the initial preliminary scoping searches conducted on Web of Science and MedLine, the review question was translated into a search syntax comprising four clusters relating to the topics of 1) anonymity, 2) gamete, 3) donation, and 4) motives. The 2–15 keywords in each cluster were connected using the Boolean operator OR, while the four clusters were connected using the Boolean operator AND. The search syntax was developed and refined through an iterative process using five key references identified in the preliminary scoping searches to check search syntaxes on sensitivity and specificity . Owing tThe search of bibliographic databases was supplemented by a manual search of major journals, and forward and backward citation chaining of key references. However, this process did not reveal any additional studies that had not been identified using the initial search algorithm.de facto did not have an actual choice concerning donor anonymity because of legal, regulatory, financial, or other restrictions, but nevertheless inquired into the recipients’  considerations concerning anonymous gamete donation were also included. Studies on recipients of non-anonymous donations were also included if they discussed the recipients’ decision-making process around donor anonymity. Purely quantitative, non-empirical, and studies published in languages other than English, German, or French were excluded. Studies focusing solely on embryo or mitochondrial donation were also excluded. This review was limited to (aspiring) parents (and their partners) who were at least already in the process of starting treatment. Thus, studies drawing on surveys of sections of the general population were excluded.The references retrieved were assessed for inclusion or exclusion. Empirical studies providing original qualitative data by means of interviews, fieldwork, questionnaires or online surveys including open-ended questions, and mixed-method studies with a significant focus on qualitative inquiry were included. The studies included involved  recipients of sperm or oocyte donation, and were focused on their (and their partners’) attitudes, experiences, preferences, and/or motivations for considering the usage of anonymous gamete donations. Studies either focused on the pre-treatment decisions of potential recipients or retrospective accounts of recipients reflecting on their former choices concerning donor anonymity and the experiences that resulted therefrom. Alternatives to choosing anonymous donors may have been the use of known donors  and non-related non-anonymous donors , or donations involving non-absolute forms of anonymity, such as identity-release donations. Special forms of known-anonymous oocyte donation where recipients provide a donor recruited among fertile friends or family members to the program and whose oocytes are shared anonymously with other participants  were conThe quality assessment of qualitative research is subject to an ongoing debate. Recent research suggests that there is still no consensus on the necessity, value, methods, and criteria of quality assessment of qualitative research in the context of systematic reviews. The “[e]xclusion of so-called inadequately reported studies had no meaningful effect on the synthesis” :1,425. TIn order to answer the review question at a high level of abstraction, data analysis was informed by sociological accounts of the functions of ignorance and the intentionality of knowledge restriction. Assuming that donor anonymity constitutes a form of non-knowledge or ignorance , recipieAmong the various methods of analyzing and synthesizing qualitative data in the context of systematic reviews , applyinAdapting Mayring’s procedural model, which proposes a segmentation of the analytical process into six consecutive steps :85–87, iBefore data analysis, the studies were screened for their major characteristics. An adequate appraisal of the findings was possible only by considering the high dependency of the results on issues such as the national context under study, the family type examined, and the type of gamete used. These characteristics were recorded with the help of a data extraction sheet. Codes of each sub-category were correlated with characteristics of the studies as possible factors impacting on recipients’ motives and experiences and the results were reported.The search identified 7,281 references in all. After removing duplicates, 3,376 studies were screened for relevance in a two-stage procedure: first by title and abstract  and second by full text (349 studies excluded). Ancillary search strategies  were performed, but did not lead to the identification of additional studies to be included in the review. The remaining 53 studies were included in the review. In six studies, the same three datasets were analyzed by different sets of authors. In two studies, the same dataset was presented by the same author. All these studies were included in the review as the different forms of publication  resulted in distinct differences in the extent of materials presented and levels of analysis. The 53 studies included in this review therefore represent 49 datasets. The literature search and study selection process is indicated using a PRISMA flow chart .n = 45), and as PhD dissertations (n = 5) and book chapters (n = 3). Further, two studies were published in French, one in German, and 50 in English. The countries of study included the US (n = 15), the United Kingdom (n = 8), Belgium (n = 8), Canada (n = 6), France (n = 2), Germany (n = 2), and Israel, Sweden, and the Netherlands (n = 1 each). Some focused on multiple countries in the context of online surveys (n = 4), or on forms of CBRS (n = 4). One compared the donor choices of recipients in Sweden and Ireland  studies focused on sperm donation, and fewer on oocyte donation (n = 11). Two studies on sperm donation also included some cases of combinations of sperm and oocyte donations (n = 33) while other participants were exclusively recipients of anonymous (n = 19) or open-identity (n = 1) donations. All studies were conducted in specific contexts determined by certain national legal frameworks or institutional regulations that restricted the actual reproductive options available to the participants and/or had a significant influence on the choice of donor. Changing legislation concerning donor anonymity in many countries made it imperative to clarify these circumstances diligently at the time and place of data collection for each study. These are reported in Most (onations . Particin = 5), during (n = 3), and after treatment (n = 32), and some included participants at different stages of treatment (n = 13). In most of the studies conducted post-treatment, the donor-conceived children had not yet reached adolescence; the maximum age of the children reported was 29 years : the recipients themselves, the child, the donor, or the nuclear family’s social environment. A fifth main category covered pragmatic reasons underlying the recipients’ preference for donor anonymity. The main categories, categories and sub-categories are summarized in A considerable proportion of motives for choosing anonymous donation pertained to the recipients’ intention to remain themselves ignorant of the donor’s identity. As reported by the studies reviewed, by choosing not to know the donor’s identity, recipients expected to protect their psychological well-being and their relationship with their partners by controlling the distance to the donor. They also aimed to unburden their relationship with the child. These motives are summarized in the first main category.Recipients perceived donor anonymity as a means to create and maintain a psychological distance from the donor. By remaining unaware of the identity of the donor, recipients could avoid acknowledging the donor as a person, classifying them as “not a real person” or a “non-person” :710 and Ignorance of the donor’s identity also helped recipients to establish a sense of normality. Recipients considered donor anonymity a benefit as it allowed them to repress or alleviate thoughts about their infertility, as “a known donor could be an unwanted reminder” of the recipients’ infertility :2,069, aThe data showed that anonymity allowed recipients to make a full and truthful disclosure to the child by revealing all information available on the mode of conception and the donor. Donor anonymity made the disclosure of the fact of donor conception to the child easier for the recipients :944. It The second main category comprised the motive to keep the child ignorant of donor identity. Motives and experiences in this category were concerned with protecting the well-being of the child by controlling the donor-child relationship. They also included motives concerned with the child-recipient relationship and protection of the donor.The studies showed that recipients considered donor anonymity as a means to create distance between the donor and their child. Donor anonymity is seen as a guarantee that precludes the child from contacting the donor: “[A]nonymous donation made it impossible for the child to be able to find or contact the donor, which was a comforting thought for some parents” :15. The Recipients also indicated that the child-recipient relationship benefits from donor anonymity, as it allows recipients to consolidate their parentage. Keeping a child ignorant of the donor’s identity is believed to prevent the child from picturing and personalizing the donor as a parent figure: “Discovering information about the donor entails the risk that it will become a big issue for the children, as she  uses the term ‘father’. She claims that the construct of the donor will change from ‘cell’ to ‘person’ to ‘father’ as soon as the children know more about him. Therefore, she clearly states her preference for the anonymous system” :1,252. DThe third main category comprised motives and experiences centered on the ignorance of the donor, as the third stakeholder (the other two being the recipients and the child). In contrast to the intentionality of ignorance described in the previous category, here recipients’ anticipated or actual benefits revolve around the donor’s ignorance of the child’s identity. The main motives associated with this form of ignorance were expectations of being able to control the influence of the donor on the child and the child’s well-being, on the recipients’ parenting, and on family affairs, thus ensuring long-term stability of family. A large part of the motives and experiences summarized in this main category are related to the recipients’ decision to avoid a known donor, as this donor type would entail immediate issues of donor involvement and legal dimensions, which were often perceived by the recipients as undesirable, threatening, or as a potential source of conflict. However, accounts of recipients to whom identity-release donors were available as an alternative to anonymous donation e.g.,  also felPrecluding donor interference in child-related issues was frequently identified in the studies included. Donor anonymity was considered by recipients to protect the child from harmful consequences of the donor interfering in the child’s life, such as the disruption of the stability of a child’s family environment by introducing a “potentially harmful multiple-parent situation” :482 and The fourth main category focused on keeping the family’s social environment ignorant of the donor’s identity. The motives underlying this category pertained on the one hand to maintaining secrecy and privacy against the social environment of the family in general, and on the other to avoiding conflict with the extended family in particular, which may have arisen if a known person, especially a family member were to have been used as donor. The studies included showed that recipients preferred anonymous donation to keep the circumstances of their child’s conception a secret from those in their social environment :385. DonThe fifth main category comprising pragmatic reasons focused on motives and experiences pertaining to donor anonymity that were not directly grounded in expectations arising out of anonymity, that is, expected benefits from keeping certain stakeholders in ignorance. Rather, it focused on secondary aspects accompanying anonymous donations. This category comprised various factors. The studies included revealed that the lack of alternatives to anonymous donation left some recipients with no choice other than to opt for an anonymous donation. One reason for this could be legal or regulatory constraints such as “the sperm bank they went to did not offer non-anonymous donors or it was contrary to regulations in their country” :713, or Some recipients reported that they preferred an anonymous donation not based on an explicit consideration of benefits of anonymity per se, but rather because of medical, financial, and other aspects linked to an anonymous donation. Narratives mentioned in the literature showed that some recipients chose anonymous donations because of the accompanying advantages, such as a higher medical success rate of anonymous treatment :385. OthCorrelating the various motives and experiences in relation to donor anonymity with characteristics of the studies as possible factors impacting the relevance of particular motives and experiences for certain recipients in specific situations did not lead to conclusive results in form of distinct patterns being identified. The analysis rather showed that the majority of motives and experiences were relevant for example to both recipients of sperm and oocyte donation, and to both lesbian and heterosexual recipients. However, the following observations could be made.for an anonymous donor than to the choice against other donor types, in particular against a known donor, such as the motives and experiences of the main category “intention to keep the donor ignorant of the child’s identity” or the “intention to keep oneself ignorant of donor identity” in order to “avoid the emotional challenges of using a known donor.” This highlighted the importance of the alternatives to anonymous donation available to recipients in a specific context for their choice of donor type . Further, the fact that only heterosexual couples were identified as preferring to keep themselves ignorant of donor identity in order to “preclude the donor from interfering in establishing feelings of normality” and solely lesbian couples chose anonymous donors to “avoid conflicts within the extended family” arguably points to social expectations, family norms, and notions of infertility forming the background against which the recipients of these specific family types lay out their respective strategies of family making. Additionally, the fact that the “intention to keep the child ignorant of donor identity” in order to “protect the child from the harmful consequences of seeking to contact the donor” was almost exclusively identified by studies on sperm donation might suggest that recipients tend to have differing images of sperm and oocyte donors concerning their openness and willingness to attend to the needs of their genetic children in case these choose to contact them. Finally, the impact of social and legal circumstances on the recipients’ motives for anonymous donation is reported for example by a longitudinal study by Some motives and experiences identified in this review pertained less to the choice The results showed a broad of spectrum of motives and experiences on the part of recipients in relation to donor anonymity. They also pointed out some limitations of the existing research and helped identify topics and approaches for future research. These areas will be discussed in this section in detail. The implications and limitations of this review and its methodology will also be addressed critically.The integrated overview of the studies included in this review strongly suggests that recipients’ motives and preferences pertaining to donor anonymity are not necessarily fixed and unchangeable, but are subject to reconsideration and revision over time. Some studies framed the recipients’ pre-treatment considerations of donor types as an ongoing and multifaceted process, in which initially preferred and imagined routes to parenthood can be abandoned in favor of alternatives, such as by reconsidering and gradually replacing an original preference for a known donor with the eventual decision to use an anonymous one . Pre-treOnly some studies explicitly expounded on the change in the recipients’ attitudes over time. This review was able to identify a number of studies incorporating (or being part of) a longitudinal approach. However, most studies restricted their analysis to the preferences and experiences of recipients at a specific point in time. Understanding the recipients’ needs and preferences concerning the desire to know or not know—not only the donor’s identity, but also donor information in general —as constAs demonstrated by this review, the intention to protect the parental status of the non-biological parent is a recurring motive underlying the preference for anonymous donation. However, motives and experiences of non-biological parents in relation to sperm donor anonymity remain under-researched. Most studies on DI settings either did not explicitly collect data from non-biological parents e.g., , or did As mentioned above, only some of the studies focused specifically on the questions pertaining to preferences regarding the donor type as formulated in the review question , detailsThe approach chosen for this review led to the introduction of four main categories of motives and experiences pertaining to the intention to keep the recipients themselves, the child, the donor, or the family’s social environment in ignorance of the donor’s identity . A closer examination of the contents of each of these main categories shows that the intention to keep a certain stakeholder in ignorance is not limited to controlling just this specific stakeholder. Most forms of ignorance seem to have been employed with the aim  of simultaneously influencing multiple stakeholders and relationships among them. For example, categories summarized under the main category “intention to keep the child ignorant of donor identity” relate to issues of protecting the child and controlling the relationship between the donor and the child by creating a distance between them, and to matters of donor protection and benefiting the relationship between the recipients and the child.This finding points to alternatives to common framings of the discussion on donor anonymity in terms of competing rights (the child’s right to know their genetic origins vs. the parents’ right to privacy and autonomy) . From thFurther, more than the terms “anonymity” and “secrecy” convey, the term “ignorance” underscores the fact that a significant proportion of the recipients’ preference for donor anonymity is related to the intention to keep the recipients themselves unaware of the donor’s identity, as shown in the first main category. This also puts into perspective frameworks that aim to reduce donor anonymity to the recipients’ intention to keep the child ignorant and to place the child’s infringed right to know in opposition to the recipients’ interests.Although this review—consistent with its aim to carve out the entire unabridged spectrum of possible motives and experiences involved—was able to collate data from the current literature, the findings presented are limited by the fact that motives and experiences are strongly influenced by the particular circumstances of the participants involved in each individual study. The decision to include studies on both sperm and oocyte donation and on recipients of various sexual orientations and forms of partnership in this review contributed to a thorough understanding of the full spectrum of motives and experiences concerning donor anonymity in general. Although relevant features of the studies reviewed, such as the time of assessment, the types of donors and gametes, and the socio-legal background, were thoroughly reported in this review  and wereAlthough some of the studies reported on participants’ sociodemographic characteristics such as religion or ethnicity and inquired into the influence of these factors on recipients’ choices of anonymous donation e.g., , future Another direction for future research comprises investigations of contexts that allow recipients to exert their choices concerning donor anonymity in situations that are comparatively free of legal constraints. Such a free and unrestricted choice between anonymous and non-anonymous donors in double-track systems or in non-clinical settings such as Internet-facilitated sperm donations  seems prThis review was limited to a specific kind of information concerning the donor that the recipients preferred to keep themselves or others ignorant of, namely the donor’s identity. However, donor identity is not the only information that recipients have to deal with. Even where anonymous donations are made, recipients relying on sperm banks may have access not only to basic biometric information on the donor, but also to an extensive range of additional non-identifying information such as their “background, physical characteristics, education, profession, personality, health and family history, photos, handwritten greeting, voice sample, staff impression, and EQ tests” . In suchThis systematic review provided an overview of the spectrum of possible motives and experiences of recipients in relation to a preference for anonymous gamete donation by examining the findings of qualitative studies. To interpret these motives and experiences as a form of ignorance directed toward particular stakeholders involved allows the reframing of the discourse on donor anonymity; this is a fruitful approach that can be refined further and applied in future research. Potential directions for future research on motives for donor anonymity as identified by this review include the need for more thorough inquiries into the change in recipients’ preferences over time, such as by making comparisons of pre- and post-treatment attitudes toward donor anonymity in the form of longitudinal studies, and focusing on the perspective of non-biological parents.


Among recurrent implantation failure (RIF) patients, the rate of successful implantation remains relatively low due to the complex etiology of the condition, including maternal, embryo and immune factors. Effective treatments are urgently needed to improve the outcomes of embryo transfer for RIF patients. In recent years, many researchers have focused on immunotherapy using granulocyte colony-stimulating factor (G-CSF) to regulate the immune environment. However, the study of the G-CSF for RIF patients has reached conflicting conclusions. The aim of this systematic review and meta-analysis was performed to further explore the effects of G-CSF according to embryo transfer cycle (fresh or frozen) and administration route (subcutaneous injection or intrauterine infusion) among RIF patients.2 index were analyzed. Stata 15 was used for statistical analysis.The PubMed, Embase and Cochrane Central Register of Controlled Trials (CENTRAL) databases were searched for literature published from the initial to October 2020. The meta-analysis, random-effects model and heterogeneity of the studies with I2 = 0.0%, n = 410; frozen RR: 1.44, 95% CI: 1.14–1.81, I2 = 0.0.%, n = 366), and for both subcutaneous injection and intrauterine infusion , but the biochemical pregnancy rate of the RIF group was also higher than that of the control group . There were no significant differences in the miscarriage rate  and live birth rate  when a random-effects model was employed.A total of 684 studies were obtained through the databases mentioned above. Nine RCTs included 976 RIF patients were enrolled in this meta-analysis. Subgroup analysis indicated that G-CSF improved the clinical pregnancy rate for both the fresh and frozen embryo transfer cycles (fresh RR: 1.74, 95% CI: 1.27–2.37, IThe administration of G-CSF via either subcutaneous injection or intrauterine infusion and during both the fresh and frozen embryo transfer cycles for RIF patients can improve the clinical pregnancy rate. However, whether G-CSF is effective in improving livebirth rates of RIF patients is still uncertain, continued research on the utilization and effectiveness of G-CSF is recommended before G-CSF can be considered mainstream treatment for RIF patients. After more than 4 decades of development, assisted reproductive technology (ART) has become one of the main ways to treat infertility. However, recurrent implantation failure has consistently been a bottleneck preventing infertile patients from achieving a better pregnancy during in vitro fertilization and embryo transplantation (IVF-ET). RIF usually refers to a history of embryo transplantation failure following several ART-based procedures. A positive blood HCG test indicates the initial implantation of the embryo , and theGranulocyte colony stimulating factor (G-CSF) is a glycoprotein secreted by endothelial cells, macrophages and other immune cells that functions as a growth factor and cytokine. The functional sites of G-CSF are widespread in the human body; some studies have reported –7 that tThe first use of G-CSF in ART was reported by Gleicher et al. , who revThe PubMed, Embase and Cochrane Central Register of Controlled Trials (CENTRAL) databases were searched for literature published before October 2020. The protocol of this systematic review was registered with INPLASY (registration number INPLASY202170040). The obtained studies were screened in accordance with the PICOS rule . Participants: Patients with RIF; intervention group: G-CSF; control group: placebo or no treatment; main outcome: clinical pregnancy rate; study type: RCT. The search strategy for PubMed is as follows: ((((((recurrent implantation failure [Title/Abstract]) OR (repeated implantation failure [Title/Abstract])) OR (mif [Title/Abstract])) OR (rif [Title/Abstract])) OR (((repeated [Title/Abstract]) AND (implantation [Title/Abstract])) AND (failure [Title/Abstract]))) OR (((multiple [Title/Abstract]) AND (implantation [Title/Abstract])) AND (failure [Title/Abstract]))) AND ((((((((Granulocyte Colony-Stimulating Factor [Mesh]) OR (G-CSF [Title/Abstract])) OR (Granulocyte colony stimulating factor [Title/Abstract])) OR (Granulocyte-colony stimulating factor [Title/Abstract])) OR (colony-stimulating factor [Title/Abstract])) OR (CSF [Title/Abstract])) OR (Lenograstim [Title/Abstract])) OR (Filgrastim [Title/Abstract])).A study was included when it met all of the following criteria: 1. Patients under the age of 40 years who failed to achieve clinical pregnancy after the transfer of at least four good-quality embryos in a minimum of two IVF fresh or frozen cycles. 2. Subcutaneous injection or intrauterine infusion of G-CSF before a fresh or frozen embryo transfer cycle. 3. RCT with a control group that received placebo or no treatment. 4. Reporting of one of the following outcomes: clinical pregnancy rate, live birth rate, miscarriage rate, biochemical pregnancy rate. 5. Patients in RCT with uterine factors such as uterine anomalies, myoma, endometrial polyps were excluded. 6. Patients in RCT with systemic diseases and contraindications of G-CSF were excluded. Studies that did not meet all the criteria above were excluded.Data extraction was performed by two review authors (Hou and Jiang) independently, and disagreement was resolved by discussion or decided by a third review author (Meng). Randomized controlled trials were assessed for risk of bias using the Cochrane ‘Risk of bias’ tool according to the criteria outlined in the Cochrane Handbook for Systematic Reviews of Interventions (Version 5.1.0). The following domains were assessed by two researchers (Hou and Jiang) independently:1.Selection bias ;2. Performance bias (blinding of participants and personnel);3. Detection bias (blinding of outcome assessors);4. Attrition bias (incomplete outcome data);5. Reporting bias (selective reporting);6. Other bias .The evaluations were categorized as having ‘low risk’, ‘unclear risk’ or ‘high risk’ of bias; a detailed list is shown in Table 2 statistic was examined, and I2 > 50% was considered to represent significant heterogeneity between studies. Subgroup analysis was performed in terms of embryo transplantation cycle and administration route. We did not assess publication bias across studies since the number of included studies was under 10.Stata software  was employed to perform all data analysis. The influence of G-CSF treatment on the outcomes of IVF-ET for RIF patients was assessed with pooled risk ratios (RRs) and their 95% confidence intervals (CIs). The pooled RRs were calculated through a Mantel–Haenszel fixed-effects model if there was no heterogeneity; otherwise, a random-effects model was adopted. Statistical heterogeneity across studies was formally tested using Cochran’s Q test. The ITable .A total of 684 studies were obtained by primary search: 230 from PubMed, 393 from Embase, and 61 from CENTRAL. After a thorough screening, 10 RCTs , 19–24 p2 = 0.0%, n = 976). When subgrouped by embryo transfer cycle , the result indicated that G-CSF treatment improved the clinical pregnancy rate for RIF patients for both cycles ; see Fig. 2 = 0.0%, n = 497; intrauterine RR: 1.39, 95% CI: 1.09–1.78, I2 = 0.0%); see Fig. Nine studies reported the clinical pregnancy rate and included 976 RIF patients, 462 of whom were in the study group and 524 of whom were in the control group. A fixed-effects model was used for data synthesis, and the result indicated that G-CSF treatment improved the clinical pregnancy rate for RIF patients ; see Fig. Four studies reported the biochemical pregnancy rate and included 469 RIF patients, 228 of whom were in the study group and 241 of whom were in the control group. A fixed-effects model was used for data synthesis, and the results indicated that G-CSF treatment improved the biochemical pregnancy rate for RIF patients ; see Fig. 2 = 52.5%); see Fig. 2 of 52.5% was observed in this synthesis, indicating considerable heterogeneity, which might have been caused by the small number of included patients.Three studies reported the live birth rate and included 372 RIF patients, 166 of whom were in the study group and 206 of whom were in the control group. A random-effects model was used for data synthesis, and no significant difference was found between the study and control groups , which can lead to reproductive disorders such as endometriosis, recurrent abortion, and premature ovarian failure . It is kBiochemical pregnancy tends to occur when the endometrial environment cannot maintain the continuation of the established pregnancy. Many previous studies , 23 repoThe routine G-CSF administration routes in ART include subcutaneous injection and intrauterine infusion. However, whether one route is better than the other remains obscure. Zeyneloglu et al. reported a better embryo transfer outcome by combining subcutaneous and intrauterine G-CSF treatment for RIF patients than by subcutaneous injection alone . In thisAs mentioned above, G-CSF might be beneficial for the establishment and maintenance of pregnancy. The results of this review and meta-analysis show no significant effect of G-CSF treatment on the miscarriage or live birth rate. Due to the limited number of included patients, the exact effect of G-CSF on these rates. Miscarriage for RIF patients remains unclear, and more high-quality, larger and multicenter RCTs are needed to provide more authoritative evidence for clinical practice.In this review and meta-analysis, the results of data synthesis for 976 RIF patients indicate that G-CSF treatment improved the clinical pregnancy rate; however, the biochemical pregnancy rate increased as well, which is coincident with previous studies. In addition, subgroup analysis indicated that the administration of G-CSF by either subcutaneous injection or intrauterine infusion during both fresh and frozen embryo transfer cycles for RIF patients improved the clinical pregnancy rate. The influence of G-CSF on the miscarriage and live birth rates of RIF patients is unclear, and more studies are needed before G-CSF become mainstream treatment for RIF patients.


Albuminocytologic dissociation in cerebrospinal fluid (CSF) is a diagnostic hallmark of Guillain–Barré syndrome (GBS). Compared to CSF total protein (TP), the CSF/serum albumin quotient  has the advantage of method-independent reference ranges. Whether the diagnostic yield differs between Qalb and CSF-TP is currently unknown.We retrospectively analyzed the diagnostic yield  of Qalb and CSF-TP levels in patients with GBS. We evaluated two different equations (Reiber’s and Hegen’s) for age-adjusted URLs of Qalb and compared results to CSF-TP using the standard URL of 0.45 g/L as well as age-adjusted URLs (by decade of age). Additionally, multivariable logistic regression analysis was used to assess the effect of clinical factors on the diagnostic yield.We analyzed 110 patients  with sensorimotor (68), motor (16), sensory (12) and localized (8) GBS as well as Miller Fisher syndrome (6).r = 0.95, p < 0.001). The diagnostic yield of Qalb was 65% with Reiber’s and 47% with Hegen’s age-adjusted URLs compared to 66% with the fixed CSF-TP URL of 0.45 g/L and 49% with age-adjusted CSF-TP URLs. A longer duration from clinical onset to lumbar puncture was associated with a higher diagnostic yield.Qalb and CSF-TP were highly correlated (Qalb strongly correlates with CSF-TP in patients with GBS with a similar diagnostic yield for the detection of a blood–nerve barrier dysfunction. However, the diagnostic yield of both values is lower when using more recent age-adjusted URLs and at earlier timepoints. The Guillain–Barré syndrome (GBS) is an acute immune-mediated neuropathy with a range of clinical subtypes . DiagnosMoreover, protein levels in the CSF have been shown to be age-dependent  and sensIn this study, we retrospectively analyzed CSF results in a cohort of clinically well-characterized patients with different GBS subtypes.whether using Qalb increases diagnostic yield  in comparison to CSF-TPby comparing two different equations for age-adjusted upper reference limits (URLs) for Qalb andto evaluate clinical factors that may have an effect on the diagnostic yield of Qalb and CSF-TP.The aims of the study were to investigateWe retrospectively examined clinical data of adult patients with GBS who were treated at the Department of Neurology of the Medical University of Vienna between January 2000 and December 2019. The study was approved by the ethics committee of the Medical University of Vienna (EK Nr. 1927/2016). The requirement to obtain patient consent was waived for this retrospective study.We extracted clinical data from patient charts and grouped patients clinically into sensorimotor or pure motor classical GBS, pure sensory GBS and localized variants  as well as Miller Fisher syndrome . We retrospectively calculated the Medical Research Council (MRC) sum score at admission from patient charts. The score ranges from 0  to 60  points. Nerve conduction studies were analyzed according to Rajabally’s criteria . GanglioCSF/Albserum) with age-adjusted URL  as previously suggested by Reiber  were subsequently analyzed. Patients were clinically classified as sensorimotor (68), motor (16), sensory (12) and localized (8) GBS as well as Miller Fisher syndrome (6). The MRC sum score at baseline was 54 (IQR 46–60). Baseline characteristics are shown in Table The median time from clinical onset to LP was 5 days .All patients had cell counts below 50 cells/µL  as required by the Brighton criteria [r = 0.95, 95% CI 0.93–0.97; p < 0.001; Fig. r = 0.41, 95% CI 0.23–0.55; p < 0.001) and CSF-TP . Figure The median Qalb was 9.3  and median total protein levels were 0.58 g/L  with a high linear correlation between the two variables (p = 0.75) and CSF-TP values (p = 0.054). Table There was no statistically significant difference between GBS subgroups for Qalb  was 65% (71/110 patients) with Reiber’s equation for age-adjusted URLs of Qalb and 47% 52/110) with Hegen’s equation. For CSF-TP, the diagnostic yield was 66% (72/110) using the fixed URL of 0.45 g/L and 49% (51/110) using the age-adjusted URLs. 45% (49/110) of patients had CSF-TP and Qalb values above all 4 analyzed URLs and 32% (35/110) had consistently values within the normal range. Table /110 withtime from symptom onset to LP per day) for a value above the URL  were 1.22 with Reiber’s equation , 1.11 with Hegen’s equation , 1.63 for a CSF-TP URL of 0.45 g/L  and 1.09 for age-adjusted CSF-TP URLs . No significant adjusted effects were found for age at onset, sex, MRC sum score at admission and GBS subgroups. Of note, all six patients with MFS had Qalb and CSF-TP values within the normal range but all had their LP done within the first 5 days after clinical onset.The multivariate logistic regression model showed that only time from symptom onset to LP had a significant adjusted effect on the diagnostic yield. Specifically, the estimated odds ratios  of Qalb compared to CSF-TP in patients with GBS. The main finding was that Qalb highly correlated with CSF-TP and had a similar diagnostic yield. The latter was, however, lower for both values at earlier timepoints and when using more recent age-adjusted URLs.To our knowledge, no previous study investigated Qalb in patients with GBS in comparison to CSF-TP. With regard to the detection of a blood–nerve barrier dysfunction in patients with GBS, we found no clear superiority of Qalb compared to CSF-TP as demonstrated by the high number of patients with concordant findings regardless of the used URL (77%). Nevertheless, given the high correlation of Qalb and CSF-TP, it might be preferable to use Qalb in scientific settings—e.g., in multicenter studies—due to its laboratory-independent URLs that facilitate pooling of data, respectively, comparison between sites .The diagnostic yield of Qalb and TP was lower with more recently suggested age-adjusted URLs. For CSF-TP, this has already been recently shown in a large cohort of patients with classical sensorimotor GBS in a Canadian study, where age-adjusted reference limits led to a markedly lower proportion of patients with albuminocytologic dissociation compared to the conventional fixed cut-off of 0.45 g/L, especially in the first week after clinical onset . While aAs expected, we found that the time from clinical onset to LP correlated, albeit moderately, with CSF-TP and Qalb. A value above the URL in the first week was found in only a third to a half of patients depending on the measure and URL. Beyond 2 weeks, the vast majority of patients had elevated values, which is in line with other reports , 9, 18.Qalb and CSF-TP values did not differ significantly between GBS subgroups, but levels in pure motor GBS were numerically lower compared to sensorimotor GBS. Indeed, this was also reported in a recent study that showed higher CSF-TP levels in primary demyelinating compared to axonal subtypes or MFS . The intLimitations of this study are the retrospective nature and non-standardized CSF collection.Furthermore, no information was available on which proportion of the CSF was used for analysis which could have influenced CSF measurements since a previous study showed that protein concentrations decrease between the first (0–4th) milliliters and the last (21st–24th) milliliters . HoweverIn conclusion, we found that Qalb correlates well with CSF-TP in patients with GBS and has a similar diagnostic yield regarding the detection of a blood-nerve barrier dysfunction. However, the diagnostic yield for both values is lower at earlier timepoints and when using more recent age-adjusted URLs.


We compared the effectiveness and safety of pegylated granulocyte colony-stimulating factor (peg-G-CSF) vs. non-peg-G-CSF for hematopoietic stem cell mobilization in allogeneic hematopoietic stem cell transplantation in a real-world setting. We included 136 consecutive healthy donors treated with non–peg-G-CSF (n = 53) or peg-G-CSF (n = 83), and 125 consecutive recipients  in this study. All harvesting was completed successfully. No significant difference in leukapheresis number and adverse events frequency was observed, nor were there severe adverse events leading to discontinuation of mobilization. The leukapheresis products mobilized by peg-G-CSF had higher total nucleated cells (p < 0.001), monocytic myeloid-derived suppressor cells (p < 0.001), granulocytic myeloid-derived suppressor cells (p = 0.004) and B cells (p = 0.019). CD34+ cells and other lymphocyte subsets  were similar in both apheresis products. Patients who received grafts mobilized by peg-G-CSF exhibited a lower incidence of grade III-IV acute graft-versus-host disease (p = 0.001). The 1-year cumulative incidence of chronic graft-versus-host disease and relapse, 1-year probability of graft-versus-host disease-free relapse-free survival, and overall survival did not differ significantly between subgroups. Our results suggest that collecting allogeneic stem cells after the administration of peg-G-CSF is feasible and safe. Peg-G-CSF mobilized grafts may reduce severe acute graft-versus-host disease compared with non-peg-G-CSF mobilized grafts after allogeneic stem cell transplantation. The beneficial effects of a peg-G-CSF graft might be mediated by increased numbers of monocytic myeloid-derived suppressor cells. Mobilization with recombinant human granulocyte colony-stimulating factor (G-CSF) followed by leukapheresis has become the standard procedure for obtaining CD34+ peripheral blood stem cells (PBSCs) for hematopoietic stem cell transplantation (HSCT). Some studies optimizing the dosage and schedule of G-CSF have reported an association between G-CSF trough blood levels and mobilization efficacy –3. PegylMyeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population that includes immature myeloid cells and the progenitor cells of macrophages, dendritic cells (DCs), monocytes, and neutrophils. MDSCs include two major subpopulations: monocytic MDSCs (M-MDSCs) and granulocytic (polymorphonuclear) MDSCs (G-MDSCs). G-MSDCs share phenotypic and morphologic features with neutrophils, whereas M-MDSCs are similar to monocytes and are characterized by high plasticity. MDSCs are involved in tumor-associated immunosuppression and play immune-regulatory roles in pathologic conditions associated with chronic inflammation or stress . PreviouThe study was approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, in accordance with the Declaration of Helsinki. The medical data of 136 consecutive healthy donors and 125 consecutive patients with hematological diseases who received allogeneic HSCT at our transplantation center from November 2016 to June 2019 were collected. Among the 136 donors, 11 unrelated donors were treated with G-CSF and underwent standard apheresis harvesting at our center. However, the 11 patients received HSCT at other centers, so there were no relevant clinical outcome data. All donors had undergone pre-donation health examinations, including medical history, physical examination, abdominal ultrasound, echocardiography, electrocardiogram, full blood count, blood chemistry analysis, and search for infectious disease markers. Adverse events were classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0 .®, Shijiazhuang Pharmaceutical Group Co., Ltd., single subcutaneous injection: <60 kg, 9 mg; 60–100 kg, 12 mg; >100 kg, 15 mg) were used to mobilize hematopoietic stem cells on day 0. Donor bone marrow and/or peripheral blood cells were collected using standard mobilization protocols. G-CSF mobilized bone marrow (day 3 after G-CSF) and/or peripheral blood progenitor cells (day 4 after G-CSF) were harvested. Peg-G-CSF mobilized bone marrow (day 3 after peg-G-CSF) and/or peripheral blood progenitor cells (day 4 after peg-G-CSF) were harvested. Successful stem cell collection was defined as total nucleated cells > 6 × 108/kg recipient weight and CD34+ cells > 2 × 106/kg recipient weight. If the donor failed to produce a sufficient yield of CD34+ progenitor cells after the first leukapheresis, they were treated with an additional 2×5 μg/kg non-peg-G-CSF.G-CSF  or peg-G-CSF -total body irradiation (TBI)-based or busulfan (Bu)-based myeloablative regimens. Patients with nonmalignant diseases, such as SAA received fludarabine (Flu)/Cy/antithymocyte globulin (ATG) regimens. The day of the stem cell infusions was defined as day 0 when it came to transplantation outcomes. GVHD prophylaxis mainly combined cyclosporine A (CsA), methotrexate (MTX), and mycophenolate mofetil (MMF). Diagnosis and grading of aGVHD were based on the Mount Sinai Acute GvHD International Consortium (MAGIC) criteria . ChronicMDSCs were analyzed using monoclonal antibodies against the following proteins: CD45-PerCP, CD14-APC, CD33-PE, and HLA-DR-FITC . For each blood sample, background staining and non-leukocytes were corrected by isotype controls and CD45 staining. The percentage of MDSCs in leucocytes was calculated as MDSCs/CD45+ cells. The absolute count of MDSCs in the grafts was calculated as the infused leucocytes per kilogram multiplied by the percentage of MDSCs. The gating strategy for MDSC analysis is shown in 9/L for three consecutive days and platelet counts ≥ 20 × 109/L without transfusion for 7 days. Chimerism analyses were routinely evaluated by PCR of short tandem repeat sequences and/or fluorescence in situ hybridization (FISH) analysis for the X and Y chromosomes (XY-FISH). Donor chimerism status was defined based on previous reports (3 copies/mL) in blood. Epstein-Barr virus (EBV) viremia was defined as positive results of reverse transcriptase PCR (>1 × 103 copies/mL or continuous increase) in the blood. CMV disease diagnosis requires clinical symptoms plus CMV documented in tissue by histopathology, virus isolation, rapid culture, immunohistochemistry, or DNA hybridization techniques. Invasive fungal disease (IFD) was defined according to the revised EORTC/MSG criteria , GraphPad Prism Version 7  and R software 4.0.2. The survival rates were analyzed using the Kaplan-Meier method. Survival differences between groups were estimated by the log-rank test. Cumulative incidences were estimated for GVHD and relapse in which death from any cause was a competing risk for GVHD and relapse. Gray’s test was used in the cumulative incidence analyses. To confirm the outcomes and adjust for potential confounding factors, multivariate Cox models were assessed for the proportional hazards assumption, and interaction terms with covariates were tested. Variables with a p-value that was less than 0.15 in the univariate analyses were included in the Cox model, and the number of variables did not exceed 20% of the size of the valid endpoint. The final multivariate models were constructed using a forward stepwise selection approach. The characteristics among the groups were compared using the Chi-square test for categorical variables and the Mann–Whitney U test for continuous variables. The final model of significance attained ≤ 0.050.Donor characteristics are listed in Increased white blood cell count and monocyte count in the peripheral blood were observed after mobilization with peg-G-CSF, peaking 3 days after administration, while G-CSF mobilization peaked 4 days after administration , 29.2% vs. 7.3% (p = 0.001), and 19.5% vs. 32.9% (p = 0.444), respectively Figure 2In the standard G-CSF group, 31 patients were alive at the median follow-up of 17.8 months , and the actuarial 1-year OS was 80.5% ± 6.2%. In the peg-G-CSF group, 67 patients were alive at the median follow-up of 13.6 months , and the 1-year OS was 83.8% ± 4.1%. There was no significant difference in the 1-year OS, 1-year GRFS, 1-year probability of relapse, and severe infection between the two groups. The outcomes of the two groups are shown in detail in p = 0.047). There were no significant variables on multivariable analysis for the risk of relapse . Due to the superior mobilization with peg-G-CSF compared with standard G-CSF, the predicted rate of failure to mobilize might be similar to or lower than that seen with standard G-CSF. The stem cell products mobilized by peg-G-CSF might have stronger immunomodulatory effects than that mobilized by G-CSF, as the frequency of MDSCs especially M-MDSCs in the pegylated group was much higher than that in the non-pegylated group. Our previous data and many studies have confirmed that MDSCs especially M-MDSCs can prevent GVHD without disabling GVL effect , 27–29. Bone pain and headache are the main adverse effects during the administration of mobilization agent. The long half-life of peg-G-CSF could induce the concern of prolonged or excessive leukocytosis, splenic enlargement, and even the potential risk of splenic rupture . AlthougIn our cohort, we observed similar outcomes for OS and GRFS. Furthermore, there were similar relapse rates. Our results for the survival outcomes are, in part, in line with a phase I/II clinical trial, showing satisfactory survival rates in the peg-G-CSF group. Although no comparison for the clinical outcomes between groups was found in that cohort , the resWe acknowledge important limitations of our study, which are mainly related to the relatively small sample size and single-center, retrospective nature, including the short median follow-up of 13.6 months for the peg-G-CSF group and the lack of the aforementioned data (donor blood biochemistry test etc.). The function test of MSDC in different harvests was not performed. In our future research, we will supplement relevant experiments to further elucidate the underlying mechanism. Experiences with peg-G-CSF in healthy donors are still very limited. A prospective randomized clinical trial (ChiCTR2000032370) is ongoing at our center to yield better insight into peg-G-CSF mobilization and to provide more valid data. Although our data support the premise that the higher MDSCs in grafts mobilized by peg-G-CSF are associated with lower risk of severe aGVHD, the complete mechanism remains to be explored by more functional assays in the future.In conclusion, although the mobilization effectiveness of peg-G-CSF appears comparable to that of G-CSF, less discomfort after administration and the lower incidence of severe aGVHD represent arguments for the use of peg-G-CSF. Peg-G-CSF mobilized grafts might ameliorate severe aGVHD by enriching M-MDSCs in the graft. These findings require validation in large prospective randomized trials and real world data.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LLi, JY, and YL. The first draft of the manuscript was written by LLi and all authors commented on previous versions of the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by grants from the National Natural Science Foundation of China .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.


Trapa taiwanesis Nakai, TT-hull) was extracted by either steeping them in cold 95% ethanol (C95E), refluxing 95E, refluxing 50E, or refluxing hot water (HW) to obtain C95EE, 95EE, 50EE, and HWE, respectively. These four extracts showed acetylcholinesterase (AChE) inhibitory activities and free radical scavenging activities, as well as anti-non-enzymatic protein glycation in vitro. Eight compounds were isolated from TT-hull-50EE and were used to plot the chromatographic fingerprints of the TT-hull extracts, among which tellimagrandin-I, tellimagrandin-II, and 1,2,3,6-tetra-galloylglucose showed the strongest AChE inhibitory activities, and they also exhibited anti-amyloid β peptide aggregations. The scopolamine-induced amnesiac ICR mice that were fed with TT-hull-50EE or TT-hull-HWE (100 and 200 mg/kg) or tellimagrandin-II (100 and 200 mg/kg) showed improved learning behavior when evaluated using passive avoidance or water maze evaluation, and they showed significant differences (p < 0.05) compared to those in the control group. The enriched hydrolysable tannins of the recycled TT-hull may be developed as functional foods for the treatment of degenerative disorders.Agricultural waste from the hulls of water caltrop ( The global prevalence of dementia is increasing continuously , and it ε-(carboxymethyl)lysine (CML) , galactose, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), horse radish peroxidase-conjugated goat anti-rabbit IgG (A6154), phosphate-buffered saline (PBS), scopolamine hydrobromide, and thioflavin T (ThT) were purchased from Sigma Chemical Co. . The OxiSelect™ Assay Kit (STA-345) was from Cell Biolabs Inc. . The glutathione (GSH) was measured using glutathione assay kits . The BCA The dried TT-hulls were provided by a farmer from Guantian District, Tainan city, Taiwan. The dried, powdered TT-hulls were extracted by either steeping them in cold 95% ethanol (C95E), refluxing 95E, refluxing 50E, or refluxing hot water (HW) to obtain C95EE, 95EE, 50EE, and HWE, respectively. The 3-L C95E was added to one kg powdered TT-hulls and placed at room temperature for one week. After being filtered, the residue was extracted following the same procedure twice. The filtrates were collected and concentrated as C95EE. Each of 3.5-L of 95E, 3.5-L of 50E, or 5-L of HW was added to 700 g powdered TT-hulls and extracted under refluxing for 3 h. Each filtrate was collected, concentrated, and lyophilized as 95EE, 50EE, and HWE, respectively. The recovery of C95EE, 95EE, 50EE, and HWE, respectively, was approximately 8.6%, 6.5%, 10.2%, and 12.8%.2O and MeOH sequentially, which were collected, concentrated, and lyophilized for further purification. The MeOH eluates (26 g) were chromatographed on a Toyopearl HW-40 (C) column (2.5 cm i.d. × 55 cm) and eluted batchwise with one liter of H2O, 50% MeOH, 60% MeOH, 70% MeOH, 80% MeOH, and 70% acetone to obtain T-H, T-50M, T-60M, T-70M, T-80M, and T-70A fractions, respectively. The T-50M fraction was purified using a LiChroprep RP-18 column (2.5 cm i.d. × 50 cm) and eluted with 0.05% trifluoroacetic acid (TFA):acetonitrile (CH3CN) (92:8) to obtain 1,6-di-O-galloyl-β-D-glucopyranoside  and gallic acid . The T-60M fraction was purified with a LiChroprep RP-18 column (2.5 cm i.d. × 50 cm) and eluted with 0.05% TFA:CH3CN (90:10) to obtain 1,2,3-tri-O-galloyl-β-D-glucopyranoside  and 1,2,6-tri-O-galloyl-β-D-glucopyranoside . The T-70M fraction was purified with a LiChroprep RP-18 column (2.5 cm i.d. × 50 cm) and eluted with 0.05% TFA:CH3CN (82:18) to obtain 1,2,3,6-tetra-O-galloyl-β-D-glucopyranoside . The T-80M fraction was purified with a LiChroprep RP-18 column (2.5 cm i.d. × 50 cm) and eluted with 0.05% TFA:CH3CN (83:17) to obtain 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranoside  and tellimagrandin I . The T-70A fraction was purified with a LiChroprep RP-18 column (2.5 cm i.d. × 50 cm) and eluted with 0.05% TFA:CH3CN (85:15) to obtain tellimagrandin II . The purity of each compound was determined by HPLC and was shown to exceed 95%. All structures were determined by 1H-NMR and 13C-NMR, including 2D-NMR techniques, and by comparison of those data with authentic compounds as follows.The 50EE (77 g) was dissolved in water and chromatographed over a Diaion HP-20 column (10.5 cm i.d. × 35 cm) and then eluted with 5-L of H(a) Tellimagrandin II (TT-1):1H-NMR , δ, 7.12 , 7.01 , 6.97 , 6.66 , 6.48 , 6.20 , 5.84 , 5.60 , 5.36 , 5.22 , 4.55 , 3.89 . 13C-NMR , δ, 167.3, 166.9, 165.5, 164.9, 164.2 (C=O), 145.3, 145.1, 144.9, 144.4, 144.3, 143.6, 138.9, 138.5, 138.3, 135.7, 135.5, 125.6, 125.0, 119.6, 119.5, 118.9, 114.9, 114.8, 109.4, 0.9.3, 109.2, 107.3, 107.0, 92.8 (Glc C-1), 72.4 (Glc C-3), 72.2 (Glc C-5), 71.0 (Glc C-2), 69.9 (Glc C-4), 62.2 (Glc C-6).(b) 1,2,3,6-tetra-O-galloyl-β-D-glucopyranoside (TT-2):1H-NMR , δ, 7.16 , 7.080 , 7.076 , 7.00 , 6.15 , 5.67 , 5.47 , 4.63 , 4.52 , 4.15 , 4.08 . 13C-NMR , δ, 166.9, 166.5, 166.2, 165.3(C=O), 146.2, 146.1, 146.0 , 139.8, 139.3, 139.0 , 121.6, 121.3, 120.6, 120.0 , 110.3, 110.19, 110.16, 110.0 , 93.6 (Glc C-1), 76.2 (Glc C-5), 76.0 (Glc C-3), 72.0 (Glc C-2), 69.4 (Glc C-4), 63.9(Glc C-6).(c) 1,2,3-tri-O-galloyl-β-D-glucopyranoside (TT-3):1H-NMR , δ, 7.08 , 7.07 , 6.99 , 6.07 , 5.60 , 5.39 , 3.98 , 3.92 , 3.82 , 3.78 . 13C-NMR , δ, 166.6, 166.2, 165.3 (C=O), 146.2, 146.0 , 139.7, 139.2, 139.0 , 121.4, 120.7, 120.3 , 110.3, 110.2, 110.1 , 93.5 (Glc C-1), 78.7, 76.3, 72.0, 69.3, 61.9 (Glc C-6).(d) 1,2,6-tri-O-galloyl-β-D-glucopyranoside (TT-4):1H-NMR , δ, 7.14 , 7.09 , 7.06 , 5.95 , 5.24 , 4.59 , 4.44 , 3.97 , 3.92 , 3.73 . 13C-NMR , δ, 166.9, 166.4, 165.4 (C=O), 146.13, 146.10, 146.0 , 139.6, 139.0, 138.9 , 121.7, 121.4, 120.3 , 110.3, 110.2, 110.0 , 93.7 (Glc C-1), 76.2 (Glc C-5), 75.5 (Glc C-3), 74.0 (Glc C-2), 71.2 (Glc C-4), 64.1 (Glc C-6).(e) 1,6-di-O-galloyl-β-D-glucopyranoside (TT-5):1H-NMR , δ, 7.15 , 7.11 , 5.70 , 4.55 , 4.37 , 3.77 , 3.62-3.53 . 13C-NMR , δ, 166.9, 165.8 (C=O), 146.11, 146.07 , 139.4, 138.9 , 121.7, 121.0 , 110.4, 110.0 , 95.8 (Glc C-1), 77.8, 76.1, 73.9, 70.9, 64.4 (Glc C-6).(f) gallic acid (TT-6):1H-NMR , δ, 7.13 . 13C-NMR , δ, 168.0(C=O), 146.0, 138.6, 122.1, 110.1 .(g) 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranoside (TT-7):1H-NMR , δ, 7.17, 7.11, 7.05, 7.01, 6.97 , 6.33 , 6.01 , 5.65 , 5.62 , 4.56 , 4.53 , 4.40 . 13C-NMR , δ, 166.5, 166.0, 165.8, 165.7, 165.1 (C=O), 146.3, 146.1, 146.1, 146.1, 146.0 , 139.9, 139.4, 139.4, 139.2, 139.1 , 121.5, 120.8, 120.7, 120.0 , 110.5, 110.4, 110.3, 110.2 , 93.4 (Glc C-1), 74.0 (Glc C-3), 73.4 (Glc C-5), 71.8 (Glc C-2), 69.4 (Glc C-4), 62.9 (Glc C-6).(h) Tellimagrandin I (TT-8):1H-NMR , α-anomer, δ, 7.01 , 6.93 , 6.61, 6.50 , 5.83 , 5.49 , 5.31 , 5.11 , 5.09 , 4.64 , 3.83 . β-anomer, δ, 7.00 , 6.89 . 6.61 , 6.46 , 5.59 , 5.34 , 5.20 , 5.14 , 4.96 , 4.20 , 3.91 . 13C-NMR  δ: 169.9, 169.8, 169.5, 169.4, 168.1, 167.9, 167.6, 167.3, 146.6, 146.5, 146.4, 146.3, 146.1, 146.0, 145.0, 140.3, 140.2, 140.1, 137.8, 126.50, 126.47, 126.1, 126.0, 121.1, 120.9, 120.8, 116.9, 116.8, 116.61, 116.56, 110.56, 110.51, 110.5 , 108.80(HHDP), 108.76(HHDP), 108.4 (HHDP), 97.3 (β-Glc C-1), 91.9 (α-Glc C-1), 75.0 (β-Glc C-2), 74.5 (β-Glc C-3), 73.8 (α-Glc C-4), 72.9 (β-Glc C-5), 72.1 , 71.8 (α-Glc C-3), 67.8 (α-Glc C-5), 64.43 (β-Glc C-6), 64.36 (α-Glc C-6).Ratio of α-anomer/β-anomer = 6: 4. 3CN . The mobile phase was pumped at the flow rate of 1.0 mL/min, and 10 μL was injected for analysis. The column temperature was kept at 40 °C, and the wavelength was set at 280 nm for monitoring.For fingerprinting analysis, each extract was prepared at 10 mg/mL. Analysis was performed using the LiChrospher 100 RP-18e column  using a Waters Chromatography system. A gradient elution program of mobile phase was set as follows: 0.05% TFA:CHblank−A405sample)/(A405blank)] × 100%. The concentration for 50% inhibition (IC50) of AChE activity of each TT-hull extract or purified compound was calculated from each linear equation by three test concentrations and their corresponding AChE inhibitions. For C95EE, 95EE, and 50EE, 125, 250, and 500 μg/mL were used; for HWE, 200, 250, and 500 μg/mL were used; for TT–1 and TT–8, 12.5, 25, and 50 μM were used; for TT–2, 50, 100, and 200 μM were used; for TT–3 and TT–6, 100, 200, and 400 μM were used.The acetylthiocholine iodide and DTNB were used for in vitro AChE inhibitory activity assays following the previous report . The totThe antioxidant index of free radical scavenging activities of the extracts (1.5 or 2.5 μg/mL) and purified compounds (10 μM) of TT-hull were determined by a commercial OxiSelect™ Assay Kit for oxygen radical absorbance capacity (ORAC) determination . The staThe non-enzymatic glycation model of BSA/galactose was used following the previous experiments ,32. The 440nm/Em486nm were recorded at a time of 0 h and 24 h, and changes in fluorescence over a 24 h period were expressed as ΔE. The inhibition of Aβ aggregation (%) was calculated as [(ΔEcontrol−ΔEsample)/(ΔEcontrol)] × 100%.The ThT fluorescent assay was used to monitor the isolated compounds from the TT-hull on inhibition of Aβ (1-42) peptide aggregations . For the® RMH2500, Jefferson City, MO, USA) and water were provided during the animal experiments. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Taipei Medical University (LAC-2016-0275). After one week of acclimation, for treatments of TT-hull extracts to test prevention, the mice were randomly divided into seven groups (6 heads/group), including the blank, the control, the donepezil-positive control, and the four extract-administered groups. The ICR mice were pre-administered orally with 50EE or HWE at concentrations of 100 and 200 mg/kg once a day by gavage from day 1 to day 10; for the blank, the control, and the donepezil (5 mg/kg)-positive control, the mice received an equal volume of distilled water for parallel experiments. From day 11 to day 20, the same procedure was performed in the 50EE- and HWE-administered groups once a day by gavage; in the blank and the control groups, the same equal volume of distilled water was orally administered once a day by gavage; in the donepezil-positive control, the mice were administered orally once a day with donepezil (5 mg/kg). Except for the blank group, the mice in each group were intraperitoneally injected with scopolamine (1 mg/kg) 30 min after the oral sample administration, and the mice in the blank group were injected with an equal volume of PBS. After being injected with scopolamine or PBS for another 30 min, each mouse was evaluated by a passive avoidance test at day 13 to day 14 and by the Morris water maze from day 17 to day 19. At the end of the experiments at day 20, the mice were sacrificed. For oral administration of purified compounds to test prevention, the ICR mice were pre-administered orally with TT-1  or TT-2 (300 mg/kg) once a day by gavage from day 1 to day 5; for the blank, the control, and the donepezil (5 mg/kg)-positive control, the mice received an equal volume of distilled water for the parallel experiments. From day 6 to day 11, the procedure was the same for the oral administration of the TT-hull extracts. Each mouse was evaluated by a passive avoidance test at day 6 to day 7 and by the Morris water maze from day 8 to day 10. At the end of the experiments at day 11, the mice were sacrificed, and the brain tissue was isolated and stored at −80 °C for further measurements.The oral administration of TT-hull extracts or purified compounds in scopolamine-induced amnesiac ICR mice was performed according to the previous report , with soThe passive avoidance test and Morris water maze were used to evaluate the learning and memory functions of scopolamine-treated mice after the treatments with TT-hull extracts or purified compounds, by following the previous report . The appsample−A405sample blank]/μg protein. The glutathione assay kit  was used to determine the reduced form GSH levels based on the reaction of GSH with DTNB reagents to generate a yellowish 5-thio-2-nitrobenzoic acid and expressed as A412 nm/μg protein.At the end of the TT-1 and TT-2 treatments, mice were sacrificed, and the whole brain of each ICR mouse was isolated and immediately stored at −80 °C for the measurement of residual AChE activity and the reduced GSH levels. The whole brain of each mouse was ground into powder by means of a mortar and pestle in liquid nitrogen and then extracted by adding 1 mL of 100 mM phosphate buffer (pH 7.5) to the ice bath. The residual AChE activity determination followed the previous report , except post hoc Tukey’s test. The different lowercase letters in the anti-Aβ peptide aggregates of purified compounds under the fixed ratio  treatment, the different uppercase letters in step-through latency of the acquisition trial, or the different lowercase letters in the retention trial were considered significantly different among groups (p < 0.05). The two-group comparison was analyzed using Student’s t-test, and any difference in comparison with the control group was considered statistically significant when p < 0.05 (*), or p < 0.01 (**), or p < 0.001 (***). Statistical analysis was performed using the GraphPad Prism 5.0 software .The quantitative data were presented as mean ± SD of three independent experiments. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) and the 50 of AChE activity of C95EE, 95EE, 50EE, and HWE, respectively, were calculated to be 453.7, 280.5, 344.9, and 326.2 μg/mL in the present assays. The order of AChE inhibition was 95EE > HWE > 50EE > C95EE. The ORAC values of the four TT-hull extracts of C95EE (1.5 μg/mL), 95EE (1.5 μg/mL), 50EE (1.5 μg/mL), and HWE (2.5 μg/mL) in vitro, respectively, were 3.3, 2.8, 3.0, and 3.3 μM Trolox equivalents. Therefore, the order of ORAC antioxidant activities of TT-hull extracts was C95EE > 50EE > 95EE > HWE. 50 of AChE inhibitory activity of TT-1, TT-2, TT-3, TT-6, and TT-8, respectively, was calculated to be 18.6, 84.2, 371.5, 246.2, and 18.4 μM in the present assays. The TT4 at concentration of 800 μM did not reach 50% AChE inhibition; therefore, the IC50 of AChE inhibition of TT-4 should be higher than 800 μM. The order of AChE inhibition was TT-8 ≅ TT-1 > TT-2 > TT-6 > TT-3 >> TT-4. The ORAC values of TT-1, TT-2, TT-3, TT-4, TT-6, and TT-8 in vitro at a concentration of 10 μM, respectively, were 5.1, 18.7, 15.1, 18.1, 23.7, and 7.5 μM Trolox equivalents. The ORAC activities of the ellagitannin class of TT-1 and TT-8, respectively, were 50% and 75% of those of Trolox; the ORAC activities of the gallotannin class of TT-2, TT-3, and TT-4 were close to two-fold higher than those of Trolox. The gallic acid (TT-6) showed 2.4-fold higher ORAC activities than those of Trolox. p < 0.05) compared to the other purified compounds.Owing to the small amounts of TT-5 and TT-7, only six compounds were used to determine the biological activities. p > 0.05) among the groups (the same uppercase letters in the black column). However, the scopolamine-induced amnesiac mice treated with TT-hull-50EE, TT-hull-HWE, and donepezil, and the mice in the blank group, remained for a longer period of time in the light box (the step-through latency) in the retention trial, and all had significant differences (p < 0.05) compared to those in the control (lowercase letters in the white column), while the learning and memory functions of mice in the groups treated with TT-hull extracts were improved and comparable to those in the blank group. The different doses of TT-hull-50EE and TT-hull-HWE treatments showed no significant difference (p > 0.05) in the step-through latency in the retention trial. p > 0.05) in the latency (sec) of the reference memory task of the Morris water maze (data no shown). However, the scopolamine-induced amnesiac mice treated with TT-hull-50EE, TT-hull-HWE, and donepezil, and the mice in the blank group, spent more time in the target quadrant  compared to those in the control group.The order of the AChE inhibition of TT-hull extracts was 95EE > HWE > 50EE > C95EE A. The requadrant C and hadquadrant D, and alp > 0.05) among groups (the same uppercase letters in the black column). However, the scopolamine-induced amnesiac mice treated with TT-1 (100 and 200 mg/kg) and donepezil, and the mice in the blank group, but not TT-2 (300 mg/kg), showed longer step-through latency (sec) in the light box of the retention trial and showed significant differences (p < 0.05) compared to those in the control (lowercase letters in the white column) group, in which the learning and memory functions of mice in the groups treated with TT-1 were improved and comparable to those in the blank group. The different doses of TT-1 treatment and the donepezil-positive control (5 mg/kg) showed no significant difference (p > 0.05) in the step-through latency in the retention trial. Although the TT-2 intervention showed a longer step-through latency (sec) in the retention trial, there were no significant differences (p > 0.05) compared to those in the control group. p > 0.05) in the latency (sec) of the reference memory task of the Morris water maze (data no shown). However, the scopolamine-induced amnesiac mice treated with a high dose of TT-1 (200 mg/kg) and donepezil, and mice in the blank group, showed a longer time spent in the target quadrant (p < 0.05) compared to those in the control group.quadrant C, higherquadrant D, and sip < 0.05, ** p < 0.01, *** p < 0.001) compared to those in the control group.This study is the first in which the oral administration of TT-hull extracts and the isolated compound of TT-1 showed a significant improvement in the learning and memory functions in amnesiac mice models , compareWang et al.  reportedThe scopolamine-induced amnesia was used in AD animal models and human clinical trials according to the cholinergic hypothesis, and the scopolamine occupied the muscarinic receptors to block acetylcholine and then temporarily ceased the neurotransmission. The scopolamine-induced amnesia could also enhance the AChE activity and oxidative stress in the brain extracts . The 8-mIn conclusion, the agricultural waste of TT-hulls was recycled to investigate AChE inhibition and then validate the proof-of-concept using scopolamine-induced amnesiac mice, while the learning and memory functions were evaluated by the passive avoidance test and the Morris water maze. The protective functions of TT-hull extracts (such as 50% ethanol extracts and hot water extracts) and the marker constituent TT-1 were shown to improve the learning and memory functions. The Japanese water caltrop hull extracts were shown to lower the blood glucose in fasting mice by an oral carbohydrate tolerance test . Therefo