How do parents understand and feel about identity-release egg donation?Almost one-third of mothers and fathers did not understand the identifiable nature of their egg donation; mothers expressed complex and sometimes difficult feelings about the prospect of future donor–child contact.Identity-release egg donation has been the only treatment option available to patients wishing to pursue this route to parenthood in the UK since 2005. However, little is known about how well parents understand this legislation, and how they feel about potential donor–child contact.This qualitative interview study included 61 mothers and 51 fathers whose 5-year-old children were conceived via identity-release egg donation. Interviews were conducted between April 2018 and December 2019. 44) were then asked about their thoughts and feelings regarding the prospect of future donor–child contact. Mothers’ narratives were analysed using thematic analysis.Data are reported from phase two of a longitudinal study of families created using open-identity egg donation. In-depth, semi-structured interviews were conducted with mothers and fathers. The interviews contained a section on what parents understood about the identifiable nature of the donor. These data were analysed using qualitative content analysis. Mothers who understood the identifiable nature of their egg donation (n = 16) did not understand the identifiable nature of their egg donation. Mothers’ and fathers’ misunderstandings about identity-release egg donation fell into two categories: (i) Unclear about identity-release and (ii) Belief that the donor is anonymous. Reflexive thematic analysis revealed that egg donation mothers’ feelings about identity-release donation could be understood via three organizing themes: (i) identity-release as a threat, (ii) acceptance: it is what it is and (iii) embracing identity-release. The findings indicated that egg donation mothers utilized various strategies to manage their feelings about identity-release egg donation in day-to-day life, and each theme was associated with at least one coping strategy.Almost one-third of parents (28% of mothers, n = 17; 31% of fathers, n =Participants were predominantly from White, middle-class backgrounds. Further research with a more diverse sample is needed to improve generalizability.These findings indicate that parents would benefit from more comprehensive provision of information, both at time of treatment and following conception, to ensure they have fully understood the nature of the donation. Parents may also benefit from follow-up care to help manage any complex or difficult feelings about donor–child contact.This research was supported by a Wellcome Trust Collaborative Award [208013/Z/17/Z]. The authors have no conflicts of interest to declare.N/A. Egg donation is an increasingly common form of fertility treatment offered to women who are unable to conceive using their own eggs. Recent years have seen an increase in the number of families created using egg donation, with the number of IVF cycles using donor eggs in the UK rising from 2263 in 2014 to 4212 in 2018, representing 6.1% of all IVF cycles in 2018 . LegislaConcerns have been raised about the impact on family functioning of the prospect of future donor–child contact, as the donor may be perceived as an ongoing, salient presence within family life  mothers demonstrated at least a basic understanding of identity-release egg donation; however, 17 (28%) mothers did not understand that their egg donor would be identifiable to their child in the future. Thirty-five fathers (69%) understood the principles of identity-release donation, whilst 16 (31%) did not. Categories and illustrative quotations are presented in  7) and fathers  did not know whether their child could access further information about the donor in the future. Some parents phrased this in terms of not being able to remember or needing to do some ‘research’ by looking at their medical paperwork from the treatment stage. Others said they did not know because they had not investigated the question, with one mother stating that she had not found out what information about the donor her child was entitled to as she did not intend to tell her child about her method of conception.Of the 33 parents who did not understand identity-release donation, most mothers  demonstrated vague but incomplete knowledge about identity-release donation. For example, two parents knew that age 18 is a threshold for children to seek out information, but they were unsure of precisely what information they would be entitled to access.A small number of parents  of mothers and 13% (n = 2) of fathers believed that their egg donor was fully anonymous and that their child would be unable to find out any further information about the donor.This was the second most common type of misunderstanding about identity-release egg donation. Thirty-five per cent  acceptance: it is what it is and (iii) embracing identity-release. The findings indicated that egg donation mothers utilized various strategies to manage their feelings about identity-release egg donation in day-to-day life, and each theme was associated with at least one coping strategy. Organizing themes and subthemes are depicted in a thematic map in This analysis included the mothers who understood what was meant by identity-release donation only. Three organizing themes were produced from the analysis: (i) threat to the mother–child relationship, a threat to the child and a threat to the donor. Mothers adopted at least one of three strategies to cope with this threat: emphasizing parenthood, distancing and reluctant engagement. Mothers adopted one or all of these strategies, although those who principally adopted a ‘reluctant engagement’ strategy were less likely to adopt a ‘distancing’ strategy, and vice versa.Many mothers viewed the prospect of knowing the donor’s identity, and potential donor–child contact, as threatening to some degree. This perceived threat was represented in three ways: as a not done my job properly’, whilst another expressed a deep fear that the donor–child relationship would be more ‘real’ than the one she shared with her child:‘I’d be worried that they’d suddenly have this unbelievable connection that perhaps is truer of a biological mother, and perhaps what we’ve got isn’t a true mother-son relationship? I don’t know, it worries me’. (Coleen)A prominent perspective was that the child’s ability to access the donor’s identifying information constituted a direct threat to the mother–child relationship. Identity-release was represented as having the power to weaken, or interfere with, mothers’ sense of ownership of their child, and as posing a challenge to their identity as a mother. One mother explained that her child’s interest in the donor would be an indicator that she’d ‘probably a fear of mine really, is that he might really like her and her family and, like, in future Christmases decide to spend time with her rather than us’. (Wendy)Many mothers expressed the hope that their child would not seek contact with the donor in the future and stated that any interest their child was to show in the donor would be emotionally painful to them. Some mothers also expressed a fear of rejection, not just at the personal but also at the family-wide, level: ‘‘That’s probably why I nurture and care for her so much more, because I want her to be, to know that I, you know, absolutely adore the ground she walks on. I don’t want her to then say ‘well you were rubbish, I’m going to go and find who’s biologically mine’ or whatever. I want to overcompensate I suppose because I don’t want her to go and find [the donor]’.Conversely, other mothers were concerned about the donor’s interest in the child. For example, one mother said that despite her curiosity about the donor, she would not want her child to initiate the identity-release process in case it piqued the donor’s interest in her child. Mothers were concerned that the identity-release process itself would have a negative, potentially long-term, impact on their relationship with their child. For example, Valerie felt that the prospect of identity-release influenced her parenting due to the fear that her child would one day use it as a weapon against her:limit or influence’ their own perception of their child, with concerns that this may then impact their relationship with their child.Some mothers also worried that access to information about the donor’s identity or physical appearance may ‘… take advantage of him in some way or hurt him’. A related concern for several mothers was that they would have a personal reaction to the donor and that these reactions might disrupt their child’s experience of contact with the donor. This ranged from one mother creating an ‘ideal vision’ of the donor to mothers worrying that they may dislike the donor upon making contact. Others focussed on the possibility that their child may be disappointed or face rejection from their donor after attempting to make contact:‘I wouldn’t like to rule [donor contact] out completely, but then obviously you couldn’t guarantee that’s what she was going to get, and she could be rebuffed and get really upset’. (Agatha)Identity-release was perceived by some as posing a threat to their child. Mothers expressed concerns about how the donor may behave, or what the donor might want from their child after making contact. Mothers worried that the donor might ‘upsetting’ for their child, and they expressed regret that their child may have to go through a difficult process. For some, there was a tension between advised early disclosure of identity-release donor conception to their child, and their child’s inability to access the donor’s identifying information until the age of 18. These mothers implied that this waiting period may cause their child emotional or psychological distress. These concerns were often compounded by the fact that because the identity-release system is so new in the UK, very little is known about the rates and quality of donor–child contact. Mothers, therefore, expressed a fear of the unknown, observing that ‘even the experts don’t know how it would go’.Mothers also anticipated the identity-release process taking a toll on their children as young adults. Many thought that the process may be difficult or ‘suddenly appear’ in the donor’s life ‘out of nowhere’ was seen as a threat hanging over the donor’s future. Similarly, some mothers viewed not initiating the identity-release process as a way of expressing gratitude to their donor; the value of the donor’s gift was so great that despite their curiosity, mothers wanted to ‘do the right thing … for her’ and respect the donor’s boundaries and not ‘rock the boat’.Though less prominent, this subtheme relates to some mothers’ concerns that the identity-release process might negatively impact the donor’s life or discourage potential donors altogether. For several mothers, the potential for donor offspring to ‘‘I know that there will come a time where [child] legally will gain the right to gain access to the information. Part of me worries about that. And I think that I try to say to myself that this is stupid because I’m the only mum she knows. I’ve done everything for her, I carried her, I’ve given birth to her, I’ve done everything for her. I’m the only mum she knows’.Three strategy subthemes were identified as ways in which mothers managed their feelings of threat in day-to-day life. The first strategy was to emphasize the role of parenthood, thereby minimizing the impact that the donor’s identity could have on the parent–child relationship. This was expressed in several different ways; most commonly, the biological relationship shared by mother and child (through gestation) was considered to counteract, or neutralize, the threat of any potential bond between the child and the donor. Many mothers directly contrasted identity-release egg donation with adopted children seeking out their birth families, believing that because the donor did not gestate or give birth to them, their child would feel no imperative to seek a connection with them in the future. Mothers also referred to the experience of parenting as a protective factor; some expressed fears of their child developing a strong bond with the donor, but then reassured themselves by referring to their experiences of carrying and parenting their child. Noreen observed that:The belief that the experience of parenting trumps the genetic connection shared by donor and child was further underlined by the view taken by a small number of mothers who suggested that the donor’s experiences of parenting her own children would negate any desire to build a relationship with her donor offspring. Some mothers also believed that the quality of the mother–child relationship was a key factor in determining whether their child would wish to pursue the donor’s identity in the future. Whilst this strategy was comforting for most mothers, a few remained concerned that changes in mother–child relationship quality as children entered adolescence would result in their child’s increased interest in seeking the donor’s identity.dangerous’, viewing identifying information about the donor as a potential threat to her psychological well-being. Other mothers expressed the view that it was ‘important that you don’t know too much’, to avoid knowledge of the donor intruding upon their perception of, and feeling of connectedness to, their child. Mothers who used this strategy also tended to create for themselves a picture of an uninterested, indifferent donor, to reduce the level of perceived threat inherent in identity-release:‘I don’t think the person would want to meet up to be perfectly honest. I can’t imagine that the egg donor would actually want to meet her’. (Lilian)A second strategy employed by mothers to manage their feelings of threat was to distance themselves from identity-release. Some mothers distanced themselves from the donor as a person, suggesting that ignorance of the donor’s identity was protective in some way. For example, one mother described her curiosity about the donor as ‘‘It’s up to him really whether he wants to find out really, hopefully I’ll be dead and buried by then [laughs], by the time he’s interested’. (Georgina)Amongst mothers who accepted the possibility of contact between their child and the donor, there was a tendency to frame contact with the donor in purely functional terms and in doing so, reject the prospect of having a relationship with the donor. Some mothers also distanced themselves from the identity-release process itself, particularly by situating the identity-release process in the future. Several mothers said that the identity-release process was still too distant a prospect to concern themselves with, embracing an ‘out of sight, out of mind’ attitude. Others distanced themselves from the idea of their child wanting to initiate donor–child contact. This ranged from expressing a hope that the child would not wish to initiate contact, to the overt desire to be absent from the process:I wouldn’t particularly relish it, but I would also completely understand it’, to expecting to feel ‘utterly broken-hearted’ by their child’s interest in the donor. Some mothers expressed a resignation to disclosing the details of identity-release to their children, despite a strong preference not to. These attitudes were based on both mothers’ sense of obligation to their child, and to beliefs that, as information about identity-release is ‘so accessible’ in a digital information age, the ‘sensible thing’ would be to inform their child of identity-release, despite their own, sometimes quite marked, concerns:‘At some point, we’ll have to have that conversation with her and with [sibling]. But again, I don’t really want to have that. I know she’s entitled to it, and it’s splashed all over her notes so she’s going to find out, but if there was any way of her not finding out I would do that. I would do anything for her not to find that out.’ (Martha)Some mothers who perceived identity-release as a threat approached it with a certain level of resignation; typically, mothers asserted the right of their child to request the identity of their donor, despite their own negative feelings about it and subsequently reluctantly engaged with the prospect of identity-release. Mothers’ feelings ranged in strength, from feeling that ‘‘I think if [child] was in touch with [the donor] then I would want to at least have some sort of distal contact about that like I wouldn’t-, I would feel uncomfortable if [child] got in touch with her and then just didn’t tell me anything and that that was a secret.’ (Delphine)Several mothers suggested that, should their child ever contact the donor, they would wish to be involved in the process despite their negative feelings about identity-release. Some mothers explained this as a need to be protective of the child, whereas others characterized their involvement as protective of the mother–child relationship:that’s fine’ and ‘I don’t mind’. Some mothers referred to their knowledge of identity-release, and how this helped them accept it. Similarly, some recounted scenarios wherein they had intentionally sought out more information about identity-release from healthcare professionals, which in turn led to a greater acceptance of identity-release. Several mothers, like Marie, expressed the feeling that they had come to terms with identity-release over time:‘Well I don’t feel like threatened by it, I would be quite happy if she wanted to so … if it comes to it and she wants to find out more then yeah. I don’t feel, I wouldn’t say ‘oh no, no, you can’t do that.’ If she wants to then she will … I’m probably a bit more chilled out about it now than I was’.The second theme describes a small subgroup of mothers’ narratives that displayed few feelings of perceived threat but stopped short of embracing the prospect of future donor–child contact. Mothers demonstrated an acceptance of identity-release, which ranged from expressions of indifference, through to becoming accepting of the realities of identity-release over time. Responding to questions about how they felt about future donor–child contact, these mothers responded with neutral responses such as ‘it’s not my decision really is it, it’s [child]’s decision … it’ll be fine, it’s up to him really’ (Georgina). Often, part of this strategy was to emphasize the importance of providing support to their child, whatever they chose to do. For example, Bonnie explained that:‘I’m just going to take it from [child]’s lead and respect whatever he wants to do, because it’s, you know … it’s his call, it’s his call and I just … all I need to do is support him to do whatever he wants to do’.A strategy related to mothers’ acceptance of identity-release was to take a child-led approach to donor–child contact. When asked about their child initiating the identity-release process, mothers tended to respond that it was up to their child. In doing so, mothers deprioritized their own feelings about the prospect of donor–child contact, and instead assumed a neutral stance whereby they asserted the rights of their child. For example, when asked how they felt about possible donor–child contact, several mothers answered with responses such as, ‘knowledge of genetic origins as essential to the child, expressing a personal desire for information about the donor, and viewing identity-release as a gateway to a broader family network. This embracing attitude was associated with one strategy subtheme whereby mothers framed themselves as potential facilitators of donor–child contact.Another prominent perspective was that identity-release constitutes an opportunity to be embraced, with mothers expressing positive feelings about potential donor–child contact. This was captured by three subthemes: representing it would be wrong to keep it from’ their children and that their children had a right to know ‘where they came from’. Some mothers viewed the donor’s identity as information that fundamentally belonged to their child:‘Although they’ll have me as their mum, genetically there’ll always be, you know, some things, questions they might ask or just information that they want about themselves, and I think it’s important that, you know, they have access to that information’. (Gabby)The first subtheme describes a tendency to represent identity-release as a benefit by conferring significance onto their child’s genetic origins. Many mothers emphasized their children’s right to access their donor’s identifying information, stating that ‘feel that there’s something missing’ As such, identity-release represented an opportunity for their child to seek information ‘for their own identity’. Some mothers expressed regret that the donor’s information was not available to their child at a younger age, stressing the importance of their child knowing ‘who they’re part of’ as ‘a matter of course’. Similarly, Henrietta reflected on the importance of her own experiences with her family for her own identity development, drawing comparisons between her own and her children’s situation:‘This is something they need to know, it’s for their future when they grow. I come from quite a mixed family … and part of, for me, growing up was getting to know my half family. So I kind of think of it in those terms, that actually it probably is going to be, for them, making sense of themselves and where they came from’.Others stressed that knowledge of their genetic origins was essential for proper identity development and suggested that, without it, their child would ‘intrigued’ about the donor’s identity and several mothers expressed a desire to meet the donor. For example, Jemima explained that she wanted ‘probably more than just to know who she is, but to actually meet her’; these mothers expressed clear enthusiasm towards the possibility of meeting the donor, describing it variously as a ‘nice’ and ‘exciting’ prospect. The desire to meet the donor was sometimes intense; one mother recounted her experience of trying to resist the temptation of attempting to trace the donor using online databases and social media platforms, before ultimately accepting that it was up to her children to decide. This was echoed by other mothers who, whilst acknowledging that contacting the donor was their child’s decision, admitted that they’d be ‘disappointed if [child] doesn’t [contact the donor], because I can’t make that decision, but I would like to meet the person’. (Hannah).The majority of mothers who viewed identity-release as an opportunity expressed a desire to have a level of contact with the donor. Mothers reported being ‘magnitude’ of the donor’s contribution. For example, Susanne commented that ‘I probably would [want to meet the donor] because it would be, you know, I’d want them to see [child] really to see what, you know, what they’ve given us’.Some mothers also gave distinct reasons why they were interested in contact with the donor. In some cases, they wished to get to know the donor to discover if there were similarities between their child and the donor or donor siblings. Other mothers expressed a wish to meet the donor so that they could ask about their medical history. Most mothers who expressed a wish to contact the donor were motivated by a desire to express gratitude to the donor. This ranged from somewhat cursory references to thanking the donor, to a heartfelt desire to acknowledge the ‘slightly fascinating’ and ‘exciting’ prospect. Other mothers focussed on the importance of tracing same-donor offspring for their child’s benefit, discussing the opportunity to do so as ‘a very important thing’ for their child.An important feature of identity-release donation for many mothers who expressed an ‘embracing’ attitude was the potential for accessing a wider family network. For some mothers, the value of identity-release was not only in enabling their child to contact the donor but also to trace same-donor offspring. Some mothers spoke of their own interest in finding out about same-donor offspring, representing it variously as a ‘tragic’ if she and her husband were to die without their child having made contact with the donor, as she anticipated that ‘[child] would need contact then … and there might be somebody there who’s got a little space for her’. Another mother went as far as to say that the donor could represent ‘some kind of parent figure’ to her child in the case of her death.A minority of mothers viewed the donor and donor siblings as a potential support network in the event of their own death, often after reflecting on their own older age as parents. One mother observed that it would be ‘‘I always think about, you know, a lot of women went to [country] and were getting egg donations in [country] and were going there specifically because [country’s] law keeps the details of donors anonymous and then I’m just thinking you couldn’t do that to a child, you know, that’s part of them, but they’re never ever to know or never ever to find out must be really difficult you know? So I think, you know, I think it’s important that they do know that it’s you know, just looking after them as well’. (Gabby)Embracing the prospect of identity-release was associated with one distinct strategy for managing these positive feelings in day-to-day life. Mothers viewed themselves as uniquely placed to facilitate donor–child contact and saw it as a responsibility of egg donation parenthood to help their child have as positive an experience as possible. For some mothers, the very choice to undergo treatment in the UK was a conscious and active one that was based on the perceived benefits of the identity-release system. These mothers often compared UK treatment favourably to the policies governing egg donation in other countries where donors are anonymous. Giving their child the opportunity to trace the donor was seen as taking proper care of their child:‘Just whatever details he wants, I’ll provide them … once he’s eighteen, if he wants to contact the donor, then I’ll be able to facilitate that. I’ll guide him in the right direction’. (Roberta)Mothers also expressed their desire to facilitate the identity-release process by pursuing additional information about the donor. One mother, when explaining why she chased her clinic for a letter the donor had written, observed that she was motivated by the desire to have information in case her child asked for it in the future. This desire to provide their child with plentiful information was shared by several mothers, and doing so was often considered to be an important part of their role as parents:‘You hope that you could say, “I’ll give you the skills now to go your own way and make your own decisions”. So I think when the time comes then, you know, my job and [partner]’s job is to lay the groundwork so that when the opportunity arises for some contact … that that can be a wholesome, balanced thing, you know? So [she] can explore her relationships in an unconfusing way’. (Tabitha)Additionally, some mothers expressed their intention not only to inform their child of their right to access identifying information about the donor, but to encourage them to do so, due to the belief that it would benefit their child. Moreover, some mothers viewed it as their responsibility to equip their child with the personal and emotional skills necessary to cope with the identity-release process in the future:Unexpectedly, around one-third of both mothers and fathers did not understand identity-release donation. Parents were either uncertain about what, if anything, their child could learn about the donor in the future, or they thought that they had used a fully anonymous egg donor to conceive. It is plausible that these parents’ focus had been on falling pregnant after, often, many years of trying, and they did not have the psychological resources available to research the specifics of egg donation legislation. In a Dutch study, many parents of donor-conceived children reported that they had not considered issues of disclosure or future donor–child contact during the treatment process . HoweverThis study is the first to explore UK egg donation mothers’ perspectives on identity-release donation when children are in early childhood, and the findings offer insights into the complex nature of navigating non-genetic parenthood, as well as the specific challenges of parenting after using identity-release egg donation. The findings suggest that mothers use multiple strategies to make sense of, and manage their feelings about, their use of identity-release egg donation in day-to-day life. These findings are in line with the limited evidence from studies of families formed through identity-release sperm donation, with some parents variously demonstrating comfort with, concerns about, and ambivalent feelings towards future donor–child contact (That a substantial number of mothers perceived identity-release donation as threatening is important and suggests that for some mothers, identity-release contributes to a perception of the donor as an ongoing and salient presence that may put pressure on relationships within the family unit. This finding is consistent with studies of mothers’ motivations for choosing anonymous egg donors, where mothers expressed a preference for anonymous donation to protect the mother–child relationship . The minEgg donation mothers’ narratives demonstrated considerable ambivalence about the prospect of future donor–child contact, with mothers expressing complex and sometimes contradictory feelings. Many mothers expressed concern that the future relationship between their child and the donor may be stronger or more meaningful than their own relationship with their child. In the first phase of the present study, some mothers expressed concern that the donor would want to claim the child in the future . These pMothers’ emphasis of the importance of the parenting experience has been acknowledged elsewhere in the identity-release donation literature. Mothers who viewed identity-release donation as an opportunity to be embraced often positioned themselves as facilitators of donor–child contact. A similar role has been reported amongst mothers of children born through identity-release sperm donation; when discussing their approach to the disclosure process, mothers represented themselves as ‘process managers’, demonstrating the belief that a responsibility of gamete donation parenthood is to initiate the disclosure process and keep it moving forward . This suMothers who felt positively about their child discovering the donor’s identity considered the donor’s identity as essential information for their child to develop a full and positive self-concept. This is in line with theories of identity development that position knowledge of one’s biological origins as an essential factor that contributes to identity development . Donor-cA further noteworthy finding is the representation of identity-release by some mothers as a gateway to a broader family network. Most commonly, mothers represented identity-release donation as a link to a network of same-donor offspring. This was often, but not always, discussed in the context of their child being an only child, and accompanied by the hope that a network of same-donor offspring might provide support and comfort to their child in the future. Research from the broader parenting literature also attests to concerns amongst many parents about raising only-children, with siblings often positioned as providing children with the social skills and support necessary for optimal adjustment (cis-gender, heterosexual, White, highly educated couples; the findings will therefore have limited generalizability to populations of different gender identities, sexual orientations, ethnicities or socioeconomic statuses. For example, LGBTQIA+ identifying parents have been found to form, make meaning of and enact relationships with gamete providers in qualitatively different ways to cis-gender heterosexual identifying parents (cis-gender, heterosexual couples who can currently access ARTs in the UK (The present sample consisted primarily of  parents , IVF andCurrent HFEA guidelines state that clinics should inform parents of the fact that their child will be able receive identifying information about the donor, and they recommend that this be shared with the child at an early age. However, the findings of this study suggest that there is still a considerable lack of awareness regarding accessing information about the donor, and about the possibility of donor–child contact in the future. A significant proportion of mothers viewed the donor as threatening 5 years after the birth of their child. This suggests that, for some mothers, the challenges of parenting a child conceived using an identifiable egg donor may not be limited to the pre- and post-natal periods, and that feelings of threat, opportunity and ambivalence may develop and subside over the family life course. Rather than simply encouraging parents to access additional information about the donor, clinics may better serve their patients by also acknowledging and normalizing the possibility that parents may feel ambivalence towards the availability of donor information and the prospect of donor–child contact. Patients seeking treatment with donor gametes in the UK may benefit from the provision of counselling following the birth of their child.


Delivery of effective anti-leukemic agents to the central nervous system (CNS) is considered essential for cure of childhood acute lymphoblastic leukemia. Current CNS-directed therapy comprises systemic therapy with good CNS-penetration accompanied by repeated intrathecal treatments up to 26 times over 2–3 years. This approach prevents most CNS relapses, but is associated with significant short and long term neurotoxicity. Despite this burdensome therapy, there have been no new drugs licensed for CNS-leukemia since the 1960s, when very limited anti-leukemic agents were available and there was no mechanistic understanding of leukemia survival in the CNS. Another major barrier to improved treatment is that we cannot accurately identify children at risk of CNS relapse, or monitor response to treatment, due to a lack of sensitive biomarkers. A paradigm shift in treating the CNS is needed. The challenges are clear – we cannot measure CNS leukemic load, trials have been unable to establish the most effective CNS treatment regimens, and non-toxic approaches for relapsed, refractory, or intolerant patients are lacking. In this review we discuss these challenges and highlight research advances aiming to provide solutions. Unlocking the potential of risk-adapted non-toxic CNS-directed therapy requires; (1) discovery of robust diagnostic, prognostic and response biomarkers for CNS-leukemia, (2) identification of novel therapeutic targets combined with associated investment in drug development and early-phase trials and (3) engineering of immunotherapies to overcome the unique challenges of the CNS microenvironment. Fortunately, research into CNS-ALL is now making progress in addressing these unmet needs: biomarkers, such as CSF-flow cytometry, are now being tested in prospective trials, novel drugs are being tested in Phase I/II trials, and immunotherapies are increasingly available to patients with CNS relapses. The future is hopeful for improved management of the CNS over the next decade. Childhood acute lymphoblastic leukemia (ALL) is a curable disease with more than 90% of children achieving long term survival , 2. ThisCNS staging is usually performed by counting white blood cells in the cerebrospinal fluid (CSF) along with microscopy of a cyto-centrifuged CSF sample to morphologically identify leukemic blasts. This is used to assign patients to CNS1, CNS2 or CNS3 status  rates and less CNS relapse , 19. HowIn keeping with the large variability in CNS2/3 rates across different protocols, and conflicting data on CNS2 status as an independent prognostic factor  MRD quantification. Sensitive response biomarkers capable of accurately quantifying CSF MRD and thus the dynamics of CSF clearance are clearly needed.All modern ALL protocols employ a combination of systemic therapy and intrathecal (IT) therapy against CNS leukemia, with some trial groups also using radiotherapy for selected high-risk groups. This upfront CNS therapy is sometimes called “CNS prophylaxis”, which reflects the aim to prevent CNS relapse. However, it can be wrongly misconstrued as meaning it is preventing dissemination of leukemia to the CNS, although CNS leukemia is likely to be present at the time of diagnosis. Thus, “CNS-directed therapy” is a more appropriate term than “CNS prophylaxis”. The current CNS-directed therapy approaches taken by selected study groups are summarized in Table The main agents with significant CNS activity are intrathecal and systemic glucocorticoids (not least dexamethasone), intrathecal or intravenous (high-dose) methotrexate and cytarabine, and asparaginase. When choosing therapy, it is also important to consider potential neurotoxicity. Interested readers are referred to comprehensive reviews on this topic , 26. BriWhilst all modern protocols achieve low rates of CNS relapse, the optimal treatment regimen to maximize CNS control whilst minimizing toxicity is currently unknown. One consistent observation is that trials that intensify CNS-directed therapy often have reduced CNS relapse rates but an excess of later BM relapses. This results in no differences in EFS or OS, or sometimes worse OS . This has led to the concept of BM and CNS relapses being “competing events”. Poor risk ALL may relapse early (often “on treatment”) in the CNS, due to less chemotherapy exposure and immune surveillance in this compartment. If CNS-therapy is intensified this risk is reduced, but the same poor risk features make the patient at higher risk of BM relapse once intensive systemic treatment has finished.The most intensively studied areas are around (i) choice of glucocorticoid, (ii) systemic methotrexate regimen, (iii) the use of single versus triple intrathecal therapy, and (iii) the safety of omitting radiotherapy.Dexamethasone is considered to be the most effective oral glucocorticoid to treat CNS ALL. This is based on several randomized clinical trials comparing prednisolone and dexamethasone, including the collaborative study group l’Associazione Italiana di Ematologia e Oncologia Pediatrica – Berlin Frankfurt Münster’s (AIEOP-BFM) ALL2000  and the 2) or Capizzi regimen  intravenous methotrexate during interim maintenance. Am improvement in 5-year EFS was reported with the high-dose regimen. This was associated with a reduced, but non-significant, CNS relapse rate without excess toxicity. Converstly when studied in T-ALL, in COG AALL0434, the Capizzi regimen was superior to high-dose methotrexate by 5-year EFS and OS [Intravenous methotrexate regimen, and glucocorticoid choice, were the subjects of the Children’s Oncology Group’s (COG) AALL0232 trial . 3154 hiS and OS .Given the poor CSF penetration of many systemic anticancer agents Table , direct COG AALL1131 hypothesized that prophylactic triple intrathecal therapy (TIT)  would improve EFS, due to reduced CNS relapses, compared with single intrathecal therapy (methotrexate) (IT MTX) post-induction . The triIn the last decade many ALL trials have explored reducing or omitting the use of cranial irradiation and, therefore, the associated toxicities without sacrificing event-free survival. The consistent finding of these trials is that in the frontline setting cranial irradiation can be safely excluded in conjunction with an appropriately augmented systemic regimen. A meta-analysis, published in 2016, included over 16,000 patients, aged 1–18 years, enrolled across 10 study groups, and concluded that cranial irradiation does not impact CNS relapse risk in modern protocols . This men = 6) as was the total number of any CNS relapse events (n = 10). However, the investigators found a significantly reduced risk of iCNS relapse in the dasatinib arm, but no difference in any CNS relapse. This difference could potentially reflect the higher dasatinib-imatinib dose relation used compared to dosing currently used in other protocols  or other extramedullary sites, or combined relapse involving two or more of these sites, most being BM + CNS. Even in the case of iCNS relapses submicroscopic marrow involvement is often seen , and it The optimal therapy of iCNS relapse remains controversial. Early iCNS relapses have poor outcomes with EFS/OS rates of 41%/52% on COG AALL0433 and very similar outcomes from other groups . RandomiHistorically, late iCNS relapses have had an excellent outcome on protocols using high dose chemotherapy and cranial radiotherapy of 24 Gy. In the UKALL R3 trial this group had a 5 yr EFS of 81% and OS of 85% . UnfortuNovel immunotherapies including bi-specific T cell engagers and antibody-drug conjugates have revolutionized the management of relapsed and refractory ALL in the last decade and are starting to be used for high-risk patients in front line settings. Evaluation of the CNS activity of these agents has been difficult as patients with CNS relapses were often excluded from early trials due to concerns of enhanced risk of neurotoxicity and the difficulties in measuring disease response in the CNS. Recently prospective Phase 3 studies from Europe  and the Despite use of all the agents described above there are a small but significant population of patients who present with recurrent or refractory CNS relapses. Clinical trials are hampered by the absence of sensitive biomarkers to monitor disease response similar to BM MRD. To this adds lack of access to precision medicine umbrella trials due to the challenges in obtaining material for genomic profiling and uncertainty regarding drug penetration into the CNS. As a result, iCNS relapses are excluded from current Phase I/II trials for relapsed ALL, and no randomized trials have addressed the optimal approach for this group. There are however isolated case reports and small case-series indicating efficacy of some novel treatment approaches including intrathecal rituximab, thiotepa and liposomal cytarabine (Depocyte) –64. In aAdvances in treatment of CNS leukemia are hampered by two main issues. Firstly, the lack of sensitive and specific diagnostic, response, and prognostic biomarkers able to risk-stratify CNS-directed therapy and to measure the efficacy of novel treatment approaches. Secondly, the lack of non-toxic novel agents with activity against CNS leukemia. Recent progress in these two vital areas is discussed below.The ability to accurately diagnose and quantify CNS ALL and its response to treatment will unlock the door to risk-adapted therapy as well as facilitate Phase I/II trials for novel CNS-active agents. Options include more sensitive and reliable detection of leukemic cells in CSF, or measuring soluble biomarkers secreted from leukemic cells in situ. Figure 9/L) leading to a much higher sensitivity than conventional cytology [Recently, multicolor flow cytometric analysis of CSF has been applied as a more sensitive method for detection of leukemic blasts in the CSF compared to cytospin. In flow cytometry, blasts are identified based on their aberrant expression of leukemia-associated immunophenotype markers, making this technique highly specific . Furthercytology . Furthercytology .n = 673) by the NOPHO group that CSF flow positivity at diagnosis was an independent risk factor for relapse among children and adolescents with ALL [At diagnosis, CNS involvement has been detected by CSF flow cytometry in 17–41% of cases compared to only 3–10% of cases classified as CNS2 and CNS3 by cytospin in children with ALL , 68–70. with ALL . This asTLP with blasts (TLP+) has previously been associated with increased risk of CNS relapse in childhood ALL , 71–73. Another technique that has been proposed for detection of submicroscopic levels of CNS involvement is PCR on cell-free DNA in CSF. PCR on CSF DNA is typically performed with the patient-specific primers against variable regions in immunoglobulins and the T-cell receptor generated for bone marrow MRD. In pediatric ALL patients, PCR detected CNS dissemination in 20–47% of cases compared to 5–17% by cytospin –77. The Given that leukemic cells in the CNS are often adherent to stroma rather than free-floating in CSF it is hypothesized that measurement of a CSF biomarker that is released or taken-up by leukemic cells may provide a more relevant and quantifiable measure of disease burden than cell-based methods. Biomarkers may reflect a variety of biological mechanisms, such as transmigration and adhesion to CNS-stroma, metabolic plasticity, and cellular cross-talk. It may, however, prove difficult to reduce this complexity into single biomarkers, and combinatorial risk-scores incorporating multiple biomarkers and/or genetic/demographic features may be needed.Currently, evidence from mass spectrometry-based proteomics of CSF is sparse, with only a few small cohort studies published. However, characterization of alterations in the CSF proteome and/or metabolome may convey measures of CNS malignancy. Here, aberrant catalase levels have been reported in the CSF proteome of B- and T-ALL patients with cytospin CNS2-status . CatalasRecently, blast-derived extracellular vesicles have been hypothesized to foster malignant transformation of leukemic cells and facilitate transmigration across the blood-CSF-barrier by priming of choroid plexus cells , 83. TecMetabolic plasticity may be pivotal for adapting to the low nutrient microenvironment in CSF, and metabolites and metabolic regulators are potential biomarkers for CNS-ALL. Distinct biological mechanisms have been described, including hypoxic adaptation by upregulation of vascular endothelial growth factor A (VEGFA)  and metaCirculating microRNA (miRNA) in CSF have been linked to CNS involvement. Many miRNAs have been reported, yet not validated, but may include high expression of the miRNA-181-family, miR-34a, miR-128a, miR-128b, and miR-146a, in CSF positive relative to CSF negative patients , 89.Finally, cfDNA is released from cells into the surrounding body fluids by a variety of mechanisms including active release and secondary to apoptosis and cell turnover. Measurement of cfDNA in plasma (and/or CSF for brain tumors ) detectsReal advances in treating CNS leukaemia will require new therapeutics. Replacement of conventional agents such as methotrexate with less neurotoxic alternatives will benefit all patients. Novel agents are also needed to unlock the potential of biomarker driven approaches to identify high-risk patients and for those with refractory disease. An overview of agents in preclinical development and clinical testing are shown in Fig. Chimeric antigen receptor T (CAR-T) cells targeting CD19 on B-ALL have demonstrated convincing evidence of activity in the CNS. In early-stage clinical trials, investigators have reported the presence of CAR-T cells within the CSF of treated ALL patients . SubsequOne strategy is to target the leukemic cells that have already entered the CNS by disrupting the molecular mechanisms that support their survival in the CNS microenvironment. In the leptomeninges the leukemic cells are in direct contact with the CSF that has low oxygen and nutrient levels compared to the blood . Recent Two interesting therapeutic strategies that have not yet progressed to clinical testing, include disruption of metabolic adaption to the CNS microenvironment and contact-induced quiescence. Accumulating evidence supports that the low oxygen and nutrient availability in the CSF induce metabolic adaptations in the leukemic cells that promote their survival in the CNS microenvironment . SeveralLeukemic cells predominantly reside within the meninges that cover the surface of the brain and spinal cord and only invade the brain parenchyma in late-stage disease . Due to Normal lymphocytes express numerous chemokine receptors, including CXCR3, CXCR4 and CCR7, which promote migration into the CNS through expression of the corresponding ligands in the brain endothelium and choroid plexus epithelium . In T-ALSince a large proportion of children with ALL already have CNS involvement at diagnosis, drugs blocking the entry of leukemic cells into the CNS may have limited effect in the clinical setting. Currently, it is not known if continuous trafficking of leukemic cells from the blood or bone marrow into the CNS occurs after initial CNS seeding. The therapeutic benefit of targeting CNS invasion in childhood ALL thus needs to be confirmed in clinical studies and carefully designed xenograft studies where treatment is initiated only after CNS involvement has been established.Finally, one other barrier to effective new therapies is whether the drug can enter the CSF compartment. Intrathecal delivery circumvents the need to cross the blood brain/blood-CSF barrier, but it is onerous for families and repeated general anesthesia in children may provide an added neurocognitive burden . Oral smOptimizing treatment of the CNS remains a challenge in childhood ALL. Current approaches are intensive, non-discriminative, and cause significant morbidity, whilst treatment options for patients with relapsed/refractory CNS-ALL are limited. Solutions lie in new drugs and better biomarkers. The lack of accurate biomarkers is most critical. Without a means to measure treatment response the testing of novel agents becomes difficult. Moreover, biomarkers will help identify patients at low or high risk for CNS relapse. For low-risk children who currently receive large amounts of CNS-directed therapy, reducing this treatment burden would have significant health and economic benefits. For high-risk children novel approaches are needed, facilitated by new drugs. Fortunately, research into CNS-ALL has increased over the last decade and is starting to provide a better understanding of disease biology as well as putative drug targets and biomarkers. Some biomarkers, such as CSF-flow cytometry, are now being tested in prospective trials. Novel drugs are also being tested in Phase I/II trials, although wider access for iCNS relapse patients is needed. The future is hopeful for improved management of the CNS over the next decade.


Aquaporin-4 (AQP4) is a channel protein that plays a fundamental role in glymphatic system, a newly described pathway for fluid exchange in the central nervous system, as well as a central figure in a fascinating new theory for the pathophysiology of neurodegenerative diseases such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD). In this study, cerebrospinal fluid (CSF) concentration of AQP4, amyloid-β, total tau and P-tau were determined in 103 CSF samples from patients affected by neurodegenerative dementias (AD and FTD) or psychiatric diseases and 21 controls. Significantly higher levels of AQP4 were found in AD and FTD patients compared to subjects not affected by neurodegenerative diseases, and a significant, positive correlation between AQP4 and total tau levels was found. This evidence may pave the way for future studies focused on the role of this channel protein in the clinical assessment of the glymphatic function and degree of neurodegeneration. Interstitial fluid and solute clearance are crucial for homeostasis. Although in peripheral tissues this function is carried out by the well-characterised lymphatic system, the central nervous system (CNS)—which is one of the organs with the highest metabolic rate —was thouSince AQP4 plays a key role in both the physiological and the pathological glymphatic system, one of the most interesting and appealing approaches, as well as the purpose of this study, is to investigate the changes in AQP4 concentration in the CSF, obtained by lumbar puncture, that is routinely performed when a neurodegenerative disease is suspected.A total of 124 subjects were recruited for this study, 103 of whom were patients undergoing neurological workup at the Neurodegenerative Diseases Unit of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico in Milan , between January 2012 and February 2020, whereas 21 were healthy controls. The clinical workup included past medical history; general and neurological examination; neurocognitive assessment—including neuropsychological tests such as Mini-Mental State Examination (MMSE); magnetic resonance imaging; and lumbar puncture. Control subjects underwent lumbar puncture in suspicion of pathologies of the CNS and were discharged with no evidence of CNS involvement. Therefore, we considered them as controls.Subjects were assessed at baseline and during the follow-up and diagnoses were established according to multiparametric criteria: the International Working Group 2 (IWG-2) Criteria for Alzheimer’s Disease Diagnosis  for diagLumbar puncture was performed in the L3/L4 or L4/L5 interspace between 8 and 10 am after a night of fasting. The samples were later centrifuged at 1500 × g for 10 min at 4 °C. The supernatants were aliquoted in polypropylene tubes and stored at − 80 °C until use. Before freezing them, the samples were analysed and CSF cell count, glucose, and total protein were calculated. CSF levels of Aβ1–42, Aβ1–40, tau, and P-tau of 101 subjects were assessed using the method ChemiLuminescence Enzyme ImmunoAssay (CLEIA) by a Lumipulse G600II platform (Fujirebio).These evaluations allowed to stratify patients according to their pathophysiological background basing on the amyloid/tau/neurodegeneration (ATN) classification of the National Institute on Aging – Alzheimer's Association (NIA-AA) Research Framework. This classification is based on dichotomous categories  of Aβ (A), tau (T), and neurodegeneration (N) biomarkers . CSF conwww.cusabio.com).AQP4 concentration in the CSF was determined with immunoassay from Cusabio for the whole population considered for the study; quantitative enzyme immunoassay technique includes an AQP4 antibody pre-coated into a microplate, about 100 μl of standards and samples are pipetted into the wells and each AQP4 present is bound by the immobilised antibody. After removing any unbound substances, a biotin-conjugated antibody specific for AQP4 is added and then, after washing, avidin conjugated Horseradish Peroxidase (HRP) is also added followed by the substrate solution and the colour developed is in proportion to the amount of AQP4 bound in the initial step. The optical density was determined using a micro reader set to 450 nm. The sensitivity of this assay or Lower Limit of Detection of human AQP-4 is typically less than 39 pg/ml as reported by the manufacturer (https://www.jamovi.org) and JASP v 0.16.3 (https://jasp-stats.org/). AQP4 and other CSF biomarker levels, demographic parameters such as age and disease at the time of the lumbar puncture, and educational stage as well as MMSE score were all considered continuous variables and expressed as mean ± standard error of the mean (SEM). Conversely, patients’ sex, diagnostic group, and the dichotomised classification on AQP4 levels compared to the median value were all considered categorical variables. Significant statistical threshold was set at 0.05. Shapiro–Wilk’s test of normality was performed for continuous variables, and intergroup comparisons were carried out using the non-parametric Kruskal–Wallis test and Mann–Whitney test. Spearman’s correlation was used to assess the association between CSF biomarkers. Multivariate linear regressions were used to explore the relation between different CSF biomarkers, and demographics were used as covariates. Categorical variables were analysed using the chi-squared test. With respect to the patients’ progression in MMSE scores for at least 4 years of follow-up, Kaplan–Meier estimators were realised, considering the conversion to dementia (from a MMSE score ≥ 24 to a MMSE score < 24) as the main event.Data were analysed using the statistical spreadsheets Jamovi v 1.8.1 , patients with cognitive deficits but with no diagnosis of neurodegenerative disease (MCI and psychiatric patients), and controls. Finally, only two groups were analysed and compared to each other: one was composed of patients with a diagnosis of neurodegenerative disease, whereas the other was obtained by gathering all the other subjects. Furthermore, the NIA-AA ATN classification was considered, and 101 of the 124 patients—the ones whose concentrations of specific proteins measured using CLEIA were available—were divided into groups according to A, T, and N parameters .p = 0.034) and in MMSE score between AD patients and the not neurodegenerative groups , between subjects affected by FTD and controls , as well as between MCI groups and controls (p = 0.035). These differences maintained their significance when the main groups were considered .Principal sociodemographic and clinical characteristics of studied subjects are summarised in Table p < 0.001) with a mean of 296.6 ± 16.0 pg/ml. There was no correlation between AQP4 concentration and demographic parameters  except for MMSE score at the time lumbar puncture was performed (Spearman’s correlation: rho =  − 0.160 p = 0.038).AQP4 levels were not normally distributed (Shapiro–Wilk p = 0.046), but no other statistically significant variations were obtained   and the same result was obtained when only two groups were considered, i.e., the neurodegenerative (mean = 337.9 ± 24.9 pg/ml) versus not neurodegenerative subjects (mean = 243.1 ± 15.3 pg/ml). Mann–Whitney test between these two groups was significant, with a p = 0.010    Fig. .Fig. 3Bo10); however the distribution was still non-normal; hence, non-parametric testing was preferred.At visual inspection, the distribution of CSF AQP4 concentrations was positively skewed; hence, for correlation and regression analysis, data were log-transformed , but no other significant correlations were observed, neither positive nor negative .A significant positive correlation was observed between Logχ2 = 7.99, p = 0.005).The same evidence was demonstrated with chi-squared association test, when comparing the ATN groups with the AQP4-High and AQP4-Low group, defined as previously mentioned . Log10 AQP4 levels were positively correlated with total tau (Log10 AQP4 and Tau p = 0.026). No significant correlation was obtained when considering Log10 AQP4 and Aβ protein in any of its subunits  , 41 AD, and 32 non-AD dementia . The stuFor what concerns the causes behind this evidence, not enough is known, so only assumptions can be made. One possible explanation is that reactive gliosis, a common feature in glymphatic system degeneration , 25, migBoth these interpretations are reinforced by the association between AQP4 levels and total tau concentration found in this study, which is the very indicator for neurodegeneration in the ATN classification , 27, 28.No significance was found when comparing AQP4 and Aβ. Currently, the reasons behind this evidence are not known and probably involve a pathogenetic link between the two molecules. Another possible explanation may lie in the fact that patients suffering from FTD, who show hallmarks of neurodegeneration, do not necessarily have amyloid burden as well , so in sThe Kaplan–Meier indicators, instead, proved no significant difference in predicting the rate of conversion to dementia. This may be the consequence of setting the median value, an arbitrary cut-off, as a discriminant for the AQP4 groups, instead of a real pathological threshold, which has not yet been identified.Our findings are consistent with previous results concerning the expression of AQP4 in postmortem frontal cortex of cognitively healthy and histopathologically confirmed individuals with AD, in particular increasing AQP4 expression and loss of perivascular astrocytic endfeet localisation of AQP4 are associated to AD pathology .Nevertheless, it is correct to underline that, although it is widely acknowledged that CSF exchanges with the brain interstitial fluid through the perivascular spaces , the exiThe limitations of this study are the rather small sample size for the statistical analysis, although considering that we studied CSF, numbers seem reasonable. In particular, the small number of control subjects did not allow to identify an effective AQP4 cut-off value; therefore, we limited to use AQP4 median value to artificially stratify the population into subjects with high and with low levels of AQP4. A study with larger groups of patients and controls is needed to confirm these preliminary data and to identify an AQP4 cut-off value.In addition, the choice of a case–control study to investigate a potential biomarker, undoubtedly not as effective as a prospective cohort study would be, could explain the lack of significance in the Kaplan–Meier indicators. Furthermore, despite being two of the most common causes of neurodegeneration, AD and FTD are not the only diseases with that pathological feature , 36. TheThis study has several positive aspects as well: firstly, it is one of the first analyses that tries to identify an easily accessible biomarker to characterise the newly described glymphatic system, as lumbar puncture is already being used in the diagnostic workup for people with suspected dementia . FurtherOverall, these data show that AQP4 is in fact a very good potential biomarker for neurodegeneration and possibly, given its fundamental role in the glymphatic pathway , for theAnother interesting perspective is that using AQP4 as a marker of glymphatic functionality could help identify those common neurological diseases in which the system is compromised: for example, this study showed that, compared to controls, patients suffering from MCI or psychiatric disorders displayed a slightly, yet not significantly, increased levels of AQP4 Fig. . StudiesFuture perspectives also include the description of normal values for this protein, since there are very few studies which focused on this task –24, and,This study proves that a fundamental protein for glymphatic system function and CSF and ISF flow, AQP4, is increased in CSF of patients suffering from different types of dementia compared to healthy controls, and strongly correlates with a marker for established neurodegeneration such as tau. This piece of evidence leads to a hypothesis that AQP4 could represent a fascinating potential biomarker for future studies on dementia as well as glymphatic function, and analysing this protein and its level in body fluids may help explain the pathogenesis of these diseases and hopefully provide a new tool for both diagnosis and prognosis in clinical practice.


Xeruborbactam (XERU) is a member of a new class of cyclic boronic acid β-lactamase inhibitors with inhibitory activity against major members of Class A, B, C, and D beta-lactamases. This report describes the safety and pharmacokinetics of XERU following multiple doses alone and in combination with meropenem.Twenty-four healthy subjects were enrolled into one of 3 cohorts with XERU alone. XERU was administered as a 500 mg loading dose followed by 250 mg q8h or 1000 mg loading dose followed by 500 mg q8h. 15 healthy subjects were enrolled in a single cohort of meropenem alone administered as a 4000 mg loading dose followed by 2000 mg q8h or meropenem in combination with XERU administered as a 500 mg loading dose followed by 250 mg q8h for 10 days. Intensive plasma sampling was obtained after dosing and assayed for drug content using validated LC-MS/MS methods.XERU and Meropenem Steady State pharmacokinetic parameters are shown in the table below.No subjects discontinued due to AEs and no SAEs were observed. There was no evidence of increasing numbers or severity of AEs with increasing dose. All AEs were mild or moderate in severity.XERU was safe and well-tolerated when administered alone and in combination with meropenem. XERU has unique PK properties that includes a long elimination half-life that provides sustained plasma concentrations. The loading dose of XERU allows for rapid achievement of steady state plasma levels of XERU within the first day of treatment. XERU plasma PK properties, along with a broad spectrum of inhibitory activity, facilitates its use with multiple beta-lactam antibiotics including meropenem.David Griffith, n/a, Qpex Biopharma: Employee Jason Roberts, BPharm(Hons), PhD, FSHP, FISAC, British Society of Chemotherapy: Grant/Research Support|Cipla: Honoraria|Gilead: Advisor/Consultant|MSD: Advisor/Consultant|MSD: Honoraria|Pfizer: Board Member|Pfizer: Honoraria|Qpex: Grant/Research Support|Sandoz: Board Member|Summit: Advisor/Consultant|Wolters Kluwer: Advisor/Consultant Elizabeth Morgan, n/a, Qpex Biopharma: Employee Shawnee Gehrke, n/a, Qpex Biopharma: Employee Michael Dudley, n/a, ArrePath: Board Member|Qpex Biopharma: Board Member|Qpex Biopharma: Employee Jeff Loutit, MBChB, Qpex Biopharma: Employee.


There was a strong positive correlation between the number of T2 lesions and CSF NfL level  in the MS group. There was excellent consistency between ELISA and SIMOA for CSF samples, but not for serum samples. Our results indicated that CSF NfL levels may also be used in the management of NDCNS and that SIMOA is the most reliable method for serum NfL determination.The neurofilament light chain (NfL) is a promising biomarker in the diagnosis, prognosis, and treatment response evaluation of neurological diseases. The aims of this study were to compare the cerebrospinal fluid (CSF) NfL levels in multiple sclerosis (MS) and certain non-demyelinating diseases of the central nervous system (NDCNS); to determine the relationship between clinical and radiological features and CSF NfL levels in patients with MS; and to compare the enzyme-linked immunosorbent assay (ELISA) and single molecule array (SIMOA) methods for NfL measurement using paired CSF and serum samples. We retrospectively analyzed the clinical data and performed NfL measurements in CSF and serum samples of newly diagnosed and treatment-naive patients with CNS diseases evaluated between 1 January 2019 and 1 January 2020. Eligible patients were divided into three groups: MS (n = 23), differential diagnosis of MS (n = 19), and NDCNS (n = 42). First, we compared the CSF NfL levels among the three groups using the previously validated CSF ELISA assay. Next, we evaluated the relationship between CSF NfL levels and the clinical and radiological findings in MS group. Finally, we compared CSF and serum samples from patients of the MS groups  using two different methods (ELISA and SIMOA). The CSF NfL level was the highest in the NDCNS group (1169.64 [535.92−5120.11] pg/mL,  In the management of neurological diseases, reliable and easily accessible biomarkers are necessary for establishing the diagnosis, evaluating the prognosis, and monitoring the response to treatment . IdeallyMultiple sclerosis (MS) is a chronic inflammatory, autoimmune, demyelinating disease of the CNS, which has several subtypes, including relapsing-remitting MS (RRMS) and progressive MS (primary progressive MS (PPMS), secondary progressive MS (SPMS)). Even though the presentation of the disease is mostly relapsing-remitting (85%), ~15% of it is progressive from the onset. Both oxidative stress and neuroinflammation play a pivotal role in the pathophysiology of MS. As a result of these pathological events, axonal damage is unavoidable in the end ,3.The NfL is a subunit of neurofilaments, which are cylindrical proteins found in the neuronal cytoplasm that maintain axonal stability. Although neurofilaments are present in dendrites and neuronal soma, their expression is particularly high in axons . BecauseAs CSF and blood levels of NfL have been shown to increase particularly in neurodegenerative diseases and NfL has been shown to be associated with some disease characteristics, there is an increasing number of studies investigating this relationship. Although CSF is the most valuable specimen type in terms of reflecting physiological or pathological events in the CNS due to its neighborhood with the brain parenchyma, blood samples are more useful in terms of being more easily accessible. Therefore, there is a need for reliable methods to determine blood NfL levels.Sandwich enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence, and high-sensitive single molecule array (SIMOA) methods have been used in different studies for NfL measurements. While ELISA has limited sensitivity and can reliably measure NfL levels only in the CSF, SIMOA measures serum NfL levels with high sensitivity even in healthy people . NonetheThe role of NfL in the diagnosis, prognosis, and treatment response evaluation has been investigated mostly in patients with MS, which is one of the neuroinflammatory diseases ,10-12, aTherefore, in this study, we employed clinical and biochemical approaches to investigate the role of NfL both in MS and non-demyelinating diseases of the CNS. The aims of this study were: (1) to compare the CSF NfL levels in MS and non-demyelinating diseases of the CNS, with emphasis on malignant and benign CNS tumors, using a previously validated CSF ELISA kit ; (2) to This study included a retrospective analysis of the clinical data and post-hoc NfL measurements in CSF and serum samples of newly diagnosed and treatment-naive patients with CNS diseases evaluated between 1 January 2019 and 1 January 2020. Patients who had received steroid treatment in the last month or immunomodulatory therapy in the past three months before sample collection were excluded from the study.Based on the diagnosis, patients were divided in three groups: newly diagnosed MS, differential diagnosis of MS, and non-neuroinflammatory and non-demyelinating diseases. Since appropriate group selection, proper uniform definitions, and terminology are critical in CSF biomarker studies, the groups were renamed. Definitions in the previously published consensus guideline were used to identify and rename control groups . The difThe following criteria were used for the diseases included in the differential diagnosis of MS group (inflammatory disease controls (INDCs)), respectively: (1) Patients who did not comply with the diagnosis of MS clinically and radiologically; and (2) did not meet the McDonald’s diagnostic criteria. In their first clinical attack, these patients prospectively applied to our neurology clinic and were included in this study. First, anti-aquaporin-4 and anti-myelin-oligodendrocyte-glycoprotein antibodies tests were performed in all of these patients whose diagnosis was suspected, and neuromyelitis optica spectrum disorder and myelin oligodendrocyte glycoprotein antibody-associated disease were excluded. Then, diseases that could be confused with MS radiologically were investigated. The main ones were systemic lupus erythematosus, CNS manifestations of primary antiphospholipid syndrome, rheumatoid arthritis, Sjögren’s syndrome, and sarcoidosis. While some of this groups were diagnosed with CNS involvement of certain rheumatological diseases , the rest could not be included in any group and possible clinical isolated syndrome (n = 7) recovered after the first clinical attack and are still being followed up.MS diagnosis was determined based on the revised McDonald criteria , and disWe reviewed the patients’ medical records and collected the following data: demographic ; clinical ; laboratory (serum and CSF examinations); and radiological data (number of T2 lesions). All MRI images were analyzed by an experienced neurologist. The number of T2 lesions was counted on T2-weighted and/or fluid-attenuated inversion-recovery magnetic resonance images. In the brain, T2 lesions >3 mm in diameter were counted in fluid-attenuated inversion-recovery (FLAIR) images using T2-weighted and proton density-weighted spin-echo images as an aid. T2 lesions were also counted in the spinal cord if they were present. Gadolinium enhancing lesions were not counted in all patients, and there are missing data in our data set.Serum and CSF samples were obtained from the Department of Neurology of the Hacettepe University School of Medicine. All lumbar puncture procedures were performed at the same time of the day to exclude the influence of the circadian rhythm. CSF samples with blood contamination were excluded from the study.Anti-aquaporin-4 and anti-myelin-oligodendrocyte-glycoprotein antibodies were confirmed twice (>1:40 titer) using a commercial fixed cell-based assay . This analysis was performed at the time of first diagnosis of MS to exclude certain diseases, such as neuromyelitis optica spectrum disorder and myelin oligodendrocyte glycoprotein antibody-associated disease.CSF samples were centrifuged at 400 g for 10 min, and blood samples at 2.000 g for 10 min at room temperature within an hour. After centrifugation, samples were stored as aliquots in 1.5-mL polypropylene Eppendorf tubes at −80°C at the Department of Biochemistry of the Gazi University Faculty of Medicine until the time of analysis. Paired CSF and serum samples were transferred to the Neuroimmunology Laboratory of the University Hospital Basel, Switzerland for SIMOA analysis. Other analyzes were performed using ELISA at the Medical Biochemistry Research Laboratory of the Gazi University Faculty of Medicine.http://www.turkbiyokimyadernegi.org.tr/turkbiyokimyadernegi/vucut-sivilari-analizi-calisma-grubu).Previously published biobanking procedures were considered as references for processing the CSF samples from all patients and the paired serum samples from certain patients . In Turk® ELISA Kit , which has been previously validated for CSF samples. Intra- and inter-assay coefficients of variability (CV%) were <5 and <10, respectively. The measuring range was 100−10.000 pg/mL, with a limit of detection at 33 pg/mL. Before the analyses, all CSF samples were diluted with sample diluent at a ratio of 1:2.For ELISA analysis of CSF samples, we used the NF-lightFor ELISA analysis of serum samples, we used the Human Neurofilament Light Polypeptide ELISA Kit . Intra- and interassay CV% were <10 and <12, respectively. The measuring range was 15.6−1.000 pg/mL, and the limit of detection was < 6.2 pg/mL.® NF-Light assay  was used for both serum and CSF analysis. All analyses were performed on the HD-X Analyzer™ by running the samples in duplicate. Intra- and interassay CV% were within acceptable limits, with a limit of detection at 0.038 pg/mL. SIMOA analyzes were performed at the Neuroimmunology Laboratory of the University Hospital Basel, Switzerland.For SIMOA analysis, high-sensitivity SIMOAThis research involving human subjects complied with all relevant national regulations and institutional policies and was conducted in accordance with the tenets of the Helsinki Declaration (as revised in 2013). This study was reviewed and approved by the Ethics Committee of the Gazi University Faculty of Medicine . Written informed consent was obtained from all participants included in this study.All analyzes were performed using IBM SPSS Statistics for Windows, version 21.0 . GraphPad Prism software  was used for graphical demonstrations.p < 0.05 were considered significant.Categorical variables were presented as frequencies (n) and percentages (%), while ordinal variables were described by medians and interquartile ranges or means and standard deviations for Gaussian distributed data. The normality of the distribution of numerical variables was examined using the Shapiro-Wilk’s tests. Spearman’s correlation test was used to analyze the correlations of serum and CSF biomarker levels with numeric clinical variables in all patients and within each disease. Mann–Whitney’s test was used to compare two independent groups on a quantitative variable. The Kruskal–Wallis test was used to compare independent k groups (k > 2) on a quantitative variable. The patients’ baseline demographic characteristics were compared using Fisher’s exact test or Wilcoxon’s test. Analysis of covariance (ANCOVA) was performed considering CSF NfL levels as dependent variables, groups  as fixed variables, and age as a covariate to examine differences between serum biomarker levels among the various groups. Variables with a two-tailed For comparison of the ELISA and SIMOA methods, the NfL results obtained by the two methods were compared using the paired samples t-test. Consistency was evaluated using Passing-Bablok regression analysis and the relationship between them was expressed as the correlation coefficient. The results were evaluated visually using a Bland–Altman plot.The comparison of CSF NfL levels among the three groups and the analysis of their relationship with the clinical and radiological features in patients with MS was performed in a total of 84 CSF samples. There were 23 patients in the newly diagnosed MS group . None of these patients were using immunomodulatory therapy. In the second group, there were 19 patients with a differential diagnosis of MS. The non-neuroinflammatory and non-demyelinating diseases group (n = 42) was divided into five subgroups: malignant brain tumors (n = 4), benign brain tumors (n = 12), hydrocephalus (n = 7), differential diagnosis of headache (n = 9), and other  (n = 10). For the comparison of the ELISA and SIMOA methods, a total of 19 paired serum and CSF samples (RRMS = 19) were used.p = 0.012), and the patients in the non-demyelinating diseases of the CNS group had the highest mean age (45.85±15.62 years). The index immunoglobulin G levels were higher in the MS group than in the differential diagnosis of MS group . The CSF NfL level was the highest in the non-demyelinating diseases group , with subgroup values as follows: malignant brain tumors, 13099.84 ± 4507.57 pg/mL; benign brain tumors, 3033.50±3028.41 pg/mL; hydrocephalus, 3049.94±2937.34 pg/mL; differential diagnosis of headache, 3502.91 ± 4364.67 pg/mL; and other, 940.60±945.89 pg/mL. The mean CSF NfL level of the malignant brain tumors subgroup was significantly higher than those of the other subgroups (p = 0.005). When age was considered as a covariate variable, the main effect of the groups on the CSF NfL value was still found to be statistically significant . The CSF NfL results and the values of some biochemical parameters are presented in The demographic characteristics of the patients are shown in p < 0.05; r = 0.547, p < 0.05; and r = 0.525, p < 0.05, respectively; p < 0.05; r = 0.388, p < 0.05; and r = 0.598, p < 0.05, respectively). There was a strong positive correlation between the number of T2 lesions and CSF NfL levels . The correlation between biochemical markers and clinical features is shown in A statistically significant positive correlation was found between age at sampling and the EDSS score, CSF protein levels, and CSF NfL levels , p < 0.001). The intercept A and slope B values were 169.0023 (95% CI 38.1851−469.4667) and 0.7790 (95% CI 0.5870−1.0792), respectively. The paired samples t-test result indicated that there was no statistically significant difference between the two methods for CSF samples (p = 0.752). There was consistency between SIMOA and ELISA for CSF NfL according to the slope, intercept, and confidence interval values pertaining to these values. The mean CSF NfL values were 1269.96± 991.10 and 1239.52± 693.05 for SIMOA and ELISA, respectively. For serum samples, based on the regression analysis, the equation was found as y = -126.798808 + 22.433071 x , p = 0.531). The intercept A and slope B values were -126.7989 (95% CI −693.7511–−26.3872) and 22.4331 (95% CI 12.3235−76.4909), respectively. The paired samples t-test results indicated that there was a statistically significant difference between the two methods for serum samples (p < 0.001). There was no consistency between SIMOA and ELISA for serum NfL according to the slope, intercept, and confidence interval values belonging to these values. The mean serum NfL values were 16.83 ± 23.53 and 97.35±71.58 for SIMOA and ELISA, respectively.Regression analysis for CSF samples yielded the equation: y = 169.002271 + 0.779003 x (rIn this study, we found that CSF NfL levels were highest among patients with non-demyelinating diseases of the CNS. Furthermore, there was a strong positive correlation between the number of T2 lesions in patients with MS and CSF NfL levels. In addition, our results indicated that SIMOA is the most reliable method for serum NfL determination.Although many studies have investigated the NfL and its role in disease diagnosis, prognosis, and treatment response -23, studFurthermore, we found that CSF NfL levels in patients evaluated for differential diagnosis of headache were lower in patients who were pathologically diagnosed with a benign lesion. This suggests that CSF NfL levels may be used to differentiate benign from malignant lesions. In addition, CSF and serum NfL level monitoring may be helpful to indicate the existence of a pathological brain lesion in patients with clinical but no radiological findings. This may enable early suspicion and diagnosis of malignant tumors that may be overlooked radiologically based on clinical findings and NfL levels.Our results also showed that CSF NfL levels were correlated with age, EDSS scores, the number of relapses, and the number of T2 lesions in patients with MS, which is in agreement with the findings of previous studies -26. ThisAt present, there are many commercially available ELISA kits from different companies that measure serum NfL levels. In this study, we compared the ELISA and SIMOA methods and found that there was very good consistency between them in terms of CSF NfL levels. However, there was no consistency between the two methods for serum samples. This indicated that commercially available ELISA kits for serum NfL measurement are not reliable and that SIMOA remains the most reliable and precise method for serum NfL measurement. SIMOA’s superiority has been shown in previous studies .It has been shown in the previous studies that NfL is released in increased amounts with aging ,28. EspePathologically, MS is characterized by different types of lesions that can be staged by the presence and morphology of microglia/macrophages in relation to demyelination. Previous studies investigated the role of CSF and serum/plasma NfL measurements in MS by combining the conventional MRI and clinical features. What is new about axonal damage is the question if neuropathological properties of inflammatory lesion activity, neuroaxonal injury, or neurodegeneration correlate with axonal damage, as reflected by CSF NfL levels in MS ,30. Van The importance of neurobiomarkers was highlighted in a recent article . In thisOur study had some limitations. First, we could analyze only a proportion of the CSF samples using SIMOA due to the related financial burden. However, we plan to expand this study in a larger patient cohort by introducing this ultrasensitive method, which has a great role in neurobiomarker research, in our country. Second, due to ethical considerations in our country, we could not obtain CSF samples from healthy controls for comparison to those of different patient groups. Third, in patients with brain tumors, only CSF samples obtained before surgery were available. Therefore, we could not compare the pre- and post-operative CSF NfL levels. Besides, active Gadolinium enhancing lesions represent a more robust radiological activity parameter. We could not include these parameters because of missing data.This was the first study in Turkey to examine NfL levels in CSF and serum samples using the SIMOA method. Our results indicated that CSF NfL levels may be used not only in the follow-up of demyelinating, but also in the management of non-demyelinating CNS diseases. Moreover, we have once again shown that SIMOA is the most reliable method for serum NfL levels determination. Nonetheless, further studies with larger samples are needed to investigate NfL levels in the serum and CSF and their role in non-demyelinating CNS diseases, such as metastatic or non-metastatic brain lesions.


Background: Muscular-activity timing is useful information that is extractable from surface EMG signals (sEMG). However, a reference method is not available yet. The aim of this study is to investigate the reliability of a novel machine-learning-based approach (DEMANN) in detecting the onset/offset timing of muscle activation from sEMG signals. Methods: A dataset of 2880 simulated sEMG signals, stratified for signal-to-noise ratio (SNR) and time support, was generated to train a hidden single-layer fully-connected neural network. DEMANN’s performance was evaluated on simulated sEMG signals and two different datasets of real sEMG signals. DEMANN was validated against different reference algorithms, including the acknowledged double-threshold statistical algorithm (DT). Results: DEMANN provided a reliable prediction of muscle onset/offset in simulated and real sEMG signals, being minimally affected by SNR variability. When directly compared with state-of-the-art algorithms, DEMANN introduced relevant improvements in prediction performances. Conclusions: These outcomes support DEMANN’s reliability in assessing onset/offset events in different motor tasks and the condition of signal quality (different SNR), improving reference-algorithm performances. Unlike other works, DEMANN’s adopts a machine learning approach where a neural network is trained by only simulated sEMG signals, avoiding the possible complications and costs associated with a typical experimental procedure, making this approach suitable to clinical practice. Assessing muscle-recruitment timing is relevant in different fields, including clinical gait analysis and electromyography-driven assistive devices ,2. TradiMachine/deep learning has proven to be effective in interpreting sEMG signals for different purposes , such asIn spite of the presence of a large literature on the machine-learning based interpretation of sEMG signals, this approach is scarcely adopted to face the challenge of assessing the timing of muscle activation. The problem to solve is essentially an sEMG-based prediction of a transition between the period when the muscle is silent and the period when the muscle is active, i.e., to discriminate between actual sEMG activity and noise. Given that, the possibility of adopting a machine learning approach that learns to interpret the shape of the sEMG signals for assessing muscle-activation onset and offset seems to be a feasible solution. A very recent study proposed by Ghisleri et al. adopted long short-term memory (LSTM) recurrent neural networks (RNN) for detecting muscle activity . Very enTo develop a novel high-performance approach (DEMANN) that contributes to support the use of machine learning for muscle activity detection;To highlight the advantages of the proposed machine-learning approach, such as the possibility of real-time applications, achieved without loss of accuracy and with respect to existing, non-machine-learning-based systems;To limit the deterioration of event assessment associated with low SNRs and the large inter-signal variability of SNR, typical of clinical environments, by training the model with simulated sEMG signals with a large range of SNR values;To reduce the complexity of the experimental protocol associated with model training, since no signal acquisition is needed to provide real time activation predictions.The goal of the present study was to investigate the suitability of a novel machine-learning-based approach in assessing the onset-offset timing of muscle activation, i.e., the Detector of Muscular Activity by Neural Networks (DEMANN). Specifically, the present approach aimed to predict both onset and offset timing using only simulated sEMG signals with a large range of SNR values for neural network training in order to explore a large range of SNRs without deterioration, which is often encountered in clinical environments. This aspect, together with the simple architecture of the neural network (based on a multi layer perceptron), should help to provide fast training and prediction, making this approach very suitable for clinical purposes. Thus, the main contributions that the present study would like to provide could be summarized as follows:The robustness of DEMANN was evaluated by a test bench of simulated sEMG signals and two datasets of real sEMG signals. Simulated and real sEMG signals underwent the same procedure described in the following sections. DEMANN was validated by a direct comparison with reference approaches on both simulated and real data.A simulation study, using a test bench of signals, was carried out for assessing the performance of the DEMANN approach in predicting onset and offset events of muscular activity. sEMG signals acquired during cyclic movements could be modeled as the superimposition of the actual signal produced by muscle contraction and the background noise . In thiser level . This dier level . This GaSimulated sEMG signals were generated from all the different combinations of the values adopted for σ , for α , and SNR values from 1 dB to 30 dB, with step = 1. In  range, and a concatenation of outputs of the different processing procedures . These cThe classifier was a hidden single-layer (32 units) fully-connected neural network. A Rectified Linear Unit (ReLU) activation function was used, and a sigmoid function was adopted to map the network output to a 0–1 interval. The binary output was achieved by using a standard threshold of 0.5. The model was trained with a learning rate of 0.001, a batch size of 512 for 40 epochs using the standard stochastic gradient descent (SGD) optimization algorithm, and by adding a L2 regularization penalty set to 0.0001. The training set was composed of only simulated sEMG signals: 8 signals for each combination of σ  and α  were chosen, for a total of 96 signals for each SNR. Considering 30 SNR values , a total of 2880 simulated signals were included.The classifier performances were evaluated on three different testing sets. The first one was composed of only simulated sEMG signals. Eight signals were generated for each combination of σ, α, and SNR. Nine different SNRs were considered, specifically 3, 6, 10, 13, 16, 20, 23, 26, and 30 dB, as suggested in [The second test set was composed of 103 real sEMG signals proposed in [The third test set included foot–floor contact and sEMG data collected during 30 healthy adults walking, as described in For all the three test sets, model validation and performance were computed in signals never used during the training of the model.T of 100 ms was adopted, as suggested in [pt if an event of the same kind occurred in the ground-truth signal at time gt, such that To achieve the model output, segmented sEMG signals were provided as input to the trained model. Thus, the model output was composed of sequences of 0 (no muscle activity) alternating with sequences of 1 (muscle activity). This signal was chronologically scanned to identify the transitions between the two conditions: the transition from 0 to 1 identified the onset event and the transition from 1 to 0 detected the offset event. This was achieved by the following procedure: a time tolerance t motion . Thus, st-test was applied to verify the significance of difference between the normally-distributed samples. The Mann-Whitney test was applied to verify the significance of difference between the non-normally-distributed samples. Statistical significance was established at 5%.The Shapiro-Wilk test was adopted to appraise the normality of data distribution. A two-tailed, non-paired Student’s The mean classification accuracy computed in the testing set stratified for different SNR is shown in While in the present study, a shallow neural network was used as a classifier, the DEMANN approach can be flexibly modified to embed a different machine-learning model. Support vector machines (SVM) are identified in literature as suitable modeling tools ,23,24,25p < 0.05) was provided by the DEMANN approach for both onset and offset timing. No significant differences were detected in precision, recall, or F1-score between the performances of the two models.A significantly lower mean MAE  of recall and F1-score was provided by DEMANN, for onset and offset prediction. This improvement (p < 0.05) was preserved also considering signals from a single muscle, for both TA and for GL. No significant differences (p > 0.05) were identified in the VL signals and for all the prediction parameters.A second validation was performed on the sEMG dataset acquired during walking , with a The present study was designed to test the capability of a novel machine-learning-based approach of estimating onset and offset timing of muscle activation. One of the main advantages of the present DEMANN approach is that the neural network was trained by means of only simulated sEMG signals , thus avoiding all the possible complications and costs associated with a typical experimental procedure. A further advantage was the running time. Without considering the processing time, which depends on the processing capability of the running device , once the model was trained, the maximum delay of activation prediction was 10 ms (the size of the windows). Although this paper did not explicitly target real-time applications, such a delay can be acceptable even under real-time constraints , making In the present study, DEMANN proved to provide high performances in three different datasets: (1) a test bench of 864 simulated sEMG signals; (2) 103 real sEMG signals acquired in vastus lateralis during knee extension and in biceps brachii during elbow flexion; and (3) real sEMG signals from gastrocnemius lateralis, tibialis anterior, and vastus lateralis collected during 30 subjects walking. Details are reported in the following two sections.DEMANN provided a high classification performance, quantified by a mean accuracy (±SD) of 97.8 ± 3.0% and supported by the accuracy = 95.3% in the worst-case scenario . This was true also considering each single SNR value (The efficiency of the DEMANN approach was firstly proved versus a different machine-learning model. The support vector machine (SVM) was chosen among the models proposed in the literature as a suitable tool for this purpose ,23,24,25NR value D; the reNR value C, no sigOne of the most reliable sEMG timing detectors reported in the literature is the wavelet-based approach described in . In thatThe dataset introduced in  was mainp < 0.05, p < 0.05) also considering the mean values over the signals from the same muscle, in the case of TA and GL. Otherwise, for VL, no significant difference was detected between the two approaches. TA and GL are mainly ankle flexor muscles and VL is a knee extensor; it is acknowledged that ankle muscles are typically more involved in the walking task [Since walking is one of the most useful tasks to obtain insights on human movement, DEMANN was tested also on a dataset of sEMG data collected during 30 healthy adults walking. Despite the high data variability due to curves, reversing, deceleration, and acceleration during the eight-shaped path, prediction performances were >90% for both the onset and offset prediction . Performing task . Given tThe present outcomes suggest the feasibility of predicting onset-offset timing of muscular recruitment of the proposed machine-learning-based method, which was able to provide high performances also in condition of large variability of the sEMG signal. The adoption of DEMANN introduced several further advantages, such as a running time compatible with real time applications, a small deterioration of event detection due to low SNR values and to a large within-signal variability of SNR, and reduced complexity of the experimental protocol associated with model training, since no real signal is needed. All these advantages make this approach suitable for clinical practice and for being included in the procedure for controlling sEMG-driven assistive devices, such as orthoses and exoskeletons.The DEMANN approach was validated in simulated sEMG signals and in real sEMG signals acquired in young able-bodied subjects, but not in elderly and pathological populations. This is acknowledged as a limitation of the present study. Future studies will be focused on assessing the reliability of the DEMANN approach to provide a robust prediction of activation events also in these populations and on the possible improvements to implement for adapting the model to different conditions and environments. While the present study showed that relatively simple supervised methods, such as shallow neural networks, can be suitable for muscle activation detection, further experiments should be made to determine an optimal classifier to embed in the detecting system.